Effects of azithromycin on human neutrophils
Transcript of Effects of azithromycin on human neutrophils
University of Calgary
PRISM: University of Calgary's Digital Repository
Graduate Studies Legacy Theses
1999
Effects of azithromycin on human neutrophils
Koch, Crystal Coco
Koch, C. C. (1999). Effects of azithromycin on human neutrophils (Unpublished master's thesis).
University of Calgary, Calgary, AB. doi:10.11575/PRISM/13027
http://hdl.handle.net/1880/25122
master thesis
University of Calgary graduate students retain copyright ownership and moral rights for their
thesis. You may use this material in any way that is permitted by the Copyright Act or through
licensing that has been assigned to the document. For uses that are not allowable under
copyright legislation or licensing, you are required to seek permission.
Downloaded from PRISM: https://prism.ucalgary.ca
THE UNNERSITY OF CALGARY
Effects of Azitbromycin on Human Neutrophils
by
Crystal C . Koch
A THESIS
SUBMITTED TO THE FACULTY OF GRADUATE STUDIES
IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE
DEGREE OF MASTER OF SCIENCE
DEPARTMENT OF BIOLOGICAL SCIENCES
CALGARY, ALBERTA
JULY, 1999
O Crystal C. Koch 1999
National Library 1*1 of Canada Biblioth&que nationale du Canada
Acquisitions and Acquisitions et Bibliographic Services services bibliographiques
395 Wellington Street 395. rue Weflington Ottawa ON K IA ON4 OnawaON K l A W Canada Canada
Your N. vmro rlhrmcr
Our rVs Norrr remmtx
The author has granted a non- exclusive licence allowing the National Library of Canada to reproduce, loan, distribute or sell copies of this thesis in microform, paper or electronic formats.
The author retains ownership of the copyright in this thesis. Neither the thesis nor substantial extracts fkom it may be printed or otherwise reproduced without the author's permission.
L'auteur a accorde une licence non exclusive pennettant a la Bibliotheque nationale du Canada de reproduire, preter, distribuer ou vendre des copies de cette these sous la forme de microfiche/film, de reproduction sur papier ou sur format electronique .
L'auteur conserve la propriete du droit d'auteur qui protege cette these. Ni la these ni des extraits substantiels de celle-ci ne doivent &re imprimes ou autrement reproduits sans son autorisation.
ABSTRACT
Pathogen virulence factors and the host inflammatory response cause tissue injury
associated with respiratory tract infections. Azithromycin has demonstrated efficacy in
treating these infections. We hypothesized that azithromycin may have anti-
inflammatory properties, perhaps through the induction of neutrophil apoptosis. This
study assessed the effects of azithromycin on human neutrophii 1) apoptosis, 2)
phagocytosis by macrophages, 3) oxidative function, and 4) interleukin-8 production in
the presence or absence of Streptococcus pneumoniae compared with penicillin,
erythromycin, dexamethasone or saline. Azithromycin significantly induced neutrophil
apoptosis but this effect was abolished in the presence of ' s pneumoniae. Macrophage
phagocytosis of azithromycin-treated neutrophils was significantly enhanced.
Azithromycin did not affect neutrophil oxidative metabolism or interleukin-8 production.
In summary, azithromycin induces neutrophil apoptosis without affecting neutrophil
function, and enhances phagocytic clearance of these cells by macrophages. These
properties may confer anti-inflammatory benefits to this antibiotic.
ACKNOWLEDGEMENTS
I would like to sincerely thank Dr. Buret for allowing me to work in his lab and
giving me the opportunity to learn many new things and make many lifelong friends.
I would also like to extend a special thanks to AIex Chin and David Esteban for
their invaluable assistance.
Additionally, I would like to thank the multitude of people who gave me help and
advice, including Carol Ann Stremick, Kathy Oke, Kevin Scott, Michaela Waiter, Wilson
Lee, Kathy Murrin, all the other members of the Biofilm Research Group, and alf of my
blood donors.
A very special thanks goes out to Michael Lang for keeping me happy and sane
while conducting this project.
Finally, I would like to thank the Natural Sciences and Engineering Research
Council of Canada, the Alberta Heritage Foundation for Medical Research and Pfizer
Canada for fhding my research.
I dedicate this to my Mom and Dad
who always support everything I do
TABLE OF CONTENTS
APPROVAL PAGE
ABSTRACT
ACKNOWLEDGEMENTS
DEDICATION
TABLE OF CONTENTS
LIST OF FIGURES
LIST OF ABBREVIATIONS
CHAPTER 1: INTRODUCTION
1.1 The Pathogenesis of Respiratory Tract Infection
1.1.1 Bacterial Virulence Factors
1 . 1 . 1 . 1 Adhesion and Uptake
1 . 1 . 1 -2 Cytotoxins
1.1.1.3 Other Factors
1.1.2 Inflammation
I . 1.2.1 Mediators
1.1.2.2 Neutrophils
1.1.2.3 Macrophages
1.2 Pneumonia
v i
iii
1.2.1 Streptococcus pneumoniae
1.3 Treatment
1.3.1 Antibiotics and Antibacterial Agents 15
1.3.1.1 Beta-lactarns I 5
1.3.1.1.1 Penicillin 16
1.3.1.1.2 Cephalosporins 16
1.3.1.2 Sulfonamides I 7
1.3.1.3 Fluoroquinolones 17
1.3.1.4 Tetracyclines 17
1.3.1 -5 Macrolides 18
1.3.1.5.1 Structure 18
1.3.1.5.2 Mode of action (antimicrobial activity) 19
1.3.1 S . 3 Resistance 19
1.3.2 Azithromycin 20
1.3 -2.1 Structure 21
1 -3 -2.2 Pharmacodynamics and Antimicrobial Activity 2 1
1.3.2.3 Pharmacokinetics 23
1 -3 -2.4 Efficacy in Animal Models 27
1.3 -2.5 Clinical Efficacy and Dosage 28
1.3.2.6 Tolerability 30
1.3.2.7 Drug Interactions 3 1
1.3.3 Anti-inflammatory Therapies 32
1.3.4 Immunomodulation by Antibiotics
1.4 Apoptosis
1.4.1 Induction
1.4.2 Neutrophil Apoptosis
1.4.3 Regulation of Apoptosis in Neutrophils
1.5 Research Hypothesis and Objectives
CHAPTER 2: METHODS AND MATERLALS
2.1 Drugs
2.2 Bacteria
2.3 Subjects
2.4 Neutrophils
2.5 Moaocytes/M~crophages
2.6 CFU Counts
2.7 Annexin-Vff ropidium Iodide Labelling
2.8 Cell Death ELISA
2.9 Macrophage Pbagocytosis
2.10 Nitro Blue Tetrazolium Assay
2.1 1 Interleukin-8 Assay
2.12 Statistical Analysis
CHAPTER 3: RESULTS viii
3.1 Cell Isolation
3.2 CFU Counts
3.3 Neutrophil Apoptosis
3.4 Macrophage Phagocytosis
3.5 Oxidative Function and IL-8 Production
CHAPTER 4: DISCUSSION
REFERENCES
APPENDIX A: Structure of Azithromycin
and Erythromycin
LIST OF FIGURES
FIGURE:
1. S. pneumonia recovery after incubation with drugs
2. Light micrographs demonstrating annexin-V/PI staining:
A) bright field; B) fluorescent apoptotic and necrotic PMNs
3. Apoptosis in PMNs treated with TNFa
4. Apoptosis in PMNs treated with drugs via annexin-V/PI:
A) without lysate; B) with S. pneumonia lysate
5. Necrosis in PMNs treated with drugs
6. Apoptosis in PMNs treated with drugs via Cell Death ELISA:
A) without S. pneumonia; B) with live S. pneumonia
7. Light micrograph of nonspecific esterase staining of mononuclear cells 62
8. Light micrograph of macrophages incubated with PMNs 64
9. Phagocytosis of azithromycin-treated PMNs by macrophages 66
10. Oxidative activity of PMNs: A) without lysate; B) with S. pneumonia lysate 68
1 1. IL-8 production by PMNs treated with drugs 70
PAGE
50
52
LIST OF ABBREVIATIONS
A R D S
CBA
CFU
ELISA
fMLP
HBSS
Ig
IL
LPS
LT
MDM
MHC
'NBT
PAF
PARP
PBS
PI
PMA
PMN(s
TGF
W a
adult respiratory distress syndrome
Columbia blood agar
colony forming unit
enzyme-linked immunosorbent assay
formyl methionine leucine phenylalanine
Hank's balanced salt solution
immunoglobulin
interleukin
1ipopoIysacc haride
leukotriene
Iscove's modified Dulbecco's medium
major histocompatibility complex
nitro-blue tetrazoliurn
platelet activating factor
pol y- ADP-ribose-pol ymerase
phosphate-buffered saline
propidium iodide
phorbol 12-myristate 1 3-acetate
polymorphonuclear neutrophil(s)
transforming growth factor
tumor necrosis factor alpha
xi
CHAPTER 1: INTRODUCTION
1.1 The Pathogenesis of Respiratory Tract Infection
The lungs are exposed to more pathogenic organisms than virtually any other
tissue [I ] . Fortunately, the lungs have many mechanisms to remove, neutralize or kill
pathogens such as mechanical, secretory and immune systems 12, 31. Mechanical
defenses include filtration by nasal vibrissae, the ciliated epithelial barrier, and the cough
reflex. The mucociliary lining constitutes the secretory defense system. If a respiratory
pathogen manages to evade these mechanisms and reaches the lungs, it will encounter
resident pulmonary alveolar macrophages and other immune components such as
polymorphonuclear neutrophils (PMNs) as a third line of defense.
Despite these defense mechanisms, bacterial colonization of the respiratory tract
is a common cause of morbidity and mortality. The pathogenesis of bacterial infection in
the respiratory tract involves both bacterial virulence factors and the host inflammatory
response [4, 51. The host inflammatory response is primarily driven by neutrophils.
Despite being necessary for host defense, there is increasing evidence of the contribution
by polymorphonuclear neutrophils to the severity of a number of inflammatory diseases
such as streptococcal pneumonia [5- 121. In such pathologic circumstances, the potential
for host tissue damage is clear: cytotoxic compounds such as elastase, hypochlorous acid,
and oxygen radicals released by neutrophils at sites of inflammation can damage
surrounding tissues, and induce necrosis in neighboring neutrophils [7, 1 1 1. Necrotic
neutrophils swell and burst as a result of metabolic collapse and failure to maintain ionic
2
homeostasis, releasing more cytotoxic compounds and pro-inflammatory mediators [I 31.
This can lead to severe host tissue damage and inflammation.
1.1.1 Bacterial Virulence Factors
Bacterial virulence factors initiate the host inflammatory response. Gram-
negative bacteria have lipopolysaccharide (endotoxin) on the outermost layer of their cell
wall that has antigenic properties and activates the inflammatory process [14]. Gram-
positive bacteria have a large peptidoglycan layer that, along with associated lipoteichoic
acids, can exert endotoxin-like effects. Virulence factors aid in bacterial colonization and
protect the microorganism from host defenses enabling bacterial invasion and infection to
occur [I 51.
1.1.1.1 Adhesion and Uptake
Adhesion of bacteria in the host localizes pathogenic bacteria to target tissues
[I 51. Adherence of bacteria to host cells and extracellular matrix components is aided by
adhesins such as pili, fimbriae, and other adhesion molecules such as M protein of
Streptococci and fibronectin-binding proteins of Streptococci and Staphylococci.
P hosphory lcholine, a cell wall component of Streptococcus pneurnoniae, binds the
platelet-activating factor receptor on activated endothelial cells leading to adherence to
and invasion of these cells. Teichoic acids aid adhesion of many Gram-positive bacteria
to host epithelium fibronectin molecules [ 161. Many pathogens produce virulence factors
(such as EspA and EspB of Escherichia coli) that activate host cell signal transduction
3
pathways leading to expression of and activation of receptors on the host cell to which
the bacteria can then bind.
Some bacteria such as Salmonelfa and Shigella enhance their uptake into
nonphagocytic cells, enabling them to enter a protected environment or allowing them to
Pass through the epithelium [IS]. Yersinia enterocolitica and Yersinia
pseudotuberculosis avoid phagocytic uptake by injecting Yop proteins into phagocytic
cells, paralyzing the actin cytoskeleton and dephosphorylating host proteins which
impairs phagocytosis. Pseudornonas aeruginosa has this same effect on phagocytic cells
via secretion of exoenzyrne S which modifies G proteins involved in actin regulation.
1.1.1.2 Cytotoxins
Bacteria produce toxins which damage host cells by interfering with
transmembrane signalling, by producing pores that alter the permeability of membranes
resulting in lysis of host cells, or by altering cytosolic components of the host ceil [is].
Some bacteria can induce apoptosis (programmed cell death) in host phagocytes, thereby
preventing bacterial clearance (15, 171. Shigella protein IpaB binds and activates
interleukin-1 p-converting enzyme (ICE), a cysteine protease which induces apoptosis
when activated via cleavage of host cell substrates [15]. Mycobacterium avium induces
apoptosis in human macrophages possibly via the generation of oxygen free radicals 1171.
Attachment of M. avium to host cell surface receptors may activate second messengers
resulting in oxidant production which could alter the redox status and activity of host
enzymes. Diphtheria toxin, Pseudomonas exotoxin A, and cholera toxin induce apoptosis
in vitro, although the mechanism differs in each case [I 51. Staphylococcus aureus toxic
4
shock syndrome toxin-1 induces T cell apoptosis and inhibits immunoglobulin G (IgG)
production. S. pneumoniae has extracellular toxins such as pneumolysin, neuraminidase,
and purpura-producing factor which kill phagocytes and interfere with chemotaxis [3,
181.
1 . I -1 -3 Other Factors
Many intracellular organisms can prevent fusion of phagosomes with lysosomes
[15]. Some intraceliular pathogens, such as Mycobacterium avium, can inhibit
acidification of the phagocytic vacuole preventing activation of Iysosomal enzymes.
Streptococcus pneumoniae and Haemophilus influenzae, two common causative agents of
pneumonia, produce proteases that digest IgA [ I 81. The thick polysaccharide capsule of
S pneurnoniae and the M protein within the cell wall of Streptococcus pyogenes help
protect these organisms from being phagocytosed by macrophages and neutrophils [3,
181.
1.1.2 Inflammation
Inflammation is classified as acute on the basis of the persistence of injury,
clinical symptoms and the nature of the inflammatory response [19]. Acute inflammation
is characterized by the accumulation of fluid and plasma constituents in tissues,
intravascular stimulation of platelets, and the presence of neutrophils. Chronic
inflammation, on the other hand, is characterized by the presence of lymphocytes, plasma
cells and macrophages.
5
The hallmarks of inflammation include redness, swelling, heat, pain and loss of
function [20]. Inflammatory mediators released by cells at sites of infection cause
vasodilation and increased blood flow and, in conjunction with increased vascular
permeability, result in formation of inflammatory exudate and local edema.
The host immune system responds to bacterial products such as
lipopolysaccharide (LPS) and formyl methionine leucine phenylalanine (fMLP) and an
inflammatory response is generated [14]. A humoral response is produced that is mainly
mediated via immunoglobulin G, which opsonizes the organism [2]. In conjunction with
complement, IgG can also neutralize many viruses and toxins and lyse certain Grarn-
negative bacilli. Secretory IgA, localized at mucosal surfaces where most pathogenic
microbes gain access to the body, serves an important protective fhction [2 11. Secretory
IgM is also present in mucosal secretions but in Iower amounts than IgA. Secretory
antibodies cross-link antigens and block attachment of bacteria to mucosal cells. IgM is
the first immunoglobulin produced in response to infection (5 to 7 days after antigen
exposure) but its concentration declines rapidly and IgG and IgA become more
predominant. Previous exposure or immunization to an antigen results in a more rapid
and stronger response by lymphocytes which produce antigen-specific immunoglobulins.
A cell-mediated immune response is also elicited and alveolar macrophages are
the main effector cells. Lipopolysaccharide (endotoxin) activates macrophages,
monocytes, endothelial cells, platelets, mast cells, neutrophils, lymphocytes and
fibroblasts [14]. Alveolar macrophages process antigens that reach the lower respiratory
tract and kill many microbes [2]. As discussed further, leukocyte recruitment is mediated
by the generation of cytokines by lymphocytes, macrophages and other cells [IZ].
6
Leukocytes adhere to the endothelium and emigrate fiom the vasculature into the tissues
in response to chemotactic gradients.
Spontaneous phagocytosis can occur when neutrophils come into contact with
bacteria. Neutrophils also recognize and attach to pathogens via Fc receptors and
complement receptors on the neutrophil surface [12]. Fc receptors bind to the Fc portion
of immunoglobulin G molecules and complement receptors bind to C3b and C3bi found
on the surface of opsonized bacteria. Phagocytosis of the pathogen ensues and
microbicidal killing occurs within the phagolysosome of the neutrophil.
Mild acute idammation caused by bacteria is usually resolved when the infection
is cleared via host defense mechanisms (including the inflammatory response) or with
antibiotics [20]. In pneumonia, the lung are filled with exudate, fibrin, bacteria and
inflammatory cells. During the resolution phase of inflammation, this abnormal material
is removed (mainly via the action of macrophages), and normal structure and h c t i o n
can be restored.
1.1.2.1 Mediators
Interleukin-1 (IL-l), tumor necrosis factor a (TNFa), leukotriene Bq (LTB4), and
interleukin-8 (IL-8) are some of the major pro-inflammatory mediators involved in
inflammation. Bacterial antigens, such as LPS, as well as host immune components
stimulate the production and release of inflammatory mediators fiom many cell types
[22]. Lipopolysaccharide activates macrophages and they secrete peptide and lipid
mediators such as IL- 1, TNFa, IL-6, thromboxane, leukotrienes, and platelet activating
factor [14]. Reactions to LPS, infection and other inflammatory stimuli are mediated by
7
IL-1 and TNF [23]. JL-1 and TNF induce acute inflammation by inducing gene
expression and synthesis of proteins in other cell types found in the lungs [23]. Both IL-
la and TNFa activate neutrophils. IL-1 is produced by various cell types in response to
bacterial antigens and toxins, products of activated lymphocytes, complement and
clotting components [23]. TNFa is produced by activated monocytes and macrophages,
lymphocytes, endothelial cells and keratinocytes 1231. Neutrophils also release IL- 1,
TNFa and IL-8 [20]. IL-1 and TNF both cause an increase in IL-8 [24].
Mediators such as IL-8 and LTBJ strongly amplify inflammation. IL-8 plays an
important role in inflammation and clearance of pathogens by stimulating neutrophil
chemotaxis and degranulation, and enhancing phagocytosis of opsonized particles [25-
271. Although individual neutrophils produce only small amounts of IL-8, their large
number at sites of inflammation make neutrophils the most significant source of IL-8 in
inflammatory processes 1283. Neutrophils also release LTBJ, another potent neutrophil
chemoattractant responsible for potentiating inflammation [29].
Inflammatory mediators can be involved in the pathogenesis of many disease
states. IL-1, TNFa, IL-8, and LTB4 play a role in the pathogenesis of cystic fibrosis
patients infected with Pseudomonus aeruginosa [30-341. Increased levels of IL- 1, TNFa,
and IL-8 have been implicated in adult respiratory distress syndrome (ARDS) [35, 361.
1.1.2.2 Neutrophils
Neutrophils (polymorphonuclear leukocytes) originate and mature in bone
marrow, are then reteased into the blood where they circulate for approximately ten
hours, and can migrate fiorn the vasculature into sites of infection where they can live up
8
to three days [20, 37-39]. Neutrophils are terminally differentiated cells that retain a
smaller number of homeostatic pathways than their bone marrow precursors 1401. These
cells are highly specialized for degranulation and oxidative metabolism.
Neutrophils are the principal cell type involved in acute inflammation. These
cells are involved in phagocytic clearance of pathogens fiom infected tissues and thus are
important in the resolution of infection. They are necessary in the removal of pathogens
fiom the Lung and their influx is regulated by chemoattractants and upregulation of
adhesion molecules [7, 11, 371. In humans, neutrophils comprise approximately 65% of
circulating leukocytes but only 2% of leukocytes recovered from healthy broncho-
alveolar spaces [37]. In healthy lungs, more than 90% of lung leukocytes are alveolar
macrophages, while monocytes constitute only 5% of leukocytes in the circulation. This
distribution of leukocytes in the lung reverses upon exposure to bacterial infection.
Neutrophils carry out their anti-bacterial t'unction via oxygen-dependent or
independent mechanisms. Firstly, when neutrophils are activated by pro-inflammatory
mediators such as IL-I or TNFa, membrane enzymes including NADPH oxidase,
myeloperoxidase, and esterase are activated and result in the initiation of the respiratory
burst [ l l , 411. Reactive oxygen intermediates such as superoxide anion, hydrogen
peroxide, hypochlorous acid and hydroxyl radical are produced during the respiratory
burst of activated neutrophils. Oxygen is converted to superoxide, which is then
converted to hydrogen peroxide. Neutrophil granules contain myeloperoxidase, which
converts most of the hydrogen peroxide to hypochlorous acid if chloride is also present.
Some hydrogen peroxide is also convened to water via catalase or to hydroxyl radicals if
9
iron is present These reactive oxygen intermediates are involved in oxygen-dependent
bacterial killing-
In addition, neutrophils have intracellular granules that contain proteolytic
enzymes such as lysozyme, elastase, gelatinase, collagenase, cathepsin G, proteinase 3
and bactericidal proteins such as lacto ferrin, de fensins, bactericidaYpermeability - increasing protein, and azurocidin [ I , 1 1 1.
When neutrophils become activated during infection these enzymes, proteins and
oxidants are released into the phagosome and the extracellular environment and aid the
neutrophil in killing bacteria. Bacteria are killed by the oxidation and digestion of
membrane phospholipids and glycoproteins 1121. Some of these same compounds can
also act on host tissue components causing damage. In order to prevent such injury,
antiproteases and antioxidants are produced by the host [I 1, 411. Severe inflammatory
states overwhelm this system and cause injury to host tissues. For example, patients with
acute respiratory distress syndrome (ARDS) display abnormal antioxidant capacity in
association with severe lung damage [4 1 1. Oxygen radicals generated by neutrophils
inactivate antiproteases and W e r increase proteolytic damage to the lung. Massive
neutrophil infiltration seen in pneumonia results in the release of large quantities of
oxidants, overwhelming antioxidants and antiproteases, which in turn leads to lung injury
1.1.2.3 Macrophages
Bone marrow precursors differentiate into blood monocytes and migrate into
tissues where they develop into macrophages [42]. Local production of chemotactic
10
factors, including fMLP and the complement-derived peptide CS des arg, at the site of
infection induces the immigration of monocytes into tissues [43]. Macrophages are
important in the inflammatory process and the resolution of inflammation [44]. These
cells are major effectors of the immune response against pathogens [12, 441. Resident
alveolar macrophages are the host's first line of cellular defense against infections. Their
defensive capabilities can be overwhelmed, however, by large numbers or highly virulent
bacteria, and recruited neutrophiis help defend the host in this case [12]. Besides their
role in phagocytosis of bacteria, rnacrophages also function in antigen presentation to T
lymphocytes [453. Fragments of internalized pathogens bind to class I1 major
histocompatibility complex proteins (MHC) which are then expressed on the cell surface
[46]. Helper T cells with receptors specific for the antigen bind the MHC-antigen
complex, become activated and secrete cytokines (such as IL-4) that stimulate B cells that
have already encountered that particular antigen and display antigen fragments bound to
MHC 11. T cell receptors also bind to the antigen-MHC complex of these B cells. This
results in activation of antigen-specific B cells. Activated B cells give rise to plasma
cells that produce specific antibodies against the particular antigen. The phagocytosis of
bacteria activates macrophages, which can then recruit neutrophils to sites of infection by
secretion of neutrophil chemotactic factors such as IL-8 [2]. Macrophages are also
responsible for clearing cellular debris from inflamed sites. Macrophage phagocytosis of
dying celIs is another mechanism whereby acute inflammatory reactions during
pathological processes are physiologically controlled [47]. Indeed, macrophages have
been implicated in the regulation of neutrophil-mediated damage as they rapidly
phagocytose dead and dying neutrophils before the cell lyses and releases its cytotoxic
1 I
and pro-inflammatory compounds into the tissues, thus preventing damage to host
tissues, inflammatory reactions and autoimmune responses (autoantibodies against
nucleosomal DNA released from dead cells) [47]. Moreover, recent information suggests
that during phagocytosis of apoptotic neutrophils, in contrast to phagocytosis of
opsonized particles including bacteria, the production by macrophages of pro-
inflammatory mediators, such as IL-IP, TNFa and IL-8, is inhibited [48]. These
observations suggest that opsonin-mediated phagocytosis by macrophages activates
inflammatory processes, while vitronectin/phosphatidylserine-mediated phagocytosis
does not activate such processes. It is not yet known whether the inhibition of the
production of pro-inflammatory mediators is due to binding of apoptotic cells to receptors
on macrophages or to the uptake process. In the same study, increased production of
transforming growth factor+ 1 (TGF-P I), platelet activating factor (PAE'), and
prostaglandin E2 (PgE2) were also reported and appeared to mediate the decrease in pro-
inflammatory cytokines. The mechanism by which phagocytosis of apoptotic cells
stimulates production of TGF-B 1, PAF, and PgE2 is not known nor is the mechanism by
which these products reduce production of pro-inflammatory mediators.
1.2 Pneumonia
Pneumonia is one the most common community-acquired and nosocomial
infections, and despite improved treatment strategies, the mortality rate remains high
[49]. It is the fifth leading cause of death in the USA. In pneumonia, fibrin deposition
congests the alveoli and bronchioles of the lungs [SO, 511. Severe inflammation causes
pulmonary tissue damage that can ultimately lead to respiratory failure and death. Fever,
12
chills, headache, cough and chest pain accompany pneumonia. A number of pathogens
are known to cause pneumonia, including Streptococcus pneumoniae, Streptococcus
pyogenes, S faphylococcus aure us, HaernophiZus influenzae, Kle bsiella pneumoniae,
Escherichia coZi, Pseudomonas aeruginosa, Mycoplosma pneumoniae, Legionefla
pnettmophila, adenovirus type 111, influenza A and B viruses, rickettsiae, and fungi [50-
521. Combined infections of multiple respiratory pathogens are ofien seen 153, 541. The
most common bacterid lung infection in adults is streptococcal pneumonia caused by
SIreptococcus pneumoniae 131. Bacteria are spread via contact with an infected person.
A 7 to 10 day incubation period precedes the clinical manifestations of streptococcal
pneumonia (chills, fever* and pain) [55] . The bacteria invade the body through the
airways and reach the lungs where they multiply rapidly in the alveolar tissue. The
antiphagocytic capsule of S. pneumoniae prevents phagocytosis by alveolar macrophages
and other phagocytes, and the infection proceeds resulting in purulent exudate-filled
aiveoli. Neutrophil influx ensues and they accumulate in the infected alveoli and
contribute to exudate formation. This proceeds for about a week during which
anticapsular antibody is formed which usually results in recovery due to adequate
phagocytosis and killing of the organism unless the disease is too severe or the host is
immunocomprornised. Particularly virulent strains of bacteria may overwhelm host
defenses leading to respiratory failure.
Streptococcus was isolated in 55% of patients with pneumonia in one study [52].
Many normal healthy individuals carry streptococci in their upper respiratory tract,
however, only a small proportion develop streptococcal pneumonia [52]. A study by
Cherry et al. isolated both pathogenic bacteria and rhinovirus fiom 45% of patients,
13
perhaps indicating a viral infection as the initiating cause in bacterial pneumonia [53].
The large volume of mucous secretions in the nose and pharynx increase the chance of
aspiration of bacteria past the epiglottaf barrier where gravity can carry the bacteria into
the alveoli. In another study, 46% of patients had an upper respiratory tract infection
(possibly viral) in the week or so prior to the onset of bacterial pneumonia [52]. A viral
source of the initial infection was difficult to confirm in most cases since viruses may
only be shed for a few days and may not have been detectable by the time of
hospitalization for the secondary bacterial pneumonia.
The annual rate of pneurnococcal infections in the USA is approximately 500,000
cases in all age groups, but this rate can increase in certain populations [3, 551. A major
risk factor for contracting pneumonia is age; those over forty have a three to four times
greater chance than those under twenty [3]. Hospitalization also increases the risk of
contracting pneumonia [49]. Pneumonia causes 8% to 33% of all nosocomial infections
making it the third most common nosocomial infection after urinary tract and surgical
wound infections. Nosocomial infections are often caused by Gram-negative bacteria
and are fatal in more than 50% of cases. The rate of respiratory tract infection is greatest
in developing countries [56-581.
1.2.1 Streptococcus pneumoniae
Streprococcus pneumoniae is the most common causative agent of bacterial
pneumonia in humans [50]. This organism appears as a lancet-shaped Gram-positive
diplococcus that is found in the human nasopharynx and has no known environmental or
animal reservoir [3]. S. pneumoniae is encapsulated and a-hemolytic. Asymptomatic
14
carriers are often more important in spreading the disease than those with acute infection
since they are not isolated and treated [3]. A number of factors determine whether
bacterial invasion and infection will occur such as degree of challenge, virulence factor
expression, primary viral infection, host defenses and immune competence, nutritional
status, alcohol intoxication [3]. S. pneumoniae has extracellular toxins such as
pneumolysin, neurarninidase, and purpura-producing factor but, although proposed, the
exact role of these in the pathology of pneumonia remains unclear [3, 551. Hydrolytic
enzymes such as neurarninidase and IgAI protease may contribute to the colonization of
the host [3, 18, 591. Neurarninidase acts to kill phagocytes and interfere with their
cheinotaxis and the destruction of secretory IgA impairs opsonization and phagocytic
clearance of the bacteria. S. pneumoniae strains canying mutations in genes encoding for
pneurnolysin, autolysin and surface protein A demonstrate reduced virulence. Purpura-
producing factor is a glycan-teichoic acid fragment generated by hydrolysis of the cell
wall by autolysin [60]. Lipoteichoic acids aid in bacterial adhesion to host cells [16].
The primary determinant of pathogenicity is the polysaccharide capsule, which helps
protect S. pneumoniae from being phagocytosed by macrophages and neutrophils [3].
1.3 Treatment
Several options are available for the treatment of bacterial pneumonia. Besides
vaccines for the prophylaxis of pneumonia, therapeutic strategies such as antibiotics and
anti-inflammatory drugs are available 131. Drugs that modulate the immune response to
infection have been studied experimentally, and several anti-inflammatory strategies
15
focus on preventing neutrophil-mediated tissue injury. Of course, the treatment of choice
remains the use of antibiotics to eradicate the bacterial infection.
1.3.1 Antibiotics and Antibacterial Agents
There are many classes of antibiotic and/or antibacterial drugs available to treat
pneumonia including beta-lactamases, sulfonarnides, fluoroquinolones, tetracyclines, and
macrolides. The choice of drug depends on the specific organism isolated from sputum
or blood, the pharmacodynamics of the drug and its antibacterial spectrum, as well as
clinical experience and an assessment of the patient's history.
1.3.1.1 Beta-lactams
Beta-lactam antibiotics include penicillins and cephaiosporins 161 1. They contain
a beta-lactam ring with various substituents that lend different properties to the antibiotic.
Beta-lactam antibiotics bind to penicillin-binding proteins and interfere with bacterial cell
wall synthesis by inhibiting transpeptidase cross-linkage of adjacent peptidoglycans [62].
Additionally, beta-lactarns inactivate an inhibitor of an autolytic enzyme in the bacteria1
cell wall, leading to lysis of the bacterium [61]. Bacterial resistance to beta-lactams is
mainly due to the production of beta-lactamases, reduced outer membrane permeability
(decreasing drug penetration; particularly in Gram-negative organisms), or modified
penicillin-binding sites. Many strains of S. pneumoniae are resistant to p-lactams due to
modification of the penicillin-binding proteins; S. pneumoniae does not produce a P-
lactamase.
1.3.1.1. I Penicillin
Penicillin G (benzylpenicillin) has been one of the most widely used antibiotics
for treating streptococcal pneumonia, but growing bacterial resistance to penicillin has
forced the development of other drugs [SO, 511. Penicillin contains a thiazolidine ring
linked to the beta-lactam ring, which is the target of beta-lactamases and acylases fiom
penicillin-resistant organisms [62]. Penicillin G is a natural penicillin that is produced by
mold cultures. It is acid labile and bactericidal [61]. Semisynthetic penicillin derivatives
(such as nafcillin, amoxicillin, ampicillin and piperacillin) have been produced which are
acid-stable, more resistant to cleavage by beta-lactarnases, or have an enhanced spectrum
of activity against both Gram-positive and Gram-negative bacteria.
1.3.1 .1.2 Cephalosporins
Cephalosporins are derived fiom Cephalosporium h g i . They are water-soluble
and relatively acid-stable. Cephalosporins vary in their susceptibility to beta-lactamases
[6 11. Their usefulness in pneumonia may be limited since they have poor lipid solubility
which hinders their diffusion into tissues. Many semisynthetic broad-spectrum
cephalosporins, such as cefuroxirne, have been produced by varying the side chains of the
beta-lactam ring. They are effective against most Gram-positive and some Gram-
negative organisms. Resistance to this class of drugs is increasing due to both plasmid-
encoded and chromosome-encoded beta-lactamases. In fact, nearly all Gram-negative
bacteria produce a beta-lactamase that hydrolyzes cephalosporins to a greater extent than
penicillins.
17 1 -3. I -2 Sulfonamides
Sulfonamides, such as sulfadiazine and sulfamethoxazole, are derived fiom
sulfanilamide by substitution at the amide group [61]. Sulfanilimide is a smxctural
analogue of p-aminobenzoic acid that is needed for folic acid synthesis in bacteria. DNA
and RNA synthesis require folic acid. Sulfonamides compete with p-aminobenzoic acid
for the enzyme dihydro-pteroate synthetase and are bacteriostatic rather than bactericidal
since they only inhibit bacterial growth. Sulfamethoxazole, in combination with
trimethoprim (an anti biotic which acts as a folate antagonist), is called co-trimoxazole
and is effective at much lower dosages. Co-trimoxazole is sometimes used to treat
pneumonia.
1.3.1 -3 Fluoroquinolones
Fluoroquinolones, such as ciprofloxacin and norfloxacin, are synthetic antibiotics
that inhibit topoisomerase I1 (a DNA gyrase) and thus inhibit DNA transcription [61].
Although they are active against Gram-positive organisms, these antibiotics are
especially active against Gram-negative bacteria. Ciprofloxacin is used for pneumonia
caused by Haemophilus influemae. One of the new drugs of choice for pneumonia is
levofloxacin.
1 -3.1 -4 Tetracyclines
Originally, tetracyclines, such as oxytetracycline, were obtained from
Streptomyces [6 1 1. Newer tetracyclines, such as minoc ycline and tetracycline, are
semisynthetic or synthetic. They are taken up by active transport and are distributed
18
throughout the body. They act by inhibiting protein synthesis by competing with
aminoacyl tRNA for the A site on ribosomes. Tetracyclines have a very broad spectrum
of activity against both Gram-positive and Gram-negative organisms but they are only
bacteriostatic. Their usefulness is limited as many strains of bacteria have become
resistant [61]. Resistance occurs due to defective uptake by bacteria; plasmid encoded
genes lead to the development of energy-dependent efflux mechanisms which transport
the drug out of the bacterium. Some bacteria also alter the ribosome binding site so the
drug can not bind.
1.3.1.5 Macrolides
Macrolides have been used for many years for the treatment of various upper and
Iower respiratory infections [63]. The first macrolide, erythromycin, was introduced in
1952. Many derivatives of erythromycin have since been developed. One of these is
azithromycin which will be discussed in detail in a later section.
1.3.1.5.1 Structure
Structurally, macrolides consist of a lactone ring structure with attached
carbohydrate moieties. Macrolides have a variety of lactone ring structures but three
main groups are the 1 4-membered macrolides (erythromycin, clarithromycin and
roxithromycin), the 1 5-membered macrolides (azithrornycin) and the 16-membered
macrolides (josamycin, lincosamides, and streptogramins) [64]. Azithromycin is more
correctly classified as an azalide due to the presence of a nitrogen atom into the
macrolide ring.
1.3.1.5.2 Mode of action (antimicrobial activity)
Macrolides are generally considered to be bacteriostatic rather than bactericidal,
and are active against a variety of Gram-positive and Gram-negative organisms [64]. The
antibacterial activity of macrolide antibiotics is due to reversible binding of the drug to
the bacterid 50s subunit of the 70s ribosome, thus inhibiting protein synthesis within the
bacterial cell [65]. Macrolides stimulate dissociation of peptidyl tRNA from the
ribosome by a variety of mechanisms including inhibition of translocation or peptidyl
transferase reactions [66]. Some nonbacterial organisms such as Entamaeba hisrolyrica
and Toxoplasrna gondili are also affected by macrolides but the mechanism of action is
not known [64].
1.3.1 S . 3 Resistance
Cross-resistance to azithromycin and erythromycin has been shown in many
studies [64]. Enzymatic methylation of the ribosome binding site of macrotides is
postulated to be responsible for resistance of organisms to macrolides by preventing
macrolides from binding to the ribosome [67-701. The methylase modifies the 50s
ribosome subunit by ~ ~ - r n e t h ~ l a t i o n of adenine in 23s ribosomal RNA [70]. In addition
to macrolides, this also confers resistance to lincosamides and streptogramin B
antibiotics. The methylase can be constitutively produced by some bacteria such as
Staphylococcus aureus and Staphylococcus epidermidis. Subinhi bitory concentrations of
the macrolide can also induce expression of the methylase in such bacteria as
20
Streptococcus pneumoniae and S~reptococcus pyogenes. Plasrnid pE 1 94 specifies this
type of resistance to macrolides.
Another mechanism of resistance involves the presence of the plasmid pNE24,
which results in energy dependent efflux of 14- and 15-membered macrolides (but not
16-membered macrolides) from bacteria [71]. The energy dependent macrolide efflux
pump maintains intracelluiar antibiotic concentrations below those necessary for binding
of the macrolide to ribosomes. The efflux pump recognizes specific structural
components of 14- and 15-membered macrolides and is able to remove several thousand
macrolide molecules per second. Bacteria are thus able to maintain growth in the
presence of antibiotic. Plasmid pNE24 has been detected in Staphylococcus epidermidis.
Other forms of resistance to macrolide antibiotics include hydrolysis of the
macrolide ring by esterases, and phosporylation or glycosylation of the macrolide [7 11.
1.3.2 Azithromycin
Standard treatments for lower respiratory tract infections include penicillins,
cephalosporins, or erythromycin [61, 721. Other agents such as sulfonamides,
ciprofloxacin, or tetracyclines may also be prescribed. Penicillins are limited in their
usage due to patient hypersensitivity reactions and bacterial resistance, and
cep halosporins have poor activity against intracellular pathogens. Erythromycin was '
introduced over thirty years ago and has been considered the representative macrolide.
Its disadvantages include need for fiequent administration, acid instability,
gastrointestinal disturbances and poor activity against Haemophilus species. This has led
to the development of new macrolides. hthromycin (CP-62,993; XZ-450) is a
21
semisynthetic macrolide antimicrobial drug of the azalide class. It is more acid-stable
than erythromycin and demonstrates improved pharmacokinetic parameters [64]. Like
other macrolides, the activity of azithromycin does, however, decrease in an acidic
environment 1731.
1.3.2.1 Structure
Azithromycin is derived from erythromycin. Its chemical structure is 9-deoxo-
9A-methyl-9A-aza-9A-homo-erythromycin A [74]. Azithromycin has a 1 5-membered
lactone ring structure with a tertiary amino group, attached to two sugars (see Appendix
A) [64]. The amino substituent and expanded ring strvcture differentiates it fiom
erythromycin (1 4-membered lactone ring). The enhanced acid-stability of azithromycin
(300-fold increase over erythromycin) is related to these structural differences [75].
1 -3 -2.2 Pharrnacodynamics and Antimicrobial Activity
The antimicrobial activity of azithromycin, like that of other macrolides, is due to
inhibition of protein synthesis fiom the drug binding to the 50s subunit of the bacterial
ribosomes [65]. Azithromycin has a similar spectrum of activity to other macrolides; it is
active against many Gram-positive and Gram-negative bacteria. In addition to its
bacteriostatic activity, azithromycin is also bactericidal against a number of bacterial
species. Bactericidal activity is a decrease in "CFU concentration of at least 3 loglo
CFU/mL or a 99.9% kill rate within 24 hours"[64]. Azithromycin i s bactericidal against
Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pneumoniae,
Haernophilus influenzae, Klebsiella pneurnoniae, Borderella species, Legionella
22 pne umophila, Moraxella catarrhalis, Chlamydia trachoma t is, Chlamydia peumoniae,
Mycobacterium avium complex, and Borrelia burgdorferi [64]. Bacteria resistant to
erythromycin are also usually resistant to azithromycin.
All macrolides exhibit a postantibiotic effect (PAE) which means bacterial
regrowth is suppressed for a short period of time after exposure to an antimicrobial drug.
The PAE is important to consider because drug ievels may become lower than the
minimum inhibitory concentration (MIC) in the interval between doses. Azithromycin
has shown a postantibiotic effect of 1.7 to 3.9 hours for many respiratory tract pathogens
~761.
The d i d e azithromycin has proven to be more effective in the treatment of a
number of respiratory tract infections compared to erythromycin, although both
antibiotics have comparable antibacterid properties [77, 781. Azithrornycin has greater
activity than erythromycin against Gram-negative bacteria and this is thought to be due to
the extra positive charge from the methyl substituted nitrogen (structure-activity
relationship) [79]. It may also be due to the high binding affinity for ribosomes [80].
Azithromycin also interacts with magnesium binding sites on outer membrane
phospholipids, which serves to increase its membrane permeability and enhance its
uptake 1791. The exact mechanism of uptake, however, has not been determined but is
postulated to involve passive diffusion and lysosomal trapping [8 1, 821.
Clinically, the antimicrobial activity of azithromycin appears to be enhanced by
the ability of this compound to reach high tissue concentrations 1641. Azithromycin is
more concentrated in tissues than in blood and accumulates intracellularly within
monocytes, polymorphonuclear Ieukocytes and alveolar macrophages to concentrations
23
much greater than those in the extracellular environment [8 1-84]. This may enhance the
antimicro bid activity of azithrom ycin against intracelluiar pathogens. Azithromycin has
been shown to accumulate inside phagocytes much more effectively than erythromycin,
and is primarily located within lysosomes [8 1, 85, 861. Azithromycin does not impair the
phagocytic h c t i o n of cells and even enhances phagocytosis of S~aphyZococcus aureus at
subinhibitory concentrations [81, 871. Synergistic killing between azithromycin and
phagocytes has been demonstrated in vifro for S. aureus. L. pneumophila and H.
influenzae [84]. Azithromycin is released fiom phagocytes more slowly than
erythromycin (within 24 hours versus 3 hours, respectively) and it is suggested that
phagocytes deliver the drugs to sites of infection increasing tissue concentrations [88].
Fibroblasts also accumulate azithromycin (and release it more slowly than they release
erythromycin) and may act as a slow release reservoir of antibiotic within tissues 1891.
1 -3.2.3 Pharrnacokinetics
Animal models, healthy volunteers, and patients requiring antibiotics have been
used to investigate the pharmacokinetics of azithromycin. As discussed previously,
azithromycin is more acid-stable than erythromycin as the structural modifications block
the normal degradation pathway. At pH 2, 10% decay takes 20 minutes for azithromycin
but only 3.7 seconds for erythromycin [75].
Azithromycin is rapidly taken up from the blood into intracellular compartments
of p hagoc ytes and fibroblasts and is slowly released. Unlike other macrolides,
azithromycin achieves high concentrations in tissues, including the lungs. High tissue
concentrations are prolonged due to the long half-life of azithromycin [90]. This permits
24
a single daily dose regimen for most infections, which enhances compliance. Product
labeling states that azithromycin should be taken on an empty stomach (as food decreases
the oral bioavailability of the drug), but subsequent studies have found that food has no
effect on the bioavailability of azithromycin 191, 921. In healthy fasting volunteers, an
oral bioavailability of 37% was shown when given a single 500 mg dose (931. The low
oraI bioavailability is attributed to incomplete absorption rather than acid degradation or
extensive first-pass metabolism 1941. A 500 mg dose of azithromycin gave a mean peak
plasma or serum concentration (C,,) of 0-4 to 0.45 mg/L when measured 2.5 hours after
administration to healthy fasting volunteers [93, 951. Higher mean peak concentrations
were reported for other macrolides and were achieved in a shorter time. The mean C,,
in healthy volunteers given 500 mg twice on day 1 and once on days 2 to 5 increased
from 0.41 to 0.62 mg/L over five days 1931.
Macrolides are dicationic yet they are still lipid soluble [64]. This results in
extensive distribution in body fluids [96, 971. The volume of distribution (Vd) is the
volume of fluid required to contain the total amount of drug in the body at the same
concentration as is present in the plasma [61]. The Vd for azithromycin has been
reported to be 23 to 3 1 L k g body weight, which is greater than the total amount of body
water (0.55 Lkg body weight) indicating that the drug is stored in tissues and cells [97].
Delivery of azithromycin to tissues is accomplished via two mechanisms - direct uptake
by tissues and uptake by phagocytic cells (targeted delivery) [90]. As discussed above,
azithromycin is rapidly taken up and slowly released from many cell types leading to
high local concentrations of drug [98].
25
In patients who received a single 500 mg dose, serum concentrations declined in a
biphasic manner over three days post-administration and were between 1 and 9 mg/kg in
gynecological tissue (ovary, fallopian tube, uterus, cervix, fibroid tissue), lung, peritoneal
fluid, prostate, tonsil and urologicd tissue (testis, epididymis, vas deferens) [93, 99, 1001.
Lower concentrations (0.2 to 1 mg/kg) were found in bone, fat, gastric mucosa, and
muscle, but all concentrations were higher than those found in serum, reflecting the rapid
uptake and concentration of azithromycin in tissues versus bIood. Patients given a single
500 mg dose of azithromycin orally were also used to assess concentrations of
azithromycin in the respiratory tract [ 10 1 - 1031. Broncho-alveolar macrophages,
bronchial epithelial lining fluid, bronchial mucosa and sputum all achieved a maximum
concentration within 48 hours, while this was reached in lung tissue within 96 hours, with
concentrations ranging fiom 1.56 to 26.59 mg/L. These values represent high tissue or
fluid : senun concentration ratios (52 : 1 to 11 50 : 1). In patients, with respiratory
infection, who received 500 mg on day 1, and 250 mg on days 2 to 5, the mean peak
sputum concentration of azithromycin was 3.7 mg/L at 15 hours which represents a
sputum : serum concentration ratio of 5.8 : 1 [74].
Equilibrium dialysis at pH 7.4 and 25°C was used to determine in vitro protein
binding of [14~]-azithromycin in human senun [104]. Protein binding decreased as the
azithromycin concentration increased; binding was 50% for azithromycin concentrations
of 0.02 and 0.05 mg/L, 37% at 0.7 mgL and 7% at 1 mg/L. Erythromycin and
roxithromycin demonstrated higher binding than azithromycin (72% at 0.4 mg/L
erythromycin and 96% at 2.5 mg/L roxithromycin) [I 04, 1051. Therefore, azithromycin
has a greater proportion of drug in the free form that is then available for distribution to
26
infected tissues. Azithromycin, erythromycin, and roxithromycin are predominantly
bound to a,-acid glycoprotein as suggested by low concentrations of drug displaying
saturability 1105, 1061.
Azithrornycin is mostly eliminated in the feces via transintestinal excretion, but a
small amount leaves in the urine 1107, 1081. Azithromycin is primarily eliminated in an
unchanged form, although some biotransformation does occur. Biotransfonnation
primarily occurs via N-demethylation of the desoamine sugar or the 9a position of the
macrolide ring but hydrolysis and hydroxylation also occur [ 1 081.
Azithromycin (500 rng) showed a mean terminal elimination half-life (tlQ) fiom
serum of 9.6 to 14 hours when given orally and 35 to 40 hours when given intravenously
[95, 1041. The tin for oral azithromycin (500 mg twice on day 1 and 500 mg once on
days 2 to 5) increased from 11 to 14 hours on day 5 to 48 hours over 1 to 3 days after the
last dose and to 57 hours over 1 to 6 days after the last dose [104].
The effect of age was compared in a nonblind parallel study using healthy elderly
and young volunteers given 500 mg on day 1 and 250 mg on days 2 to 5 [109]. Mean
maximum serum concentrations (-0.4 mg/L on day 1 and -0.25 mg/L on day 5 ) and
minimum serum concentrations (0.05 mg/L) were similar for young and elderly
volunteers and there was no difference in urinary excretion. Area under the plasma
concentration-time curve (AUC) and t,, were significantly increased in the elderly. An
inverse relationship was found between creatinine clearance and AUC indicating that
dosage adjustment is not required in elderly people with mild renal impairment, only
those with more severe renal impairment. Subsequent studies have confirmed that
azithromy cin can be safely used in children of all ages [ 1 1 01.
27
Healthy volunteers and patients with mild to moderate hepatic dysfunction did not
display differences in pharmacokinetic parameters except for an increase in t , ~ (68 versus
54 hours) suggesting that dosage adjustment, when mild to moderate hepatic impairment
is present, is not necessary since azithromycin treatment is of short duration and tolerated
favorably [ 1 1 1 1.
1.3 -2.4 Efficacy in Animal Models
The efficacy of azithromycin in vivo has been assessed using animal models of
infection. Mice infected with S. pneumoniae showed a higher rate of survival when given
subcutaneous or oral azithromycin (25 - 50 mglkg) either before or after infection than
when given erythromycin [112]. Bacteria were completely cleared from lungs and blood
in mice given two subcutaneous injections of azithromycin (25 mg/kg) at 48 and 65 hours
post-infection but were not cleared in mice given erythromycin, clarithromycin,
roxithromycin or spindamycin [113]. This result was consistent with greater
concentrations of azithromycin that also remained longer in the lung. When
azithromycin (25 mg/kg) was given orally 24 hours before infection with pneumoniae,
recovery of bacteria after 6 hours of infection was reduced by greater than 99.9%
compared to untreated controls [77]. In the same study, erythromycin (100 mg/kg)
produced no difference compared to controls and roxithromycin (50 mglkg) produced a
96% reduction in bacterial recovery compared to control values.
Intraperitoneal delivery of azithrornycin (3.6 mg/kg/day) resulted in greater
therapeutic efficacy than erythromycin (96 mg/kg/day) in a lethal L. pneumophila
pneumonia model in guinea pigs. Survival rates were 100% with azithrornycin and 83%
28
with erythromycin and clearance of bacteria was complete after two days with
azithromycin whereas erythromycin did not clear the bacteria after six days 11 141. Oral
administration for two days in the same animal model resulted in 100% survival in the
azithromycin group (3.6 mg/kg once daily) and in the clarithromycin group (28.8 mgkg
twice daily). Although the higher dose of clarithromycin resulted in similar survival
rates, clarithrornycin did not eradicate bacteria fiom the lungs as did azithromycin [115].
In another study, mice infected with K pneumoniae showed a 99% reduction in bacteria
recovery from the lungs when given azithromycin (50 mg/kg/day) 24 hours before
infection compared to the untreated control [77].
Taken together, the results generated fiom these animal studies demonstrate that
the efficacy of azithromycin in vivo is greater than that of many other macrolides.
1 -3.2.5 Clinical Efficacy and Dosage
The clinical efficacy of azithromycin has been demonstrated in the treatment of
lower and upper respiratory tract infections, skin and soft tissue infections,
urethritis/cervicitis (1 g over 1 to 3 days), early Lyme disease and M. avium complex
infections (500 mg/day for 10 to 30 days) [64]. The efficacy of azithromycin is
correlated with high tissue concentrations. Typical treatment regimens are a single or
divided 500 mg dose on day one followed by either 250 mg/day for four more days or
500 mg/day for three more days. In both regimens, the azithromycin concentrations in
respiratory tract tissues (> 2 mg/L) are above the minimum inhibitory concentrations
(MIC) for key pathogens (Haemophilus injluenrae, azithromycin MICgO of 0.5-2.0 mg/L;
Streptococcus pyogenes, azithromycin MICgO of 0.12 mgk; Streptococcus pneumoniae,
29
MormceZZa catarrhalis, Mycoplasma pneumoniae, ChZamydia pneumoniae, Legionella
pneumophila, all with an azithromycin MICgO of I 2.0 rng/L) for up to ten days following
treatment [98]. Small children should be given 10 mgkg on day one and 5 mg/kg on
days two to five and children weighing over 45 kg should be given the adult dosage via
either regimen.
Standard regimens of azithromycin have been shown to have comparable efficacy
compared to erythromycin, amoxicillin, cefaclor, amoxicillin.clavulanate, roxithromycin,
and clarithromycin in the treatment of tower respiratory tract infections, although all the
other drugs required more frequent and prolonged regimens than azithromycin to achieve
this same eficacy [72, 1161. This suggests that azithromycin has a more potent clinical
effect than the other drugs to which it was compared. In a study of patients with upper or
lower respiratory tract infection given azithromycin (500 mg on day one and 250 mg on
days 2 to 5), a 95% clinical cure rate and a 91% clearance of sputum bacteria was
demonstrated [117]. A 9 1% clinical cure rate was recorded for patients with acute
bacterial pneumonia given azithromycin (500 mg/day for 3 days) 11181. Studies have
found that azithromycin is more effective than erythromycin in eradicating H. injluenzae
[74]. Clinical cure rates for azithromycin in comparative studies of lower respiratory
tract infections ranged from 36% to 80% and clinical success rates (cure + improvement)
ranged from 92% to 100% [64]. Azithromycin was better tolerated and showed a lower
incidence of adverse effects compared to roxithromycin 172, 1 191. Upper respiratory
tract infections treated with azithromycin (500 mg on day 1 and 250 muday on days 2-5)
showed clinical cure rates of 98% and a 96% clearance of bacteria [117]. Clinical cure
rates for azithromycin ranged from 74% to 95% and clinical success rates ranged fiom
30
98% to 100Y0 in comparative studies on the eficacy of azithromycin in upper respiratory
tract infections [64]. Eradication rates for bacteria were greater than 89% and the clinical
eficacy of azithromycin was demonstrated to be equivalent to the other antibiotics tested.
Children given a totai of 30 mgkg of azithromycin over three or five days demonstrated
clinical cure rates of 78% to 95% for those with acute otitis media and 86% to 93% in
those with pharyngitis and/or tonsillitis [64]. The clinical cure rate of azithromycin was
greater but clinical success rates were not different than those of amoxiciilin in children
with acute otitis media [120].
Overall, clinical efficacy and pharmacological studies suggest that azithromycin
displays comparable or greater efficacy in eradicating certain infections compared to
other antibiotics, and that azithromycin requires a shorter and less frequent dosage
schedule.
1.3.2.6 Tolerability
The overall adverse effect rate is 12% for azithromycin with the most frequent
being gastrointestinal disturbances (9.6%) [diarrhea (2.6%), nausea (2.6%) and
abdominal pain (2.5%)] and central and peripheral nervous system effects such as
headaches and dizziness (1 -3%) [121]. This rate is lower than those seen for
arnoxicillin/probenecid (54.5%), cefaclor (1 6.7%), doxycycline (1 5.8%) and
erythromycin (20.4%). A study comparing the numbers of discontinuations of therapy
demonstrated that azithromycin had a lower percentage of discontinuations of therapy
compared to all the other drugs tested except for doxycycline [121]. There were no
neurological, audiometric or ophthalmoscopic abnormalities detected nor did
3 1
phospholipidosis occur in this study. Patients with hypersensitivity to other macrolides
should not be given azithromycin [121]. The adverse effects of azithromycin are similar
in young and elderly people 112 I].
1 -3 2 . 7 Drug Interactions
Drug metabolism in the body involves enzyme systems such as the mixed
h c t i o n oxidase system [61]. The enzyme cytochrorne P450 is one of the key enzymes
involved in this system. Hydroxylation by P450 often produces a reactive intermediate.
Some drugs can induce this enzyme which can iead to an increase or a decrease in the
metabolism of a second drug. Unlike erythromycin and many other macrolides,
azithromycin does not induce or inhibit cytochrome P450 IIIA enzymes [l22]. In part
because of this, no pharmacokinetic interactions have been found for patients
concomitantly receiving theophylline, cydosporine, warfarin, cimetidine, carbarnazepine,
methyl prednisolone, terfenadine, oral contraceptives or zidovudine [ 1 2 1, 123- 1251.
Azithromycin should be taken 1 hour before or 2 hours after antacids containing
aluminum and magnesium as this has been shown to reduce the peak serum
concentrations but not the total absorption of azithromycin [104, 1 2 11. Interactions
between azithromycin and either ergotamine or digoxin have not been investigated but
since erythromycin csuses ischemia and peripheral vasospasm when given with
ergotarnine and erythromycin causes increased serum digoxin levels, co-administration is
not recommended for ergotamine and azithrornycin and digoxin levels must be monitored
[641.
1.3.3 Anti-inflammatory Therapies
Anti-inflammatories can firnction to reduce the inflammation associated with
pneumonia and prevent host-mediated damage but they do not eradicate the source of the
infection. A number of anti-inflammatory strategies aim at down-regulating the
neutrophil response to infection and inflammation. One example of anti-inflammatory
drugs are the glucocorticoids that inhibit neutrophil emigration out of the vasculature and
into sites of inflammation by decreasing neutrophil adherence to endotheliurn, inhibiting
chemotaxis and inhibiting production of neutrophil chemotactic factors [126]. Inhibition
of phospholipase A2 by glucocorticoids prevents the production of arachidonic acid
derivatives such as leukotriene B4 that attract and activate neutrophils.
The anti-inflammatory glucocorticoid dexarnethasone inhibits neutrophil
superoxide generation, release of Iactoferrin and lysozyme, emigration, chemotaxis, and
production of LTB4 and IL-1 [127, 1281. Dexamethasone induces apoptosis of rat
thymocytes, however, it inhibits apoptosis in human neutrophils [129, 1301.
Dexamethasone decreases IL-8 production by macrophages pretreated with
dexamethasone before exposure to lipopolysaccharide [ 1 3 1 1.
1.3.4 Immunomodulation by Antibiotics
Respiratory tract infections are commonly treated with macrolide antibiotics.
Besides having antibacterial effects, many macrolides have been postulated to have anti-
inflammatory properties that may enhance their efficacies in reducing illness.
Azithromycin has proven to be more effective in the treatment of a number of respiratory
33
tract infections compared to erythromycin, another macrolide with comparable
antibacterial properties [64].
A number of reports have suggested that macrolides, such as erythromycin,
roxithromycin, and clarithromycin, have anti-inflammatory effects [7, 10, 28, 132- 1361.
Azithromycin may also have anti-inflammatory properties. Macrolides may exert this
effect by modulating the production of cytokines including IL- 1, IL-6, IL-8 and TNFa 17,
28, 132-1 391. Macrolides may also alter fhctions of inflammatory cells 120, 140-1421.
Some macrolides, such as azithromycin, roxithromycin, and clarithromycin, inhibit the
respiratory burst of neutrophils [143-1471. It was recently demonstrated that induction of
bovine neutrophil apoptosis by the veterinary macrolide tilmicosin may be associated
with anti-inflammatory benefits [ 1 481. Erythromycin, roxithromycin, clarithrornycin, and
midecarnycin have also been demonstrated to induce neutrophil apoptosis [149].
Apoptotic neutrophils, d i k e necrotic neutrophils, remain intact and are removed from
tissues by phagocytes, thus preventing the release of pro-inflammatory products in siru
148, 1501. The induction of apoptosis by azithromycin has not been previously studied
and its biological significance has yet to be uncovered.
1.4 Apoptosis
Cells can die via one of two forms of cell death, necrosis or apoptosis. Necrotic
cell death results in release of intracellular contents leading to inflammation and
extensive tissue damage [47].
Apoptosis, or programmed cell death, is a distinct form of cell death, both
morphologically and biochemically. In apoptosis, the cell actively participates in its own
34
destruction. Apoptosis is characterized by a number of features: the membrane of an
apoptotic celI remains intact, the cytoplasm and the nucleus becomes condensed, and the
DNA is cleaved into mono- and oligo-nucleosomes, which are commonly used as
indicators of apoptosis [150, 1511. In vivo, apoptosis is rapidly followed by uptake of
apoptotic cells into phagocytic cells [48]. This process is involved in the regulation of
cell turnover, tissue remodeling, regulation of the immune response, and resolution of
inflammation [48, 1 521.
1.4.1 Induction
Apoptosis can be induced by the binding of an appropriate ligand to either a
membrane receptor or a cytoplasmic receptor, which activates a second messenger
system [I 73. Various signals can induce apoptosis, such as the activation of receptors for
Fas ligand or TNF, glucocorticoid treatment, UV irradiation, and serum or growth factor
withdrawal (such as IL-3 or platelet-derived growth factor) [ I 48, 1 53 - 1 5 71. Although the
stimuli may vary, one mechanism consistently implicated in apoptosis is the activation of
a cascade of cytosolic proteases called caspases that initiate the changes associated with
apoptosis [Mi]. In human cells, many substrates for caspases have been identified as
DNA repair enzymes @oly-ADP-ribose-polymerase [PARP], and DNA-protein kinase),
homeostatic proteins (protein kinase C, D4-GDI, and sterol regulatory element binding
proteins), and structural proteins (nuclear lamins A and B, fodrin, and the nuclear mitotic
apparatus protein PUMA]) [40]. It is not known exactly how cleavage of these and other
substrates leads to the apoptotic phenotype except that multiple homeostatic pathways are
interrupted [40].
1.4.2 Neutrophil Apoptosis
Accumulation and bursting of necrotic neutrophils at the site of inflammation
contribute to the propagation of inflammation during infectious pneumonia and other
respiratory tract infections. Neutrophils recruited to sites of infection can undergo either
necrosis or apoptosis (programmed cell death). Cytotoxic compounds such as elastase,
hypoch1orous acid, and oxygen radicals reieased by necrotic neutrophils recruited to sites
of infection can damage surrounding tissues, and induce necrosis in neighbouring
neutrophils [7, 1591. Neutrophils injured in this fashion further release cytotoxic
compounds and pro-inflammatory mediators [13]. In contrast, apoptotic neutrophils may
be eliminated without spilling pro-inflammatory and cytotoxic products in siru [48, 1501.
Apoptosis is a less harmful process than necrosis since the membrane of an apoptotic
neutrophil remains intact. In addition, apoptotic cells are more rapidly phagocytosed by
macrophages, through the use of various surface-signaling molecules, including the
exposure of phosphatidylserine on the outer leaflet of the cell membrane 1160, 1611.
Thus, apoptosis is a more desirable form of cell death since the preservation of membrane
integrity and rapid clearance of apoptotic cells from the site of infection minimize host
tissue damage and inflammation [132, 162, 1631. This could be important in the
resolution of lung inflammation resulting fiom diseases such as streptococcal pneumonia.
1.4.3 Regulation of Apoptosis in Neutrophils
Activation of caspases (interleukin l Ptonverting enzyme homologues) initiates
apoptosis [40]. After isolation fiom blood, neutrophils undergo spontaneous apoptosis,
36
and thus neutrophil age is one factor involved in inducing apoptosis 1401. Forty-five
percent of neutrophils incubated for 24 hours are apoptotic. Caspase-3 is the primary
caspase involved in the initiation of the apoptotic cascade [40]. Active caspase-3 is
detected in apoptotic neutrophils but not in fieshly isolated neutrophils. Neutrophils
contain a number of caspase substrates including D4-GDI, gelsolin, lamin B, and fodrin
[40]. D4-GDI is a negative regulator of Rho GTPases, which may regulate the
cytoskeletal and membrane alterations accompanying cell death [164, 1651. Cleaved D4-
GDI is unable to bind GTPases thus allowing these GTPases to exert their effects.
Cleavage and consequent activation of gelsolin, an actin-severing protein, results in a
reduction in F-actin content and a disruption of cell structure leading to apoptosis [166].
Gelsolin cleavage also contributes to nuclear fragmentation. Lamin B is a nuclear matrix
protein which when cleaved leads to the chromatin condensation characteristic of
apoptosis [ 1671. Proteolytic processes during apoptosis make the surface of neutrophils
pro-phagocytic and more readily identifiable by macrophages. Fodrin is a cytoskeletal
component and its cleavage may be involved in membrane blebbing of apoptotic cells
[168]. Fodrin cleavage has been linked to the exposure of phosphatidylserine on the
outer membrane, which appears to be one mechanism by which phagocytic cells
recognize and clear apoptotic neutrophils [169]. Phosphatidylserine, normally localized
to the inner half o f the bilayer of the cell membrane of neutrophils, is translocated to the
outer half of the bilayer through the activation of scramblases and the downregulation of
aminophospholipid translocase (which normally reinternalizes any phosphatidylserine
that reaches the outer membrane) [161]. The receptor, 61D3, on macrophages may be
involved in phosphatidylserine-dependent phagocytosis [ I 701. Uptake of apoptotic cells
37
also occurs via the vitronectin receptor and CD36 on macrophages, which interact with
neutrophils via thrombospondin and an as yet unidentified molecule on the surface of
apoptotic neutrophils, which does not appear to be phosphatidylserine [47, 1 70, 1 7 13.
1 .S Research Hypothesis and Objectives
While the clinical effectiveness of azithromycin may be due in part to its
pharmacokinetic properties, it may have anti-inflammatory properties in addition to its
antibacterial effects. It is my hypothesis that azithromycin may induce apoptosis in
human neutrophils more effectively than other antibiotics and hence help the host to
dispose of these cells without perpetuating local inflammation via enhanced macrophage
cIearance of apoptotic neutrophils. The aim of this study was to determine the effects of
azithromycin on human neutrophil apoptosis, oxidative function and IL-8 production, in
the presence or absence of S. pneurnoniae.
Specific objectives of this study are:
1. To assess the induction of neutrophil apoptosis by azithromycin in comparison to
other drugs, in the presence or absence of Srreptococcus pneumoniae.
2. To investigate the effect of azithromycin on the phagocytosis of neutrophils by
macrophages.
3. To determine the effects of azithromycin on neutrophil fhction (oxidative
metabolism, IL-8 production) in comparison to other drugs, in the pcn ,,sence or
absence of Srreptococcus pneurnoniae.
38
CHAPTER 2: METHODS AND MATERIALS
2.1 Drugs
Azithromycin (Pfizer Canada Inc., Montreal, PQ, Canada), penicillin-G (Sigma
Diagnostics, St. Louis, MO, USA), and erythromycin (Sigma) were dissolved in pyrogen-
free phosphate buffered saline (PBS, pH 7.1) and diluted to a concentration of 0.05
@mL. Dexamethasone (Schering Canada Inc., Pointe-Claire, PQ, Canada) was diluted
in PBS (pH 7.1) to a concentration of 0.02 &mL.
2.2 Bacteria
Sfreptococcus pneumoniae 14259 (human clinical isolate obtained from Dr. R. R.
Read, Foothills Hospital, Calgary, AB, Canada) was grown overnight on Columbia blood
agar (CBA) plates at 37°C. Bacterial cells were collected, suspended in PBS (pH 7.1),
and diluted to approximately 1.5 x lo8 bacterialml using a 0.5 McFarland standard
(Dalynn Laboratory Products, Calgary, AB, Canada). The final concentration of viable
bacteria (- 1 x 10' CFU/mL) was calculated via colony forming unit (CFU) counts of
serial dilutions on CBA after a 24 h incubation at 37OC. Bacteria were used either live or
as sonicates (4 h, on ice, 85% duty cycle, output control setting 4, W-350 sonifiera,
Branson Ultrasonics Corporation, Danbury, CT, USA).
2.3 Subjects
Male and female subjects were recruited from volunteers at the University of
Calgary. Only those not taking medications were included in these studies. Throughout
39
this study more than 10 different donors were used and at least three different donors
were used to obtain data for the individual experiments.
2.4 Neutrophils
Blood was collected by venipuncture from healthy human volunteers and
collected in sodium heparin vacutaine? tubes (Becton Dickinson, Franklin Lakes, NJ,
USA). Neutrophils were purified by density gradient centrifugation using HistopaqueTM
1 11911 077 (Sigma). Whole blood was layered onto a gradient of Histopaquem 1077 (3
mL) that was underlayered with Histopaquem 1 1 19 (3 mL). AAer centrifugation
( I SOOxg, 30 min, Beckman J-6B centrifuge, Beckrnan Instruments, Pa10 Alto, CA, USA),
the mononuclear cell layer was removed and discarded. The neutrophil layer was
removed, washed in 10 mL of lx Hanks' balanced salt solution without calcium and
magnesium (HBSS, Gibco BRL, Life Technologies Inc., Grand Island, NY, USA), and
centrifbged (1 500xg, 1 0 min). Contaminating erythrocytes were lysed by resuspending
the pellet in 1 mL of sterile distilled water for 30 seconds, and osmolarity was restored by
adding 1 mL of 2 x HBSS (Gibco BRL). AAer washing in 10 mL of lx HBSS, purified
cells were resuspended in either HEPES-buffered RPMI 1630 (Sigma) supplemented
with 0.05pg/mL L-glutarnine, ca2+/Mg2+-free PBS (pH 7.1) (for NBT assay) or 1 x HBSS
(for macrophage phagocytosis) to a final concentration of 10' to 1 o6 cells/ml depending
on the experiment. Cells were counted in a haemacytometer (Spencer Bright-Line
Improved Neubauer, American Optical Corp., Buffalo, NY, USA), and viability was
assessed by 0.1 % trypan blue (Gibco BRL) exclusion. The cell suspension (1 00 pL) was
40
centrifuged (20xg, 10 min) onto a microscope slide with a cytospin (Shandon Southern
Producers Ltd., Cheshire, England), fixed and stained with Diff Quick (Baxter Healthcare
Corp., Miami, FL, USA) and neutrophil purity was assessed by direct microscopy
differential leukocyte counts. Neutrophils were incubated (37"C, 5% C 0 2 ) for various
times with PBS (pH 7.1) (controls), azithromycin (0.05 pg/mL), penicillin (0.05 pg/mL),
erythromycin (0.05 pg/mL), dexamethasone (0.02 pg/mL), or tumor necrosis factor-a
(TNFa, R&D Systems, Minneapolis, MN, USA, 5 ng/mL), with or without live
Streptococcus pneumoniae or lysate fiom S. pneumoniae.
2.5 Monocytes/Macrophages
Human peripheral blood monocytes can be cultured in vifro. They become
activated both by the isolation process fiom whole blood and by attachment to tissue
culture plastic surface 11721. This adherence in the presence of serum mimics the
immigration of monocytes through the vasculature into the site of infection and primes
the monocyte for inflammatory responses [173]. The activated monocytes differentiate
into macrophages when serum is present in the medium [174]. As the monocytes
differentiate they increase in size and phagocytic activity, and many biochemical and
morphological changes occur [172]. Human leukocytes contain esterases which
hydrolyze aliphatic and aromatic esters [45, 1751. Macrophages contain a nonspecific
esterase that can be detected by addition of the substrate a-naphthyl acetate coupled to a
diazonium salt, followed by counter-staining with methyl green. Hydrolysis of the
substrate produces an insoluble coloured azo dye. Macrophages show a diffuse
41
r e a r o w n staining of the cytoplasm with a green nucleus (positive for nonspecific
esterase), while monocytes, which produce very littie nonspecific esterase, appear green
(negative for esterase).
Blood was collected by venipuncture from healthy human volunteers and
collected in Vacutainea C P V cell preparation tubes with sodium citrate (Becton
Dickinson). Monocytes were purified by density centrifbgation (2000xg, 30 min). The
mononuclear cell layer was removed, washed in 1 x HBSS (1 0 mL), and centrifuged
(300xg, 15 min). The pellet was resuspended in 1 x HBSS (10 mL) and centrifuged
(3OOxg, 10 min). The pellet was resuspended in Iscove's modified Dulbecco's medium
(MDM) (Gibco BRL) to a final concentration of 6 x lo6 cells/mL. Cells were counted in
a haemacytometer, and viability was assessed by trypan blue (0.1%) exclusion.
Monocytes (1 mL) were incubated (37"C, 5% CO2) in 24-well tissue culture plates
(Costar, Cambridge, MA, USA) for 60 min after which non-adherent cells (mainly
lymphocytes) were removed by washing 3 times with l x HBSS (37OC, 1 mL) [176].
Monocyte purity was assessed by staining cytospin preparations with Diff Quick and
direct microscopy differential leukocyte counts. Monocytes were cultured in 1 mL
Iscove's MDM containing 10% fetal bovine serum (Sigma) and were allowed to
differentiate into macrophages for seven days. The media was replaced every 2-3 days
by aspiration.
Macrophage differentiation was ascertained by mature cell morphology (size and
shape changes) after being stained with Diff Quick and by a positive reaction for
nonspecific esterase. Nonspecific esterase staining was done as previously described
42
[45]. Briefly, after cells were fixed, they were incubated with esterase substrate solution
containing a-naphthyl acetate (Sigma) and hexazoatized pararosalanine (Sigma) for 45
min at 37°C. Slides were counter-stained for 2 min with 1% methyl green and examined
by light microscopy.
2.6 CFU Counts
S. pneumoniae was incubated with PMNs and drugs (37"C, 5% COz) at a ratio of
10:l for 2 h and 6 h, plated on CBA plates after serial dilutions, and colonies were
counted after 24 h incubation at 37°C.
2.7 Annexin-VIPropidium Iodide Labelling
Neutrophil apoptosis and necrosis were assessed by double staining with FITC-
conjugated mexin-V and propidium iodide (PI) using an Annexin-V FLUOS kit
(Boehringer-Mannheim GmbH, Germany), according to manufacturer's instructions.
Annexin binds with high affinity to phosphatidylserine which is translocated from the
inner half of the bilayer to the outer half of the membrane bilayer during both apoptosis
and necrosis 11 591. Necrotic ceils are differentiated by staining with propidium iodide.
After 1 hour co-incubation with or without drugs and with or without bacterial lysate (4: 1
bacteria to cell ratio), PMNs (5.5 x lo6 cells/mL) were centrifuged (Baxter Canlab
Biofuge A, Heraeus Sepatech GmbH. Germany) (200xg, 5 min), then washed with I mL
PBS (pH 7.1). Cells were centrihged again, resuspended in 100 pL of FITC-conjugated
annexin-V and propidiurn iodide staining solution, and incubated for IS minutes in the
43
dark. Samples were then centrihged (200xg, 5 min) and resuspended in PBS (pH 7.1),
and a wet mount of 15 pL was observed by epifluorescent microscopy (450 m and 535
nm) (Leitz Aristoplan, Germany) at 400x magnification and the percentages of apoptotic
and necrotic cells were determined from ten different fields per preparation. The high
and low counts were discarded and the other 8 were averaged.
As a positive control for the annexin-V/PI labeIling system, PMNs were incubated
with 5 ng/mL (approximately 100 U/mL) recombinant human TNFa in PBS (pH 7.1)
with 0.1% bovine serum albumin for 1, 2, and 3 hours. TNFa at this concentration is
known to induce neutrophil apoptosis [177].
2.8 Cell Death ELISA
Neutrophil apoptosis was also assayed via ELISA. Neutrophils (lo5 cells/mL)
were co-incubated for 6 hours with or without drugs and with or without live bacteria
(10: 1 bacteria to cell ratio) and stored at -70°C for later analysis. Samples were assayed
for apoptosis with a Cell Death Detection ELISA kit (Boehringer Mannheim), which
detects the histone region (H 1, H2A, H2B, H3, and H4) of mono-nucleosomes and oligo-
nucleosomes that are formed during apoptosis. The plates were read at 405 nrn
(THERMOmaxTM microplate reader, Molecular Devices Corp., Menlo Park, CA, USA) at
intervals for 58 min, starting 1 rnin after addition of substrate. Results were expressed as
the ratio of the sample absorbance versus the mean absorbance of the vehicle-treated
control.
44
2.9 Macrophage Phagocytosis
PMNs (6 x 1 o6 cells/mL) were incubated for 1 hour with or without azithromycin
and then centrifuged (minimum speed, 5 min, microcentrifuge). The pellet was washed
by resuspending in 1 x HBSS (1 mL) and centrifuged (minimum speed, 5 min). This
pellet was resuspended in l x HBSS to the original ceH volume of 300 pL. The PMNs
were acided to the wells containing 7 day old cultured macrophages and incubated for 1
hour (37"C, 5% C02). PMNs were obtained from the same donor as described above.
The wells were washed three times with 1 x HBSS (37OC, 1 mL) and stained with Diff
Quick. Three counts of 100 cells were done on each well to determine the number of
macrophages containing phagocytosed PMNs. A total of 2400 macrophages were
counted. Only macrophages that had completely surrounded a PMN were counted.
2.10 Nitro Blue Tetrazolium (NBT) Assay
The oxidative function of PMNs (lo6 cells/mL) co-incubated for 1 hour with or
without drugs and with or without bacterial lysate (10:l bacteria to cell ratio) was
assessed by NBT reduction. Cells were incubated in chamber slides (Nalge Nunc
International, Naperville, IL, USA) for 20 minutes with nitro blue tetrazoliwn (NBT, 2
mg/mL, Sigma) in the presence or absence of 2 pg/mL phorbol 12-myristate 13-acetate
(PMA) (Sigma). Cells were fixed with methanol and counterstained with safianin (1%).
The percentage of oxidatively active cells with blue formazan crystals in their cytoplasm
was calculated following direct microscopic examination and counting under oil
immersion ( 1 000x) with a light microscope (Zeiss, Germany).
45
2.1 1 Interleukin-8 Assay
PMNs (2x10' cells/mL) were incubated for 2 hours with drugs, then washed in
PBS (pH 7.1 ) and incubated (24 h, 3 7OC, 5% C02) with or without S. pneumoniae lysate
(10:l bacteria to cell ratio). Cells were then centrifbged (800xg, 10 min) and the
supernatant collected. IL-8 production was assessed by a Quantikinem human IL-8
sandwich enzyme immunoassay (R&D Systems) according to the manufacturer's
instructions. This assay is specific for natural and recombinant human IL-8. No
significant cross-reactivity or interference has been observed for other human or mouse
interleukins or other factors tested. The sensitivity of this kit is 10 pg/rnL.
2.12 Statistical Analysis
Results were expressed as mean + SEM and compared by one-way analysis of
variance (ANOVA) followed by Tukey's test for multiple comparison except for the
macrophage phagocytosis results which were analyzed using Fisher's Exact test with a 2-
sided P value and a chi-square approximation. P<0.05 was considered significant.
CHAPTER 3: RESULTS
3.1 Cell Isolation
The success of the cell isolation procedures was determined via examination of
the viability and purity of the resultant populations. The viability of purified PMNs
incubated in PBS (pH 7.1), HBSS, or RPMI 1640 was >95%, and cell purity was >98%.
The purity of the isolated mononuclear cells was >98% and viability was >97%.
Monocytes accounted for -40% of the mononuclear cells. Contaminating cells in the
purified monocytes were mainly Lymphocytes and the occasional neutrophil, but due to
the short lifespan of neutrophils none remained after the third day of incubation [44].
Contaminating lymphocytes in the monocyte preparation were removed by washing
following the initial 1 hour incubation in the well plates, as lymphocytes do not adhere to
the wells [ I 761.
3.2 CFU Counts
CFU counts were done to determine whether the antibacterial activity of the
antibiotics was altered by the presence of neutrophils and whether S. pneumoniae was
susceptible to the antibiotics tested. After 6 hours of co-incubation with PMNs,
azithromycin, penicillin, and erythromycin significantly decreased S. pneumoniae
numbers cor~pared to controls (Figure I). At 6 hours, bacterial numbers in preparations
containing azithromycin were significantly lower than those exposed to other antibiotics.
Incubation with dexamethasone did not affect bacterial numbers.
3.3 Neutrophil Apoptosis
To assess the anti-inflammatory potential of azithromycin, I looked at the
induction of apoptosis in neutrophils. To determine whether azithromycin induces
neutrophil apoptosis two systems were used - annexin-V/PI labelling and Cell Death
Detection ELISA. The annexin-VPI fluorescent labelling system allowed discrimination
of apoptotic fiom necrotic cells (Figure 2A and Figure 28). TNFa induced neutrophil
apoptosis in a time-dependent fashion (Figure 3). In the absence of S. pneumoniae lysate,
azithromycin induced neutrophil apoptosis at levels similar to those seen in cells
incubated with TNFa for 3 h (Figure 4A). Cells incubated with penicillin, erythromycin,
or dexarnethasone were not different fiom vehicle controls. Addition of bacterial lysate
inhibited the induction of apoptosis by azithromycin (Figure 4B).
In an attempt to explain this inhibition of apoptosis by bacterial lysate, cell
necrosis was assessed to see if neutrophil necrosis was higher in the samples containing
lysate. As shown in Figure 5, S. pneumoniae did not afCect neutrophil necrosis. In
addition, neutrophil recovery was not significantly different between cells incubated with
saline ( 7 . 4 3 ~ 1 o6 f 4.61 x 10' cells/mL), azithromycin (6 .06~ 1 o6 f 4.8 1 x 10' cells/ml), S.
pneumoniae (6 .88~ lo6 f 7 . 0 4 ~ I 0' cellslml) or azithromycin and S. pneumoniae together
( 7 . 1 5 ~ 1 o6 i 3.3 1 x 10' cells/mL).
The induction of neutrophil apoptosis by azithromycin was also detected by
ELISA at 6 h in the absence of S. pneumoniae (Figure 6A). The colorimetric reaction
was rapid enough to be detected after 1 minute of incubation with reagent. As was
observed with annexin-V labelling, co-incubation with S. pneimoniae abolished this
48
effect (Figure 6B). Incubation of neutrophils with penicillin, erythromycin, or
dexamethasone did not affect neutrophil apoptosis in the presence or absence of S.
pneumoniae.
3.4 Macrophage Phagocytosis
Apoptotic neutrophils are rapidly phagocytosed in vivo by phagocytes, a process
that disposes of these cells without perpetuating Iocal inflammation. To determine
whether the increased number of apoptotic cells seen in azithromycin-treated neutrophil
populations contributed to an increased uptake of these cells by macrophages, I used
monocyte-derived macrophages. Monocyte differentiation into macrophages was
determined using two techniques: mature cell morphology and nonspecific esterase
staining. On day 0, all cells looked like typicai monocytes (cell size Iess than 13 pm, a
round nucleus, and a thin ring of cytoplasm). Cell morphology (cell size greater than 13
pm, a bean shaped nucleus, and much larger cytoplasmic area) revealed differentiation of
70% on day 2, 96% on day 5, and 98% on day 7. Nonspecific esterase staining showed
differentiation of 79% on day 2, 92% on day 5, and 98% on day 7 (Figure 7). These
measures indicate that a large population of differentiated macrophages was present on
day seven
Macrophages which had ingested neutrophils were identified via tight microscopy
(Figure 8). Macrophage phagocytosis of neutrophils after 1 hour was found to be
significantly increased for azithromycin-treated PMNs compared to control PMNs
(Figure 9).
49
3.5 Oxidative Function and LC-8 Production
To determine whether the induction of apoptosis by azithromycin affects the
function of neutrophils, I looked at oxidative function and 1L-8 production. The ability to
produce reactive oxygen intermediates for microbial killing is an essential function of
neutrophils. None of the drugs affected oxidative h c t i o n in resting (not exposed to
PMA) or stimulated (exposed to PMA) neutrophil populations (Figure IOA). Similarly,
the drugs did not affect oxidative function when co-incubated with bacterial lysate
(Figure 10B).
Neutrophils produce chemotactic IL-8 in order to recruit more neutrophils to sites
of infection. Exposure of neutrophils to S. pneumoniae lysate promoted synthesis of IL-8
(Figure 1 1). IL-8 secretion was not affected by any of the antibiotics in the presence or in
the absence of S. pneumoniae lysate compared to controls (Figure 11). In contrast,
dexamethasone significantly inhibited S. pneumoniae lysate-induced IL-8 synthesis.
Figure 1. S. pneumoniae recovery (loglo CFU) after 2 h and 6 h co-incubation of PMNs
with azithromycin (---.em- -*), penicillin (- -@ - -), erythromycin (- X - -) and
dexamethasone (- -A-). Antibiotic treatments are significantly different from
control (-*-) and dexarnethasone at both times (n=5; experiment was repeated 3
times). * PcO.05 compared with control. A Pc0.05 azithromycin compared with all
groups.
1 2 3 4 5 6 7 Time (h)
Figure 2. Light micrographs (400x) of PMNs stained with the annexin-V/propidiurn
iodide system. A) Bright field micrograph illustrating several PMNs, and B)
fluorescence micrograph of the same field showing an apoptotic PMN (arrow) and two
necrotic PMNs (arrowheads); viable neutrophils seen on the bright field micrograph were
not labelled.
Figure 3. Apoptosis in PMNs treated with 5 ng/mL TNFa for 1 , 2, and 3 h (n=4;
experiment was repeated 2 times), determined by annexin-VIP1 labelling. The bar
labelled control represents the mean of the control values (n=6) from 1, 2, and 3 h. *
P<0.05 compared with control.
CONTROL TNF I H TNF2H TNF3H SAMPLE
Figure 4. Apoptosis in neutrophils determined by annexin-VffI labelling after 1 h
incubation with azithromycin (Azi), penicillin (Pen), erythromycin (Ery), or
dexamethasone (Dex). A) Without lysate, B) with S. pneumoniae lysate (4 CFU:I
PMN). TNFa sample is the 3 h incubation value from Figure 2. * P<0.05 compared
with control. ** Pc0.05 compared with control and other drugs (n25; experiment was
repeated 5 times).
TNFa CONT AZI PEN ERY DEX
Figure 5. PMN necrosis in samples incubated with azithromycin (Azi), penicillin (Pen),
erythromycin (Ery), or dexamethasone (Dex) and with (black bars) or without (white
bars) S. pneumoniae Iysate determined by annexin-V/PI labelling. No significant
difference between any samples (n25; experiment was repeated 5 times).
CONT AZI PEN ERY D M CONT AZI PEN E R Y DEX
Figure 6. Apoptosis detected by Cell Death ELISA, afier 6 h incubation. A) Without S.
pneurnoniae, B) with live S. pneurnoniae. Absorbance readings were plotted as a ratio of
the sampIe absorbance to the control, which was set at 1 . Azithromycin (-==C--• ==),
penicillin (- -0 - -), erythromycin (- =X- -) and dexamethasone (- -A-),
control (-*-). *Pc0.05 compared with control and dexamethasone (n 2 5;
experiment was repeated 4 times).
Figure 7. Light micrograph (400~) of monocytes!macrophages stained for nonspecific
esterase. Positively stained cells (monocyte-derived macrophages) appear red and
negatively stained cells (undifferentiated monocytes) appear green.
Figure 8. Light micrograph (400~) of macrophages incubated with PMNs for one hour.
A macrophage that has phagocytosed a neutrophil (arrow) and a non-phagocytosed PMN
(arrowhead) are seen.
Figure 9. Phagocytosis of azithromycin-treated PMNs by macrophages afier 1 h,
following a 1 h incubation of PMNs with azithromycin or HBSS (control). *P<0.05
versus control (experiment was repeated 3 times; a total of 2400 macrophages were
counted). No error bars are shown as values are discrete numbers and not a mean.
control azithromycin
Sample
Figure 10. Oxidative activity determined by the percentage of resting (white bars) and
PMA-stimulated (black bars) PMNs able to reduce NBT. Samples incubated 1 h with
azithrornycin (Azi), penicillin (Pen), erythromycin (Ery), or dexamethasone (Dex) and
A) without S. pneumoniae Iysate or B) with S. pneumoniae lysate. In A) and B), all
stimulated groups are significantly different from a11 resting groups. No significant
difference between drug-treated and control samples within any group (n=5; experiment
was repeated 3 times).
CONT AZl PEN ERY DEX C O W AZI PEN ERY DU(
CONT AZI PEN ERY DEX CONT AZI PEN ERY DEX
Figure 1 1 . IL-8 production by PMNs incubated 24 h with (black bars) or without (white
bars) S. pneumoniae lysate following a 2 h incubation with azithromycin (Azi), penicillin
(Pen). erythromycin (Ery), or dexamethasone (Dex). No significant difference was
observed between antibiotic-treated and control samples within groups treated with or
without lysate (1124; experiment was repeated 2 times). *P<0.05 versus sham-treated
control.
Cont Azi Pen Ery D e w Cont Ati Pen Ery Dex
CHAPTER 4: DISCUSSION
My study investigated the effects of azithromycin on human circulating
pol ymorphonuclear neutrophils. My results indicate that azithromycin induces neutrophil
apoptosis without affecting the oxidative metabolism or IL-8 production of these cells in
vib-o (Figures 4A, 6A, 10, and I 1) Exposure of neutrophils to azithromycin significantly
increased in vitro phagocytic uptake of these cells by macrophages (Figure 9). Co-
incubation of neutrophils with S. pneumoniae inhibited the induction of apoptosis by
azithromycin (Figure 4B, and 68).
Co-incubation of live S. pneumoniae with antibiotics (azithromycin,
erythromycin, and penicillin) and neutrophils confirmed that the antibacterial effects of
these antibiotics are not altered by the presence of neutrophils, and that azithromycin
exhibits effective antibacterial properties (Figure 1). After six hours of incubation
azithromycin was more effective in eradicating S. pneumoniae than the other antibiotics.
My observations are consistent with those published in other reports [77, 781.
Previous studies have suggested that macrolides have anti-inflammatory effects,
but the mechanisms of this action remain unclear 17, 10, 28, 132-1361. A number of
these studies have focused on the effects of macrolides on cytokine production [7, 28,
132-1391. Other studies have suggested that macroiides modulate the hc t i ons of
polymorphonuclear leukocytes, macrophages, and lymphocytes, as well as altering the
functions of airway secretory cells and epithelial cells [lo, 140-1421. Although still
controversial, findings from these previous studies indicate that erythromycin,
roxithromycin and clarithromycin may reduce the production of pro-inflammatory IL- 1,
73
IL-6, IL-8 and TNF. In addition, erythromycin derivatives, including azithromycin,
roxithromycin and clarithromycin, are known to inhibit the oxidative response of
neutrophils in a time and concentration dependent fashion [I 43- 1471. My results indicate
that after two hour exposure to azithromycin, the release of IL-8 by a population of
human neutrophils remains unchanged, despite the commitment of some cells to undergo
programmed cell death (Figure 11). Moreover, results from my study indicate that one
hour exposure to azithromycin is insufficient to reduce oxidative metabolism of human
neutrophils (Figure 10). Taken together, these observations indicate that the effects of
azithromycin on IL-8 production as well as oxidative metabolism are time and
concentration dependent (as the other studies used different drug concentrations and
exposure times than my study).
Dexamethasone is known to reduce IL-8 production by LPS-treated human
peripheral blood monocytes and alveolar macrophages [131]. In my study,
dexarnethasone significantly inhibited Srreprococcus pneumoniae lysate-induced IL-8
synthesis by neutrophils (Figure 11). In vivo, a reduction in IL-8 could be an important
strategy to reduce the inflammatory lung injury caused by neutrophils. IL-8 is a major
chemotactic cytokine for neutrophils, and macrophages and monocytes are the major
producers of this cytokine during the inflammatory response 1281. The large numbers of
neutrophils at sites of inflammation, however, make these cells an additional important
contributor of IL-8 [28, 1781. The results of my study, however, indicate that the eficacy
of azithromycin is not dependent on the reduction of pro-inflammatory IL-8 (Figure I 1).
Accumulation and necrosis of neutrophils in tissues contribute to the propagation
of inflammation in a number of inflammatory diseases, including infectious pneumonia
74
[I, 11, 551. In contrast, apoptotic neutrophils may be eliminated without spilling pro-
inflammatory products in siru. A recent report indicates that erythromycin,
roxithrom ycin, clari throm ycin, and midecarn ycin shorten neutrophil survival by
accelerating apoptosis 11491. The biological significance of this observation has yet to be
uncovered. It is shown in my study that after one hour exposure to azithromycin,
apoptosis may be detected in human neutrophils via annexin-V labelling of exposed
phosphatidylserine (Figure 4A). Additionaily, neutrophils exposed to azithrornycin for
one hour exhibited an increased level of phagocytic uptake by human monocyte-derived
macrophages (Figure 9). Only activated macrophages are able to phagocytose apoptotic
neutrophils. Human peripheral blood monocytes cultured in vitro become activated both
by the isolation process from whole blood and by attachment to tissue culture plastic
s u ~ a c e s in the presence of serum [172]. The adherence mimics the immigration of
monocytes through the vasculature into the site of infection and primes the monocyte for
inflammatory responses [173]. Macrophages use phosphatidylserine to recognize and
phagocytose certain populations of apoptotic cells. The macrophage receptor, 61D3,
binds to phosphatidylserine on apoptotic cells [170]. Additionally, the vitronectin
receptor and CD36 on macrophages interact with apoptotic neutrophils via
thrombospondin and a surface molecule (which does not appear to be phosphatidylserine)
[47, 170, 1711. The results of my study suggest that apoptotic neutrophils may also use
the p hosphatidy lserine-dependent mechanism for uptake into macrophages since both
phosphatidylserine exposure (which annexin-V binds) and phagocytic uptake of
neutrophils by macrophages were increased in azithromycin-treated neutrophils.
75
I hypothesize that azithromycin may induce apoptosis in human neutrophils more
effectively than other antibiotics and hence help the host dispose of these cells without
perpetuating local inflammation. Consistent with this hypothesis, it was recently shown
that tilmicosin, a macrolide used in the treatment of bovine pneumonic pasteurellosis, has
anti-inflammatory benefits which are associated with neutrophil apoptosis and inhibition
of local leukotriene B4 release [148]. In addition, it is well established in the literature
that physiological removal of apoptotic neutrophils in the inflamed lung in vivo promotes
the resolution of inflammation [179, 1801. Neutrophil influx must be limited and
neutrophils that are no longer necessary must be removed in order for the resolution of
inflammation to occur [179]. Macrophage clearance of apoptotic neutrophils prevents
tissue destruction by the cytotoxic intracellular contents of neutrophils.
Annexin-V labelling detects the exposure of phosphatidylserine on the outer
leaflet of the cell membrane, an early event in cell death [159, 1811. In this system,
apoptosis is distinguished fiom necrosis by propidium iodide labelling, which enters only
cells with a compromised membrane. The experimental assessment of apoptosis was
validated by using TNFa as a positive control (Figure 3) [177]. My results indicate that
human neutrophils exposed to azithromycin for one hour in vitro were committed to
apoptotic death, while erythromycin, penicillin and dexamethasone showed no induction
of apoptosis in neutrophils (Figure 4A). In addition, the formation of mono- and oligo-
nucleosomes, a late event in apoptotic cell death, was detected in neutrophils via ELISA
after six hours of incubation with azithromycin (Figure 6A).
In another study, erythromycin was shown to induce apoptosis in neutrophils in
virro [149]. The fact that this was not observed in my study may be explained by the
76
different incubation times and antibiotic concentrations; the previous study assessed the
induction of neutrophil apoptosis afier twenty-four hours of incubation, and used a
minimum concentration of 1 pg/mL [149]. My findings suggest that azithromycin
induces neutrophil apoptosis more quickly and at lower drug concentrations than
erythromycin. Neutrophils have a higher affinity for azithromycin than erythromycin.
Additional studies will help assess whether this difference in affinity accounts for the
more rapid induction of neutrophil apoptosis by lower concentrations of azithromycin
compared to erythromycin 1641. The anti-inflammatory glucocorticoid dexamethasone
has been reported to inhibit neutrophil apoptosis [130], but this effect was not seen in my
study. Again, as inhibition of apoptosis in human PMNs was observed after twenty-four
hours of exposure to dexarnethasone, the six hour incubation used in my study may not
have been sufficient to significantly inhibit apoptosis [130]. Regardless, my study
demonstrates that azithrornycin induces apoptosis in human neutrophils to levels greater
than those observed for other drugs and that azithromycin-treated neutrophils show a
greater uptake by macrophages (Figures 4A, 6A, and 9). The induction of apoptosis may
be responsible for the increased phagocytic uptake of azithromycin-treated neutrophils
via recognition of apoptotic cells by a phosphatidylserine-dependent mechanism. I
postulate that this mechanism may at least in part contribute to the clinical efficacy of this
macrolide in the treatment of infectious pneumonia by limiting the ability of neutrophils
to damage surrdunding tissues and preventing further amplification of inflammation.
The mechanism by which azithrom ycin indwes apoptosis in neutrophils remains
unknown and is an area that requires W e r study. It may be that azithromycin binds to
and activates receptors such as the Fas receptor or the TNF receptor that have been
77
implicated in the induction of the apoptotic cascade of events [153, 154, 1821.
Alternatively, azithromycin may enter neutrophils and induce apoptosis by activating a
nuclear or cytosolic receptor. It has been suggested that the loss of lipid asymmetry is
linked to activation of the Fas system [161]. The mechanism by which the enzymes
involved in phosphatidylserine translocation become activated or inhibited is not known
but has been postulated to involve increased cytosolic calcium concentrations in
erythrocytes and platelets [183]. Alternatively, the mechanism by which TNFa results in
neutrophil apoptosis is postdated to involve the generation of reactive oxygen
intermediates [ 1 841.
The effect of azithromycin on other cell types involved in the mediation of
inflammatory lung injury is another area that may give additional information about the
clinical efficacy of azithromycin in resolving pulmonary infection.
My results suggest that the apoptosis-inducing effect of azithromycin is abolished
when neutrophils are co-incubated with Streptococczrs pneumoniae lysate (Figures 4B,
and 6B). Levels of necrosis were not different between samples with or without lysate,
indicating that the low apoptotic level was not due to an increase in necrosis (Figure 5).
The mechanisms whereby S. pneumoniae products may reverse or inhibit the apoptotic
signal remain to be investigated. In one study, the LPS of Gram-negative bacteria has
been shown to inhibit TNFa-induced apoptosis and spontaneous apoptosis in neutrophils
in virro, possibly by inducing synthesis of proteins which are involved in regulating the
apoptotic pathway or by production of cytokines such as IL-I P and TNFa [185]. TNFa
(in concentrations under 0.1 U/mL), and IL-1P inhibit apoptosis. In another study,
Staphylococcal enterotoxins (superantigens) have been shown to increase production of
78
LTBJ, and LTB4 is able to delay neutrophil apoptosis [186]. Thus, superantigen may act
indirectly to inhibit neutrophil apoptosis. In vivo, superantigen may also inhibit
neutrophil apoptosis through its effects on cytokine production by other immune cells
such as monocytes and lymphocytes [186]. It remains to be shown whether other Gram-
positive bacteria have components that also inhibit apoptosis, and whether this effect may
contribute to the difficulty in treating infections caused by these organisms. Further
studies are also needed to assess whether the inhibition of azithromycin-induced
apoptosis observed in my study occurs in the presence of other bacterial species,
including Haernophilus injluemae, another respiratory tract pathogen which azithromycin
is more effective against than S. pneurnoniae [64]. A paper recently suggested that
macrolides might significantly reduce the inflammatory injury associated with the
presence of H. influenzae in the lower respiratory tract, via unknown mechanisms [134].
In summary, azithrornycin induces apoptosis in human neutrophils more
effectively than erythromycin or penicillin (Figures 4A and 6A). Phagocytic uptake of
azithromycin-treated neutrophils by macrophages is enhanced (Figure 9), which in turn
may confer a significant anti-inflammatory benefit to this drug. The oxidative function of
resting or stimdated neutrophils and the production of IL-8 remain unchanged (Figures
10 and l l ) , indicating that these functions are unaffected in the early stages of
azithromycin-induced apoptosis. I postulate that the clinical efficacy of azithromycin in
the treatment of lower respiratory tract infections is due, at least in part, to committing
neutrophils to apoptotic death, hence reducing the amount of cytotoxic and pro-
inflammatory compounds released by these cells in the infected lung. Whether inhibition
79
of neutrophil apoptosis by S. pneumoniae contributes to the difficulty in treating
infections caused by this pathogen requires further investigation.
Results from my study open the way to a number of directions for future research.
A summary of these include:
1 ) Exploring the mechanism by which azithromycin induces apoptosis in neutrophils.
2) Determining if the higher affinity of azithrornycin for neutrophils is responsible for
inducing apoptosis more rapidly and at lower drug concentrations than erythromycin.
3) Examining the effect of azithromycin on other inflammatory cells.
4) Determining whether azithromycin-induced neutrophil apoptosis and enhanced
phagocytosis by macrophages occur in vivo.
5) Exploring the biological significance and clinical importance of azithromycin-
induced apoptosis in v iva
6) Elucidating the mechanism by which Streptococcus pneumoniae reverses or inhibits
the induction of apoptosis by azithromycin and determining whether this inhibition
occurs in the presence of other species of bacteria.
7) Determining whether the inhibition of apoptosis by Streptococcus pneumoniae
contributes to the difficulty in treating streptococcal pneumonia.
REFERENCES
Slauson, D.O., The mediation of pulmonary in$kmrnatory injury. Advances in
Veterinary Science and Comparative Medicine, 1982- 26: p. 99-1 53.
Murphey, S.A., Host defenses in the respiratory tract, in The Pneumonias, M.E.
Levison, Editor. 1984, John Wright - PSG Inc.: Boston. p. 8-22.
Lentnek, A., J.L. LeFrock, and A. Molavi, Streptococcus pneumoniae, in The
Pneumonias, M.E. Levison, Editor. 1984, John Wright - PSG Inc.: Boston. p.
261-271.
Baarsch, M.J., R.W. Scamurra, K. Burger, D.L. Foss, S.K. Maheswaran, and M.P.
Murtaugh, Inflarnmatoiy cytokine expression in swine experimentally infected
with Actinobucillus pleuropneumoniae. Infection and immunity, 1995. 63(9): p.
3587-3594.
Collins, M.K.L. and A. Lopez-Rivas, The control of apoptosis in mammalian
cells. Trends in Biochemical Sciences, 1993.18: p. 307-309.
Buret, A., M.L. Dunkley, G. Pang, R.L. Clancy, and A.W. Cripps, Pulmonary
immunity to Pseudomonas aeruginosa in intestinally immunized rats: roles of
alveolar macrophages, furnor necrosis factor alpha, and interleukin-l a Infection
and Immunity, 1994.62(12): p. 5335-5343.
Leff, J.A. and J.E. Repine, Neufrophil-mediated rissue injury, in The Neutrophil,
J.S. Abramson and J.G. Wheeler, Editors. 1993, IRL Press at Oxford Univ. Press:
Oxford. p. 229-262.
Mizgerd, J.P., B.B. Meek, G.J. Kutkoski, D.C. Bullard, A.L. Beaudet, and C.M.
Doerschuk, Selectins and neutrophil traflc: margination and Streptococcus
8 1
pneumoniae-induced emigration in murine lungs. Journal of Experimental
Medicine, 1996. 184: p. 639-645.
9. Simons, R.K., R.V. Maier, and S . Lemard, Neutrophil firnction in a rat model of
endotoxin-induced lung injury. Archives o f Surgery, 1 987. 122: p. 197-203.
10. Tamaoki, J., N. Sakai, E. Tagaya, and K. Komo, Macrolide antibiotics protect
against endotoxin-induced vascular leakage and ne utrophil accumulation in rat
trachea. Antimicrobial Agents and Chemotherapy, 1994.38(7): p. 164 1-1 643.
11. Weiss, S.J., Tissue destruction by neutrophils. The New England Journal o f
Medicine, 1989.320(6): p. 365-376.
1 2. Sharar, S.R., R.K. Winn, and J.M. Harlan, Endotoxin-induced interactions of
inflammatory cells with the lungs, in Endotoxin and the Lungs, K.L. Brigham,
Editor. 1994, Marcel Dekker, Inc.: New York. p. 229-265.
13. Dive, C., C.D. Gregory, D.J. Phipps, D.L. Evans, A.E. Milner, and A.H. Wyllie,
Analysis and discrimination of necrosis and apoptosis @rogrammed cell death)
by mulfiparameterflorv cytomerry. Biochimica et Biophysica Acta, 1992. 1133: p.
275-285.
14. Sonesson, H.R.A., U. Zahringer, H.D. Grimmecke, 0. Westphal, and E.T.
Rietsc hel, Bacterial endof oxin - chemical structure and biological activity, in
Endotoxin and the Lungs, K.L. Brigham, Editor. 1994, Marcel Dekker, Inc.: New
York. p. 1-20.
15. Finlay, B.B. and P. Cossart, Exploitation of mammalian host cell functions by
bacterial pathogens. Science, 1 997.276: p. 7 1 8-725.
82
Beachey, E.H. and H.S. Courtney, Bacterial adherence ofgroup A streptococci to
mucosal surfaces. Respiration, 1 989. SS(Supp1.1) : p. 3 3 -40.
Hayashi, T., A. Catanzaro, and S.P. Rao, Apoptosis of human monocytes and
macrophages by Mycobacterium avium sonicate. Infection and Immunity, 1997.
65(12): p. 5262-527 1 .
Rote, N.S., Infection and Alterations in Immunity and Inf7ammation, in
Pathophysiology - The Biologic Basis for Disease in Adults and Children, K.L.
McCance and S.E. Huether, Editors. 1998, Mosby: Toronto. p. 237-285.
Fantone, J.C. and P.A. Ward, Inflammation, in Pathology, E. Rubin and J.L.
Farber, Editors. 1994, J. B. Lippincott: Philadelphia. p. 33-66.
Hurley, J.V., Acute Inflammation. 2 ed. 1 983, New York: Churchill Livingstone.
p. 151.
Kuby, J., Immunoglobulins: structure and function, in Immunology. 1994, W. H.
Freeman and Company: New York. p. 109- 132.
Morrison, D.C. and R.J. Ulevitch, The effects of bacterial endotoxin on host
mediation systems. American Journal o f Pathology, 1978. 93: p- 527-6 17.
Dinarello, C.A., Role of interleukin-l and tumor necrosis factor in systemic
responses to infection and infammation, in Inflammation: Basic Principles and
Clinical Correlates, J.I. GalIin, I.M. Goldstein, and R. Snyderman, Editors. 1992,
Raven Press, Ltd.: New York. p. 2 1 1-232.
Kunkel, S.L., S.W. Chensue, T.J. Standiford, and R.M. Stricter, Endotoxin-
dependent cytokine networks, in Endotoxin and the Lungs, K.L. Brigham, Editor.
1994, Marcel Dekker, Inc.: New York. p. 305-320.
83
25. Detmers, P.A., S.K. Lo, E. Olsen-Egbert, A. Walz, M. Baggiolini, and Z.A. Cohn,
Neutrophil-activating protein ]/interleukin 8 stimulates the binding activity of the
leukocyte adhesion receptor CDllb/CDI8 on human neutrophils. Journal of
Experimental Medicine, 1990. 171: p. 1 1 55-1 162.
26. Kunkel, S.L., T. Standiford, K. Kasahara, and R.M. Strieter, interleukin-8 (IL-8):
the major neutrophil chemotactic facror in the lung. Experimental Lung Research,
1991. 17: p. 17-23.
27. Yoshimura, T., K. Matsushima, J.J. Oppenheim, and E.J. Leonard, Neutrophil
chemotact ic factor produced by lipopolysaccharide (LPS) -slim ulated human
blood mononuclear leukocytes: partiaZ characterization and separation from
interleukin I (IL I). The Journal of Immunology, 1987. 139(3): p. 788-793.
28. Fujishima, S., A.R. Hoffman, T. Vu, K.J. Kim, H. Zheng, D. Daniel, Y. Kim, E.F.
Wallace, J.W. Larrick, and T.A. R a f i , Regulation of neutrophil interleukin 8
gene expression and protein secretion by LPS, W F - a and IL- IP Journal of
Cellular Physiology, 1993. 154: p. 478-485.
29. Buret, A., M. Dunkley, R.L. Clancy, and A.W. Cripps, Effector mechanisms of
intestinally induced immuniw to Pseudomonas aeruginosa in the rat lung: role of
neutrophils and leukotriene Bd. Infection and Immunity, 1993. 61(2): p. 671 -679.
30. Dean, T.P., Y. Dai, J.K. Shute, M.K. Church, and J.O. Warner, Interleukin-8
concenh*alions are elevated in bronchouZveolar lavage, sputum and sera of
children with cysticfzbrosis. Pediatric Research, 1993.34(2): p. 1 59- 16 1.
3 1. Lawrence, R. and T. Sorrell, Eicosapentaenoic acid in cysticfibrosis: evidence of
a pathogenetic role for leukotriene Bg. Lancet, 1993.342: p. 465-469.
84
32. Greally, P., M.J. Hussein, A.J. Cook, A.P. Piper, and J.F. Price, Sputum tumour
necrosis factor-a and leukotriene concentrations in cystic fibrosis. Archives of
Disease in Childhood, 1993.68(3): p. 389-392.
33. Suter, S., U.B. Schaad, P. Roux-Lombard, E. Girardin, G. Grau, and J.M. Dayer,
Relation between tumor necrosis factor-alpha and granulocyte elastase- al-
proteinase inhibitor complexes in the plasma of patients with cystic fibrosis.
American Review of Respiratory Disease, 1 989. 140: p. 1640- 1644.
34. Wilmott, R.W., J.T. Kassab, P.L. Kilian, W.R. Benjamin, S.D. Douglas, and R.E.
Wood, Increased levels of interleukin-1 in bronchoalveolar washings porn
children with bacterial pulmonary infections. American Review of Respiratory
Disease, 1990. 142: p. 365-368.
35. Demling, R.H., The modern version of adult respiratory distress syndrome.
m u d Review of Medicine, 1995. 46: p. 193-202.
36. Movat, H.Z., M.I. Cybulsky, I.G. Colditz, M.K. Chan and C.A. Dinarello, Acute
inflammation in gram-negative infection: Endotoxin, interleukin I , tumor necrosis
factor, and neutrophils, Federation Proceedings, 1987.46(1): p. 97- 104.
37. Martin, T.R., G. Raugi, T.L. Merritt, and W.R. Henderson, Relative contribution
of leukotriene Bq to the neutrophil chemotactic activity produced by the residenr
human alveolar macrophage. Journal o f Clinical Investigation, 1987. 80: p. 1 1 14-
1124.
3 8. Golde, D. W., Production, distribution and fate of neutrophils, in Hematology,
W.J. Williams, E. Beutler, A.J. Ersler, and M.A. Lichtman, Editors. 1990,
McGraw-Hill: New York. p. 795-806.
8 5
Cartwright, G.E., J.W. Athens, and M.M. Wintrobe, 77te kinetics of neuirophilic
cells. Blood, 1964.24: p. 780-798.
Sanghavi, D.M., M . Thelen, N.A. Thornberry, L. Casciola-Rosen, and A. Rosen,
Caspase-mediated proteolysis during apoptosis: insights @om apoptotic
neutrophils. FEBS Letters, 1998.122: p. 1 79- 1 84.
Repine, J. E. and P. E. Parsons, Oxidant-antioxidant balance in endotoxin-induced
oxidative injury and tolerance to oxidalive injury, in Endotoxin and the Lungs,
K.L. Brigham, Editor. 1994, Marcel Dekker, Inc.: New York. p. 207-227.
Van Furth, R., Current view on the mononuclear phagocyte system.
Immunobiology, 1982. 161: p. 178-1 85.
Doherty, D.E., C. Haslett, M.G. Tonnesen, and P.M. Henson, Human monocyte
adherence: a primary eflect of chemotactic factors on the monocyte ro stirnula~e
adherence to human endothelium. The Journal of Immunology, 1987. 138(6): p.
1762-1771.
Johnson, W.D.J., B. Mei, and Z.A. Cohn, The separation, long-term cultivation,
and maturation of the human monocyte. The Journal of Experimental Medicine,
Partridge, L.J. and I. Dransfield, Isolation and characterization of mononuclear
phagocytes, in Tumor Immunobiology: A Practical Approach, G. Gallagher, R.C .
Rees, and C.W. Reynolds, Editors. 1993, IRL Press: New York. p. 91-1 18.
Campbell, N.A., The body's defenses, in Biology. 1996, The Benjamin/Cummings
Publishing Company, Inc.: Don Mills, Ontario. p. 852-878.
86
Savill, J., V. Fadok, P. Henson, and C. Haslett, Phagocyre recognition of cells
undergoing apoptosis. Immunology Today, 1 993. 14(3): p. 1 3 1 - 1 36.
Fadok, V.A., D.L. Bratton, A. Konowal, P.W. Freed, J.Y. Westcott, and P.M.
Henson, Macrophages that have ingested apoptotic cells in vitro inhibit
proinflomrnatory cytokine production through autocrine/paracrine mechanisms
involving TGF-fl PGE2, and PAF Journal of Clinical Investigation, 1998. 101: p.
890-898.
Levison, M. E., ed. The Pneumonias - Clinical Approaches to Infectious Diseases
of the Lower Respiratory Tract. 1984, John Wright - PSG hc.: Boston. p. xi.
Abrutyn, E., Strategies in the approach to treatment of pneumonia, in The
Pneumonias, M.E. Levison, Editor. 1984, John Wright - PSG Inc.: Boston. p.
110-120.
Anderson, K.N., Mosby 's Medical. Nursing, and Allied Health Dictionary. 4 ed.,
ed. K.N. Anderson, L.E. Anderson, and W.D. Glanze. 1994, Toronto: Mosby. p.
1235- 1237.
Fiala, M., A Study of the Combined Role of Vincses, Mycoplasmas and Bacteria in
Adult Pneumonia. The American Journal of the Medical Sciences, 1969. 257: p.
44-5 1.
Cherry, J.D., J.A. Diddams, and E.C. Dick, Rhinovirus infections in hospitalized
children: Provocative bacterial interrelationships. Archives of Environmental
Health, 1967. 14: p. 390.
Lepow, M.L., N. Balassanian, J. Emmerich, R.B. Roberts, M.S. Rosenthal, and E.
Wolinsky, Interrelationships of viral, mycoplasmal, and bacterial agents in
87 uncomplicated pneumonia American Review of Respiratory Disease, 1 968.97: p.
55. Miller, J.N. and E.J. Baron, Streptococcal and enterococcal infection and disease.
and Listeriosis, in Medical Microbiology - A Short Course, E.J. Baron, R.S.
Chang, D.H. Howard, J.N. Miller, and J.A. Turner, Editors. 1994, Wiley-Liss:
Toronto. p. 137- 165.
56. Sow, O., M. Frechet, A.A. Diallo, S. Soumah, M.K. Conde, P. Diot, E. Boissinot,
and E. Lemarie, Community acquired pneumonia in adults: a study comparing
clinical features and outcome in Afiica (Republic of Guinea) and Europe
(France). Thorax, 1996.51: p. 385-388.
5 7. Anonymous, Acute respiratory infections: the forgotten pandemic. The
International Journal of Tuberculosis and Lung Disease, 1998.2(1): p. 2-4.
58. Merchant, M., D.R. Karnad, and A.A. Kanbur, Incidence of nosocomial
pneumonia in a medical intensive care unit and general medical ward patients in
a public hospital in Bombay, India. Journal o f Hospital Infection, 1998. 38: p.
143- 148.
59. Paton, J.C., P.W. Andrew, G.J. Boulnois, and T.J. Mitchell, Molecular analysis of
the pathogenicity of Streptococcus pneumoniae: the role of pneumococcal
proteins. Annual Review of Microbiology, 1993.47: p. 89- 1 15.
60. Chetty, C. and A. Kreger, Generation of purpura-producing principle f iom
pneumococcal ceN walls. Journal o f Bacteriology, 1985. 163(1): p. 389-39 1.
6 1. Rang, H.P., M.M. Dale, J.M. Ritter, and P. Gardner, Pharmacology. 1995, New
York: Churchill Livingstone. 855.
88
Weinstein, L., Peniciilins and cephalosporins, in The Phrmacological Basis of
Therapeutics, L.S. Goodman and A. Gilman, Editors. 1975, Macmillan
Publishing: New York. p. t 130-1 166.
Carbon, C., Pharmacodynarnics of macrolides, aralides, and streptogramins:
effect on extracefldar pathogens. Clinical infectious Diseases, 1998.27: p. 28-32.
Peters, D., H. Friedel, and D. McTavish, Azithrornycin: A review of its
antimicrobial activity, pharrnacokinetic properties and clinical eflcacy. Drugs,
1992.44(5): p. 750-799.
Neu, H.C., Antibacterial therapy: problems and promises. Hospital Practice,
1990.23(Part I): p. 1 8 1 - 194.
Mazzei, T., E. Mini, A. Novelli, and P. Periti, Chemisw and mode of action of
macrolides. Journal of Antimicrobial Chemotherapy, 1993.31(Suppl. C): p. 1-9.
Dubnau, D., Translational attenuution: the regulation of bacterial resistance to
[he macrolide-lincosamide-streptogramin B antibiotics. C RC Critical Reviews in
Biochemistry, 1984. 16: p. 103- 132.
Weisblum, B., C. Siddhikol, C.J. Lai, and V. Demohn, Erythromycin-inducible
resistance in SIaphylococcus aureus: requirements for induction. Journal of
Bacteriology, 197 1 . 106: p. 835-847.
WeisbIurn, B., S.B. Holder, and S.M. Halling, Deoxyribonucleic acid sequence
common to staphylococcal and streptococcal plasmids which specifi
erythromycin resistance. Journal of Bacteriology, 1979. 138: p. 990-998.
Weisblum, B., Inducible erythromycin resistance in bacteria. British Medical
Bulletin, 1984. 40: p. 47-53.
89
7 1. Goldman, R.C. and J.O. Capobianco, Role of an energy dependent egux pump on
plasmid pNE21-mediated resistance to 14- and I 5-membered macrolides in
Staphylococcus epidermidis. Antimicro bid Agents and Chemotherapy, 1 990. 34:
p. 1 973- 1980.
72. Laurent, K., Eflcacy, safety and tolerability of azirhromycin versus roxithromycin
in the treatment of acute lower respiratory tract infictions. Journal of
Antimicrobial Chemotherapy, 1996.37(Suppf. C): p. i 15- 124.
73. Barry, A.L., R.N. Jones, and C. Thornsberry, In vitro activities of azirhromycin
(CP62.993), clarirhromycin (A-56268; TE-03 I) , erythromycin, roxithromycin and
clindamycin. Antimicrobial Agents and Chemotherapy, 1988.32: p. 752-754.
74. Davies, B.I., F.P.V. Maesen, and R. Gubbelmans, Azithromycin (CP-62.993) in
acute exacerbations ofchronic bronchitis: an open clinical, microbiologiical and
pharmacokinetic study Journal of Antimicrobial Chemotherapy, 1 989.23: p. 743-
751.
75. Fiese, E.F. and S.H. Steffen, Comparison of the acid stability of azithromycin and
erythromycin A. Journal of Antimicrobial Chemotherapy, 1990. 2S(Suppi. A): p.
39-47.
76. Debbia, E.A., G. Molinari, P. Paglia, and G.C. Schito, Post-antibiotic effect of
arithrornycin on respiratory tract pathogens. Drugs Under Experimental and
Clinical Research, 1990. 14: p. 6 15-6 19.
77. Retsema, J.A., A.E. Girard, D. Girard, and W.B. Milisen, Relationship of high
tissue concentrations of arirhromycin to bactericidal activity and ejicaacy in vivo.
Journal of Antimicrobial Chemotherapy, 1990. 25(Suppl. A): p. 83-89.
90
78. Retsema, J., A. Girard, W. Schelkly, M. Manousos, M. Anderson, G. Bright, R.
Borovoy, L. Breman, and R. Mason, Spectrum and mode of action of
azithromycin (CP-62,993). a new IS-membered-ring macrolide with improved
potency against gram-negative organisms. Antimicrobial Agents and
Chemotherapy, 1987.31(12): p. 1939- 1947.
79. Li, 2. and R.E.W. Hancock, Mechanism of uptake of the dibasic rnacrolide
antibiotic azithromycin across the o uter membrane of Escherichia coli. Abstract.
Proceedings of the 6th International Congress of Infectious Diseases, Montreal,
July. 1990.
80. Goldman, F.C., S.W. Fesik, and C.C. Doran, Role of protonated and neutral
forms of macrolides in binding to ribosomes from Gram-positive and Gram-
negafive bacteria. Antimicrobial Agents and Chemotherapy, 1990. 34: p. 426-
431.
8 1 . Gladue, R.P., G.M. Bright, R.E. Isaacson, and M.F. Newborg, in vifro and in vivo
uprake of azithromycin (CP-62.993) by phagocytic cells - possible mechanism of
delivery and release at sites of infection. Antimicrobial Agents and
Chemotherapy, 1989.33: p. 277-282.
82. Laufen, H., A. Wildfeuer, and P. Lach, Mechanism of azithrornycin uptake in
human polymorphonuclear leucocytes. Arzneimiffel-Forschung/Dmg Research,
1990.40: p. 686-689.
83. Wildfeuer, A., H. Laufen, and T. Zimmermann, Uptake of azithromycin by
various cells and its intracellular activity under in vivo conditions. Antimicrobial
Agents and Chemotherapy, 1996.40(1): p. 75-79.
91
84. Wildfeuer, A., H. Laufen, D. Muller- Wening, and 0. Haferkamp, Interaction of
azithromycin and human phagocytic cells. (Iptakz of the antibiotic and the effect
on the survival of ingested bacteria in phagocytes. Armeimittel- Forsc hung/D rug
Research, 1989.39: p. 755-758.
85. Carlier, M.B., B. Scorneaux, and P.M. Tulkens, Abstract 510. Accumulation,
subcellular distribution and activity of azithromycin (At) compared to
roxithromycin (Rx) and ciprofloxacin (Cp) in J774 macrophages (M#, in
Programs and Abstructs of the Thirty-First Interscience Conference on
Antimicrobial Agents and Chemotherapy, Chicago, IL. 1991, American Society
for Microbiology: Washington, DC. p. 184.
86. Shepard, R.M., D.E. Amacher, M.A. Mullins, R.A. Ferraina, C.V. Higgins, A.B.
Jakowski, et al, Abstract 21 7. Mechanism of azithromycin tissue uptake and
accumulation: subcellular localization of azithromycin in rat liver, in Programs
and Abstracts of the First International Confirence on Macrolides, Azalides and
Streptogramins, Santa Fe, NM. 1 992, Wallace Communications: Atlanta. p. 50.
87. Pascual, A., G. Lopez-Lopez, J. Aragon, and E.J. Perea, Eflect of azithromycin,
roxithromycin and erythromycin on human polymorphonuclear leukocyte finction
against Staphylococcus aureus. Chemotherapy, 1990.36(6): p. 422-427.
88. Bonnet, M. and P. Van der Auwera, In vitro and in vivo intraleukocytic
accumularion of azithromycin (CP-62, 993). Antimicrobial Agents and
Chemotherapy, 1992.36(6): p. 1 302- 1 309.
92
Gladue, R.P. and M.E. Snider, lnrracellular accumulation of azithromycin by
cultured human fibroblasts. Antimicrobial Agents and Chemotherapy, 1 990. 34:
p. 1056-1060.
Lode, H., K. Borner, P. Koeppe, and T. Schaberg, Azithromycin - review ofkey
chemical, pharmacokineric and microbiological features. Journal of
Antimicrobial Chemotherapy, 1996.37(Suppl. C): p. 1-8.
Schlossberg, D., A-ithromycin and clarithromycin, Medical Clinics of North
America, 1995. 79(4): p. 803-8 15.
Foulds, G., D. Luke, R. Teng, S. Willavize, H. Friedman, and W. Curatolo, The
absence of an effect of food on the bioavailability of azithromycin administered as
tablets, sachet or suspension. Journal of Antimicrobial Chemotherapy, 1996.
37(Suppl. C): p. 37-44.
Foulds, G., R.M. Shepard, and R.B. Johnson, The pharmacokinetics of
azithromycin in human serum and tissues. Journal of Antimicrobial
Chemotherapy, 1990. 25(Suppl. A): p. 73-82.
Luke, D. and G. Foulds, Disposition of oral azithromycin in humans. Clinical
Pharmacology and Therapeutics, 1997.6l(June): p. 64 1 -648.
Cooper, M.A., K. Nye, J.M. Andrews, and R. Wise, The pharmacokinetics and
inflammatory fluid penetration of orally administered azithromycin. Journal of
Antimicrobial Chemotherapy, 1990.26: p. 533-538.
Johnson, R.B., The role of azalide antibiotics in the treatment of chlamydia.
American Journal o f Obstetrics and Gynecology, 199 1. 164: p. 1 794- 1 796.
93
97. Shepard, R.M., D.J. Weidler, D.C. Garg, P.O. Madsen, C.E. Cox, et al, Abstract.
Human pharmacokinetics of azithromycin (CP-62.993/XZ-450). A new macrolide
with an extended hav-Zije and high tissue penetration. Proceedings of the 27th
Interscience Conference on Antimicrobial Agents and Chemotherapy, October,
1987.
98. Foulds, G. and R.B. Johnson, Selection of dose regimens of azithromycin. Journal
of Antimicrobial Chemotherapy, 1993.31(Suppl. E): p. 39-50.
99. Foulds, G., K.H. Chan, J.T. Johnson, R.M. Shepard, and R.B. Johnson,
Concentrations of azithromycin in human tonsillar tissue. European Joumal of
Clinical Microbiology and Infectious Disease, 199 1. 10: p. 853-856.
100. Krohn, K., Gynaecological tisszte levels of mithromycin. European Journal of
Clinical Microbiology and Infectious Disease, 199 1. 10: p. 864-868.
10 1. Moms, D.L., A. De Souza, J.A. Jones, and W.E. Morgan, High and prolonged
pulmonary tissue concentrations of azithromycin following a single oral dose.
European Journal of Clinical Microbiology and Infectious Diseases, 199 1. 10: p.
859-86 1.
102. Baldwin, D.R., J.P. Ashby, J.M. Andrews, R. Wise, and D. Honeybourne,
Pulmonary disposition of azirhromycin foZZowing a single 500 mg oral dose.
Thorax, 1990.45: p. 324P.
103. Baldwin, D.R., R. Wise, J.M. Andrews, J.P. Ashby, and D. Honeybourne,
Azithromycin concentrations at the sites of pulmonary infection. European
Respiratory Journal, 1990.3: p. 886-890.
94
1 04. Foulds, G., D.M. Hilligoss, E.B. Henry, and N. Gerber, The effects of an antacid
or cimeridine on the serum concentrations of azithromycin. Journal of Clinical
Pharmacology, 199l. 31(2):164-167.
105. Zini, R., M.P. Fournet, J. Barre, D. Tremblay, and J.P. Tiliernent, In vitro study of
roxirhromycin binding to serum proteins and erythrocytes in man. British Journal
o f Clinical Practice, 1988. 42(Suppl. A): p. 54.
1 06. Dette, G. A. and M. Knothe, The binding protein of erythromycin in human serum.
Biochemical Pharmacology, 1986.35: p. 959-966.
107. Schentag, J.J. and C.H. Ballow, Tissue directed pharmacokinetics. American
Journal of Medicine, 199 2.91(Suppl. 3A): p. 5- 1 1.
108. Shepard, R.M., H.G. Fouda, R.A. Ferraina, and M.A. Mullins, Abstract.
Disposition and metabolism of azithromycin in rats, dogs and humans.
Proceedings of the 6th International Congress of Infectious Diseases, Montreal,
July, 1990.
109. Coates, P., R. Daniel, A.C. Houston, J.H.L. Antobus, and T. Taylor, An open
study to compare the pharmacokinetics, safety and tolerabili~ of a multiple-dose
regimen of mi t hrornycin in young and eldedy volunteers. European Journal of
Clinical Microbiology and Infectious Diseases, 1991. 10: p. 850-852.
110. Treadway, G. and D. Pontani, Paediatric safery of azithromycin: worldwide
experience. Journal of Antimicrobial Chemotherapy, 1996. 37(Suppl. C): p. 143-
149.
95
1 11. Mazzei, T., C. Surrenti, A. Novelli, A. Crispo, S. Fallani, V. Carla, E. Surrenti,
and P. Periti, Pharmacokinetics of azithromycin in patients with impaired hepatic
firnction. Journal of Antimicrobial Chemotherapy, 1 993. 3 l(Supp1. E): p. 57-63.
112. Azoulay-Dupuis, E., E. Vallee, J.P. Bedos, M. Muffat-Joly, and J.J. Pocidalo,
Prophylactic and therapeutic activities of azithromycin in a mouse model of
pneumococcal pneumonia. Antimicrobial Agents and Chemotherapy, 1 99 1. 35: p.
1024- 1028.
1 1 3. Veber, B., E. Azoulay-Dupuis, E. Vallee, and J.J. Pocidalo, Abstract. Correlation
between eflcacy and pharmacokinetics of rnacrolides in infected lung.
Proceedings of the 3 1 st Interscience Conference on Antimicrobial Agents and
Chemotherapy, Chicago, 199 1.
1 14. Fitzgeorge, R.B., A.S.R. Featherstone, and A. BaskerviIle, Eficacy of
azithromycin in the rreatment of guinea pigs infected with Legionella
pneumophih by aerosol- Journal o f Antimicrobial Chemotherapy, 1 990.
25(Suppl. A): p. 101-1 08.
115. Fitzgeorge, R.B., S. Lever, and A. Baskerville, A comparison of the eflcacy of
azithromycin and clarithromycin in oral therapy of experimental airborne
Leggionnaires ' disease. Journal of Antimicrobial Therapy, 1 993. 3 l(Supp1. E): p.
171-176.
1 1 6. Mertens, J.C.C., P. W.C. van Barneveld, H.R.G. Asin, E. Ligtvoet, M.R. Visser, T.
Branger, and A. I. Hoepelman, Double- blind, randomized study comparing the
eflcacies and safeties of a short (3-dayl course of a7ithromycin and a Zday
96
course of amoxicillin in parients with acute exacerbations of chronic bronchitis.
Antimicrobial Agents and Chemotherapy, 1992.36(7): p. 1446- 1459.
1 17. Gunjaca, M., I. Klinar, M. Gosev, and B. Bogic, Abstract. Eficacy of
azithromycin in the treatment of respiratory tract infections in general practice.
Proceedings of the 6th International Congress of Infectious Diseases, Montreal,
July, 1990.
1 1 8. Mybergh, J. G. J. Nagel, and E. Petschel, The eflcacy and tolerance of a three-day
course of azithromycin in the treatment of community-acquired pneumonia.
Journal of Antimicrobial Chemotherapy, 31(Suppl. E): p. 163-1 69.
1 19. Muller, O., An open cornparafive study of azithromycin and roxithromycin in the
treatment of acute upper respiratory tract infections. Journal o f Antimicrobial
Chemotherapy, 1996.37(Suppl. C): p. 83-92.
120. Mohs, E. A. Rodriguez-Solares, E Rivas and Z. el Hoshy, A comparative study of
azithromycin and amoxicillin in paediatric patients with acute otitis media.
Journal of Antimicrobial Chemotherapy, 1993.31(Suppl. E): p. 73-79.
121 - Hopkins, S., Clinical toleration and safety of uzithromycin. American Journal of
Medicine, 1991. 91(SuppI. 3A): p. 40s-45s.
122. Arnacher, D.E., S.J. Schomaker, and J.A. Retsema, Comparison of the effects of
the nerv aralide antibiotic, azithromycin and erythromycin estolate on rat liver
cytochrome P-450. Antimicrobial Agents and Chemotherapy, 199 1. 35: p. 1 1 86-
1190.
97
123. Harris, S., D.M. Hilligoss, P.M. Colangelo, M. Eller, and R. Okerholm,
Arithrornycin and terfenadine: lack of drug interactions. Clinical Pharmacology
and Therapeutics, 1995.5%: p. 3 10-3 1 5.
124. Hopkins, S.J., Clinical toleration and safety of azithromycin in children and
adults. Reviews in Contemporary Pharmacotherapy, 1 994. 5: p. 3 83-3 89.
125. Chave, J.P., A. Munafo, J.Y. Chatton, P. Dayer, M.P. Glauser, and J Biotlaz,
Once-a-week arirhromycin in AIDS patients: tolerability, kinetics, and eflects on
ridovudine disposition. Antimicrobial Agents and Chemotherapy, 1992. 36(5): p.
1013-1018.
126. Goldstein, R.A., D.L. Bowen. and A.S. Fauci, Adrenal corticosteroiak, in
Inflammation: Basic Principles and Clinical Correlates, J.I. Gallin, I . M.
Goldstein, and R. Snyderman, Editors. 1992, Raven Press: New York. p. 1061 - 1081.
127. Coates, T.D., B. Wolach, D.Y. Tzeng, C. Higgins, R.L. Baehner, and L.A. Boxer,
The mechanism of action of the anti-inflammatory agents dexamethasone and
auranofin in human polymorphonuclear leukocytes. Blood, 1983. 62: p. 1 070-
1077.
128. Groneck, P., D. Reuss, B. Gotze-Speer, and C.P. Speer, Effects of dexamethasone
on chemotactic activity and inflammatory mediators in trucheo bronchial
aspirates of preterm infants at risk for chronic lung disease. Journal of Pediatrics,
1993.122: p. 93 8-944.
129. Sikora, E., E. Grassilli, E. Radziszewska, E. Bellesia, D. Barbieri, and C.
Franceschi, Transcription factors DNA-binding activity in rat thymocytes
98
undergoing apoptosis after heat-shock or dexamethasone treatment. Biochemical
and Biophysical Research Communications, 1993. 197(2): p. 709-7 15.
1 30. Liles, W.C., D.C. Dale, and S.J. Klebanoff, Glucocorticoi~ inhibit apoptosis of
human neutrophils. Blood, 1 995.86(8): p. 3 1 8 1-3 1 88.
I 3 1. Standiford, T.J., S.L. Kunkel, M. W. Rolfe, H.L. Evanoff, R.M. Allen, and R.M.
Stricter, Regulation of human macrophage- and blood monocyte-derived
interleukin-8 by prostaglandin E - 7 and dexarnethasone. American Journal of
Respiratory CetI and Molecular Biology, 1992. 6: p. 75-8 1.
132. Moms, R.G., A.D. Hargreaves, E. Duvall, and A.H. Wyllie, Hormone-induced
cell death 2. Surface changes in thymocytes undergoing apoptosis. American
Journal of Pathology, 1984. 115(3): p. 426-436.
133. Scaglione, F. and G. Rossoni, Comparative anti-inflammatory effects of
roxithromycin, azithromycin and clurithromycin. Journal of Antimicrobial
Chemotherapy, 1998.41(Suppl. B): p. 47-50.
134. Khair, O.A., J.L. Devalia, M.M. Abdelaziz, R.J. Sapsford, and R.J. Davies, Eflect
of erythromycin on Haemophil us influenzae endotoxin-induced re lease of IL-6.
IL-8 and SICAM-I by cultured human bronchial epithelial cells. European
Respiratory Journal, 1995.8: p. 145 1-1 457.
1 3 5. Labro, M.T., Anti-inflammatory activity of macrolides: a new therapeutic
potential? Journal of Antimicrobial Chemotherapy, 1998. Ql(Supp1. B): p. 37-46.
136. Oishi, K., F. Sonoda, S. Kobayashi, A. Iwagaki, T. Nagatake, K. Matsushima, and
K. Matsumoto, Role of interleukin-8 (IL-8) and an inhibitory effect of
99
erythromycin on I.-8 release in the airways of patients with chronic airway
diseases. Infection and Immunity, 1994. 62(10): p. 41454152.
137. Iino, Y., M. Toriyarna, K. Kudo, Y. Natori. and A. Yuo, Erythromycin inhibition
of lipopolysaccharide-stimulated tumor necrosis factor alpha production by
human monocytes in vitro. Annds of Otology, Rhinology and Laryngology, 1992.
101: p. 16-20.
138. Morikawa, K., H. Watabe, M. Araake, and S. Morikawa, Modulatory effect of
antibiotics on wokine production by human monocytes in vitro. Antimicrobial
Agents and Chemotherapy, 1 996.40(6): p. 1 366- 1 370.
139. Takeshita, K., I. Yamagishi, M. Harada, S. Otomo, S. Nakagawa, and Y.
Mizushima, Immunological and anti-inflammatory effects of clarithromycin:
inhibition of interleukin 1 production of murine peritoneal macrophages. Drugs
Under Experimental and Clinical Research, 1989. 15: p. 527-533.
140. Roche, Y., M.A. Gougerot-Pocidalo, M. Fay, N. Forest, and J.J. Pocidalo,
Macrolides and immunity: efects of erythromycin and spiramycin on human
mononuclear cell proliferation. Journal of Antimicrobial Chemotherapy, 1986.
17(2): p. 195-203.
141. Goswami, S.K., S. Kivity, and Z. Marom, Erythromycin inhibits respiratory
glycoconjugate secretion porn human airways in vitro. American Review of
Respiratory Diseases, 1 990. 141 : p. 72-78.
142. Umeki, S., Anti-inflammatory action of erythromycin: Its inhibitory effect on
neutrophil NADPHoxidase activity. Chest, 1993. 104: p. 1 191-1 193.
1 00
143. Anderson, R., Erythromycin and roxithromycin potentiate human neufrophil
Zocomotion in vitro by inhibition of leukoattractant-activated superoxide
generation and autooxidation. Journal o f Infectious Diseases, 1 989. 159(5): p.
966-973.
144. Hand, W.L., D.L. Hand, and N.L. King-Thompson, Antibiotic inhibition of the
respiratory burst response in human polymorphonuclear leukocytes.
Antimicrobial Agents and Chemotherapy, t 990.34(5): p. 863-870.
145. Labro, M.T., J.E. Benna, and C. Babin-Chevaye, Comparison of the in-vitro eflect
of several macrolides on the oxidative burst of human neutrophils. Journal of
Antimicrobial Chemotherapy, 1989.24: p. 56 1-572.
146. Mitsuyarna, T., T. Tanaka, K. Hidaka, M. Abe, and N. Hara, Inhibition by
erythromycin of superoxide anion production of human polymorphonuclear
neutrophils through the action of cyclic AMP-dependent protein kinase.
Respiration, 1995.62: p. 269-273.
147. Wenisch, C., B. Parschalk, K. Zedtwitz-Leibenstein, A. Weihs, I. el Menyawi,
and W. Graninger, Eflect of single oral dose of azithromycin, clarithromycin, and
roxithrornycin on polymorphonuclear leukocyte finc t ion assessed ex vivo by flow
cytometry- Antimicrobial Agents and Chemotherapy, 1996.40(9): p. 2039-2042.
148. Chin, A.C., D.W. Morck, J.K. Memll, H. Ceri, M.E. Olson, R.R. Read, P. Dick,
and A.G. Buret, Anti-inflammatory benefits of tilmicosin in calves with
Pasteurella haemolytica-infected lungs. American Journal of Veterinary
Research, 1 998.59(6): p. 765-77 1.
10 1
Aoshiba, K., A. Nagai, and K. Komo, Erythromycin shortens neutrophil survival
by acceleraring apoptosis. Antimicrobial Agents and Chemotherapy, 1995. 39(4):
p. 872-877.
Squier, M.K.T., A.J. Sehnert, and J.J. Cohen, Apoptosis in leukocytes. Journal of
Leukocyte Biology, 1995.57: p. 2-10.
Whyte, M.K., B.L.C. Meagher, J. MacDermot, and C. Haslett, Impairment of
ficnction in ageing neutrophils is associated with apoptosis. Journal of
Immunology, 1993. 150: p. 5 124-5 134.
Wyllie, A.H., J.F. Kerr, and A.R. Cume, Cell death: the significance of apoptosis.
International Review of Cytology, 1980.68: p. 25 1-306.
Beutler, B., The complex regulation and biology of TNF (cachectin). Critical
Reviews in Oncogenesis, 1990.2: p. 9-1 8.
Brunner, T., R.J. Mogil, D. LaFace, N.J. Yoo, A. Mahboubi, F. Echeverri, A.J.
Martin, W.R. Force, D.H. Lynch, C.F. Ware, and D.R. Green, Cell-autonomous
Fas (CD9j'/Fas-ligand interaction mediates activation-induced apoptosis in T-
cell hybridomas. Nature, 1995.373: p. 44 1-444.
Martin, S.J. and T.G. Cotter, Ultraviolet B irradiation of human leukemia HL-60
cells in vitro induces apoptosis. International Journal of Radiation Biology, 1991.
59: p. 1001-1016.
Telford, W.G., L.E. King, and P. J. Fraker, Evaluation of glucocorticoid-induced
DNA fragmentation in mouse thymocytes by flow cytometry Cell Proiiferation,
1991.24: p. 447-459.
1 02 Collins, M.K.L. and A. Lopez-Rivas, The control of apoptosis in mammalian
cells. Trends in Biochemical Sciences, 1993. 18: p. 307-309.
Froelich, C. J., V. M. Dixit, and X. Yang, Lymphocyte granule-mediated apoptosis:
matters of viral mimicry and deadly proteases. Immunology Today, 1998. 19(1):
p. 3 1-36.
Vermes, I., C. Haanen, H. SteRens-Nakken, and C. Reutelingsperger, A novel
assay for apoptosis - j70w cyiomerric detection of phospharidylserine expression
on early apoptotic cells using fluorescein labelled annexin V . Journal of
ImmunologicaI Methods, 1995. 184: p. 39-5 1 .
Savill, J.S., A.H. Wyllie, J.E. Henson, M.J. Walport, P.M. Henson, and C. Haslett,
Macrophage phagocytosis of ageing netrtraphils in inflammation. Journal of
Clinical Investigation, 1989. 83: p. 865-875.
Verhoven, B., R.A. SchlegeI, and P. Williamson, Mechanisms of
phosphatidylserine exposure, a phagocyte recognition signal, on apoproric T
lymphocytes. Journal of Experimental Medicine, 1995. 182: p. 1 597- 160 1 .
Savill, J., I. Dransfield, N. Hogg, and C. Haslett, Vifronectin receptor-mediated
phagocytosis ofcells undergoing apoptosis. Nature, 1990.343: p. 1 70- 1 73.
Aliprantis, A.O., G. Diez-Roux, L.C.F. Mulder, A. Zychlinsky, and R.A. Lang,
Do macrophages kill through apoptosis? Immunology Today, I 996. 17(12): p.
573-576.
Chong, L., A. Traynor-Keplan, G.M. Bokoch, and M.A. Schwartz, The smaN
GTP-binding protein Rho regulates a phosphatidylinosifol I-phosphate 5-kinase
in mammalian cells. Cell, 1994. 79: p. 507-5 13.
103
165. Na, S., T.H. Chuang, A. Cunningham, T.G. Tun, J.H. Hanke, G.M. Bokoch, and
D.E. Danley, D4-GDI. a subshwate of CPP32, is proteolyzed during Fas-induced
apopfosis, Journal o f Biological Chemistry, 1996.271 : p. 1 1209- 1 12 1 3.
166. Geng, Y.J., T. Azuma, J.X. Tang, J.H. Hartsvig, M. Muszynski, Q. Wu, P. Libby,
and D.J. Kwiatkowski, Caspase-3-induced gelsolin fragmentation contributes to
actin cyroskeletal collapse, nucleolysis, and apoptosis of vascular smooth muscle
cells exposed to proinflammatory cyto kines. European Journal of Cell Biology,
1998. 77(4): p. 294-302.
167. Oberhammer, F.A., K. Hochegger, G. Froschl, R. Tiefenbacher, and M. Pavelka,
Chromatin condensation during apoptosis is accompanied by degradation of
Iamin A+& without enhanced activation of cdc2 kinase. Journal of Cell Biology,
2994. 126(4): p. 827-837.
1 6 Martin, S.J., G.A. O'Brien, W.K. Nishioka, A.J. McGahon, A. Mahboubi, T.C.
Saido, and D.R. Green, Proteolysis of fodrin (non-erythroid spectrin) during
apoptosis. Journal of Biological Chemistry, 1995.270(12): p. 6425-6428.
169. Fadok, V.A., D.R. Voelker, P.A. Campbell, J.J. Cohen, D.L. Bratton, and P.M.
Henson, Exposure of phosphatidylserine on the surface of apoptotic lymphocytes
triggers specific recognition and removal by macrophages. The Journal of
Immunology, 1992. 148(7): p. 2207-22 16.
170. Flora, P.K. and C.D. Gregory, Recognition of apoptotic cells by human
rnacrophages: inhibition by a monocyte/macrophage-specific monoclonal
antibody. European Journal of Immunology, 1994.24: p. 2625-2632.
104
Fadok, V.A., J.S. Savill, C. Haslett, D.L. Bratton, D.E. Doherty, P.A. Campbell,
and P.M. Henson, Different populations of macrophages use either the vitronectin
receptor or the phosphatidylrerine receptor to recognize and remove apoptotic
cells. Journal of Immunology, 1 992. 149( 1 2): p. 4029-403 5.
Kelley, J.L., M.M. Rozek, C.A. Suenram, and C.J. Schwartz, Activation of human
blood monocytes by adherence to tissue culture plastic surfaces. Experimental
and hlolecular Pathology, 1987. 46: p. 266-278.
Haskill, S., C. Johnson, D. Eierman, S. Becker, and K. Warren, Adherence
induces selective mRNA expression of rnonocyte mediators and proto-oncogenes.
The Journal o f Immunology, 1988. 140(5): p. 1690-1694.
Krause, S.W., M. Rehli, M. Kreutz, L. Schwarzfischer, J.D- Paulauskis, and R.
Andreesen, Differential screening identifies genetic markers of rnonocyte to
macrophage maturation. Journal of Leukocyte Biology, 1996.60: p. 540-545.
Li, C.Y., K. W. Lam, and L.T. Yam, Esterases in human leukocytes. The Journal
of Histochemistry and Cytochemistry, 1973.2 l(1): p. 1 - 12.
Sanderson, R.J., F.T. Shepperdson, A.E. Vatter, and D.W. Talmage, Isolation and
enumeration of peripheral blood monocyfes. The Journal of Immunology, 1977.
118(4): p. 1409-1414.
Takeda, Y., H. Watanabe, S. Yonehara, T. Yarnashita, S. Saito, and F. Sendo,
Rapid acceleration of neutrophil apoptosis by tumor necrosis factor-a
International Immunology, 1993.5(6): p. 69 1-694.
Bazzoni, F., M.A. Cassatella, G. Rossi, M. Ceska, B. Dewaid, and M. Baggiolini,
Phagocytosing neutrophiis produce and release high amounts of the neutrophil-
105
activating peptide ilinterleukin-8. Journal o f Experimental Medicine, 1 99 1 . 173:
p. 771 -780.
179. Cox, G., J. Crossley, and 2. Xing, Macrophage enguljinent of apoptotic
neutrophils contributes to the resolution of acute puZmonary inflammation in vivo.
knerican Journal of Respiratory Cell ar.d Molecular Biology, 1995. 12: p. 232-
237.
180. Savill, J., Apoptosis in resolution of inflammation. JournaI of Leukocyte Biology,
1997.61: p. 375-380.
1 8 1 . Homburg, C.H.E., M. de Haas, A.E.G. von dem Borne, A.J. Verhoeven, C.P.M.
Reutelingsperger, and D. Roos, Human neutrophils lose their surface FcyRllI and
acquire annexin V binding sites during apoptosis in vitro. Blood, 1 995. 85(2): p.
532-540.
182. Tsuchida, H., Y. Takeda, H. Takei, H. Shinzawa, T. Takahashi, and F. Sendo, In
vivo regulation of rot neutrophil apoptosis occurring spontaneously or induced
with TNF-a or cycloheximide. The Journal of Imunology, 1995. 154: p. 2403-
2412.
183. Williamson, P., A. Kulick, A. Zachowski, R.A. Schlegel, and P.F. Devaux, c a t C
induces transbilayer redistribution of all major phospholipiak in human
erythrocytes. Biochemistry, 1992.31: p. 6355-6360.
184. Roller-Labelle, E., M.4 . Grange, C. Elbim, C. Marquetty, M.-A. Gougerot-
Pocidalo, and C. Pasquier, Hydroxyf radical as a potential intracellulur mediator
of polymorphonuclear neutrophil apoptosis. Free Radical Biology and Medicine,
1998.24(4): p. 563-572.
106
185. Hachiya, O., Y. Takeda, H. Miyata, H. Watanabe, T. Yamashita, and F. Sendo,
Inhibition by bacterial lipopolysaccharide of spontaneous and TNFa induced
human neutrophil crpoptosis in vitro. Microbiology and Immunology, 1995.39: p.
7 1 5-723.
186. Moulding, D.A., C. Walter, C.A. Hart, and S.W. Edwards, Effects of
staphylococcal enferotoxins on human neunophil functions and apoptosis.
Infection and Immunity, 1999. 67(5): p. 23 12-23 18.
107
APPENDIX A: Structure of Azithrornvcin and Ewthromvcin
Azithromycin
Erythromycin
(Adapted fiom reference 64)