Leukocytes Using an In Vitro Mode1 Aashish · 2020. 4. 7. · The Interaction Between Human...
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The Interaction Between Human Cytomegalovhs and
Polymorphonuclear Leukocytes Using an In Vitro Mode1 of Study
Aashish Chakravertty
A thesis submitted in conformity with the requirernents
for the degree of Master of Science
Graduate Department of Laboratory Medicine and Pathobiology
University of Toronto
(9 Copyright by Aashish Chakravertty, 1999
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The Interaction Between Human Cytomegalovirus and Polymorphonuclear Leukocytes Using an In Vitro Mode1 of Study
Aashish Chakravertty Master of Science, 1999
Department of Laboratory Medicine and Pathobiology University of Toronto
ABSTRACT
Background: Human cytomegdovirus (HCMV) is a cornmon causative agent of
congenital viral infections in humans and an important pathogen in allograft recipients.
HCMV transmission has been associated with a number of body fluids, particularly
blood. The use of leukocyte filtered blood and blood products for transbion has
resulted in significantly reduced HCMV infection in allograft recipients. In patients with
active HCMV infection, detection of HCMV phosphoprotein pp65 inside
polymorphonuclear leukocytes (PMNL) is often used to follow the virus load. It is as yet
unresolved whether detection of pp6S and other viral antigens is a result of active HCMV
infection within PMNL, or detection is due to uptalce and accumulation of viral antigens
from infected cells through phagocytosis. The purpose of this study was to investigate
the interaction between HCMV and PMNL, usïng an in vitro mode1 of study.
Methods: H L 6 0 cells (human promyelocytic leukemia ce11 line) which are of rnyeloid
lineage, were stimulated with DMSO to obtain more mature cells. Unstimulatecl and
stimulated cens were inoculated with live and heat inactivated HCMV at various
multiplicities of infection (MOI). Cells were tested for the praence of HCMV
imrnediate-early (IE) antigen (Ag), pp65 Ag, and late Ag by immunofluorescence assays
(IFA).
They were M e r tested for HCMV DNA, mRNA, inféctiou viral progeny, and
examined for virai particles by electron microscopy (EM) and immunoelectron
microscopy (IEM).
Results: HCMV IE, pp65, and late Ag were detected in unstimulated and DMSO
stimulated HL60 cells inoculated with live or heat inactivated virus. Inoculated cultures
were positive for viral DNA. NASBA positive signals suggesting the presence of HCMV
late pp67 mRNA transcripts were detected withui inoculated cultures, as well as in the
original viral inoculurn. There was no evidence of infectious progeny virus within
inoculated cultures. Virus-like particles were detected by EM, but not to leve1s
characteristic of HCMV infection. Attempts at employing IEM for fÙrther identification
of viral particles was unsuccessful.
Conclusion: Our results provide some support for replication of HCMV in HL-60 cells
as demonstrate- by virai antigen and DNA detection, and support for the contrary (no
transmission of virus, EM work). Without M e r investigation, it is not possible to
definitively conclude the extent to which HCMV replicates in HL-60 cells.
ACKNOWLEDGMENTS
I would like to thank the following people in recognition of their contribution to this
thesis.
Firstly, my supervisor Dr. Tony Mamilli, who has provided me the opportunity to work
under his tutelage. Over the past few years, 1 have gained so much insight into the field
of virology and research, and would like to express my gratitude for the wealth of
h o wledge and guidance Dr. Maznilli has provided.
1 also wish to thank my cornmittee members, Drs. Joyce de Azavedo, Kelly MacDonald
and Martin Petric for their valuable advice and suggestions throughout my project.
Special thanks to George Moussa and Robert Chua for their laboratory expertise, Anna
Smargassio and the entire Department of Microbiology at Mount Sinai Hospital for ail
their assistance.
1 would like to close by thanking my parents Rita and Anin Chakravertty and my brother
Raja for their encouragement and support throughout my studies.
TABLE OF CONTENTS
LIST OF FIGURES ............................................................................................. *0008
LIST OF TABLES ............................................................................................... *.**9
........................................................................................ INTRODUCTION 10
............................................................................... PROJECT HISTORY ..IO
CYTOMEGALOVIRUS
............................................................................................ Characterization 1 4
................................................................................................. Epidemiology 14
............................................................................................. Clinical Disease 15
........................................................................................................ Latency 1 6
.............................................................................. HCMV Replicative Cycle 17
............................................................................... HCMV Gene Expression 18
...................................................................................... HCMV Inactivation -20
....................................................................... HCMV Permissive Ce11 Lines 22
TBE MYELOID LiNEAGE
.............................................................................................. Granulopoiesis -23
........................................................................................ Granule Production 25
....................................................................................... Neutrophil Function 27
Cytokines IL-8. IL.6. TNF-a and HCMV ..................................................... 30
.............................................................................. Culturing of Neutrophils 30
HL40 CELL LINE
............................................................. Establishment of HL-60 Ce11 Line 1
Cytologie Characteristics of HL-60 Cultured Cells ....................................... 31
.................................................................................. 1.4.3 General Differentiation 32
1.4.4 ModelofStudy .............................................................................................. 33
1 .4.5 Infectivity of HL-60 Cells .............................................................................. 35
2.0 OBJECTIVES m.m.......m..m...m........................m..mm...m.....m......m..m.....m..m....... ~ ~ o m o m m m o o ~ 6
MATERIALS AND METHODS
Propagation of MRC-5 Cells ......................................................................... 37
............................................................................. Propagation of HFF Cells -37
Propagation of HL-60 Cells ........................................................................... 37
.................................................. Induction of Differentiation of HL60 Cells 37
......................................................................................... Virus Propagation -38
........................................ Determination of Stock Viral Titre in TCIDSdml -38
...................................................... Inoculation of HL-60 Cells With HCMV 39
.............................................................................. HCMV Heat Inactivation -39
Detennination of Viral Titre in HCMV hoculated Cultures of .......................................... Unstimulated or DMSO Stimulateci HL-60 Cells 39
Immuno fluorescence Assa y (FA) For Detection of HCMV ........................................................... 1mmediate.Early. pp65. Late Antigen 40
HCMV Nucleic Acid Isolation and PCR ....................................................... 41
Determinhg the Lower Limit of Detection of PCR ...................................... 41
HCMV pp67 mRNA Detection ..................................................................... 42
........... Electron Microscopy (EM) Analysis of HCMV Inoculated Cultures 43
Emmunoelectron Microscopy (IEM) Analysis of HCMV Inocdated ......................................................................................................... Cultures -44
IL.6, IL.8. TNF-a Stimulation ...................................................................... 46
RESULTS
Effect of HCMV Inoculation on HL-60 Ce11 Cultures .................................. 47
Dose Dependent F A Response ..................................................................... 48
Variable lFA Success ..................................................................................... 48
pp67 mRNA Transcript Detection ................................................................. 48
hfectious Progeny Not Produced in IFA Positive Ce11 Cultures .................. 49
............................................................................ Electron Microscopy (EM) 54
............................................................. Immunoelectron Microscopy (IEM) .59
.................................... Effect o f IL-6, IL-8, TNF-a on HL-60 Ce11 Cultures 60
Figure t Proposeci differentiation sequence (restriction potentialities) of ............................. the pluipotential hematopoietic stem ce11 CFU-S 26
Figure 2 The effect of increasing MOI on percentage of cells IFA positive .......................................................................... for late HCMV Ag ..S 1
Figure 3 Success rate of HCMV late antigen detection within inoculated ............................................................................ HL-60 ce11 cultures 52
Figure 4 Electron micrograph of a HCMV inoculated HFF ceIl 4 days ................................................................................ ps t inoculation. -57
Figure 5 Electron micrograph of a HCMV inoculated HL-60 ce11 4 days .................................................................................. ps t inoculation 58
Figure 6 Immunoelecm>n micrograph dernonstrating non-specific binding ......................................... of conjugated gold particles to HFF cells. 62
Figure 7 Proposed mode1 of interaction between HCMV and a HL-60 ce11. ... 68
LIST OF TABLES
Table 1 Detection of HCMV antigens and DNA within inoculated HL-60 .................................................. ce11 cultwes 4 days post inoculation 50
Table 2 Sumrnary of IFA, PCR, and NASBA analysis using live or heat inactivated HCMV as inoculum with HL-60 ce11 cultures 4 days
........................................................ post inoculation. .................... ... 53
Table 3 Determination of viral titre in HCMV inoculated IFA positive HL-60 cells and DMSO stimulated HL-60 cells 4 days
................................................................................ post inoculation.. 55
Table 4 Defining features of HCMV infection by electron mimswpy.. ....... 56
Table 5 Summary of IEM atternpts using HFF cells uninoculated and Inoculated with HCMV, 4 days p s t inoculation ............................... 6 1
1 . Q ) I N T R O D U C T I O N
Background knowledge relating to the studies performed in this thesis is selectively
summarized in this introduction under the following headings :
Project History
Cytomegalovirus
The Myeloid Lineage
HL-60 Ce11 Line
1.1) HCMV PROJECT HISTORY
Leukocytes have been shown to be the main vehicle through which human
cytomegalovinis (HCMV) is transmitted during transfusion. The use of leukocyte
filtered blood and blood products for transfusion results in significantly reduced rate of
HCMV infection in the recipient (De Witte et al., 1990; Winston et al., 1980). In patients
with active HCMV infection, the presence of HCMV phosphoprotein 65 (pp65) inside
the nuclei of pol ymorphonuclear leukocytes (PMNL) (antigenemia assay) (The et al.,
1990; Veal et ai., 1996) is often used to assess the viral load (The et al., 1995).
Furthmore, quantitative detedon of pp65 antigen has been shown to be an effective
diagnostic approach for monitoring the progress of HCMV disease (Ranger-Rogez et al.,
1996). A number of studies have been wnducted on the detection of HCMV pp65 Ag in
PMNL fkom transplant patients and H N positive patients, with symptomatic HCMV
infection (Boeckh et al., 1992; Erice et al., 1992; Veal et al., 1996; Spaete et al., 1994;
van der Bij et al., l988a; Grefle et al., 1992; van der Bij et al., l988b; Zipeto et al., 1992;
Boland et al., 1992; Grefle et al., 1994). Since pp65 Ag was detected during active
HCMV infection within PMNL, it was hypothesized that HCMV wuld infect PMNL,
which would lead to active gene expression and production of viral pp65 Ag. Along with
detection of the early-late pp65 Ag, immediate-early (IE) Ag and HCMV late Ag have
been detected in PMNL from patients with active HCMV infection (Revello et al., 1992;
Gema et al., 1990; Sinzger et al., 1995). In addition, HCMV IE Ag has been detected in
fibroblast cells, inoculated with PMNL f5om viremic irnmunosuppressed patients (The et
al., 1990; Dankner et al, 1990). A more recent study has found pp65 Ag in PMNL upon
CO-culturing with HCMV infected fibroblasts or infect4 endothelial cells, and these
PMNL were shown to transmit infectious virus upon CO-culturing with fksh fibroblast
cells (Revello et al., 1998). Virus has also been isolated fiom PMNL preparations h m
infected patients (Gema et al., 1992).
Additional support for the concept of active HCMV infection of PMNL is
provideci by detection of viral DNA in the cells. HCMV DNA has been detected in
PMNL of post transplant recipients (VeaI et al., 1996; Dankner et al., 1990; von Laer et
al., 1995; Gerna et al., 1992; Saltzman et al., 1988; Zipeto et al., 1992; Boland et al.,
1992) as well as those h H N positive patients (Veal et aL, 1996; Gema et al., 1992;
Boivin et al., 1998; Zipeto et al., 1992; Gozlan et al., 1993). Detection of HCMV DNA
alone, does not correlate with active HCMV infection, since the presence of the virus in a
latent state is always a possibility. However, deteftion of viral DNA, dong with
detection of antigens or mRNA transcripts, provides stmng evidence for active Wal gene
expression. The best indicator for expression of the HCMV genome is the presence of
mRNA transcripts in PMNL. Gozlan and colleagues (1993) detected HCMV late mRNA
in PMNL fiom HIV-infected patients. In a subsequent study, IE mRNA and late mRNA
was detected in PMNL of rend transplant patients with active HCMV infection (von Laer
et al., 1995). Dankner and colleagues (1990) were able to detect HCMV IE, early and
late mRNA transcripts in PMNL of irnmunosuppressed patients, thus demonstrating gene
expression f?om al1 three phases of HCMV replication.
Along with detection of HCMV DNA, mRNA transcripts and antigens h m d l
phases of the replication cycle, intact virus particles have been obsewed in PMNL by
elecwn mimscopy (Martin et ai., 1984). Although the literature Qted thus far provides
strong support for active HCMV gene expression and viral replication within PMNL of
the susceptible host, other studies on the interaction of HCMV and PMNL, have fond
conflicting results.
In a study by Sinzger et al. (1995) only the 72K IE Ag but not early or late Ag
were detected in PMNL consistent with an abortive infection. Turtinen and colleagues
(1987) were able to detect HCMV DNA in the cytoplasm of PMNL but were unable to
detect any viral mRNA transcripts. In a study reported by Grefie et al. (1992) both IE Ag
and pp65 Ag were detected in PMNL, but only IE mRNA was detected in PMNL and
there was no detection of pp65 -A. By contrast, both IE and pp65 mRNA were
detected in late stage HCMV infected endothelid cells (Grefte et al., 1994). Since pp65
is an early-late HCMV protein, this finding is suggestive of restricted expression of the
viral genome and acquisition of pp6S by means other than de novo synthesis. As
mentioned previously, HCMV virions have been detected in PMNL by electron
microscopy (Martin et al., 1984; Turtinen et al., 1987). Specifically, detection has been
localized to phagosomes and there has been no detection of intranuclear virions or
nucleocapsids, suggesting HCMV was not actively repticaîing inside PMNL.
These studies have provided important insights in our attempt to trying to explain
the observed in vivo interaction between PMNL and HCMV. in this context, there are
findings which support the concept bat PMNL are conducive for complete HCMV
replication and gene expression (Revel10 et al., 1992; Gerna et al., 1990; Suizger et al.,
1 995; Gozlan et al., 1 993; von Laer et al., 1 995; Danhier et al., 1990). Other f idhgs
support the concept that PMNL are conducive for only resmcted viral gene expression
(Sinzger et al., 1995; Gerna et al., 1992; Grefte et al., 1994). Therefore, while the
interactions between HCMV and P M N L rernains undear, m e r investigations are
warrant ed .
1.2.1) CHARA CTERI24 TION
Cytomegalovinrs (CMV), formally designated as human herpesvirus 5 (HHV-5),
is a rnember of the betaherpesvinis group. This DNA virus has the largest gemme of the
herpesvinises (230kb-240kb). It is characterized by a relatively slow replication cycle
which lads to the production of large, often multinucleated cells (Mocarski, 1996).
Similar to other herpesvinises, primary infection is followed by a latent infection.
Ubiquitous in nature, CMV is highly species-specific. A wide range of animal species
including humans, other primates, domestic animals and rodents have an associated CMV
(Mocarski, 1996). Humans are the only known reservoir for human cytomegalovinis
(HCMV). Transmission of HCMV has been associated with close personal contact
(saliva, sexual contact), vertical transmission (in utero, breast milk), organ and tissue
transplant, including blood transfusion. HCMV has been isolated fiom a number of
body fluids including oropharyngeal secretions, urine, cervical and vaginal secretions,
semen, breast milk, tears, feces and blood (Britt and Alford, 1996).
1.2.2) EPIDEMIOLOGY
In North Arnerica, HCMV infects approximately 50% of the population, with
infection rates rising to 90% within some urban centres. HCMV is the most common
congenital viral infection in humans. Data fkom the United States indicates that 0.2%-
2.2% (-40 000) of infants bom each year are infected in ufero. In cases where HCMV
disease ensues, damage to perceptual organs accounts for the majority of morbidity
associated with intrauterine HCMV infection. Long-term neurologic sequeiae may occur
in up to 10- 15% of these infected infants, with hearing loss comprising the most cornmon
neurologic abnorrnality. Between 8% and 60% of al1 infants are infeçted during the first
6 months of life, as a result of HCMV transmission via breast milk (Bntt and Alford,
1996).
1.2.3) CUNICAL D I S E S E
HCMV infection is usually asymptomatic in the healthy host. Occasionally,
infection may present as a mononucleosis-like syndrome, similar to that resulting from
infection with Epstein-Barr Virus (EBV) @ritt and Alford, 1996). Infiequent
complications of HCMV mononucleosis include pneunonia, hepatitis, CNS involvement
(Guillan-Barré syndrome) as well as aseptic meningitis. HCMV disease, resulting fiom
primary infection or reactivation of latent infection, within the immunocompromised and
irnmunosuppressed host, is a serious concern. With severe disseminated disease, HCMV
involvement can be identified in virhially al1 organ systems. Vinuia, resulting fkom
urinary tract viral replication is a consistent feature of HCMV infkction in al1 age groups.
Viruria occurs in both noxmal or immunosuppressdi~~l~~luno~~rnpromised hosts and can
occur during primary HCMV infection, or reactivation of latent HCMV.
HCMV is an important pst-transplant pathogen in allograft recipients. Sources
of infecting virus include the transplanted organ, blood products used during the
procedure, and reactivation of the recipient's endogenous virus. The degree of
immunosuppression in the recipient correlates with an increased risk of HCMV infection.
HCMV infections have been described in solid organ allografts involving the
kidney, liver, heart and heart-lung. Between 60% and 100% of renai, cardiac and hepatic
allograft recipients develop HCMV infection (Bntt and Alford, 1996). A numba of
clinical syndromes can arise due to HCMV infection in the pst-transplant period.
Prolonged fevers, leukopenia, as well as life-threatening complications such as severe
infection of the GI tract, hepatitis and pneumonia have been documenteci. Within bone
marrow transplant @MT) patients, HCMV is a significant cause of death. Mortality rates
can approach 80-90%. Pneumonîa is the most commonly associateci syndrome resulting
in mortality when left untreated (Bntt and Alford, 1996).
HCMV is a major opportunistic infëction encountered by patients with AIDS. In
the era before highly active antiretroviral therapy (HAART), autopsy studies have shown
90% of patients with AIDS develop active HCMV infection and up to 40% may develop
sight andor life-threatening HCMV disease. HCMV has been proposed as a cofactor in
the pathogenesis of HIV infections (Barry et al., 1990). Clinical syndromes with HCMV
have included disease in essentially every organ; the most clinically significant involve
the eyes, Iung, CNS, and GI system.
1.2.4) LA TENCY
ï o date, much work has been conducted in trying to identie the site(s) of latency
of HCMV. Early studies suggested T-lymphocytes were the primary site of latency (Rice
et al., 1984). The wai genome has been detected in peripheral blood monocytes and
macrophages of healthy carriers, thus suggesting these cells rnay also be a primary site of
latency (Taylor-Wiedeman et ai., 1991, Fish et al., 1995). Animal models have been
used to try and characterize CMV latency. Using a mouse model, endothelid cells, lung
alveolar macrophages and bone marrow cells have been identified as harbouring murine
CMV DNA in latently infected animals (KofEon et al., 1998). A recent study has
identified HCMV latency-associated transcripts within a small percentage of myeloid-
type progenitor cells and dendritic cell progenitors in the healthy seropositive host @abn
et al., 1998). Thus, hematopietic cells may be an important reservoir for HCMV and of
significant importance in reactivation of latent virus.
1.2.5) H C W REPLICA TIVE CYCLE
There are a number of key steps involved in the HCMV replicative cycle, which
have been elucidated from studies of HCMV infection of permissive fibroblast cell lines.
This process is initiated by attachment of the virus to a host cell receptor followed by
penetration and uncoating of the viral nucleocapsid (White et al., 1994). In fibroblast
cells, viral envelope glycoprotein B (gB) and glycoprotein H (gH) have been shown to
interact with host proteins annexin 11, heparan sulfate proteoglycans, and a 92.5 kDa
receptor (Compton, 1995; Pietropaolo and Compton, 1997; Keay et al., 1989; Keay and
Baldwin 1982). Virus penetration proceeds by pH independent fusion of the viral
envelope and the host cell plasma membrane, thus releasing the nucleocapsid into the
cytoplasm (Compton, 1995). The HCMV genome has three temporally distributed
classes of genes. Once the HCMV DNA is released inside the cell, transcription of
HCMV immediate early genes (IE) within the nucleus is initiated by the host cell RNA
polymerase 11. The temporal distribution of HCMV gene expression is M e r discussed
in section 1.2.6. Transcription of late genes is dependent on viral DNA replication. In
fibroblast cells, HCMV DNA replication is observed 14-1 6 hours post inoculation @.i.)
and is mediated by a v d l y encodeci DNA polymerase (Mocarski, 1996). Transcription
and translation of late proteins provides the structural components necessary for virion
assembly. The HCMV genome is packaged into prefomed nucleocapsids in the nucleus
(Mocarski, 1 996).
Vinons budding fiom the nucleus probably acquire an envelope from either the
i ~ e r nuclear membrane and accumulate in the perinuclear space, then move into
cytoplasmic vesicles (Mocarscki, 1996). The mechanism by which infectious virions are
released upon assembly and maturation remains unclear. It is known that HCMV is a
relatively cell-associated virus and at very late stages post infection, roughly half of the
progeny virions may be found in the culture media, with the remainder being cell-
associated.
1.2.6) HCW GENE EXPRESSION
Specific genes of HCMV are expressed at different times throughout infection.
These genes fa11 into three major classes. The earliest genes to be expressed are the a or
imrnediate-early (IE) genes which are transcribed independent of both viral DNA
replication and translation of other viral gene products. Similar to al1 herpesvinises,
expression of f3 (early) genes, and y (late) genes requires synthesis of fünctional IE gene
products. The principle XE gene product is a 491 amino acid phosphoprotein of molecdar
weight 72 kd (pp72). pp72 regulatory activities include autostimulation of its own
synthesis as well as a tram-activator of early and late gene expression (Mocarski, 1996).
In fibroblast cells, pp72 has been detected as early as 20-30 minutes (pi.). It has been
shown to associate with cellular chromatin and to be synthesized throughout infection.
However, transcript levels are lowest at the onset of viral DNA replication.
Generally, f3 gene transcripts are expressed later and expression continues into
late stages of infection. This class of genes cm be divided into subclasses based on their
time of expression. pi genes have been detected as early as 4-8 hours p.i. while $2 genes
can be detected 8-24 hours pi. Phosphoprotein 65 @p65), e n d e d by a f3 gene is an
early-late protein produced in the replicative cycle of HCMV. It is located in the
tegument region between the capsid and envelope of HCMV virions (Mocarski, 1996). It
has 561 amino acids and has been shown to be highly immunogenic. Furthemore, it is
the most abundant tegument protein, accounting for 95% of the mass of this structure.
Within infected cells, pp65 serves as a major phosphate acceptor and it is the primary
target for phosphorylation in vitro by the viral protein kinases. in addition, pp65 has
been hypothesized to have an important role in vivo since it is the principle target of
cytotoxic T lymphocytes (Da1 et ai., 1996). pp65 carries two nuclear localization signais
and its accumulation within the nucleus suggests a role within the nuclei of infected cells
(Mocarski, 1996). In vitro studies using fibroblast cells have shown that pp65 is
nonessential for growth of HCMV (Schmolke et al., 1 995). However, studies involving
astrocytoma cells indicate that detection of extracellular progeny was alrnost completely
blocked by inhibiting pp65 expression @al et al., 1996). Thus, the intracellular h c t i o n
of pp6S remains unclear.
As with early genes, y genes c m also be divided into subclasses. y1 transcript~ can
first be detected 12-36 hours p.i. and y2 transcripts at 2448 hours p.i. The majority of y
genes encode structural proteins. At least 30 proteins have been detected in mature
HCMV vinons, distributed in the envelope, tegument and capsid. Phosphoprotein 67
(pp67) is an example of a viral structural protein produced late in infection. The role of
pp67 is unclear, however this 102 amui0 acid tegument protein may have some
involvement with protein kinase activity of the Mnis (Mocarski, 1996).
The virion envelope carries a minimum of 8 glycoproteins organized in three or 4
giycoprotein complexes while the tegument contains as many as 20 proteins (Mocarski,
1996). In HCMV the major capsid protein (MCP) is a 1,370 amino acid protein of
molecular mass 150 kd. While MCP shares substantial arnino acid sirnilarity with the
major capsid protein (VPS) of HSV- 1, the HCMV MCP exhibits low imrnunogenicity.
1.2.7) HCMV INACTIVA TION
The HCMV envelope is a lipid bilayer membrane composed of host lipids and
viral proteins. The envelope is derived fiom either the nuclear or cytoplasmic
membranes, including early endosomal membranes (Mocarski, 1996). Biological
membranes are not rigid; lipids and membrane proteins are constantly in lateral motion
(Stryer, 1988). Membrane fluidity is mediatecl by the fatty acid composition and
cholesterol content of the membrane. Lipid bilayer membranes c m exist in an ordered or
rigid state, as well as a disordered or fluid state. The transition between the two states is
determined by the transition temperature, which is dependent upon the length of the fatty
acyl chains, and their degree of unsaturation. Shorter fatty acyl chains md double bonds
interfere with a highly ordered packing of fatty acyl chains and thus the transition
temperature is lowered. Membrane fluidity is moderated by choloesterol. Cholestml
prevents the crystallization of fatty acyl chains by fitting between them, and sterically
inhibiting large motions of fatty acyl chains, remdering the membrane more rigid (Stryer,
1988).
Heat treatment has been used as a mode of inactivating a wide variety of viruses.
Hurnan imrnunodeficiency virus (HTV) infectivity has been reduced to minimal levels
(several log decrease in titre) when subjected to 56OC heat treatment for 20-30 minutes
(McDougal et al., 1985; Spire et al., 1985; Resnick et al., 1986). Similarly, Sindbis
virus, polio virus, pseudorabies and H N were inactivated when subjected to heaî (Estep
et al., 1988). Heat treatment has been used to inactivate HCMV viral stocks (Almeida-
Porada et ai., 1997; Valyi-Nagy et al., 1988). Heat treatment of 56°C for 30 minutes
results in membrane fluidization of H N (Aloia et al., 1988). increasing the membrane
fluidity above a critical minimum decreases HIV viability. in a study conducted using
vesicular stomatitis virus, treatment which removed cholesterol resulted in fluidization of
the viral membrane and reduced infectivity of the virus (Moore et al., 1 978). Sirnilarily,
butylated hydroxytoluene (BHT)' a fluidizing drug, inactivates lipid enveloped DNA and
RNA viruses in vitro (Aloia et al., 1988). BHT is an additive wideIy used in human and
animal foods to maintain freshness and avoid spoilage by delaying degradation of lipid
components of food (Kim et al., 1978). BHT has been previously shown to greatly
rduce numbers of infectious HSV Miions (Kun et al., 1978). BHT, used at
concentrations known to have no biologically adverse effects when used as an additive,
was able to inactivate HCMV. Thus, heat treatment, reducing the cholesterol content, or
the use of BHT results in membrane fluidization leading to a loss of integrity of the viral
envelope (and associated receptors and ligands), and the observed inactivation of virai
infectivity .
1.2.8) HCMV Pennksive Cell tines
Depending on its nature, the host ce11 may be eitber permissive or restrictive for HCMV
infection (Burd et al., 1996). In a restrictive or non-permissive ceii line, the viral
replication cycle may be blocked at any point nom attachment through to the final stage
of assembly and release (White and Fenner, 1994). HCMV exhibits a highly restricted
host range in ce11 culture with skin or lung fibroblasts being the preferred host ce11
(Mocarski, 1 996). Undi fferentiated as well as transformed ceil lines are generaily non-
permissive (Mocarski, 1 996). A number of reports have identified otha permissive ce11
lines for HCMV infection in vim, however, ce11 types other than fibroblasts do not
support viral replication to the same degree (Mocarski, 1996). Brain endothelid cells
have been shown to support complete viral gene expression and cytopathic effect (Poland
et al., 1990) whereas astroglial lines Vary in their ability to support HCMV replication.
For exarnple, the T98G ce11 line supported incomplete (immediate-early) gene
expression, HS-683 supported extensive virus replication with minimal viral CPE, while
A-172 did not support any detectable gene expression (Poland et al., 1990). Neuronal
ce11 lines (SK-N-MC) were found to be fully permissive to HCMV infection (Poland et
al., 1990), and this was probably related to the state of differentiation of the cell, and to
the transcriptional regdatory molecules present in the ce11 at the time of infection (Burd
et al., 1996). Infectious virus was detected in glioma and neuroblastoma cells inoculated
with HCMV in vitro (Ogura et al., 1 986). HCMV infection of astrocytoma cells has been
shown with expression of IE, early and late antigens as well as production of extracellular
infectious virus (Duclos et al, 1989). While HCMV antigens have been detected within
PMNL, it is unclear whether these cells support active HCMV replication.
13) THE MYELOID LINEAGE
Z. 3.1) GRANClzOPOIESIS
In the life of the neutrophil, it passes through three phases: bone m m w , blood
and tissue. Within the bone marrow, there is a mitotic cornpartment and a non-mitotic
storage compartment. Most of the neutrophils in the body reside in the bone m m w
(Newburger and Parmley, 1 99 1 ). Once neutrophils are released into the blood, they have
a half-life of 6-9 hours and irreversibly move fiom circulating to marginating pools
(Newburger and Pannley, 1991). These granulocytes irreversibly leave the blood by way
of diapedesis between endo thelial cells and penetration of the basement membrane.
Arnong the mechanisms responsible for neutrophil ce11 death is programmed ceIl death or
apoptosis (Newburger and Parmley, 199 1).
Granulopoiesis begins with the pluripotential stem ce11 CFU-S (an acronym for
colony foxming unit-spleen) (Keeling, 1987). Most of these cells are in the resting phase,
but of these, a small fiaction differentiate into committed stem cells. A proposed
differentiation sequence of the pluripotential stem ce11 is outlined in Figure 1.
Granulopoiesis follows successive differentiation fiom the pluripotential stem ce11 to
committed stem ce11 CFU-GM (colony fonning unit-granulocyte-macrophage), followed
by further differentiation to committed stem cell CFU-G (colony fonning unit-
granulocyte).
The myeloblast is the first recognizable ceIl of the granulocytic series (McDonald
et al, 1 98 8; Keeling, 1 987; Jandle, 1 987). Differentiation of the myeloblast results in the
successive development of the promyelocyte, myelocyte, metamyelocyte, band
neutrophil and finaliy the terminal1 y di fferentiated segrnented neutrophil. The myeloid
maturation sequence is characterized by progressive decrease in nuclear size, early
presence of nucleoli and subsequent disappearance, early appearance of azuophilic
granules, late appearance of specific granules, as well as nuclear indentation and
segmentation (Beck, 1985)-
Myeloblasts represent the earliest stage of myeloid development within the
mitotic cornpartment of the bone mamw (Newburger and Panniey, 1991). They can
give rise to neutrophils as well as eosinophils and basophils and hence lack morphologic
feahires that predict their differentiation into one of the granulocytic lines (Newburger
and Parmley, 199 1). Myeloblasts do not contain any granules and have a roundoval
nucleus occupying 415 of the total ce11 area (McDonald et al., 1988). Pmmyelocytes are
at the midstage of development within the mitotic cornpartment, and resemble
myeloblasts except for the presence of azurophilic granules in the cytoplasm (McDonald
et al., 1988). Myelocytes are the first cells containing secondary or specific cytoplasmic
granules which are either neutrophilic, eosinophilic or basop hilic in nature (McDonald et
al., 1988; Keeling, 1987; Beck, 1985). Myelocytes also represmt the 1 s t stage of
development in the mitotic cornpartment (Newburger and Parmley, 1991). The
myelocyte is smaller than the promyelocyte and nucleoli are no longer present (Beck,
1985). Metamyelocytes as well as band and segmented neutrophils represent nondividing
cells within the bone m m w (Newburger and P a d e y , 1991). Metamyelocytes are
characterized by a slightly indented nucleus and persistence of secondary granules
(McDonald et al., 1988; Keeling, 1987). Further nuclear diffaentiation is evident in the
band neutrophil by the presence of a "u-shaped" nucleus. The band neutrophil is the first
of the neutrophilic line that can be identified in normal circulating blood (Keeling, 1987).
Finally, the temiinally differentiated segrnented neutrophil is the end product of the
granulocytic series. It is distinguished fiom its predecessor by its multi-lobular nucleus
~ ~ ~ e ~ t e d by thh strands of chromatin (keiing, 1987).
1.3.2) GRANULE PRODUCTION
D i f f m t types of hurnan neutrophil granules are formed sequentially d u d g
maturation of neutrophils h m the promyelocyte stage, to the band ce11 stage, and thus
c m be used as markers of myeloid differentiation. Specifically, two types of proteins are
stored within these granules: intragranular proteins which are liberated fiom the ce11
during exocytosis of the granule, and granule membrane proteins which are incorporated
into the plasma membrane (Le Cabec et al., 1997).
In a classification system based upon the content of myeloperoxidase, there are
two types of granules that are distinguishible in human neutrophils: peroxidase-positive
granules and peroxidase-negative granules (Beck, 1985). The peroxidase-positive
granules, othenvise termed azurophilic or primary granules, contain lysosomal hydrolytic
enzymes, neutral proteases, myeloperoxidase, glycoaminoglycans, acid phosphatase,
cationic bactericidal proteins, and lysozyme (Beck, 1 985).
1 Diff eren tiation
CFU-E
CFU-M
Figure 1. Proposed differentiation sequence (restriction of potentialities) of the pluripotential hematopoietic stem cell CFU-S. Diffmtiation follows a specific sequence of successive restriction of potentialities with each of the unlabeled cells on the left retaining al1 potentialities except those lost above it. CFU, colony forming unit; M, macrophage; EO, eosinophil; GM, granulocyte-macrophage; G, granulocyte; MEG, megakaryoc yte; E, erythroid. (Modi fied fiom Nicola NA and Johnson GR: Blood l982;60(4): 101 9- 1029.)
They are subdivided into those which are defensin-rich or defain-poor (Le C a b et
al., 1997). There are two subsets of peroxidase-negative grandes, classifieci according to
their lactoferrin and gelatinase content: specific or secondary granules, and gelatinase or
tertiary granules (Le Cabec et al., 1997). Specific grandes contain lysozyme, l ac to feh
and vitamin Bit-binding protein while the gelatinase or teritiary granules are named as
such due to their gelatinase content (Le Cabec et al., 1997; Bak, 1985).
Synthesis of granular proteins is dependent upon the matunty of the myeloid d l .
Azurophilic granular proteins are synthesized at the promyelocyte stage, specific granular
proteins are synthesized at the myelocyte/metamyelocyte stage, and gelatinase is
synthesized mainly at the band ce11 stage (Le Cabec et al., 1997; Newburger and
Parrnley, 199 1 ).
1.3.3) NEUTROPHIL FUNCTION
Neutrophils are responsible for much of the body's defense against bacterial
invaders through the process of phagocytosis (Keeling, 1987; Beck, 1985). The essential
hc t ion of the neutrophil is to rapidly move to a site of rnicrobial invasion, engulf, and
kill the microorganism (Newburger and Pannley, 1991). Phagocytosis is a cornplex
series of events involving chemotaxis, rewgnition, engulhent, and killing of the
organism (Keeling, 1987). Chernotaxis of neutrophils is an inducible interaction arising
fkom immunologie as well as nonimmunologic pathways (Keeling, 1 987). Activated
complement wmponents C3a and CSa, generated as part of the cascade of the classical
complement pathway or the alternate complement pathway, are chernotactic for the
neutrophil (Keeling, 1987; Abbas et al., 199 1 b).
The kinin-generating system, as well as the clotting system dso provide appropriate
stimuli for neutrophil recniitment (Jandle, 1987). Neuîrophils respond to chernotactic
stimuli by following a direct path towards the stimulus with ameboid movement
(Keeling, 1987).
A critical step in the phagocytic process is recognition of the foreign particle.
Phagocytosis occurs more rapidly and efficiently when particles to be engulfed are
opsonized (Keeling, 1987). Neutrophils possess surface receptorj for c~mplement C3 as
well as receptors for the Fc fiagrnent of s e m antibodies (Gallagher et al., 1979). Fc
receptors appear to trigger ingestion while C3 receptors are more involved in binding of
the opsonized particle to the neutrophil (Beck, 1985). Once attachrnent occurs, the
neutrophil extends pseudopods which surround the foreign particle thereby engulfhg it in
a temporary storage vacuole or phagosome (Keeling, 1987; Beck, 1985). Killing of
ingested organisms is achieved by the fusion of cytoplasrnic granules with the
phagosorne, and subsequent discharge of granular contents into the phagosome, a process
known as degranulation (Newburger and Parmley, 199 1 ; Keeling, 1987; Beck, 1985).
A number of pathways exist for killing ingested organisms. There are oxygen-
independent mechanisms which are darnaging to various bacteria. Some include the
acidic pH of the phagosome, exposure to lysozyme which hydrolyzes the glycosidic
bonds of ce11 wall peptidoglycans, and secretion of bactericidal proteins which can alter
the membrane permeability of bacteria (Newburger and Parmley, 199 1 ; Beck, 1985).
Also, defensins, which are cysteine-nch cyclic polypeptides, are cytotoxic to
metabolically active bacteria, fun@ and enveloped Wuses (Newburger and Pannley,
199 1 ). By in large however, the killing of ingested organisms is primarily achieved by an
oxygen-dependent process (Newburger and Parmiey, 199 1; Keeling, 1987; Beck, 1985).
During ingestion, neutrophils actively metabolize oxygen to produce reactive
products that are toxic to ingested bacteria (Newburger and Pannley, l99 1 ; Beck, 1985).
Upon activation of neutrophils, by chernotactic stimuli or attachment of particles, there is
a dramatic increase in oxygen consumption referred to as the "respiratory burst"
(Keeling, 1987). Al1 of the oxygen taken up during the respiratory burst initially foxms
superoxide (O2>. 0; is the first reduction product of oxygen and is highly reactive
resulting in m e r reduction to hydrogen peroxide (H202) (Beck, 1985). While
superoxide inactivates Mnxses, damages plasma membranes and is capable of killing
cells, hydrogen peroxide is probably more important for bactericidal activity (Be&
1985). H202 is directly bactericidal and is also involved in bacterial killing via the
myeloperoxidase-halide ion system (Keeling, 1987). Reactions between hydrogen
peroxide, myeloperoxidase and chloride produces toxic hypochlorous acid as well as
toxic chloroamines (Newburger and Pannley, t 99 1 ; Keeling, 1987). Therefore, the
neutrophil is structurally and fùnctionally equipped for its important task of rnigrating to
infections, recognizing the infectious agent, and killing the invading organism. Defects
in any of these pathways result in altered defense against pathogens (Newburger and
Parmley, 199 1).
1.3.4) CYTOKlh?ES IL-8, IL-6, TNF- a AhD ACMV
Cytokine production in vivo has been associated with increased pathogenesis of
HCMV infection and disease (Humar et al., 1999). HCMV infection of fibroblasts and
endothelid cells have shown upregulation of interleulrin-8 (IL-8) levels (Gnindy et ai.,
1998; Craigen et al., 1997). increased IL-8 levels have resulted in recruitment of
neutrophils to sites of endotheliai infection as well as significantly enhanced
transendothelial migration (Gnrndy et al., 1 998; Craigen et ai., 1 997; Humar et al., 1999).
IL-8 induction has been described in vitro with a number of viruses such as hepatitis B,
influenza A, respiratory syncytial virus, measles virus, as well as herpes simplex virus
(Craigen et ai., 1997). HCMV Section has been shown to upregulate interleukin-6 (IL-
6) production. Primarily fhctioning as a B lymphocyte growth and diflerentiation factor
and an activator of T lymphocytes (Abbas et al., 199 1 a; Geist and Dai, 1 W6), IL-6 dong
with HCMV have been irnplicated in lung allograft rejections. Turnor necrosis factor
alpha (TNF-a) is a potent activator of neutrophils as part of the uiflamrnatory response
(Abbas et ai., 199 1 a). TNF-a induces the HCMV IE gene promoter and thus may be
involved in reactivation of latent HCMV infection (Humar et al., i999; Fietze et al.,
1 994; Stein et ai., 1993; Docke et al., 1994). Therefore, cytokine induction/production
appears to be closely associated to HCMV infection.
1.3.5) CUL TURING OF NEC/TROPHILS
Band neutrophils and segmented neutrophils can be detected in nomal circulating
blood (Keeling, 1987). Once neutrophils are released into the peripheral blood, they
have a half-life of 6-9 hours (Newburger and Parmiey, 1991), and have a defined
lifespan; 4-5 days in nomal tissue, shorter in sites of infection and inflammation
(Keeling, 1987). Since the replication cycle of HCMV in a known permissive fibroblast
ce11 line is slow, 48-72 hours (Mocarski, 1996), culNnng of neutrophils for in vitro
studies is problematic. Thus, identification and use of a myeloid ce11 line possessing
similar morphological and fimctional characteristics as neutrophils would be best suiteù
for conducting in vitro HCMV inoculation studies. The HL-60 ce11 line allows for the
development of such a modet.
1.4) KL-60 CELL LINE
1.4.1) ESTABLISHMENT OF HL-60 CELL LllVE
Peripheral blood leukocytes were obtained by leukophoresis fiom a 36-year-old
caucasian female diagnosed with acute promyelocytic leukemia (Gallagher et al., 1977).
Leukocytes were initially cultured in the presence of conditioned medium fiom human
ernbryonic lung cet1 cultures, but it was subsequently found that continued growth of
leukocytes did not require conditioned medium supplements (Collins et al., 1 977;
Gallagher et al., 1 979).
1.4.2) CYTOLOGIC CHARA CTERISTICS OF HL-60 CUL TURED CE=
HL-60 cells grow in single-ce11 suspension without a tendency to clump or adhere
to plastic or g las (Gallagher et al., 1979). These cells are generally round or ovoid with
a diameter varying fiom 9p to 25p; larger cells are generally binucleate. Wright-Giemsa
staining has shown that the predominant ce11 type within the HL-60 ce11 culture, is a
promyelocyte (Gallagher et al., 1979). This ce11 contains a large round nucleus with
distinct margins, fine chromatin and 2-4 nucleoli. The cytoplasm is deeply basophilie
containing multiple azurophilic granules. HL40 cells stain with myeloid lineage specific
stains such as myeloperoxidase, ASD chloroacetate esterase and Sudan black B however
they do not stain with alkaline phosphatase, a stain characteristically positive in normal
neutrophilic granulocytes (Gallagher et al., 1979). While the majority of cells in this
culture are promyelocytes, approximately 1 O- 12% are more mature myeloid cells,
predominantly myelocytes, with rare fully mature segmenteci neutrophils (Collins et al.,
1978). HL-60 cells express surface receptors for Fc hgment and complernent (C3)
which have been associated with di fferentiated granulocytes (Gallagher et al., 1 979).
1.4.3) GENERAL DIFFERENTC4 TION
HL-60 cells allow for the developrnent of a well-dehed in vitro experimental
system for examining aspects of the differentiation process (Yen and Albright, 1984).
Precursor cells and differentiated cells differ in two significant ways. Differentiated cells
are usually restricted to the Gil0 ceIl cycle phase while precursor cells are proliferating
and distributed throughout the ce11 cycle. in addition, differentiated cells are capable of
specialized metabolism, characteristic of the diffixentiated phenotype, whereas p r e c m r
cells are not (Yen and Albright, 1984). HL-60 cells exempli@ these characteristics
They are proliferatively active and have the capability to differentiate dong either the
monocytic or myeloid ce11 lineages, in response to different soluble factors (Yen and
Albright, 1984).
Agents such as 1,25-(OH)2 vitamin D3, sodium butyrate, and 12-0-
tetradecanolphorbol-13-acetate (TPA) induce terminal monocytic differentiation of HL-
60 cells (Yen and Albright, 1984; Yen et al., 1987; Fischkoff and Rossi, 1990). Butyric
acid stimulation of HL-60 cells has resulted in stable sublines consisting of neutrophils,
monocytedmacrophages, eosinophils, as well as a sublines containing a mixture of these
celi types (Collins et al., 1978; Fischkoff and Rossi, 1990). Retinoic acid, dimethyl
formamide and dimethyl sulfoxide (DMSO) induce myeloid differentiation in HL-60
cells (Yen et al., 1987; Skubitz and August, 1983). Addition of DMSO results in a
marked increase in the proportion of mature myeloid cells: myelocytes, metamyelocytes,
band neutrophils, and segmente- neutrophils (Collins et al., 1978). DMSO has been
shown to induce hct ional as well as morphological maturation in HL-60 cells as
demonstrated by an increase in the rate of superoxide production, degranulation,
ingestion of opsonized particles, and bacterial killing (Newburger et al., 1 979;
Newburger et al., 1984). The phagocytic and chemotactic functions of the HL-60 cells
were grealy enhanced by induction for differentiation with DMSO (Gallagher et al.,
1979; Collins et al., 1978). HL-60 cells exhibit phagocytic activity and responsiveness to
chemotactic stimulus cornmensurate with the proportion of mature cells in the culture
(Gallagher et al., 1979; Collins et al., 1978).
1.4.4) MODEL OF STUDY
HL-60 cells have been widely used as a mode1 of study for myeloid ce11 growîh
and differentiation (Skubitz and August, 1983; Collins et al., 1977). Their ability to
acquire characteristics of more mature cells of the myeloid fineage upon induction for
differentiation have made HL-60 cells an important ce11 line for investigation. Collins
and colleagues (1978) have show that mature HL40 cells are capable of phagocytosis.
Thus, this ce11 line may be used as a model to study neutrophil function. Studies trying to
determine lineage direction and cornmitment stages for differentiation in the myeloid
lineage have utilized HL-60 cells as an appropriate model of study (Fischkoff and Rossi,
1990). A study interestecl in understanding the involvement of adherence glycoproteins
on the surface of poIymorphonucleat leukocytes (Pm) with mobilization of PMNL to
sites of inflammation ernployed the HL-60 ce11 line as a model for their investigation
(Schrnalstieg et al., 1 986). HL-60 cells have also been used to study monocytes and their
interaction with endothelial cells involving HCMV infection at sites of vascular injury
(Guetta et al., 1997). This ce11 line has also been widely used for studying the
mechanism of apoptosis (Shimura et al., 1997). As well, HL-60 cells have been used to
study the rffects of ce11 aging in vitro (Levi et al., 1997). It has been shown that prion
protein (PrPC), involved in prion diseases in humam and animals, is present on ~ ~ 3 4 +
bone marrow stem cells (Dodelet and Cashman, 1998). Induction of HL-60 cells with
retinoic acid mimics the suppression of PrP in granulocyte difkentiation and thus the
HL-60 ce11 line provides a potential model to study PrP gene regulation and protein
function (Dodelet and Cashman, 1998). Potential for differentiation dong monocytic or
granulocytic pathways bas made HL-60 cells a model system for studying HIV-1
infection in early myeloid cells (Pise et al., 1992; Cannon et al., 1993).
1.4.5) INFECTIVITY OF HL60 C E U S
HL-60 cells have been shown to be permissive for infection by a number of
in fectious agents. Human granulocytic ehrlichiosis (HGE), s recently recognized disease
close1 y related to Ehrlichia equi and Ehrlichia phagocytophiïa and îransmiîîexi by ticks,
has been shown to replicate within HL-60 cells (Bedner et al., 1998). Infection r d t e d
in growth arrest of the cells and induction of apoptosis. Thus, HL-60 cells provide a
usefùl mode1 for invatigating the reproductive cycle and cellular changes induced by this
pathogen. Measles virus (MV) has been shown to infect bone marrow progenitor cells as
well as HL-60 cells. These ceIls were used to assess the effect of maturation on MV
infection (Helin et al., 1999). A number of studies have been conducted in which HL-60
cells were infected with human immunodeficiency virus type 1 (HIV-1). These studies
investigated the effect of HIV infection on di fferentiation of myeloid progenitor wlls and
whether chronically infected cells could differentiate upon stimulation, resulting in ce11
lineaga expressing productive HIV infection (Pise et ai., 1992; Cannon et aï., 1993;
Semmel et al., 1994). A study conducted by Jerome and colleagues (1998) has show
that herpes simplex virus 1 (HSV-1) infection of HL-60 cells inhibits DNA
fragmentation, a characteristic feature of apoptosis ofien studied in HL-60 -11s.
Therefore, this ce11 line has been shown to be permissive for infection with a mernber of
the herpesvirus family.
2 . 0 ) O B J E C T I V E S
The infection of a human promyelocytic leukemia ce11 line (HL-60) with HCMV was
investigated. Untreated and dimethyl sulfoxide (DMSO) shulated HL-60 cells were
infected with HCMV. The airn of this project was to study the extent to which HCMV
replicates in these cells, which are considered as a mode1 for virus Section of P m .
Specifically, this investigation included the following aspects.
The expression of viral antigens within HCMV inoculated HL-60 cells and DMSO
stimulated HL-60 cells was examined by irnmunofluorescense assay.
The presence of the viral genome and viral mRNA transcripts within HCMV
inoculated ce11 cultures was investigated using PCR and nucleic acid sequence-based
amplification (NASBA).
HCMV inoculated HL-60 cells and DMSO stirnulated HL-60 cells were screened for
the presence of viral particles by electron microscopy/immunoelectron microswpy.
The production of infectious virus in HCMV inoculated HL-60 and DMSO
stimulated HL-60 ce11 cultures was studied.
3 . O ) M A T E R I A L S A N D M E T H O D S
3.1) Propagation of MRC-5 Cells
Human ernbryonic lung fibroblast cells (MRC-5) (Biowhittaker) were grown to
confluency in tissue culture flasks using Eagles minimal essential medium (MEM)
(B iowhittaker) supplemented with 200 rnM L-glutamine, 1 m g h l gentamich, 250 pg/ml
fungizone, I O mg/ml vancomycin and 5% fm bovine serum (FBS). Media was replaced
every 5-7 days.
3.1.1) Propagation of WFF Ce&
Human foreskin fibroblast cells (HFF) (Biowhittaker) were purchased in tissue culture
tubes as confluent monolayers. For maintenance of ce11 cultures, Eagles minimal
essential medium (MEM) (Biowhittaker) supplemented with 200 mM L-glutamine, 1
mg/ml gentamicin, 250 p g / d fungizone, 10 m g h l vancomycin and 5% fetal bovine
serum (FBS) was use& Media was replaced every 5-7 days.
3.1.2) Propagation of HL-60 Cells
Human promyelocytic leukemia ce11 line HL-60 (ATCC #CCL-240) was cultured in
Iscove's modified Dulbecco medium supplernented with 200 mM L-glutamine, 1 m g / d
gentamicin, 250 &ml fungizone, 10 m g h l vancomycin and i 0% FBS. Cultures were
split every 2-3 days malntaining ce11 densities between 2x10' and 1 x106 cells/rnl.
3.1.3) Inducrioit tif Differentiation of HL-60 Celh
HL-60 cells were induced to differentiate into more mature cells of the myeloid lineage
by stimulation of 5x1 O' celldnil with 1.5% DMSO for 6 days (Collins et al., 1978).
Maturïty was assessed by microscopie determination of nuclav differentiation. Cytospin
slide preparations of 0.1 ml of ce11 suspension were made using a Shandon Cytospin 2
centrifuge (2ûûxg, 3 minutes). The preparations were subjected to Romanowsky staining
(Sakura RSG-6 1 stainer) and slides were observed by light rnicroscopy.
3.2) Virus Propagufi'on
HCMV Davis strain (ATCC #VR-807) was propagated in confluent monolayers of MRC-
5 cells. Maintenance media was decanted fiom the monolayer, viral inoculum was
added, followed by adsorption of the virus for 1 hour at 37OC. When infected ce11
cultures showed greater than 80% cytopathic effect (4+ CPE), HCMV infected MRC-5
cells were harvested, and cell-free supernatants were frozen at -70°C. Virus was titrated
on confluent monolayers of MRC-5 cells in a 96 well microtitre piate (6 welldten fold
dilution) and concentration expressed as TCIDso.
3.2.1) Determination of Stock Viral Titre in TCIDsdml
MRC-5 cells were grown to confluency in a 96 well microtitre plate. A h z e n aiiquot of
virus stock was thawed rapidly in a 37°C water bath and 100 pl of virus suspension was
added to 900 pl of MEM, resulting in a 1 : 10 dilution of virus inoculum. Serial ten-fold
dilutions were perfonned. For each dilution of virus, 100 pl of suspension was
inoculated into 1 well of MRC-5 cells (media fiom well was aspirated prior to
inoculation). This procedure was repeated 5 times for each dilution of virus thus
resulting in a total of 6 inoculations per dilution of virus. The plate was incubated (37OC
x 1 hour), virus suspension aspirated, and 200 pl of fiesh media was added. The plate
was observed for detection of CPE at successive 24 hour tirne points until no fiuther
increase in CPE was observed. Detexmination of TCIDso was performed by the Reed and
Muench method (1 938) for quantifjing viral titre. Stock virus titre ranged between
30 000 TCiD5~ml - 40 000 TCIDsdml. One infectious unit (IU) was dehed as 0.7
TCIDso (Poisson distribution).
3.3) Inocuiation of HL60 C e k WM W C W
Unstimulated HL-60 cells or DMSO stimulated HL-60 cetls were seeded in shell vials,
inoculated at multiplicity of infection (MOI) ranging from 0.1 to 5 infectious unitslcell
(IUIcell), and centrifùged (3500xg, 15 minutes) in a DAMON DPR-6000 centrifige.
CelIs were resuspended in tiesh media (Iscove's modifieci Dulbecco medium + 5% FBS)
and incubated for 4 days at 37°C in an atmosphere of 5% CO2.
3.3.1) H C W Heat Inactivation
An aliquot of viral supernatant from the original inoculum was heated to inactivate the
virus in a water bath (56°C x 30 minutes). MRC-5 shell viais were iiioculated with the
preparation pnor to and der heat inactivation to determine if infectious virus rernained
after heating.
3.4) Determination of Virai Titre in HCMYlnocufated Cultures ofClirstimitia&d or
DMSO Stimulated HL60 Ceils
Immediately afier inoculation and centrifugation with HCMV, 1 x 1 o4 cells were removed.
The rernaining cells were resuspended in fiesh media and incubated for 4 days at 37OC in
an atrnosphere of 5% CO2. The harvested cells were niptured by vortexing with glass
beads, and the suspension was titrated in MRCd cells to determine the initial viral load
present within the inoculated culture (as described in 3.2.1) . Following the incubation
period, cells were again harvested and virus was titrated to determine the fiaal viral load.
The lower limit of detection of the assay was 10 infkctious units.
3.5) Immuno/luorescence Aswy (TFA) For Deteciibn of R C W Immediritc-Eurb,
pp65, Late Antigen
Four days post inoculation (pi.), HL-60 cells were deposited on slides using a cytospin
(as described in 3.1.2). The preparations were initially fixed for 10 minutes in phosphate
buffered saline (PBS) containhg 5% formaldehyde, 2% sucrose, washed in PBS
containing 1% FBS for 5 minutes, and exposed to PBS containing 0.5% Nonidet, 10Y0
sucrose, 1% FBS for 5 minutes to pemieabilize them, and then washed with PBS
containing 1% FBS for 5 minutes. Monoclonal antibody directed against HCMV
immediate-early (IE) antigen (Bartels, Inc.), pp65 (Biotest Clonab), or late antigen
(Bartels, Inc.) was applied to the slide which was then incubated at 37OC for 30 minutes
in a humidified chamber. Slides were washed 3x in PBS, the secondary antibody (FITC
conjugate of goat-anti-hurnan immunoglobulin with Evans Blue) was applied and the
slides were incubated at 37°C for 30 minutes in a humidified chamber. Slides were
washed, mounted and examïned with a fluorescence microscope.
3.6) HCMV Nucieic Acid Isoluîion und PCR
Total nucleic acid was isolated fiom HL-60 cells and DMSO stimulateci HL-60 cells
inoculated with HCMV, 4 days pi. using the ~ u c l i ~ e n s ~ Isolation kit. Nucleic acid was
stored at -70°C until use. For amplification of a designated HCMV genome sequence, 2
primers were used (Pl: 5'-GGA TTC GCA TGG CAT TCA CGT ATG T-3', P2: 5'-
GAA TTC AGT GGA TAA CCT GCG GCG A-3') resulting in a 406-bp DNA amplicon
whose sequence corresponded to that of the unique short region of HCMV. As a positive
control for nucleic acid extraction, amplification of a 536 bp thgrnent of the human p-
globin gene was performed using primers HBG-l: 5'-GGT TGG CCA ATC TAC TCC
CAG G-3', HBG-2: 5'-GCT CAC TCA GTG TGG CAA AG-3'. DNA amplification was
conducted using the Perkin-Elmer GeneAmpQ3 PCR Reagent Ki t Total nucleic acid was
arnplified in a 100 pl reaction volume containhg 2.5 U of AmpliTaq@ DNA Polymerase,
200 p M each of the four deoxynucleoside triphosphates and 1.0 p M of each primer
buffered with 10X PCR buffer [50 mM KCI, 10 mM Tris-HC1 (pH 8.3), 1.5 mM MgC12,
and 0.01% (wt/voI) gelatin]. Each sample was amplified in 40 cycles of 94"C, 56OC.
72"C, with a final 5 minute extension at 72OC. Amplified products were visualized by
electrophoresis on 1.5% agarose gel and ethidium bromide staining.
3.6.1) Determining the Lower Lhif of Deteclion of PCR
A previousl y titrated virai preparation was used to determine the lower limit of sensitivity
of the PCR assay. Total viral nucleic acid was extracted ( ~ u c l i ~ e n s ~ Isolation kit) fkom
200~1 of virus infected culture supematant, which had a defined number of infectious
units, fiom which 30p1 was used as template for PCR. Amplification parameters are
those describeci in 3.6. The lower limit of detection of PCR was determineci to be
between 2 12-228 infectious units, as determined fiom three separate trials.
3.7) WCMVpp67 mRNA Deîecîion
Total nucleic acid was extracted h m HCMV inoculated HL-60 cells and DMSO
stimulated HL-60 cells as described in section 3.5. Nucleic acid sequence based
amplification (NASBA) and detection of HCMV pp67 mRNA transcripts was carried out
using reagents and protocols provided by ~ u c l i ~ e n s ~ ~ . The NASBA reaction mixture is
comprised of 3 enzymes (reverse transcriptase (RT), RNase H, and T7 RNA polymerase)
and two primers. Primer 1 (Pl) wntains a 3' temiinal sequence compiementary to a
sequence on pp67 mRNA (+), and a 5' temiinal sequence of a prornoter recognized by T7
RNA pol ymerase. Primer 2 (P2) contains a sequence complementary to the P 1 -primeci
DNA strand. NASBA proceeds by binding of Pl to pp67 mRNA (+) and extension by
RT and formation of a cDNA copy. The RNA strand of the RNA:cDNA hybrid is
degraded by RNase H, followed by annealing and elongation of P2. This results in
double stranded (ds) DNA containing a transcriptionally active promoter recognized by
T7 RNA polymerase. T7 RNA polymerase produces multiple copies of RNA banscripts
which are antisense to the original target RNA sequence @RNA (-). Each newly
synthesized antisense RNA can bind P2 and act as a ternplate for synthesis of a
complimentary cDNA strand. RNase H degradation of the RNA strand in the
RNA:cDNA hybrid, followed by Pl binding and elongation results in ds DNA with a T7
RNA pol ymerase promoter. Therefore, N ASB A al10 ws for exponential synthesis of
RNA products.
To briefly describe the protocol for pp67 mRNA amplification, 5 pl of nucleic
acid was incubated with 10 pl of primer solution (6S°C x 5 minutes) and allowed to cool
(41 OC x 5 minutes). 5 pl of enzyme solution was added to the mixture and incubated
(4 1 OC x 90 minutes). Amplification of mRNA transcripts was complete and detection
was conducted as follows. 10 pl of each amplification product was mixeci with 10p1 of
detection diluent. 5 pl of diluted amplfication product was mixed with 20p1 of system
control (SC) hybridization solution and 20 pl of wild type (WT) hybridization solution.
Hybridization mixtures were incubated (41°C x 30 minutes), 300 pl o f assay buffer was
added to each mixture and detection of mRNA transcripts was wnducted using the
~ u c l i ~ e n s ~ Reader which ernploys the electrochemiluminescense (ECL) principle.
NASBA was perfomed in triplicate for HL-60 cells and DMSO stirnulated HL-60 cells
inoculated with live or heat-inactivated HCMV. Uninoculated HL-60 cells and HFF cells
were used as negative controls while HFF cells inoculated with HCMV s m e d as a
positive control for pp67 mRNA production and detection.
3.8) Efectron Microscopy (EM) Anaiysik of HCMVInoculated Cultures
EM was performed on cells infected for 4 days with HCMV. HCMV inoculated HL-60
cells and DMSO stimulated HL-60 cells were fixed in a solution of 2% glutaraldehyde in
O. 1 M PO4 b u f k (pH 7.3) for 1 hour. Preparations were washed in buffer (O. 1 M Po4 / 5
minutes) and p s t - fixed in 1 % osmium tetroxide ( 1 hour). After washing in buffer (O. 1 M
PO4 / 5 minutes), samples were dehydrateci in a graded ethanol series followed by
propylene oxide, then ernbedded in Spurr epoxy resin. Ultrathin (100 nm) sections were
placed on copper grids and stained with m y I acebte (10 minutes) and lead citrate (10
minutes). Sections were then exarnined by electron rnicroscopy.
3.8.1) Immunoelecîron Microscorn (IEM) Anahsis of HCMVlnoculeted Cultvres
Infected ce11 cultures were prepared for IEM examination 4 days pi. with HCMV. Three
separate fixation techniques were utilized for IEM. 1. Standard EM Fixation as
described in Section 3.8. A modification of this procedure was dso wployed which
excluded a pst-fixation in osmium tetroxide. This cornpouad is a strong fixative and
may affect antigenicity of viral epitopes. 2. Immuno-Fixation: Inoculated cell cultures
were fixed in a solution of 1% glutaraldehyde, 4% paraformaidehyde in a 0.1 M sodium
cacodylate buffer (pH 7.3) for 1 hour. Samples were washed in buffer, dehydrated in a
graded ethanol series and embedded in K4M Lowicryl resin under UV light at -20°C.
Ultrathin sections were placed on fonnvar coated nickel grids. This procedure was also
modified by lowering the concentration of glutaraldehyde and fixing samples in O. 1%
glutaraldehyde, 4% paraformaldehyde in a 0.1 M sodium cacodylate buffer (pH 7.3) for 1
hou. 3. Ctyosubstitution: The technique employed was modified fiom Haller et al.
( 1992). Inoculated ce11 cultures were washed in 0.1M Po4 buffer (2 x 5 minutes) and
fixed in a solution of O. 1% glutaraldehyde, 4% paraformaldehyde in a O. 1M PO4 buffer
for 2-4 hours. Cells were washed in buffer (3 x 10 minutes) and infused with 2.3M
sucrose at 4OC oveniight. Samples were subject to plunge fieezing in liquid nitrogen,
dehydrated and stained with 0.5% uranyl acetate in 100% methanol at -80°C for 48-72
hours. Samples were flushed at -80°C with 100% methanol to rernove excess uranyl
acetate. Temperature was brought up to -20°C oveniight. Methanol was exchanged with
1 : 1 HM20 Lowicry1:rnethanol oveniight at -20°C. Pure HMSO Lowicryl was introduced
to the samples and stored at -20°C for 1-2 hours. A fresh change of Lowicryl was made
and stored at -20°C for an additional 1-2 hours. UV polymerization was conducted at
-20°C and samples were processed as ultrathin sections were placed on formvar coated
nickel grids.
Imrnunolabeling of grids was conducted utilizing two separate protoculs.
1. Grids were rinsed in a solution of 0.15% glycine, 0.5% BSA in PBS (2 x 10 minutes)
then washed in a solution of 0.5% BSAPBS (4 x IO minutes). Grids were labeled with
monoclonal antibodies to the IE antigen, pp65 antigen or monoclonal aotibody 284,
which recognizes the major capsid protein (MCP) of HCMV (1 hour). The following
dilutions of antibody were tested: 1 : 1, 1 : 10, 1 :20, 1 : 1 0 . Unbound primary antibody was
removed by washing in 0.5% BSA/PBS (4x10 minutes). Grids were labeled with a 1:20
dilution of rabbit anti-mouse IgG conjugated to Arnersham 10 nm gold particles, for 1
hour. Unbound gold conjugate was removed by washing in 0.5% BSAIPBS (10
minutes), PBS (4 x 10 minutes) and in distilled water (4 x 10 minutes). Samples were
then stained with uranyl acetate (1 0 minutes) and Iead citrate (5 minutes). Monoclonal
antibody 28-4 was kindly provided by W.Britt, Birmingham, Alabama.
2. Grids were incubated for 10 minutes in 2 5 ~ 1 of a 10% solution of heat-inactivated
goat serurn in 0.1% BSA/Tris. Grids were labelled with monoclonal antibodies as
described above. Unbound primary antibody was removed by tramferring the grid
through a series of 5 droplets of phosphate buffer for 5 minutes. Grids were then labelled
with a 1 : 10 dilution of rabbit anti-mouse IgG conjugated to Biocell 20 nm gold particles,
for 1 hour. Unbound gold conjugate was removed by subsequent washes in distilled
water (5 x 2 minutes). After immunolabelling, samples were stained with uranyl acetate
(10 minutes) and lead citrate (5 minutes). Sections were then examined by electron
microscopy.
3.9) IL-6, IL-8, TNFu Sk'mulaa'on
Cytokines are produced during the effector phase of natural and specific
immunity. They are produced by a number of different ce11 types and rapidly secreted, in
order to help mediate the immune response. Cytokines IL-6, IL-8 and TNF- a a p p r to
be closely related to active HCMV infection in the host. Thus, the effect of these
cytokines on HCMV inoculation of HL-60 cells and DMSO stimulated HL-60 cells was
investigated.
HL-60 cell cultures and DMSO stimulated HL60 ce11 cultures were inoculated
with HCMV as previously described (3.3). The effect of cytokine stimulation on the
percentage of IFA positive (late antigen) cells, was determined by resuspending HCMV
inoculated cells in media containing various concentrations of cytokines IL-6, IL-8, and
TNF-a. Cytokine containing media was prepared and tested in triplicate over the
following ranges for IL-6, IL-8, and TNF-a: (IL-6: 0,O. 1,0.5, 1,5, 10 ng/ml), (IL-8: 0,
1 0, 20, 50, 75, 1 00 ng/ml) (TNF-a: 0, 0.05, 0.1, 1, 5, 1 0, 20 ng/ml). Following use of
only individual cytokines, media was prepared using a combination of the three cytokines
(IL-6: 10 ng/ml, IG8: 100 nghl , TNF- a 20 ng/ml) and (n-6: 5 ng/ml, IL-8: 50
n g h l , T N F a : 10 ng/ml), and tested over six trials. HL-60 and DMSO stimulated ce11
cultures inoculated with HCMV, resuspended in media without cytokines, served as
controls. HCMV inoculated ce11 cultures were assayed four days pi. by IFA for presence
of late viral antigen, as previously described (3.5).
4.1) Effect of HCWlnocu[an'on on 112-60 Cell Culiures
HL-60 ce11 cultures and DMSO stimulated HL-60 ce11 cultures were inoculated
with HCMV at a multiplicity of infection (MOI) of 1 infectious unit/cell. Using an
indirect immunofluorescence assay (TFA), HCMV inoculated cultures were positive for
viral antigens fkom al1 three phases of HCMV replication 4 days pi. (Table 1). Similar
levels of antigen were detected in unstimulated and DMSO stimulated HLdO cells
suggesting that differentiation may not influence HCMV antigen detection in this ce11
line.
HCMV antigens were detected by IFA in HL-60 and DMSO stimulated HL-60
ce11 cultures inoculated with heat inactivated HCMV. There was no significant
difference in the quantity of cells positive for HCMV Ag when using live or heat
inactivated virus inoculum. No viral antigens were detected in control MRC-5 cells
inoculated with heat inactivated HCMV, 4 days pi.
PCR was conducted to determine whether the viral genome was present within
inoculated cultures. A 406bp ffagment fiom the unique short (Us) region of HCMV was
amplified from HL-60 and DMSO stimulated HL-60 ce11 cultures, inoculated with live
and heat inactivated HCMV, 4 days pi . (Table 1). Fibroblast cells inoculated with live
or 56OC heat inactivated HCMV were PCR positive for viral DNA 4 days pi.
4.1.1) Dose Dependent IFA Response
Experiments were conducted to determine if the presence of virai antigen in HL-
60 ce11 cultures was directly related to the inoculating dose of HCMV. Unstimulated and
DMSO stimulated HL-60 ce11 cultures were inoculated at an MOI of O. 1, 0.3, 0.5, 1 and
5. Four days p.i., cells were tested by IFA for detection of late HCMV Ag, to determine
the percentage of IFA positive cells. As the infectious dose increased, the percentage of
LFA positive cells also increased for al1 inoculated cultures tested (Fig.2). HCMV
antigens were detected in cultures inoculated at MOI'S of 0.5 or greater- There was a
significant increase in percentage of IFA positive cells when the inoculating dose was
increased fiom an MOI of 1 to an MOI of 5.
4.1.2) Variable IFA Success
While HCMV antigen detection appeared to depend on the size of the inoculum,
detection of viral antigen by IFA was not feasible on every occasion in contrast to
infected MRC-5 cells (Fig.3). As the MOI was increased, the percentage of IFA positive
cells also increased in al1 inoculated cultures, with the highest yields achieved at an
MOI= 1.
4.2) pp6 7 mRNA Transcript Detedon
To m e r characterize the interaction between HCMV and inoculated cultures of
HL-60 cells, pp67 mRNA transcript detection was conducted ushg nucleic acid sequeme
based analysis OJASBA). Results are outlined in Table 2. Positive signals suggesting
detection of pp67 mRNA transcripts were found in both IFA positive and IFA negative
HL60 cells and DMSO stimulated HL-60 ce11 cultures, using live or heat inactivated
HCMV. Viral mRNA transfnpts were also detected in the original viral iwculum. Viral
transcripts were not detected in uninoculated HL-60 cells and DMSO stimulated HL40
cells (data not shown).
4.3) Infctious Progeny Not Produced in IFA Positive Cell Culncns
Upon detection of Wal antigens and gmetic material in HCMV i n d a t e d HL60 ce11
cultures, it was necessary to detemine whether infectious viral progeny was being
produced in these cells. For this determination, virus nom the infected cultures was
titrated. Inoculated HL-60 cells and DMSO stimulated HL-60 ceils were hawested after
centrifugation and again after a 4 day incubation period. In al1 trials, no virus could be
detected at the limit of detection of the assay afier 4 days (Table 3). No virus could be
detected in HL-60 cells and DMSO stimulated HL-60 cells inoculated with heat
inactivated HCMV.
Y0 Cell IFA (+)
O. 1 0.3 0.5 1 5 MOI
Fig.2. The effect of increasing MOI on percentage of cells F A positive for late HCMV Ag. For each determination, between 1 x 1 o3 and 1 xlo4 cells were counted and 0-4 cells were IFA positive. Data points represent the mean of six independent trials f SD at each infectious dose. MRC-5 cells inoculated at the above MOI result in -100% IFA positivity.
O. 1 0 .3 0.5 1 MOI
Fig.3. Success rate of HCMV late antigen detection within inoculated HL-60 ce11 cultures. HL-60 cells and DMSO stimulated HL-60 cells inoculated with HCMV (live or heat inactivated) were assayed for late viral antigen, 4 days p i . Inoculations were conducted over the complete range of infectious doses, for each trial. Bars represent successfbl antigen detection expressed as a percentage, based upon six independent trials. As the infectious dose was increased, the percentage of trials positive for late HCMV antigen detection by IFA also increased to a maximum at a MOI=l.
4.4) Electron Microscopy
in order to explore the possibility that viral replicative processes may have k e n
initiated but at some point were prematurely inhibited resulting in production and
assembl y of noninfectious virions, electron microscopy (EM) was çonducted to
determine if HCMV nucleocapsids could be detected inside infected HL-60 ce11 cultures
positive for HCMV antigens by iFA. The HCMV permissive human foreskin fibroblast
(HFF) cells were used as a control for HCMV replication. EM examination of HCMV
inoculated cultures showed typical characteristics of HCMV infected cells (viral
nucleocapsids in the nucleus, budding fiom the nuclear membrane, multivesicular bodies
containing viral nucleocapsids) as had been shown in HCMV infected fibroblasts and
endothelid cells (Pande et al., 1 990; Depto and Stenberg, 1 989; Sing and Ruscetti, 1 990).
HCMV virus particles were detected in inoculated HFF cells (Fig.4A-C) and particles
consistent with viral morphology within inoculated HL-60 cells (FigSA-B). These
particles were approximately 120nm in HFF cells and 150nm in HL-60 cells. No HCMV
nucleocapsids were detected in uninoculated cultures. Observations of EM analysis are
summarized in Table 4.
Table 3. Determination of viral titre in HCMV inoculated IFA positive HL-60 cells and DMSO stimulated HL-60 cells, 4 days post inoculation.
HL-60 + HCMV DMSO Stimulated HL-60 + HCMV
Initial' Titre Final' Titre Initial Titre Final Titre
121 - 1 04 -
208 - 1 04 -
178 - 222 ..
103 - 78 -
Ti tre infectious unitslml h i a l = virus titre akr HCMV adsorption (-) Below 10 IU inal al = virus titre after 4 days post inoculation
Fig.4. Electron micrograph of a HCMV inoculated HFF ce11 4 days post inoculation. (A) Presence of virus particles in the nucleus as well as a degenerated nuclear membrane. (B) Vinons budding fiom the nuclear membrane. (C) Presence of a virus particle inside a cytoplasmic multivesicular body. Arrows indicate virus particles. Q = nucleus, Q= cytoplasm.
Fig.5. Electron micrograph of a HCMV inoculated HL-60 ce11 4 days post inoculation. (A) Cytoplasmic detection of a virus particle. (B) Presence of a virus particle inside a cytoplasrnic multivesicular body. Arrows indicate virus-like particles.
4.4.1) Immunoefeciron Mlcroscopy (IEW
In order to c o n w whether particles detected within inoculated HL-60 cells were
viral nucleocapsids, imrnunoelectron rnicroscopy (IEM) was attempted. Specificaliy,
three separate sample preparation techniques, as well as modifications within the
procedwe, were attempted (Section 3.8.1 ). immunocytochemical staining was also
perfomed using two different techniques. IEM analysis was perfomed initially upon
HFF celis, those inocuiated with HCMV, as well as uninoculated, to serve as a positive
and negative control respectiveiy. The findings are sumrnarized in Table 5. Prior to
processing for IEM analysis, HFF ce11 cultures were subject to IFA analysis to test the
sensitivity of monoclonal antibodies to be used for IEM. Positive control samples stained
with each of the antibodies and negative control sarnples did not stain (data not shown).
Al1 of the fixation protocols including modifications have shown detection of virus
particles within positive controls, and a lack of detection in negative control samples
(Table 5). None of the techniques were able to yield a positive result with
imrnunostaining for detection of virus particles. Figure 6 depicts an immunoelectron
micrograph of uninoculated and HCMV inoculated HFF cells processed under the
conditions highlighed in Table 5. Figure 6A shows the presence of gold particles within
the nucleus and cytoplasm of an uninoculated HFF cell. Virions were not detected in this
cell. Figure 6B shows detection of both virus particles and gold particles within the
nucleus of a HCMV infected HFF c d , however no gold staining of virions was observed.
Similar observations were found with each of the techniques and antibodies tested.
Since, it was not possible to obtain adequate controls for immunolabelling, HCMV
inoculated cultures of HL-60 cells were not tested.
4.5) Eneeî of IL-6, LL-8, TNF- a riRL60 Cell Cultures
Cytokines IL-6, IL-8 and TNF- a pztr to be closely related to active HCMV
infection in the host. These cytokines were utilized in HCMV inoculations of HL40 and
DMSO stimulated HL-60 ce11 cultures, to observe if there were any stimulatory effects
resulting in increased numbers of [FA positive cells. Individual cytokines were tested
over a range of their observeci biological effects (IL-6: O. lng/ml - 10 ng/ml, IL-8: 10
ng/ml- 1 00 @ml, RilF-cr : 0.05 ngM - 20 ng/ml) (Chernicon International inc.). There
was no significant increase in percentage of IFA positive cells using concentrations of the
cytokines within their specified range (+/O cytokines: 0-4 positive cells / 10 00). HCMV
inoculations of HL-60 cells and DMSO stimulated HL-60 cells were conducted, and cells
were incubated in the presence of the three cytokines (IL-6: 1 O ng/rnl, IL-8: 100 ng/ml ,
T N F a : 20 ng/rnl) or (IL-6: 5 ng/ml, IL-8: 50 nglml , TNF- a : îû nghl). There was
no increase in the percentage of IFA positive cells for late HCMV Ag with either
combination of cytokines, in HL-60 cells and DMSO stimulated H L 6 0 cells, when
compared to cytokine negative controls (0-4 positve cells / 10 000).
Table 5. Summary of IEM attempts using HFF cells uninoculated and inoculated with HCMV, 4 days post inoculation
Fixation Standard Immuno Fixation Cryosubstitution Protocol EM Fixation
Virion detection pp65 Pl
PZ IE Pl
PZ Late Pl
P2 MCP Pl
P2 OSO.,: osm
(+) (-) Control Control
lm tetroxide glut : glutaldeh yde
(+) (4 Control Control
(+) Control: HCL
(+) Control
1% glut.
(-) Control: uninocu
0.1% glut.
1 1
V inoculated HFF cell Pl : labelling protocol 1 lated HFF cell P2: labelling protoc012
Fig.6. Imrnunoelectron micrograph demonstrating non-speci fic binding of conjugated gold particles to HFF cells. (A) Uninoculated HFF cell. Presence of gold particles in both the nuclear and cytoplasmic regions, with no detection of virus particles within the cell. (B) HCMV inoculated HFF cell. Detection of numerous virus particles, few gold particles and non-specific association of the two within the nucleus. Arrows indicate gold particles. a = nucleus, Q = cytoplasm.
5.O)DI S C U S S I O N
HCMV IE, pp65 and late antigen were detected in HL-60 cells and DMSO
stirnulated HL-60 cells inoculated with live and heat inactivated virus, 4 days p.i. Under
similar conditions, cultures of the known HCMV susceptible fibroblast ce11 line (MRC-
5) were 100% infected with live virus and uninfectexi with heat inactivated inoculum,
while in the HL-60 ce11 line, viral antigens were detected in very few cells using both live
and heat inactivated HCMV. To M e r investigate these findings, experiments were
conducted with the MRC-5 fibroblast ce11 line and HCMV (data not shown). In
fibroblasts inoculated with HCMV, there was detection of IE, pp65, and late Ag 1 hour
post inoculation (pi.). Considering the infectious cycle of HCMV in fibroblast cells,
virus penetration has been shown to occur 5 minutes p.i. and IE protein has been detected
as early as 20 minutes p.i. (Mocarski, 1996). The switch fiom early gene transcription to
late gene transcription has been shown to occur approximately 24-36 hours pi.
Therefore, detection of IE, pp65, and late viral antigens in HCMV inoculated fibroblasts
1 hour p.i. are probably due to antigens acquired fiom the initial virus inoculum, and not
fiom de novo synthesis. Similar findings have been reportai by 0th- investigators
(Revello et al., 1992; Grefte et ai., 1992; Revello et al., 1998). Antigens were also
detected at 24,48, 72, and 96 hours p.i. as gene hanscription and translation were taking
place within infected fibroblast cells.
Investigating antigen detection in MRC -5 cells using 56OC heat inactivated virus
inoculum, it was observed that at 1 hour p.i., al1 3 viral antigens were detected. However,
observation at 24, 48, 72, and 96 hours p.i., viral antigens could not be detected. Thus,
viral antigens present as part of the inoculum can be initially detected in fibroblasts upon
56°C heat treatment of the inoculurn. The disparity between why Wal antigeas can be
detected 1 hour p.i. using heat inactivated inoculum but not at later times may be
explained as follows. Heat treatment results in inactivation of the virus (see section
1.1.5) rendering the virus incapable of gene transcription and translation. The initial
detection of viral antigens (1 hour p.i .) is due to uptake, and localizatioa/conceatration of
specific antigens. Over tirne, the initial antigens present may disperse, become extmded
fiom the nucleus, or degraded by enzymes present within the c d , and become
undetectable. Since transcription and translation of the viral genome are not occurring,
new viral antigens are not being produced. T'us, after initial viral antigen detection at 1
hour p.i, Wal antigens are not detected in fibroblasts inoculated with heat inactivated
virus inoculum.
halogous flndings have been described with HCMV antigen detection in
polymorphonuclear leukocytes (antigenernia assay) (The et al., 1990). Delayed
processing of blood samples has shown a significant decrease in the number of HCMV
antigen positive cells detected (Gema et al., 1 992). Quantitative antigenemia has been
shown to drop between 40% to 55% when samples have been stored for 24 hours; with a
more significant decrease in samples stored at room temperature, than at 4OC (Bush and
Sluchak-Carlsen, 1998; Boeckh et al., 1994; Landry et al., 1995; Schafer et al., 1997).
While the process/mechanism(s) involved in accounting for the di fferential antigen
counts in polymorphonucIear ledcocytes is unclear, these studies suggest that HCMV
antigens may not be indefinitely stable within these cells. Thus, sirnilar processes may be
invo lved for explaining initial deteaion of HCMV antigens, followed b y their subsequent
disappearance, in fibroblast cells using heat inactivated virus inoculum.
In the case of HL-60 cells and DMSO stimulated HL-60 cells inf'ted with
HCMV, the maximum number of cells positive for viral antigens was found to occur 4
days p.i. using live or heat inactivated virus inoculum. No Wal antigen was detected
when cells were examined at 1,24, 48 or 72 hours p.i. No increase in the number of cells
containing viral antigen could be seen by incubation beyond four days. A distinct
difference was evident when comparing fibroblast cells with HL-60 cells and their
interaction with heat inactivated HCMV. HL-60 cells and DMSO stimulated HL-60 cells
were HCMV Ag positive while fibroblast cells were negative for HCMV Ag by F A 4
days pi . (Table 1). Apart fkom the fact that these are two different ce11 lines challenged
with HCMV, the observed antigen detection in HL-60 cells may be accounted for by the
possibility that the interaction between HL-60 cells and HCMV may be a longer process
than that described for fibroblast cells (Mocarski, 1996). The route of virus entry into
HL-60 cells is unclear. Receptor binding as well as phagocytosis of virus particles
remain possible routes of entry. Considering phagocytosis of virus particles, perhaps
only a small fraction of HL-60 cells are in a state in which they can actively phagocytose
these particles. Accordingly, it takes time for these few cells to acquire enough viral
antigen fiom the inoculum to produce a signal detected by FA. Specuiative and not
previously reported in the literature is the idea that the binding/phagocytic interaction
between HL-60 cells and HCMV involves very few virions at a time. Thus, the entire
process of virus uptake may require 4 days before any appreciable detection of viral
antigens can take place by IFA. Furthemore, upon virus entry, perhaps virions remah
stable within the cytoplasrn for a period of tirne. Secondary signalling systems may not
have been triggered and enzymes involved in degrading foreign particles may not have
been activated (Keeling, 1987; Newburger and Pannley, 1991; Beck, 1985). Such
signailing systems when finally triggered result in breakdown of the virus particle and
subsequent nuclear locaiization of viral antigens. These ideas are illustrated in Figure 7.
Observing HL60 cells and DMSO stimulated HL-60 cells, virai antigen detection
(IFA) in HCMV inoculated cultures did not consistently yield positive results, as was
observed with inoculated fibroblast cells (Fig.3). Since the route of virus entry is unclear,
explanation of this observation can be attempted by considering both reccptor-ligand
binding and phagocytosis, as a means of virus entry.
Consider k t a binding interaction between HCMV and HL40 cells. Perhaps
there are very few putative host cell receptors for HCMV ligands to bind on the surface
of the cell. Furthemore, these receptors may be distributed widely apart. This is of
concern if productive binding and attachment of the virion requires binding to multiple
receptor(s) thus involvhg a cascade of interactions between viral and cellular
components (Compton, 1995; White and Femer, 1994).
In accordance with the describeci postulates the following analysis of Fig.3 can be made.
There is no detection of viral antigen when inoculating HL-60 cells at an MOI of 0.1-0.3.
Since the initial interaction between the virus and host ce11 is one of chance, bringing the
virion into close vicinity with the host c d , at this low MOI, opportunity for interaction is
very limited. ïncreasing the infeftious dose to an MOI of 0.5, the probability of
interaction between virions and receptors increases, allowing for attachment and
penetration of the virus (Fig. 7A) followed by Ag detection by IFA. Our results indicate
that HCMV inoculation at an MOI. of 1 allows for optimal binding and subsequent
detection of Wal antigens, with the most wnsistency. Increasing the infectious dose and
inoculating at an MOI of 5, there is a distinct drop in fiequency of trials successfùi for Ag
detection. It is unclear why this correlation exists.
Similady, if a phagocytic interaction was involved, what may be o c c u r ~ g is that
inoculating at an MOI of 0.1-0.3, the viral load is t w low and appreciable phagocytosis,
degradation and antigen detection does not occw. Increasing the infectious dose to an
MOI of 0.5 and 1 increases the chances of this interaction occuning by virtue of a greater
number of virions in the surroundings. By increasing the infectious dose to an MOI of 5,
there is an unexplainable observai drop in frequency of trials successful for Ag detection.
An important finding fkom this set of experiments was at the best of thes,
HCMV Ag detection only appproached -80% with a range of percent positive trials fiom
-20% - -80% for al1 HL-60 ce11 cultures. Fibroblasts are a known HCMV permissive
ce11 line in vitro (Compton, 1 995; Mocarski, 1 996). Inoculation with an infectious dose of
0.1 MOI was sufficient to observe Ag detection in each trial (100% success, Fig.3).
Observing less than 100% Ag detection success with HL-60 ce11 cultures is not entirely
unexpected as HCMV has a highly restricted host range in ce11 culture (Momki , 1996).
What is worthy of investigation is that employing the same technique(s) for inoculating
cells, viral antigen detection which occurred in one trial, did not necessarily occur the
ma-
l 2 3 4
Fig.7. Proposed mode1 of interaction between HCMV and a HL-60 ceIl. (A) Receptor binding interaction between HCMV and a HL-60 d l . (B) Phagocyîic interaction between HCMV and a mature HL-60 cell. 1. HCMV virion with intact envelope binds to a receptor on HL40 d l / DMSO stimulated HL60 ceIl (A), HCMV virion is phagocytosed by a mature HL-60 ceIl (B). 2. Vims is internalized and rernains stable wiWwithout envelope. 3. Additional virions attach (A), phagocytosed (B) and penetrate into the cell. 4. Signalling pathways are activated, virions are degraded releasing viral antigens which localize in the nucleus and are detected by IFA 4 days post inoculation.
following time the experïment was conducteci. These factors as welI as other techniques
which could be used will be addresseci towards the end of this section.
Detection of HCMV DNA within PMNL has been described using DNA
hybridization techniques (Veal et al., 1996; Dankner et al., 1 990) and PCR. (Gerna et al.,
1992; Boivin et al., 1998; Zipeto et al., 1992). in this study, PCR was used to detect viral
DNA in HL-60 ce11 cultures and fibroblast cells 4 days pi., using live virus inoculurn as
well as heat inactivated virus inoculum. Heat inactivation of HCMV (section 1.2.6)
results in membrane fluidization (Aloia et al., 1988). Viral ligands implicated in early
events of HCMV infection such as binding and penetration are associated with the
envelope of the virion (Compton, 1995). Thus, heat inactivation results in loss of
integrity of the viral envelope and correspondhg ligands for host ce11 receptors.
Detection of the viral genome in fibroblast cells and HL-60 ce11 cultures inoculated with
heat inactivated virus inoculum, cannot be explained by the conventional mode1 of virus
attachent and penetration (Compton, 1995). It was necessary to determine other
possible routes of entry of HCMV.
Binding of beta-2 microglobulin (P2m) to the tegument of HCMV (Stannard,
1989) could provide an altemate route of entry into permissive cells for unenveloped
HCMV nucleocapsids. P2m has been identifie. as the light chah (12 000 MW) of class 1
HLA (Cresswell et al., 1974). Class 1 HLA is a dimeric molecule composed of a
pol ymorp hic, trammembrane a-chah (44 000 MW, heavy chain) noncovaientl y linked
with a B-chah (12 000 MW, light chain) anchored outside the plasma membrane
(Cresswell et al., 1974; Hyafil and Strominger, 1979). Heat inactivation of HCMV at
56°C resuits in fluidization of the lipid membrane envelope (Aloia et al., 1988) exposing
the nucleocapsid and associated tegument (see section 1.2.6). Synthesis of &m by cells
of the body (Gnindy et al., L 98%) as well as exogenous addition of &m in fetal bovine
s e m (Grundy et al., 1987a) allows for binding of B2m to the tegument of HCMV
vinons (Stannard, 1989). Binding of Ptm-coated nucleocapsids to class I HLA molecules
on the surface of host cells (Grundy et al., 1987b) followed by displacement of the fb
chain of HLA and association with P2m nom the f32m-coated nucleocapsid (Hyafil and
Strominger, 1979) could provide an alternate route for attachent and entry for
unenveloped HCMV parricles. While it is unclear what interaction is occurring between
HCMV and HL-60 ce11 cultures, attachent of heat treated virus rnay occur in this
manner.
Detection of viral DNA in HL-60 cells and DMSO stimulated HL-60 cells has
significant implications. Promyelocytes are normally found only in the bone marrow and
our results raise the possibility that in vivo, HCMV may penetrate into these neutrophil
precursor cells. Other reports have shown HCMV DNA detection in bone marrow cells
including C ~ 3 4 ' hematopoietic progenitor cells (Maciejewski et al., 1992; Meyer-Konig
et al., 1997). The replication cycle of CMV is slow, 48-72 hours to yield detectable levels
of viral progeny (Mocarski, 1996). Neutrophils have a defined lifespan, 4-5 days in
normal tissue, shorter in sites of infection and inflammation (Keeling, 1987). Thus, virus
may penetrate into promyelocytes and other immature cells in the bone marrow and
following fûrther maturation and release into circulation, mature HCMV DNA harbonng
cells may possibly transmit the gemme to other susceptible cells (Gmdy et al., 1998) or
upon proper stimulation, active transcription may ensue within the mature PMNL. Thus,
promyelocytes could be an important site of HCMV latency.
Upon detection of viral antigens and viral DNA withui inoçulated HL-60 cell
cultures, search for viral mRNA transcripts was conducted using NASBA. Studies have
shown detection of late viral mRNA transcripts within PMNL using hybndization
techniques (Gozlan et al., 1993) and RT-PCR (von Laer et al., 1995; Dankner et al.,
1990). Recent studies have shown that detection of HCMV mRNA using NASBA is
more sensitive than RT-PCR (Blok et al., 1998) and thus qualitative NASBA was
perfoxmed on HCMV inoculated ceIl cultures. Our results indicate that a positive signal
was obtained using NASBA suggesting that mRNA was present within inoculated HL60
cell cultures as well as in the inoculum (uninoculated ce11 cultures were NASBA
negative). Since the NASBA assay for CMV pp67 mRNA detection is a qualitative
assay, it cannot be concluded whether detection of pp67 transcripts within inoculated ce11
cultures is a result of transcription within inoculated cells. Of significant importance is
that mRNA transcripts were detected in the inoculum, prior to and fier heat inactivation
(56°C/30minutes), while the instability of mRNA has been well documented. Total
nucleic acid was extracted (Section 3.6) and used for pp67 mRNA detection. It is
unlikely that mRNA would be present in the inoculum due to tirne and temperature
effects. The stability of HCMV DNA is unknown. NASBA has been show to amplie
both DNA and RNA sequences, with accumulation of both products occuning during the
first 45 minutes. AAer 90 minutes, the arnount of RNA product exceeds DNA product
20-fold (Sooknanan et al., 1995). Therefore, what may account for the positive signal
detected in tested samples is in fact single-stranded viral DNA which has entered the
cyclic amplification process. To firrther elaborate, viral DNA may dissociate during the
amplification process (6S°C), allowing for binding of Primer 1 to single stranded DNA.
Alternatively, pieces of fiagmented viral genome may be found within the stock virus
inoculum, origïnating h m ruphued, infeaed fibroblast cells. Binding of Primer 1 and
extension by reverse transcriptase would provide a template for binding Primer 2. As
discussed in Section 3.7, synthesis and subsequent detection of pp67 transcripts would
result. While measures were taken to ensure that contamination did not occur,
nonetheless contamination of reactions with only a few molecules of nucleic acids can
lead to false positive results. Thus, positive NASBA signals may have resulted h m
carry-over or contamination during nucleic acid isolation and amplification procedures.
Studies evaluated NASBA pp67 mRNA detection according to specificity and
predictive value for onset of HCMV infection, by cornparison to other assays such as
antigenernia and RT-PCR. However, these studies were conducted with whole blood
sarnples and NASBA has not been tested on ce11 culture. Thus the NASBA procedure
may be an inappropnate diagnostic tool for investigating whether active transcription was
occurring within inoculated HL-60 ce11 cultures. Confirmation of r e d t s employing RT-
PCR or the use of radio-labeled nucleotides for detection of progeny mRNA transcripts is
necessary in determinhg the extent to which HCMV replicates in HL-60 cells.
Studies have been conducted whereby IFA positive PMNL have been shown to
transmit infectious vinis (Grundy et al., 1998; Craigen et al., 1997). In this study, we
have shown that IFA positive HL-60 cells and DMSO stimulated HL-60 cells did not
produce v ins progeny that could be transmitted to fibroblast cells. This firrther
substantiates the hypothesis that active gene transcription and viral replication does not
occur within HL-60 cells.
To determine if HCMV virions could be detected in HL-60 cells, samples positive
for late HCMV Ag were processed for EM analysis and showed detection of particles of
approximate size and similar morphology to HCMV virions, within the cytoplasm
(FigSA), and within a multivesicular body (FigSB) of HL-60 cells inoculated with
HCMV. A dense concentration of nucleocapsids and virions within the nucleus was
observed in HFF cells inoculated with HCMV (Fig.4A, 4B). This finding is consistent
with Severi et al. (1988) who showed that starting h m 3-4 àays pi., there were a
multitude of nucleocapsids, both containhg an electron-dense core and coreless, in
HCMV infected fibrobhsts. Nuclmcapsid maturation has been shown to occur within
the nucleus where the HCMV genome is packaged into preformed nucleocapsids
(Mocarski, 1996). Thus, accumulation of nucleocapsids in the nucleus of infected cells is
characteris tic of HCMV infection and virion morphogenesis.
In cornparison to HFF cells, there appears to be no detection of intranuclear
nucleocapsids in HL-60 cells inoculated with HCMV. This finding suggests that HCMV
replicative processes may not be occurring in HL-60 cells, and thus further corroborates
the hypothesis that HL40 cells are not conducive for Wal replication. EM detection of
HCMV virions in PMNL has been previously described (Martin et al., 1984). Martin and
colleagues (1984) have shown that detection of HCMV virions has been iocalized to
phagosomes. There was no detection of intranuclear virions or nucleocapsids, suggesting
HCMV was not actively replicating inside PMNL.
In order to c o n m detection of virus particles, immunoelectron microscopy
(IEM) was utlilized. IEM was initially tested on the HCMV permissive HFF ceIl line.
HFF cells infected with HCMV were assayed by IFA and dernonstrateci typical nuclear
staining using antibodies directed against E, pp65, MCP antigens. Utilizing a number of
fixation and immunolabelling combinations we were unsuccessNl in obtainuig positive
immunolabelling of infected HFF cells. Our results do provide a foundation for
development of a fixation procedure for IEM detection of HCMV in culture.
Immunoelectron microscopy has been used for diagnosing herpes virus infections
(Folkers et a[., 199 1 ; Vreeswijk et al., 1988; Fokers et al., 1992). In these studies, virus
has been isolateci h m a lesion and characterized by binding of the antibody-gold
conjugate to core and envelope antigens. There was no need to utilize fixation
procedures which may denature protein molecules (Hayat M.A, 1970). Although
atternpts were made to minimize the toxicity (remove 0s04) and concentration of
fixatives (0.1 % glutaraldehyde), antigenicity could not be maintained. Cryosubstitution
has been shown to preserve pst-embedding antigenicity (Haller et al., 1992) which
appears to be problernatic with HCMV antigens. Thus, cryosubstitution was performed
and antigenecity to vira1 epitopes was not retained.
Cytokine production in vivo has been associated with increased pathogenesis of
HCMV infection and disease (Humar et al., 1999). Attempting to stimulate HCMV
inoculated HL-60 cells and DMSO stimulated HL-60 cells with various wmbinations of
cytokines IL-6, IL-8 and TNF-a, our studies have indicated that there is no affect on the
percentage of HCMV Ag positive cells. Simulation of the in vivo condition is not
entirely possible to replicate under in vitro studies, however, testing of inoculation
conditions utilizing cytokine concentrations fonsistently reported in clinically significant
HCMV infections, allows for some insight into interactions which may be occurring.
According to the published literature, this is the first study to our knowledge
investigating the interaction between the human promyelocytic ce11 line HL60 and
HCMV. Our results suggest that HCMV does enter HL-60 cells, while the exact physical
association remains unclear. Viral antigens have been detected fiorn al1 three phases of
replication, however antigen positive ce11 cultures were not capable of transmitting
infectious virus. Viral DNA was detected within inoculated ce11 cultures but
confirmation for active gene expression codd not be made, since the Wal mRNA
detection assay (NASBA) did not provide conclusive results. Finally, EM analysis has
shown that vinidvirus-like particles were not detected at levels which would characterize
HCMV infection. Therefore, there is some support for replication of HCMV in HL-60
ce1Is provided by viral antigen and DNA detection, and support for the mntrary (no
transmission of virus, EM work). It is not possible to provide a definitive conclusion
conceming the extent to which HCMV replicates in HL-60 cells without fùrther
investigation.
Our results indicate that there appears to be some interaction between HCMV and
HL-60 cells in vitro. For further experimentation, it is necessary to obtain a greater level
of consistency using this mode1 for investigation. Thus, fbture studies should focus
initially on detmining factors to enhance antigen detection within HL-60 cells and to
obtain consistent results. Factors accounting for discrepant obsmations can be broken
d o m into three broad categones; ce11 culturing, virus harvesting, and technique. HL-60
cells are a continuously proliferathg human myeloid ce11 line predominantly consisting
of promyelocytes (Gallagher et al., 1 979) with approximately 1 0- 12% spontaneously
differentiating to more mature cells of the myeloid lineage (Collins et al., 1978).
Culturing HL-60 cells involves splitting and passaging in culture media to maintain ce11
densities between 2x 1 0' and 1 x 1 o6 cells/ml (Section 3.1.1). Therefore, a few factors
arise which may result in slight differences during HCMV inoculations. Some of these
include, the passage numbedage of cells which cm affect ce11 viability, membrane
composition, susceptibility to stress, as well as receptor interaction. A study conducteci by
Shimura and colleagues ( 1997) has shown that passaging of HL-60 cells predisposes the
ce11 population to apoptosis at room temperature. Rutter and colleagues (1W6) employed
pro ton magnetic resonance spectroscropy to show that earl y passage MRC-5 cells
compared with late passage MRC-5 cells demonstrate an increase in cholesterol and lipid
content. Yuan et al. (1 996) has shown that late passage MRC-5 cells are more susceptible
to oxidative stress than early passage cells. Thiele and colleauges (1987) first
demonstrated the influence of culture age on the susceptibility of MRC-5 cells to HCMV
infection. They found that monolayers beyond 3-1 1 days old were less susceptible to
HCMV, possibly due to older monolayers losing receptors for HCMV. Such findings
were confirmed by Fedorko et al. (1989).
The length of time cells had been in culture before medium renewal may affect
membrane properties and thus interaction with HCMV. A study by Levi et ai. (1997) has
shown that in HL-60 cells, there is decreased membrane lipid fluidity due to increase in
cholesterol concentration, upon incubation in culture medium. Another factor to consider
may be that, at the time of harvesting cells for inoculation, ce11 cultures which have
proIiferated to the upper limit of suggested cell density may behave differently than cells
which are cultureci at roughly 1 log lower ce11 density.
Virus culturing rnay also affect individual innoculation trials. HCMV was
propagated in confluent monolayers of MRCJ cells, and harvested when infected ce11
cultures showed greater than 80% CPE (4+), which was evident 4-6 days p.i (Section
3.2). Thus, virus hawested, titrated, and fiozen at different times rnay be a factor.
Finally, slight differences in technique or experimental conditions rnay affect
independent inoculation trials. For example, ce11 pelleting, resuspension, and transfer
during the inoculation procedure can provide stress upon the cells. Thus, cells in a given
tnal rnay respond differently to such situations, possibly amibutecl to their age and
cultunng specificities, as described earlier. Another factor accounting for differences
between independent trials is visualization by immunofluorescent microscopy. Since the
number of cells showing fluorescence is quite low, it is possible that an aliquot observed
rnay not contain any positive cells, or while scanning, positive cells rnay be missed.
Once this mode1 is working consistently, an important direction would be to
elucidate the route of virus entry. Is uptake of vinislvinis constituents occurring by way
of phagocytosis or is there a specific binding interaction between the virion and a host
ce11 receptor. Addressing the issue of a phagocytic phenornenon, HL-60 cells exhibit
phagocytic and chemotactic fiinctions cornmensurate with the percentage of mature cells
in the culture (Gallagher et al., 1979; Collins et al., 1978), however our studies have
indicated that there is no significant difference in IFA positivity between immature and
mature HL-60 cells. This suggests that phagocytosis rnay not be involved in at least the
early stages of myeloid ce11 differentiation. Considering viral ligand-host receptor
binding between HL-60 cells and HCMV, a number of viral envelope proteins which rnay
act as putative receptors have been studied in depth using known susceptible fibroblast
ce11 lines. Glycoprotein B (gB) is the major envelope protein and has been the focus of
most attention as a viral receptor (Mocarski, 1996). gB binds to heparin sulfate
proteoglycans (HSPG) (Compton, 1995; Compton et al., 1993) as well as annexin II
(Pietropaolo and Compton, 1997; Adlish et al., 1990) and is involved in virus
penetration, cell-to-ce11 spread, fusion of infected cells however is not involved in
attachent of the virus (Compton, 1995; Boyle and Compton, 1998; N a v m et al.,
1993). Similarily, studies involving glycoprotein H (gH) have shown that this envelope
protein binds to a 92.5 kDa receptor on fibroblast cells (Compton, 1995; Mocarski, 1996;
Keay et al., 1989; Keay and Baldwin, 1992) and it too is primarily involved in virus
penetration, and fusion of infected cells with no apparent involvement with virus
attachent (Compton, 1995; Fuller et al., 1989; Keay and Baldwin, 199 1; Keay and
Baldwin, 1995). Thus, it is still unclear which Wal receptor(s) are involved in
attachent of HCMV to fibroblast cells. Defining putative receptors involved in
attachent to HL60 cells may provide insight into virus attachent occuring in vivo.
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