Understanding the Role of Fungi in the Pathophysiology And
-
Upload
lussievareta -
Category
Documents
-
view
212 -
download
0
description
Transcript of Understanding the Role of Fungi in the Pathophysiology And
According to the Centers for Disease Control and Prevention (CDC), the
current prevalence of sinusitis in the US is up to 13.4% of the population
(29.5 million adults).1 A clear understanding of the underlying
pathophysiology and effective medical treatment options is still lacking for
chronic rhinosinusitis (CRS). Patients with CRS suffer from various
symptoms, including nasal congestion, copious mucus production, cough,
and acute exacerbations with facial pain, the latter presumably secondary to
bacterial infections.2 Furthermore, the socioeconomic burden of CRS is
profound considering that the current estimated number of office-based
visits is 12.5 million a year.1 Given the great impact of CRS, it is imperative
to better understand the mechanisms underlying the disease process so that
appropriate treatment can be employed.
Bacterial Infections in Chronic Rhinosinusitis
The fact that patients with CRS experience recurrent bacterial exacerbations
fueled the notion that the disease process is a bacterial infection resistant to
antibiotic intervention and surgery. However, bacterial infections always
produce a neutrophilic inflammation and, in contrast, the chronic
inflammation in CRS is predominantly mediated by eosinophils.3–6 Thus,
bacterial infections likely play a role in the acute exacerbations—when
neutrophils can also be identified—but it is unlikely that they induce the
underlying inflammatory process.
It Is All About Eosinophils
Eosinophils are typically associated with immunoglobulin E (IgE)-mediated
allergies. They are recruited into affected tissue by cytokines and
chemokines during the late phase of the allergic reaction. However, in CRS
only 40–60% of patients have a positive allergy test, and the eosinophilic
inflammation is present with the same intensity in those patients who have
detectable IgE-mediated allergies as in those that do not.7 Also patients with
IgE-mediated allergies develop a different symptom complex from those
with CRS, including sneezing and itching eyes. In other words, patients with
CRS who have elevated IgE levels may have allergic rhinitis as a comorbid
disease, but the mechanism behind the eosinophilic inflammation appears
to be independent of the IgE-mediated allergic response.
However, previous studies found that eosinophils were not present in all CRS
cases. This could have been due to the use of systemic corticosteroids prior
to surgery when tissue samples are collected. Steroids are potent inhibitors
of eosinophilic inflammation and can lead to the lack of eosinophils found in
these samples. In addition, the eosinophilic inflammation is extremely
heterogenous. It is absent in large areas of harvested specimen, only to be
present in abundance in other areas only millimeters away.8 Thus, by avoiding
pre-operative steroids and by sampling larger or multiple sites one can
appreciate better the eosinophilic nature of the inflammation in CRS.
Cytokines and Immunoglobulins
As explained above, CRS cannot be adequately explained by theories of IgE-
mediated hypersensitivity, nor is there sufficient evidence to show that CRS
is due to bacterial inflammation. However, a prominent IgE independent
and cytokine-driven eosinophilic inflammation has been found in these
patients. Interleukin (IL)-5, IL-13 (both TH-2), and interferon (IFN)-gamma
(TH-1) activate and prolong the lifespan of eosinophils, induce their
migration from the vasculature to sinus tissue, and enhance their destructive
ability, respectively.9–11 All of these cytokines are present in CRS tissue and
absent in healthy controls.12 In order to determine the causative agent
responsible for inciting the eosinophilic inflammation, one has to find the
trigger for the cytokine production.
An Immune Response to Fungi
To study whether certain fungi could incite the production of cytokines,
peripheral blood mononuclear cells (PBMCs) from CRS patients—which
a report by
Niv Mor , BA ,1 David A Sherr is , MD ,1 Hirohito Kita , MD 2 and Jens U Ponikau , MD 1
1. Department of Otorhinolaryngology, The State University of New York at Buffalo;
2. Department of Internal Medicine, Division of Allergic Diseases, Mayo Clinic Rochester, Minnesota
Understanding the Role of Fungi in the Pathophysiology and Treatment of Chronic Rhinosinusitis
68 © T O U C H B R I E F I N G S 2 0 0 7
Allergic Disorders
Niv Mor, BA, is a third-year medical student at The State University of New York at Buffalo, wherehe is pursuing an interest in otolaryngology. He graduated from Phi Beta Kappa with a BA inmathematics from Brandeis University in Waltham, Massachusetts. He studied the role of extra-cellular matrix proteins in smooth muscle relaxation of the lower esophageal sphincter at theHospital for Special Surgery in New York.
David A Sherris, MD, is Professor and Chair of the Department of Otolaryngology at The StateUniversity of New York at Buffalo, the Chief of Otolaryngology Services for the Kaleida HealthSystem, and a Director of the Gromo Institute and Sinus Center in Buffalo, New York. Hespecializes in rhinology and facial plastic surgery. He graduated from the University of Rochester School of Medicine in Rochester, New York, followed by a otolaryngology residency atthe University of Rochester Medical Center in Rochester. After a facial plastic fellowship at theUniversity of Washington in Seattle, he joined the Mayo Clinic Faculty in Rochester, Minnesota,and became the Chair of Facial Plastic Surgery.
Hirohito Kita, MD, is a Professor of Immunology and Medicine and the Director of the AllergicDisease Research Laboratory at the Mayo Clinic and Mayo College of Medicine. He specializes ineosinophil cell biology and airway inflammation. He graduated from the Mie University School ofMedicine, Japan. After a residency in pediatrics and a fellowship in pediatric allergy, he became apost-doctoral research fellow at the Mayo Clinic in Rochester, Minnesota.
Jens U Ponikau, MD is an Assistant Professor and Chair of Otolaryngology Research at The StateUniversity of New York at Buffalo, and a Director of the Gromo Institute and Sinus Center. After aresidency in otolaryngology he became a research fellow in otolaryngology and joined the MayoClinic faculty in Rochester, Minnesota. His research focuses on the role of fungi in the upper(chronic rhinosinusitis) and lower airways (asthma). He graduated from Medical School inHamburg, Germany.
Sherris_edit.qxp 10/10/07 15:22 Page 68
Understanding the Role of Fungi in the Pathophysiology and Treatment of Chronic Rhinosinusitis
contain lymphocytes and antigen-presenting cells—were exposed to
different fungal antigens from species commonly found in the nasal and
paranasal sinuses. Ninety percent of CRS patients produced large amounts
of IL-5 in response to one particular fungus called Alternaria, while all CRS
patients produced large amounts of IL-13. This immune reaction was
specific for CRS since healthy controls did not react. In addition, IFN-gamma
(TH-1) production in response to Alternaria species was significantly
elevated compared with healthy controls.12
To further clarify the nature of the immune response to the fungi, serum
IgE and IgG antibodies to Alternaria were analyzed relative to the amount
of IL-5 production that was induced by Alternaria. Alternaria IgE levels did
not correlate with the level of IL-5 produced, confirming that this immune
response occurred independently of an IgE-mediated mechanism.
However, specific Alternaria IgG levels were strongly correlated with the
level of IL-5 produced. The production of IgG was significantly increased
in CRS patients compared with healthy controls, and the response was
independent of whether or not allergy was detected in the serum.13 Since
IgG levels are an indicator of the degree of exposure to antigen, this
indirectly suggests that the severity of this immune reaction is correlated
with exposure to Alternaria.
Thus, CRS patients, but not healthy controls, produce the cytokines
necessary to induce the eosinophilic inflammation, and certain
fungi—specifically Alternaria—trigger this reaction. Furthermore, this
pattern of cytokine production challenges the previously held belief that a
strictly inverse relationship exists between TH1 and TH2 immune responses.
Where Are the Fungi?
Given the role of fungal antigens in inciting this immune response in CRS
patients, previous studies that found negative fungal cultures and
negative fungal histopathology must be looked at in perspective.
Improved methods, such as collecting as much mucus as possible from
each specimen and removing the fungi from the mucus before incubating
the sample for culture, have increased the sensitivity of these tests.14
Results from these improved methods showed cultures positive for fungi
in 96% of patients with CRS; the healthy control group was also positive
for fungi (96–100%).14,15 These findings demonstrate that fungi are
present in the nasal and sinus cavity of all humans; however, only patients
with CRS seem to respond to them with cytokine production leading to
an eosinophilic inflammation.
In addition, the varying negative results from previous pathology
studies were due to the limitations of the Gomori methenamine silver
(GMS) stain used in these studies.16 GMS relies on the polysaccharide
present in the fungal cell wall to visualize the organism; however, the
fungi in CRS are present in the mucin, which is known to contain a
high concentration of polysaccharides. Chitin, a monosaccharide
derivative of glucose that is present in the cell wall of most fungi and not
present in mammals, appears to have greater sensitivity and specificity
for detecting fungal organisms (see Figure 1).17 More specifically, the
presence of Alternaria was further elucidated by more sensitive
Alternaria-specific methods such as polymerase chain reaction (PCR) or
antigen detection. These methods verified the presence of Alternaria
DNA or antigens in 100% of CRS patients.18 This is not surprising given
the fact that Alternaria alternata-specific antigens are found in 95–99%
of US households.19
What Do Eosinophils Do in the Sinuses?
Eosinophils were thought to infiltrate the sinus tissue of CRS patients and
accumulate below the basal membrane. Recently, however, it was
appreciated that they are actually only traveling through the tissue and
towards the mucus. On the luminal side of the epithelium the eosinophils
form in cell clusters, where they degranulate.20 Eosinophils in the tissue
were found largely intact. In contrast, the eosinophils in the luminal
clusters were releasing their cytotoxic eosinophilic protein, including
major basic protein (MBP), in concentrations that appear to far exceed
those necessary to induce epithelial damage (see Figure 2).20 Importantly,
69U S R E S P I R A T O R Y D I S E A S E 2 0 0 7
Figure 1: A Method to Identify Eosinophils in the Mucus
Panel A shows mucus from a patient with chronic rhinosinusitis that has been stained withGomori methenamine silver (GMS). No fungi can be visualized. Panel B is a serial section ofpanel A, which is stained with a fungal-specific chitinase immunofluorescence stain. Thepresence of the fungal elements (white arrows) can clearly be visualized in the mucus. The redarrows depict clusters of eosinophils in the mucus. Note that the fungal elements are localizedin the center of the eosinophilic clusters (GMS and Chitinase immunofluorescence staining;original magnification x400).
A. B.
Figure 2: Eosinophils Clusters in the Mucus
Panel A is a photomicrograph of mucus that is attached to tissue from a patient with chronicrhinosinusitis. The eosinophils form clusters (black arrows) in the mucus, and the epitheliumis eroded from the luminal side (yellow arrows). Panel B is a serial section of panel A. Theeosinophilic clusters are the site of diffuse major basic protein (MBP) release (white arrows).In contrast, the MBP in the tissue is confined within the intact eosinophils (red arrows). Thissuggests that the eosinophils in the tissue are in transit towards their final target in themucus. (Hematoxylin-and-Eosin and MBP immunofluorescence staining; originalmagnification x400).
A. B.
Given the great impact of chronic
rhinosinusitis, it is imperative to better
understand the mechanisms underlying
the disease process so that appropriate
treatment can be employed.
Sherris_edit.qxp 16/10/07 10:47 Page 69
the epithelial damage is inflicted from the luminal side since the upper
layers of the epithelium are affected from the outside.8
Serial sections of the mucus using chitinase stains showed fungal
elements clearly localized in the center of crowded eosinophils. The
eosinophils form in clusters and surround the fungi in the same way
that they target and attack parasites (see Figure 1, red arrows and
Figure 3).17
The damage to the epithelium is explained by the high concentration
of eosinophilic toxic proteins, and the injured tissue presumably provides
an entry port for colonizing bacteria to invade the nasal mucosa of the
sinus cavity.5,8,21–25 In healthy control subjects, the absence of cytotoxic
MBP in the mucus explains the lack of epithelial damage and,
consequently, the lack of bacterial infections despite the presence of
bacteria. All of these findings explain why antibiotics, IgE-mediated
immunotherapy, and antihistamines have limited effectiveness as long-
term treatments of CRS.26,27
Does Alternaria Cause Eosinophils to Degranulate and
Release Its Toxic Proteins?
The observations that eosinophils target fungi in the mucus
and that the fungi are the sites of degranulation sparked further
investigations as to whether the fungi are actually causing the
eosinophils to degranulate. When seven fungi were tested (Aspergillus,
Penicillium, Alternaria, Candida, Curvularia, Bipolaris, and Cladosporium
spp.), only Alternaria and Penicillium induced a calcium-dependant
degranulation of eosinophils. In addition, only Alternaria caused
activation of eosinophils, as measured by the expression of CD63 and
CD11b, the production of IL-8, and an increase in intracellular calcium by
eosinophils. The specific activity was centered on an Alternaria antigen
with a molecular weight of 61kDa that is heat-labile and works through
a G-protein coupled receptor. In summary, Alternaria, but not other
fungi, caused activation and degranulation of eosinophils, whereas
neutrophils did not respond.13
Treatment of Chronic Rhinosinusitis
Glucocorticoids reduce inflammatory cytokines, inhibit the production,
migration and activation of eosinophils, and ultimately lead to eosinophilic
apoptosis.28 However, the administration of systemic glucocorticoids does
not address the trigger for the inflammation, and provides only temporary
relief with numerous undesirable side effects.29,30 Topical steroid sprays are
limited by constraints on delivery into occluded nasal and paranasal cavities.
However, they recently demonstrated efficacy in patients with nasal polyps,
where they reduced the nasal obstruction and the size of the polyps in two
independent trials.31,32
Another therapeutic target would be to reduce the antigenic stimulus in the
sinus and nasal cavities that activates the inflammatory response. Due to the
extra-mucosal, non-invasive nature of the fungi, systemic antifungals would
have to be given at a high dose in order to reach effective concentrations in
the mucus. In view of the potential risk of renal, liver, and cardiac toxicity of
a long-term treatment, more safety and efficacy data are necessary to justify
broad use of systemic antifungals.
Recent studies have tested the effects of direct administration of
antifungal agents. Intranasal treatment with amphotericin B reduced
both the inflammatory mucosal thickening (as seen by computed
tomography (CT) scan) and improved the disease stage (as seen by
Allergic Disorders
70 U S R E S P I R A T O R Y D I S E A S E 2 0 0 7
Figure 3: Eosinophils Forming Around a Fungal Element
The above image is an transmission electron micrograph of eosinophils (black arrows) formingaround a fungal element (red arrow). Note the intimate relationship of the eosinophils engulfingthe fungi in preparation to release their toxic major basic protein (yellow arrow).
Figure 4: Before and After Treatment of Chronic Rhinosinusitis
Coronal computed tomography scans of a patient with chronic rhinosinusitis before and after treatment with intranasal amphotericin B. Panel A shows inflammatory mucosalthickening partially occluding the nasal cavity and the paranasal sinuses, which is significantly reduced in panel B after intranasal treatment with amphotericin B. The challengeof intranasal delivery is further illustrated by residual disease in the hard to reach upperethmoid sinuses (*).
A. B.
The administration of systemic
glucocorticoids does not address the
trigger for the inflammation, and
provides only temporary relief with
numerous undesirable side effects.
Sherris_edit.qxp 16/10/07 10:50 Page 70
endoscopy) (see Figure 4).26,27 In addition, patients receiving amphotericin
B had reduced levels of eosinophil-derived neurotoxin (EDN)—an
intranasal marker of eosinophilic inflammation—compared with patients
in the placebo group. Interestingly, the daily intranasal administration of
the placebo solution, containing colored sterile water, worsened the
inflammation when seen by CT scans, did not improve the endoscopic
staging, and increased levels of EDN.26 These results suggest that the
therapeutic benefit of an intranasal amphotericin B solution is not due to
the mechanical cleansing of the paranasal sinuses, but due to a
therapeutic effect of the amphotericin B.
Nasal polyposis is considered to be the late and ultimate stage of CRS
that presents with edematous swellings in the sinus tissue. Treatment
with nasal lavage of amphotericin B resulted in the total disappearance
of the polypoid mucosa.33,34 The efficacy of antifungal treatment on nasal
polyps was related to the severity of disease: 62% of patients in stage I
polyposis and 42% of patients in stage II were cured using this
treatment. None of the patients in stage III were cured, presumably due
to reduced delivery of the topical medication in severely obstructed
patients.33,34 Previously, lysine acetylsalicylate (LAS) was used to treat nasal
polyps due to its antiproliferative effects on fibroblasts and its ability to
reduce the recurrence of nasal polyps more effectively than surgery.
However, when patients were treated with a combination therapy of
intranasal LAS and amphotericin B, they had significantly lower rates of
recurrence of nasal polyps than those patients who were treated with
LAS alone.35
Conclusion
Alternaria, a ubiquitous airborne mold, is present in CRS patients
as well as in healthy controls. However, only the immune cells of CRS
patients react to the presence of Alternaria with the production of
cytokines crucial for inciting the eosinophilic inflammation seen in this
disease. This cytokine-driven immune response occurs independent of IgE
production. In addition, Alternaria induces the degranulation of
eosinophils and the release of their toxic protein. In CRS patients one can
appreciate the migration, cluster formation, degranulation, and targeting
of extramucosal fungi by the eosinophils. Antifungal therapy shows
promise in reducing the trigger for the eosinophilic inflammation, and
consequently the inflammatory mucosal thickening. However, drug
delivery, dosage, and frequency and duration of treatment must be
further optimized. ■
1. Pleis JR, Lethbridge-Çejku M, Summary health statistics for U.S.adults: National health interview survey, 2005, National Center forHealth Statistics, Vital Health Stat 10, 2006;232:1–153.
2. Bishai WR, Issues in the management of bacterial sinusitis,Otolaryngol Head Neck Surg, 2002;127:S3–S9.
3. Bryson JM, Tasca RA, Rowe-Jones JM, Local and systemiceosinophilia in patients undergoing endoscopic sinus surgery forchronic rhinosinusitis with and without polyposis, Clin Otolaryngol,2003;28:55–8.
4. Bhattacharyya N, Chronic rhinosinusitis: is the nose reallyinvolved?, Am J Rhinol, 2001;15:169–73.
5. Harlin SL, Ansel DG, Lane SR, et al., A clinical and pathologic studyof chronic sinusitis: the role of the eosinophil, J Allergy ClinImmunol, 1988;81:867–75.
6. Stoop AE, van der Heijden HA, Biewenga J, van der Baan S,Eosinophils in nasal polyps and nasal mucosa: animmunohistochemical study, J Allergy Clin Immunol,1993;91:616–22.
7. Collins M, Nair S, Smith W, et al., Role of local immunoglobulin Eproduction in the pathophysiology of noninvasive fungal sinusitis,Laryngoscope, 2004;114:1242–6.
8. Ponikau JU, Sherris DA, Kephart GM, et al., Features of airwayremodeling and eosinophilic inflammation in chronicrhinosinusitis: is the histopathology similar to asthma?, J AllergyClin Immunol, 2003;112:877–82.
9. Hamilos DL, Leung DY, Wood R, et al., Evidence for distinctcytokine expression in allergic versus nonallergic chronic sinusitis,J Allergy Clin Immunol, 1995;96:537–44.
10. Hamilos DL, Leung DY, Wood R, et al., Eosinophil infiltration innonallergic chronic hyperplastic sinusitis with nasal polyposis(CHS/NP) is associated with endothelial VCAM-1 upregulation andexpression of TNF-alpha, Am J Respir Cell Mol Biol, 1996;15:443–50.
11. Sanchez-Segura A, Brieva JA, Rodriguez C, T lymphocytes thatinfiltrate nasal polyps have a specialized phenotype and produce amixed TH1/TH2 pattern of cytokines, J Allergy Clin Immunol,1998;102:953–60.
12. Shin SH, Ponikau JU, Sherris DA, et al., Chronic rhinosinusitis: anenhanced immune response to ubiquitous airborne fungi, J AllergyClin Immunol, 2004;114:1369–75.
13. Inoue Y, Matsuwaki Y, Shin SH, et al., Nonpathogenic,environmental fungi induce activation and degranulation ofhuman eosinophils, J Immunol, 2005;175:5439–47.
14. Ponikau JU, Sherris DA, Kern EB, et al., The diagnosis andincidence of allergic fungal sinusitis, Mayo Clin Proc, 1999;74:877–84.
15. Braun H, Buzina W, Freudenschuss K, et al., Eosinophilic fungalrhinosinusitis: a common disorder in Europe?, Laryngoscope,2003;113:264–9.
16. Allphin A, Strauss M, Abdul-Karim F, Allergic fungal sinusitis:problems in diagnosis and treatment, Laryngoscope, 1991;101:815–20.
17. Taylor MJ, Ponikau JU, Sherris DA, et al., Detection of fungalorganisms in eosinophilic mucin using a fluorescein-labeled chitin-specific binding protein, Otolaryngol Head Neck Surg, 2002;127:377–83.
18. Gosepath J, Brieger J, Vlachtsis K, Mann W, Fungal DNA is presentin tissue specimens of patients with chronic rhinosinusitis, Am JRhinol, 2004;18:9–13.
19. Salo PM,Yin M, Arbes SJ Jr, et al., Dustborne Alternaria alternataantigens in US homes: results from the National Survey of Leadand Allergens in Housing, J Allergy Clin Immunol, 2005;116:620–22.
20. Ponikau JU, Sherris DA, Kephart GM, et al., Striking deposition oftoxic eosinophil major basic protein in mucus: implications forchronic rhinosinusitis, J Allergy Clin Immunol, 2005;116:362–9.
21. Abu-Ghazaleh RI, Dunnette SL, Loegering DA, et al., Eosinophilgranule proteins in peripheral blood granulocytes, J Leukoc Biol,1992;52:611–18.
22. Gleich GJ, Adolphson CR, Leiferman KM, The biology of theeosinophilic leukocyte, Annu Rev Med, 1993;44:85–101.
23. Fujisawa T, Kephart GM, Gray BH, Gleich GJ, The neutrophil andchronic allergic inflammation: immunochemical localization ofneutrophil elastase, Am Rev Respir Dis, 1990;141:689–97.
24. Frigas E, Loegering DA, Gleich GJ, Cytotoxic effects of the guineapig eosinophil major basic protein on tracheal epithelium, LabInvest, 1980;42:35–43.
25. Hisamatsu K, Ganbo T, Nakazawa T, et al., Cytotoxicity of humaneosinophil granule major basic protein to human nasal sinusmucosa in vitro, J Allergy Clin Immunol, 1990;86:52–63.
26. Ponikau JU, Sherris DA, Weaver A, Kita H, Treatment of chronicrhinosinusitis with intranasal amphotericin B: a randomized,placebo-controlled, double-blind pilot trial, J Allergy Clin Immunol,2005;115:125–31.
27. Ponikau JU, Sherris DA, Kita H, Kern EB, Intranasal antifungaltreatment in 51 patients with chronic rhinosinusitis, J Allergy ClinImmunol, 2002;110:862–6.
28. Schleimer RP, Bochner BS, The effects of glucocorticosteroids onhuman eosinophils, J Allergy Clin Immunol, 1994;94:1202–13.
29. Kondo H, Nachtigal D, Frenkiel S, et al., Effect of steroids on nasalinflammatory cells and cytokine profile, Laryngoscope, 1999;109:91–7.
30. Friedman WH, Katsantonis GP, Bumpous JM, Staging of chronichyperplastic rhinosinusitis: treatment strategies, Otolaryngol HeadNeck Surg, 1995;112:210–14.
31. Stjarne P, Mosges R, Jorissen M, et al., A randomized controlledtrial of mometasone furoate nasal spray for the treatment of nasalpolyposis, Arch Otolaryngol Head Neck Surg, 2006;132:179–85.
32. Small CB, Hernandez J, Reyes A, et al., Efficacy and safety ofmometasone furoate nasal spray in nasal polyposis, J Allergy ClinImmunol, 2005;116:1275–81.
33. Ricchetti A, Landis BN, Maffioli A, et al., Effect of anti-fungal nasallavage with amphotericin B on nasal polyposis, J Laryngol Otol,2002;116:261–3.
34. Ricchetti A, Landis BN, Giger R, et al., Effect of local antifungaltreatment on nasal polyposis, Otorhinolaryngol Nova,2002–2003;12:48–51
35. Corradini C, Del Ninno M, Buonomo A, et al., Amphotericin B andlysine acetylsalicylate in the combined treatment of nasalpolyposis associated with mycotic infection, J Investig Allergol ClinImmunol, 2006;16:188–93.
Antifungal therapy shows promise
in reducing the trigger for the
eosinophilic inflammation, and
consequently the inflammatory
mucosal thickening.
Understanding the Role of Fungi in the Pathophysiology and Treatment of Chronic Rhinosinusitis
71U S R E S P I R A T O R Y D I S E A S E 2 0 0 7
Sherris_edit.qxp 10/10/07 15:24 Page 71