Document Detail

Aberrant cytokine pattern of the nasal mucosa in granulomatosis with polyangiitis.
Jump to Full Text
MedLine Citation:
PMID:  23031229     Owner:  NLM     Status:  Publisher    
Abstract/OtherAbstract:
ABSTRACT: INTRODUCTION: In granulomatosis with polyangiitis (GPA), a complex autoimmune small-vessel vasculitis frequently associated with chronic necrotizing inflammation of the nasal mucosa, elevated nasal Staphylococcus (S.) aureus carrier rates are a risk factor for relapse. As cytokines are primarily involved in the regulation of defense against potentially pathogenic microorganisms, the aim of this study was to compare healthy individuals and GPA patients with respect to their baseline cytokine expression of nasal epithelial cells (NEC), which form the first barrier against such triggers. The ability of S. aureus to influence the nasal microenvironment's cytokine secretion was assessed by exemplary stimulation experiments. METHODS: Baseline expression of 19 cytokines of primary NEC of GPA patients and normal controls (NC) was quantified by a multiplex cytokine assay. Stimulation experiments were performed with supernatants of S. aureus and expression of interleukin-8 was determined by ELISA. RESULTS: In GPA, an altered pattern of baseline cytokine expression with significantly up-regulated G-CSF and reduced interleukin (IL)-8 concentrations was observed. Both NEC of GPA patients and NC responded to stimulation with S. aureus, but GPA patients displayed a significantly lower IL-8 secretion and a diminished dynamic range of response towards the stimulus. CONCLUSIONS: The data presented underline the hypothesis of a disturbed epithelial nasal barrier function in GPA. The dysregulated baseline expression of G-CSF and IL-8 and the reduced response to microbial stimulation may facilitate changes in the composition of the nasal flora and favour an imbalanced inflammatory response, which might be relevant for the disease course.
Authors:
Janet Wohlers; Katrin Breucker; Rainer Podschun; Jurgen Hedderich; Peter Lamprecht; Petra Ambrosch; Martin Laudien
Related Documents :
18535199 - Inflammation-associated lysophospholipids as ligands for cd1d-restricted t cells in hum...
10200559 - Jnk/sapk activity contributes to trail-induced apoptosis.
24633619 - Cytokine profiles in allergic rhinitis.
15326479 - Death receptor signaling regulatory function for telomerase: htert abolishes trail-indu...
15572759 - Death receptor regulation and celecoxib-induced apoptosis in human lung cancer cells.
10218979 - Demonstration of functional oxytocin receptors in human breast hs578t cells and their u...
6305049 - Immunosuppressive effect of entamoeba histolytica extract on hamsters.
21496499 - Tetrandrine suppresses amyloid-β-induced inflammatory cytokines by inhibiting nf-κb p...
14618099 - Characterization of three human oligodendroglial cell lines as a model to study oligode...
Publication Detail:
Type:  JOURNAL ARTICLE     Date:  2012-10-2
Journal Detail:
Title:  Arthritis research & therapy     Volume:  14     ISSN:  1478-6362     ISO Abbreviation:  Arthritis Res. Ther.     Publication Date:  2012 Oct 
Date Detail:
Created Date:  2012-10-3     Completed Date:  -     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  101154438     Medline TA:  Arthritis Res Ther     Country:  -    
Other Details:
Languages:  ENG     Pagination:  R203     Citation Subset:  -    
Export Citation:
APA/MLA Format     Download EndNote     Download BibTex
MeSH Terms
Descriptor/Qualifier:

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine

Full Text
Journal Information
Journal ID (nlm-ta): Arthritis Res Ther
Journal ID (iso-abbrev): Arthritis Res. Ther
ISSN: 1478-6354
ISSN: 1478-6362
Publisher: BioMed Central
Article Information
Download PDF
Copyright ©2012 Wohlers et al.; licensee BioMed Central Ltd.
open-access:
Received Day: 7 Month: 12 Year: 2011
Revision Received Day: 11 Month: 6 Year: 2012
Accepted Day: 16 Month: 8 Year: 2012
Print publication date: Year: 2012
Electronic publication date: Day: 17 Month: 10 Year: 2012
Volume: 14 Issue: 5
First Page: R203 Last Page: R203
PubMed Id: 23031229
ID: 3580515
Publisher Id: ar4041
DOI: 10.1186/ar4041

Aberrant cytokine pattern of the nasal mucosa in granulomatosis with polyangiitis
Janet Wohlers1 Email: janet.wohlers@gmx.de
Katrin Breucker1 Email: kati.breucker@t-online.de
Rainer Podschun2 Email: podschun@infmed.uni-kiel.de
Jürgen Hedderich3 Email: hedderich@medinfo.uni-kiel.de
Peter Lamprecht4 Email: peter.lamprecht@uk-sh.de
Petra Ambrosch1 Email: ambrosch@hno.uni-kiel.de
Martin Laudien1 Email: laudien@hno.uni-kiel.de
1University of Kiel, Department of Otorhinolaryngology, Head and Neck Surgery, Arnold-Heller-Straße 14, D-24105 Kiel, Germany
2Institute for Infection Medicine, University Hospital Schleswig-Holstein, Campus Kiel, Brunswiker Straße 4, D-24105 Kiel, Germany
3University of Kiel, Department of Medical Informatics and Statistics, Brunswiker Straße 10, D-24105 Kiel, Germany
4University of Lübeck, Department of Rheumatology, University Hospital of Schleswig-Holstein, Campus Lübeck, and Rheumaklinik Bad Bramstedt, Ratzeburger Allee 160, D-23538 Lübeck, Germany

Introduction

Granulomatosis with polyangiitis (GPA) is characterized by chronic necrotizing granulomatous inflammation with a predilection of the upper and lower respiratory tract and proteinase 3 (PR3) specific cytoplasmic anti-neutrophil cytoplasmatic antibodies (C-ANCA) [1,2].

So far, the pathogenetic mechanisms resulting in inflammation and autoimmunity in GPA are poorly elucidated. A complex interaction between genetic susceptibility and environmental factors is discussed [3-6], whereas low familiar clustering [7] stresses the importance of the latter.

Epidemiological studies revealed higher Staphylococcus (S.) aureus colonisation rates in GPA-patients compared to healthy and diseased controls [8-10]. Moreover, nasal carriage was shown to be associated with significantly elevated relapse rates, endonasal disease activity and first manifestation in the upper respiratory tract [8,11,12]. In addition, treatment with trimethoprim/sulfamethoxazole reduces the annual number of respiratory infections and the incidence of relapses in GPA-patients in remission [10,13,14].

Various staphylococcal superantigens were demonstrated to exert strong stimulatory effects on immunocompetent cells [15]. Furthermore, T-cells were shown to exhibit irregular modes of differentiation and cytokine expression upon stimulation with S. aureus [16,17], and S. aureus specificity was found in T-cell clones generated from peripheral blood lymphocytes of GPA-patients [17].

Remarkably, recent findings suggest a dysbalanced microbiom of the nasal cavity rather than a distinct microbial trigger comparable to the dysbiosis in inflammatory bowel diseases like Morbus Crohn [18]. Kain et al. demonstrated that in ANCA-associated systemic vasculitides highly prevalent auto-antibodies to lysosomal membrane protein-2 can cross-react with adhesins of gram-negative pathogens [19].

Another hint at microbial triggering of GPA is given by Pendergraft et al. demonstrating that patients harbouring PR3-ANCA also produce auto-antibodies to complementary PR3 (cPR3), a peptide translated from the antisense DNA strand of PR3. Conversely, the presence of cPR3 leads to production of both anti-cPR3 and anti-PR3 auto-antibodies. Genetic sequences that could be translated to cPR3-peptides were identified in numerous microbial and fungal organisms, including S. aureus, Entamoeba histolytica and Ross-River virus [20].

Taken together, the evidence is increasingly pointing to an imbalanced inflammatory response to microbial challenge as a potentially relevant process in the pathogenesis of GPA or vice versa.

Cytokines are significantly involved in the regulation of immune and inflammatory processes [21]. They enable inter-cellular communication and initiate immune responses by recruiting and activating specific immune cells, thus playing a decisive role in successful local defense against microorganisms [22]. Although aberrant cytokine patterns in GPA-patients in serum or plasma [23-25], mononuclear cells [26,27], pulmonary lesions [28,29] and bronchoalveolar lavage fluid [30] are verified by numerous studies, so far no study has been performed to assess altered cytokine expression in nasal epithelial cells, which form the first barrier against potential exogenous triggers.

We hypothesized an alteration in the prevailing pattern of cytokine expression of nasal epithelial cells especially focusing on microbial defense. The main objectives of this study were, therefore, to determine: i) the basal expression levels of 19 cytokines closely linked to microbial defense mechanisms on protein level in GPA-patients and normal controls and, furthermore, ii) the ability of S. aureus to influence the nasal microenvironment's cytokine secretion by exemplary stimulation experiments.


Materials and methods
Patients and biopsies

For analysing baseline cytokine secretion, nasal mucosa biopsies were obtained from 20 patients with GPA and 19 normal controls (NC). For stimulation experiments, biopsies of 10 GPA-patients and 10 NC were generated. Biopsies were taken from nasal turbinates at sites that were visually free of disease activity and suspect alterations like granuloma, edema, bloody patches, purulent secretion or crusts.

The study protocol was approved by the ethics committee of the University of Kiel, Germany (AZ A101/07) and was in accordance with the principles of the Declaration of Helsinki (latest revision October 2008). All patients provided written informed consent prior to enrolment. Exclusion criteria included pregnancy, haemostatic disorder and age of less than 18 years.

GPA was diagnosed according to the American College of Rheumatology classification criteria and Chapel Hill definitions for GPA as recommended by the European League Against Rheumatism (EULAR) [31]. GPA-subgroups were determined according to the European Vasculitis Study Group definitions and recent EULAR recommendations [31]. Extent of the disease was assessed by the Disease Extent Index (DEI) [32], disease activity was classified using the Birmingham Vasculitis Activity Score (BVAS) [33]. The Vasculitis Damage Index (VDI) was applied to record organ damage as a consequence of granulomatous inflammation and vasculitis [34,35]. All patients underwent a standardized interdisciplinary evaluation [36] and were examined endoscopically by an ear, nose and throat - specialist to evaluate endonasal GPA activity [2].

In order to assess the presence of systemic inflammation, white blood cell counts (WBC) as well as measurement of C-reactive protein (CRP) concentration and erythrocyte sedimentation rate (ESR) were performed. Besides slightly elevated ESR, as typical in GPA patients [37], all serologic parameters were within the normal range. In case of GPA, serologic titers of PR3-specific C-ANCA and myeloperoxidase-specific P-ANCA were determined according to Savige et al. [38].

In the normal control group, biopsies were taken while doctors were performing airway passage improving surgery. No individual of this group showed anamnestic or endoscopic signs of acute or chronic inflammatory or autoimmune alterations of the nasal mucosa or was under immunomodulating medication.

Detailed description of patient groups is given in Tables 1 and 2.

Primary cell culture

Nasal epithelial cells (NEC) were enzymatically isolated from biopsies using dispase (Invitrogen, Karlsruhe, Germany). The cells were grown in Airway Epithelial Cell Growth medium (Promocell, Heidelberg, Germany) in a 96-well plate until pre-confluence with approximately equal numbers of cells per well were obtained. After a mean cultivation time of 13 days, supernatants were collected and stored at -80°C.

Multiplex cytokine assay

The supernatants of NEC were analysed by performing a Bio-Plex™ Cytokine Assay according to the manufacturer's instructions (Bio-Rad Laboratories, Munich, Germany) allowing quantification of multiple cytokines with broad range standard curves in small-sample-sizes. Pro-inflammatory (interleukin (IL)-1α, IL-1β, IL-5, IL-6, IL-7, IL-8, IL-17, tumour necrosis factor-alpha (TNF-α)) and anti-inflammatory (IL-4, IL-10, IL-13) mediators, proteins mainly associated with adaptive immune responses (IL-2, IL-4, IL-12p70, IL-13, IFN-γ) or with recruitment of immune cells (IL-8, MCP-1, MIP-1), cytokines predominantly responsible for proliferation of inflammatory effector cells (G-CSF, GM-CSF) and control of apoptotic procedures associated with inflammation (TNF-β) were investigated.

Briefly, samples of 50 µl NEC supernatant were incubated with anti-cytokine antibody-coupled beads. After washing, the complexes were incubated with biotinylated anti-cytokine detection antibodies, and finally with streptavidin-phycoerythrin. Cytokine concentrations were measured using a Luminex 96-well plate reader (Bio-Plex™ 100-Multiplex-Suspension-Array-Reader; Bio-Rad Laboratories, Munich, Germany) and the Bio-Plex™ Manager Software 4.1.1 (Bio-Rad Laboratories, Munich, Germany). Human recombinant cytokines were used as standards.

Stimulation of human nasal epithelial cells with S. aureus

For stimulation of NEC, supernatants containing the bacterial secretory products of S. aureus strain T190-2 (kindly provided by B.M. Bröker, University of Greifswald, Germany), which has been described to predominate in nasal isolates in Western Europe, was chosen. The S. aureus supernatants were not analysed for virulence factors secreted into the growth medium. However, in PCR analysis the test strain was positive for toxic shock and enterotoxin genes [39]. Experiments were performed corresponding to Sachse et al. [40]. According to the results of preliminary experiments concerning time and dose dependency (8, 12, 16, 24 hours; data not shown), stimulation experiments were performed with S. aureus supernatants diluted 1:5 in a final volume of 300 µl for 24 hours. An impact of the bacterial growth medium tryptic soy broth on the stimulation was ruled out before and NEC incubated in fresh cell culture medium without bacterial supernatants served as controls. Cell viability greater than 95% was verified by trypan blue dye exclusion test, and vital cell morphology was controlled in phase contrast microscopy. Moreover, 10 µl of cell culture supernatants were incubated on Columbia blood agar to prove absence of bacterial contamination. After stimulation, cell culture supernatants were collected and stored at -80°C until analysis.

Quantification of IL-8 via ELISA

IL-8 concentrations in cell culture supernatants after stimulation were determined in duplicate by standard ELISA using the BD OptEIA human IL-8 set (BD Biosciences, San Diego, CA, USA).

Statistical analysis

All statistics were performed in an exploratory manner using SPSS statistical software for Windows, version 18 (SPSS Inc., Chicago, IL, USA). Based on the Shapiro-Wilk-Test, normal distribution could be assumed for neither the entire Bio-Plex™ data nor for the delta values (stimulated - basal) of the stimulation experiments. Therefore, differences between the two groups (GPA versus NC) were evaluated by the nonparametric Mann-Whitney-U-Test. Normally distributed stimulation data were analysed by means of a general linear model for repeated measurement procedures in order to test the effects of within-subject factors (effect of stimulation within each group) and of between-subject factors (comparison of GPA- and NC-group). A P-value of ≤0.05 was regarded as statistically significant.


Results
Altered pattern of baseline cytokine expression in GPA-patients

For 17 of the analysed 19 cytokines in the supernatants of NEC, no difference between GPA-patients and normal controls could be detected (see Table 3 for details).

In contrast, NEC of GPA-patients showed significantly higher protein expression of granulocyte colony-stimulating factor (G-CSF, P = 0.050). Furthermore, concentrations of interleukin-8 (IL-8, CXCL8) were remarkably reduced in NEC of GPA-patients compared to NC (P = 0.009), as illustrated in Figure 1.

Diminished response to stimulation with S. aureus culture supernatants in GPA-patients

Based on the results of the multiplex cytokine assay, a more detailed functional study of IL-8 expression of NEC upon stimulation with secretory products containing S. aureus-culture-supernatants was performed.

While both groups (GPA and NC) showed a statistically significant response to the stimulus (NC: P = 0.005, GPA: P = 0.005), baseline IL-8 expression (mean value 1,247.9, SD 881.6, maximum 2,735.3, minimum 379.0 pg/ml) as well as IL-8 expression after stimulation (mean value 2,753.0, SD 1,938.2, maximum 6,744.4, minimum 776.5 pg/ml) was significantly lower (P = 0.006) in GPA-patients compared to NC (basal: mean value 2,876.1, SD 1,498.3, maximum 4,935.5, minimum 500.0 pg/ml; stimulated: mean value 5,647.4, SD 2,407.9, maximum 9,516.1, minimum 1,521.9 pg/ml), as demonstrated in Figure 2A. Remarkably, also the dynamic range of the reaction (stimulated minus basal) was considerably more restricted in NEC of GPA-patients (P = 0.029, Figure 2B).

No correlation between nasal S. aureus-colonisation, endonasal GPA-activity or prednisolone dosages and these results could be detected (data not shown).


Discussion

Cytokines' elaborate interplay enables inter-cellular communication and constitutes a crucial part in the regulation of immune and inflammatory processes - two important components in the pathogenesis of GPA - thus allowing an adequate response to microbial challenges.

Apart from Balding et al., who described an altered Th2-cytokine milieu in nasal biopsies [41], no comprehensive analysis of baseline cytokine expression in the nasal epithelium of GPA-patients has been performed so far.

For GPA, alterations in cytokine levels in pulmonary lesions have been described [28,29], but none of the 19 cytokines relevant for microbial defense included in this study were examined. Results of Richter et al., who reported elevated concentrations of the pro-inflammatory cytokines IL-1α, IL-1β and IL-6 in bronchoalveolar lavage fluid of GPA-patients [30], are difficult to compare to our findings obtained from a distinct compartment but hint at an altered cytokine spectrum.

Once induced by an inflammatory stimulus, almost all tissues within the human body are capable of producing G-CSF, thus leading to an increase in number and activation of neutrophils [42]. G-CSF serum levels are markedly elevated in response to infection and usually fall in parallel with the recovery process [43], whereas they remain elevated and correlate with disease activity in chronic inflammatory conditions, such as rheumatoid arthritis and Behçet disease [44,45]. Locally elevated G-CSF concentrations have also been observed in inflammatory bowel disease [46]. These findings match our results of locally elevated concentrations in NEC reasonably.

A prolonged life time of neutrophils resulting from a delayed apoptosis induced by G-CSF [47] increases the possibility of being primed and expressing PR3 on the cell surface [23,48]. Further activation through binding of ANCA can result in damage and lysis of endothelial cells [49]. In endothelial cells, G-CSF has been shown to down-regulate lipopolysaccharide-induced IL-8 expression [50], which would be conceivable for epithelial cells as well. Besides, the possible ability of G-CSF to reduce neutrophil killing of S. aureus [51] could have tremendous negative impacts on an effective immune defense.

IL-8 chemoattracts polymorphonuclear neutrophils (PMN) and monocytes and further promotes their activation [52], thus creating and maintaining an inflammatory microenvironment at the site of infection.

Lamprecht et al. suggested a down-regulation of monocytic production of IL-8 during the course of GPA [26], which is in line with our findings. Stimulation of PMN with IL-8, especially after previous treatment with TNF-α, leads to PR3 translocation to the cell surface [53], thus providing the prerequisite for interactions with PR3-ANCA, which directly activates diverse inflammatory reactions in PMN [54]. Variation of IL-8 expression levels is fine-tuned by graduated activation of at least three signaling pathways: NF-κB, JNK (JUN-N terminal protein kinase) and p38-MPK (mitogen-activated protein kinase) [55]. In order to evaluate whether this complex interplay is operating effectively in GPA-patients, we stimulated NEC with culture supernatants of S. aureus, which apart from its particular role in the pathogenesis of GPA has been shown to be a potent inducer of IL-8 expression in nasal epithelial cells [56,57]. The imbalance of IL-8 with a reduced baseline expression and a diminished response to bacterial stimulus of GPA-NEC could reasonably lead to a shift in the microbial flora towards an overbalance of facultative pathogenic microorganisms.

These data, in addition to the observation of a severely impaired ciliar beat frequency [58] and a reduced production of the antimicrobial peptide human β-defensin 3 of NEC upon stimulation with S. aureus [59], as well as an imbalanced regulation of genes involved in epithelial barrier function [18], fortify the hypothesis of considering GPA a disease with a disturbed epithelial barrier function as was also discussed for other chronic inflammatory diseases with nasal involvement [60].

However, this study has its limitations. Patients with GPA received immunomodulating therapy, for which negative impact on cytokine expression has been described [61,62]. Differential expression of the investigated cytokines (most of them without differences between NC and GPA) argues for successful prevention of such effects by cultivating NEC for an average of 13 days prior to the investigation.

The fact that the pattern of cytokine expression of NEC can be influenced by external stimuli paves the way for local pharmacological interference. Shifting the basal cytokine balance towards a higher IL-8 level, for example, by applying recombinant human IL-8 [63] or non-pathogenic bacteria components [64] could be a future therapeutic option.


Conclusions

Taken together, our data suggest a specifically altered pattern of baseline cytokine expression of the nasal epithelium in patients with GPA compared to NC. This can facilitate changes in the composition of microbial colonisation and favour an imbalanced inflammatory response to microbial challenge and thus disease exacerbation. Our findings of an aberrant response to S. aureus stimulation in patients with GPA further underline this hypothesis. It remains to be investigated whether our results exemplify a general alteration in the reactive cytokine response to external stimuli. Taken into account the results of all 19 examined cytokines, we assume pathophysiological relevance of IL-8 and G-CSF in GPA, which could have potential therapeutic implications.


Abbreviations

BVAS: Birmingham Vasculitis Activity Score; C-ANCA: cytoplasmic anti-neutrophil cytoplasmatic antibodies; cPR3: complementary PR3; CRP: C-reactive protein; DEI: Disease Extent Index; ELISA: enzyme-linked immunosorbent assay; ESR: erythrocyte sedimentation rate; EULAR: European League Against Rheumatism; G-CSF: granulocyte colony-stimulating factor; GM-CSF: granulocyte macrophage colony-stimulating factor; GPA: Granulomatosis with polyangiitis; IFN-γ: interferon-γ; IL: interleukin; JNK: JUN-N terminal protein kinase; MAPK: mitogen-activated protein kinase; MCP-1: monocyte chemotactic protein-1; MIP-1: macrophage inflammatory protein-1; NC: normal controls; NEC: nasal epithelial cells; P-ANCA: perinuclear anti-neutrophil cytoplasmatic antibodies; PMN: polymorphonuclear neutrophils; PR3: proteinase 3; S. aureus: Staphylococcus aureus; TNF: tumor necrosis factor; VDI: Vasculitis Damage Index; WBC: white blood cell count.


Competing interests

The authors declare that they have no competing interests.


Authors' contributions

ML, RP, JH and PA designed and coordinated the study. JW carried out the experiments. ML, JW, KB and PL were involved in data acquisition and interpretation. JH, JW and ML performed the statistical analysis. JW, KB, PL, RP, JH, PA and ML drafted and revised the manuscript. All authors provided final approval of the submitted manuscript.


Acknowledgements

This study was supported by the German Research Foundation (DFG) funded Clinical Research Unit (KFO 170). We thank A. Hölzgen, U. Kreutz, T. Görögh and A.-M. Röen for their expert technical assistance. We gratefully acknowledge participation of all patients and healthy volunteers who took part in this study.


References
Bacon PA,The spectrum of Wegener's granulomatosis and disease relapseN Engl J MedYear: 200535233033215673799
Paulsen JI,Rudert H,Manifestations of primary vasculitis in the ENT regionZ RheumatolYear: 20016021922511584719
Hamidou MA,Audrain M,Ninin E,Robillard N,Muller JY,Bonneville M,Staphylococcus aureus, T-cell repertoire, and Wegener's granulomatosisJoint Bone SpineYear: 20016837337711707002
Jagiello P,Gross WL,Epplen JT,Complex genetics of Wegener granulomatosisAutoimmun RevYear: 20054424715652778
Laudien M,Ambrosch P,Till A,Podschun R,Lamprecht P,[Diagnosis, therapy and current research aspects of selected chronic inflammatory diseases with head and neck involvement]Z RheumatolYear: 20086739740618600330
Tervaert JW,Popa ER,Bos NA,The role of superantigens in vasculitisCurr Opin RheumatolYear: 19991124339894627
Wieczorek S,Holle JU,Epplen JT,Recent progress in the genetics of Wegener's granulomatosis and Churg-Strauss syndromeCurr Opin RheumatolYear: 20102281419864953
Laudien M,Gadola SD,Podschun R,Hedderich J,Paulsen J,Reinhold-Keller E,Csernok E,Ambrosch P,Hellmich B,Moosig F,Gross WL,Sahly H,Lamprecht P,Nasal carriage of Staphylococcus aureus and endonasal activity in Wegener s granulomatosis as compared to rheumatoid arthritis and chronic rhinosinusitis with nasal polypsClin Exp RheumatolYear: 201028515520412703
Stegeman CA,Tervaert JW,Sluiter WJ,Manson WL,De Jong PE,Kallenberg CG,Association of chronic nasal carriage of Staphylococcus aureus and higher relapse rates in Wegener granulomatosisAnn Intern MedYear: 199412012178250451
Stegeman CA,Tervaert JW,De Jong PE,Kallenberg CG,Trimethoprim-sulfamethoxazole (co-trimoxazole) for the prevention of relapses of Wegener's granulomatosis. Dutch Co-Trimoxazole Wegener Study GroupN Engl J MedYear: 199633516208637536
Popa ER,Stegeman CA,Abdulahad WH,van der Meer B,Arends J,Manson WM,Bos NA,Kallenberg CG,Cohen Tervaert JW,Staphylococcal toxic-shock-syndrome-toxin-1 as a risk factor for disease relapse in Wegener's granulomatosisRheumatology (Oxford)Year: 2007461029103317409134
Zycinska K,Wardyn KA,Zielonka TM,Demkow U,Traburzynski MS,Chronic crusting, nasal carriage of Staphylococcus aureus and relapse rate in pulmonary Wegener's granulomatosisJ Physiol PharmacolYear: 200859Suppl 682583119218710
De Groot K,Reinhold-Keller E,Tatsis E,Paulsen J,Heller M,Nolle B,Gross WL,Therapy for the maintenance of remission in sixty-five patients with generalized Wegener's granulomatosis. Methotrexate versus trimethoprim/sulfamethoxazoleArthritis RheumYear: 199639205220618961911
Reinhold-Keller E,de Groot K,Rudert H,Nölle B,Heller M,Gross WL,Response to trimethoprim/sulfamethoxazole in Wegener's granulomatosis depends on the phase of diseaseQJMYear: 19968915238730339
Popa ER,Stegeman CA,Kallenberg CG,Tervaert JW,Staphylococcus aureus and Wegener's granulomatosisArthritis ResYear: 20024777911879541
Abdulahad WH,Stegeman CA,Limburg PC,Kallenberg CG,CD4-positive effector memory T cells participate in disease expression in ANCA-associated vasculitisAnn N Y Acad SciYear: 20071107223117804529
Mayet WJ,Marker-Hermann E,Schlaak J,Meyer Zum Büschenfelde KH,Irregular cytokine pattern of CD4+ T lymphocytes in response to Staphylococcus aureus in patients with Wegener's granulomatosisScand J ImmunolYear: 19994958559410354370
Laudien M,Häsler R,Wohlers J,Böck J,Lipinski S,Bremer L,Podschun R,Ambrosch P,Lamprecht P,Rosenstiel P,Till A,Molecular signatures of a disturbed nasal barrier function in the primary tissue of Wegener's granulomatosisMucosal ImmunolYear: 2011456457321412229
Kain R,Exner M,Brandes R,Ziebermayr R,Cunningham D,Alderson CA,Davidovits A,Raab I,Jahn R,Ashour O,Spitzauer S,Sunder-Plassmann G,Fukuda M,Klemm P,Rees AJ,Kerjaschki D,Molecular mimicry in pauci-immune focal necrotizing glomerulonephritisNat MedYear: 2008141088109618836458
Pendergraft WF III,Preston GA,Shah RR,Tropsha A,Carter CW Jr,Jennette JC,Falk RJ,Autoimmunity is triggered by cPR-3(105-201), a protein complementary to human autoantigen proteinase-3Nat MedYear: 200410727914661018
Tosi MF,Innate immune responses to infectionJ Allergy Clin ImmunolYear: 200511624124916083775
Medzhitov R,Janeway C Jr,Innate immunityN Engl J MedYear: 200034333834410922424
Csernok E,Szymkowiak CH,Mistry N,Daha MR,Gross WL,Kekow J,Transforming growth factor-beta (TGF-beta) expression and interaction with proteinase 3 (PR3) in anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitisClin Exp ImmunolYear: 19961051041118697616
Krumbholz M,Specks U,Wick M,Kalled SL,Jenne D,Meinl E,BAFF is elevated in serum of patients with Wegener's granulomatosisJ AutoimmunYear: 20052529830216242914
Torheim EA,Yndestad A,Bjerkeli V,Halvorsen B,Aukrust P,Froland SS,Increased expression of chemokines in patients with Wegener's granulomatosis - modulating effects of methylprednisolone in vitroClin Exp ImmunolYear: 200514037638315807865
Lamprecht P,Kumanovics G,Mueller A,Csernok E,Komocsi A,Trabandt A,Gross WL,Schnabel A,Elevated monocytic IL-12 and TNF-alpha production in Wegener's granulomatosis is normalized by cyclophosphamide and corticosteroid therapyClin Exp ImmunolYear: 200212818118611982607
Ludviksson BR,Sneller MC,Chua KS,Talar-Williams C,Langford CA,Ehrhardt RO,Fauci AS,Strober W,Active Wegener's granulomatosis is associated with HLA-DR+ CD4+ T cells exhibiting an unbalanced Th1-type T cell cytokine pattern: reversal with IL-10J ImmunolYear: 1998160360236099531324
Coulomb-L'Hermine A,Capron F,Zou W,Piard F,Galateau F,Laurent P,Crevon MC,Galanaud P,Emilie D,Expression of the chemokine RANTES in pulmonary Wegener's granulomatosisHum PatholYear: 20013232032611274642
Zhou Y,Huang D,Farver C,Hoffman GS,Relative importance of CCR5 and antineutrophil cytoplasmic antibodies in patients with Wegener's granulomatosisJ RheumatolYear: 2003301541154712858455
Richter AG,Stockley RA,Harper L,Thickett DR,Pulmonary infection in Wegener granulomatosis and idiopathic pulmonary fibrosisThoraxYear: 20096469269719359270
Hellmich B,Flossmann O,Gross WL,Bacon P,Cohen-Tervaert JW,Guillevin L,Jayne D,Mahr A,Merkel PA,Raspe H,Scott DG,Witter J,Yazici H,Luqmani RA,EULAR recommendations for conducting clinical studies and/or clinical trials in systemic vasculitis: focus on anti-neutrophil cytoplasm antibody-associated vasculitisAnn Rheum DisYear: 20076660561717170053
Reinhold-Keller E,Kekow J,Schnabel A,Schmitt WH,Heller M,Beigel A,Duncker G,Gross WL,Influence of disease manifestation and antineutrophil cytoplasmic antibody titer on the response to pulse cyclophosphamide therapy in patients with Wegener's granulomatosisArthritis RheumYear: 1994379199248003065
Luqmani RA,Bacon PA,Moots RJ,Janssen BA,Pall A,Emery P,Savage C,Adu D,Birmingham Vasculitis Activity Score (BVAS) in systemic necrotizing vasculitisQJMYear: 1994876716787820541
Exley AR,Bacon PA,Luqmani RA,Kitas GD,Gordon C,Savage CO,Adu D,Development and initial validation of the Vasculitis Damage Index for the standardized clinical assessment of damage in the systemic vasculitidesArthritis RheumYear: 1997403713809041949
Luqmani RA,Assessing disease activity in the systemic vasculitidesCurr Opin RheumatolYear: 200214232811790992
Reinhold-Keller E,Beuge N,Latza U,De Groot K,Rudert H,Nolle B,Heller M,Gross WL,An interdisciplinary approach to the care of patients with Wegener's granulomatosis: long-term outcome in 155 patientsArthritis RheumYear: 2000431021103210817555
Fauci AS,Haynes BF,Katz P,Wolff SM,Wegener's granulomatosis: prospective clinical and therapeutic experience with 85 patients for 21 yearsAnn Intern MedYear: 19839876856336643
Savige J,Gillis D,Benson E,Davies D,Esnault V,Falk RJ,Hagen EC,Jayne D,Jennette JC,Paspaliaris B,Pollock W,Pusey C,Savage CO,Silvestrini R,van der Woude F,Wieslander J,Wiik A,International Consensus Statement on Testing and Reporting of Antineutrophil Cytoplasmic Antibodies (ANCA)Am J Clin PatholYear: 199911150751310191771
Holtfreter S,Grumann D,Schmudde M,Nguyen HT,Eichler P,Strommenger B,Kopron K,Kolata J,Giedrys-Kalemba S,Steinmetz I,Witte W,Broker BM,Clonal distribution of superantigen genes in clinical Staphylococcus aureus isolatesJ Clin MicrobiolYear: 2007452669268017537946
Sachse F,von Eiff C,Stoll W,Becker K,Rudack C,Induction of CXC chemokines in A549 airway epithelial cells by trypsin and staphylococcal proteases - a possible route for neutrophilic inflammation in chronic rhinosinusitisClin Exp ImmunolYear: 200614453454216734624
Balding CE,Howie AJ,Drake-Lee AB,Savage CO,Th2 dominance in nasal mucosa in patients with Wegener's granulomatosisClin Exp ImmunolYear: 200112533233911529927
Eyles JL,Roberts AW,Metcalf D,Wicks IP,Granulocyte colony-stimulating factor and neutrophils--forgotten mediators of inflammatory diseaseNat Clin Pract RheumatolYear: 2006250051016951705
Kawakami M,Tsutsumi H,Kumakawa T,Abe H,Hirai M,Kurosawa S,Mori M,Fukushima M,Levels of serum granulocyte colony-stimulating factor in patients with infectionsBloodYear: 199076196219641700729
Kawakami T,Ohashi S,Kawa Y,Takahama H,Ito M,Soma Y,Mizoguchi M,Elevated serum granulocyte colony-stimulating factor levels in patients with active phase of sweet syndrome and patients with active behcet disease: implication in neutrophil apoptosis dysfunctionArch DermatolYear: 200414057057415148101
Nakamura H,Ueki Y,Sakito S,Matsumoto K,Yano M,Miyake S,Tominaga T,Tominaga M,Eguchi K,High serum and synovial fluid granulocyte colony stimulating factor (G-CSF) concentrations in patients with rheumatoid arthritisClin Exp RheumatolYear: 20001871371811138333
Ina K,Kusugami K,Hosokawa T,Imada A,Shimizu T,Yamaguchi T,Ohsuga M,Kyokane K,Sakai T,Nishio Y,Yokoyama Y,Ando T,Increased mucosal production of granulocyte colony-stimulating factor is related to a delay in neutrophil apoptosis in inflammatory bowel diseaseJ Gastroenterol HepatolYear: 199914465310029277
Maianski NA,Mul FP,van Buul JD,Roos D,Kuijpers TW,Granulocyte colony-stimulating factor inhibits the mitochondria-dependent activation of caspase-3 in neutrophilsBloodYear: 20029967267911781253
Hellmich B,Csernok E,Trabandt A,Gross WL,Ernst M,Granulocyte-macrophage colony-stimulating factor (GM-CSF) but not granulocyte colony-stimulating factor (G-CSF) induces plasma membrane expression of proteinase 3 (PR3) on neutrophils in vitroClin Exp ImmunolYear: 200012039239810792393
Kallenberg CG,Stegeman CA,Heeringa P,Autoantibodies vex the vasculatureNat MedYear: 2008141018101918841136
Schneider EM,Lorenz I,Ma X,Weiss M,G-CSF modulates LPS-induced apoptosis and IL-8 in human microvascular endothelial cells: involvement of calcium signalingAnn N Y Acad SciYear: 20031010788515033698
Leavey PJ,Sellins KS,Thurman G,Elzi D,Hiester A,Silliman CC,Zerbe G,Cohen JJ,Ambruso DR,In vivo treatment with granulocyte colony-stimulating factor results in divergent effects on neutrophil functions measured in vitroBloodYear: 199892436643749834243
Apostolakis S,Vogiatzi K,Amanatidou V,Spandidos DA,Interleukin 8 and cardiovascular diseaseCardiovasc ResYear: 20098435336019617600
Csernok E,Ernst M,Schmitt W,Bainton DF,Gross WL,Activated neutrophils express proteinase 3 on their plasma membrane in vitro and in vivoClin Exp ImmunolYear: 1994952442508306499
Brouwer E,Huitema MG,Mulder AH,Heeringa P,van Goor H,Tervaert JW,Weening JJ,Kallenberg CG,Neutrophil activation in vitro and in vivo in Wegener's granulomatosisKidney IntYear: 199445112011318007582
Hoffmann E,Dittrich-Breiholz O,Holtmann H,Kracht M,Multiple control of interleukin-8 gene expressionJ Leukoc BiolYear: 20027284785512429706
Damm M,Quante G,Rosenbohm J,Rieckmann R,Proinflammatory effects of Staphylococcus aureus exotoxin B on nasal epithelial cellsOtolaryngol Head Neck SurgYear: 200613424524916455372
Rudack C,Sachse F,Albert N,Becker K,von Eiff C,Immunomodulation of nasal epithelial cells by Staphylococcus aureus-derived serine proteasesJ ImmunolYear: 20091837592760119917683
Ullrich S,Gustke H,Lamprecht P,Gross WL,Schumacher U,Ambrosch P,Laudien M,Severely impaired respiratory ciliar function in Wegener's granulomatosisAnn Rheum DisYear: 2008681067107119028765
Hui Y,Wohlers J,Podschun R,Hedderich J,Lamprecht P,Ambrosch P,Laudien M,Antimicrobial peptides in nasal secretion and mucosa with respect to S. aureus colonization in Wegener's granulomatosisClin Exp RheumatolYear: 2011294956
Tan BK,Schleimer RP,Kern RC,Perspectives on the etiology of chronic rhinosinusitisCurr Opin Otolaryngol Head Neck SurgYear: 201018212619966566
Sachse F,von Eiff C,Becker K,Rudack C,Anti-inflammatory effects of ciprofloxacin in S. aureus Newman induced nasal inflammation in vitroJ Inflamm (Lond)Year: 200851118664275
Szekanecz Z,Vegvari A,Szabo Z,Koch AE,Chemokines and chemokine receptors in arthritisFront Biosci (Schol Ed)Year: 2010215316720036936
Zwierzina H,Holzinger I,Gaggl S,Wolf H,Schollenberger S,Lam C,Bammer T,Geissler D,Lindley I,Recombinant human interleukin-8 restores function in neutrophils from patients with myelodysplastic syndromes without stimulating myeloid progenitor cellsScand J ImmunolYear: 1993373223288382840
Iwase T,Uehara Y,Shinji H,Tajima A,Seo H,Takada K,Agata T,Mizunoe Y,Staphylococcus epidermidis Esp inhibits Staphylococcus aureus biofilm formation and nasal colonizationNatureYear: 201046534634920485435

Figures

[Figure ID: F1]
Figure 1 

Baseline cytokine expression. Significantly altered baseline cytokine expression of primary nasal epithelial cells concerning G-CSF (A) and IL-8 (B) of granulomatosis with polyangiitis (GPA) patients compared to normal controls (NC). Data are represented as box plots showing the median as a straight line; the boxes correspond to the 75th and 25th percentiles and the whiskers indicate normal variation limits.



[Figure ID: F2]
Figure 2 

Effect of stimulation with S. aureus. Significant differences in IL-8 expression of primary nasal epithelial cells (NEC) comparing granulomatosis with polyangiitis (GPA) patients and normal controls (NC) before and after stimulation with S. aureus supernatants (A, P = 0.006). Furthermore, the dynamic range (Δ IL-8, secretion after stimulation minus basal secretion) is significantly reduced in GPA-patients (B, P = 0.029).



Tables
[TableWrap ID: T1] Table 1 

Patients' characteristics


Baseline cytokine secretion Stimulation
Normal control Granulomatosis with polyangiitis Normal control Granulomatosis with polyangiitis
(n = 19) (n = 20) (n = 10) (n = 10)

Age (yrs, mean and range) 39.50 (18 to 76) 51.55 (24 to 71) 43.30 (23 to 56) 50.80 (24 to 71)
Sex 11 male, 8 female 12 male, 8 female 7 male, 3 female 7 male, 3 female
First GPA manifestation till study entry (yrs, median and range) 3.8 (19 to 0) 3.3 (9 to 0)
First GPA diagnosis till study entry (yrs, median and range) 1.6 (19 to 0) 3.1 (9 to 0)

Serologic parameters

mean value SD mean value SD mean value SD mean value SD
WBC (cells/nl) 6.74 1.55 8.22 2.82 7.87 1.97 7.77 2.33
ESR 1st h (mm) 8.14 3.94 33.00 27.60 8.00 2.92 52.40 35.61
median range median range median range median range
CRP (mg/dl) 1.30 0.9 to 5.8 0.70 0 to 8.6 1.50 1.3 to 3.4 0.85 0.1 to 8.6
C-ANCA-titre (1:) 80 0 to 2560 640 0 to 2560
P-ANCA-titre (1:) 0 0 to 640 0 0 to 61

[TableWrap ID: T2] Table 2 

Characteristics of patients with GPA


Baseline cytokine secretion Stimulation
n % n %

Biopsy proof GPA 12 60 7 70

EULAR subgroups generalized 14 70 8 80
early systemic 5 25 1 10
local 1 5 0 0
severe 0 0 1 10

EULAR disease activity remission 6 30 2 20
response 0 0 1 10
relapse, minor 8 40 2 20
relapse, major 4 20 3 30
refractory 1 5 0 0
low-activity 1 5 2 20

Endoscopy: endonasal activity none 12 60 4 40
mild 7 35 4 40
moderate 0 0 2 20
not evaluated 1 5 0 0

Disease scores median (range) median (range)
DEI 2 (0 to 5) 2 (0 to 5)
BVAS-1 3 (0 to 13) 1.5 (0 to 12)
BVAS-2 0 (0 to 4) 0 (0 to 4)
VDI 0.5 (0 to 3) 2 (0 to 4)

Immunomodulating therapy n (%) mean value (mg) n (%) mean value (mg)
prednisolone 19 (95) 12.89 9 (90) 10.67
methotrexate 10 (50) 22.25 2 (20) 20
cyclophosphamide regular 3 (15) 133.33 1 (10) 100
cyclophosphamide bolus 2 (10) 1000 2 (20) 1000
azathioprine 2 (10) 125 2 (20) 125
leflunomide 2 (10) 25 2 (20) 25
mycophenolate mofetil 1 (5) 2000 0 (0) 0
cummulative cyclophosphamide 6 (30) 45.17 (g) 8 (80) 14.88 (g)

[TableWrap ID: T3] Table 3 

Baseline cytokine expression of nasal epithelial cells (pg/ml)


Normal control (NC) Granulomatosis with polyangiitis (GPA) NC vs. GPA
median range median range P-value

IL- 1α 71.74 <1.51 to 319.24 92.38 7.90 to 477.11 0.322
IL-1β <2.67 <2.67 to 8.67 2.70 <2.67 to 12.74 0.224
IL-2 <1.42 <1.42 to 6.46 3.60 <1.42 to 6.00 0.070
IL-4 <0.32 <0.32 to 0.80 <0.32 <0.32 to 0.72 0.283
IL-5 below detection limit (<2.51)
IL-6 26.22 2.81 to 788.29 29.61 3.70 to 139.47 0.607
IL-7 below detection limit (<2.43)
IL-8 1388.14 <1.77 to 5188.14 319.16 <1.77 to 3708.72 0.009
IL-10 below detection limit (<1.78)
IL-12 <2.55 <2.55 to 3.75 <2.55 <2.55 to 4.03 0.283
IL-13 below detection limit (<2.68)
IL-17 2.77 <1.87 to 6.41 3.54 <1.87 to 6.01 0.101
G-CSF 19.60 <1.31 to 204.41 61.30 1.53 to 663.58 0.050
GM-CSF 10.50 <0.74 to 28.70 12.50 7.74 to 21.56 0.084
IFN-γ 4.61 <1.61 to 21.41 5.92 2.27 to 18.18 0.089
MCP-1 3.66 <1.81 to 8.21 4.12 <1.81 to 7.90 0.667
MIP-1β <1.41 <1.41 to 2.37 1.43 <1.41 to 2.79 0.141
TNF-α <6.42 <6.42 to 13.46 <6.42 <6.42 to 11.00 0.296
TNF-β 9.44 <2.14 to 24.46 14.20 3.35 to 23.40 0.054


Article Categories:
  • Research Article


Previous Document:  Survey of UK radiology trainees in the aftermath of 'Modernising Medical Careers'.
Next Document:  Optimization of a direct spectrophotometric method to investigate the kinetics and inhibition of sia...