interleukin-8 and Granulomatosis-with-Polyangiitis

Wegener's granulomatosis (WG) is characterized by a predominance of the type 1 T-helper cell (Th1) response. We have studied monocytic cytokine expression in untreated patients and in patients who did not respond to prior methotrexate or trimethoprim-sulphamethoxazole therapy, i.e. patients with active disease. Intracytoplasmic IL-12 and TNF-alpha expression was significantly increased in WG compared with healthy controls. IL-8 expression was not increased. Two and 12 weeks of daily standard oral cyclophosphamide and corticosteroid (CYC + GC) treatment induced a stable remission of the disease. Elevated IL-12 and TNF-alpha expression of monocytes was normalized. The active metabolite of CYC was shown to down-regulate IL-12 mRNA in vitro. Monocytic cytokines, especially IL-12, may have a role in the early determination and skewing of the immunoregulatory response towards a Th1 profile. It appears that CYC + GC exerts its effect by normalizing the Th1-driving cytokine pattern, and CYC may maintain this mode of action. Normalization of the skewed cytokine pattern may be a prerequisite and an indicator of inducing a remission in WG.

Topics: Adrenal Cortex Hormones; Adult; Cells, Cultured; Cyclophosphamide; Cytokines; Cytoplasm; Down-Regulation; Female; Granulomatosis with Polyangiitis; Humans; Interleukin-12; Interleukin-8; Kinetics; Longitudinal Studies; Male; Middle Aged; Monocytes; Remission Induction; RNA, Messenger; Treatment Outcome; Tumor Necrosis Factor-alpha

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.. 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.. 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.. 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.

Topics: Adolescent; Adult; Aged; Case-Control Studies; Cells, Cultured; Cellular Microenvironment; Cytokines; Epithelial Cells; Female; Granulocyte Colony-Stimulating Factor; Granulomatosis with Polyangiitis; Humans; Interleukin-8; Male; Middle Aged; Nasal Mucosa; Staphylococcus aureus; Young Adult

There remains a need for biomarkers to guide therapy in antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis. Our objective was to determine whether measures of platelet activation or inflammation are associated with disease activity in Wegener's granulomatosis (WG).. Study subjects were participants in a clinical trial. Soluble CD40 ligand (sCD40L), C-reactive protein, interleukin 6 (IL-6), IL-8, monocyte chemoattractant protein 1 (MCP-1), P-selectin, vascular endothelial growth factor, and proteinase 3 (PR3)-specific ANCA were measured by ELISA using plasma samples obtained at baseline (active disease), at remission, and prior to, during, and after first flares. Disease activity was assessed by the Birmingham Vasculitis Activity Score for WG (BVAS/WG). Association of biomarkers with disease activity was determined with conditional logistic and linear regression.. Over a mean followup of 27 months, 180 subjects underwent 2044 visits; markers were measured in 563 samples. Longitudinally, all markers other than IL-6 were associated with disease activity. The strongest associations for active disease at baseline versus remission were observed for sCD40L (OR 4.72, 95% CI 2.47-9.03), P-selectin (OR 6.26, 95% CI 2.78-14.10), PR3-ANCA (OR 9.41, 4.03-21.99), and inversely for MCP-1 (OR 0.36, 95% CI 0.22-0.57). BVAS/WG increased by 0.80 (95% CI 0.44-1.16), 0.83 (95% CI 0.42-1.25), and 0.81 (95% CI 0.48-1.15) per unit-increase in PR3-ANCA, sCD40L, and P-selectin, respectively; and decreased by 1.54 (95% CI 0.96-2.12) per unit-increase in MCP-1.. Cytokines arising from within the circulation, including those of platelet activation, correlate with disease activity in WG.

Topics: Antibodies, Antineutrophil Cytoplasmic; Biomarkers; C-Reactive Protein; CD40 Ligand; Chemokine CCL2; Cytokines; Follow-Up Studies; Granulomatosis with Polyangiitis; Humans; Inflammation; Interleukin-8; Myeloblastin; P-Selectin; Platelet Activation; Retrospective Studies; Severity of Illness Index; Vascular Endothelial Growth Factor A

The presence of antineutrophil cytoplasmic antibodies in granulomatosis with polyangiitis (Wegener's) (GPA) implicates the neutrophil as a key effector cell. Previous studies have reported elevated neutrophil counts in the lung, although the determinants of neutrophil chemotaxis in the GPA lung are unknown. Bronchoalveolar lavage fluid (BALF) cell counts, myeloperoxidase (MPO) and chemokines were measured in 27 patients with GPA, 20 disease controls with idiopathic pulmonary fibrosis (IPF) and six healthy controls. CXC chemokine ligand (CXCL)8, interleukin (IL)-1β, epithelial neutrophil-activating protein 78, granulocyte colony-stimulating factor (G-CSF) and granulocyte-macrophage colony-stimulating factor were measured by ELISA. The neutrophil chemotactic potential of BALF was investigated using the under-agarose method, and specific antibodies were used to examine the role of CXCL8 and IL-1β. GPA BALF had an increased neutrophil percentage, and elevated MPO, CXCL8 and G-CSF concentrations compared with healthy controls. Chemotaxis of control neutrophils towards BALF from patients with active (p=0.006) and remission (p=0.077) GPA, and IPF (p=0.001) patients was increased compared with normal controls. BALF-induced chemotaxis correlated with BALF IL-1β (r=0.761, p=0.001) and CXCL8 (r=0.640, p=0.012) in GPA, and was inhibited by anti-CXCL8 (85%; p<0.001) and anti-IL-1β (69%; p<0.001). Our study confirms a neutrophilia and pro-inflammatory alveolar milieu that persists in clinical remission. CXCL8 and IL-1β appear to play important roles in the neutrophil chemotactic response to BALF.

Topics: Aged; Bronchoalveolar Lavage Fluid; Chemokine CXCL5; Chemotaxis, Leukocyte; Female; Granulocyte Colony-Stimulating Factor; Granulocyte-Macrophage Colony-Stimulating Factor; Granulomatosis with Polyangiitis; Humans; Idiopathic Pulmonary Fibrosis; Interleukin-1beta; Interleukin-8; Male; Middle Aged; Neutrophils

The use of cardiopulmonary bypass (CPB) triggers a systemic inflammatory response (IR), but it is not known if a similar response occurs in an immunosuppressed patient with autoimmune disease.. Observational study in a 56-year-old man receiving immunosuppressive therapy for Wegener's granulomatosis (WG) who underwent aortic valve replacement on CPB. The following markers for IR were studied in the perioperative period: C3a, C5a, neutrophil elastase (NE), interleukin eight (IL-8), white cell count (WCC) and C-reactive protein (CRP). Results were compared with published literature on the IR in patients undergoing coronary revascularisation with and without the use of CPB. All inflammatory markers increased in the perioperative period. The intensity of IR was markedly reduced compared to published literature for patients undergoing coronary revascularisation on CPB and the temporal patients and extend resembled that for off-pump.. In a patient with WG on immunosuppressive therapy the CPB-related IR is reduced.

Topics: Aortic Valve Insufficiency; Blood Vessel Prosthesis Implantation; Cardiopulmonary Bypass; Complement C3a; Complement C5a; Granulomatosis with Polyangiitis; Humans; Immunosuppression Therapy; Interleukin-8; Leukocyte Elastase; Male; Middle Aged; Systemic Inflammatory Response Syndrome

It has been shown that interaction of anti-PR-3 antibodies with human endothelial cells (EC) leads to an activation of EC in vitro, i.e. induction of adhesion molecules like E-selectin, VCAM-1 and tissue factor. The aim of this study was to investigate the effect of anti-PR-3 antibodies on endothelial IL-8 expression.. EC were cultured in 96-well plates and stimulated with TNF-alpha and IL-1beta for 1 h to induce membrane expression of endothelial PR-3. Anti-PR-3 antibodies were purified from sera from patients with clinically active Wegener's granulomatosis. Purified anti-Ro, anti-centromere, anti-dsDNA antibodies and a monoclonal anti-PR-3 antibody (WGM2) served as controls. Induction of IL-8 mRNA was detected by RT-PCR. IL-8 was measured in the supernatant of EC by ELISA. In addition, induction of NFkappaB was investigated by PAGE of nuclear extracts of EC and Western blot with ab against p65.. In contrast to controls, interaction of anti-PR-3 antibodies (patients and WGM2) with cytokine activated EC led to the highest amount of IL-8 synthesis. Priming of EC with cytokines alone induced a markedly lower IL-8 level. The lowest levels of IL-8 could be measured after incubation of unprimed EC with anti-PR-3 antibodies. Anti-PR-3 antibody induced endothelial IL-8 expression could be inhibited by cycloheximide. In addition, we established that the activation of NF-kappaB is critically involved in anti-PR-3 antibody induced endothelial IL-8 production.. In summary, we were able to show that anti-PR-3 antibodies induce endothelial IL-8 synthesis by activating NF-kappaB. As IL-8 represents a powerful neutrophil chemoattractant, our data provide further evidence for a direct pathogenic effect of anti-PR-3 antibodies in ANCA related diseases.

Topics: Antibodies, Antineutrophil Cytoplasmic; Autoantibodies; Autoantigens; Cells, Cultured; Endothelium, Vascular; Granulomatosis with Polyangiitis; Humans; Immunoglobulin G; Interleukin-1; Interleukin-8; Myeloblastin; NF-kappa B; Reverse Transcriptase Polymerase Chain Reaction; Serine Endopeptidases; Transcription, Genetic; Tumor Necrosis Factor-alpha; Umbilical Veins

Cytoplasmic antineutrophil cytoplasmic antibodies (cANCA) that accompany the neutrophilic vasculitis seen in Wegener's granulomatosis (WG), are directed against proteinase-3 (PR-3), a serine proteinase which is located in azurophilic granules of neutrophils and monocytes. PR-3, when expressed on the surface of TNFalpha-primed neutrophils, can directly activate neutrophils by complexing cANCA and promoting concomitant Fcgamma receptor (FcgammaR) cross-linking. Although the neutrophil's pathogenic role in WG has been studied, the role of the monocyte has not been explored. The monocyte, with its ability to release cytokines and regulate neutrophil influx, also expresses PR-3. Therefore, the monocyte may play a significant role in WG via the interaction of surface PR-3 with cANCA, inducing cytokine release by the monocyte. To test this hypothesis, monocytes were studied for PR-3 expression and for IL-8 release in response to cANCA IgG. PBMC obtained from healthy donors displayed dramatic surface PR-3 expression as detected by immunohistochemistry and flow cytometry in response to 0. 5-h pulse with TNFalpha (2 ng/ml). Purified monoclonal anti-PR-3 IgG added to TNFalpha-primed PBMC induced 45-fold more IL-8 release than an isotype control antibody. Furthermore, alpha 1-antitrypsin (alpha1-AT), the primary PR-3 antiprotease, inhibited the anti-PR-3 induced IL-8 release by 80%. Importantly, Fab and F(ab')2 fragments of anti-PR-3 IgG, which do not result in Fcgamma receptor cross-linking, do not induce IL-8 release. As a correlate, IgG isolated from cANCA positive patients with WG induced six times as much PBMC IL-8 release as compared to IgG isolated from normal healthy volunteers. Consistent with PR-3 associated IL-8 induction, alpha1-AT significantly inhibited this effect. These observations suggest that cANCA may recruit and target neutrophils through promoting monocyte IL-8 release. This induction is mediated via Fcgamma receptor cross-linking and is regulated in part by alpha1-AT.

Topics: alpha 1-Antitrypsin; Antibodies, Antineutrophil Cytoplasmic; Antibodies, Monoclonal; Cells, Cultured; Cross-Linking Reagents; Cycloheximide; Dose-Response Relationship, Drug; Granulomatosis with Polyangiitis; Humans; Immunoglobulin Fab Fragments; Immunohistochemistry; Interleukin-8; Monocytes; Myeloblastin; Receptors, IgG; Serine Endopeptidases; Tumor Necrosis Factor-alpha

Proteinase 3 is the major target antigen of antineutrophil cytoplasmic autoantibodies (ANCA) in Wegener's granulomatosis and is contained in the azurophilic granules of polymorphonuclear neutrophils, the dominant cell type in vascular lesions during the early stages of systemic vasculitis. This study questioned whether neutrophil lysosomal enzymes, once released at the site of inflammation, are able to potentiate the influx of additional neutrophils by enhancing the production of the chemotactic cytokine interleukin-8 (IL-8) by endothelial cells. Therefore, human umbilical vein endothelial cells in culture were incubated with varying concentrations of highly purified proteinase 3, human neutrophil elastase, and cathepsin G for different time periods. The supernatants were subsequently assessed for IL-8 antigen by using a sandwich ELISA. The presence of both proteinase 3 and elastase resulted in an increased production of IL-8, up to 15.6- and 4.2-fold, respectively, in a dose- and time-dependent fashion. Cathepsin G did not influence IL-8 production. Although the addition of an alpha 1-proteinase inhibitor completely abrogated elastase-mediated IL-8 production, it did not significantly influence the effect of proteinase 3. Both proteinase 3-and elastase-mediated production of IL-8 was inhibited by cycloheximide, indicating de novo synthesis. This was supported by the finding of increased IL-8 mRNA levels in proteinase 3-treated human umbilical vein endothelial cells by using Northern blot analysis. Taken together, the neutrophil lysosomal enzymes proteinase 3 and human neutrophil elastase may contribute to a self-perpetuating process of neutrophil recruitment in acute inflammation by increasing de novo synthesis of IL-8 by endothelial cells. The studies presented here also show that proteinase 3 mediates its effect independently of its enzymatic activity, indicating a hitherto unknown mode of action on endothelial cells.

Topics: alpha 1-Antitrypsin; Autoantigens; Autoimmune Diseases; Cathepsin G; Cathepsins; Cells, Cultured; Chemotaxis, Leukocyte; Cycloheximide; Endothelium, Vascular; Gene Expression Regulation; Granulomatosis with Polyangiitis; Humans; Interleukin-8; Lysosomes; Myeloblastin; Neutrophils; Protein Synthesis Inhibitors; RNA, Messenger; Serine Endopeptidases; Stimulation, Chemical; Umbilical Veins

We report a case of Wegener's granulomatosis (WG), with neutrophil accumulation in bronchoalveolar lavage fluid (BALF). Peripheral blood neutrophilia was present but the anti-neutrophil cytoplasmic antibody (ANCA) was negative. The serum and BALF levels of neutrophil-related cytokines, including interleukin (IL)-8, granulocyte colony-stimulating factor (G-CSF) and IL-1 beta, were increased, particularly in BALF. Plasma and BALF levels of neutrophil elastase and defensins, which are released by neutrophils and are potentially toxic to cells, were also elevated. Our findings suggest that neutrophils and neutrophil-related cytokines may play an important role in the pathogenesis of anti-neutrophil cytoplasmic antibody negative as well as anti-neutrophil cytoplasmic antibody positive Wegener's granulomatosis.

Topics: Antibodies, Antineutrophil Cytoplasmic; Blood Bactericidal Activity; Blood Proteins; Bronchoalveolar Lavage Fluid; Cytokines; Defensins; Granulocyte Colony-Stimulating Factor; Granulomatosis with Polyangiitis; Humans; Interleukin-1; Interleukin-8; Leukocyte Elastase; Male; Middle Aged; Neutrophils

Apart from the diagnostic value of anti-neutrophil cytoplasmic antibodies (ANCA), their detailed characterization and that of their corresponding antigens have opened new ways for the exploration of the pathogenesis of primary systemic vasculitis. ANCA are now thought to play an important functional role via activation of phagocytic cells (e.g. polymorphonuclear neutrophils (PMN)). In this study we examined the mechanisms by which ANCA could gain access to proteinase 3 (PR3) in intact PMN, at two levels: ex vivo by analysing the presence of PR3 on the plasma membrane of PMN from patients with ANCA-associated vasculitis, and in vitro by stimulation of PMN using different cytokines, including recombinant tumour necrosis factor-alpha (rhTNF-alpha) and two forms of IL-8 (produced by monocytic and endothelial cells). Using immunocytochemical staining techniques (FACS and immunoelectronmicroscopy) PR3 has been detected on the plasma membrane of PMN from patients with active ANCA-associated vasculitis. However, this phenomenon is also seen in patients with sepsis who do not have ANCA. In addition, TNF-alpha and both forms of IL-8 act synergistically and induce a translocation of PR3 from the intragranular loci to the cell surface of PMN. These results provide strong evidence for the hypothesis that ANCA are directly pathogenic by binding to PR3 which is expressed on the cell surface of primed/activated PMN.

Topics: Antibodies, Antineutrophil Cytoplasmic; Antigens, CD; Autoantibodies; Cell Membrane; Granulomatosis with Polyangiitis; Humans; Interleukin-8; Myeloblastin; Neutrophils; Platelet Membrane Glycoproteins; Serine Endopeptidases; Tetradecanoylphorbol Acetate; Tetraspanin 30; Tumor Necrosis Factor-alpha

The expression of cytokines that are potentially involved in the pathogenesis of vasculitis was studied in patients with primary systemic vasculitis (PSV). In extension of earlier reports, we detected an overexpression of transforming growth factor beta (TGF beta), interleukin 6 (IL6), and interleukin 8 (IL8), indicating that the whole cytokine cascade is activated to a significant extent in PSV.

Topics: Churg-Strauss Syndrome; Cytokines; Gene Expression; Granulomatosis with Polyangiitis; Humans; Interleukin-6; Interleukin-8; RNA, Messenger; Transforming Growth Factor beta; Vasculitis