Expression of collagenase and potential transcriptional factors in the MRL/l mouse arthropathy.

Typical erosions of articular joint structures in rheumatoid arthritis and in the spontaneous destructive hind-limb arthropathy of autoimmune MRL-lpr/lpr (MRL/l) mice occur predominantly in areas contiguous with proliferating synovial lining cells, suggesting release of proteolytic enzymes from these cells. Synovial lining cells were isolated from arthritic MRL/l mice, and the spontaneous expression of the interstitial procollagenase and its potential transcriptional factors, egr-1 and c-fos, was examined in vitro. The data indicate that basal collagenase RNA expression was stronger in MRL/l cells than in virus-transformed cells. Moreover, elevated RNA levels of the c-fos gene could be detected in the collagenase-expressing synovial lining cells in vitro. In a related immunohistochemical study, collagenase was detected in situ in proliferating synovial lining cells as well as in chondrocytes of the first stage of pathological changes in the MRL/l mouse arthropathy.

Oncogene activation in rheumatoid synovium.

Rheumatoid arthritis (RA) is a chronic systemic disorder that is dominated by the debilitating sequelae associated with the progressive destruction of articular joints. The molecular and cellular basis of rheumatoid joint destruction is characterized by an abnormal expression of oncogenes modulating cellular proliferation and the induction of lysosomal and metalloproteinases. Based on the observation that the synovial hyperplasia in RA is associated with the proliferation of transformed-appearing synovial lining cells and an overexpression of such oncogenes, the possibility that a hitherto unknown HTLV related retrovirus is involved in the etiopathogenesis of RA is discussed.

New perspectives in pulmonary angiitis. From pulmonary angiitis and granulomatosis to ANCA associated vasculitis.

Traditionally clinical and histopathological features were mainly relied on for classification of vasculitis and granulomatosis of the lung. These can be complemented by immunodiagnostic features which contribute to the classification as well as to the understanding of the pathogenesis of these disorders. Previously five conditions were classified together under the heading "pulmonary angiitis and granulomatosis" (PA & G), mainly on the basis of histological similarities. These conditions have in common a granulomatous histopathology together with necrosis of varying degree, pulmonary vasculitis and occasionally systemic vasculitis. The introduction of novel immunodiagnostic methods led to different approaches of classification, specifically a separation between a group of disorders associated with antinuclear antibodies (ANA), referred to as collagen vascular diseases, and a group of systemic autoimmune diseases unrelated to ANA, referred to as primary systemic vasculitides. Among the latter, Wegener's granulomatosis (WG), Churg-Strauss syndrome (CSS) and microscopic polyangiitis (MPA) are associated with a group of autoantibodies (antineutrophil cytoplasmic antibodies--ANCA) which separate them from other members of the PA & G group. The granulomatous and vasculitic disorders WG and CSS together with the non-granulomatous small-vessel vasculitis MPA now form a new group of diseases ('ANCA-associated vasculitides') which have many clinical, serological and immunohistochemical features in common. Collagen vascular diseases (CVD) are serologically characterized by distinct subspecificities of antinuclear antibodies (ANA), sometimes pronounced hypergammaglobulinaemia, complement consumption and immune deposits (antigen-antibody-complement complexes) which are common in situ in immune-complex vasculitis. In this article newer aspects of the clinical course, the immunodiagnostic procedure, and the immunopathogenesis of the relatively large group of pulmonary angiitis will be described.

Circulating cytokines and soluble CD23, CD26 and CD30 in ANCA-associated vasculitides.

OBJECTIVE: To assess circulating immunoregulatory cytokines and soluble surface markers of T and B cell activation in the plasma of patients with Wegener's granulomatosis (WG), Churg-Strauss syndrome (CSS) and microscopic polyangiitis (MPA) during active and inactive disease, in order to establish their value in discriminating between disease entities and as markers of disease activity. METHODS: Plasma levels of IL-4, IL-5, IL-10, IL-12, IL-13, IFN-gamma and soluble CD23, CD26 and CD30 were determined by enzyme-linked immunosorbent assay in patients with WG (n = 21), CSS (n = 19) and MPA (n = 14) during active disease and remission. RESULTS: Concerning cytokines, no differences were observed for IFN-gamma, IL-4, IL-5 and IL-13. Plasma levels of IL-12 were decreased in all subgroups of patients. On the contrary, IL-10 levels were significantly elevated only in patients with CSS. Levels of sCD30 were significantly increased in patients with active generalized WG and CSS, but not in those with MPA and localized WG, correlating with the disease extent and activity. sCD26 levels were markedly decreased in patients with generalized WG, CSS and MPA and increased towards remission. sCD23 levels were slightly, but not significantly increased in CSS and generalized WG. CONCLUSION: Regarding the investigated immunoregulatory cytokines (Th1/Th2 type), only the measurement of plasma levels of IL-10 discriminated CSS from WG and MPA. The reported data could indicate a similar status of T cell activation in generalized WG and CSS, and possibly a shift in peripheral immunity towards a more humoral dominated immune response. The differences observed between patients with the localized and generalized forms of WG seem to reflect the clinically known biphasic course of this disease.

Autoantibodies to prothrombin and phosphatidylserine/prothrombin-complexes: do they contribute to the serodiagnosis of primary and secondary anti-phospholipid syndrome?

The diagnostic and clinical relevance of Ab to pure and phosphatidylserine-complexed prothrombin for primary and secondary APS was investigated in a total of 357 patients with (n = 169) and without (n = 188) connective tissue diseases. The overall frequency of anti-prothrombin Ab in sAPS, pAPS and patients without APS-related symptoms were found to be 50.0, 37.5 and 22.0%, respectively. From a total of 72 anti-prothrombin-positive samples, 12.5% were specific for pure prothrombin, 31.9% for phosphatidylserine/prothrombin-complexes and 55.6% recognized both antigenic forms. The simultaneous occurrence of other anti-phospholipid Ab was observed in 84% of all sera. Both types of anti-prothrombin Ab are significantly associated with lupus anticoagulant activity, but only Ab to pure prothrombin display such a relationship to clinical manifestations of APS. Based on these results, it cannot be recommended at present to include anti-prothrombin assays in the routine procedure for the serodiagnosis of APS. However, patients negative for lupus anticoagulant and typical APS-related anti-phospholipid Ab should be tested for anti-prothrombin reactivity, favoring, mainly due to its higher specificity, the ELISA containing pure prothrombin as antigen.

Reactivity profiles of autoantibodies to different phospholipids and the phospholipid-binding protein beta2-glycoprotein I in patients with clinical symptoms related to thromboembolic and/or vasculopathic events with or without connective tissue diseases.

To study the antigenic and epitope specificities of anti-phospholipid Ab in detail, we investigated 177 patients without (62 with APS-related systemic clinical symptoms, 115 with microangiopathies) and 164 patients with connective tissue diseases (CTD). Ab associated with primary APS (pAPS) seem to show a restricted specificity (phospholipid/beta2-GPI-complexes), whereas those in secondary APS (sAPS) react additionaly with pure beta2-GPI. Simultaneously, beta2-GPI-independent Ab were also frequently present in both conditions (50% of all Ab-positive sera). In CTD patients, the reactivity profile "pure beta2-GPI + phospholipid/beta2-GPI-complexes" is significantly associated with clinically manifest sAPS. Comparing cardiolipin and phosphatidylserine as antigenic target, the overall concordance (crossreactivity?) between both assays was lower than expected (52%), being highest in pAPS (87%) and sAPS (65%). Based on these results, a two-step procedure for reliable serological diagnosis of APS could be recommended: Ab-screening using a mix of phospholipids complexed with beta2-GPI (sensitivity > 90% for Ab concentrations above 20 U/ml) followed by an assay allowing the simultaneous detection of all relevant antigenic and epitope specificities.

Anti-dsDNA antibody subtypes and anti-C1q antibodies: toward a more reliable diagnosis and monitoring of systemic lupus erythematosus and lupus nephritis.

The putative distinct diagnostic and pathogenic potential of aDNA-Ab subtypes, differing in their affinity or epitope specificity, was subject of several studies with controversial results. Comparing five assays, characterized by different reaction conditions and nature/source of dsDNA, we investigated the abovementioned problem in a retrospective study on 100 systemic lupus erythematosus (SLE) patients and 100 controls (other CTD, autoimmune hepatopathies). As demonstrated, only assay 3 (Farrzyme, TBS, UK) and 5 (Farr-RIA, Trinity Biotech, Ireland) are really suitable to detect primarily high avidity aDNA-Ab. Both were significantly linked to lupus nephritis (specificity 84%) and highly specific for SLE (95 and 96%). Thereby, assay 3 was found to be the first solid phase ELISA probably suitable to replace the Farr-RIA. Classical ELISAs (assay 1, Orgentec, Germany, and 2, Bindazyme, TBS, UK), detecting aDNA-Ab more or less independent from their avidity, or tests with only intermediate specificity for high avidity Ab (assay 4, ELIAdn, Sweden Diagnostics, Germany), were less specific for SLE (83, 79, 91%, respectively) and not associated with renal involvement (specificity 54-57%). At least in the patients studied here, obvious antigen-related differences could not be observed. With slight differences, all assays were suitable to monitor disease activity and therapy in SLE, agreeing with the ECLAM score in about 70-80% of cases. For lupus nephritis, aC1q-Ab are as specific as high avidity aDNA-Ab and capable to close a diagnostic gap in some cases. Thus, to enhance the specificity (up to 98%) and to consider the distinct diagnostic/pathogenic potential of aDNA-Ab subtypes in SLE, under routine clinical laboratory conditions it should be recommended to combine a sensitive screening test with a more specific second assay.

Immunogenetic aspects of ANCA-associated vasculitides. Editorial overview.

The objective of this issue is to review the genetic bases and immunogenetic variations of antineutrophil cytoplasmic antibody (ANCA)-associated vasculitides (AAV), with particular emphasis on major histocompatibility complex class I and II molecules, the polymorphisms in the intercellular adhesion molecule-1 gene, tumor necrosis factor genes, alpha-1-antitrypsin (alpha1AT) and Fcgamma receptor (FcgammaR) genes. Although the aetiologies of the various AAV are unknown, the available genetic epidemiological data support the importance of genetic factors in susceptibility to these diseases. Moreover, there is considerable reason to believe that immunologic mechanisms contribute to the pathogenesis of AAV. The data presented here indicate that, in addition to immunospecific genes, such as HLA alleles, genes determining the level of the immune response (i.e. cytokines, adhesion molecules, protease inhibitors, FcgammaR) may contribute to AAV.

Pathogenesis of Wegener's granulomatosis.

Although a single disease entity, Wegener's granulomatosis (WG) displays a set of clinical manifestations, each with a different immunopathogenesis. Granuloma formation, "pauci-immune" vasculitis and glomerulonephritis (= renal vasculitis) are the histologic hallmarks of WG which can occur together (WG triad) in full-blown disease, or separately in "initial phase" disease or the formes frustes. The different clinical manifestations are characterised by multiple immune abnormalities that culminate in the over-production of autoantibodies directed mainly against proteinase 3 (PR3-ANCA). A number of in vitro observations point to the potential mechanisms by which ANCA could induce neutrophil-mediated vascular injury, i.e. vasculitis. The most commonly postulated scenario for ANCA-mediated vasculitis involves the interaction of polymorphonuclear neutrophils (PMN) and endothelial cells (EC) via cell adhesion molecule interactions. The initiating event is ANCA-induced leukocyte activation, in which PMN-derived mediators (i.e. cytokines, lipid metabolites, etc.) are intimately involved. The result is necrotizing inflammation of blood vessel walls. However, the clinical and pathological hallmark of WG is the coexistence of vasculitis and granuloma. The causative agent(s) leading to granuloma formation, predominantly in the respiratory tract, is still unknown, but the presence of T cells in the granulomatous inflammation indicates T-cell hyperactivity. Immunohistochemical studies have shown that the cellular infiltrations in renal and pulmonary lesions of WG primarily contain CD4+ T-cells and macrophages. Recent investigations have demonstrated that CD4+ T-cells from granulomatous lesions in the nose and from bronchoalveolar lavage (BAL) mainly express the Th1 cytokine profile, which stimulates predominantly cell-mediated immune responses. This result supports the hypothesis that due to the two-phase course of WG there occurs a polarisation of the T-cell sub-population (Th1 versus Th2 type) which may explain the transition from the initial (granulomatous) phase of WG (so-called localized or locoregional restricted WG) to the generalized (vasculitic) phase. In conclusion, although the initiating events in the development of WG are still unknown, remarkable advances have been made in characterizing the infiltrating inflammatory cells and their products, which is of major importance in understanding the pathogenesis of this disease. In this regard, the use of specific mediators (i.e. cytokines, adhesions molecule antagonists, anti-Id ANCA, etc.) to modulate the inflammatory response may prove beneficial in the therapy of WG.

Cathepsin B in synovial cells at the site of joint destruction in rheumatoid arthritis.

Based on the concept that proteolytic enzymes, like cathepsins, are associated with tissue destruction, we investigated the expression of the matrix-degrading cysteine proteinase cathepsin B in synovial tissues from the joints of patients with rheumatoid arthritis. The data indicate an enhanced transcription of cathepsin B in synovial cells when compared with normal fibroblasts, cathepsin B-producing epithelial tumor cells (SW1116), or fibroblasts derived from inflamed tonsils. Immunolocalization of cathepsin B appeared to be restricted mainly to the synovial cells attached to cartilage and bone at sites of rheumatoid joint erosion.

Enzymatic detection systems for non-isotopic in situ hybridization using biotinylated cDNA probes.

The feasibility of various non-isotopic enzymatic detection systems was tested for in situ hybridization using biotin-labelled, nick-translated cDNA probes. For this purpose, we isolated and prepared cDNA restriction fragments encoding the proteolytic cysteine proteinase cathepsin L and analysed Kirsten murine sarcoma virus-transformed BALB/3T3 cells, which have been shown to express high amounts of cytoplasmic RNA of this ras oncogene-induced proteinase. When compared on a semiquantitative basis, colorimetric non-isotopic detection of cDNA hybrids with avidin-biotin-peroxidase conjugates visualized by silver intensification of the nickel-diaminobenzidine end-product was superior to that obtained with avidin-biotin-alkaline phosphatase using different substrates for development. When the peroxidase staining technique was applied for RNA detection, it was found that overnight incubation in methanol containing hydrogen peroxide followed by deproteination with HCl was the most effective method for inhibition of endogenous peroxidase activity. For DNA detection, non-specific nucleic staining was completely abolished when heat treatment (100 degrees C) of the cell specimens was performed prior to hybridization.

Expression of proteolytic cathepsins B, D, and L in periodontal gingival fibroblasts and tissues.

BACKGROUND: A major feature of gingivitis and periodontitis is the destruction of the collagenous matrix of the surrounding connective tissue. The hypothesis that proteolytic enzymes release from cells adjacent to the site of destruction was recently supported by the presence of increased levels of cathepsin B, D, and L in the gingival crevicular fluid of patients suffering from periodontal disease (PD). EXPERIMENTAL DESIGN: We studied the expression of mRNA of cathepsins B,D, and L in early passaged, adherent gingival fibroblasts of patients with chronic adult PD. In addition, we examined the presence of cathepsin D and L mRNA-expressing cells in periodontal tissue specimen by in situ hybridization, and we localized the respective enzymes by immunohistochemistry. RESULTS: Strong gene expression of the three cathepsin types could be detected by dot hybridization in all PD-derived gingival cells. Immunohistochemical distribution of cathepsins and mRNA for cathepsin D and L could be demonstrated mainly in PD tissue specimen, largely in the area in between the epithelium and the adjacent subepithelial connective tissue. Examination of the cell types revealed multiple macrophage- and fibroblast-shaped cells expressing positive staining for cathepsins. CONCLUSIONS: The present data support the the concept that cathepsins play a major role in tissue destruction and may represent a target for therapeutic interventions.

Expression of collagenase and potential transcriptional factors c-fos and egr-1 in periodontal gingival fibroblasts.

In view of the important role of fibroblast-type collagenase in the pathogenesis of periodontal disease (PD), we investigated the expression of this metalloproteinase in primary cultures of non-stimulated fibroblasts dissected from gingival tissues of patients with generalized moderate and localized severe chronic adult PD. Enhanced hybridization signals for collagenase RNA were observed in 8/8 PD-cases when compared with equivalent RNA amounts extracted from normal fibroblasts. Since both the proto-oncogene c-fos and the "early growth response" gene egr-1 might be involved in the transcriptional regulation of the collagenase gene expression in vivo, we also compared the relative expression of both potential transcriptional factors with collagenase RNA in the same fibroblast cytoplasmic extracts. Hybridization signals indicated elevated RNA amounts for c-fos in 8/8 PD-cases and for egr-1 in 7/8 PD-cases when compared with the cells from non-inflamed tissue. In periodontitis gingival tissue specimens, immunolocalization of collagenase could be confirmed in fibroblasts, macrophages and epithelial cells in situ. Collagenase label was not widely distributed within the tissues, but concentrated at the interface between epithelium and connective tissue. The data provide the first evidence that gingival fibroblasts producing elevated levels of collagenase RNA amounts express also c-fos and egr-1 indicating a crucial role for both genes in cellular proliferation and collagenase expression in gingival and periodontal tissue destruction in vivo.

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 vitro.

The theoretical risk of triggering vasculitis resulting from administration of G-CSF and GM-CSF to patients with anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitides (AAV), such as Wegener's granulomatosis (WG), who develop agranulocytosis due to cytotoxic therapy, is unknown. Since there is strong evidence that activation of polymorphonuclear neutrophils (PMN) induced by binding of ANCA to PR3 or myeloperoxidase (MPO) expressed on their plasma membrane is involved in the pathogenesis of systemic vasculitides (SV), we studied the surface expression of PR3 and MPO on PMN from healthy donors in response to G-CSF and GM-CSF in vitro by flow cytometric analysis. Increasing doses of G-CSF did not alter PR3 expression on either untreated or tumour necrosis factor-alpha (TNF-alpha)-primed donor PMN significantly. In contrast, GM-CSF significantly increased PR3 membrane expression on both intact PMN and neutrophils primed with TNF-alpha. MPO expression was not significantly altered by either G-CSF or GM-CSF. In summary, these data demonstrate that GM-CSF, but not G-CSF, induces plasma membrane expression of PR3 on PMN in vitro. Since in AAV accessibility of the antigen (PR3 or MPO) to the antibody (ANCA) on the plasma membrane of PMN is thought to be essential for neutrophil activation by ANCA, the results of the present study suggest that administration of GM-CSF to patients with WG with neutropenia implies a definite theoretical risk of deterioration of vasculitis via this mechanism.

Elevated interleukin-4 and interleukin-13 production by T cell lines from patients with Churg-Strauss syndrome.

OBJECTIVE: To investigate cytokine production patterns of T cell lines (TCL) from patients with Churg-Strauss syndrome (CSS). METHODS: Short-term polyclonal TCL were generated from peripheral blood of patients with CSS or Wegener's granulomatosis (WG) and healthy controls (HC). TCL were established in the presence of interleukin-2 (IL-2) and phytohemagglutinin and were phenotypically characterized by flow cytometry. Th1/ Th2 cytokine production by stimulated TCL (72 hours) was analyzed by enzyme-linked immunosorbent assay. RESULTS: TCL that represented the progeny of in vivo-activated T cells from CSS patients displayed a heterogeneous immunophenotype, with a predominance of CD4+ T cells when compared with WG TCL, which were predominantly CD8+. All CSS TCL shared the ability to produce large amounts of interferon-gamma (IFNgamma), IL-4, and IL-13 compared with HC (P = 0.014 for all 3). Production of IL-4 and IL-13 was higher in CSS TCL than in WG TCL (P = 0.014 for both). IL-5 production was up-regulated in WG TCL compared with CSS TCL (P = 0.014). Compared with HC, WG TCL showed increased production of IFNgamma (P = 0.021), IL-5 (P = 0.043), and IL-13 (P = 0.021). CONCLUSION: Our results indicate that, while there is evidence for both a type 1 and a type 2 response in CSS, type 2 cytokine production pattern appears to predominate in this disease when compared with WG and HC.

Intracellular secretory pathway and ultrastructural localization of interstitial procollagenase in human gingival fibroblasts.

Fibroblast-type collagenase, a neutral secretory metalloproteinase capable of cleaving interstitial collagen types I-III, is expressed by a number of different cell types including fibroblasts, macrophages, osteoblasts, and keratinoyctes. To elucidate the secretory pathway of this enzyme, we examined the ultrastructural localization of this metalloproteinase in cultured human gingival fibroblasts, particularly the routing of the enzyme from the Golgi cisternae to the cell surface utilizing rabbit polyclonal antibodies raised against human fibroblast (pro) collagenase. For this purpose, one percent glutaraldehyde followed by gentle permeabilization with saponin gave superior preservation of both cellular morphology and intracellular antigenicity. At the light microscopic level, the reacting antibodies visualized by immunofluorescence and immunoperoxidase staining were localized intracellularly in the perinuclear region reflecting the Golgi apparatus. Immunoelectronmicroscopy using the pre-embedding technique and peroxidase or immunogold staining revealed electron dense label in large vacuoles indicating extended cisternae of the Golgi field. Vesicles were noted leaving the plasma membrane in long extensions. Moreover, intact vesicle containing the antibody reaction product appeared outside the membrane. In addition, most extracellular vesicular structures appeared empty of label suggesting that the collagenase had been liberated into the extracellular space. The latter observation was supported by the fact that the label was found also on the extracellular surface of the cells indicating a (re)association of collagenase with the outer cell membrane. These data demonstrate that the pathway of interstitial collagenase in human gingival fibroblasts is similar to that of other secretory proteins.

Elevated monocytic IL-12 and TNF-alpha production in Wegener's granulomatosis is normalized by cyclophosphamide and corticosteroid therapy.

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.

Spontaneous expression of immediately-early response genes c-fos and egr-1 in collagenase-producing rheumatoid synovial fibroblasts.

In view of the important role of interstitial collagenase in the pathogenesis of rheumatoid arthritis (RA), we studied the expression of fibroblast-type collagenase in rheumatoid synovium and searched for its potential transcription factors, namely the oncoprotein c-fos and the early-growth-response gene-1 (egr-1), an inducible zinc-finger encoding gene. Elevated levels of RNA sequences complimentary to c-fos and egr-1 cDNA probes could be detected in cytoplasmic extracts of collagenase-expressing synovial fibroblast-like cells when compared to equivalent RNA amounts isolated from control fibroblasts. Utilizing immunocytochemistry, immunoreactivity for c-fos oncoprotein was found in 13 of 19 joint specimens obtained from patients with active RA. These oncoprotein data were positively correlated to the collagenase expression in the same specimens. Moreover, immunohistochemical analysis confirmed the localization of both oncoprotein c-fos and fibroblast-type collagenase within synovial fibroblast-like cells attached to bone erosions.

Cytokine profiles in Wegener's granulomatosis: predominance of type 1 (Th1) in the granulomatous inflammation.

OBJECTIVE: To determine whether a specific cytokine pattern (type 1 [Th1] or type 2 [Th2]) predominates in Wegener's granulomatosis (WG), by evaluating interferon-gamma (IFNgamma) and interleukin-4 (IL-4) expression in different compartments of the body (i.e., biopsied nasal mucosal tissue [NBS], bronchoalveolar lavage [BAL] fluid, and peripheral blood [PB]) and comparing the findings with those in disease and healthy control subjects. METHODS: Competitive reverse transcriptase-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay were used to assess IFNgamma and IL-4 expression in T cell clones (TCC), T cell lines (TCL), and polyclonal CD4+ and CD8+ cells derived from NBS, BAL, and PB. RESULTS: Patients with WG and chronic rhinitis were found to share in situ production of messenger RNA (mRNA) specific for IFNgamma (Th1). Only 2 patients with WG expressed IL-4, whereas IL-4 mRNA PCR products were found in inflamed tissues of the disease control patients. The granuloma-derived T cells of WG patients produced only IFNgamma, while TCC, TCL, and CD4+ and CD8+ T cells from BAL and PB produced mainly IFNgamma. CONCLUSION: Our data indicate that a Thl cytokine pattern predominates in the granulomatous inflammation in patients with WG.