Proteinase-3 as the major autoantigen of c-ANCA is strongly expressed in lung tissue of patients with Wegener's granulomatosis

Proteinase-3 (PR-3) is a neutral serine proteinase present in azurophil granules of human polymorphonuclear leukocytes and serves as the major target antigen of antineutrophil cytoplasmic antibodies with a cytoplasmic staining pattern (c-ANCA) in Wegener's granulomatosis (WG). The WG disease appears as severe vasculitis in different organs (e.g. kidney, nose and lung). Little is known about the expression and distribution of PR-3 in the lung. We found that PR-3 is expressed in normal lung tissue and is upregulated in lung tissue of patients with WG. Interestingly, the parenchymal cells (pneumocytes type I and II) and macrophages, and not the neutrophils, express PR-3 most strongly and may contribute to lung damage in patients with WG via direct interaction with antineutrophil cytoplasmic antobodies (ANCA). These findings suggest that the PR-3 expression in parenchymal cells of lung tissue could be at least one missing link in the etiopathogenesis of pulmonary pathology in ANCA-associated disease.     

Autoimmunity is triggered by cPR-3(105-201), a protein complementary to human autoantigen proteinase-3

It remains unclear how and why autoimmunity occurs. Here we show evidence for a previously unrecognized and possibly general mechanism of autoimmunity. This new finding was discovered serendipitously using material from patients with inflammatory vascular disease caused by antineutrophil cytoplasmic autoantibodies (ANCA) with specificity for proteinase-3 (PR-3). Such patients harbor not only antibodies to the autoantigen (PR-3), but also antibodies to a peptide translated from the antisense DNA strand of PR-3 (complementary PR-3, cPR-3) or to a mimic of this peptide. Immunization of mice with the middle region of cPR-3 resulted in production of antibodies not only to cPR-3, but also to the immunogen's sense peptide counterpart, PR-3. Both human and mouse antibodies to PR-3 and cPR-3 bound to each other, indicating idiotypic relationships. These findings indicate that autoimmunity can be initiated through an immune response against a peptide that is antisense or complementary to the autoantigen, which then induces anti-idiotypic antibodies (autoantibodies) that cross-react with the autoantigen.     

Staphylococcus aureus and Wegener's granulomatosis

Wegener's granulomatosis (WG) is a form of systemic vasculitis. It is characterized by granulomatous inflammation in the upper and lower airways, vasculitis and necrotizing glomerulonephritis, and is strongly associated with antineutrophil cytoplasmic antibodies against proteinase 3. Since the etiology of the disease is not clear, treatment, consisting of corticosteroids and immunosuppressives, is nonspecific and associated with severe side effects. Pinpointing the trigger(s) of the disease would highly improve treatment. Clinical evidence shows that an infectious agent, the bacterium Staphylococcus aureus, is a risk factor for disease relapse, suggesting its involvement in the pathogenesis of WG. Here we review both clinical and experimental data that either indicate or support a role for S. aureus in WG.     

Staphylococcus aureus and Wegener's granulomatosis

Wegener's granulomatosis (WG) is a form of systemic vasculitis. It is characterized by granulomatous inflammation in the upper and lower airways, vasculitis and necrotizing glomerulonephritis, and is strongly associated with antineutrophil cytoplasmic antibodies against proteinase 3. Since the etiology of the disease is not clear, treatment, consisting of corticosteroids and immunosuppressives, is nonspecific and associated with severe side effects. Pinpointing the trigger(s) of the disease would highly improve treatment. Clinical evidence shows that an infectious agent, the bacterium Staphylococcus aureus, is a risk factor for disease relapse, suggesting its involvement in the pathogenesis of WG. Here we review both clinical and experimental data that either indicate or support a role for S. aureus in WG.     

Lung Fibroblasts Share Mesenchymal Stem Cell Features Which Are Altered in Chronic Obstructive Pulmonary Disease via the Overactivation of the Hedgehog Signaling Pathway

Background

Alteration of functional regenerative properties of parenchymal lung fibroblasts is widely proposed as a pathogenic mechanism for chronic obstructive pulmonary disease (COPD). However, what these functions are and how they are impaired in COPD remain poorly understood. Apart from the role of fibroblasts in producing extracellular matrix, recent studies in organs different from the lung suggest that such cells might contribute to repair processes by acting like mesenchymal stem cells. In addition, several reports sustain that the Hedgehog pathway is altered in COPD patients thus aggravating the disease. Nevertheless, whether this pathway is dysregulated in COPD fibroblasts remains unknown.

Objectives and Methods

We investigated the stem cell features and the expression of Hedgehog components in human lung fibroblasts isolated from histologically-normal parenchymal tissue from 25 patients—8 non-smokers/non-COPD, 8 smokers-non COPD and 9 smokers with COPD—who were undergoing surgery for lung tumor resection.

Results

We found that lung fibroblasts resemble mesenchymal stem cells in terms of cell surface marker expression, differentiation ability and immunosuppressive potential and that these properties were altered in lung fibroblasts from smokers and even more in COPD patients. Furthermore, we showed that some of these phenotypic changes can be explained by an over activation of the Hedgehog signaling in smoker and COPD fibroblasts.

Conclusions

Our study reveals that lung fibroblasts possess mesenchymal stem cell-features which are impaired in COPD via the contribution of an abnormal Hedgehog signaling. These processes should constitute a novel pathomechanism accounting for disease occurrence and progression.     

Wegener's granulomatosis: clinico-radiological finding at initial presentation

Diagnosis of Wegener's granulomatosis at the early stage is difficult because of the nonspecific symptoms which mimic other disorders. The aim of this paper is to describe clinical and radiological features of Wegener's granulomatosis (WG) in a Serbian population at initial presentation. A retrospective review of 37 patient's case records was carried out. All those patients were diagnosed with WG and they attended the Institute for lung diseases in Belgrade over the period of 15 years. There were 20 males and 17 females, ranging in age from 18 to 73 years (mean age 46.2 years). The mean period from the onset of the first symptoms to diagnosis of WG was 4.59 +/- 6.15 months. The criteria of American College of Rheumatology were fulfilled in all patients. Twenty-five of 37 patients had systemic, generalized form of WG and while 12 of them had a limited involvement of upper and lower respiratory system. The frequency of different system involvement was: upper respiratory tract 64.8%, lower respiratory tract 100%, kidneys 67.5%, musculoskeletal system 40.5%, skin 27.2%, eyes 8.1%, and nervous system two patients. ANCA (antineutrophil cytoplasmic antibodies) test was positive in 32 ((86.5%) patients, and negative in 5 (13.5%). All patients were ANA negative. Histological evidence of granulomatous vasculitis was obtained in 34 (91.9%), whereas in three patients the diagnosis was based on clinical manifestations and positive c-ANCA test. There are minor variations in our data when compared with those reported in literature.     

Lung CD57+ cell density is increased in very severe COPD

Among all inflammatory cells involved in COPD, those with a cytolytic or elastolytic activity are thought to play a key role in the pathogenesis of the disease. However, there is no data about the infiltration of cells expressing the CD57 marker in small airways and parenchyma of COPD patients. In this study, surgical specimens from 43 subjects undergoing lung resection due to lung cancer (9 non-smokers, 18 smokers without COPD and 16 smokers with moderate COPD) and 16 patients undergoing double lung transplantation for very severe COPD were examined. CD57+ cells, neutrophils, macrophages and mast cells infiltrating bronchioles (epithelium, smooth muscle and connective tissue) and parenchymal interstitium were localized and quantified by immunohistochemical analysis. Compared to the other groups, the small airways of very severe COPD patients showed a significantly higher density of CD57+ cells, mainly infiltrated in the connective tissue (p=0.001), and a significantly higher density of neutrophils located characteristically in the epithelium (p=0.037). Also, the density of neutrophils was significantly higher in parenchyma of very severe COPD patients compared with the rest of the groups (p=0.001). Finally, there were significant correlations between the bronchiolar density of CD57+ cells and the FEV1 values (R=-0.43, p=0.022), as well as between the parenchymal density of neutrophils and macroscopic emphysema degree (R=0.43, p=0.048) in COPD groups. These results show that CD57+ cells may be involved in COPD pathogenesis, especially in the most severe stages of the disease.     

Heterogeneity of CD4 and CD8+ memory T cells in localized and generalized Wegener's granulomatosis

Memory T cells display phenotypic heterogeneity. Surface antigens previously regarded as exclusive markers of naive T cells, such as L-selectin (CD62L), can also be detected on some memory T cells. Moreover, a fraction of CD45RO+ (positive for the short human isoform of CD45) memory T cells reverts to the CD45RA+ (positive for the long human isoform of CD45) phenotype. We analyzed patients with biopsy-proven localized Wegener's granulomatosis (WG) (n = 5), generalized WG (n = 16) and age- and sex-matched healthy controls (n = 13) to further characterize memory T cells in WG. The cell-surface expression of CD45RO, CD45RA, CD62L, CCR3, CCR5 and CXCR3 was determined on blood-derived T cells by four-color flow cytometric analysis. The fractions of CCR5+ and CCR3+ cells within the CD4+CD45RO+ and CD8+CD45RO+ memory T cell populations were significantly expanded in localized and generalized WG. The mean percentage of Th1-type CCR5 expression was higher in localized WG. Upregulated CCR5 and CCR3 expression could also be detected on a fraction of CD45RA+ T cells. CD62L expression was seen on approximately half of the memory T cell populations expressing chemokine receptors. This study demonstrates for the first time that expression of the inducible inflammatory chemokine receptors CCR5 and CCR3 on CD45RO+ memory T cells, as well as on CD45RA+ T cells ('revertants'), contributes to phenotypic heterogeneity in an autoimmune disease, namely WG. Upregulated CCR5 and CCR3 expression suggests that the cells belong to the effector memory T cell population. CCR5 and CCR3 expression on CD4+ and CD8+ memory T cells indicates a potential to respond to chemotactic gradients and might be important in T cell migration contributing to granuloma formation and vasculitis in WG.     

Clinical utility of fine needle aspiration biopsy in the diagnosis of Wegener's granulomatosis. A report of two cases.

In two patients, pulmonary lesions of Wegener's granulomatosis (WG) were sampled by fine needle aspiration biopsy: one with the clinical diagnosis of primary pulmonary malignancy and the other with a clinical suspicion of WG. In the latter case the smears showed distinctive eosinophilic, collagen necrosis (pathergic necrosis), poorly formed granulomata composed of loose aggregates of elongated, often palisading epithelioid histiocytes, and multinucleate histiocytes. A cell block preparation in this case contained minute tissue fragments illustrating the distinctive, pathergic-type necrosis. In the former case, many of these features were present, but additionally there were several groups, atypical bronchial epithelial cells that, in light of the clinical impression, initially led to an incorrect diagnosis of bronchoalveolar carcinoma. Subsequent review of this case led to the diagnosis of WG. Antineutrophil cytoplasmic antibody (ANCA) serology was later obtained, confirming the diagnosis of WG in both cases. In our experience, the cytomorphologic findings of granular collagen necrosis, granulomata and multinucleate cells, although not specific, should alert the cytopathologist to consider the diagnosis of WG, especially when special stains for microorganisms are negative. A recommendation for ANCA serology testing early in the disease process, particularly in the limited forms of the disease, may lead to early recognition of WG, resulting in prompt institution of immunosuppressive therapy, greatly improving the patient's prognosis.     

Free radical generation induces epithelial-to-mesenchymal transition in lung epithelium via a TGF-β1-dependent mechanism

Fibrotic remodelling of lung parenchymal and airway compartments is the major contributor to life-threatening organ dysfunction in chronic lung diseases such as idiopathic pulmonary fibrosis (IPF) and Chronic Obstructive Pulmonary Disease (COPD). Since transforming growth factor-β1 (TGF-β1) is believed to play a key role in disease pathogenesis and markers of oxidative stress are also commonly detected in bronchoalveolar lavage (BAL) from such patients we sought to investigate whether both factors might be interrelated. Here we investigated the hypothesis that oxidative stress to the lung epithelium promotes fibrotic repair by driving epithelial-to-mesenchymal transition (EMT) via the augmentation of TGF-β1. We show that in response to 400μM hydrogen peroxide (H(2)O(2)) A549 cells, used a model for alveolar epithelium, and human primary bronchial epithelial cells (PBECs) undergo EMT displaying morphology changes, decreased expression of epithelial markers (E-cadherin and ZO-1), increased expression of mesenchymal markers (vimentin and α-smooth muscle actin) as well as increased secretion of extracelluar matrix components. The same oxidative stress also promotes expression of TGF-β1. Inhibition of TGF-β1 signalling as well as treatment with antioxidants such as phenyl tert-butylnitrone (PBN) and superoxide dismutase 3 (SOD3) prevent the oxidative stress driven EMT-like changes described above. Interventions also inhibited EMT-like changes. This study identifies a link between oxidative stress, TGF-β1 and EMT in lung epithelium and highlights the potential for antioxidant therapies to limit EMT and its potential contribution to chronic lung disease.     

Antineutrophil cytoplasmic antibody (ANCA)-associated autoimmune diseases induced by antithyroid drugs: comparison with idiopathic ANCA vasculitides

Clinical and serological profiles of idiopathic and drug-induced autoimmune diseases can be very similar. We compared data from idiopathic and antithyroid drug (ATD)-induced antineutrophil cytoplasmic antibody (ANCA)-positive patients. From 1993 to 2003, 2474 patients were tested for ANCA in the Laboratory for Allergy and Clinical Immunology in Belgrade. Out of 2474 patients, 72 (2.9%) were anti-proteinase 3 (PR3)- or anti-myeloperoxidase (MPO)-positive and their clinical and serological data were analyzed. The first group consisted of ANCA-associated idiopathic systemic vasculitis (ISV) diagnosed in 56/72 patients: 29 Wegener's granulomatosis (WG), 23 microscopic polyangiitis (MPA) and four Churg-Strauss syndrome. The second group consisted of 16/72 patients who became ANCA-positive during ATD therapy (12 receiving propylthiouracil and four receiving methimazole). We determined ANCA and antinuclear (ANA) antibodies by indirect immunofluorescence; PR3-ANCA, MPO-ANCA, anticardiolipin (aCL) and antihistone antibodies (AHA) by ELISA; and cryoglobulins by precipitation. Complement components C3 and C4, alpha-1 antitrypsin (α1 AT) and C reactive protein (CR-P) were measured by nephelometry. Renal lesions were present in 3/16 (18.8%) ATD-treated patients and in 42/56 (75%) ISV patients (p <0.001). Skin lesions occurred in 10/16 (62.5%) ATD-treated patients and 14/56 (25%) ISV patients (p <0.01). ATD-treated patients more frequently had MPO-ANCA, ANA, AHA, aCL, cryoglobulins and low C4 (p <0.01). ISV patients more frequently had low α1 AT (p = 0.059) and high CR-P (p <0.001). Of 16 ATD-treated patients, four had drug-induced ANCA vasculitis (three MPA and one WG), while 12 had lupus-like disease (LLD). Of 56 ISV patients, 13 died and eight developed terminal renal failure (TRF). There was no lethality in the ATD-treated group, but 1/16 with methimazole-induced MPA developed pulmonary-renal syndrome with progression to TRF. ANCA-positive ISV had a more severe course in comparison with ATD-induced ANCA-positive diseases. Clinically and serologically ANCA-positive ATD-treated patients can be divided into two groups: the first consisting of patients with drug-induced WG or MPA which resemble ISV and the second consisting of patients with LLD. Different serological profiles could help in the differential diagnosis and adequate therapeutic approach to ANCA-positive ATD-treated patients with symptoms of systemic disease.     

PR-39 coordinates changes in vascular smooth muscle cell adhesive strength and locomotion by modulating cell surface heparan sulfate-matrix interactions

PR-39 is proline-rich peptide produced at sites of tissue injury. While the functional properties of this peptide have not been fully defined, PR-39 may be an important regulator of processes related to cell-matrix adhesion since it reportedly upregulates syndecan-4, which is a critical determinant of focal adhesion formation. The ability of PR-39 to modulate the adhesion and chemokinetic migration behavior of arterial smooth muscle cells (SMCs) in a fashion coordinated with syndecan-4 expression was investigated. Treatment of SMCs with PR-39 did not alter syndecan-1 mRNA, but did induce a two-fold increase in syndecan-4 mRNA (P < 0.0001) and significantly enhanced cell surface expression of both syndecan-4 (P < 0.01) and heparan sulfate (HS) (P < 0.05). These observations were consistent with an observed increase in cell-matrix adhesive strength (P < 0.05) and a reduction in cell speed (P < 0.01) on fibronectin-coated substrates. Incubation of PR-39 treated cells with a soluble fibronectin derived heparin-binding peptide, as a competitive inhibitor of heparan sulfate/matrix interactions, abolished these effects. These data suggest that PR-39 mediated alterations of cell adhesion and motility may be related, in part, to the increased expression of heparan sulfate glycosaminoglycans (GAGs) that accompany the upregulation of cell surface syndecan-4. Furthermore, this investigation supports the notion that factors which control syndecan-4 expression may play an important role in regulating adhesion related cell processes.     

Differential glycosylation of polyclonal IgG, IgG-Fc and IgG-Fab isolated from the sera of patients with ANCA-associated systemic vasculitis

Post-translational modifications (PTMs) of proteins produced in vivo may be tissue, developmentally and/or disease specific. PTMs impact on the stability and function of proteins and offer a challenge to the commercial production of protein biotherapeutics. We have previously reported a marked deficit in galactosylation of oligosaccharides released from polyclonal IgG isolated from sera of patients with the anti-neutrophil cytoplasmic antibodies (ANCA) associated vasculitides; Wegener's granulomatosis (WG) and microscopic polyangiitis (MPA). Whilst normal polyclonal IgG molecules are glycosylated within the IgG-Fc region, approximately 20% of molecules also bear oligosaccharides attached to the variable regions of the light or heavy chain IgG-Fab. It is of interest, therefore to compare profiles of oligosaccharides released from the IgG-Fc and IgG-Fab of normal IgG with that isolated from the sera of patients with WG or MPA. This study shows that whilst the oligosaccharides released from ANCA IgG-Fc are hypogalactosylated those released from IgG-Fab are galactosylated and sialylated. These results show that hypogalactosylation of IgG-Fc is not due to a defect in the glycosylation or processing machinery. It rather suggests a subtle change in IgG-Fc conformation that influences the addition of galactose. Remarkably, this influence is exerted on all plasma cells. Interestingly, a licensed monoclonal antibody therapeutic, produced in Sp2/0 cells, is also shown to be hypogalactosylated in its IgG-Fc but fully galactosylated in its IgG-Fab.     

Apelin: a new plasma marker of cardiopulmonary disease

OBJECTIVES: Dyspnea is a major symptom of both parenchymal lung disease and chronic heart failure. Underlying cardiac dysfunction can be assessed by measurement of cardiac-derived B-type natriuretic peptide or its precursor in plasma. However, no specific endocrine marker of the lung parenchyma has so far been identified. We therefore examined whether plasma concentrations of apelin, a novel inotropic hormone, is affected in patients with chronic parenchymal lung disease without cardiac dysfunction. METHODS AND RESULTS: Patients with severe chronic parenchymal lung disease and normal cardiac function (n=53), idiopathic pulmonary hypertension with increased right ventricular pressure (n=10), and patients with severe left ventricular systolic dysfunction (n=22) were enrolled. Plasma apelin-36 and proBNP concentrations were measured with radioimmunoassays. While proBNP plasma concentrations were unaffected in chronic parenchymal lung disease patients compared to normal subjects, the apelin-36 concentration was reduced 3.3-fold (median 35 pmol/l (0-162 pmol/l) vs. 117 pmol/l (55-232 pmol/l), P<0.001). Moreover, the apelin-36 concentration was decreased in chronic heart failure patients (2.1-fold, P<0.01) and in patients with idiopathic pulmonary hypertension (4.0-fold, P<0.001). In contrast, the proBNP concentration was highly increased in both chronic heart failure and idiopathic pulmonary hypertension patients. CONCLUSION: Plasma concentrations of apelin-36, a novel inotropic peptide, are decreased in patients with chronic parenchymal lung disease and preserved cardiac function. Combined measurement of apelin-36 and proBNP may be a new diagnostic approach in distinguishing pulmonary from cardiovascular causes of dyspnea.     

Evaluation of the findings of computed tomography of the lungs in patients with nonspecific pneumothorax in late periods after surgical treatment

By using the results of examination of 54 patients operated on for nonspecific spontaneous pneumothorax (NSP), the authors conclude that X-ray computed tomography (CT) is of high value. In addition to external respiratory function that identifies pulmonary functional changes, it is recommended that a comprehensive examination should include X-ray CT with parenchymal densitometry of the upper, middle and lower lung to reveal lung tissue structural changes. The examination of patients should be performed as soon as possible when the disease is detected and in the late postoperative period for early surgical correction of an emphysematous process in the lung.     

What you should know about PR3-ANCA: Evidence for the role of T cells in the pathogenesis of systemic vasculitis

The pathogenesis of systemic vasculitis is complex and is likely to involve many mechanisms. There is a growing body of evidence that T cells may contribute to the pathogenesis of anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitides. Predominantly, T cells and monocytes are found in inflammatory infiltrates in patients with Wegener's granulomatosis (WG). The production of ANCA appears to be T-cell-dependent. T lymphocytes from the peripheral blood of patients with ANCA-associated vasculitis have been shown to proliferate in response to proteinase 3 (PR3). These and other findings outlined in this review indicate T-cell involvement, although further studies are still needed to elucidate the exact contribution of T cells to the pathogenesis of systemic vasculitis.     

Heterogeneity of CD4 and CD8+ memory T cells in localized and generalized Wegener's granulomatosis

Memory T cells display phenotypic heterogeneity. Surface antigens previously regarded as exclusive markers of naive T cells, such as L-selectin (CD62L), can also be detected on some memory T cells. Moreover, a fraction of CD45RO⁺ (positive for the short human isoform of CD45) memory T cells reverts to the CD45RA⁺ (positive for the long human isoform of CD45) phenotype. We analyzed patients with biopsy-proven localized Wegener's granulomatosis (WG) (n = 5), generalized WG (n = 16) and age- and sex-matched healthy controls (n = 13) to further characterize memory T cells in WG. The cell-surface expression of CD45RO, CD45RA, CD62L, CCR3, CCR5 and CXCR3 was determined on blood-derived T cells by four-color flow cytometric analysis. The fractions of CCR5⁺ and CCR3⁺ cells within the CD4⁺CD45RO⁺ and CD8⁺CD45RO⁺ memory T cell populations were significantly expanded in localized and generalized WG. The mean percentage of Th1-type CCR5 expression was higher in localized WG. Upregulated CCR5 and CCR3 expression could also be detected on a fraction of CD45RA⁺ T cells. CD62L expression was seen on approximately half of the memory T cell populations expressing chemokine receptors. This study demonstrates for the first time that expression of the inducible inflammatory chemokine receptors CCR5 and CCR3 on CD45RO⁺ memory T cells, as well as on CD45RA⁺ T cells ('revertants'), contributes to phenotypic heterogeneity in an autoimmune disease, namely WG. Upregulated CCR5 and CCR3 expression suggests that the cells belong to the effector memory T cell population. CCR5 and CCR3 expression on CD4⁺ and CD8⁺ memory T cells indicates a potential to respond to chemotactic gradients and might be important in T cell migration contributing to granuloma formation and vasculitis in WG.     

What you should know about PR3-ANCA. Evidence for the role of T cells in the pathogenesis of systemic vasculitis

The pathogenesis of systemic vasculitis is complex and is likely to involve many mechanisms. There is a growing body of evidence that T cells may contribute to the pathogenesis of anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitides. Predominantly, T cells and monocytes are found in inflammatory infiltrates in patients with Wegener's granulomatosis (WG). The production of ANCA appears to be T-cell-dependent. T lymphocytes from the peripheral blood of patients with ANCA-associated vasculitis have been shown to proliferate in response to proteinase 3 (PR3). These and other findings outlined in this review indicate T-cell involvement, although further studies are still needed to elucidate the exact contribution of T cells to the pathogenesis of systemic vasculitis.