Histologic analysis of renal leukocyte infiltration in antineutrophil cytoplasmic antibodyassociated vasculitisImportance of monocyte and neutrophil infiltration in tissue damage.код для вставкиСкачать
ARTHRITIS & RHEUMATISM Vol. 50, No. 11, November 2004, pp 3651–3657 DOI 10.1002/art.20607 © 2004, American College of Rheumatology Histologic Analysis of Renal Leukocyte Infiltration in Antineutrophil Cytoplasmic Antibody–Associated Vasculitis Importance of Monocyte and Neutrophil Infiltration in Tissue Damage Sven Weidner,1 Marina Carl,1 Regine Riess,2 and Harald D. Rupprecht1 ular infiltration of CD68-positive macrophages with serum creatinine concentration at the time of biopsy (P ⴝ 0.001 and P ⴝ 0.006, respectively). Conclusion. These data underscore a major role of monocytes in addition to neutrophils in the tissue damage of AAV. Objective. The histopathologic lesions in antineutrophil cytoplasmic antibody (ANCA)–associated vasculitis (AAV) have been studied extensively, but the exact composition of the cellular infiltrate is unclear. We undertook this study to analyze renal leukocyte infiltration and the cellular distribution within glomeruli and interstitium in 65 renal biopsy samples obtained from patients newly diagnosed as having AAV. Methods. Renal cellular tissue infiltration was assessed with an immunoperoxidase method. Furthermore, the infiltrating cell types were correlated with clinical and histopathologic data. Results. The predominant interstitial infiltrating cells were T lymphocytes, while monocytes and, to a lesser extent, granulocytes constituted the dominant infiltrating cell types in glomeruli. Interestingly, lymphocyte infiltration was predominantly periglomerular, especially around glomeruli with sclerosis or heavy crescent formation, while interstitial monocyte and neutrophil infiltration was diffusely distributed over the interstitial tissue. A significant correlation was found for the glomerular infiltration of CD68-positive macrophages with the presence of glomerular necrosis as well as with the number of glomeruli with crescents (P < 0.0001 and P ⴝ 0.005, respectively). No correlation was found for interstitial fibrosis with the infiltration of any leukocyte subset. Furthermore, a significant correlation was found for the interstitial as well as for the glomer- The antineutrophil cytoplasmic antibody (ANCA)–associated vasculitides, Wegener’s granulomatosis (WG), microscopic polyangiitis (MPA), and renal limited vasculitis (RLV) are chronic autoimmune diseases with the development of fibrinoid necrosis and tissue inflammation of histologic heterogeneity in several organ systems. The lesions seem to occur predominantly in vessel-rich tissues involved in filtration processes or are located at mucous membranes (1). Consequently, one of the hallmarks of these diseases is renal involvement, which is also linked to a higher mortality (2,3). Renal involvement in ANCA-associated vasculitis (AAV) is characterized by focal and segmental necrotizing glomerulonephritis coupled with extracapillary proliferation and crescent formation of extremely variable intensity. The most characteristic feature distinguishing this glomerular disease from other types of glomerulonephritis is the scarcity or absence of immune complex deposition (termed “pauci-immune”). This inevitably focuses our attention on the infiltrating cells as initiators of tissue destruction. The analysis of crescentic glomerulonephritis of various causes has demonstrated a significant increase of glomerular granulocytes and monocytes, while the interstitial infiltrates have been found to be primarily composed of T lymphocytes and monocytes (4–6). The leukocyte subpopulations in the inflamma- 1 Sven Weidner, MD, Marina Carl, PhD, Harald D. Rupprecht, MD: Ludwig-Maximilians-University, Munich, Germany; 2 Regine Riess, MD: Klinikum Nürnberg, Nuremberg, Germany. Address correspondence and reprint requests to Sven Weidner, MD, Medizinische Poliklinik, Klinikum der Universität München–Innenstadt, Pettenkoferstrasse 8a, D-80336 Munich, Germany. E-mail: email@example.com. Submitted for publication May 7, 2004; accepted in revised form August 2, 2004. 3651 3652 WEIDNER ET AL tory cell infiltrates within glomeruli and interstitium in renal biopsy samples from patients with AAV have not been completely analyzed. Although some data pointed to a cell-mediated type of immunity with lymphocytes as the supposed initiators of the inflammatory process (7), there is increasing evidence for a dominant role of monocytes in the tissue damage of AAV. In smaller biopsy studies, the cell infiltrates of vasculitic tissue lesions were primarily composed of monocytes (8,9). Further characterization of these cells in the proliferative lesions and crescents of necrotizing glomerulonephritis demonstrated the accumulation of activated monocytes (10). In this study, we analyzed the pattern of renal leukocyte infiltration and the distribution of leukocyte subpopulations in the interstitial and glomerular compartment. Furthermore, we performed a correlation of the infiltrating cell types with clinical and histopathologic data. PATIENTS AND METHODS Patient selection. Sixty-five renal biopsy samples from 65 patients newly diagnosed as having small-vessel vasculitis according to the Chapel Hill Consensus Conference (CHCC) criteria (11) were selected for this study. All patients had clinical features of renal involvement and histologically focal necrotizing glomerulonephritis with few or no immunoglobulin deposits (pauci-immune). Only patients diagnosed as having WG, MPA, or RLV were included. Biopsy samples from patients with immune complex vasculitides such as HenochSchönlein purpura or cryoglobulinemic vasculitis were not analyzed. Furthermore, biopsy samples from patients with secondary vasculitis due to autoimmune diseases or from patients with Goodpasture’s syndrome or anti–glomerular basement membrane nephritis were excluded from this analysis. Biopsy samples were obtained before or within 24 hours of the initiation of immunosuppressive treatment. All patients received induction treatment with orally administered cyclophosphamide and corticosteroids. In 32 patients, treatment with corticosteroids was preceded by intravenous (IV) pulse methylprednisolone for 3 consecutive days. Plasmapheresis was additionally used in 3 patients. These 3 patients were included in the MEPEX (randomized trial of adjunctive therapy for severe glomerulonephritis in ANCAassociated systemic vasculitis: plasma exchange versus intravenous methylprednisolone) trial of the European Vasculitis Study Group, comparing plasmapheresis with IV pulse methylprednisolone for treatment of severe renal failure in systemic vasculitis. Patient classification. Patients were retrospectively reclassified according to the CHCC criteria (11) as having WG, MPA, or RLV. Patients were classified as having WG if they had systemic vasculitis and the presence of granulomatous inflammation in a biopsy specimen or the presence of clinical signs strongly suggestive of granulomatous disease. These Table 1. Primary antibodies used for immunohistochemistry Antibody Specificity Dilution Anti-CD3 Anti-CD4 Anti-CD8 Anti-CD15 Anti-CD68 Anti-CD79 T lymphocytes T helper cells T cytotoxic/suppressor cells Granulocytes Macrophages B lymphocytes 1:150 1:20 1:50 1:50 1:150 1:50 comprised involvement of the upper respiratory tract with nasal inflammation (purulent/bloody nasal discharge), sinusitis, or otitis media or lower respiratory tract manifestation with pulmonary nodules or fixed infiltrates. Patients were classified as having MPA if they had systemic vasculitis and the absence of granuloma formation in a biopsy specimen and the absence of clinical signs strongly suggestive of granulomatous disease. Patients were classified as having RLV if they had biopsy-proven pauci-immune necrotizing glomerulonephritis without symptoms of systemic vasculitis. According to the CHCC, noninvasive evaluations could be used to identify abnormalities that adequately predicted the presence of granulomatous inflammation without having to perform a histologic examination. As required by the CHCC nomenclature (11), ANCA antigen specificity was not used as a definition criterion. Periglomerular inflammation or heavy crescentic destruction of the glomerulus often appears as granuloma-like lesions on renal biopsy. This phenomenon was not used to classify patients as having WG, since it appears as a nonspecific feature of any crescentic glomerulonephritis. ANCA analysis. All patients had been tested for the presence of ANCA by indirect immunofluorescence (IIF) as well as for the presence of proteinase 3–ANCA (PR3-ANCA) and myeloperoxidase-ANCA (MPO-ANCA) by enzymelinked immunosorbent assay (ELISA). The IIF tests and ELISA systems used for ANCA detection were manufactured by Euroimmun (Lübeck, Germany). Renal biopsy tissue preparation. Renal tissue was fixed in 4% paraformaldehyde immediately after biopsy and embedded in paraffin according to standard techniques. Sections were stained with hematoxylin and eosin and mounted with glycerol gelatin (Merck, Darmstadt, Germany). Immunohistochemistry. To assess renal cellular tissue infiltration, a 3-step immunoperoxidase method was used. Staining for CD3, CD4, and CD8 demonstrated T lymphocytes as well as T cell subsets. CD79 staining was performed for B lymphocytes. CD15 was used as a marker for granulocytes and CD68 for the demonstration of macrophages. Sections were deparaffinized, rehydrated, and fixed in methanol–H2O2 (Merck) to suppress endogenous peroxidase activity. For CD3 staining, sections were pretreated with 0.1% Pronase (Dako, Hamburg, Germany) for 10 minutes. Sections stained for CD4, CD8, and CD79 required microwave pretreatment in citrate buffer for 4 minutes at 600W, followed by 10 minutes at 250W. Staining for CD15 and CD68 was performed without pretreatment. After washing in phosphate buffered saline (Biochrom, Berlin, Germany), sections were incubated with the primary monoclonal anti-human antibodies (see Table 1) for 1 hour. CELLULAR INFILTRATION IN ANCA-ASSOCIATED VASCULITIS Anti-CD3, anti-CD8, anti-CD15, anti-CD68, and anti-CD79 were from Dako. Anti-CD4 was from Novocastra (Newcastleupon-Tyne, UK). For CD3 staining, peroxidase-labeled goat anti-rabbit IgG (Dianova, Hamburg, Germany) and horseradish peroxidase–labeled rabbit anti-goat IgG (Dako) were used as second and third antibodies, respectively, at a dilution of 1:100. All other stainings were performed with rabbit antimouse IgG (Dako) and goat anti-rabbit IgG (Dianova) as subsequent antibodies at a dilution of 1:100. Staining was visualized with aminoethylcarbazole (Dako). Sections were counterstained with Mayer’s hematoxylin (Sigma, Deisenhofen, Germany) and mounted with glycerol gelatin. Tissue analysis and evaluation of leukocyte infiltration. Tissue analysis was evaluated with a blinded protocol. Routine histopathologic assessment was performed by an experienced nephropathologist in accordance with a previously standardized scoring protocol (12). Each glomerulus was evaluated separately for the presence of fibrinoid necrosis, extracapillary proliferation, sclerosis (local, segmental, or global), and rupture of Bowman’s capsule. The number of glomeruli with any of these lesions was calculated as a percentage of the total number of glomeruli in the individual biopsy sample. For the correlation of cellular infiltration with histologic lesions, these percentages were recoded on a 5-point scale as follows: 0 for 0%; 1 for ⱕ25%; 2 for ⱕ50%; 3 for ⱕ75%; and 4 for ⱕ100%. The interstitial lesions were scored on a 4-point scale (⫺ ⫽ absent; ⫹ ⫽ mild; ⫹⫹ ⫽ moderate; and ⫹⫹⫹ ⫽ strong). The interstitial infiltrating cells were counted in 8 fields at 400⫻ magnification, referring to an area of 0.16 mm2/field. The results were expressed as the number of positive cells per square millimeter. Glomerular infiltration was assessed as the mean number of infiltrating cells per glomerular cross-section (gcs). Statistical analysis. Statistical analysis was performed with SPSS 11.0 for Windows (SPSS, Chicago, IL). The mean ⫾ SD was reported for normally distributed data. The chi-square test was used for comparison of categorical data. Bivariate Spearman’s correlation was applied to determine an association of leukocyte infiltration with histopathologic lesions and of leukocyte infiltration with renal function. Since several parameters were used for these calculations, the level of significance was set to P ⬍ 0.008 according to the Bonferroni adjustment. For all other analyses, P values less than 0.05 were considered significant. All tests were 2-tailed. 3653 Table 2. Frequency of ANCA subspecificities in association with diseases, as determined by ELISA* PR3-ANCA MPO-ANCA ANCA negative Total (n ⫽ 65) WG (n ⫽ 17) MPA (n ⫽ 27) RLV (n ⫽ 21) 31 (48) 28 (43) 6 (9) 14 (82) 3 (18) 0 (0) 9 (33) 17 (63) 1 (4) 8 (38) 8 (38) 5 (24) * Values are the number (%) of patients. ANCA ⫽ antineutrophil cytoplasmic antibody; ELISA ⫽ enzyme-linked immunosorbent assay; WG ⫽ Wegener’s granulomatosis; MPA ⫽ microscopic polyangiitis; RLV ⫽ renal limited vasculitis; PR3 ⫽ proteinase 3; MPO ⫽ myeloperoxidase. Renal function and histopathologic lesions. The mean ⫾ SD serum creatinine concentration at the time of biopsy was 423 ⫾ 270 moles/liter. Proteinuria was present in all patients, with a mean ⫾ SD level of 1,700 ⫾ 1,916 mg protein/24 hours. Crescentic glomerulonephritis was present in 60 biopsy samples (92%), with fibrinoid necrosis in 54 biopsy samples (83%). Interstitial infiltration. There was a mean ⫾ SD of 1,085.6 ⫾ 1,152.2 leukocytes/mm2 infiltrating the interstitium. The values for interstitial leukocytes are listed in Table 3. Interstitial infiltration was most prominent for CD3-positive lymphocytes, with a mean ⫾ SD level of 328.3 ⫾ 376/mm2. The numbers of CD4-positive lymphocytes and CD8-positive cells were approximately the same (117.9 ⫾ 158.4/mm2 and 164 ⫾ 191.7/mm2, respectively). Only 6 biopsy samples were negative for CD3-positive cells, and 3 biopsy samples were completely negative for T cells. B lymphocytes were also observed. There was a considerable number of CD68positive macrophages (241.3 ⫾ 401.9/mm2). Lymphocyte infiltration was predominantly periglomerular, especially around glomeruli with sclerosis or heavy crescent formation, while interstitial monocyte and neutrophil infiltration was diffusely distributed over the interstitial tissue (Figures 1A–F). Vascular infiltration was rare. There was no difference in interstitial RESULTS Patient classification and ANCA subspecificities. Seventeen patients (26%) were classified as having WG and 27 patients (42%) were classified as having MPA. RLV was present in 21 patients (32%). By ELISA, PR3-ANCA or MPO-ANCA were found in 59 patients (91%). PR3-ANCA were detected in 31 patients (48%) and MPO-ANCA were found in 28 patients (43%). Six patients were ANCA negative. Table 2 shows the frequency of ANCA subspecificities in association with diseases. Table 3. Interstitial and glomerular leukocyte infiltration* Marker Interstitium, cells/mm2 Glomerulus, cells/gcs CD3 CD4 CD8 CD15 CD68 CD79 Total leukocyte count 328.3 ⫾ 376 117.9 ⫾ 158.4 164 ⫾ 191.7 48.4 ⫾ 80.5 241.3 ⫾ 401.9 185.5 ⫾ 258.9 1,085.6 ⫾ 1,152.2 1.3 ⫾ 2.6 0.3 ⫾ 1.2 0.6 ⫾ 1.6 3.2 ⫾ 7.4 4.7 ⫾ 11.1 0.1 ⫾ 0.3 10.2 ⫾ 17.6 * Values are the mean ⫾ SD. gcs ⫽ glomerular cross-section. 3654 WEIDNER ET AL Figure 1. Renal leukocyte infiltration in antineutrophil cytoplasmic antibody–associated vasculitis. Infiltration was assessed using a 3-step immunoperoxidase method (see Patients and Methods). A, Periglomerular accumulation of CD3-positive T cells with residues of heavy crescent formation. B, Prominent infiltration of CD4-positive T lymphocytes around a glomerulus with evident sclerosis. C, Diffuse interstitial infiltration of CD8-positive T lymphocytes. D, A pattern similar to that in C with periglomerular accumulation near a sclerosed glomerulus is seen for CD79-positive B lymphocytes. E, Intense interstitial infiltration of CD15-positive neutrophils with diffuse distribution over the interstitial tissue. F, Diffuse distribution of infiltrated CD68-positive macrophages in the interstitial tissue, similar to the pattern in E. G, Glomerular infiltration of CD15-positive neutrophils. H, Prominent glomerular infiltration of CD68-positive cells with presence of macrophages within crescents. (Original magnification ⫻ 200 in A–C, E, and G; ⫻ 400 in D, F, and H.) infiltration according to ANCA or diagnostic subgroups, either for leukocyte subsets or for the total number of infiltrating cells. Glomerular infiltration. In all biopsy samples, a mean ⫾ SD of 15.35 ⫾ 9 glomeruli (range 4–45) were analyzed. The mean ⫾ SD total infiltrating leukocyte CELLULAR INFILTRATION IN ANCA-ASSOCIATED VASCULITIS count was 10.2 ⫾ 17.6/gcs. CD68-positive macrophages were the dominant glomerular infiltrating cell type (4.7 ⫾ 11.1/gcs), followed by granulocytes (3.2 ⫾ 7.4/gcs) (Table 3, Figures 1G and H). Twenty-seven biopsy samples were completely negative for glomerular macrophages. Glomerular lymphocyte infiltration, represented by CD3-positive cells, was rare. When present, mean lymphocyte infiltration was slightly higher for CD8positive lymphocytes than for CD4-positive cells (0.6/gcs versus 0.3/gcs) (Table 3). A significant correlation was found for simultaneous glomerular infiltration of CD15positive granulocytes with CD68-positive macrophages (r ⫽ 0.52, P ⬍ 0.0001) and for CD4-positive lymphocytes with CD8-positive lymphocytes (r ⫽ 0.611, P ⫽ 0.001). There was no significant difference in glomerular infiltration according to ANCA or diagnostic subgroups, either for leukocyte subsets or for the total number of infiltrating cells. Comparison of cellular infiltration with histopathologic lesions. All patients had pauci-immune glomerulonephritis. We analyzed 998 glomeruli in the 65 biopsy samples for the presence of histopathologic lesions. Twenty-eight percent of glomeruli were normal, and 15.8% had sclerosis. Crescents were found in 41.3% of glomeruli, and fibrinoid necrosis was present in 31.3%. A significant correlation was found for the glomerular infiltration of CD68-positive macrophages with the presence of glomerular fibrinoid necrosis as well as with the number of glomeruli with crescents (r ⫽ 0.856, P ⬍ 0.0001 and r ⫽ 0.773, P ⫽ 0.005, respectively). No correlation was found for interstitial fibrosis with the infiltration of any leukocyte subsets. Renal function and cellular infiltration. The total number of infiltrating glomerular leukocytes per individual biopsy sample showed a significant correlation with initial serum creatinine concentration (r ⫽ 0.624, P ⫽ 0.012), but not with serum creatinine concentration after 1 year. A significant correlation was found for the interstitial as well as the glomerular infiltration of CD68positive macrophages with serum creatinine concentration at the time of biopsy (r ⫽ 0.646, P ⫽ 0.001 and r ⫽ 0.754, P ⫽ 0.006, respectively), but not with serum creatinine concentration after 1 year. No significant correlation was found for the interstitial or glomerular infiltration of other leukocyte subsets, either with initial serum creatinine concentration or with serum creatinine concentration after 1 year. DISCUSSION The pathogenetic concept of AAV involves the ANCA-induced activation of neutrophils and monocytes 3655 with the release of oxygen radicals and granule components, resulting in endothelial necrosis. Further cellular activation leads to cytokine and chemokine release with the subsequent vascular and perivascular infiltration of inflammatory cells. However, the precise composition and distribution of the inflammatory infiltrate have not been analyzed in detail. In this study, the predominant interstitial infiltrating cells were T lymphocytes, while monocytes and, to a lesser extent, granulocytes constituted the dominant infiltrating cell types in glomeruli. Interestingly, interstitial lymphocytes were predominantly periglomerular, especially around glomeruli with sclerosis or heavy crescent formation, while monocyte and neutrophil infiltration was diffusely distributed over the interstitial tissue. The serum creatinine concentration at the time of biopsy correlated significantly with the interstitial as well as the glomerular infiltration of CD68-positive macrophages. The presence of monocytes as the predominant glomerular infiltrating cell type in renal tissue in AAV has also been reported by other investigators (8,10). Ferrario and Rastaldi were able to identify macrophages mainly in areas of necrotizing crescentic lesions and granulomatous infiltrates. The further analysis of these cells revealed their acute activation status and the expression of proinflammatory cytokines such as tumor necrosis factor ␣ (TNF␣) and interleukin-1 (IL-1) at the protein and messenger RNA levels (9). The major role of neutrophils in the induction of injury in AAV remains unchallenged. However, our data provide important information on the possible initiation and amplification of tissue injury by ANCA-stimulated monocytes acting in concert with neutrophils. This is demonstrated by the significant correlation of glomerular necrosis as well as the number of crescentic glomeruli with glomerular monocyte infiltration. It is evident that monocytes play an important pathogenetic role in a wide range of chronic inflammatory processes including vasculitis (1) and glomerulonephritis. In vitro, monocytes are activated by ANCA, resulting in the release of oxygen radicals and chemokine secretion (13–15). Oxygen-dependent mechanisms have been implicated in the tissue damage of various diseases. Several experimental models for inflammatory hepatic and pulmonary diseases have demonstrated that tissue injury occurs in part via macrophage-derived reactive oxygen intermediates (16). This is consistent with studies of experimental models of both immune and nonimmune glomerulonephritis, in which macrophages and their secretory products have been implicated in glomerular tissue injury (17,18). Macrophages accumulating in animal models of proliferative glomerulone- 3656 phritis in rabbits and rats produced significant amounts of reactive oxygen species (19,20). Furthermore, macrophage activation also results in the secretion of a wide range of molecules, including proinflammatory cytokines as well as collagenases and proteases responsible for tissue degradation. The local release of TNF␣ and IL-1 has the capacity to induce or further amplify tissue damage. In accordance with the data obtained by Ferrario and Rastaldi, Noronha et al demonstrated the in situ production of TNF␣ and IL-1␤ by mononuclear cells in renal biopsy samples in AAV. The number of TNF␣- and IL-1␤–positive cells was markedly increased in biopsy samples with active lesions. Positive cells were also present in crescents surrounding tuft necrosis and in the walls of arteries and arterioles with acute vasculitic lesions (21). Given the correlation of glomerular macrophages with glomerular necrosis as well as the correlation of interstitial and glomerular infiltration of macrophages with serum creatinine concentration at the time of biopsy, our data underscore the importance of monocyte infiltration for tissue damage in AAV. Investigators in some studies have suggested pauci-immune glomerulonephritis to be a disease of delayed-type hypersensitivity due to the prominent presence of T cells (7,8). Delayed-type hypersensitivity, a manifestation of cell-mediated immunity, is the immunologic profile of granuloma formation that is induced by the deposition of a relatively indigestible antigenic material within tissue. Mediated by a variety of cytokines, the initiating event is a complex interaction between antigen-presenting mononuclear phagocytes and predominantly T helper cells, resulting in T lymphocyte proliferation and activation. Oxygen radicals and nitric oxide seem to play important roles in the initiation and amplification of these processes. Although the release of oxygen radicals by neutrophils and monocytes is considered to be the primary event in the necrotizing inflammation of AAV, to date it is not clear how these mechanisms ultimately lead to granuloma formation. However, secondary activation of monocytes after the initiation of the inflammatory process cannot be excluded. This may occur by direct cellular contact between stimulated T cells and monocytes. Contactmediated signaling of monocytes by stimulated T lymphocytes is a potent proinflammatory mechanism that triggers massive up-regulation of the proinflammatory cytokines IL-1 and TNF␣ (22,23). Thus, activated monocytes contribute to the secretion of proinflammatory cytokines, further trapping inflammatory cells at these sites. Macrophages start to proliferate locally and par- WEIDNER ET AL ticipate in granuloma formation (24,25). It remains a matter of speculation whether the infiltrative pattern observed in the present study with periglomerular lymphocytes and intraglomerular monocytes represents the attempt to obtain direct cellular contact. Our data support the notion of an important role of monocytes in the tissue damage of AAV. These data may provide a basis for the development of monocytetargeted interventions or for the potential of TNF␣directed treatment modalities. Further analysis with cytokine and chemokine staining of renal biopsy samples will be required to determine the immunologic mechanisms of cell infiltration and pattern of distribution. ACKNOWLEDGMENT The authors are very grateful to Birgit Hausknecht for excellent technical assistance. REFERENCES 1. 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