Antineutrophil cytoplasmic autoantibodies (ANCA) and their target antigens in Chinese patients with lupus nephritis

Background: ANCA have been found in patients with systemic lupus erythematosus (SLE); however, the prevalence of ANCA and their target antigens is still not certain. This study is to investigate the prevalence of ANCA and their target antigens in Chinese patients with lupus nephritis. Methods: Ninety-five serum samples were collected from 95 renal-biopsy-proven lupus nephritis patients. Indirect immunofluorescence using ethanol-fixed leukocytes as substrate and ELISA using six highly purified known ANCA antigens as solid-phase ligands were performed. The specific ANCA antigens included proteinase 3, myeloperoxidase, bactericidal/permeability-increasing protein, human leukocyte elastase, cathepsin G, and lactoferrin. The prevalence of ANCA in patients with (n=65) and without (n=30) active renal pathological lesions was also compared to reveal whether ANCA correlates with disease activity. Results: (i) None of the sera recognized proteinase 3, myeloperoxidase, and human leukocyte elastase, and only one serum recognized bactericidal/permeability-increasing protein. The striking finding was that 59/95 (62.1%) sera recognized cathepsin G and the titres of some sera reached 1/3200. Eight of 95 sera (8.4%) recognized lactoferrin. (ii) The percentage of anticathepsin G antibody positive samples in patients with active renal lesions was significantly higher than in patients without active lesions (73.4 vs 36.7%, P<0.0001), whereas, anti-lactoferrin antibodies had no correlation with active renal lesions. (iii) By indirect immunofluorescence, only 22% of the 95 sera were ANCA positive. Conclusions: Our results suggest that the majority of lupus nephritis patients have ANCA and that the major target antigens is cathepsin G. Anti-cathepsin G antibodies seem to be correlated with renal disease activity. Key words: ANCA; autoantibodies; autoantigen; autoimmune disease; cathepsin G; lupus nephritis; SLE; vasculitis      

Antineutrophil cytoplasmic autoantibodies antigen specificity.

The results presented during the Third International ANCA Workshop, Washington, DC, 1990, allowed a better definition of the antigenic specificity of the antineutrophil cytoplasmic autoantibodies (ANCA). The large predominance of two major antigen specificities for proteinase 3 (PR3) and myeloperoxidase (MPO), in the group of vasculitic patients, was confirmed. PR3 and MPO are colocalized in the azurophilic granules of neutrophils and translocated to the cell surface during activation and thus are able to interact with ANCA after neutrophil preactivation. Furthermore, by comparison of amino acid and DNA sequences, the agreement was reached that PR3 was identical to AGP7, p29, and myeloblastin, described independently and involved in the control of growth and differentiation of leukemic cells. In addition to the two major ANCA antigens, a number of neutrophil cytoplasmic antigens recognized by ANCA have been previously identified (human leukocyte elastase [HLE], lactoferrin). It was established during the Third Workshop that these rare ANCA specificities, occurring in a limited number of patients, include a cationic antimicrobial protein (CAP57) and cathepsin G. However, the variety of ANCA antigen specificities contrasts with the fact that the vast majority of ANCA-positive sera are monospecific for a single ANCA antigen. Finally, the fine specificity of granulocyte-specific antinuclear antibodies (GS-ANA) occurring in rheumatoid arthritis and ulcerative colitis is still unknown, but clearly a substantial proportion of GS-ANA belongs to the ANCA family.     

抗中性粒细胞胞浆抗体相关小血管炎的靶抗原及其临床病理特点

目的明确中国人抗中性粒细胞胞浆抗体（ANCA）相关小血管炎的检出率、主要的特异性靶抗原及临床病理特点。方法对近年来送检的820份不同患者的血清行IIF-ANCA检测，阳性血清则进一步经抗原特异性ELISA明确靶抗原，并探讨ANCA相关小血管炎患者的临床病理特点。应用的6个高度纯化的ANCA靶抗原包括蛋白酶3（PR3）、髓过氧化物酶（MPO）、杀菌／通透性增高蛋白（BPI）、组蛋白酶G（CG）、乳铁蛋白（LF）和人白细胞弹力蛋白酶（HLE）。结果43/820（5．2％）为IIF-ANCA阳性，其中36/820（4．4％）为原发性小血管炎。c-ANCA8例，其中7/8识别PR3，1例同时识别LF；p-ANCA28例，其中24例识别MPO，5例识别BPI，2例识别CG，1例识别LF，部分血清可同时识别二到三个不同的抗原。无1例血清识别HLE。ANCA相关小血管炎临床上多表现为多器官损害，肾、肺最易受累，肾脏病理呈活动性病变。ANCA相关小血管炎患者多有中重度贫血及明显的血沉快。结论ANCA相关小血管炎在我国并不少见；其主要的特异性靶抗原为MPO和PR3；临床呈多系统损害。     

Autoantibodies to GBM and neutrophil cytoplasm in rapidly progressive glomerulonephritis

The incidence of autoantibodies to glomerular basement membrane (AGBMA) and neutrophil cytoplasmic antigens (ANCA) in the initial sera of 889 consecutive patients with a suspected diagnosis of rapidly progressive glomerulonephritis, was determined by prospective study. Forty-seven (5%) were positive for AGBMA alone, 246 (28%) were positive for ANCA alone, 576 (65%) had neither autoantibodies while 20 (2%) had both. Clinical and pathological data collected from patients with both autoantibodies suggested the coexistence of anti-glomerular basement membrane disease and systemic vasculitis. Together, assays for AGBMA and ANCA are important in the diagnosis and management of rapidly progressive glomerulonephritis and may help its further classification.     

Anti-endothelial cell antibodies in antineutrophil cytoplasmic antibodies negative pauci-immune crescentic glomerulonephritis

Aim: Pauci‐immune crescentic glomerulonephritis (CrGN) is frequently associated with circulating anti‐neutrophil cytoplasmic antibodies (ANCA). However, in patients with ANCA‐negative pauci‐immune CrGN, the pathogenesis is not clear. Anti‐endothelial cell antibodies (AECA) have been implicated in the pathogenesis of vasculitis. The purpose of this study is to investigate the prevalence of AECA and their possible clinical significance in ANCA‐negative pauci‐immune CrGN. Methods: Sera from 19 patients with ANCA‐negative pauci‐immune CrGN, 26 patients with ANCA‐positive pauci‐immune CrGN and 10 healthy blood donors were collected. Soluble proteins extracted from cultured human umbilical vein endothelial cells were used as antigens and western blot analysis was carried out to detect AECA. Results: In ANCA‐negative pauci‐immune CrGN, 10 of 19 patients were serum IgG‐AECA positive and seven bands reactive with endothelial antigens could be blotted. The prevalence of skin rash and thrombocytosis was significantly higher in patients with anti‐76 kDa and anti‐123 kDa autoantibodies than in patients without, respectively. Birmingham Vasculitis Activity Scores of patients with anti‐200 kDa AECA were significantly higher than in patients without. In the sera of 26 ANCA‐positive cases, 23 were AECA positive and 11 bands could be recognized. The prevalence of total AECA and anti‐90 kDa AECA was significantly lower in patients with ANCA‐negative pauci‐immune CrGN than in patients with ANCA‐positive pauci‐immune CrGN. Conclusion: Anti‐endothelial cell antibodies could be found in sera of patients with ANCA‐negative pauci‐immune CrGN; some AECA might have some clinical significance. The discrepancies of AECA might be a possible contributor to the differences between ANCA‐negative and ANCA‐positive pauci‐immune CrGN.     

Severe pulmonary hemorrhage and systemic vasculitis in association with circulating anti-neutrophil cytoplasm antibodies of IgM class only

We report an association of severe pulmonary hemorrhage with circulating autoantibodies to neutrophil cytoplasmic antigens (ANCA) restricted to IgM class in three patients with systemic vasculitis. ANCA were detected by indirect immunofluorescence and isotype specific solid phase radioimmunoassay (SPRIA). Institution of immunosuppressive therapy was accompanied by an isotype switch to IgG ANCA and recovery in all three patients. In an associated study, ANCA activity was found in eluates from the washed glomeruli of two postmortem cases, with the same isotype distribution as was present in the sera.     

Autoantibodies against glomerular mesangial cells and their target antigens in lupus nephritis

Mesangial proliferation and deposition of immunoglobulins and complement components within glomerular mesangium was one of the important pathological features of lupus nephritis. Autoantibodies against human mesangial cells could be detected in the sera of patients with IgA nephropathy (IgAN) and Henoch-Sch?enlein nephritis. We speculated that autoantibodies against human glomerular mesangial cells might play a role in the development of lupus nephritis. OBJECTIVE: To screen autoantibodies against human glomerular mesangial cells in sera from patients with lupus nephritis and to identify their target antigens. METHODS: Sera were collected from 96 patients with lupus nephritis as well as 25 patients with IgAN and 20 patients with idiopathic membranous nephropathy (IMN). Cell lysates of in vitro cultured human glomerular mesangial cells were used as antigens in Western-blot analysis to detect autoantibodies against human mesangial cells in sera from patients with lupus nephritis as well as IgAN and IMN. The clinical and pathological significance of the autoantibodies were further investigated. RESULTS: Autoantibodies against human mesangial cells could be detected in 94/96 (97.9%) of the sera from patients with lupus nephritis in Western-blot analysis. Twelve protein bands could be blotted by the sera from patients with lupus nephritis. The prevalence of autoantibodies against human mesangial cells in IgAN was 14/25 (56.0%) and only seven protein bands could be blotted. Five autoantibodies (anti-18, 24, 36, 46, and 91 kD) could be detected only in sera from patients with lupus nephritis. In patients with lupus nephritis, some autoantibodies might have some relationship with gender, hematuria, ANA, anti-dsDNA or anti-ENA antibodies. CONCLUSIONS: There are autoantibodies directly against heterogeneous antigens of human glomerular mesangial cells in sera from patients with lupus nephritis, and some of them might be associated with different clinical manifestations.     

Autoantibodies Against Glomerular Mesangial Cells and Their Target Antigens in Lupus Nephritis

Mesangial proliferation and deposition of immunoglobulins and complement components within glomerular mesangium was one of the important pathological features of lupus nephritis. Autoantibodies against human mesangial cells could be detected in the sera of patients with IgA nephropathy (IgAN) and Henoch-Schöenlein nephritis. We speculated that autoantibodies against human glomerular mesangial cells might play a role in the development of lupus nephritis. Objective. To screen autoantibodies against human glomerular mesangial cells in sera from patients with lupus nephritis and to identify their target antigens. Methods. Sera were collected from 96 patients with lupus nephritis as well as 25 patients with IgAN and 20 patients with idiopathic membranous nephropathy (IMN). Cell lysates of in vitro cultured human glomerular mesangial cells were used as antigens in Western-blot analysis to detect autoantibodies against human mesangial cells in sera from patients with lupus nephritis as well as IgAN and IMN. The clinical and pathological significance of the autoantibodies were further investigated. Results. Autoantibodies against human mesangial cells could be detected in 94/96 (97.9%) of the sera from patients with lupus nephritis in Western-blot analysis. Twelve protein bands could be blotted by the sera from patients with lupus nephritis. The prevalence of autoantibodies against human mesangial cells in IgAN was 14/25 (56.0%) and only seven protein bands could be blotted. Five autoantibodies (anti-18, 24, 36, 46, and 91 kD) could be detected only in sera from patients with lupus nephritis. In patients with lupus nephritis, some autoantibodies might have some relationship with gender, hematuria, ANA, anti-dsDNA or anti-ENA antibodies. Conclusions. There are autoantibodies directly against heterogeneous antigens of human glomerular mesangial cells in sera from patients with lupus nephritis, and some of them might be associated with different clinical manifestations.     

Antineutrophil cytoplasmic autoantibody-associated diseases: a rheumatologist's perspective

Autoantibodies against neutrophil cytoplasmic antigens (ANCA) produce two major immunofluorescence (IF) patterns on ethanol-fixed granulocytes: the "classical" (centrally accentuated) C-ANCA, associated with Wegener's granulomatosis (WG), and P-ANCA (perinuclear), which mainly occur in renal vasculitis. Rheumatic manifestations are an important clinical finding in systemic vasculitis, often preceding a fulminant course and sometimes imitating various rheumatic disorders. We analyzed the incidence of ANCA in rheumatic patients and looked for the frequency of rheumatic symptoms in systemic vasculitis. In WG (n = 186), we found rheumatic symptoms in 55% (myalgia, 45%; arthritis, 21%); in 90%, rheumatic complaints were associated with active vasculitis. In 730 patients with various rheumatic conditions (eg, 268 rheumatoid arthritis, 130 systemic lupus erythematosis [SLE], 32 sharp-S, 50 ankylosing spondylitis, 43 systemic sclerosis) no C-ANCA were found. On the contrary, the P-ANCA pattern was seen in seven of 62 giant cell arteritis, five of 27 Felty's/Still's syndrome, and four of 130 SLE patients in addition to renal vasculitis (21/74). We demonstrated that 95% of C-ANCA-positive sera react with proteinase 3 (PR3 or myeloblastin). Using monoclonal antibodies, we showed that PR3 is expressed on the plasma membrane of neutrophil granulocytes and monocytes; thus, PR3 autoantigens are accessible for circulating antibodies. The detection of ANCA in sera from vasculitis and other rheumatic diseases is of immunodiagnostic value and provides new insight in the pathogenesis of systemic vasculitides.     

抗肾小球基底膜抗体伴抗中性粒细胞胞浆抗体阳性肾炎(四例报告及文献复习)

目的　了解抗肾小球基底膜抗体（ Ａｎｔｉ Ｇ Ｂ Ｍ） 伴抗中性粒细胞胞浆抗体（ Ａ Ｎ Ｃ Ａ） 阳性患者的临床病理特点。方法　对我科近４ 年来６８ 例 Ａｎｔｉ Ｇ Ｂ Ｍ 和（ 或） Ａ Ｎ Ｃ Ａ 阳性患者进行 Ａｎｔｉ Ｇ Ｂ Ｍ 及 Ａ Ｎ Ｃ Ａ 检测,对其中４ 例两者均阳性患者进行临床病理分析。结果　６８ 例患者中４ 例两者均阳性,占全部 Ａｎｔｉ Ｇ Ｂ Ｍ 阳性患者的２４ ％ ,占全部 Ａ Ｎ Ｃ Ａ 阳性患者的７ ％ 。该４ 例患者 Ａｎｔｉ Ｇ Ｂ Ｍ 的百分结合率较单纯 Ａｎｔｉ Ｇ Ｂ Ｍ 阳性患者低。４ 例中３ 例髓过氧化物酶 Ａ Ｎ Ｃ Ａ（ Ｍ Ｐ Ｏ Ａ Ｎ Ｃ Ａ） 阳性,１ 例蛋白酶３ Ａ Ｎ Ｃ Ａ（ Ｐ Ｒ３ Ａ Ｎ Ｃ Ａ） 阳性,全身系统表现较多,与单纯性 Ａ Ｎ Ｃ Ａ 阳性患者相似。所检病例肾脏病理多为新月体肾炎,免疫荧光检查多为 Ｉｇ Ｇ、 Ｃ３ 呈细颗粒样分布于 Ｇ Ｂ Ｍ。虽经积极治疗,多数患者预后较差,类似单纯 Ａｎｔｉ Ｇ Ｂ Ｍ 肾炎。结论　 Ａｎｔｉ Ｇ Ｂ Ｍ 伴 Ａ Ｎ Ｃ Ａ 阳性患者全身表现类似单纯 Ａ Ｎ Ｃ Ａ 相关小血管炎患者,但治疗效果及预后相对较差又类似于 Ａｎｔｉ Ｇ Ｂ Ｍ 肾炎患者     

Anti-Ro(SS-A) 52 kDa and 60 kDa specificities in undifferentiated connective tissue disease

Autoantibodies to Ro(SS-A) may recognize two different polypeptides, of 52 kDa and 60 kDa, respectively. We used an ELISA with purified human recombinant antigens to conduct a detailed analysis of the specificities of anti-Ro(SS-A) antibodies from 170 patients with definite diagnoses (systemic lupus erythematosus [SLE], n = 55; primary Sj?gren's syndrome [PSS], n = 39; systemic sclerosis, n = 9; rheumatoid arthritis [RA], n = 10) or undifferentiated connective tissue disease (UCTD, n = 57). Most of the patients with SLE or PSS had both anti-52 kDa and -60 antibodies; isolated anti-60 kDa antibodies were found in 13% of the SLE patients and in none of the PSS patients, whereas high titers of anti-52 kDa were more common in the PSS than in the SLE patients. In the UCTD patients, the anti-Ro(SS-A) profile showed no significant correlations with clinical features but was associated with the clinical outcome. Over the mean follow-up of five years, definite SLE developed in four of the five UCTD patients with isolated anti-60 kDa vs only one of the remaining 52 patients (P < 0.0001); progression to PSS was seen in seven of the 34 patients with both anti-52 kDa and anti-60 kDa vs none of the remaining 23 patients (P = 0.03); none of the 12 patients with isolated anti-52 kDa developed a definite connective tissue disease. CONCLUSION: Our study suggests that analysis of anti-Ro(SS-A) specificity may provide useful information for predicting the course of UCTD.     

Does the presence of ANCA in patients with ulcerative colitis necessarily imply renal involvement?

BACKGROUND.: ANCA are thought to play a pathogenic role in renal vasculitis.ANCA may also be detected in patients with diseases not usuallyassociated with renal pathology, such as ulcerative colitis.Our study was conducted to determine if the presence of ANCAin patients with ulcerative colitis is associated with renalpathology. METHODS.: Eight ANCA-positive and five ANCA-negative patients with a histologicaland endoscopic diagnosis of active ulcerative colitis were investigated.Repeated complete urinalyses and determination of microalbuminuriaand creatinine clearance were performed. Serum IgG and IgA ANCAwere evaluated in all patients by indirect immunofluorescenceand ELISA, and when detected the antibodies were further characterizedby alpha granules preparation, myeloperoxidase, lactoferrin,and cathepsin G. RESULTS.: In both ANCA-positive and ANCA-negative patients renal functionwas normal or near normal and urinalyses (including microalbuminuria)failed to disclose any abnormalities. ANCA exhibited a perinuclearpattern in all ANCA-positive patients. Interestingly, none ofthe ANCA-positive patients had antibodies to myeloperoxidaseor to alpha granules which are usually found in the sera ofpatients with ANCA-associated vasculitis, and only one had antibodiesto lactoferrin. The ANCA specificity remained undetermined inthe remaining seven patients. At the end of the 1-year observationperiod, all ANCA-positive patients remained ANCA-positive withoutdeveloping symptoms, signs or laboratory abnormalities consistentwith renal involvement. CONCLUSIONS.: Renal damage was not observed in ANCA-positive patients withulcerative colitis even after 1 year of follow-up, suggestingthat the ANCA found in these patients do not share the antigenictargets with the ANCA commonly found in renal vasculitis. Thereforethe potential of ANCA of inducing renal lesions (if any) isdependent on their own antigenic specificity.     

Antimyeloperoxidase antibodies in patients with extracapillary glomerulonephritis

The sera of 64 patients with extracapillary glomerulonephritis were investigated by enzyme-linked immunosorbent assay for the presence of antibody to human neutrophil myeloperoxidase (MPO). In all, circulating anti-MPO were found in 30% and antineutrophil cytoplasm antibodies (ANCA) detected by indirect immunofluorescence in 44% of the patients. Autoantibody to components of neutrophil granulocytes was not found in patients with other forms of glomerulonephritis. The incidence of ANCA (16/23) was higher than that of anti-MPO (5/23) in patients with a diagnosis of Wegener's granulomatosis. By contrast, anti-MPO was found in a majority of vasculitis patients without extrarenal symptoms (6/9), including 3 patients treated with hydralazine. One of the patients treated with hydralazine had circulating ANCA in combination with anti-MPO. Anti-MPO was also found in 1 out of 6 patients with Goodpasture's syndrome. The findings emphasize that autoantibodies to distinct components of neutrophil granulocytes partly differ with regard to diagnostic specificity.     

Spécificité 52 kDa et 60 kDa des anti-Ro(SS-A) dans les connectivites inclassables

Anti-Ro(SS-A) 52 kDa and 60 kDa specificities in undifferentiated connective tissue disease. — Objective. Autoantibodies to Ro(SS-A) may recognize two different polypeptides, of 52 kDa and 60 kDa, respectively. Methods. We used an ELISA with purified human recombinant antigens to conduct a detailed analysis of the specificities of anti-Ro(SS-A) antibodies from 170 patients with definite diagnoses (systemic lupus erythematosus [SLE], n = 55; primary Sjögren's syndrome [PSS], n = 39; systemic sclerosis, n = 9; rheumatoid arthritis [RA], n = 10) or undifferentiated connective tissue disease (UCTD, n = 57). Most of the patients with SLE or PSS had both anti-52 kDa and -60 antibodies; isolated anti-60 kDa antibodies were found in 13 % of the SLE patients and in none of the PSS patients, whereas high titers of anti-52 kDa were more common in the PSS than in the SLE patients. Results. In the UCTD patients, the anti-Ro(SS-A) profile showed no significant correlations with clinical features but was associated with the clinical outcome. Over the mean follow-up of five years, definite SLE developed in four of the five UCTD patients with isolated anti-60 kDa vs only one of the remaining 52 patients (p < 0.0001); progression to PSS was seen in seven of the 34 patients with both anti-52 kDa and anti-60 kDa vs none of the remaining 23 patients (ρ = 0.03); none of the 12 patients with isolated anti-52 kDa developed a definite connective tissue disease. Conclusion. Our study suggests that analysis of anti-Ro(SS-A) specificity may provide useful information for predicting the course of UCTD.     

The Diagnostic and Prognostic Significance of ANCA

Antineutrophil cytoplasmic antibodies (ANCA) constitute a family of autoantibodies directed against various components of the neutrophil cytoplasm. Their identification and association with vasculitis and rapidly progressive glomerulonephritis has led to considering these diseases as possible autoimmune disorders. In addition, ANCAs constitute a diagnostic tool and a guideline for therapy during follow-up. Originally identified by Davies et al. in 1982 in 8 patients who had necrotizing glomerulonephritis but no immune deposits or systemic vasculitis (1), ANCA are now regarded as a serological marker for active pauci-immune necrotizing and crescentic glomerulonephritis, either in their renal-limited form or associated with systemic vasculitis such as Wegener's granulomatosis (WG), microscopic polyangütis (mPA), and Churg-Strauss syndrome (CSS) (2-9). The usefulness of ANCA detection for the diagnosis of these forms of vasculitis is now established but its usefulness on follow-up remains disputed (10-13). Two major ANCA antigens have already been identified. Proteinase 3 (PR3) in a serine protease of 29 kDa initially identified by Kao and producing a cytoplasmic staining pattern termed cANCA by indirect immunofluorescence (IIF) (14,15). Myeloperoxidase (MPO) is another myeloid lysosomal enzyme producing an artefactual perinuclear staining of ethanol-fixed neutrophils termed pANCA (2,16). Both are localized in the azurophilic granules of neutrophils and monocytes, are translocated to the cell surface during cell activation (17), and are able to interact directly with ANCA. Despite this common location, MPO and PR3 are associated with a broad spectrum of clinical conditions.     

Membrane proteinase 3 and Wegener's granulomatosis

Proteinase 3 (PR3) is found in neutrophil and monocyte lysosomal granules. Anti-neutrophil cytoplasmatic antibodies (ANCA) with specificity for PR3 are characteristic for patients with Wegener's granulomatosis. The interaction of ANCA with neutrophilic ANCA antigens is necessary for the development of ANCA-associated diseases. ANCA bind to membrane-expressed PR3 and induce full-blown activation in primed neutrophils. We discuss two different aspects of membrane PR3 (mPR3). The first aspect is the amount of PR3 and mechanisms controlling this issue. The second aspect is the presence of two neutrophil subsets that differ in the mPR3 expression phenotype.     

The pathogenic role of antineutrophil cytoplasmic autoantibodies.

Antineutrophil cytoplasmic autoantibodies (ANCA) are found in the sera of patients with systemic necrotizing vasculitis and glomerulonephritis. Their role in the pathogenesis of these diseases is not clearly understood; however, there is a growing body of data that supports a pathogenic function for these antibodies. In vitro they can activate neutrophils and monocytes to produce reactive oxygen species (ROS), degranulate, and damage target cells. The antigens to which they are directed stimulate T lymphocytes from patients with these diseases. The ANCA directed against proteinase 3 (PR3) may also play a role in growth regulation of monocytes by inactivating the enzymatic function of its antigen. The proposed model of ANCA-induced disease takes into account both the in vitro data and the natural history of these diseases.     

Anti-LAMP-2 antibodies are not prevalent in patients with antineutrophil cytoplasmic autoantibody glomerulonephritis

Lysosomal membrane protein 2 (LAMP-2) is a target of antineutrophil cytoplasmic autoantibodies (ANCA) in addition to the more commonly known targets proteinase 3 and myeloperoxidase. The prevalence of anti-LAMP-2 antibodies and their relationship to disease in ANCA glomerulonephritis are not well described. We measured anti-LAMP-2 reactivity in 680 sera samples (two academic centers) from patients with ANCA glomerulonephritis (n=329); those with ANCA-negative glomerulonephritis (n=104); those with fimbriated, gram-negative Escherichia coli urinary tract infection (n=104); disease controls (n=19); and healthy volunteers (n=124). With levels in healthy controls used to define a reference range, anti-LAMP-2 reactivity was present in 21% of ANCA sera from two of the centers; reactivity was present in 16% of the control group with urinary tract infection. Western blotting and immunofluorescence microscopy did not verify positivity. Titers of anti-myeloperoxidase and anti-proteinase 3 antibodies were 1500-fold and 10,000-fold higher than anti-LAMP-2 titers, respectively. There was no correlation between anti-LAMP-2 antibodies and disease activity. Furthermore, Wistar Kyoto rats injected with anti-LAMP-2 antibodies did not develop glomerulonephritis. In conclusion, antibodies that react with LAMP-2 may exist at very low titers in a minority of patients with ANCA disease. These data do not support a mechanistic relationship between anti-LAMP-2 antibodies and ANCA glomerulonephritis.     

Enhanced Formation and Disordered Regulation of NETs in Myeloperoxidase-ANCA–Associated Microscopic Polyangiitis

Microscopic polyangiitis (MPA) is an ANCA-associated vasculitis that affects small vessels, especially renal glomeruli. We recently demonstrated that the abnormal formation and impaired degradation of neutrophil extracellular traps (NETs) may be crucially involved in the generation of myeloperoxidase (MPO)-ANCA and subsequent development of MPA. This study assessed the formation and regulation of NETs in patients with MPO-ANCA–associated MPA. Peripheral blood samples were obtained from 38 patients with MPO-ANCA–associated MPA, 23 patients with systemic lupus erythematosus (SLE), and 8 healthy controls. IgG eluted from MPO-ANCA–associated MPA sera demonstrated the highest ability to induce NETs, and this ability correlated with disease activity and paralleled ANCA affinity for MPO. Moreover, addition of recombinant human MPO to these IgG samples reduced NET induction. Additionally, MPO-ANCA–associated MPA sera exhibited lower rates of NET degradation that recovered partially upon depletion of IgG. The activity of DNase I, an important regulator of NETs, was also lower in MPO-ANCA–associated MPA and SLE sera. IgG depletion from MPO-ANCA–associated MPA sera partially restored the rate of NET degradation, and addition of DNase I synergistically enhanced this restoration. Addition of anti-MPO antibodies did not inhibit DNase I activity, and some MPO-ANCA–associated MPA sera contained anti-NET antibodies at levels not correlated with MPO-ANCA titers, suggesting the involvement of unidentified autoantibodies as well. The collective evidence suggests a vicious cycle involving MPO-ANCA and the regulation of NETs could be critically involved in the pathogenesis of MPO-ANCA–associated MPA.     

Autoantibodies to neutrophil cytoplasmic antigens (ANCA) do not bind to polymorphonuclear neutrophils in blood

BACKGROUND: Autoantibodies to neutrophil cytoplasmic antigens (ANCA), particularly to proteinase 3 (PR3), are found in the majority of patients with systemic Wegener's granulomatosis. The autoantibodies are widely used as diagnostic markers. Their role in the development and progression of the disease, however, is still under investigation. The primary target of ANCA, PR3, is located in the cytoplasm of polymorphonuclear neutrophils (PMN) or monocytes and is translocated to the cell surface upon stimulation. In patients with Wegener's granulomatosis PR3 is up-regulated most prominently during active disease. Despite the fact that both autoantibodies to PR3 and PMN expressing PR3 are present in patients with Wegener's granulomatosis, there is no evidence for binding of the autoantibodies to PMN. The present study was designed to analyze binding characteristics of autoantibodies to PR3 on PMN. METHODS AND RESULTS: PMN of patients with active Wegener's granulomatosis (N= 10) were tested for autoantibody binding. Despite high autoantibody titer and PR3 expression on the PMN, no surface-bound IgG was found on PMN ex vivo. When ANCA-containing plasma from patients was incubated with isolated PMN, stimulated to express PR3, again no specific binding of the autoantibody could be detected. Also keeping the samples on ice did not allow surface detection of IgG, ruling out degradation or internalization of the autoantibodies. Only when purified IgG fractions were used, binding to PMN was seen in 14 of 25 patients. Already 1% of plasma, however, was sufficient to greatly reduce the IgG binding. Reduced binding of the IgG fraction was also seen when a larger reaction volume was used. CONCLUSION: Our data indicate that autoantibodies to PR3 have a rather low affinity for surface-associated PR3 on PMN. This, in turn, argues against the hypothesis that ANCA contributes to the pathogenesis of the disease by stimulating viable PMN in whole blood.