Leukocyte gene expression signatures in antineutrophil cytoplasmic autoantibody and lupus glomerulonephritis

Leukocytes play a major role in the development and progression of autoimmune diseases. We measured gene expression differences in leukocytes from patients that were antineutrophil cytoplasmic autoantibody (ANCA) positive, patients with systemic lupus erythematosus (SLE) or rheumatoid arthritis (RA), and healthy donors to explore potential pathways for clinical intervention. Leukocyte gene expression profiles were determined on Affymetrix U133A/B chips in 88 autoimmune patients, 28 healthy donors, and healthy donor leukocyte cell subtypes that were activated in vitro. Comparison of gene expression in leukocytes identified differentially expressed signature genes that distinguish each donor source. The microarray expression levels for many signature genes correlated with the clinical activity of small vessel vasculitis in the ANCA patients; a result confirmed by quantitative real time-polymerase chain reaction for 16 relevant genes. Comparison with in vitro-activated leukocyte subtypes from healthy donors revealed that the ANCA signature genes were expressed by neutrophils while the SLE signature genes were expressed in activated monocytes and T cells. We have found that leukocyte gene expression data can differentiate patients with RA, SLE, and ANCA-related small vessel vasculitis. Monitoring changes in the expression of specific genes may be a tool to help quantify disease activity during treatment.     

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.     

Outcome analysis of patients with vasculitis associated with antineutrophil cytoplasmic antibodies

BACKGROUND: Objective scoring systems of disease activity and disease-associated damage have proven useful in the management of patients with systemic vasculitis. PATIENTS AND METHODS: We used the recently designed Birmingham vasculitis activity score (BVAS; maximum score 63) and vasculitis damage index (VDI; maximum score 59) to assess initial activity and long-term damage, respectively, in ANCA positive patients from one center over a 3-year period. Thirty-two patients with ANCA vasculitis were identified and analyzed as an historic cohort. The median BVAS for all vasculitis patients at first presentation was 19 (range 6 - 36). Patients with Wegener's granulomatosis had a significantly higher total score and respiratory BVAS score compared to the 15 with microscopic polyangiitis. The majority of patients received standard cyclophosphamide/steroid treatment. RESULTS: At the end of follow-up (mean 24.9 months), 4 patients had died; all patients had evidence of permanent organ damage. The median total VDI score at last follow-up was 4.0 (range 0-11), with no differences between patients with Wegener's granulomatosis and microscopic polyangiitis. The VDI was not associated with the number of relapses. A high initial BVAS was found to correlate with a later high vasculitis damage index (r = 0.56). Initial renal or respiratory involvement was also associated with longterm damage in the same organ system. CONCLUSION: Although mortality from ANCA-associated vasculitis has decreased, morbidity remains a common problem. High early-disease activity may identify patients at high risk of long-term organ damage, allowing more effective individualized therapy. This hypothesis requires validation in a prospective, controlled study.     

A pediatric occurrence of crescentic glomerulonephritis associated with antineutrophil cytoplasmic antibodies and mesangial IgA deposits

Antineutrophil cytoplasmic antibody-(ANCA) associated glomerulonephritis usually shows histopathologic features of pauciimmune crescentic glomerulonephritis and occurs late in life. We report a 14-year-old Japanese girl presenting with proteinuria, hematuria and mildly elevated serum creatinine. A renal biopsy specimen demonstrated crescentic glomerulonephritis, immunofluorescence showed mesangial IgA staining. Electron microscopic examination disclosed paramesangial deposits. Serum ANCA against myeloperoxidase (MPO) were detected at high titers. Myeloperoxidase-ANCA-related nephritis accompanied by IgA nephropathy is considered rare in childhood and teen years. Yet, if ANCA assays and detailed electron microscopic examination of renal specimens were performed routinely in patients with rapidly progressive glomerulonephritis, the diagnosis might be more frequent in young patients.     

Iga nephropathy, antineutrophil cytoplasmic antibodies and crescentic glomerulonephritis in a patient with the Hermansky-Pudlak syndrome

Hermansky-Pudlak syndrome is an uncommon cause of renal dysfunction. Because of the risk of bleeding in this condition, few patients have undergone a renal biopsy. Renal dysfunction has been attributed to the deposition of ceroid pigment in the tubules and interstitial fibrosis. We report a case with renal biopsy findings of ceroid deposition and interstitial fibrosis, but also of mesangial IgA deposition, crescentic glomerulonephritis, and an interstitial lymphocytic infiltrate. Furthermore, perinuclear antineutrophil cytoplasmic antibodies of the IgG subclass were detected in a blood sample. It is well known that ceroid pigment in this syndrome accumulates in monocytes, macrophages and T lymphocytes and it has been suggested that this may affect their function. We suggest that this novel combination of renal changes might be explained on the basis of alterations in immune mechanisms in the Hermansky-Pudlak syndrome.     

Cytoplasmic antineutrophil cytoplasmic antibody positive pauci-immune glomerulonephritis associated with infectious endocarditis

Renal deterioration often occurs in cases of infectious endocarditis (IE), but, IE- associated nephritis with rapidly progressive glomerulonephritis (RPGN) is rare. Patients with severe infection (e.g., IE) sometimes show positivity for cytoplasmic antineutrophil cytoplasmic antibodies (C-ANCA). Therefore, diagnosis and treatment are very difficult in cases of RPGN with IE and positivity for C-ANCA. Such cases are rare, only 12 have been reported in the English literature. Herein, we describe the case of a 50-year-old man who presented with RPGN with IE and tested positively for C-ANCA. He was referred to our hospital because of leg edema, purpura and renal dysfunction. Laboratory tests revealed serum creatinine elevation and positivity for C-ANCA and proteinase 3-specific (PR3)-ANCA. RPGN and acute renal failure were diagnosed. Hemodialysis and steroid therapy were started. Streptococcus oralis was isolated by blood culture. Transthoracic echocardiography revealed grade III mitral valve insufficiency with two vegetations. Therefore, IE was diagnosed. The steroid therapy was stopped, and antibiotic therapy was begun. Because there was no improvement, surgical therapy was performed. The operation was successful, but the patient died of brain hemorrhage. Our experience in this case indicates C/PR3-ANCA positive RPGN must be ruled out in patients with infectious disease, particularly IE, together with renal symptoms, and renal biopsy should be performed.     

How are antineutrophil cytoplasmic autoantibodies detected?

Antineutrophil cytoplasmic autoantibodies (ANCA) are present in patients with systemic vasculitis with or without renal involvement. These antibodies were first seen using indirect immunofluorescence (IIF). Two types of patterns are seen on ethanol-fixed neutrophils: the cytoplasmic and the perinuclear pattern. The cytoplasmic pattern is called C-ANCA (classical or cytoplasmic ANCA) and the perinuclear, P-ANCA. Antibodies to a serine proteinase, called proteinase 3 or myeloblastin, give rise to the C-ANCA pattern, while antibodies to myeloperoxidase give rise to the P-ANCA pattern. Proteinase 3, as well as myeloperoxidase, is present in the primary granules of neutrophils, and the P-ANCA pattern is thus an artifactual staining pattern. Myeloperoxidase, which is a basic protein, redistributes during ethanol fixation from the primary granules to the negatively charged nucleus. As an alternative to the IF technique, several solid-phase assays have been developed using either 125I or enzyme-labeled secondary antibodies. Depending on the degree of purification of the antigens used, such assays may be used for screening or as a complement to the IF method. Today it is possible to directly screen for both types of ANCA using enzyme-linked immunosorbent assay (ELISA). Simultaneous screening for antiglomerular basement membrane (GBM) antibodies (Goodpasture antibodies) increases the diagnostic yield, especially in patients with renopulmonary syndromes.     

Interleukin-8: A pathogenetic role in antineutrophil cytoplasmic autoantibody-associated glomerulonephritis

BACKGROUND: In neutrophil trafficking, the role of interleukin-8 (IL-8) is location dependent. Tissue IL-8 directs transmigration, whereas intravascular IL-8 frustrates this process. The bystander damage of glomerular endothelium by antineutrophil cytoplasmic autoantibody (ANCA)-activated neutrophils is believed to be an early event in the pathogenesis of ANCA-associated glomerulonephritis. We have studied the role of IL-8 in this process. METHODS: Intraglomerular expression of IL-8 in patients with ANCA-associated glomerulonephritis was studied by in situ hybridization and immunohistochemistry and location of neutrophils by serial section immunohistochemistry. In vitro, we analyzed ANCA-stimulated neutrophil IL-8 production by enzyme-linked immunosorbent assay, and the IL-8 attributable effect of ANCA-stimulated neutrophil supernatant by chemotactic and transendothelial assays. RESULTS: There was intraglomerular expression of IL-8 at segmental, crescentic, and parietal epithelial sites. IL-8 protein expression colocalized to intraglomerular neutrophils; many localized within glomerular capillary loops, suggesting failed trafficking to tissue IL-8. ANCAs differentially stimulated time- and dose-dependent neutrophil IL-8 production, and ANCA-stimulated neutrophil supernatant demonstrated potent IL-8-dependent chemotactic activity and inhibited transendothelial migration of normal human neutrophils toward an IL-8 gradient. CONCLUSION: Despite heavy tissue expression of IL-8 in ANCA-associated GN, the production of IL-8 by ANCA-stimulated neutrophils within the intravascular compartment may frustrate neutrophil transmigration, encourage intravascular stasis, and contribute to bystander damage of glomerular endothelial cells.     

抗中性粒细胞胞浆抗体相关性血管炎患者白细胞介素18的水平及意义

目的检测抗中性粒细胞胞浆抗体相关性血管炎(antineutrophil cytoplasmic antibodies associated vasculitis,AAV)患者白细胞介素18(interleukin-18,IL-18)的水平,探讨IL-18在AAV发病中的意义。方法选择2013年4月至2014年7月在第三军医大学附属新桥医院肾内科住院诊治的从V患者27例设为AAV组,选择17例有蛋白尿、镜下血尿且肾活检明确诊断为轻度系膜增生性肾小球肾炎(mesangial proliferative glomerulonephritis,MsPGN)患者设为MsPGN组,另外选择17名健康者设为健康对照组,收集3组血清及其临床资料,采用酶联免疫吸附试验(enzymelinked immunosorbent assay,ELISA)方法检测3组患者血清IL-18浓度;Pearson积矩相关分析AAV患者血清IL-18水平与AAV活动性指标伯明翰血管炎活动性评分(birmingham vasculitis active scoring systen,BVAS)、C反应蛋白(C-reaction protein,CRP)水平、红细胞沉降率(erythrocyte sedimentation rate,ESR)、估算肾小球滤过率(estimated glomerular filtration rate,eGFR)、血肌酐(SCr)水平及24 h尿蛋白定量的相关性;并对活动期和缓解期AAV患者的血清1I-18、CRP和ESR水平行受试者工作特征(receiver operating characteristic,ROC)曲线的曲线下面积(area under the curve,AUC)评价IL-18作为评估疾病活动性指标的准确性。结果①活动期AAV患者IL-18浓度[(183.85±57.0)ng/L]高于缓解期AAV患者IL-18水平[(92.0±34.29)ng/L](P0.05),MsPGN组水平为[(63.0±17.7)ng/L](P0.01)和健康对照组水平为[(39.2±6.3)ng/L](P0.01);②从V患者IL-18水平与BVAS(r=0.572,P0.01)、ESR水平(r=0.394,P0.05)呈正相关;与CRP水平、eGFR、SCr水平及24 h尿蛋白定量(r=0.117,-0.250,0.290,0.200,均P0.05)无明显相关;③ROC曲线分析显示,相比血清CRP和ESR水平,IL-18水平能更好地区分AAV患者的活动性。结论 AAV患者活动期IL-18水平明显升高,且与疾病的活动性密切相关,提示IL-18可能参与AAV的病理损伤过程。     

Antineutrophil cytoplasmic antibodies (ANCA) in Chinese patients with anti-GBM crescentic glomerulonephritis

OBJECTIVE: To study the prevalence of ANCA and their target antigen in Chinese patients with anti-GBM crescentic glomerulonephritis (CGN), and to evaluate the possible role of ANCA in Chinese anti-GBM CGN patients with coexisting serum ANCA by studying clinicopathologic features of this disease. MATERIAL AND METHODS: Twenty-three sera were collected from 23 renal biopsy-proven anti-GBM CGN patients. According to the standardized procedures, all of the sera were determined by both, indirect immunofluorescence (IIF) ANCA, and enzyme-linked immunosorbent assay (ELISA) MPO-ANCA, PR3-ANCA and BPI-ANCA. The patients were divided into two groups according to serum ANCA positivity (Group A) or negativity (Group B). Thirty-three ANCA-associated pauci-immune CGN patients were regarded as control group (Group C). Their clinicopathologic features were compared to reveal whether ANCA correlated with disease activity. RESULTS: There were 11 (47.8%) cases with positive serum ANCA in 23 anti-GBM glomerulonephritis patients. There were 4/11 MPO-ANCA (one with positive PR3-ANCA and C-ANCA, three with negative IIF-ANCA), 1/11 PR3-ANCA (with positive MPO-ANCA and C-ANCA), 3/11 P-ANCA (with negative ELISA-ANCA) and 5/11 C-ANCA (one with positive PR3-ANCA and MPO-ANCA, and the other four with negative ELISA-ANCA). No BPI-ANCA was detected. No different clinicopathologic features were found between Groups A and B. Both were different from Group C in age, sex ratio, frequence of anuria and ESRD, variety of crescents, glomerular sclerosis, vessel lesion and prognosis. CONCLUSION: Our data demonstrate that ANCA in Chinese patients with anti-GBM CGN is not rare. The major target antigen of ANCA is MPO. ANCA seems not to be correlated with disease activity.     

Myeloperoxidase antineutrophil cytoplasmic antibody-positive necrotizing crescentic glomerulonephritis and membranous glomerulonephropathy.

In September 1997, a 68-year-old woman was found to have proteinuria and renal dysfunction. In December 1997, renal biopsy revealed necrotizing crescentic glomerulonephritis and membranous glomerulonephropathy. We diagnosed myeloperoxidase antineutrophil cytoplasmic antibody-positive necrotizing crescentic glomerulonephritis and membranous glomerulonephropathy because of the presence of necrotizing cellular crescents and spike lesions in the subepithelial region of the glomerular basement membrane. After steroid therapy, the antibody level and the incidence of cellular crescents showed a decrease. This is a rare case of myeloperoxidase antineutrophil cytoplasmic antibody-positive necrotizing crescentic glomerulonephritis associated with membranous glomerulonephropathy.     

Ceruloplasmin levels in antineutrophil cytoplasmic antibody-positive patients

Anti-myeloperoxidase (MPO) antibodies are associated with the development of anti-neutrophil cytoplasmic antibody (ANCA)-related vasculitis. The imbalance between the protease-antiprotease activity in the neutrophils has been implicated in the pathogenesis of ANCA-related vasculitis. Ceruloplasmin is an acute-phase protein that has antiproteinase and antioxidant properties and inhibits MPO activity. We attempted to study the association between serum ceruloplasmin and ANCA in childhood vasculitis. Forty-five ANCA-related diseases were included in the study. The age range was 4-16 years. Patients were divided into two groups based on indirect immunofluorescence and/or ELISA specificity (MPO). Twenty-six patients had p-ANCA- and 19 patients had c-ANCA-positive disease. Nine patients with Henoch-Sch?nlein purpura were studied as an ANCA-negative control group. Serum ceruloplasmin levels in p-ANCA-, c-ANCA-positive patients, and controls were 125.85+/-93.48 mg/dl, 59.79+/-17.60 mg/dl, and 64.34+/-18.77 mg/dl, respectively, and were significantly higher in patients with p-ANCA ( P<0.05). Ceruloplasmin levels were significantly decreased in remission ( P<0.05). Median MPO level in p-ANCA-positive patients was 15.2 (5-250) and was negative in all c-ANCA-positive patients. There was a significant positive correlation between MPO and ceruloplasmin levels ( r=0.70, P<0.05). Of 26 patients (53.8%) in the p-ANCA-positive group, 14 had renal involvement. The patients with renal disease had significantly higher ceruloplasmin levels than others (151.17+/-92.14 and 134.64+/-95.16 mg/dl respectively, P<0.05). In conclusion, the increase in ceruloplasmin levels during the acute phase suggests that this might be an activation criterion or a response to neutrophil-mediated tissue injury. Increased ceruloplasmin levels together with p-ANCA positivity may be predictive for renal involvement and a serious clinical course. The correlation between ceruloplasmin and MPO levels supports their association. Further studies are necessary to elucidate whether genetic and/or functional alterations in ceruloplasmin are effective in the pathogenesis of vasculitis.     

抗中性粒细胞胞质抗体在狼疮活动期和伴新月体肾炎诊断中的意义

本研究对132例不同时期、不同病理类型的系统性红斑狼疮(SLE)患者产生的抗中性粒细胞胞质抗体(ANCA)与SLE活动期相关自身抗体、狼疮疾病活动指数、临床表现和其他相关实验室检测指标间的关系进行分析,旨在探讨ANCA在狼疮活动期和伴新月体肾炎诊断中的意义.     

Treatment of antineutrophil cytoplasmic autoantibody-positive systemic vasculitis and glomerulonephritis with pooled intravenous gammaglobulin.

Antineutrophil cytoplasmic autoantibody (ANCA) is considered a serological marker for disease activity in patients with ANCA(+) systemic vasculitis. Recently, ANCA has been implicated as a pathogenic antibody that may be associated with neutrophil degranulation and release of lytic enzymes. Since intravenous gammaglobulin (IVIG) is known to contain antiidiotypic antibodies to ANCA, which could decrease the activity of the later, we chose to treat two patients with symptomatic ANCA(+) systemic vasculitis and glomerulonephritis with high-dose IVIG. The first patient, a 66-year-old man, developed rapidly progressive renal failure despite treatment with intravenous (IV) cyclophosphamide. The second patient, a 14-year-old boy, had relapsed 3 months after cessation of treatment with prednisone and cyclophosphamide. Both patients improved dramatically after treatment with IVIG, with the former recovering renal function within 11 days of therapy. In both patients, a concomitant reduction in serum ANCA titers was also observed. The second patient is currently in a sustained remission 14 months after his last IVIG dose on no other medication. These cases provide clinical evidence that IVIG has therapeutic benefit in modifying the immune-mediated injury associated with ANCA(+) systemic vasculitis and glomerulonephritis. In addition, IVIG may provide an additional safe therapeutic option to clinicians treating patient's with ANCA(+) vasculitis and glomerulonephritis who are not responsive to or are experiencing toxicity from conventional therapy.     

Clinical features and outcomes in children with antineutrophil cytoplasmic autoantibody-positive glomerulonephritis associated with propylthiouracil treatment.

A retrospective investigation was conducted by members of the Japanese Society for Pediatric Nephrology from 1990 to 1997 to define the clinical features and outcomes in children with antineutrophil cytoplasmic autoantibody (ANCA)-positive glomerulonephritis associated with propylthiouracil treatment. Seven Japanese pediatric patients who had myeloperoxidase-specific ANCA-positive biopsy-proven pauci-immune necrotizing crescentic glomerulonephritis associated with propylthiouracil administration were entered in the study. Three patients had nephritis alone, and four had nephritis and extrarenal organ system vasculitis. Females predominated, and the mean age at onset was 14 yr. Propylthiouracil was reduced or discontinued in all patients and was switched to methimazole in three patients. For the treatment of nephritis, five patients received corticosteroids; three had pulse methylprednisolone, one had plasma exchange, and one had plasma exchange and pulse methylprednisolone before initiating oral prednisolone. The remaining two patients received cyclophosphamide and corticosteroids, one of whom had pulse methylprednisolone before initiating oral prednisolone and cyclophosphamide. All patients achieved remission. In general, ANCA titers correlated with the response to treatment and disease activity, with some exceptions. No patient progressed to end-stage renal disease, renal dysfunction, or death during the follow-up period (58 +/- 25 mo; range, 32 to 108 mo). All but one patient remained euthyroid. In conclusion, this experience suggests that the clinical disease spectrum of ANCA-positive disease associated with propylthiouracil treatment is similar in pediatric and adult patients and that the overall prognosis may be better than that in the non-drug-induced ANCA-positive disease.     

Tubular lesions predict renal outcome in antineutrophil cytoplasmic antibody-associated glomerulonephritis after rituximab therapy

Histopathological features in renal biopsies of patients with antineutrophil cytoplasmic antibody-associated vasculitis have predictive value for renal outcome in patients who receive standard treatment with cyclophosphamide and corticosteroids; however, whether the same holds true for rituximab-treated patients is unknown. We describe associations between renal histopathology and outcomes among patients treated with a rituximab-based regimen in the Randomized Trial of Rituximab versus Cyclophosphamide in ANCA-Associated Vasculitis trial. Two pathologists, blinded to clinical data, reviewed biopsies from 30 patients according to a standardized protocol that included assessment of T cell, B cell, and plasma cell infiltration, as well as scoring for tubulitis, interstitial inflammation, and glomerulitis. We did not observe associations between immunohistology scores and age, sex, estimated GFR at entry, or requirement for dialysis. However, tubulointerstitial inflammation was more severe among patients who had a positive test for the myeloperoxidase antineutrophil cytoplasmic antibody. In a multiple linear regression model, both CD3(+) T cell tubulitis and tubular atrophy independently associated with estimated GFR at 12 months. Tubular atrophy remained an independent predictor at 24 months (P<0.01). These results suggest that in addition to anti-B cell therapy, therapy directed at T cells may improve renal outcomes in antineutrophil cytoplasmic antibody-associated vasculitis.