Jiajin Yang

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.

DRB1*15 Allele Is a Risk Factor for PR3-ANCA Disease in African Americans

Anti-neutrophil cytoplasmic autoantibody (ANCA) disease rarely occurs in African Americans and risk factors for the disease in this population are unknown. Here, we genotyped MHC class II alleles and found that, among African Americans, those with proteinase 3-ANCA (PR3-ANCA) had 73.3-fold higher odds of having HLA-DRB1*15 alleles than community-based controls (OR 73.3; 95% CI 9.1 to 591). In addition, a disproportionate number of African American patients carried the DRB1*1501 allelic variant of Caucasian descent rather than the DRB1*1503 allelic variant of African descent. Among Caucasians, those with PR3-ANCA had 2.2-fold higher odds of carrying DRB1*1501 than controls (OR 2.2; 95% CI 1.2 to 4.0). A validation study supported by the Vasculitis Clinical Research Consortium confirmed the strong association between the DRB1*15 allele and PR3-ANCA disease, among African Americans. Furthermore, we found that DRB1*1501 protein binds with high affinity to amino acid sequences of sense-PR3, purportedly an antigenic epitope, and to the amino acid sequence complementary to this epitope in vitro. Peptides of sense-PR3 and complementary-PR3 also bound to TNF-α-induced surface expression of DRB1*1501 on peripheral neutrophils. Taken together, these data suggest HLA-DRB1*15 alleles contribute to the pathogenesis of PR3-ANCA disease.

Immune complex glomerulonephritis is induced in rats immunized with heterologous myeloperoxidase.

Anti‐neutrophil cytoplasmic antibodies (ANCA), including anti‐myeloperoxidase (MPO) antibodies, are associated with pauci‐immune necrotizing small vessel vasculitis or glomerulonephritis, 1n order to substantiate a pathogenic role for ANCA, an animal model of pauci‐immune ANCA‐induced glomerulonephritis or vasculitis is required. Brouwer et al. reported pauci‐immune glomerulonephritis in rats immunized with human MPO followed by perfusion of kidneys with lysosomal enzyme extract combined with H2O2, and suggested that this could serve as a model of ANCA‐induced disease. We repeated these studies in spontaneously hypertensive rats (SHR) and Brown Norway rats (BNR). We immunized rats with human MPO, When circulating anti‐MPO antibodies were detectable by indirect immunofluorescence microscopy and ELISA, blood pressure was measured, then perfusion of the left kidney of each rat was done via the renal artery in a closed, blood‐free circuit with either MPO + H2O;, MPO, H2O2 alone or MPO + H2O2 neutral protease. Rats were killed on day 4 or day 10 after perfusion, and specimens were examined by light and immunofluorescence microscopy. Pathological lesions and deposits of IgG. C3, and MPO were found in immunized rats perfused with MPO + H2O2 with or without neutral protease, or MPO alone, in both rat strains and on both day 4 and day 10, The degree of histologic injury was proportional in intensity to the amount of IgG immune deposits. Spontaneously hypertensive rats sustained more damage and higher blood pressure than Brown Norway rats. No lesion was observed in immunized rats perfused with H2O2 or in the non‐perfused right kidneys. Some of the non‐immunized rats perfused with MPO + H2O2 developed pathological lesions. In conclusion, these rat models are examples of immune complex‐mediated glomerulonephritis, and therefore are not similar to human ANCA‐associated disease.

ANCA patients have T cells responsive to complementary PR-3 antigen

Some patients with proteinase 3 specific anti-neutrophil cytoplasmic autoantibodies (PR3-ANCA) also have antibodies that react to complementary-PR3 (cPR3), a protein encoded by the antisense RNA of the PR3 gene. To study whether patients with anti-cPR3 antibodies have cPR3-responsive memory T cells we selected conditions that allowed cultivation of memory cells but not naïve cells. About half of the patients were found to have CD4+TH1 memory cells responsive to the cPR3(138-169)-peptide; while only a third of the patients had HI-PR3 protein responsive T cells. A significant number of T cells from patients responded to cPR3(138-169) peptide and to HI-PR3 protein by proliferation and/or secretion of IFN-gamma, compared to healthy controls while there was no response to scrambled peptide. Cells responsive to cPR3(138-169)-peptide were not detected in MPO-ANCA patients suggesting that this response is specific. The HLADRB1(*) 15 allele was significantly overrepresented in our patient group and is predicted to bind cPR3(138-169) peptide with high affinity. Regression analysis showed a significant likelihood that anti-cPR3 antibodies and cPR3-specific T cells coexist in individuals, consistent with an immunological history of encounter with a PR3-complementary protein. We suggest that the presence of cells reacting to potential complementary protein pairs might provide an alternative mechanism for auto-immune diseases.

Frequency of anti-bactericidal/permeability-increasing protein (BPI) and anti-azurocidin in patients with renal disease.

The major subtypes of anti‐neutrophil cytoplasmic antibodies (ANCA) detected by indirect immunofluorescence assay (IFA) are P‐ANCA and C‐ANCA. In patients with vasculitis, myeloperoxidase (MPO) is the major P‐ANCA antigen and proteinase 3 (PR3) is the major C‐ANCA antigen. BPI and azurocidin, which are also called 57‐kD cationic antimicrobial protein (CAP 57) and 37‐kD cationic antimicrobial protein (CAP 37), respectively, have been proposed as less frequent target antigens for C‐ANCA and P‐ANCA. In patients with renal disease, we determined the frequency of antibodies against BPI and azurocidin. By IFA on alcohol‐fixed neutrophils, monoclonal and polyclonal anti‐BPI antibodies produced a C‐ANCA pattern, whereas rabbit anti‐azurocidin antibody produced a P‐ANCA pattern. By ELISA, sera from 229 P‐ANCA‐positive patients, 99 C‐ANCA‐positive patients and 48 ANCA‐negative (by IFA) patients with renal biopsies were tested for reactivity with recombinant human BPI and purified human azurocidin. Of these sera, 17.5% of P‐ANCA, 30.3% of C‐ANCA and 20.8% of IFA‐ANCA‐negative sera were positive for anti‐BPI; and 8.3% of P‐ANCA, 3.0% of C‐ANCA and 8.3% of IFA‐ANCA‐negative sera were positive for anti‐azurocidin. There was no statistical difference in frequency of anti‐BPI between pauci‐immune necrotizing and crescentic glomerulonephritis (NCGN) and other glomerular disease (OGD), and there was a lower frequency of anti‐azurocidin in NCGN samples than in OGD samples. By Western blot, anti‐BPI‐positive sera reacted with a 57‐kD BPI band and anti‐azurocidin‐positive sera with a 29‐kD azurocidin band. In conclusion, there is a low frequency of anti‐BPI and anti‐azurocidin antibodies in ANCA‐positive patient sera; however, this does not correlate with NCGN, which is a marker for ANCA‐associated small vessel vasculitis, and a similar positivity is found in IFA‐ANCA‐negative patients with renal disease. Therefore, serologic detection of anti‐BPI and anti‐azurocidin is not diagnostically specific in patients with renal disease.

High Basal Activity of the PTPN22 Gain-of-Function Variant Blunts Leukocyte Responsiveness Negatively Affecting IL-10 Production in ANCA Vasculitis

Consequences of expression of the protein tyrosine phosphatase nonreceptor 22 (PTPN22) gain-of-function variant were evaluated in leukocytes from patients with anti-neutrophil cytoplasmic autoantibody (ANCA) disease. The frequency of the gain-of-function allele within the Caucasian patient cohort was 22% (OR 1.45), compared to general American Caucasian population (16.5%, p = 0.03). Examination of the basal phosphatase activity of PTPN22 gain-of-function protein indicated persistently elevated activity in un-stimulated peripheral leukocytes, while basal activity was undetectable in leukocytes from patients without the gain-of-function variant. To examine consequences of persistently high PTPN22 activity, the activation status of ERK and p38 MAPK were analyzed. While moderate levels of activated ERK were observed in controls, it was undetectable in leukocytes expressing PTPN22 gain-of-function protein and instead p38MAPK was up-regulated. IL-10 transcription, reliant on the ERK pathway, was negatively affected. Over the course of disease, patients expressing variant PTPN22 did not show a spike in IL-10 transcription as they entered remission in contrast to controls, implying that environmentally triggered signals were blunted. Sustained activity of PTPN22, due to the gain-of-function mutation, acts as a dominant negative regulator of ERK activity leading to blunted cellular responsiveness to environmental stimuli and expression of protective cytokines.

Increased expression of the secretory leukocyte proteinase inhibitor in Wegener's granulomatosis

The secretory leucocyte proteinase inhibitor (SLPI) is a low molecular weight, tissue‐specific inhibitor of proteases, such as elastase and cathepsin G. It is the major local protease inhibitor in the upper airways. Proteinase 3, the main autoantigen in Wegeners granulomatosis (WG), can degrade SLPI proteolytically. In addition, SLPI is sensitive to oxidative inactivation by myeloperoxidase‐generated free oxygen radicals. SLPI also has an antimicrobial capacity that can be of interest, as infection is considered to play a role in the pathogenesis of WG. This study focuses on SLPI expression in patients suffering from WG, something that to our knowledge has not been explored hitherto. Serum samples and nasal biopsies were obtained from 12 Swedish WG patients, while buffy coats were obtained from 33 American WG patients. SLPI levels in serum were measured by means of ELISA and the protein was detected by means of immunohistochemistry in nasal biopsies. mRNA expression was studied by means of in situ hybridization on nasal biopsies and RT‐PCR on leucocytes. IL‐6 or ESR were measured as markers of inflammatory activity. Cystatin C or creatinine was measured as a marker of renal filtration. White blood cell counts were registered. In serum, we found close to normal SLPI levels, without any correlation to IL‐6. Two patients had greatly elevated values, both of them suffering from severe renal engagement. Strong SLPI mRNA expression was found in nasal biopsies. RT‐PCR on leucocyte mRNA showed normal or greatly elevated expression of SLPI mRNA, correlating with disease activity. Leukocyte SLPI expression seems to be up‐regulated in active WG. Serum levels were measured in a small number of patients and were found to be close to normal. Lack of correlation to the acute phase response indicates a specific regulation. This might be linked to an altered protease/antiprotease balance. These findings could indicate that SLPI locally participates in the anti‐inflammatory and perhaps antimicrobial response in WG.

A study of conformational restraints on reactivity of human PR3-specific autoantibodies (ANCA) facilitated through protein folding manipulations of a new recombinant proteinase 3 protein

Proteinase 3 (PR3)-specific antineutrophil cytoplasmic autoantibodies (PR3-ANCA) recognize conformational epitopes on PR3. This study evaluates PR3-ANCA target epitopes utilizing a novel recombinant PR3 (rPR3) produced to accommodate manipulations of the N-terminal domain. The rPR3 molecule contains an N-terminus six histidine tag, which can be removed by enterokinase (EK) cleavage of an adjacent EK cleavage site. Once cleaved the remaining amino acids correspond to the mature N-terminus of PR3. This rPR3 can be manipulated to produce three variant forms: tagged rPR3+his, EK-cleaved (his-tag removed) rPR3− his, and EK-cleaved, denatured/refolded rPR3− his/dr (the proteolytically active form). Patients with clinically positive PR3-ANCA titers (n = 40) were confirmed for reactivity against purchased native PR3 in our system. Controls included 29 healthy volunteers and 34 MPO-ANCA patients. All PR3-ANCA sera samples tested were reactive with one or more forms of the recombinant protein (greater than mean ELISA OD 405 + 2 SDs of controls). Of significance, three sera were reactive with non-active forms only and three others were more reactive with rPR3− his/dr than with native PR3. The results of our evaluation of PR3-ANCA sera for reactivity against the three forms of our rPR3 protein uniquely exemplify the diverse array of epitopes within the PR3-ANCA population. This new recombinant form of PR3 should provide a suitable approach to mapping ANCA epitopes using site-directed mutagenesis.

Gene-Specific DNA Methylation Changes Predict Remission in Patients with ANCA-Associated Vasculitis

ANCA-associated vasculitis is an autoimmune condition characterized by vascular inflammation and organ damage. Pharmacologically induced remission of this condition is complicated by relapses. Potential triggers of relapse are immunologic challenges and environmental insults, both of which associate with changes in epigenetic silencing modifications. Altered histone modifications implicated in gene silencing associate with aberrant autoantigen expression. To establish a link between DNA methylation, a model epigenetic gene silencing modification, and autoantigen gene expression and disease status in ANCA-associated vasculitis, we measured gene-specific DNA methylation of the autoantigen genes myeloperoxidase (MPO) and proteinase 3 (PRTN3) in leukocytes of patients with ANCA-associated vasculitis observed longitudinally (n=82) and of healthy controls (n=32). Patients with active disease demonstrated hypomethylation of MPO and PRTN3 and increased expression of the autoantigens; in remission, DNA methylation generally increased. Longitudinal analysis revealed that patients with ANCA-associated vasculitis could be divided into two groups, on the basis of whether DNA methylation increased or decreased from active disease to remission. In patients with increased DNA methylation, MPO and PRTN3 expression correlated with DNA methylation. Kaplan-Meier estimate of relapse revealed patients with increased DNA methylation at the PRTN3 promoter had a significantly greater probability of a relapse-free period (P<0.001), independent of ANCA serotype. Patients with decreased DNA methylation at the PRTN3 promoter had a greater risk of relapse (hazard ratio, 4.55; 95% confidence interval, 2.09 to 9.91). Thus, changes in the DNA methylation status of the PRTN3 promoter may predict the likelihood of stable remission and explain autoantigen gene regulation.

PTPN22 R620W Polymorphism and ANCA Disease Risk in White Populations: A Metaanalysis

Objective. No clear consensus has been reached on the PTPN22 R620W polymorphism and anti-neutrophil cytoplasmic antibody (ANCA) disease, especially when stratified by ANCA specificity and disease phenotypes. Methods. A metaanalysis was conducted on the PTPN22 R620W polymorphism across 4 studies in 1399 white patients with ANCA disease and 9934 normal control subjects. Results. Overall, metaanalysis showed a statistically significant association between the A allele and ANCA disease in all subjects (OR 1.44, 95% CI 1.26–1.64, p < 0.00001), and stratification by disease category indicated the A allele was associated with granulomatosis with polyangiitis (Wegener’s; GPA; OR 1.72, 95% CI 1.35–2.20, p < 0.0001) and microscopic polyangiitis (MPA; OR 1.53, 95% CI 1.08–2.15, p = 0.02) as compared to controls. However, when stratified by ANCA specificity, the association of the A allele was statistically evident among those with proteinase 3 (PR3) ANCA disease (OR 1.74, 95% CI 1.25–2.430, p = 0.001), with the same trend but not statistically associated with myeloperoxidase ANCA disease (OR 1.94, 95% CI 0.64–5.85, p = 0.24). The marked associations were also demonstrated between this allele with lung (OR 1.69, 95% CI 1.21–2.36, p = 0.002), ENT (OR 2.03, 95% CI 1.45–2.84, p < 0.0001), skin (OR 2.55, 95% CI 1.69–3.84, p < 0.0001), and peripheral neuropathy involvement (OR 2.12, 95% CI 1.39–3.22, p = 0.0005). Conclusion. The PTPN22 620W allele confers susceptibility to the occurrence and development of ANCA disease in whites, with specific evidence among subsets with GPA, MPA, and PR3 ANCA.