Gloria A. Preston

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

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

Epigenetic basis for aberrant upregulation of autoantigen genes in humans with ANCA vasculitis

Antineutrophil cytoplasmic autoantibody (ANCA) causes vascular injury that leads to small-vessel vasculitis. Patients with ANCA aberrantly express neutrophil granule-encoding genes, including 2 that encode autoantigens: proteinase 3 (PR3) and myeloperoxidase (MPO). To uncover a potential transcriptional regulatory mechanism for PR3 and MPO disrupted in patients with ANCA vasculitis, we examined the PR3 and MPO loci in neutrophils from ANCA patients and healthy control individuals for epigenetic modifications associated with gene silencing. We found that levels of the chromatin modification H3K27me3, which is associated with gene silencing, were depleted at PR3 and MPO loci in ANCA patients compared with healthy controls. Interestingly, in both patients and controls, DNA was unmethylated at a CpG island in PR3, whereas in healthy controls, DNA was methylated at a CpG island in MPO. Consistent with decreased levels of H3K27me3, JMJD3, the demethylase specific for H3K27me3, was preferentially expressed in ANCA patients versus healthy controls. In addition, we describe a mechanism for recruiting the H3K27 methyltransferase enhancer of zeste homolog 2 (EZH2) to PR3 and MPO loci mediated by RUNX3. RUNX3 message was decreased in patients compared with healthy controls, and may also be under epigenetic control. DNA methylation was increased at the RUNX3 promoter in ANCA patients. These data indicate that epigenetic modifications associated with gene silencing are perturbed at ANCA autoantigen-encoding genes, potentially contributing to inappropriate expression of PR3 and MPO in ANCA patients.

Epitope specificity determines pathogenicity and detectability in ANCA-associated vasculitis

Anti-neutrophil cytoplasmic antibody-associated (ANCA-associated) small vessel necrotizing vasculitis is caused by immune-mediated inflammation of the vessel wall and is diagnosed in some cases by the presence of myeloperoxidase-specific antibodies (MPO-ANCA). This multicenter study sought to determine whether differences in ANCA epitope specificity explain why, in some cases, conventional serologic assays do not correlate with disease activity, why naturally occurring anti-MPO autoantibodies can exist in disease-free individuals, and why ANCA are undetected in patients with ANCA-negative disease. Autoantibodies from human and murine samples were epitope mapped using a highly sensitive epitope excision/mass spectrometry approach. Data indicated that MPO autoantibodies from healthy individuals had epitope specificities different from those present in ANCA disease. Importantly, this methodology led to the discovery of MPO-ANCA in ANCA-negative disease that reacted against a sole linear sequence. Autoantibodies against this epitope had pathogenic properties, as demonstrated by their capacity to activate neutrophils in vitro and to induce nephritis in mice. The confounder for serological detection of these autoantibodies was the presence of a fragment of ceruloplasmin in serum, which was eliminated in purified IgG, allowing detection. These findings implicate immunodominant epitopes in the pathology of ANCA-associated vasculitis and suggest that autoantibody diversity may be common to other autoimmune diseases.

Internalization of Proteinase 3 Is Concomitant with Endothelial Cell Apoptosis and Internalization of Myeloperoxidase with Generation of Intracellular Oxidants

The important issue addressed by the studies presented here is the mechanism of neutrophil-mediated damage to endothelial and epithelial cells during inflammation. Binding of neutrophil-released granule proteins to endothelial cells may be involved in vascular damage in patients with inflammatory vascular diseases. We have determined whether granule proteins proteinase 3(PR3) and/or myeloperoxidase (MPO) are internalized into endothelial cells, as examined by UV light, confocal, and electron microscopy. Coincident induction of apoptosis and/or the generation of intracellular oxidants were monitored. The results indicate that human endothelial cells (human umbilical vein endothelial cells, human umbilical arterial endothelial cells, human lung microvascular endothelial cells) internalize both PR3 and MPO, which are detected on the cell surface, in the cytoplasm, and possibly nuclear. Epithelial cells (small airway epithelial cells) internalized MPO but not PR3, implying that the mechanism of PR3 internalization may be cell-type specific and different from that of MPO. Internalization of PR3, but not MPO, correlated with activation of apoptosis. Internalization of MPO correlated with an increase in intracellular oxidant radicals. The requirement for the proteolytic activity of PR3 for the induction of apoptosis was examined by generating PR3-truncated fragments that did not contain the components of the catalytic triad. An apoptotic function was localized to the C-terminal portion of PR3. These studies reveal novel mechanisms by which the neutrophil granule proteins PR3 and MPO contribute to tissue injury at sites of inflammation.

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.

Novel Effects of Neutrophil-Derived Proteinase 3 and Elastase on the Vascular Endothelium Involve In Vivo Cleavage of NF-κB and Proapoptotic Changes in JNK, ERK, and p38 MAPK Signaling Pathways

Leukocyte-derived proteases have long been considered simply degradative. However, emerging data raise possibilities of a complex and specific biologic role for these proteases in substrate processing and in signaling pathways within cells. This study reports that the release of neutrophilic and monocytic proteases, such as proteinase 3 (PR3) and human neutrophil elastase (HNE), can result in their entry into endothelial cells coincident with the activation of proapoptotic-signaling events through ERK, JNK, and p38 MAPK. Inhibition of JNK blocked PR3-induced apoptosis, and inhibition of p38 MAPK blocked PR3- and HNE-induced apoptosis, indicating that these pathways are required for activation of apoptosis. It is here shown that protease entry results in direct cleavage of p65 NF-kappaB in the N-terminal region by PR3 and in the C-terminal region by HNE. This cleavage results in diminished transcriptional activity by NF-kappaB as demonstrated by diminished levels of TNF-alpha-induced IL-8 message in the presence of PR3 or HNE. Inhibition of caspases did not block the cleavage of p65 NF-kappaB, and sequence analysis showed that the PR3 and HNE cleavage sites are unique with respect to reported caspase sites. The data demonstrate that PR3 and HNE have specific, fundamental roles in endothelial responses during inflammation. Upon entry, they can usurp the cells control of its own fate by directly intervening into caspase cascades. This provides a unique mechanism of crosstalk between leukocytes and endothelial cells at sites of inflammation that impacts both cytokine networks and cell viability.

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.

Patients With Antineutrophil Cytoplasmic Antibody–Associated Vasculitis Have Defective Treg Cell Function Exacerbated by the Presence of a Suppression-Resistant Effector Cell Population

OBJECTIVE The development of pathogenic antineutrophil cytoplasmic antibodies (ANCAs) can result in systemic small vessel vasculitis. However, the breakdown in immune tolerance that results in the induction and persistence of ANCAs is not well understood. We undertook this study to test our hypothesis that abnormal T cell regulation is central to disease pathogenesis in patients with ANCA-associated vasculitis (AAV). METHODS Peripheral blood samples were obtained from 62 patients with AAV and 19 healthy controls for flow cytometric analysis of CD4+ T cell populations. Functional T cell studies were performed with fluorescence-activated cell sorted CD4+ T cell populations stimulated with anti-CD3/anti-CD28. RESULTS We demonstrated two separate abnormalities in T cell regulation in patients with AAV. First, we showed that the Treg cell frequency was increased in the peripheral blood of patients with active disease, but Treg cells from patients with AAV had decreased suppressive function. Treg cells from patients with active disease disproportionately used a FoxP3 isoform lacking exon 2, which might alter Treg cell function. Second, we identified a CD4+ T cell population with increased frequency that was resistant to Treg cell suppression, produced proinflammatory cytokines, and was antigen experienced. CONCLUSION AAV is associated with disruption of the suppressive Treg cell network and with increased frequency of a distinct proinflammatory effector T cell subset that comprises the majority of peripheral CD4+ T cells.

ANCA-associated Vasculitis Patients Have Defective Treg Function Exacerbated by Presence of a Suppression-Resistant Effector Population

OBJECTIVE The development of pathogenic antineutrophil cytoplasmic antibodies (ANCAs) can result in systemic small vessel vasculitis. However, the breakdown in immune tolerance that results in the induction and persistence of ANCAs is not well understood. We undertook this study to test our hypothesis that abnormal T cell regulation is central to disease pathogenesis in patients with ANCA-associated vasculitis (AAV). METHODS Peripheral blood samples were obtained from 62 patients with AAV and 19 healthy controls for flow cytometric analysis of CD4+ T cell populations. Functional T cell studies were performed with fluorescence-activated cell sorted CD4+ T cell populations stimulated with anti-CD3/anti-CD28. RESULTS We demonstrated two separate abnormalities in T cell regulation in patients with AAV. First, we showed that the Treg cell frequency was increased in the peripheral blood of patients with active disease, but Treg cells from patients with AAV had decreased suppressive function. Treg cells from patients with active disease disproportionately used a FoxP3 isoform lacking exon 2, which might alter Treg cell function. Second, we identified a CD4+ T cell population with increased frequency that was resistant to Treg cell suppression, produced proinflammatory cytokines, and was antigen experienced. CONCLUSION AAV is associated with disruption of the suppressive Treg cell network and with increased frequency of a distinct proinflammatory effector T cell subset that comprises the majority of peripheral CD4+ T cells.

Microarray studies of gene expression in circulating leukocytes in kidney diseases

The molecular characterization of changes in mRNA expression in renal tissue during disease is hampered by the acquisition of sufficient mRNA to do genomewide expression profiling. In many renal diseases, such as systemic lupus erythematosus, IgA nephropathy, antineutrophil cytoplasmic autoantibody (ANCA) associated glomerulonephritis, and small-vessel vasculitis (ANCA disease), circulating leukocytes play a role in onset, progression, and severity of the condition. Circulating leukocytes are readily isolated and supply sufficient mRNA for analysis, allowing molecular investigation into their involvement in the disease process. Our laboratory has undertaken a systematic study of the genomewide expression profiles of the circulating leukocytes from patients with a variety of renal diseases (ANCA disease, IgA nephropathy, lupus nephritis, focal segmental glomerulosclerosis), using the Affymetrix high-density gene chip array technology. Analysis of the data showed clustering of expressed genes unique for each individual disease group. These results imply that significant gene expression changes occur in leukocytes that are circulating in patients with renal diseases. In addition, gene expression has been studied in leukocytes activated in vitro by mechanisms that mimic pathogenic events in vivo. The expression levels of genes identified in in vitro studies were compared with the patient leukocyte gene expression to determine whether similar pathological events were occurring in vivo.