Immunohistochemical analysis of cellular infiltrate and gamma interferon, interleukin-12, and inducible nitric oxide synthase expression in leprosy type 1 (reversal) reactions before and during prednisolone treatment.

The effects of prednisolone treatment on the cellularity and cytokine (gamma interferon, interleukin-12, and inducible nitric oxide synthase) profiles of leprosy skin type 1 (reversal) reactions were studied using immunohistochemistry. Skin biopsies were taken from 15 patients with leprosy type 1 (reversal) reactions at days 0, 7, 28, and 180 after the start of steroid treatment. Prednisolone treatment had little effect at day 7, but by day 28 significant decreases were found in cytokine levels. Some patients maintained cytokine production at days 28 and 180. These results illustrate the strong Th1 profile of type 1 reactional lesions, the slow response to steroid therapy, and continuing activity at 180 days.

Anti-neutrophil cytoplasmic antibodies (ANCA) from patients with systemic vasculitis recognize restricted epitopes of proteinase 3 involving the catalytic site.

ANCA with specificity for proteinase 3 (PR3), a neutrophil primary granule enzyme, are of diagnostic value in Wegener's granulomatosis (WG) and certain other forms of systemic vasculitis. There is evidence to suggest that they play a pathogenic role in disease, and that the interaction of ANCA with PR3 is likely to be important. We showed, using a resonant mirror biosensor, that C-ANCA from different patients recognized the same or closely related epitopes on PR3. Studies using linear peptides in the SPOT system confirmed the highly restricted nature of this interaction and identified five linear epitopes. Fluid-phase inhibition studies, using a different set of peptides, validated the sequences involved. Using a computer-generated model of the structure of PR3, four of five epitopes were shown to be intimately linked with the catalytic site. The restricted number of epitopes, and their location at the catalytic site, has important implications for the role of C-ANCA in the pathogenesis of vasculitis.

Mapping B cell epitopes in Goodpasture's disease.

The target antigen of the pathogenic autoantibodies in Goodpasture's disease is the noncollagenous domain of the alpha 3 chain of type IV collagen. A panel of membrane-bound peptides was used to identify antibody-binding regions within the protein, and the significance of the binding by inhibition studies using soluble peptides, and by a structural analysis of the antigen, was confirmed. A total of 117 overlapping 12-mer peptides spanning the entire antigen was simultaneously synthesized as individual spots on a cellulose membrane. All nine patients' sera bound to the membrane, with a conserved pattern of peptides recognized by all sera. Inhibition studies were performed using a panel of overlapping 20-mer peptides, also spanning the entire antigen. Peptides from the regions that bound IgG were able to inhibit the binding of autoantibodies to native antigen. Predictions of the secondary structure of the noncollagenous domain of the alpha 3 chain of type IV collagen were performed by a conventional hydropathy plot and by multiple alignment of homologous alpha chains of type IV collagen and comparison with a structural data base. The core peptides binding and inhibiting Goodpasture's antibodies were predicted to be surface exposed and antigenic. Thus, the conformational epitope(s) of the Goodpasture antigen can be mapped using linear peptides.

T cell responses to myeloperoxidase (MPO) and proteinase 3 (PR3) in patients with systemic vasculitis.

T cell-mediated immune responses are likely to be important in the pathogenesis of systemic vasculitis. However, identifying the T cells involved has proved difficult, and there are conflicting reports regarding T cell proliferation in response to different autoantigens. Perinuclear (P) and cytoplasmic (C) anti-neutrophil cytoplasmic antibodies (ANCA) are closely associated with systemic vasculitis, and are generally specific for MPO or PR3, respectively. We studied the proliferative responses to MPO and PR3 of peripheral blood mononuclear cells from patients with P-ANCA or C-ANCA specific for these antigens by ELISA. These responses were compared with those of normal controls, and of disease controls with P- or C-ANCA not specific for MPO or PR3. The patient group as a whole showed significant T cell proliferation in response to the autoantigens compared with controls (P = 0.005). Cells from nine of 13 P-ANCA-positive, anti-MPO-positive patients proliferated in response to MPO, compared with five of 16 controls (P = 0.04). Cells from five of eight C-ANCA-positive, anti-PR3-positive patients proliferated in response to PR3, compared with two of 11 controls (P = 0.05). These experiments demonstrate that patients with P-ANCA or C-ANCA possess T cells which respond to MPO or PR3, respectively. As in other autoimmune diseases, responses to both antigens were also seen in a proportion of healthy controls. Further analysis of these responses will be important in understanding the pathogenesis of systemic vasculitis and in designing specific immunotherapy.

Antigen specificity of circulating anti-neutrophil cytoplasmic antibodies in inflammatory bowel disease.

OBJECTIVES: To characterize the antigen specificity of circulating anti-neutrophil cytoplasmic antibodies (ANCAs) in inflammatory bowel disease (IBD). DESIGN: Analysis of the prevalence of circulating ANCAs in patients with ulcerative colitis and Crohn's disease, by both non-specific methods (immunofluorescence against fixed neutrophil leukocytes) and specific antigen techniques (against purified neutrophil leukocyte constituents). METHODS: Indirect immunofluorescence against fixed polymorphonuclear leukocytes, and solid-phase enzyme-linked immunosorbent assay (ELISA) against neutrophil constituents (alpha-granules, elastase, myeloperoxidase, cathepsin g, lysozyme and lactoferrin). RESULTS: Although results using immunofluorescence were typical of other studies (ulcerative colitis positive in 41%, Crohn's disease in 10%), ELISA studies showed antibody activity against neutrophil components in 69% of patients with ulcerative colitis and 39% of those with Crohn's disease. Antibodies in ulcerative colitis were commonly directed (in descending order) against lysozyme, cathepsin G, elastase, and lactoferrin, and in Crohn's disease against lysozyme. CONCLUSION: Correlation of indirect immunofluorescence data and ELISA results indicated that even this large panel of specific antigens fails to identify all the ANCA targets in IBD. The lack of correlation between the findings of ANCAs, either in general or versus a specific target, and disease extent or activity in ulcerative colitis supports the suggestion that ANCAs are unlikely to be of primary importance in pathogenesis.

Use of proteinase 3 purified by reverse phase HPLC to detect autoantibodies in systemic vasculitis.

The vasculitis autoantigen proteinase 3 was purified from neutrophil primary granules using reverse phase high performance liquid chromatography. It was shown to be free of important contaminants, was enzymatically active and was antigenic to sera containing antineutrophil cytoplasmic antibodies with a cytoplasmic pattern (C-ANCA) by indirect immunofluorescence. Development of an enzyme-linked immunosorbent assay showed that this preparation could be used to detect autoantibodies in Wegener's granulomatosis and microscopic polyangiitis. Assays based on reverse phase HPLC-purified proteinase 3 will be valuable in diagnosis and monitoring of treatment, and should increase our understanding of the autoimmune response in primary systemic vasculitis.