Autoantibodies to endothelial cells and neutrophil cytoplasmic antigens in systemic vasculitis.

The interaction of circulating autoantibodies with the endothelium may be an important mechanism in the pathogenesis of systemic vasculitis. In a prospective study, we looked for circulating antiendothelial cell autoantibodies (AECA) and anti-neutrophil cytoplasm autoantibodies (ANCA) in 80 patients with suspected systemic vasculitis. AECA were measured using an isotype-specific cellular ELISA incorporating human umbilical vein endothelial cells. ANCA activity was determined by indirect immunofluorescence and radioimmunoassay. Sequential studies were performed on sera from four cases with dual positivity, where autoantibody binding was compared with von Willebrand factor (vWF) concentration and disease activity. IgG AECA were significantly higher in the 27 ANCA-positive sera as compared with normal controls (P = 0.027) with IgG (P = 0.009) and IgA (P = 0.046) AECA isotypes correlating with ANCA positivity; in contrast, no differences were found between AECA levels in the ANCA-negative sera and the normal controls. Cross-inhibition studies pointed to the co-existence of two autoantibody populations. An association between autoantibody binding, disease activity and vWF concentration was found for both ANCA and AECA. Some patients with systemic vasculitis have detectable AECA that recognize different epitopes to ANCA and like ANCA, their titre correlates with disease activity and thus they may have a pathogenetic role in these conditions.     

IgG subclass distribution of autoantibodies to neutrophil cytoplasmic antigens in systemic vasculitis

The IgG subclass distribution for both ANCA-specific, and total IgG subclasses, of sera from 48 patients with active systemic vasculitis was determined by solid-phase assay using monoclonal anti-human subclass reagents. To control for varying performance of the subclass reagents, the assays were calibrated with sera affinity-depleted into subclass specific fractions, whose ANCA IgG subclass distribution was determined by a common reagent. In 10 patients whose ANCA persisted during clinical remission, the ANCA IgG subclass distributions between active and remission phases of disease were compared. The total IgG subclass distribution of sera from patients with active vasculitis was similar to that found in a normal population. The ANCA IgG subclass distribution of the same sera showed relative enrichment for IgG3 and depletion of IgG2 (P less than 0.05). When the active and clinical remission ANCA IgG subclass distributions were compared, ANCA IgG3 levels had fallen and ANCA IgG2 levels were increased during remission (P less than 0.01).     

Autoantibodies to neutrophil cytoplasmic antigens in systemic vasculitis have the same target specificity

Many patients with systemic vasculitis have antibodies to neutrophil cytoplasm antigens (ANCA) detectable by indirect immunofluorescence. We sought to characterize further the nature of these antigens. Western blots of neutrophil protein extracts indicated that nine patients' sera, positive for ANCA by immunofluorescence, all reacted with a 45 kDa and a 27-31 kDa protein. Negative control sera, and sera taken in remission, did not react with either of these antigens. The results suggest that ANCA in vasculitis have the same target specificity and may therefore permit greater accuracy of diagnosis and increase our understanding of the pathogenesis of the conditions.     

T and B cell responses to HDM allergens and antigens

House dust mites provide well-characterized proteins to study human responses to inhaled antigens. Even in the absence of allergy they induce a high frequency of T cell precursors. The healthy response manifests by T cell proliferation and Th1 cytokines with little antibody. Responses of allergic people include Th1 and Th2 cytokines and IgE, IgG1, and IgG4 antibodies. Regulatory cells limit effector responses in healthy people. About half the IgE and IgG antibodies bind the group 1 and 2 allergens and 30% bind the group 4, 5, and 7 allergens. Although HLA independent, the recognition of the group 1 allergen shows an immunodominant region and a T cell receptor bias. The major allergens are not produced in higher amounts than many of the poorly non-allergenic proteins. The non-allergenic mite ferritin antigen shows high T cell proliferative responses with mixed cytokine production.     

T lymphocyte responses to anti-neutrophil cytoplasmic autoantibody (ANCA) antigens are present in patients with ANCA-associated systemic vasculitis and persist during disease remission

ANCA with specificity for myeloperoxidase (MPO) and proteinase 3 (PR3) are present in patients with systemic vasculitis. The aim of this work was to determine whether such patients have T cell responses to these antigens and whether these responses are related to disease activity. Peripheral blood lymphocytes from 45 patients and 19 controls were cultured with ANCA antigens and proliferation measured. The antigens used were heat-inactivated (HI) MPO, HI PR3, native (non-HI) PR3, HI whole α-granules, and 25 overlapping peptides covering the entire PR3 sequence. Significant responses to both whole PR3 preparations were seen from patient and control groups, and to the α-granules from the patient group. Patients responded at all stages of disease: active, remitting, treated or untreated. Only two patients responded significantly to MPO. Responses were significantly higher with the patient group than the control group to all four whole ANCA antigens. Responses to those PR3 peptides containing epitopes known to be recognized by ANCA were detected from one patient. Thus, these studies demonstrate that T cells from vasculitis patients can proliferate to PR3 and occasionally to associated ANCA antigens. Further, responses may persist even after disease remission has been achieved.     

Precipitating antibodies to nuclear antigens in systemic vasculitis.

We have examined sera from 61 patients with systemic vasculitis for precipitating antibodies to components of saline tissue extracts. Precipitins were rare in patients with polyarteritis nodosa (PAN) and their absence helped to distinguish PAN from vasculitis associated with other connective tissue diseases. Precipitins were detected in some patients with other vasculitides. Previously described precipitating antibodies (anti-Ro [SSA] and anti-La [SSB]) were restricted to a few patients with features of systemic lupus erythematosus (SLE). A different, as yet unidentified, precipitin which reacted with a component of rabbit thymus extract but not calf thymus or human spleen extracts was detected in many patients with rheumatoid disease. This precipitin was present in all patients with active rheumatoid vasculitis (RV) as well as 52% of patients with uncomplicated but active rheumatoid synovitis. Higher titres of precipitating antibody were present in patients with active RV than those with inactive RV or uncomplicated rheumatoid synovitis, and serial studies showed a good correlation between a fall in antibody titre and healing of vasculitis with treatment. These studies suggest that this unidentified precipitin may be an important marker of RV.     

T cell responses to orbital antigens in thyroid-associated ophthalmopathy.

Thyroid-associated ophthalmopathy (TAO) is most likely to be a T cell-mediated disease, in which cytokines released in the extraocular muscles activate fibroblasts, increasing glycosaminoglycan production. The nature of the orbital antigen recognized by the infiltrating T cells is unclear, although it is possible that there is cross-reactivity between this and a thyroid autoantigen to explain the close association with thyroid autoimmunity. We have tested the ability of human and porcine eye muscle antigen preparations to stimulate proliferation of circulating T cells from healthy subjects and patients with TAO or Graves' disease without clinical TAO. Occasional responses were seen, particularly after depletion of CD8+ T cells, and two out of 10 TAO patients responded to eye muscle proteins of 25-50 kD after fractionation of antigens on gels and subsequent elution. There was no disease-specific response of T cells to R1, R14, D1 and 1D3, recombinant proteins identified from screening an eye muscle cDNA library with sera from patients with autoimmune thyroid disease. We have also found that interferon-gamma (IFN-gamma) production by T cells from TAO patients was not stimulated by eye muscle membrane antigens or by 1D3. These results suggest that the frequency of circulating T cells responding to eye muscle antigens in TAO is low, and that several candidate orbital antigens, including the 64-kD protein 1D3, are unlikely to be important T cell autoantigens in this condition.     

Sites of specific B cell activation in primary and secondary responses to T cell-dependent and T cell-independent antigens.

Techniques which identify hapten-specific B cells in tissues have been used to determine the sites of B cell activation in rat spleens in response to T cell-dependent (TD) antigens and T cell-independent type-1 (TI-1) antigens. Surface-associated hapten binding by specific memory B cells and B blasts was distinguished from the strong cytoplasmic hapten binding by specific plasma cells and plasmablasts. Blast cells in S phase were identified in tissue sections by staining cells which had been pulse labeled in vivo with 5-bromo-2'-deoxyuridine. Hapten-specific B blast cells are found in three sites: (a) around interdigitating cells in the T cell-rich zones; (b) in the follicular dendritic cell network and (c) in association with macrophages in the red pulp. Hapten-binding memory B cells, which are not in cell cycle, accumulate in the marginal zones and to a lesser extent the follicular mantles in response to TD and TI-1 antigens. The hapten-specific blast response in T zones is confined to the first few days after antigen is given and is low for primary responses to TD antigens, but massive on secondary challenge, when marginal zone memory B cells migrate to the T zones. Both the primary and secondary T zone responses to TI-1 antigens are impressive and in these responses hapten-specific B blasts are also found in the splenic red pulp. The follicular response to TD antigens starts with a small number of B blasts (fewer than five) entering each follicle. These increase in number exponentially so that by the 4th day after immunization they fill the follicle. The oligoclonality of the response is shown in simultaneous responses to two haptens where 6%-31% of the follicles on day 3 after immunization contain blasts specific for only one of the two haptens. During the 4th day classical zonal pattern of germinal centers develops. The surface immunoglobulin-positive B blasts are lost from the follicle center, while one pole of the follicular dendritic cell network fills with surface immunoglobulin-negative centroblasts. Centroblasts do not increase in numbers but divide to give rise to centrocytes, which re-express sIg and migrate into the follicular dendritic cell network. Cell kinetic studies indicate that the centrocyte population is renewed from centroblasts every 7 h. Centrocytes either leave the germinal center within this time or die in situ.(ABSTRACT TRUNCATED AT 400 WORDS)     

No association between neutrophil FcgammaRIIa allelic polymorphism and anti-neutrophil cytoplasmic antibody (ANCA)-positive systemic vasculitis.

ANCA, implicated as having a pathogenic role in systemic vasculitis, can activate tumour necrosis factor-alpha (TNF-alpha)-primed neutrophils by cross-linking surface-expressed ANCA antigens with neutrophil FcgammaRIIa receptors to release reactive oxygen species. The FcgammaRIIa receptor exists as polymorphic variants, R131 and H131, which differ in their ability to ligate human IgG2 and IgG3. Neutrophils homozygous for the FcgammaRIIa-H131 allotype bind more efficiently to IgG3 than the FcgammaRIIa-R131 allotype and are the only human FcgammaR which bind IgG2. Our aim was to determine whether the homozygous FcgammaRIIa-H131 individuals are more susceptible to developing ANCA-associated systemic vasculitis and nephritis due to differential IgG binding and activation. FcgammaRIIa allotype was determined by both allele-specific polymerase chain reaction (PCR) and Southern blotting with allele-specific oligonucleotide probes end-labelled with 32P-gammaATP, after PCR amplification of genomic FcgammaRIIa DNA in 107 Caucasian patients with ANCA+ vasculitis (of whom 89 had renal disease) and 100 ethnically matched controls. Phenotyping of neutrophil FcgammaRIIa alleles was confirmed in some patients by quantitative flow cytometry using murine MoAbs 41H16 and IV.3. Of the patients with ANCA+ systemic vasculitis, 75 had ANCA with specificity for proteinase 3 and 32 with specificity for myeloperoxidase. Overall, no skewing in FcgammaRIIa allotypes was seen in patients compared with controls. No significant increase of the FcgammaRIIa-H131 allotype was found amongst patients irrespective of ANCA specificity, and no association between the FcgammaRIIa allotype and nephritis was found. Our data suggest that the FcgammaRIIa receptor allotype is not a major factor predisposing to the development of ANCA+ systemic vasculitis, or to nephritis.     

Dendritic cells need T cell help to prime cytotoxic T cell responses to strong antigens.

Peptide-pulsed mouse dendritic cells (DC) primed peptide-specific CD8+ cytotoxic T cell responses very effectively if they expressed minor histocompatibility antigens, which could stimulate a CD4+ T helper cell response. These DC could also prime most syngeneic mice, although there was no deliberate immunization for help (the DC were prepared in syngeneic mouse serum, to avoid any response to fetal calf serum antigens). In contrast, DC were unable to prime MHC class II-deficient mice for cytotoxic responses to the classical helper-dependent antigens Qa1a and HY. More strikingly, Balb.B DC failed to prime B6 MHC class II-deficient mice for cytotoxic responses to Balb minor antigens, even though these two strains differ at more than 40 minor histocompatibility loci. When peptide-pulsed DC were prepared without enzymes (used to release DC from lymphoid tissues), they failed to prime the majority of normal syngeneic mice, even though they expressed high levels of B7 and ICAM-1 co-stimulatory molecules, suggesting that help was provided by responses to antigens in the enzyme cocktail. The enzyme treatment itself did not provide signals that could substitute for help, since DC prepared with enzymes could not prime MHC class II-deficient mice. The observation that highly immunogenic minor-incompatible DC failed to prime MHC class II-deficient mice suggests that in the absence of inflammatory signals, even strong antigens cannot stimulate CD8+ T cell responses without help.     

Titration of antibodies against neutrophil cytoplasmic antigens is useful in monitoring disease activity in systemic vasculitides.

High levels of IgG antibodies to the multi-enzyme complex bovine pyruvate dehydrogenase (PDHC) were detected by a sensitive ELISA in all patients with histologically proven primary biliary cirrhosis (PBC), in a minority of patients with systemic sclerosis without overt PBC or with chronic liver diseases of another cause and in no rheumatoid arthritis or normal controls. Most (15/21) but not all of these positive patients recognized a 70-kD (E2) component of PDHC on immunoblotting. Additional recognition of a centromere-associated 140-kD polypeptide was associated with the presence of scleroderma-like features (Raynaud's phenomenon and sclerodactyly) in PBC, further emphasizing the serological and clinical overlap between these two conditions.     

T cell : B cell collaboration--the response to polysaccharide antigens

The humoral response to polysaccharide antigens occurs as a result of T cell-B cell collaboration that is very different from the present understanding of the mechanisms whereby protein antigens are taken up, processed and exposed on the cell membrane in the context of MHC class II molecules in a manner suitable for recognition by a T cell antigen receptor. Two models, Wiskott-Aldrich syndrome and CBA/N mice bearing the xid mutation, are examined because in both cases there is a failure to respond to polysaccharide antigens.     

Neutrophil and recombinant myeloperoxidase as antigens in ANCA positive systemic vasculitis.

IL-8 is a chemotactic cytokine with proinflammatory and growth-promoting activities. The release of IL-8 was measured in supernatants of cultured peripheral blood monocytes (PBM) that were obtained from patients with glomerulonephritis (GN) and healthy controls. Spontaneous and lipopolysaccharide (LPS)-induced IL-8 release was significantly higher in PBM isolated from patients with IgA nephropathy (IgAN) and membranous nephropathy (MN) compared with normal controls. These results raise the question of whether IL-8 contributes to the ongoing pathogenesis of GN. We cannot relate IL-8 release to clinical and laboratory parameters in IgAN and MN patients. Thus, disease progression in vivo may not be accompanied by increased or sustained IL-8 release.     

Nuclear antigens in neoplastic lymphocytes of B cell and T cell non-Hodgkin''s lymphomas.

Gross nuclear morphology is a major diagnostic feature in the identification of subtypes of non-Hodgkin's lymphoma (NHL). The authors have shown that the size, shape, and chromatin distribution of the lymphocyte nuclei vary extensively both within and between samples of a subtype, and have proposed that the variations may reflect qualitative and quantitative differences in extrachromatinic components. To test this hypothesis, the organization of individual nuclear antigens in NHL and in reactive hyperplasia biopsies was examined by immunofluorescence labeling of frozen sections with previously characterized monoclonal antibodies. The results have been correlated with observations of the staining patterns produced by the antibodies in mitogenically stimulated human peripheral blood lymphocytes. Labeling pattern and intensity with each antibody were consistent between preparations of blood lymphocytes, and all four antibodies labeled all blood lymphocyte samples tested. In contrast, only 15% of the 53 biopsies were labeled by all four antibodies, although all were stained by anti-peripherin, nearly 80% by I1, and almost 60% by PI1. Antibody PI2 labeling was detected in only 20% of the samples. Variation in labeling intensity was equally extensive both within and between biopsy samples. In general, there was little homogeneity between samples of an NHL subtype as to which antigens were detected, their labeling intensity, or their pattern of intranuclear distribution. These observations are consistent with earlier reports of significant diversity in the morphology of nonchromatin components in such samples. The data support the proposition that the heterogeneity of gross nuclear morphology in nuclei of NHL biopsies may be due in part to disordered expression or abnormal organization of nuclear proteins.     

Human T cell responses to recombinant mite antigens of Dermatophagoides farinae

We studied T cell responses to four glutathione S transferase (GST)-fused mite antigens prepared in our laboratory using peripheral blood lymphocytes from mite-sensitive patients with bronchial asthma. Of the four recombinant antigens, purified GST-Mag3 had the strongest ability to cause patients' lymphocytes to proliferate, and its potency was almost comparable to that of crude mite bodies (Dfb) and faeces (Dff) antigens and a purified major antigen, Der f 2. The responder lymphocytes were mainly T cells, because the proliferative response was depleted by the treatment of lymphocytes with anti-CD3 antibody and complement, but not with anti-CD20 antibody and complement. The responsiveness of lymphocytes to GST-Mag3 correlated with that to Der f 2, but GST-Mag3 displayed slightly higher activity to stimulate lymphocytes than Der f 2. Simultaneously, the levels of Dff- and GST-Mag3-specific IgE antibodies correlated with the responsiveness of lymphocytes to GST-Mag3. These results suggest that Mag3 is a new valuable antigen for the response of T cell proliferation in mite-sensitive patients.     

Regulatory effect of monocytes on T cell proliferative responses to oral microbial antigens.

Mononuclear cell preparations isolated by Ficoll-Hypaque centrifugation from human peripheral blood were found to vary considerably in the number of monocytes they contained (mean, 20.3%; range, 13 to 33%). The regulatory role of monocytes in T cell proliferative responses to sonic extracts of a panel of oral microorganisms was therefore investigated. T cells were fractionated by anti-immunoglobulin chromatography and depleted of monocytes by treatment with a monoclonal anti-human Ia-like (DR locus antigen) antibody and complement. Purified populations of monocytes were obtained by extensive adherence procedures. The resultant cell populations were greater than 95% pure, as judged by indirect immunofluorescence on a fluorescence-activated cell sorter. Monocyte-depleted T cells failed to respond by proliferation to the nonoral antigen tetanus toxoid, as well as to any oral microorganism, but retained responsiveness to phytohemagglutinin. Readdition of monocytes in final concentrations of from 5 to 15% resulted in the restoration of maximal T cell proliferation. Monocytes in greater numbers suppressed T cell responses to all sonic extracts tested.