Induction of endothelial cell apoptosis by heat-shock protein 60-reactive antibodies from anti-endothelial cell autoantibody-positive systemic lupus erythematosus patients

OBJECTIVE: To determine whether anti-endothelial cell autoantibodies (AECAs) from systemic lupus erythematosus (SLE) patients with the antiphospholipid syndrome are involved in the initial endothelial cell (EC) membrane perturbation effect that is postulated to provide a target for antiphospholipid antibody (aPL) binding and, hence, to trigger the thrombotic cascade. To identify the AECA antigenic target on ECs and to determine the mechanism whereby the EC membrane is disrupted. METHODS: AECAs from SLE patients were assayed for binding to ECs by flow cytometry. Positive AECAs were assayed by immunoblotting, and a consensus antigen was identified by mass spectrometry. This candidate antigen was tested in recombinant form for AECA recognition. AECAs were affinity-purified on this antigen and incubated with ECs to determine their physiologic effects. Anti-Hsp60 antibody titers were determined by enzyme-linked immunosorbent assay. The relationship of anti-Hsp60 status and lupus anticoagulant (LAC) status to thrombotic manifestations between disease onset and the last followup visit were analyzed. RESULTS: Most of the SLE sera (73%) possessed IgG that bound to the surface of ECs. These positive IgG shared reactivity against a 60-kd EC surface polypeptide that was identified as human Hsp60. The presence of Hsp60 at the EC surface was established using anti-Hsp60 antibodies from commercial sources or affinity-purified from SLE sera that bound ECs. Incubation of ECs with these anti-Hsp60 antibodies induced apoptosis in a time- and dose-dependent manner, as determined by Hoechst 33342 dye staining of condensed nuclei and by annexin V binding to surface phosphatidylserine. Anti-Hsp60 antibodies were not restricted to SLE patients, but were found in patients with other autoimmune diseases. However, anti-Hsp60 antibodies were significantly associated with an increased frequency of thrombosis when present in combination with LAC in the SLE patients. CONCLUSION: The presence of Hsp60 at the surface of ECs serves as a target for the anti-Hsp60 antibodies in SLE sera. These anti-Hsp60 antibodies bind to ECs and induce apoptosis, particularly phosphatidylserine exposure, thus providing a target for the binding of aPL and inducing the subsequent thrombotic cascade.     

Induction of endothelial cell apoptosis by heat‐shock protein 60–reactive antibodies from anti–endothelial cell autoantibody–positive systemic lupus erythematosus patients

Objective

To determine whether anti–endothelial cell autoantibodies (AECAs) from systemic lupus erythematosus (SLE) patients with the antiphospholipid syndrome are involved in the initial endothelial cell (EC) membrane perturbation effect that is postulated to provide a target for antiphospholipid antibody (aPL) binding and, hence, to trigger the thrombotic cascade. To identify the AECA antigenic target on ECs and to determine the mechanism whereby the EC membrane is disrupted.

Methods

AECAs from SLE patients were assayed for binding to ECs by flow cytometry. Positive AECAs were assayed by immunoblotting, and a consensus antigen was identified by mass spectrometry. This candidate antigen was tested in recombinant form for AECA recognition. AECAs were affinity‐purified on this antigen and incubated with ECs to determine their physiologic effects. Anti‐Hsp60 antibody titers were determined by enzyme‐linked immunosorbent assay. The relationship of anti‐Hsp60 status and lupus anticoagulant (LAC) status to thrombotic manifestations between disease onset and the last followup visit were analyzed.

Results

Most of the SLE sera (73%) possessed IgG that bound to the surface of ECs. These positive IgG shared reactivity against a 60‐kd EC surface polypeptide that was identified as human Hsp60. The presence of Hsp60 at the EC surface was established using anti‐Hsp60 antibodies from commercial sources or affinity‐purified from SLE sera that bound ECs. Incubation of ECs with these anti‐Hsp60 antibodies induced apoptosis in a time‐ and dose‐dependent manner, as determined by Hoechst 33342 dye staining of condensed nuclei and by annexin V binding to surface phosphatidylserine. Anti‐Hsp60 antibodies were not restricted to SLE patients, but were found in patients with other autoimmune diseases. However, anti‐Hsp60 antibodies were significantly associated with an increased frequency of thrombosis when present in combination with LAC in the SLE patients.

Conclusion

The presence of Hsp60 at the surface of ECs serves as a target for the anti‐Hsp60 antibodies in SLE sera. These anti‐Hsp60 antibodies bind to ECs and induce apoptosis, particularly phosphatidylserine exposure, thus providing a target for the binding of aPL and inducing the subsequent thrombotic cascade.

     

Proteomic analysis of autoantibodies in neuropsychiatric systemic lupus erythematosus patient with white matter hyperintensities on brain MRI

The pathogenesis of neuropsychiatric systemic lupus erythematosus (NPSLE) may be related to autoantibody-mediated neural dysfunction, vasculopathy and coagulopathy. We encountered an NPSLE patient whose brain showed characteristic diffuse symmetrical hyperintensity lesions in the cerebral white matter, cerebellum and middle cerebellar peduncles on T2-weighted magnetic resonance (MR) images. In this study, we investigated all the antigens that reacted strongly with autoantibodies in this patient's serum by two-dimensional electrophoresis (2DE), followed by western blotting (WB) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) using rat brain proteins as the antigen source. As a result, we identified four antigens as beta-actin, alpha-internexin, 60 kDa heat-shock protein (Hsp60) and glial fibrillary acidic protein (GFAP). There are several reports on the detection of anti-endothelial cell antibodies (AECAs) in an SLE patients. Recently, one of the antigens reacting with AECAs in SLE patient's sera has been identified as human Hsp60. We speculated that the abnormal findings on brain MR images of our patient may be due to impairment of microcirculation associated with vascular endothelial cell injury mediated by the antibody against Hsp60. This proteomic analysis is a useful tool for identifying autoantigens in autoimmune diseases involving autoantibodies.     

Targets of anti-endothelial cell antibodies in pulmonary hypertension and scleroderma

Anti-endothelial cell antibodies (AECAs) have been identified in patients with systemic sclerosis (SSc) with and without pulmonary arterial hypertension (PAH) and in patients with idiopathic pulmonary arterial hypertension (iPAH). However, their target antigens remain poorly identified. Sera from 24 patients with SSc without PAH, 20 patients with SSc with PAH, 30 with iPAH and 12 healthy controls were collected. Target antigens were identified by two-dimensional electrophoresis and immunoblotting in protein extracts of human umbilical vein endothelial cells. Targeted antigens were identified by mass spectrometry. Serum immunoglobulin G from patients with SSc with or without PAH and patients with iPAH specifically recognised 110, 82 and 37 protein spots, respectively. Among others, target antigens of AECAs included lamin A/C, tubulin β-chain and vinculin. One-dimension immunoblotting experiments confirmed the identification of lamin A/C and tubulin β-chain. In conclusion, our results confirm the presence of AECA in patients with systemic sclerosis with and without pulmonary arterial hypertension and in those with idiopathic pulmonary arterial hypertension, and provide evidence for the identification of target antigens of these autoantibodies including lamin A/C and tubulin β-chain.     

Reactivity of gamma/delta T cells to human 60-kd heat-shock protein and their cytotoxicity to aortic endothelial cells in Takayasu arteritis

OBJECTIVE: Increased numbers of circulating gamma/delta T cells with a restricted T cell receptor repertoire, as well as colocalization of the expression of heat-shock protein Hsp60/65 and gamma/delta T cells in the arterial lesions of patients with Takayasu arteritis (TA), indicate that gamma/delta T cells may react to Hsp60 and cause damage to the arterial endothelium. In this study we investigated the proliferative responses of gamma/delta T cells to human Hsp60 and their cytotoxicity to human aortic endothelial cells (ECs) in patients with TA. METHODS: Blood samples were obtained from 12 patients with TA, 8 patients with systemic lupus erythematosus (SLE) (as disease controls), and 10 healthy control subjects. Proliferative responses of circulating gamma/delta T cells to human Hsp60 were detected by flow cytometry-based bromodeoxyuridine incorporation assay. Cytotoxicity of the gamma/delta T cells to human aortic ECs was analyzed by colorimetric lactate dehydrogenase release assay. RESULTS: The gamma/delta T cells of 11 of 12 patients with TA exhibited reactivity to Hsp60, whereas none of the gamma/delta T cells from patients with SLE or healthy controls showed reactivity (both P < 0.001). The mean +/- SD proliferative response of gamma/delta T cells in patients with TA was 21.4 +/- 11.3%, compared with 4.2 +/- 1.2% in patients with SLE and 4.01 +/- 1.82% in healthy controls (both P < 0.001). In addition, compared with the control groups, the gamma/delta T cells of patients with TA had increased spontaneous cytotoxicity to aortic ECs (22.1 +/- 15.0% versus 9.6 +/- 2.13% in SLE patients and 8.1 +/- 4.7% in healthy controls; both P < 0.005), which was further enhanced following stimulation of gamma/delta T cells with Hsp60. The cytotoxicity of the gamma/delta T cells was significantly inhibited by treatment of these cells with concanamycin A and anti-Fas ligand-blocking antibodies. CONCLUSION: The results show that gamma/delta T cells in patients with TA are reactive to Hsp60 and exhibit cytotoxicity to aortic ECs, suggesting a key role of Hsp60 and gamma/delta T cells in the pathogenesis of TA.     

Identification of an autoantibody to vascular endothelial cell-specific antigens in patients with systemic vasculitis

PURPOSE: Although immunologic mechanisms have been postulated in the pathogenesis of vasculitis, an autoimmune process directed against specific autologous vascular wall antigens has not been previously documented. We examined the role of an immunologic response to vascular endothelial cell (VEC) antigens in patients with vasculitis, because of the observed immunogenicity of VECs in renal and cardiac allograft recipients. PATIENTS AND METHODS: The study patients included 21 with systemic vasculitis and four with hypersensitivity vasculitis. A healthy, normal control group consisted of 51 young subjects and 61 older subjects. Thirty-two patients with connective tissue diseases were also evaluated. The presence of autoantibody to autologous monocytes and other cell types was determined with previously described standard crossmatch techniques. Patient sera exhibiting autoantibody to autologous monocytes were screened against a panel of allogeneic cells. The cell panel consisted of concordant T and B lymphocytes, monocytes, and VECs from 16 umbilical cord donors. Sera from four patients were analyzed for specificity to the vascular endothelium of frozen sections of vessels. RESULTS: An autoantibody to VEC antigens was detected in 18 of the 21 patients (86%) with confirmed systemic vasculitis, not of the hypersensitivity type. This autoantibody was highly cytotoxic, complement-fixing, and specific for antigens on the surface of the VEC. Findings on allogeneic VEC panel analysis support the existence of multiple allotypes in the VEC antigen system. In three patients, fluctuations in the titer of the autoantibody generally correlated with clinical symptoms. In the four patients screened for the specificity of the autoantibody to anatomically different cadaveric blood vessels, specific anatomic patterns of reactivity were observed. Autoantibody to VEC antigens was not found in young controls and was documented in low frequency in older controls and in patients with connective tissue diseases. The autoantibody was seen in one of four patients (25%) with hypersensitivity vasculitis. CONCLUSION: Our results indicate that an autoantibody to VEC antigens may be involved in the pathogenesis of systemic vasculitis or may be a diagnostic marker for the disease process.     

Antiendothelial cell antibodies mediate enhanced leukocyte adhesion to cytokine-activated endothelial cells through a novel mechanism requiring cooperation between Fc{gamma}RIIa and CXCR1/2

Antiendothelial cell antibodies (AECAs) are commonly detectable in diseases associated with vascular injury, including systemic lupus erythematosus (SLE), systemic sclerosis, Takayasu arteritis, Wegener granulomatosis, Beh?et syndrome, and transplant arteriosclerosis. Here, we explore the hypothesis that these antibodies might augment polymorphonuclear leukocyte (PMN) adhesion to endothelium in inflammation. Initially, we established that a mouse IgG mAb bound to endothelial cells (ECs) significantly increased PMN adhesion to cytokine-stimulated endothelium in an FcgammaRIIa-dependent manner. Neutralizing antibodies, and adenoviral transduction of resting ECs, demonstrated that the combination of E-selectin, CXCR1/2, and beta(2) integrins is both necessary and sufficient for this process. We observed an identical mechanism using AECA IgG isolated directly from patients with SLE. Assembled immune complexes also enhanced PMN adhesion to endothelium, but, in contrast to adhesion because of AECAs, this process did not require CXCR1/2, was not inhibited by pertussis toxin, and was FcgammaRIIIb rather than FcgammaRIIa dependent. These data are the first to demonstrate separate nonredundant FcgammaRIIa and FcgammaRIIIb-mediated mechanisms by which EC-bound monomeric IgG and assembled immune complexes amplify leukocyte adhesion under dynamic conditions. Furthermore, the observation that FcgammaRIIa and CXCR1/2 cooperate to enhance PMN recruitment in the presence of AECAs suggests a mechanism whereby AECAs may augment tissue injury during inflammatory responses.     

Antiendothelial cell antibodies in systemic lupus erythematosus

Objective: Anti‐endothelial cell antibodies (AECAs) are a heterogeneous group of antibodies against a variety of antigenic determinants on endothelial cells (EC). AECAs are known to play an immunopathogenic role in triggering EC activation, leading to vascular damage. The purpose of this study was to assess: (i) the incidence of AECAs in systemic lupus erythematosus (SLE) patients with nephritis (LN) and to compare this with SLE patients without clinical evidence of nephritis; and (ii) to understand the association of AECAs with disease severity based on renal histopathological findings. Method: Fifty‐three clinically and histopathologically proven cases of LN were studied along with 20 patients without evidence of nephritis. AECAs were detected by immunofluorescence using cultured human umbilical vein endothelial cells (HUVECs). The titres and immunoglobulin subclass of AECAs were also identified. Other autoantibodies were also detected. Results: In the LN group, 21 (39.6%) were AECA positive and 19 (35.8%) were antineutrophil cytoplasmic antibody (ANCA) positive. Autoantibodies to double‐stranded DNA (anti‐dsDNA) were present in 49 (92.4%) cases. In patients without nephritis, seven (35%) tested positive for AECA, five for ANCA and all were antinuclear antibody (ANA) positive. Anti‐dsDNA was detected in 16 patients (80%), higher incidence of AECAs was noted in diffuse proliferative glomerulonephritis (41.2%) as compared to focal proliferative glomerulonephritis (37.5%) and membranoproliferative glomerulonephritis (33.3%). IgG‐AECA subclass was noted in 85.7% patients, IgM‐AECA and IgG + M AECA subclasses of AECA were detected in 7.1% cases. AECAs were also found to be associated with other autoantibodies such as ANA, anti‐dsDNA and ANCA. Conclusion: No significant differences in AECA positivity was found between SLE with and without nephritis.     

Reactivity of γ/δ T cells to human 60‐kd heat‐shock protein and their cytotoxicity to aortic endothelial cells in Takayasu arteritis

Objective

Increased numbers of circulating γ/δ T cells with a restricted T cell receptor repertoire, as well as colocalization of the expression of heat‐shock protein Hsp60/65 and γ/δ T cells in the arterial lesions of patients with Takayasu arteritis (TA), indicate that γ/δ T cells may react to Hsp60 and cause damage to the arterial endothelium. In this study we investigated the proliferative responses of γ/δ T cells to human Hsp60 and their cytotoxicity to human aortic endothelial cells (ECs) in patients with TA.

Methods

Blood samples were obtained from 12 patients with TA, 8 patients with systemic lupus erythematosus (SLE) (as disease controls), and 10 healthy control subjects. Proliferative responses of circulating γ/δ T cells to human Hsp60 were detected by flow cytometry–based bromodeoxyuridine incorporation assay. Cytotoxicity of the γ/δ T cells to human aortic ECs was analyzed by colorimetric lactate dehydrogenase release assay.

Results

The γ/δ T cells of 11 of 12 patients with TA exhibited reactivity to Hsp60, whereas none of the γ/δ T cells from patients with SLE or healthy controls showed reactivity (both P < 0.001). The mean ± SD proliferative response of γ/δ T cells in patients with TA was 21.4 ± 11.3%, compared with 4.2 ± 1.2% in patients with SLE and 4.01 ± 1.82% in healthy controls (both P < 0.001). In addition, compared with the control groups, the γ/δ T cells of patients with TA had increased spontaneous cytotoxicity to aortic ECs (22.1 ± 15.0% versus 9.6 ± 2.13% in SLE patients and 8.1 ± 4.7% in healthy controls; both P < 0.005), which was further enhanced following stimulation of γ/δ T cells with Hsp60. The cytotoxicity of the γ/δ T cells was significantly inhibited by treatment of these cells with concanamycin A and anti–Fas ligand–blocking antibodies.

Conclusion

The results show that γ/δ T cells in patients with TA are reactive to Hsp60 and exhibit cytotoxicity to aortic ECs, suggesting a key role of Hsp60 and γ/δ T cells in the pathogenesis of TA.

     

Anti-endothelial cell antibodies in patients with sarcoidosis

BACKGROUND: Anti-endothelial cell antibodies (AECA) are circulating antibodies that bind to endothelial antigens and induce endothelial cell damage. These antibodies have been detected in patients with collagen vascular disease and systemic vasculitis. Sarcoidosis is a multiple granulomatous disorder of unknown etiology, and its clinical presentation, organ involvement, and prognosis are highly diverse. In this study, we aimed to investigate the expression of AECA in patients with sarcoidosis. We also examined whether these antibodies could serve as a marker for the activity, severity, and prognosis of sarcoidosis. METHODS: Forty sarcoidosis patients, whose diagnosis was established by clinicoradiologic findings and histologic confirmation of noncaseating epithelioid cell granulomas, were studied. Serum and BAL samples were examined for AECA by cellular enzyme-linked immunosorbent assay (ELISA) using human umbilical vein endothelial cells. The findings were expressed in terms of an ELISA ratio (ER). Fifty-seven subjects without any clinical, radiologic, or serologic evidence of pulmonary disease or autoimmune disorders served as the reference population to judge the positivity of AECA. RESULTS: Patients with sarcoidosis had a significantly higher positivity rate for and levels of AECA in both serum and BAL fluid than the reference population. In addition, the ER of AECA was significantly elevated in patients with multiple lesions or who required corticosteroid therapy compared with that in patients without multiple lesions or who did not need corticosteroid therapy. CONCLUSION: Expression of AECA might be a useful marker to predict the course of sarcoidosis.     

VEGF(165) promotes survival of leukemic cells by Hsp90-mediated induction of Bcl-2 expression and apoptosis inhibition

Similar to endothelial cells (ECs), vascular endothelial growth factor (VEGF) induces Bcl-2 expression on VEGF receptor-positive (VEGFR(+)) primary leukemias and cell lines, promoting survival. We investigated the molecular pathways activated by VEGF on such leukemias, by performing a gene expression analysis of VEGF-treated and untreated HL-60 leukemic cells. One gene to increase after VEGF stimulation was heat shock protein 90 (Hsp90). This was subsequently confirmed at the protein level, on primary leukemias and leukemic cell lines. VEGF increased the expression of Hsp90 by interacting with KDR and activating the mitogen-activated protein kinase cascade. In turn, Hsp90 modulated Bcl-2 expression, as shown by a complete blockage of VEGF-induced Bcl-2 expression and binding to Hsp90 by the Hsp90-specific inhibitor geldanamycin (GA). GA also blocked the VEGF-induced Hsp90 binding to APAF-1 on leukemic cells, a mechanism shown to inhibit apoptosis. Notably, VEGF blocked the proapoptotic effects of GA, correlating with its effects at the molecular level. Earlier, we showed that in some leukemias, a VEGF/KDR autocrine loop is essential for cell survival, whereas here we identified the molecular correlates for such an effect. We also demonstrate that the generation of a VEGF/VEGFR autocrine loop on VEGFR(+) cells such as ECs, also protected them from apoptosis. Infection of ECs with adenovirus-expressing VEGF resulted in elevated Hsp90 levels, increased Bcl-2 expression, and resistance to serum-free or GA-induced apoptosis. In summary, we demonstrate that Hsp90 mediates antiapoptotic and survival-promoting effects of VEGF, which may contribute to the survival advantage of VEGFR(+) cells such as subsets of leukemias.     

In vivo analysis of the apoptosis-inducing effect of anti-endothelial cell antibodies in systemic sclerosis by the chorionallantoic membrane assay

OBJECTIVE: Systemic sclerosis (SSc) is a connective tissue disease of unknown etiology characterized by mononuclear cell infiltration and fibrosis. Vascular injury occurs early in the course of disease, and previous in vitro studies suggest a primary role for anti-endothelial cell antibodies (AECAs) in mediating endothelial cell apoptosis. The aim of the present study was to analyze the apoptosis-inducing effect of AECAs in vivo. METHODS: The optimum animal model for transfer experiments was the University of California at Davis line 200 (UCD-200) chickens that spontaneously develop a hereditary disease with features closely resembling those of scleroderma in humans. AECA-positive serum samples from UCD-200 chickens were used for intravenous injection into normal CC chicken embryos on embryonic day (ED) 13 as well as for application onto chorionallantoic membranes (CAMs) of healthy control lines on ED 10. CAMs of ED 16 embryos and combs of 1-week-old CC chickens that had received the injected serum samples were analyzed for apoptotic endothelial cells by TUNEL. RESULTS: Staining of frozen CAM sections by immunofluorescence showed evidence of in vivo binding of AECAs to the microvascular endothelium. In most groups, transfer of AECA-positive sera resulted in a significant increase in endothelial cell apoptosis as compared with controls. CONCLUSION: This study is the first to demonstrate the in vivo apoptosis-inducing effects of AECAs. The findings support our hypothesis of a primary pathogenetic role of AECAs in SSc.     

Antigenic targets and pathogenicity of anti-aortic endothelial cell antibodies in Takayasu arteritis

OBJECTIVE: Anti-endothelial cell antibodies are considered to have an important role in the pathogenesis of Takayasu arteritis (TA). Previously, these antibodies were detected using human umbilical vein endothelial cells, which do not completely represent the antigenicity/functions of aortic endothelial cells, the specific targets in TA. To delineate the precise role of antigenic targets, we investigated such targets as well as the pathogenic mechanism of antibodies directed against aortic endothelial cells (AAECAs) in TA. METHODS: AAECAs were detected using a cellular enzyme-linked immunosorbent assay (ELISA), and their antigenic targets were detected by immunoblotting. AAECA-mediated induction of endothelial adhesion molecule expression and cytokine production was studied by ELISA, and apoptosis was studied using the TUNEL method. RESULTS: AAECAs were detected in 86% of patients with TA and in 9% of controls. Sera obtained from AAECA-positive patients with TA recognized a total of 9 antigens ranging in size from 18 kd to 200 kd, of which the 60-65-kd triplet was recognized most often. The aortic endothelial cell reactivity of Hsp60-absorbed sera was reduced by approximately 50% as compared with that of unabsorbed sera (mean +/- SD 0.488 +/- 0.08 versus 0.838 +/- 0.116). Sera from AAECA-positive patients with TA, compared with sera from AAECA-negative patients with TA and that from controls, induced increased expression of E-selectin (mean +/- SD 0.833 +/- 0.063 versus 0.217 +/- 0.081 and 0.221 +/- 0.101 optical density [OD] units, respectively) and vascular cell adhesion molecule 1 (0.620 +/- 0.144 versus 0.165 +/- 0.005 and 0.177 +/- 0.055 OD units, respectively) and increased production of interleukin-4 (IL-4) (6.8 +/- 2.4 versus 1.2 +/- 1.6 and 1.3 +/- 2.5 pg/ml, respectively), IL-6 (24.3 +/- 2.4 versus 4.5 +/- 6.7 and 5.9 +/- 5.1 pg/ml, respectively), and IL-8 (36.8 +/- 10.3 versus 10.1 +/- 6.7 and 7.8 +/- 2.1 pg/ml, respectively). Sera from AAECA-positive patients with TA induced 29 +/- 6% (median +/- SEM) apoptosis of aortic endothelial cells. CONCLUSION: Our data show that the AAECAs that are present in patients with TA are directed mainly against 60-65-kd antigen(s) and may cause vascular dysfunction by inducing expression of endothelial adhesion molecules, cytokine production, and apoptosis.     

Autoantibodies typical of non-organ-specific autoimmune diseases in HIV-seropositive patients.

OBJECTIVE: To analyse serological aspects of systemic autoimmunity in HIV-1-seropositive patients and in individuals at risk for AIDS. DESIGN AND METHODS: The reactivity of antibodies in the serum of 100 HIV-1-seropositive patients was investigated by enzyme-linked immunosorbent assay (ELISA) using a series of antigens known to be recognized by antibodies from patients with multisystemic autoimmune diseases, such as systemic lupus erythematosus, mixed-connective tissue disease and Sj?gren's syndrome. RESULTS: High levels of immunoglobulin G (IgG) antibodies reacting with double-stranded DNA (dsDNA), synthetic peptides of ubiquitinated histone H2A, Sm-D antigen, U1-A RNP antigen and 60 kD SSA/Ro antigen were found in 44-95% of HIV-infected patients. Among histone antibodies, the most frequent reactions were towards the carboxy-terminal region of histone H1 and to histone H2B and its amino-terminal domain 1-25. Eight HIV-1-seropositive patients at different stages of disease according to the Centers for Disease Control classification were also studied. In most cases, no obvious fluctuations were observed over several years. Antibodies were found early, and their specificity and apparent level of activity remained relatively constant. There was no evidence of such an autoimmune response in the serum of high-risk homosexual seronegative men. CONCLUSIONS: Although the aetiology of AIDS is known, in general the aetiology of multisystemic autoimmune diseases remains to be determined, and the sequence of events taking place remains obscure in both cases. It is possible that the large spectrum of antibodies found in HIV-infected patients reflects a specific stimulation of B-cells by nuclear antigens released by apoptosis during an early stage of disease.     

Hsp60 in inflamed muscle tissue is the target of regulatory autoreactive T cells in patients with juvenile dermatomyositis

OBJECTIVE: Juvenile dermatomyositis (DM) is an autoimmune disease of unknown origin characterized by muscle weakness and skin manifestations. No definite autoantigen has yet been identified. Heat-shock proteins (HSPs) can be up-regulated at sites of inflammation, and immune reactivity to Hsp60 is suggested to play a regulatory role in various chronic inflammatory diseases. The purpose of this study was to determine whether Hsp60 could serve as an autoantigen in juvenile DM. METHODS: Muscle tissue from 4 patients with juvenile DM and 1 healthy control subject without evidence of muscle disease was stained for Hsp60. Peripheral blood mononuclear cells (PBMCs) from 22 patients and 10 healthy control subjects were tested for T cell proliferation induced by human and microbial Hsp60. Cytokine production in response to Hsp60 was examined in 15 patients and 6 healthy controls. T cell reactivity to Hsp60 was determined in muscle biopsy samples from 2 patients. RESULTS: We found significantly increased T cell proliferation to human Hsp60 in PBMCs from juvenile DM patients, which was higher during disease remission. Following in vitro activation with Hsp60, significant amounts of tumor necrosis factor alpha, interleukin-1beta (IL-1beta), and IL-10 were produced. In contrast to muscle biopsy samples from healthy controls, samples from juvenile DM patients showed up-regulation of Hsp60, induction of T cell proliferation, and production of cytokines. Production of proinflammatory cytokines by muscle-derived cells in response to Hsp60 was associated with a poor clinical prognosis, whereas human Hsp60-specific induction of IL-10 was followed by clinical remission. CONCLUSION: These findings suggest that human (self) Hsp60 is a disease-relevant autoantigen in juvenile DM. The difference in T cell response with regard to disease activity indicates an immune regulatory effect of Hsp60-specific T cells, opening up perspectives for antigen-specific immunotherapy.     

In vivo analysis of the apoptosis‐inducing effect of anti–endothelial cell antibodies in systemic sclerosis by the chorionallantoic membrane assay

Objective

Systemic sclerosis (SSc) is a connective tissue disease of unknown etiology characterized by mononuclear cell infiltration and fibrosis. Vascular injury occurs early in the course of disease, and previous in vitro studies suggest a primary role for anti–endothelial cell antibodies (AECAs) in mediating endothelial cell apoptosis. The aim of the present study was to analyze the apoptosis‐inducing effect of AECAs in vivo.

Methods

The optimum animal model for transfer experiments was the University of California at Davis line 200 (UCD‐200) chickens that spontaneously develop a hereditary disease with features closely resembling those of scleroderma in humans. AECA‐positive serum samples from UCD‐200 chickens were used for intravenous injection into normal CC chicken embryos on embryonic day (ED) 13 as well as for application onto chorionallantoic membranes (CAMs) of healthy control lines on ED 10. CAMs of ED 16 embryos and combs of 1‐week‐old CC chickens that had received the injected serum samples were analyzed for apoptotic endothelial cells by TUNEL.

Results

Staining of frozen CAM sections by immunofluorescence showed evidence of in vivo binding of AECAs to the microvascular endothelium. In most groups, transfer of AECA‐positive sera resulted in a significant increase in endothelial cell apoptosis as compared with controls.

Conclusion

This study is the first to demonstrate the in vivo apoptosis‐inducing effects of AECAs. The findings support our hypothesis of a primary pathogenetic role of AECAs in SSc.

     

Antibodies to human gastric epithelial cells and heat shock protein 60 in Helicobacter pylori positive mucosa associated lymphoid tissue lymphoma

BACKGROUND: Development of gastric mucosa associated lymphoid tissue (MALT) lymphoma is thought to be closely associated with host immune reactions to Helicobacter pylori. AIM: To investigate humoral immune responses in patients with MALT lymphoma to antigens shared by H pylori and human gastric epithelial cells. METHODS: Sera were obtained from H pylori positive patients with MALT lymphoma (n = 11) or other gastroduodenal diseases (peptic ulcer, n = 40; non-ulcer dyspepsia, n = 20) and from H pylori negative healthy control subjects (n = 10). Antibodies to HGC-27 human gastric epithelial cells and human recombinant heat shock protein (Hsp) 60 were examined using an enzyme linked immunosorbent assay (ELISA) and immunoblotting. RESULTS: Antibody titres to HGC-27 cells were significantly elevated in H pylori positive patients with MALT lymphoma when compared with titres in patients with other gastroduodenal diseases and in healthy subjects. Immunoblotting of sera from patients with MALT lymphoma often detected a band with a molecular mass corresponding to Hsp60, and both ELISA and immunoblotting showed elevated antibody titres to the recombinant human Hsp60. Antigenic similarity between Hsp60 and H pylori HspB was documented by immunoblotting experiments. CONCLUSIONS: Autoantibodies reactive with host gastric epithelial cells are often increased in MALT lymphoma, and Hsp60 is a major target antigen. Immune responses induced by immunological cross reactivity between H pylori HspB and human Hsp60 in gastric epithelium may be involved in the development of MALT lymphoma.