HIV-induced IL-6/IL-10 dysregulation of CD4 cells is associated with defective B cell help and autoantibody formation against CD4 cells

To analyse CD4 cell cytokine secretion and helper/suppressor function at a clonal level we established 446 CD4⁺ T cell clones (TCC) in four healthy controls, three HIV^? haemophilia patients, four CDC II,III and four CDC IV patients. Spontaneous TCC secretion of Th1 cytokines (IL-2, interferon-gamma (IFN-γ)) and Th2 cytokines (IL-4, IL-6, IL-10) was determined by ELISA. TCC helper and suppressor functions were tested in a pokeweed mitogen (PWM)-stimulated allogeneic co-culture system using a reverse haemolytic plaque assay for assessment of B cell responses. There was a significant association of TCC surface marker expression (Leu-8, CD45RA) with TCC IL-6 secretion in healthy controls (P < 0.01), HIV^? patients (P 0.001) and CDC II,III patients (P 0.01) but not in CDC IV patients. Likewise, TCC expression of Leu-8 and CD45RA was significantly associated with TCC suppressor function in healthy controls (P 0.0005) but not in HIV-infected patients. A reduced TCC helper frequency (10% of TCC) and an enhanced TCC suppressor frequency (> 80% of TCC) were detected only in those HIV-infected patients who showed an excessively increased TCC IL-6 secretion (> 70% of TCC) together with a significantly diminished TCC IL-10 secretion (10% of TCC). CD4 cell autoantibodies also were found only in patients with this type of cytokine dysregulation. These data indicate that CD4 cell surface markers lose their functional relevance in HIV-infected patients. HIV-induced IL-6/IL-10 dysregulation of CD4⁺ T cells, i.e. the up-regulation of spontaneous IL-6 and down-regulation of spontaneous IL-10 secretion, appears to be involved in inducing CD4 helper defects and may promote autoantibody formation against CD4 cells.     

Association of stress proteins with autoantigens: a possible mechanism for triggering autoimmunity?

Patterns of autoantibody production are diagnostic of many autoimmune disorders; the recent observation of additional autospecificities towards stress-induced proteins may also provide insight into the mechanisms by which such responses arise. Grp78 (also known as BiP) is a target of autoaggressive B and T cell responses in our murine model of anti-Ro (SS-A) autoimmunity and also in rheumatoid arthritis. In this report we demonstrate reciprocal intermolecular spreading occurs between Ro52 and Grp78 in immunized mice, reflecting physiological association of these molecules in vivo. Moreover, we provide direct biochemical evidence that Grp78 associates with the clinically relevant autoantigen, Ro52 (SS-A). Due to the discrete compartmentalization of Ro52 (nucleocytoplasmic) and Grp78 (endoplasmic reticulum; ER) we propose that association of these molecules occurs either in apoptotic cells, where they have been demonstrated indirectly to co-localize in discrete apoptotic bodies, or in B cells themselves where both Ro52 and Grp78 are known to bind to immunoglobulin heavy chains. Tagging of molecules by association with Grp78 may facilitate receptor mediated phagocytotsis of the complex; we show evidence that exogenous Grp78 can associate with cell surface receptors on a subpopulation of murine splenocytes. Given the likelihood that Grp78 will associate with viral glycoproteins in the ER it is possible that it may become a bystander target of the spreading antiviral immune response. Thus, we propose a model whereby immunity elicited towards Grp78 leads to the selection of responses towards the Ro polypeptides and the subsequent cascade of responses observed in human disease.     

Simultaneous activation of natural killer T cells and autoantibody production in mice injected with denatured syngeneic liver tissue

Denatured syngeneic liver tissue prepared by mechanical procedures was intraperitoneally injected into adult C57BL/6 mice. In parallel with a decrease in the total number of lymphocytes in the liver, spleen, and thymus from days 1-7 after the injection, the proportion of the CD4+NK1.1+CD3(int) subset of these cells (i.e. natural killer T or NKT cells) increased in the liver. Even the absolute number of these NKT cells increased in the liver on days 14 and 21. In response to the injection of denatured liver tissue, tissue damage was induced in the liver, as shown by elevated levels of serum transaminases and hepatocyte degeneration observed by electron microscopy. Sera obtained on days 7 and 14 contained autoantibodies including anti-DNA antibodies. The proportion of CD1d(high)B cells in the liver was found to decrease on days 1-7. In other words, denatured liver tissue stimulated both NKT cells and certain B cells in the liver. These results suggest that liver lymphocytes might contain not only autoreactive T cells (e.g. CD3(int) or NKT cells) but also some B cells (e.g. B-1 cells) which produce autoantibodies and that the denatured tissue had the potential to stimulate these lymphocytes and to evoke an autoimmune-like state.     

Alveolar macrophages from subjects infected with HIV-1 express macrophage inflammatory protein-1 alpha (MIP-1 alpha): contribution to the CD8+ alveolitis.

A human monoclonal anticardiolipin autoantibody (ACA) of the IgA-k isotype, designated 185/12, is described. The antibody was prepared from peripheral B cells, obtained from a patient with a history of habitual abortion, by immortalization with Epstein-Barr virus (EBV). The antibody displays a strong binding activity to cardiolipin and phosphatidyl L-serine, but not to phosphatidylcholine, phosphatidylinositol, ssDNA and dsDNA. It binds to cardiolipin in a concentration-related and saturable manner (Kd = 3.0 x 10(-8) M). This reaction is dependent upon the presence of bovine serum, and is fully inhibited by cardiolipin vesicles. The 185/12 antibody exhibits different binding patterns to the solid-phase bound cardiolipin-serum complex and to its individual components (cardiolipin and bovine serum). The Bmax of 185/12 binding to the complex (0.968 OD units) is higher than the sum of the Bmax values calculated for each one of the complex components (0.352 + 0.179 = 0.531 OD units). Bovine serum as well as purified beta 2-glycoprotein I (beta 2-GPI) in suspension inhibit the binding of 185/12 to the complex. 185/12 binding capacity increases in direct relation to the rising concentration of beta 2-GPI. Collectively, these data may be interpreted to suggest that 185/12 antibody, which is an IgA isotype, exhibits characteristics usually attributed only to antiphospholipid autoantibodies (APA) of the IgG isotype, that are associated with the clinical spectrum of APA syndrome (APA-S). It is, therefore, possible that autoantibodies of the IgA isotype could play a pathogenic role, which may be different from that of the IgG isotype, in the development of autoimmune phenomena.     

Naturally occurring human IgA autoantibodies against IgE-DES myeloma protein

The prevalence and specificity of naturally occurring human IgA anti-IgE autoantibodies (a-E Ab) were studied by ELISA with anti-IgA monoclonal antibodies (mAb) and a purified myeloma IgE as solid-phase protein, i.e., IgE-DES(κ). Such detected IgA a-E Ab were common among adults, and significantly increased geometric means (GM) were found in patients with atopy (P= 0.006; n= 41; GM = 79.3 arbitrary units (AU)/ml) and filariasis (P= 0.02; n= 41; GM = 75.9 AU/ml), as compared with nonatopic controls (n= 42; GM = 48.8 AU/ml). No such difference was observed between age-matched nonatopic (n= 22; GM = 36.7 AU/ml) and atopic (n= 22; GM = 38.6 AU/ml) children. Children had significantly (P= 0.001) lower IgA a-E Ab concentrations than adults, probably as a result of age, because IgA a-E Ab concentrations and age of children were significantly correlated (n= 44; P<0.05; r_s= 0.30). IgA a-E Ab concentrations were very low in cord serum (n= 32; median <0.1 AU/ml). Sex did not influence IgA a-E Ab concentrations in any study group. The specificity of IgA a-E Ab in nine sera was studied by ELISA inhibition assay using IgE-DES myeloma as solid-phase protein and inhibitory proteins of the IgG, IgM, IgD, and IgE classes, including five different IgE myeloma proteins, as well as three enzymatic fragments of IgE-DES. The inhibitions indicated that all IgA a-E Ab tested reacted in a low-affinity reaction with determinants restricted to IgE-DES, i.e., the solid-phase protein. These epitopes were heat-resistant (2 h; 56°C) and located in the Fab_?-DES fragment. No isotype-specific IgA a-E Ab were found because none of the four other IgE proteins were inhibitory. Subclass typing indicated that most IgA a-E Ab belonged to the IgA 1 subclass. It is unlikely, for reasons of restricted specificity, low affinity, and common prevalence, that such IgA 1 a-E Ab are connected with IgE-mediated disorders. The study also raises questions on the definition of anti-IgE antibodies.     

消化道肿瘤自身抗体检测

目的探讨消化道肿瘤患者针对肿瘤细胞的自身抗体的存在。方法收集肠癌患者及胃癌患者术前血清96份和健康体检人群血清62份，分组后采用Western印迹分析法定性检测消化道肿瘤患者术前血清中的自身抗体。结果正常健康人群和正常肝细胞株96％（212／220）的反应条无显色带，而97％（60／62）的肠癌患者肠癌细胞株、89％（39／44）的胃癌患者胃癌细胞株反应条均出现多个阳性带。结论健康人群体内很少产生针对肿瘤细胞的自身抗体，而大部分消化道肿瘤患者体内产生针对肿瘤相关抗原的自身抗体，这为消化道肿瘤的病因，早期诊断，预后监测及分子靶向治疗等研究提供了一个很好的线索。     

Cytokines, thyroid autoantibody synthesis and thyroid cell survival in culture

In autoimmune thyroid disease lymphoid cells infiltrating the thyroid gland occur in conspicuous aggregates or as a diffusely distributed population invading the thyroid follicles. Consequently cytokines secreted by activated T cells or macrophages could influence neighbouring thyroid cells as well as other lymphocytes. We have investigated this possibility using recombinant cytokines. Thyroid cell survival was assessed in terms of mitochondrial dehydrogenase activity in monolayers exposed to tumour necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma), interleukin-1 (IL-1 alpha and beta) and interleukin-2 (IL-2) in the presence or absence of thyroid-stimulating hormone (TSH). Neither TNF-alpha nor IL-2 affected thyroid cell survival, IFN-gamma was usually inhibitory and IL-1 alpha slightly enhanced cell survival in some experiments. However, the effects were small and variable and were not enhanced by potentially synergistic combinations of cytokines, longer periods of exposure, or different culture conditions. In contrast, IFN-gamma, IL-2 and TNF-alpha inhibited the ability of thyroid lymphocytes from patients with Graves' disease and Hashimoto's thyroiditis to synthesize autoantibodies to thyroid peroxidase (TPO) and thyroglobulin (Tg). Comparison of lymphoid populations isolated by digestion and/or mechanical disaggregation indicated that a population of activated B cells, plasma cells and T cells, intimately associated with thyroid cells since they could only be extracted by digestion, was influenced by cytokines. Our studies suggest that in addition to its well-recognized ability to induce MHC class II antigens on thyroid cells, IFN-gamma may inhibit thyroid cell proliferation and TNF-alpha, IFN-gamma and IL-2 may down-regulate thyroid autoantibody synthesis.     

Differences in specificities of anti-centromere sera for the monomeric and dimeric C-terminal peptides of human centoromere protein C

Centromere protein-C (CENP-C), one of the centromere autoantigens and components of the inner plate of the kinetochore, is suggested to make a dimer at the C-terminus. In order to investigate the presence of conformation-specific anti-centromere antibodies (ACA) to the dimer form, the C-terminal 124 amino acids (CF-124) were expressed in Escherichia coli, affinity purified and chemically cross-linked. Immunoblotting was utilized to compare the reactivities between the dimers and the monomers against 58 ACA(+) sera. The reactivities of the dimers were obviously higher in both IgG and IgM responses. The dimer was still more reactive than the glutathione S-transferase-fused monomer in some sera. Two kinds of CF-124 mutant (each contained one amino acid change at the N-terminal region of CF-124) and two cut segments of CF-124 (67 N-terminal amino acids and 58 C-terminal amino acids) were also examined. The former two mutants decreased the dimerization activity. The latter two mutants lost both activities except for the faint dimerization activity of the N-terminal half. Affinity-purified antibodies with CF-124 in a liquid phase containing the co-purified GroE protein of E. coli, GroEL, reacted to the centromere in culture cells. In conclusion, there are heterogeneous autoepitopes including some conformational epitopes at the C-terminal CENP-C.     

改良Coombs试验对诊断自身免疫性溶血性贫血的价值

目的：比较经典Coombs试验、改良Coombs试验、单抗Coombs分型试验,寻找更灵敏的方法诊断自身免疫性溶血性贫血（AIHA）。方法：对45例临床怀疑为AIHA病人依次进行经典Coombs试验、改良Coombs试验、单抗Coombs分型试验。结果：5例经过临床观察及相关检查发现为其它疾病;30例经典Coomb试验（ ）;36例改良Coombs试验IgG( );40例单抗Coombs分型试验（ ）;IgG1( )患最多（27例,为总数,下同）,IgG3( )其次（20例）,IgG2( )较少（14例）,IgG4( )最少（9例）,1例未检出任何亚型。还发现经典Coombs试验灵,敏度为75.5%,改良Coombs试验灵敏度为90.0%,单抗Coombs分型试验灵敏2度为97.5%。结论：温抗体IgG4种亚型主要为IgG1,某些病例可见IgG3,IgG2少见,IgG4罕见;单抗Coombs分型试验比经典Coombs试验及改良Coombs试验敏感。