Induction of experimental autoimmune thyroiditis by heat-denatured porcine thyroglobulin: a Tc1-mediated disease.

Recent studies have shown that denatured exogenous antigens can prime cytotoxic T lymphocytes (CTL). To assess the contribution of CTL to experimental autoimmune thyroiditis (EAT), porcine thyroglobulin (pTg) was heat-denatured (hdpTg) and injected i.v. into CBA/J mice, without the aid of adjuvants. Both lymphocytic infiltrations of the thyroid glands and levels of Tg-specific CTL were similar to those found in conventional EAT induced by Tg and adjuvants. In contrast, proliferative responses could not be detected, and titers of antibodies to pTg were 20 times lower. These EAT-inducer CTL belong to the CD8+ cell subset and exerted their thyroiditogenic potential through release of IFN-gamma. We conclude that hdpTg-induced EAT is mediated by type 1 cytotoxic T cells (Tc1).

Curative treatment of experimental autoimmune thyroiditis by in vivo administration of plasmid DNA coding for interleukin-10.

The autoimmune response in experimental autoimmune thyroiditis (EAT) is characterized by a lymphocyte infiltration of the thyroid gland and by the appearance of circulating autoantibodies to thyroglobulin (Tg). Cytokines play a crucial role in the immunoregulation and pathology of EAT. Systemic administration of IL-10 has curative effects on EAT, but requires high doses and iterative injections due to the rapid turnover of this molecule. We have designed an original in vivo gene transfer using a mixture of liposomes and poly-L-Lysine that greatly enhanced the transfection yield, and induced a fast and long-lasting expression of IL-10 on mouse thyroid follicular cells (TFC). IL-10 expression on TFC of mice wit EAT dramatically wipe out the lymphocytic infiltration in the thyroids. A significant diminution in the proliferative anti-Tg T cell response was observed, along with a trend towards a Th2 response characterized by decreased production of IFN-gamma and by increased anti-Tg IgG1/IgG2a Ab ratios. In conclusion, local IL-10 gene therapy using non-viral vectors is a novel and promising approach for the treatment of thyroid autoimmune disorders.

Gene therapy of experimental autoimmune thyroiditis by in vivo administration of plasmid DNA coding for Fas ligand.

Fas-Fas ligand (FasL) interaction is required for the maintenance of immune homeostasis and seems to be responsible for the privileged immune status of some tissues. Experimental autoimmune thyroiditis (EAT), which is characterized by autoreactive T and B cell responses and a marked lymphocytic infiltration of the thyroid, is a model of choice to study the therapeutic effects of FasL. Here, we provide evidence that direct injection of DNA expression vectors encoding FasL into the inflamed thyroid inhibited development of lymphocytic infiltration of the thyroid and induced death of infiltrating T cells. These results were paralleled by a total abrogation of anti-Tg cytotoxic T cell response in FasL-treated animals vs controls. In summary, our results show that FasL expression on thyrocytes may have a curative effect on ongoing EAT by inducing death of pathogenic autoreactive infiltrating T lymphocytes.

Effects of murine recombinant interleukin-10 on the inflammatory disease of rats transgenic for HLA-B27 and human beta 2-microglobulin.

Rats transgenic for HLA-B27 and human beta 2-microglobulin develop a spontaneous, multisystem, inflammatory disease that resembles human B27-associated disease and that involves the gut mucosa. This model predominantly affects the colon and is characterized by an extensive infiltration of the mucosa by inflammatory cells, largely composed of mononuclear cells. In addition, an increased plasma level of nitric oxide (NO)-derived metabolites was described in this model. Deficiency in the anti-inflammatory cytokine, interleukin-10 (IL-10), leads to the development of colitis in IL-10 knockout mice, suggesting that IL-10 plays a major role in the control of gut inflammation. The objectives of this work were to study the mechanisms of the inflammatory bowel disease (IBD) in HLA-B27 rats and to determine the effects of treatment with IL-10. The 33-3 line of HLA-B27 recombinant rats with established disease was treated in two consecutive experiments with murine recombinant IL-10 for five weeks. Assessment of the effect of this treatment was performed, based on clinical, histological and biological (myeloperoxidase and inducible NO synthase activities; tumor necrosis factor-alpha, interferon-delta, CD3, iNOS and beta-actin mRNA expression. In 33-3 rats with established disease, mesenteric lymph nodes were hyperplastic, and colonic cellularity and MPO and iNOS activities in the colonic mucosa were increased without any detectable effects of IL-10 administration. IFN-gamma and iNOS mRNA were only detected in the colon of transgenic rats. Despite a lack of effect on disease expression, IL-10 strikingly reduced the level of IFN-gamma mRNA in gut mucosa. Up-regulation of IFN-gamma mRNA suggests that the IBD of HLA-B27 rats is mediated by T-helper 1 lymphocytes. Sustained administration of IL-10, in HLA-B27 rats with established disease, efficiently inhibited IFN-gamma mRNA expression but did not influence disease expression: these results indicate that IFN-gamma may exert a critical role at an earlier stage of the disease rather in the maintenance of the lesions.

Phenothiazine-induced increase in thyroid autoantigens and costimulatory molecules on thyroid cells: a pathophysiological mechanism for drug-induced autoimmunity?

We have previously demonstrated (J Immunol 1995; 154:3593) that MHC class II antigens can be induced on thyroid epithelial cells (TEC) by alimemazine, a member of the phenothiazine group. Although this expression of MHC class II antigens on TEC confers the theoretical ability to behave as antigen-presenting cells (APC), the simultaneous expression of self antigens and co-receptor(s) must also occur for efficient presentation of self antigens. Therefore, we investigated whether alimemazine applied at pharmacologic doses would modify the expression of thyroid antigens, and simultaneously, the expression of intercellular adhesion molecule-1 (ICAM-1), B7, and LFA-1 co-receptors in human TEC in culture. Using polymerase chain reaction (PCR) amplification and Northern blot analysis, we showed that alimemazine induces increases in thyroglobulin (Tg) and thyroid-stimulating hormone receptor (TSH-R) cDNA, within the first 2 h following its addition. This phenomenon is followed 48 h later by an increase of Tg and TSH-R protein expression on the surface of TEC. Furthermore, increases in the expression of ICAM-1 and B7 co-receptors were concomitantly observed. These results suggest that alimemazine, a drug currently used in paediatrics, could play a role in the induction and perpetuation of thyroid autoimmune disorders by transforming TEC into functional APC.

Experimental autoimmune thyroiditis (EAT) in mice lacking the IFN-gamma receptor gene.

To investigate the role of interferon-gamma (IFN-gamma) in experimental autoimmune thyroidits (EAT), H-2k mice with a disrupted IFN-gamma receptor (IFN-gamma R) gene were immunized with porcine thyroglobulin (pTg). We observed that EAT occurred on day 19 and remitted on day 35 in IFN-gamma R-deficient (IFN-gamma R(0/0)) mice, whereas in wild-type mice, EAT occurred on day 21 and remitted on day 42-49. Moreover, EAT in the mutant mice was attenuated and accompanied by diminished Tg-specific cytotoxic and proliferative responses and decreased titers of anti-Tg antibodies, notably of the IgG2a and IgG2b isotypes. In contrast, Tg-specific IgG1 was increased in the IFN-gamma R(0/0) mice. In supernatants from T cells further stimulated in vitro by Tg, IFN-gamma levels were higher in IFN-gamma R(0/0) than in wild-type mice throughout the course of the disease, whereas interleukin-10 was transiently increased prior to EAT onset in both groups of mice. Finally, using IFN-gamma R(0/0) mice, we demonstrate that induction of EAT does not require an intact IFN-gamma system, while progression to full-blown disease depends on the action of IFN-gamma.

Analysis of susceptibility of NOD mice to spontaneous and experimentally induced thyroiditis.

Beside diabetes, non-obese diabetic (NOD) mice develop sporadic lymphoid infiltration of the thyroid gland, mimicking Hashimoto's thyroiditis. We have examined the prevalence of those manifestations in NOD mice, the influence of the major histocompatibility complex (MHC) and the association with autoantibodies. The incidence at 1 year is of 14.3% in wild-type NOD mice versus 19.6% in congenic NOD.H2k mice. The moderate, but statistically significant difference, based on the analysis of 161 NOD and 169 NOD.H2k mice, suggests that MHC genes partially control spontaneous NOD thyroiditis. Autoantibodies against thyroglobulin (Tg) are mouse specific and their presence correlates closely with thyroiditis. The strong correlation between cellular and humoral anomalies therefore resembles Hashimoto's thyroiditis. NOD and NOD.H2k mice actively immunized against Tg develop severe chronic lesions with epithelium necrosis and interstitial tissue fibrosis. Most interestingly, those lesions do not regress spontaneously as in CBA/J mice. Paradoxically, the response to Tg of lymph node cells from NOD mice is weaker both in proliferation and cytokine production. The defect is most evident for interferon-gamma-producing T cells and is reflected in the marked deficit in IgG2a antibodies. Thus a moderate anti-Tg response seems to favor chronicity of thyroiditis. In conclusion, NOD and NOD.H2k mice offer a unique opportunity of analyzing the factors leading to immune chronicity in a genetic context which promotes autoimmune endocrinopathies.

[Predisposition to thyroid autoimmune diseases]. / Prédisposition aux maladies autoimmunes thyroïdiennes.

OBJECTIVES: Genetic predisposition is required for the expression of thyroid autoimmune disorder addition to the immune dysfunction and the environmental factors. METHODS: In order to evaluate the role of this genetic factor, we reported the results of immunological and hormonal investigations of 62 members (TD), belonging to a large Akr family, who are related to 40 patients with Graves' disease or Hashimoto's thyroiditis. RESULTS: The hormonal analyses showed that 19 subjects exhibited an infraclinical hypothyroidism, subdivided into 7 members with pathological rates of TSH evocative of thyroid insufficiency and 12 others with compensative thyroid insufficiency. Seventeen subjects of the Akr family who had solely antithyroid autoantibodies were considered as potential candidates to develop thyroid autoimmune diseases. The clinical follow-up, during two years, confirmed the diagnosis of Hashimoto's thyroiditis in 3 members among 19 subjects with infraclinical hypothyroidism (TD05, TD28 and TD54) and in only 1 member out of the 17 potential candidates (TD03). CONCLUSION: Our results showed that a serological study of hormones and/or autoantibodies directed against thyroid antigens, could allow the detection of predisposed subjects to develop a thyroid autoimmune pathology. The Akr family seems to be suitable for the study of the localization of susceptibility genes to TAID.

Influenza A virus hemagglutinin is a B cell-superstimulatory lectin.

Influenza A viruses display T cell-independent polyclonal B cell-activating properties which are mediated by the B cell-superstimulatory envelope glycoprotein hemagglutinin (HA). In this report, the receptor-binding requirements for B cell activation by influenza viruses were expected. Neuraminidase treatment of resting mature B cells from BALB/c mice abrogated late (proliferation/immunoglobulin synthesis), early (up-regulation of cell surface markers, including CD25, B220, and B7-1) and very-early events (homotypic adhesion) in virus-responding B lymphocytes. Similarly, pretreatment of murine responder cells with different inhibitors of N-glycosylation (tunicamycin, deoxymannojirimycin) significantly suppressed subsequent B lymphocyte activation by HA, but not control responses to lipopolysaccharide or anti-mu. Assays with chimeric HA transfectants, expressing the loop region of epitope B (amino acids 155-160) of the globular head of H2 (high B cell-stimulatory subtype) or H3 (medium-stimulatory subtype) on the protein backbone of a low-stimulatory subtype (H1) failed to alter the B cell-stimulatory activity of the virus, suggesting that the hypervariable loop region is not crucial in determining the B cell-activating properties of the protein. Collectively, our results imply that the B cell-superstimulatory function of influenza virus HA is not mediated by a direct protein/protein interaction, but via binding of HA to terminal sialic acid residues on cell surface receptor glycoproteins. These findings identify the influenza virus HA glycoprotein as the first viral lectin with lymphocyte-activating properties.

Induction of experimental autoimmune thyroiditis (EAT) by heat-denaturated thyroglobulin (Tg).

Experimental Autoimmune Thyroiditis (EAT) is characterized by autoreactive T and B cell responses, assessed by a marked lymphocytic infiltration of the thyroid gland by T cells and the occurrence of circulating autoantibodies (AAb) to thyroglobulin (Tg). It was recently reported that administration of denaturated exogenous antigens primes class I-restricted cytotoxic T cells in vivo. Since cytotoxic T cells are involved in EAT development, porcine Tg (pTg) was heat-denaturated, i.v. injected into CBA/J mice and features of EAT evaluated. Simultaneously, mice were immunized with pTg and adjuvants and evaluation of EAT performed. We found that heat-denaturated pTg (hdpTg) induced EAT in recipient mice similar to native pTg/adjuvants. Surprisingly, whereas Tg-specific cytotoxic T cells were regularly found in lymph node cells from hdpTg or native pTg immunized mice, proliferative responses were only detected using T cells from native pTg immunized mice. Autoantibodies to pTg were decreased by a factor 30 in sera from mice immunized with hdpTg. These data further emphasized the role of Tg-specific cytotoxic T cells in EAT.

B cell superstimulatory influenza virus activates peritoneal B cells.

We evaluated the potential of B cell "superstimulatory" influenza viruses to activate peritoneal B cells (PBC) from BALB/c mice containing both CD5+ and CD5- "sister" cells. Like conventional B cells, PBCs responded to influenza viruses in a hemagglutinin glycoprotein (HA) subtype-specific manner with proliferation and vigorous Ig synthesis. However, a number of HA subtypes that are highly stimulatory for conventional B cells failed to induce significant responses of PBC. Isotype-determination revealed a high predominance of IgM and only very low production of IgA and IgG. HA-activated CD5+ B cells showed a hyperexpression of various activation markers, including MHC class II, intercellular adhesion molecule 1 (CD54), and B7-1 molecules. In contrast to conventional B cells, where activation by HA is antagonized by phorbol esters (PMA), HA and PMA acted synergistically on PBC, suggesting differential activation requirements of B-2 cells vs PBC in response to HA. Like HA stimulation of B-2 cells, virus-triggered proliferation of PBC was abrogated by a simultaneous treatment with F(ab')2 fragments of anti-Ig Ab and exhibited synergistic effects with LPS stimulation. HA-mediated proliferative responses of PBC, but not of B-2 cells, were positively controlled by various cytokines, including IL-4 and IL-10, and to a lesser extent by IL-6. In conclusion, our data present the first example of a stimulation of peritoneal B cells by a polyclonal-activating virus, findings that call for considering infections with polyclonal B cell-stimulatory viruses as a means of expanding the pool of potentially autoreactive CD5+ B cells.

Curative and protective effects of IL-10 in experimental autoimmune thyroiditis (EAT). Evidence for IL-10-enhanced cell death in EAT.

We studied the effects of in vivo administration of rhIL-10 in two models of experimental autoimmune thyroiditis (EAT): 1)-in EAT induced by injection of mTg emulsified in adjuvant, and 2) in EAT induced by adoptive transfer of mTg-specific T lymphocytes. Furthermore, we tried to assess both the protective and curative potential of IL-10 in EAT, by administering rhIL-10 either at the time of priming and challenge with mTg, or only at the time of challenge. We demonstrated that proliferative and cytotoxic responses of splenic cells to mTg were markedly reduced by in vivo rhIL-10 treatment. Cell surface marker studies revealed a 40 to 45% reduction in CD4+ and in CD8+ lymphoblastoid spleen cells from mice treated with rhIL-10 either in the early or in the late EAT. The severity of EAT was significantly reduced in mice treated with high-dose rhIL-10, whereas levels of autoantibodies to mTg were not altered. Furthermore, when analyzing purified T lymphocytes from rhIL-10-treated animals, an increase of cells undergoing apoptotic cell death became evident in the rhIL-10 treated group, as compared with controls. This IL-10-mediated enhancement of activation-induced cell death critically depended on the applied therapeutic dose of rhIL-10. Thus, IL-10 exerts beneficial effects on the development and course of EAT through a mechanism that could imply an IL-10-mediated enhancement of activation-induced cell death in T lymphocytes, findings that call for considering IL-10 in the immunotherapy of early-phase and likewise of already established autoimmune thyroiditis.

Molecular heterogeneity of antigen- or idiotype-induced anti-thyroglobulin monoclonal autoantibodies.

To define the molecular basis of the cognitive interaction in experimental autoimmune thyroiditis (EAT), we sequenced the variable regions of monoclonal autoantibodies to thyroglobulin (Tg), specific or not for the F40D peptide, a Tg peptide capable of inducing EAT in CBA/J mice. Three MoAbs were obtained by immunization with syngeneic Tg of CBA/J (3B8G9, 2F6F2) or C57Bl/6 (4D11F4) mice. 3B8G9 was specific for F40D peptide, whereas 2F6F2 and 4D11F4 were not. Two others were raised in CBA/J mice by manipulation of idiotypic pathways: B12 resulted from the immunization with one Ab2 beta, bearing the internal image of one F40D epitope, and TA2 from the immunization with F40D-specific cytotoxic HTC2 T cells. B12 and TA2 were both specific for F40D. All hybridomas expressed different members of the J558 VH family, except 3B8G9 which expressed a Q52 VH gene segment. These data led us to hypothesize that regulatory anti-id autoantibodies used members of one VH family located in the 5'-end of the VH locus, whereas EAT-associated autoantibodies used a member of one of the most D-proximal VH family. As expected, no homologies were found when anti-F40D monoclonal autoantibodies were compared with two other monoclonal autoantibodies displaying a different epitopic specificity. Among the anti-F40D monoclonal autoantibodies, one histidine residue located in position 35 of the CDR1 region was constantly found. Moreover, TA2 and B12 exhibited two common amino acids in their CDR3 regions, one glycine and one tyrosine, in positions 98 and 99, respectively. Striking homologies were found between TA2 and one anti-polyGAT MoAb, and between 3B8G9 and some anti-phenyloxazolone (phOx) monoclonal autoantibodies. Lastly, the VK sequence from 4D11F4 was identical at the amino acid level to the VK sequence from another monoclonal autoantibody, 81B1, which was previously raised towards syngeneic Tg in CBA/J mice. Our data imply that anti-idiotypic regulatory circuits in EAT might be generated by a heterogeneous population of B cells rather than obtained by a single dominant B cell population.

Distinctive modulation by IL-4 and IL-10 of the effector function of murine thyroglobulin-primed cells in "transfer-experimental autoimmune thyroiditis".

Experimental autoimmune thyroiditis (EAT) is characterized by autoreactive T and B cell responses, a marked lymphocytic infiltration of the thyroid gland, and the occurrence of circulating autoantibodies to thyroglobulin. Direct evidence for the involvement of lymphocytes stems from the observation that EAT can be induced in naive, irradiated CBA/J mice by transfer of in vitro restimulated effector spleen cells obtained from murine thyroglobulin (mTg)-immunized donors. Using this transfer-EAT (tEAT) model, we have investigated whether addition of recombinant murine IL-4 (rIL-4) or human IL-10 (rIL-10) during the in vitro restimulation by mTg would affect the subsequent induction of the disease. To determine the modification(s) induced during the secondary in vitro incubation with mTg and cytokine, proliferative and cytotoxic responses to mTg were studied. MTg-activated cells cultured with mTg and rIL-4 exhibited only slightly decreased proliferative responses to mTg and increased cytotoxic responses toward mTg-pulsed macrophages compared to mTg-activated cells cultured in the absence of cytokine. In contrast, proliferative and cytotoxic responses to mTg were diminished by approximately 45 and 85%, respectively, when cells were cultured with mTg and rIL-10. The injection of mTg-activated spleen cells, cultured in the presence of rIL-10, into irradiated CBA/J mice induced a significant decrease (P = 0.02) in lymphocytic infiltrations of the recipient thyroid glands compared to injection of irradiated hosts with mTg-activated cells cultured without cytokines, but no reduction in anti-mTg autoantibody production in vivo. In contrast, when mice were injected with mTg-activated cells cultured with mTg and rIL-4, the lymphocytic infiltrations of the recipient thyroid glands were similar to controls, but circulating anti-mTg antibody were surprisingly significantly reduced. These results show that two typical "Th2 cytokines," IL-4 and IL-10, when added in vitro to mTg-specific spleen cells under identical experimental conditions, can have rather diverse effects in terms of an exacerbation or weakening of cytotoxic mTg-specific T cell reactivity and a maintenance or attenuation of subsequent tEAT severity.

Superantigens induce primary T cell responses to soluble autoantigens by a non-V beta-specific mechanism of bystander activation.

Superantigens have been suggested to act as powerful TCR V beta-specific inducers of T cell reactivity in autoimmune diseases. We have investigated the capacity of staphylococcal enterotoxins (SE) to prime autoreactive T cell responses in naive animals in the Lewis rat model of experimental autoimmune encephalomyelitis (EAE), where myelin basic protein (MBP)-specific CD4+ effector T cells express almost exclusively V beta 8.2 TCR elements. By taking advantage of the reactivity of V beta 8.2+ MBP-specific T cells to SEE but not to other SEs in vitro, we estimated the potential of different SEs (SEA, SEB, and SEE) to induce a primary T cell response to soluble MBP in vivo. Upon immunization of naive rats with soluble MBP alone or MBP and SEB (which is only a very weak superantigen for rat T cells), no MBP-responses could be retrieved. Similarly, when coimmunizing naive rats with MBP and V beta 8.2-activating SEE, no autoreactivity was inducible. By contrast, coimmunization of animals with soluble MBP and the superantigen SEA that is strongly activating various T cell subpopulations in Lewis rats but not V beta 8.2+ (i.e., potentially MBP reactive) T cells led to a significant primary MBP-specific T cell autoreactivity. These SEA-induced MBP-reactive T cells expressed V beta 8.2 TCRs at levels similar to those seen in autoreactive T cells conventionally induced by immunization with MBP administered in complete Freund's adjuvant (CFA) and could induce disease in a transfer model of EAE. Thus, our results are consistent with the notion that superantigens are able to induce primary T cell responses to soluble autoantigens by a non-V beta specific mechanism of bystander priming.

Phenothiazine induces de novo MHC class II antigen expression on thyroid epithelial cells. A new mechanism for drug-induced autoimmunity.

Autoimmune responses are initiated by MHC class II-restricted T cell responses directed against tissue-specific autoantigens. Furthermore, HLA-DR expression in thyroid epithelial cells is a prominent feature of autoimmune thyroid disease. In the present work, we were particularly interested in a phenothiazine, a neuroleptic and anti-depressant drug of pharmacologic importance named alimemazine. Our interest in this compound stems from previous findings of immune effects of this and other phenothiazines. We demonstrate that MHC class II Ags can be experimentally induced on thyroid cells by pharmacologic concentrations of alimemazine, a drug commonly used in psychiatry. In contrast, MHC class II Ags were not induced on the lymphoid cell lines Raji and Jurkat. Expression of MHC class II Ag on the surface of the cloned human thyroid cell hybridoma, GEJ, was demonstrated by flow cytometry. Moreover, by using Northern blot and Southern blot analyses, this finding was confirmed at the molecular level in GEJ and in murine thyroid epithelial cell cultures, respectively. The functional role of phenothiazine-, de novo-induced MHC class II Ags on thyroid cells was assessed by both syngeneic murine thyroglobulin-specific and allogeneic proliferative T cell responses. These results suggest that antidepressant drugs of the phenothiazine type could play a role in the induction and the perpetuation of thyroid autoimmune disorders, through induction of class II restriction elements on normally class II-negative thyroid epithelial cells.

One monoclonal antibody to human thyrotropin (TSH) receptor agonist of TSH hormone stimulates the proliferation of human thyroid cells.

The question of thyroid cell growth induction by monoclonal antibodies (mAbs) to human thyrotropin receptor (hTSH-R), which stimulate increases in cAMP thyroid cells, is under debate and their implication in Graves' disease (GD) is controversial. In order to address this issue, we used characterized reagents, i.e., mAbs to hTSH-R, and cloned human thyroid hybridoma cells (GEJ). Cell counting on Days 1, 2, and 3 after addition of mAb and Northern blot analysis of mRNA specific for the c-fos oncogene were used to assess the proliferation of the thyroid cells. MAbs to hTSH-R, obtained by immunization of DBA/1 mice with affinity-purified hTSH-R, were added to GEJ cells in concentrations varying from 0.66 to 660 nM. Their effects on GEJ cell growth were compared to those of human TSH, of hLH, and of control mAb. Cell counting and evaluation of GEJ c-fos transcripts showed that mAbs to hTSH-R induce significant GEJ cell growth, whereas they were ineffective on the control cell line. Among them, one mAb, 34A, exhibited an impressive activity on thyroid cell growth and induced cAMP production comparable to that induced by bovine or human TSH. Our data demonstrate the existence of immunoglobulin which stimulates thyroid growth and elevates cAMP. The higher the cAMP production, the greater the thyroid cell proliferation. Furthermore, the data suggest a possible role for agonistic anti-hTSH-R autoantibodies in the immunopathogenesis of GD.

B cell superstimulatory influenza virus (H2-subtype) induces B cell proliferation by a PKC-activating, Ca(2+)-independent mechanism.

The influenza virus hemagglutinin glycoprotein (HA) induces a vigorous B cell proliferation and Ig-synthesis by an unknown activation mechanism, which is susceptible to the inhibitory effects of anti-Ig and anti-class II mAbs. To gain further insight into the activation mode of this T cell-independent, B cell "superstimulatory" virus, we analyzed the sensitivity of H2-subtype virus-mediated B cell activation to the inhibitory effects of various signal transduction-blocking agents and compared it to the well characterized anti-mu-mediated and the LPS-employed pathway. Cyclic-AMP agonists (cAMP-analogues, pentoxifylline, cholera toxin, and forskolin) blocked HA-mediated activation of B cells only at concentrations at least 50-fold higher than required for blocking of anti-mu-induced activation. However, HA-treatment failed to induce an increase in intracellular cAMP levels in responding B cells. The B cell response to HA was highly resistant to calcineurin-inhibitory cyclosporin-A treatment and did not result in a measurable Ca2+ influx. Similarly, HA failed to induce an increase in tyrosine phosphorylations, including phosphorylation of phospholipase C gamma 2. HA-activated B cells showed an increase in membrane-associated protein kinase C activity, and depletion of protein kinase C by pretreatment of B cells with phorbol esters inhibited a subsequent activation by HA. Collectively, our results provide a new example of B cell stimulation by multivalent type-2 Ags, which seems to be mediated by a phosphatidylinositol- and Ca(2+)-independent signaling pathway.

Desensitization of the antigen receptor primes B cells for activation by superstimulatory influenza virus.

Interaction of the B cell receptor (BCR) with non-immunogenic receptor ligands can induce a specific state of B cell unresponsiveness as a result of receptor desensitization. Provided it is maintained over time, BCR desensitization may provide the molecular basis for clonal anergy. Using an in vitro model of anti-Ig-mediated BCR desensitization, we assessed the susceptibility of desensitized "anergic" B lymphocytes to activation by B cell superstimulatory influenza virus hemagglutinin (HA). Rabbit anti-mouse Ig antibodies (whole Ig molecule or F(ab')(2)-fragments) totally abolished the response of murine B cells to HA, when added simultaneously with the virus. Pretreatment with the same antibodies, however, yielding a complete unresponsiveness to a subsequent challenge with normally mitogenic anti-Ig reagents, even enhanced the subsequent proliferative response to HA. By contrast, HA-mediated high-rate immunoglobulin synthesis was suppressed after desensitization. BCR-desensitized, HA-stimulated B cells exhibited a hyperexpression of various activation markers (B7, major histocompatibility complex class II, CD25) and served as potent antigen-presenting cells (APC) in a polyclonal model for T lymphocyte activation. These observations suggest a possible scenario for the breaking of natural B cell tolerance, where infections with B cell superstimulatory viruses may lead to the clonal expansion of receptor desensitized, functionally silenced B lymphocytes in vivo.