Autoimmunity develops in lupus-prone NZB mice despite normal T cell tolerance

NZB mice spontaneously develop an autoimmune disease characterized by production of anti-RBC, -lymphocyte, and -ssDNA Abs. Evidence suggests that the NZB mouse strain has all of the immunologic defects required to produce lupus nephritis but lacks an MHC locus that allows pathogenic anti-dsDNA Ab production. The capacity to produce diverse autoantibodies in these mice raises the possibility that they possess a generalized defect in self-tolerance. To determine whether this defect is found within the T cell subset, we backcrossed a transgene encoding bovine insulin (BI) onto the NZB background. In nonautoimmune BALB/c mice, the BI transgene induces a profound but incomplete state of T cell tolerance mediated predominantly by clonal anergy. Comparison of tolerance in NZB and BALB/c BI-transgenic mice clearly demonstrated that NZB T cells were at least as tolerant to BI as BALB/c T cells. NZB BI-transgenic mice did not spontaneously produce anti-BI Abs, and following antigenic challenge, BI-specific Ab production was comparably reduced in both BI-transgenic NZB and BALB/c mice. Further, in vitro BI-specific T cell proliferation and cytokine secretion were appropriately decreased for primed lymph node and splenic T cells derived from NZB BI-transgenic relative to their nontransgenic counterparts. These data indicate that a generalized T cell tolerance defect does not underlie the autoimmune disease in NZB mice. Instead, we propose that the T cell-dependent production of pathogenic IgG autoantibodies in these mice arises from abnormal activation of T cells in the setting of normal but incomplete tolerance.     

Functional competence of T cells in the absence of glycosylphosphatidylinositol-anchored proteins caused by T cell-specific disruption of the Pig-a gene

T lymphocytes express various glycosylphosphatidylinositol (GPI)-anchored surface proteins, such as Thy-1 and Ly-6A. However, functional contribution of GPI-anchored proteins in T cell activation is as yet poorly understood. Here we report the generation of mutant mice deficient in the expression of GPI-anchored molecules exclusively in their T cells. We established mice carrying three identically oriented lox-P sites within the Pig-a gene, which encodes a component essential for the initial step of GPI anchor biosynthesis. These mice were crossed with mice carrying the Cre recombinase gene driven by the T cell-specific p56lck proximal promoter. Offspring carrying both the lox-P-containing Pig-a gene and the Cre transgene exhibited almost complete loss of the surface expression of GPI-anchored molecules on peripheral T cells. Interestingly, those T cells deficient in GPI-anchored molecules were capable of responding to T cell receptor stimulation in vitro and in vivo. These results indicate that T cells lacking the expression of GPI-anchored molecules are functionally competent in exerting TCR-mediated immune responses.     

Induction of specific T cell tolerance by Fas ligand-expressing antigen-presenting cells

Autocrine interaction of Fas and Fas ligand leads to apoptosis of activated T cells, a process that is critical for the maintenance of peripheral T cell tolerance. Paracrine interactions of Fas ligand with T cells also may play an important role in the maintenance of tolerance, as Fas ligand can create immune-privileged sites and prevent graft rejection by inducing apoptosis in T cells. We surmised that APCs that express Fas ligand might directly induce apoptosis of T cells during presentation of Ag to the T cells, thus inducing Ag-specific, systemic T cell tolerance. Here, we show that profound, specific T cell unresponsiveness to alloantigen was induced by treatment of H-2k mice with H-2b APCs that expressed Fas ligand and that profound T cell unresponsiveness specific for the H-Y Ag was induced by treatment of H-2Db/H-Y TCR transgenic female mice with H-2Db/H-Y APCs that expressed Fas ligand. The induction of this systemic T cell tolerance required the expression of Fas ligand on the APCs as well as the expression of Fas on the T cells. The tolerance was restricted to the Ag presented by the APCs. The rapid and profound clonal deletion of the Ag-specific, peripheral T cells mediated by the Fas ligand-expressing APCs contributed to the induction of tolerance. These findings demonstrate that Ag-specific T cell tolerance can be induced by APCs that express Fas ligand and suggest a novel function for APCs in the induction of T cell apoptosis. Furthermore, they indicate a novel immunointervention strategy for treatment of graft rejection and autoantigen-specific autoimmune diseases.     

Control of neonatal tolerance to tissue antigens by peripheral T cell trafficking

Self tolerance is acquired by the developing immune system. As reported here, particular properties of the neonatal tissue contribute to this process. Neonatal skin, but not adult skin, was accessible for na?ve CD8 T cells. In mouse bone marrow chimeras generated at different ages, recent thymic emigrants were tolerized to a skin-expressed major histocompatibility complex class I antigen only during a neonatal period but not during adulthood. Blockade of T cell migration neonatally prevented tolerance induction. Thus, T cell trafficking through nonlymphoid tissues in the neonate is crucial for the establishment of self tolerance to sessile, skin-expressed antigens.     

Multiplex inheritance of component phenotypes in a murine model of lupus

Histopathological identification of invasive breast carcinoma in its earliest phases is fraught with pitfalls. Preinvasive malignant lesions complicated by radial scar, sclerosing adenosis, and lobular cancerization, among other lesions, may simulate invasive carcinoma. Fibrosis, inflammatory reaction, and other stromal changes around in situ carcinoma may mask microinvasive foci on routine stains. Conventional immunohistochemistry to demonstrate basement membrane or myoepithelial cell layer may not, by itself, be unequivocally diagnostic of invasion. We performed a novel double immunoenzyme labeling technique using an avidin-biotin complex peroxidase-diaminobenzidine system for smooth-muscle actin followed by an alkaline phosphatase anti-alkaline phosphatase-new fuchsin system for cytokeratin antigen on formalin-fixed, paraffin-embedded histology sections to evaluate 32 such problematic cases. The initial histologic impression with hematoxylin and eosin staining alone was as follows-first group: microinvasive carcinoma-10; second group: carcinoma in situ--"stromal invasion cannot be ruled out"--15; third group: frankly infiltrating carcinoma of various grades and morphologic types-6. The last group served as positive control for invasion. One fibroadenoma with fine-needle-aspiration-induced artifact simulating stromal invasion was also included. The double immunoenzyme labeling technique imparted a dark brown color to the myoepithelial cells and a vivid red color to the epithelial cells, making individual or loosely cohesive groups of malignant epithelial cells infiltrating the stroma easily detectable, whereas their in situ counterparts were contained within dark brown myoepithelial boundaries. The TNM 1997 definition of pT1mic, i.e., extension of malignant cells in the stroma with no focus measuring >0.1 cm, was followed to classify microinvasion. In the first group, microinvasion was confirmed in six cases but was not demonstrable in four. In the second group, definite invasion was identified in five cases, ruled out in nine, and in one case the suspicion of early invasion could not be entirely ruled out even after double immunoenzyme labeling. Thus, it was possible to render a definite opinion regarding presence or absence of invasion in 24 of 25 (96%) cases diagnosed as or suspected to be microinvasive. The precise and simultaneous elucidation of topography between malignant cells and myoepithelial cells on a single permanent section makes this technique a useful diagnostic tool in the evaluation of those cases of breast carcinoma that exhibit equivocal invasion.     

T cell recognition and tolerance of antibody diversity

The capacity of B cells to self-present their Ab variable regions in the context of class II MHC structures suggests a potential regulatory problem. If T cells were able to recognize self-presented Ab, then T cell help might be delivered to B cells independently of a foreign carrier epitope, resulting in a chronic state of unregulated Ab synthesis. For this reason, we have proposed that T cells normally attain a state of tolerance to Ab V region diversity. Here, we tested this idea by performing direct immunizations with unmutated isologous mAb. We also identified and analyzed epitopes recognized by class II MHC-restricted T cell hybridomas that were originally generated against two physiologically mutated isologous mAb. Our results indicate that the class II MHC-restricted T cell repertoire is tolerant of germ-line-encoded Ab diversity and that the physiologic somatic hypermutation process creates immunogenic epitopes in Ab V regions, in some cases by producing class II MHC-binding peptides. In agreement with these findings, we found that germ-line-encoded Ab V regions are presented by endogenous splenic APC at a level that is physiologically significant.     

Functional CD4+ T cell subsets defined by expression of CD45RC and NTA260 antigens and age-associated polarization in murine lupus

Using two mAb, one specific to the alternative exon 6-dependent epitope of CD45 molecules (JH6.2) and one a natural thymocytotoxic autoantibody (NTA) with an unknown reactive epitope (NTA260), we subdivided splenic CD4+ T cells from 2-month-old BALB/c mice into five phenotypically distinct subsets. CD45RC+NTA260- (S I) cells were phenotypically analogous to CD4+ T cells predominating in newborn mice and produced a significant amount of IL-2, but not so IL-4, IL-10 or IFN-gamma when stimulated with immobilized anti-CD3 mAb in vitro. They appeared to consist mainly of naive ThP cells. The CD45RC+NTA260+ (S II) subset also produced IL-2, but not other cytokines; however, the IL-2 levels produced were much higher than seen with the S I subset, thereby suggesting the predominance of further maturated ThP cells. The CD45RC-NTA260+ (S III) subset mainly produced IL-4, IL-10, IFN-gamma and less IL-2, and contained memory cells that helped the secondary antibody response to a recall antigen, and hence contained Th2 and probably a mixture of Th0 and Th1 cells. The CD45RC-NTA260- (S IV) subset was a poor responder to the immobilized anti-CD3 mAb. The CD45RCbrightNTA260dull (S V) subset consisted of a small number of cells that were phenotypically analogous to activated CD4+ T cells. While an age-associated decrease in the proportion of S I and less markedly in S II and in turn increase in S III subsets of CD4+ T cells occurred in normal BALB/c mice, autoimmune disease-prone (NZB x NZW)F1 mice showed a marked age-associated decrease in the proportion of not only S I, II but also III subsets. As aged (NZB x NZW)F1 mice carry CD4+ T helper cells for IgG anti-DNA antibody production, such age-associated polarization to the S IV subset appears to be critical in the pathogenesis of autoimmune disease in these mice.     

Termination of peripheral tolerance to a T cell epitope by heteroclitic antigen analogues

Treating mice with an immunodominant T cell epitope from moth cytochrome c (MCC(88-103)) can induce T cell unresponsiveness under certain conditions of administration. In this report, we determined whether T cell tolerance to MCC(88-103) in adult animals can be overcome by immunization with cross-reactive analogues of the tolerizing Ag. A panel of analogues of the tolerogen were tested for their capacity to terminate the tolerant state following in vivo immunization. As analyzed by their stimulatory capacity for a representative MCC(88-103)-specific T cell clone, this panel covered a wide range of cross-reactivity, including nonantigenic, antagonistic, weakly, and strongly antigenic peptides. Interestingly, only heteroclitic analogues, as measured in vitro by their enhanced antigenicity for the T cell clone that was specific for MCC(88-103), were capable of breaking tolerance. Thus, an immune response to the cross-reactive, heteroclitic analogues of tolerized self Ags may represent a mechanism by which Ag molecular mimicry operates.     

CD4 T cell tolerance to nuclear proteins induced by medullary thymic epithelium

Thymic epithelium is involved in negative selection, but its precise role in selecting the CD4 T cell repertoire remains elusive. By using two transgenic mice, we have investigated how medullary thymic epithelium (mTE) and bone marrow (BM)-derived cells contribute to tolerance of CD4 T cells to nuclear beta-galactosidase (beta-gal). CD4 T cells were not tolerant when beta-gal was expressed in thymic BM-derived cells. In contrast, CD4 T cells of mice expressing beta-gal in mTE were tolerized. Tolerance resulted from presentation of endogenous beta-gal by mTE cells but not from cross-priming. mTE cells presented nuclear beta-gal to a Th clone in vitro, while thymic dendritic cells did not. The data indicate that mTE but not thymic BM-derived cells can use a MHC class II endogenous presentation pathway to induce tolerance to nuclear proteins.     

A central capacity sharing model of dual-task performance.

The authors present the central capacity sharing (CCS) model and derive equations describing its behaviors to explain results from dual-task situations. The predictions of the CCS model are contrasted with those of the central bottleneck model. The CCS model predicts all of the hallmark effects of the psychological refractory period (PRP) paradigm: -1 slope of the PRP effect at short stimulus onset asynchronies (SOAs), underadditivity of precentral Task 2 manipulations, additivity of central or postcentral Task 2 manipulations with SOA, and carry forward to Task 2 of Task 1 precentral or central manipulations at short SOAs. The CCS model also predicts that Task 1 response times increase with decreasing SOA. The model is a viable alternative to the central bottleneck model. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Glucocorticoids protect against suppression of T cell responses in a murine model of acute ethanol exposure and thermal injury by regulating IL-6

Previous reports by this laboratory demonstrated that acute alcohol exposure combined with a 15% body surface area dorsal scald injury results in significant reductions in delayed-type hypersensitivity (DTH) and splenocyte proliferative responses compared to either insult alone. Previous studies by this lab have also shown that these defects are mediated, in part, by increased production of interleukin-6 (IL-6). Because both alcohol exposure and thermal injury are known to modulate glucocorticoid (CORT) levels, and CORT regulates IL-6 gene expression, the relationship between circulating CORT and IL-6 production in burn + ethanol mice was examined. At 24 and 48 h post-burn, a positive correlation existed between circulating CORT levels and measurements of cellular immune function. Administration of exogenous CORT to burn + ethanol-treated mice resulted in significant restoration (to 60% of control) of DTH and splenocyte proliferative responses. This restoration was concomitant with a down-regulation of circulating and macrophage-derived IL-6. The specificity of CORT in modulating these responses was tested by assessing cellular immune function and IL-6 levels after glucocorticoid receptor blockade with RU486. Taken together, these data strongly suggest that under normal circumstances CORT protects burned mice from severe immune dysfunction, a protection that is not afforded to burn + ethanol-treated mice. Furthermore, the immune dysfunction observed in burn + ethanol mice may be due to a lack of glucocorticoid attenuation of IL-6.     

Targeted disruption of the FGF2 gene does not prevent choroidal neovascularization in a murine model

Choroidal neovascularization (CNV) is the major cause of severe visual loss in patients with age-related macular degeneration. Laser treatment is helpful for a minority of patients with CNV, and development of new treatments is hampered by a poor understanding of the molecular signals involved. Several lines of evidence have suggested that basic fibroblast growth factor (FGF2) plays a role in stimulating CNV. In this study, we tested this hypothesis using mice with targeted disruption of the FGF2 gene in a newly developed murine model of laser-induced CNV. One week after krypton laser photocoagulation in C57BL/6J mice, 34 of 60 burns (57%) showed fluorescein leakage and 13 of 16 (81%) showed histopathological evidence of CNV. At 2 weeks, CNV was detected in 9 of 10 burns (90%) in which a bubble had been observed at the time of the laser treatment. Electron microscopy showed fenestrated vessels with large lumens within choroidal neovascular lesions. Two weeks after laser-induced rupture of Bruch's membrane, 27 of 36 burns (75%) contained CNV in FGF2-deficient mice compared with 26 of 30 (87%) in wild-type control mice, a difference that is not statistically significant. This study demonstrates that FGF2 is not required for the development of CNV after laser-induced rupture of Bruch's membrane and provides a new model to investigate molecular mechanisms and anti-angiogenic therapy in CNV.     

Cocaine-induced suppression of saccharin intake: A model of drug-induced devaluation of natural rewards.

In Experiment 1, water-deprived Sprague-Dawley rats were given 5 min access to saccharin. This tube retracted, an empty tube advanced, and the rats were given 1 hr to lick the empty tube on a fixed-ratio 10 lick contingency to self-administer saline or cocaine (0.33 mg/infusion) via an intravenous catheter. The results showed that rats avoided intake of saccharin after saccharin-cocaine pairings and that greater avoidance of the gustatory cue was associated with greater cocaine self-administration. In Experiment 2, a similar dose-response function was obtained with either the empty tube or a lever as the operant. In Experiment 3, avoidance of the saccharin cue and the propensity to self-administer cocaine were maintained after at least 1 month of abstinence. As such, this paradigm may be useful as a model of cue-induced craving and drug-induced devaluation of natural rewards. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Induction of alloantigen-specific T cell tolerance through the treatment of human T lymphocytes with wortmannin

Signaling through the CD28 molecule on T cells by its natural ligand, B7, on APCs has recently been shown to require the presence of an active phosphatidylinositol 3-kinase pathway to mediate some of its costimulatory activities (1-7). Using the phosphatidylinositol 3-kinase inhibitor, wortmannin (WN) (8), on human and murine T cells, we have inhibited B7-1-mediated T cell activation and induced Ag-specific tolerance. The addition of WN and/or the B7-1 antagonist, CTLA4Ig, to primary human T cell cultures stimulated with B7-1-transfected allogeneic melanoma cell lines inhibited the generation of alloantigen-specific proliferative and cytolytic responses in vitro. Subsequent examination of these WN- and CTLA4Ig-treated primary T cell cultures revealed that these lymphocyte populations were tolerized to rechallenge with the priming alloantigens in secondary cultures in the absence of additional inhibitor(s). However, reactivity to a third party allogeneic stimulator remained intact. This WN-induced tolerance was reversed by the addition of high dose IL-2, but not IL-4 or IL-7, to the primary cultures, indicating that T cell anergy, not deletion, was responsible for this phenomenon. In vivo studies using a murine graft-vs-host disease (GVHD) model demonstrated that WN treatment of allogeneic donor lymphocytes in vitro failed to generate a significant GVHD in irradiated mouse recipients compared with control allogeneic donor lymphocytes. These findings suggest potentially novel therapeutic strategies for the prevention of GVHD.     

The role of macrophages in antigen presentation and T cell tolerance

Bone marrow derived cells (dendritic cells, macrophages and B cells) are involved in antigen presentation and T cell tolerance. However, the precise functions of each cell type remain unclear. To determine the role of macrophages we produced transgenic mice expressing I-E molecules only on macrophages, by introducing the hybrid gene containing the colony stimulating factor-1 (CSF-1) receptor promoter region and the structural gene encoding E alpha d into C57BL/6 mice. In these mice I-E restricted antigen presentation and T cell priming were impaired. With respect to T cell tolerance, I-E reactive T cells were anergized but not clonally deleted. These results clearly demonstrate that macrophages by themselves are defective in efficient I-E restricted antigen presentation, so that T cells exposed to antigens expressed on macrophages are led to anergy.     

Polyclonal T cell elimination by prolonged immunostimulation in an experimental model

Acute radiation and combined radiation-thermal injuries (CRTIs) were modelled in the experiments on CBA x C57B16 mice. Changes in the concentrations of endogenic and lipopolysaccharide-induced TNF-alpha in the blood serum were measured in 3 to 24 h after gamma-irradiation at a dose of 7 Gy and a combined radiation-thermal injury. The status of mature and reserve pools of the bone marrow granulocytes was determined from the reaction of leukocytes mobilization to blood in response to the administration of interleukin-1 beta (IL-1 beta) in 48 h after Irradiation and CRTI. The contents of IL-3 and IL-6 in the blood serum, the level of cytokine secretion by the bone marrow cells were determined in vitro in the peak phases of acute radiation and combined injuries. No significant changes in the reaction of the bone marrow cells and in the level of pancytopenia were revealed after acute radiation and CRTIs, which could help in understanding an increase in the death rates after CRTIs. Also, quantitative changes in the TNF and IL-6 concentrations in the blood serum were estimated along with those in the level of secretion of IL-3 by the bone marrow cells of mice exposed to radiation and CRTIs.     

Development of T cell precursor activity in the murine fetal liver

The generation of T cell precursors in the liver of murine embryos was studied. The total number of T cell precursors in the liver was measured in thymic organ cultures by a limiting dilution assay. Sixty T cell precursors were detected in the liver at day 11 of gestation. By day 12 the number of precursors showed a 20-fold increase, half of which could be explained by in situ proliferation as ascertained by a fetal liver organ culture assay. By day 13 a further 2-3-fold increase was observed. Whereas the number of total liver cells continued to increase, that of T cell precursors declined in the following days, suggesting a massive exit of these cells after day 13. The capacity to generate a TCRB repertoire in the cells was evaluated by a PCR assay. T cell precursors in day 11 fetal liver developed a TCRB repertoire at day 8 of culture. The cells from days 12-15 developed an identically diverse repertoire by day 6, suggesting that day 11 precursors are more immature than those of later days. A mechanism for yielding a single wave of T cell precursors in the fetal liver is discussed with a proposed model.     

Monocyte chemotactic protein 1 regulates oral tolerance induction by inhibition of T helper cell 1-related cytokines

Intracerebroventricular (i.c.v.) choline (50-150 microg) increased blood pressure and decreased heart rate in spinal cord transected, hypotensive rats. Choline administered intraperitoneally (60 mg/kg), also, increased blood pressure, but to a lesser extent. The pressor response to i.c.v. choline was associated with an increase in plasma vasopressin. Mecamylamine pretreatment (50 microg; i.c.v.) blocked the pressor, bradycardic and vasopressin responses to choline (150 microg). Atropine pretreatment (10 microg; i.c.v.) abolished the bradycardia but failed to alter pressor and vasopressin responses. Hemicholinium-3 [HC-3 (20 microg; i.c.v.)] pretreatment attenuated both bradycardia and pressor responses to choline. The vasopressin V1 receptor antagonist, (beta-mercapto-beta,beta-cyclopenta-methylenepropionyl1, O-Me-Tyr2, Arg8)-vasopressin (10 microg/kg) administered intravenously 5 min after choline abolished the pressor response and attenuated the bradycardia-induced by choline. These data show that choline restores hypotension effectively by activating central nicotinic receptors via presynaptic mechanisms, in spinal shock. Choline-induced bradycardia is mediated by central nicotinic and muscarinic receptors. Increase in plasma vasopressin is involved in cardiovascular effects of choline.