Endogenous interleukin-22 protects against inflammatory bowel disease but not autoimmune cholangitis in dominant negative form of transforming growth factor beta receptor type II mice.

During chronic inflammation, interleukin (IL)-22 expression is up-regulated in both CD4 and CD8 T cells, exerting a protective role in infections. However, in autoimmunity, IL-22 appears to have either a protective or a pathogenic role in a variety of murine models of autoimmunity and, by extrapolation, in humans. It is not clear whether IL-22 itself mediates inflammation or is a by-product of inflammation. We have taken advantage of the dominant negative form of transforming growth factor beta receptor type II (dnTGF-ßRII) mice that develop both inflammatory bowel disease and autoimmune cholangitis and studied the role and the biological function of IL-22 by generating IL-22(-/-) dnTGF-ßRII mice. Our data suggest that the influence of IL-22 on autoimmunity is determined in part by the local microenvironment. In particular, IL-22 deficiency exacerbates tissue injury in inflammatory bowel disease, but has no influence on either the hepatocytes or cholangiocytes in the same model. These data take on particular significance in the previously defined effects of IL-17A, IL-12p40 and IL-23p19 deficiency and emphasize that, in colitis, there is a dominant role of IL-23/T helper type 17 (Th17) signalling. Furthermore, the levels of IL-22 are IL-23-dependent. The use of cytokine therapy in patients with autoimmune disease has significant potential, but must take into account the overlapping and often promiscuous effects that can theoretically exacerbate inflammation.

Successful immunotherapy of autoimmune cholangitis by adoptive transfer of forkhead box protein 3(+) regulatory T cells.

Treatment of primary biliary cirrhosis (PBC) has lagged behind that of other autoimmune diseases. In this study we have addressed the potential utility of immunotherapy using regulatory T cells (Treg ) to treat murine autoimmune cholangitis. In particular, we have taken advantage of our ability to produce portal inflammation and bile duct cell loss by transfer of CD8(+) T cells from the dominant negative form of transforming growth factor beta receptor type II (dnTGF-ßRII) mice to recombination-activating gene (Rag)1(-/-) recipients. We then used this robust established adoptive transfer system and co-transferred CD8(+) T cells from dnTGF-ßRII mice with either C57BL/6 or dnTGF-ßRII forkhead box protein 3 (FoxP3(+) ) T cells. Recipient mice were monitored for histology, including portal inflammation and intralobular biliary cell damage, and also included a study of the phenotypical changes in recipient lymphoid populations and local and systemic cytokine production. Importantly, we report herein that adoptive transfer of Treg from C57BL/6 but not dnTGF-ßRII mice significantly reduced the pathology of autoimmune cholangitis, including decreased portal inflammation and bile duct damage as well as down-regulation of the secondary inflammatory response. Further, to define the mechanism of action that explains the differential ability of C57BL/6 Treg versus dnTGF-ßRII Treg on the ability to down-regulate autoimmune cholangitis, we noted significant differential expression of glycoprotein A repetitions predominant (GARP), CD73, CD101 and CD103 and a functionally significant increase in interleukin (IL)-10 in Treg from C57BL/6 compared to dnTGF-ßRII mice. Our data reflect the therapeutic potential of wild-type CD4(+) FoxP3(+) Treg in reducing the excessive T cell responses of autoimmune cholangitis, which has significance for the potential immunotherapy of PBC.

Escherichia coli infection induces autoimmune cholangitis and anti-mitochondrial antibodies in non-obese diabetic (NOD).B6 (Idd10/Idd18) mice.

Several epidemiological studies have demonstrated that patients with primary biliary cirrhosis (PBC) have a higher incidence of urinary tract infections (UTI) and there is significant homology of the immunodominant mitochondrial autoantigen, the E2 component of the pyruvate dehydrogenase complex (PDC-E2), between mammals and bacteria. Previous work has demonstrated that non-obese diabetic (NOD).B6 Idd10/Idd18 infected with Novosphingobium aromaticivorans developed liver lesions similar to human PBC. It was postulated that the biliary disease was dependent upon the presence of the unique N. aro glycosphingolipids in activating natural killer T (NK T) cells. To address this issue, we infected NOD.B6 Idd10/Idd18 mice with either Escherichia coli, N. aro or use of a phosphate-buffered saline (PBS) vehicle control and serially followed animals for the appearance of liver pathology and anti-mitochondrial autoantibodies (AMA). Of striking importance, the biliary disease of E. coli-infected mice was more severe than N. Aro-infected mice and the titre of AMA was higher in E. coli-infected mice. Furthermore, the immunopathology did not correlate with the ability of bacterial extracts to produce antigen-dependent activation of NK T cells. Our data suggest that the unique glycosphingolipids of N. aro are not required for the development of autoimmune cholangitis. Importantly, the data highlight the clinical significance of E. coli infection in a genetically susceptible host, and we suggest that the appearance of autoimmune cholangitis is dependent upon molecular mimicry. These data highlight that breach of tolerance to PDC-E2 is probably the first event in the natural history of PBC in genetically susceptible hosts.

Induction of autoimmune cholangitis in non-obese diabetic (NOD).1101 mice following a chemical xenobiotic immunization.

Our laboratory has suggested that loss of tolerance to pyruvate dehydrogenase (PDC-E2) leads to an anti-mitochondrial antibody response and autoimmune cholangitis, similar to human primary biliary cirrhosis (PBC). We have suggested that this loss of tolerance can be induced either via chemical xenobiotic immunization or exposure to select bacteria. Our work has also highlighted the importance of genetic susceptibility. Using the non-obese diabetic (NOD) congenic strain 1101 (hereafter referred to as NOD.1101 mice), which has chromosome 3 regions from B6 introgressed onto a NOD background, we exposed animals to 2-octynoic acid (2OA) coupled to bovine serum albumin (BSA). 2OA has been demonstrated previously by a quantitative structural activity relationship to react as well as or better than lipoic acid to anti-mitochondrial antibodies. We demonstrate herein that NOD.1101 mice immunized with 2OA-BSA, but not with BSA alone, develop high titre anti-mitochondrial antibodies and histological features, including portal infiltrates enriched in CD8(+) cells and liver granulomas, similar to human PBC. We believe this model will allow the rigorous dissection of early immunogenetic cause of biliary damage.

Increased entry into the IFN-gamma effector pathway by CD4+ T cells selected by I-Ag7 on a nonobese diabetic versus C57BL/6 genetic background.

IFN-gamma-mediated Th1 effects play a major role in the pathogenesis of autoimmune diabetes in nonobese diabetic (NOD) mice. We analyzed functional responses of CD4(+) T cells from NOD and B6.G7 MHC congenic mice, which share the H2(g7) MHC region but differ in their non-MHC genetic background. T cells from each strain proliferated equally to panstimulation with T cell lectins as well as to stimulation with glutamic acid decarboxylase 524-543 (self) and hen egg lysozyme 11-23 (foreign) I-A(g7)-binding peptide epitopes. Despite comparable proliferative responses, NOD CD4(+) T cells had significantly increased IFN-gamma intracellular/extracellular protein and mRNA responses compared with B6.G7 T cells as measured by intracellular cytokine analysis, time resolved fluorometry, and RNase protection assays. The increased IFN-gamma production was not due to an increase in the amount of IFN-gamma produced per cell but to an increase in the number of NOD CD4(+) T cells entering the IFN-gamma-producing pathway. The increased IFN-gamma response in NOD mice was not due to increased numbers of activated precursors as measured by activation/memory markers. B6.G7 lymphoid cells demonstrated an absolute decrease in IFN-gamma mRNA, an increase in IL-4 mRNA production, and a significantly decreased IFN-gamma:IL-4 mRNA transcript ratio compared with NOD cells. CD4(+) T cells from C57BL6 mice also showed significantly decreased IFN-gamma production compared with CD4(+) T cells from NOD.H2(b) MHC-congenic mice (which have an H2(b) MHC region introgressed onto an NOD non-MHC background). Therefore, the NOD non-MHC background predisposes to a quantitatively increased IFN-gamma response, independent of MHC class II-mediated T cell repertoire selection, even when compared with a prototypical Th1 strain.

T cell receptor (TCR)-mediated repertoire selection and loss of TCR vbeta diversity during the initiation of a CD4(+) T cell response in vivo.

We recently described a novel way to isolate populations of antigen-reactive CD4(+) T cells with a wide range of reactivity to a specific antigen, using immunization with a fixed dose of nominal antigen and FACS((R)) sorting by CD4(high) expression. Phenotypic, FACS((R)), functional, antibody inhibition, and major histocompatibility complex-peptide tetramer analyses, as well as T cell receptor Vbeta sequence analyses, of the antigen-specific CD4(high) T cell populations demonstrated that a diverse sperm whale myoglobin 110-121-reactive CD4(+) T cell repertoire was activated at the beginning (day 3 after immunization) of the immune response. Within 6 d of immunization, lower affinity clones were lost from the responding population, leaving an expanded population of oligoclonal, intermediate affinity (and residual high affinity) T cells. This T cell subset persisted for at least 4 wk after immunization and dominated the secondary immune response. These data provide evidence that CD4(+) T cell repertoire selection occurs early in the immune response in vivo and suggest that persistence and expansion of a population of oligoclonal, intermediate affinity T cells is involved in CD4(+) T cell memory.

Identification and characterization of the antigen-specific subpopulation of alloreactive CD4+ T cells in vitro and in vivo.

We report the identification and characterization of the small subpopulation of alloantigen-specific T cells in vitro and in vivo. This subpopulation of T cells was distinguished by up-regulation of cell surface CD4 expression. These CD4high T cells were alloantigen specific in proliferation assays in vitro, and they expressed memory/activation markers, including CD44high and CD69high. Further studies demonstrated that these allospecific CD4high cells were also present (< or = 1% of CD4+ T cells) in vivo in BALB/c (H-2d) recipients of C57BL/6 (H-2b) skin allografts. CD4high T cells isolated from regional draining lymph nodes in these skin graft recipients reacted in a donor-specific fashion to C57BL/6 splenocyte stimulator cells in mixed lymphocyte culture. Adoptive transfer of CD4high, but not CD4normal T cells, just before skin engraftment in CD4 knockout mice, reconstituted rejection. The discovery that a small subpopulation of CD4high lymph node cells contained all of the alloantigen-specific T cells may allow study of tissue-specificity and subsequent alloantigen identification in transplantation.

MHC structure and autoimmune T cell repertoire development.

Recent work has continued to clarify the relationship between MHC structure and thymic selection that leads to peripheral T cell repertoire development in the pathogenesis of autoimmune diseases. Particular attention has been focused on the nonobese diabetic model of autoimmune diabetes, in which a unique MHC class II molecule (I-Ag7) plays a central role. In the past year, reports on the biochemistry of I-Ag7-combined with analysis of the role of I-Ag7 in T cell repertoire selection--support a model of defective thymic selection as the basis of the association between particular MHC molecules and autoimmune diseases. Analogous work has been done on the structure of the human MHC disease-susceptible and -resistant alleles, DQA1*0301 DQB1*0302 and DQA1*0102 DQB1*0602, and their effect on autoimmune repertoire selection. Comparison of these results (in naturally occurring, spontaneous autoimmune human and murine diabetes), with results in a variety of transgenic and knockout models, has produced an integrated view of how avidity considerations in repertoire selection in the thymus could affect predisposition towards autoimmunity.

Analysis of the role of variation of major histocompatibility complex class II expression on nonobese diabetic (NOD) peripheral T cell response.

The current paradigm of major histocompatibility complex (MHC) and disease association suggests that efficient binding of autoantigens by disease-associated MHC molecules leads to a T cell-mediated immune response and resultant autoimmune sequelae. The data presented below offer a different model for this association of MHC with autoimmune diabetes. We used several mouse lines expressing different levels of I-Ag7 and I-Ak on the nonobese diabetic (NOD) background to evaluate the role of MHC class II in the previously described NOD T cell autoproliferation. The ratio of I-Ag7 to I-Ak expression correlated with the peripheral T cell autoproliferative phenotype in the mice studied. T cells from the NOD, [NOD x NOD. I-Anull]F1, and NOD I-Ak transgenic mice demonstrated autoproliferative responses (after priming with self-peptides), whereas the NOD.H2(h4) (containing I-Ak) congenic and [NOD x NOD. H2(h4) congenic]F1 mice did not. Analysis of CD4(+) NOD I-Ak transgenic primed lymph node cells showed that autoreactive CD4(+) T cells in the NOD I-Ak transgenic mice were restricted exclusively by I-Ag7. Considered in the context of the avidity theory of T cell activation and selection, the reported poor peptide binding capacity of NOD I-Ag7 suggested a new hypothesis to explain the effects of MHC class II expression on the peripheral autoimmune repertoire in NOD mice. This new explanation suggests that the association of MHC with diabetes results from "altered" thymic selection in which high affinity self-reactive (potentially autoreactive) T cells escape negative selection. This model offers an explanation for the requirement of homozygous MHC class II expression in NOD mice (and in humans) in susceptibility to insulin-dependent diabetes mellitus.

Following antigen challenge, T cells up-regulate cell surface expression of CD4 in vitro and in vivo.

The low precursor frequency of Ag-specific T cells has raised significant barriers to studying the T cell response in vivo. We demonstrate that T cells up-regulate the cell surface expression of CD4 following Ag recognition, which identifies Ag-specific T cells in vitro and in vivo and allows their characterization. The CD4high cell subpopulation contains the Ag-specific population as indicated by Ag-induced proliferation and limiting dilution analyses. The use of the CD4high marker will allow analysis of the dynamics of the T cell immune response in vivo, the study of the suboptimal T cell response to Ag, and the identification of T cells which are reactive to known and unknown autoantigens.

The association of MHC with autoimmune diseases: understanding the pathogenesis of autoimmune diabetes.

The current paradigm of MHC and disease association is efficient binding of autoantigens by disease-associated MHC molecules leading to a T cell-mediated immune response and resultant autoimmune sequelae. Data presented here offer a different model for this association of MHC with autoimmune diabetes. This new explanation suggests that the association of MHC with autoimmunity results from "altered" thymic selection in which high-affinity self-reactive (potentially autoreactive) T cells escape negative selection. This model offers an explanation for the requirement of homozygous MHC class II expression in NOD mice (and in man) in susceptibility to IDDM.

Breaking self-tolerance in nonobese diabetic mice.

Unresponsiveness to self is maintained through two mechanisms of immune regulation: thymic-negative selection and peripheral tolerance. Although thymic-negative selection is a major mechanism to eliminate self-reactive T cells, normal mice have readily detectable populations of T cells reactive to self-proteins but do not exhibit autoimmune responses. It has been postulated that autoimmune disease results from breakdown or loss of peripheral tolerance. We present data that demonstrate that peripheral tolerance or unresponsiveness to self can be broken in nonobese diabetic (NOD) mice. Immunization of NOD mice (but not of conventional mice) with self-peptides caused an immune response to self-peptide with resultant autoproliferation of peripheral lymphocytes. Autoproliferation of self-reactive T cells in NOD mice resulted from the recognition and proliferation of the activated T cells to endogenously processed and presented self-antigens. This loss of self-tolerance demonstrated in vitro may well be the basis of NOD autoimmune disease in vivo.

Mice with a disrupted IFN-gamma gene are susceptible to the induction of experimental autoimmune encephalomyelitis (EAE).

Experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis, is an autoimmune disorder seen in mice and rats following immunization with myelin basic protein (MBP) or MBP-derived peptides. IFN-gamma, a cytokine produced by a variety of cells, is involved in many inflammatory and immune regulatory events. Contradictory results concerning exacerbations and the disease course were seen comparing injections of IFN-gamma in humans suffering from multiple sclerosis to studies using anti-IFN-gamma Abs in mice with EAE. To study the role of IFN-gamma and IFN-gamma-producing cells in EAE, we crossed IFN-gamma knockout mice (H-2b) (unable to produce IFN-gamma due to the disruption of the IFN-gamma gene) with an EAE-susceptible mouse strain, B10.PL (H-2u). EAE was seen in IFN-gamma knockout mice, heterozygotic (IFN-gamma +/-) mice, as well as wild-type littermates following immunization with MBP. Histologic analyses of the central nervous system of IFN-gamma knockout mice with EAE revealed massive infiltrates composed of lymphocytes, macrophages, and granulocytes. We conclude that the presence of IFN-gamma is not crucial to the induction or the clinical course of EAE.

Regulation of autoimmune response.

Recent work on such apparently disparate fields as T-cell receptor peptide-induced regulation, superantigens, antigen-induced tolerance, models of peripheral tolerance, apoptosis, and T-cell receptor antagonists demonstrates a similarity in immune response from a regulatory perspective. In many systems, a 'tolerance' pathway is observed, characterized broadly as an initial disturbance in the immune system, with a resulting predominance of effector cells, followed by a homeostatic response (often requiring CD8+ cells) which leads the effector population into T-cell receptor downregulation, T-cell inactivation, anergy and, often, eventual apoptotic death. In the regulated immune response, mixed populations of anergized and apoptosing T cells can be found. In some cases, anergy appears to lead to death while, in other instances, cells revert to a functional state. This review focuses on recent papers examining each of these topics in an attempt to obtain a preliminary integrated picture of immune regulation in autoimmune diseases.

Aggressive drug therapy for rheumatoid arthritis.

Recent data indicate that rheumatoid arthritis is more often systemic, progressive, and disabling than benign, and that it reduces life expectancy. The new evidence argues for a dramatic alteration in pharmacologic management. If several months of rest, exercise, and anti-inflammatory therapy are ineffectual, aggressive treatment with disease-modifying antirheumatic and immunosuppressive agents may be in order.