Liping Kong

Autoimmunity associated with TGF-beta1-deficiency in mice is dependent on MHC class II antigen expression.

The progressive inflammatory process found in transforming growth factor beta1 (TGF-beta1)-deficient mice is associated with several manifestations of autoimmunity, including circulating antibodies to nuclear antigens, immune complex deposition, and increased expression of both class I and class II major histocompatibility complex (MHC) antigens. The contribution of MHC class II antigens to the genesis of this phenotype has been determined by crossing the TGF-beta1-null [TGF-beta1(-/-)] genotype into the MHC class II-deficient [MHC-II(-/-)] background. Mice homozygous for both the TGF-beta1 null allele and the class II null allele [TGF-beta1(-/-);MHC-II(-/-)] are without evidence of inflammatory infiltrates, circulating autoantibodies, or glomerular immune complex deposits. Instead, these animals exhibit extensive extramedullary hematopoiesis with progressive splenomegaly and adenopathy, surviving only slightly longer than TGF-beta1(-/-);MHC-II(+/+) mice. The role of CD4+ T cells, which are also absent in MHC class II-deficient mice, is directly demonstrated through the administration of anti-CD4 monoclonal antibodies in class II-positive, TGF-beta1(-/-) mice. The observed reduction in inflammation and improved survival emphasize the significance of CD4+ cells in the pathogenesis of the autoimmune process and suggest that the additional absence of class II antigens in TGF-beta1(-/-);MHC-II(-/-) mice may contribute to their extreme myeloid metaplasia. Thus, MHC class II antigens are essential for the expression of autoimmunity in TGF-beta1-deficient mice, and normally may cooperate with TGF-beta1 to regulate hematopoiesis.

FAS (CD95)-TRANSDUCED SIGNAL PREFERENTIALLY STIMULATES LUPUS PERIPHERAL T LYMPHOCYTES

Fas (CD95) is a cell surface receptor whose biological function in circulating peripheral T cells is not well understood. To address the question of abnormal T cell sensitivity to Fas stimulation in systemic lupus erythematosus (SLE), we studied Fas‐transduced stimulation and apoptosis in peripheral blood T cells from patients with SLE and normal control. Immobilized anti‐Fas monoclonal antibodies (mAb) (imCH‐11; IgM type) significantly stimulated SLE T cell proliferation compared to T cells from normal donors and patients with rheumatoid arthritis ( p < 0.003 and p < 0.005, respectively). The soluble form of CH‐11 and other immobilized anti‐Fas mAb (UB‐2, ZB‐4; IgG type) failed to stimulate lupus T cells while immobilized human Fas ligand did. Furthermore, imCH‐11 induced IL‐2 and IL‐6 mRNA expression. However, imCH‐11 activation failed to induce expression of the T cell activation surface molecules CD25 and CD69. Addition of exogenous ceramide, a second messenger for Fas‐mediated apoptosis signaling, also induced T cell proliferation in SLE and normal controls. Moreover, fumonisin B1, a specific ceramide synthase inhibitor, and caspase inhibitors markedly suppressed imCH‐11 induced T cell proliferation, suggesting that the ceramide pathway may be involved in Fas‐transduced stimulation signals in SLE T cells. These results show that SLE T cells have an alteration in the Fas signal transduction pathway leading to cell proliferation. This defect may be important in Fas‐mediated peripheral immune homeostasis.

G-protein signaling abnormalities mediated by CD95 in salivary epithelial cells

Salivary epithelial cells from patients with primary Sjögrens syndrome (SS) undergo Fas-mediated apoptosis. Bcl-2 and Bcl-xL are apoptosis suppressing oncogenes. Very little is known about the role of these oncogene molecules in salivary epithelial cells. To investigate the possible prevention of salivary glandular destruction in SS by Bcl-2 and Bcl-xL, stable transfectants expressing these molecules were made from HSY cells, a human salivary epithelial cell line. HSY cells were transfected with an expression vector for human Bcl-2 or Bcl-xL. Stable transfectants were selected and apoptosis was induced by anti-Fas antibody. Apoptosis was quantified by propidium iodide staining followed by flow cytometry. Caspase activity was detected by immunohistochemical analysis and enzyme cleavage of DEVD-AMC, a fluorescent substrate. Response to carbachol, a muscarinic receptor agonist, and EGF was measured by Ca2+ mobilization and influx. Fas-mediated apoptosis was significantly inhibited in Bcl-2 and Bcl-xL transfectants compared to wild-type and control transfectants (empty vector). Surprisingly, caspase activity was not inhibited in Bcl-2 and Bcl-xL transfectants. Activation of the Fas pathway in the Bcl-2 and Bcl-xL transfectants by antibody also inhibited carbachol and EGF responsiveness (i.e., Ca2+ mobilization and/or influx) by 50–60%. This Fas-mediated inhibition of cell activation was partially or completely restored by specific peptide interference of caspase enzyme activity. The prevention of Fas-mediated apoptosis by the overexpression of Bcl-2 and Bcl-xL in salivary gland epithelial cells results in injured cells expressing caspase activity and unable to respond normally to receptor agonists. Such damaged cells may exist in SS patients and could explain the severe dryness out of proportion to the actual number of apoptotic cells seen on salivary gland biopsy. Cell Death and Differentiation (2000) 7, 1119–1126

MONOCYTE RESCUE OF HUMAN T CELLS FROM APOPTOSIS IS CD40/CD154 DEPENDENT

The induction of T‐cell apoptosis is regulated in part by monocytes (CD14+ cells). Human peripheral blood monocytes inhibited the spontaneous cell death of activated T cells in vitro. The inhibition of T‐cell apoptosis did not require autologous monocytes. Inhibition required direct contact with monocytes and was not due to a soluble factor. Furthermore, treatment of monocytes with actinomycin D, cycloheximide and paraformaldehyde abrogated the anti‐apoptotic activity of these cells. Blocking antibody to CD40 and CD154 (CD40 ligand) decreased the ability of monocytes to aid in T‐cell survival, whereas, blocking LFA‐1/I‐CAM‐1, Fas ligand and the CD4/major histocompatibility complex class II interaction did not affect the influence of monocytes on T‐cell survival. This shows that monocytes rescue of activated T cells from apoptosis is dependent upon CD40/CD154 interaction.

TGF-β1 Null Mutation Leads to CD154 Upregulated Expression in Affected Tissues

The TGF-β1(–/–) mouse is a murine model for systemic autoimmune disease. The aim of this study is to elucidate the immunological mechanism that leads to multifocal tissue inflammation and autoantibody production in TGF-β1(–/–) mice. Heart, lung, liver, and salivary gland from TGF-β1(–/–) were assessed for CD154 expression by RT-PCR and immunohistochemistry. Compared to wild-type littermates, CD154 expression was elevated in all tissues studied. Furthermore, IL-12 mRNA was expressed in the salivary gland and heart of TGF-β1(–/–) mice and not in wild-type littermates. This suggests that the CD154 pathway is activated in these tissues. This shows that TGF-β1 regulates CD154 expression leading to spontaneous IL-12 production and autoimmunity.