Mechanisms Of Inducible Costimulator-mediated Costimulation In The Immunopathology Of Systemic Lupus Erythematosus

The outcome of T-cell responses after T-cell encounter with specific antigens is modulated by costimulatory signals, which are required for lymphocyte activation, T helper cells differentiation, and development of adaptive immunity. CD28 and CD40L costimulation are critical for initiation of immune responses. In contrast, effector immune responses are less dependent on such costimulatory molecules. Inducible costimulator (ICOS), an newly costimulator with homology to CD28, is expressed on activated, but not resting T cells and shows T-cell costimulatory function in vitro. ICOS binds specifically to its counter-receptor B7RP-1, but not to B7-1 or B7-2. ICOS is expressed similarly on both Th1 and Th2 lines after primary stimulation, but remains high only on Th2 lines after repeated activation steps. In vivo genetic evidence has shown that ICOS delivers a costimulatory signal that is essential both for efficient cooperation between T and B cells and for normal antibody responses to T-cell-dependent antigens. ICOS engagement can augment induction of both Thl and Th2 cytokines, but under some circumstances it may more effectively costimulate Th2 responses. Studies have shown that when blocking of ICOS-ICOSL interaction, cytokines, including IFN- Y and IL-10, and chemokines, including CC and CXC chemokines are reduced, the transition of antibody is defected, and apoptosis of immune cells is increased.Lupus is a chronic autoimmune inflammatory disease with complex clinical manifestations. Human patients and murine models of lupus manifest a wide range of immunological abnormalities. The most pervasive of these are: (1) the ability to produce pathogenic autoantibodies; (2) lack of T- and B-lymphocyte regulation; (3) defective clearance of autoantigens and immune complexes. Pathogenic memory T cells produced at disease initiating step following dendritic cells (DC) presentation of "danger" self antigens creates a self-perpetuating inflammatory milieu. These cells then activate naive autoimmune B cell precursors via cytokines and costimulatorymolecules. During this time, B cells undergo class switching and somatic mutation resulting in the production of high titers of IgG autoantibodies. Furthermore, the interaction of T and B cells via costimulatory molecules may generate anti-apoptotic signals in patients with lupus leading to increased numbers of autoreactive B cells producing pathogenic autoantibodies. Inquiries into the mechanism(s) causing these T cell dysfunctions have led to the identification of several new defects of signal transduction and have begun to reveal their molecular basis and possible immunopathological role in the pathogenesis of systemic lupus erythematosus (SLE). Since ICOS is induced after T-cell activation and plays important roles in T-cell-dependent antibody production, antibody transition, and Th2 cytokines production, such as IL-10, it is postulated that ICOS may play an important role in the pathogenesis of SLE. In this study, we demonstrated (1) the expression of ICOS on peripheral blood T subsets and ICOSL on peripheral blood CD19 B cells from SLE patients with three-color flowcytometry and explored the possible correlation between the level of ICOS expression and clinical manifestations of SLE, which included disease activity and levels of serum anti-dsDNA antibodies and immunoglobulin, (2) costimulatory functional role(s) of ICOS in lupus, including enhancing T-cell proliferation, cytokines and T-cell dependent autoantibody production, and (3) the possible role(s) of MAP-kinase signaling cascades in ICOS-costimulating T-cells in the pathogenesis of SLE which might provide a new way in biological therapy of autoimmune diseases.Part I Expression and clinical significance of ICOS on peripheral blood T lymphocyte subsets in patients with systemic lupus erythematosusIn present the immunological role(s) of ICOS reported mostly come from animal studies. Until now there have several reports about the hyperexpression and function of ICOS either in injured tissues or in peripheral blood from animals with autoimmune encephalomyelitis, animals with autoimmune arthritis, and animals with autoimmune myocarditis. Recently studies about ICOS in injured tissues and in peripheral blood from patients with rheumatoid arthritis (RA) and patients withsystemic sclerosis (SS) also supported the immunopathological role(s) of ICOS in the pathogenesis of these diseases. But until now, there has only one report about the expression and possible role of ICOS in patients with SLE. In that study the authors found that ICOS was important for B cell activation and autoantibody production.In this part we detected the level of ICOS-, ICOSCD45RO-, ICOSCD45RA-, and ICOSCD28-expression on peripheral blood CD4 and CD8 T cells in normal individuals, patients with SLE, and patients with RA. Then in patients with SLE we also observed the relationship between percentages of ICOS-expressing peripheral blood T subsets and disease-stage, level of serum anti-dsDNA antibodies, and concentration of serum immunoglobulin. Our results showed that the percentages of ICOS-expressing CD4 and CD8 T cells were markedly increased in patients with SLE as compared to those of patients with RA and normal individuals, but the level of ICOSL on peripheral blood B cells from patients with SLE was significantly decreased as compared to patients with RA. The relationship between SLEDAI Score and the percentages of ICOS-expressing CD4 or CD8 T cells was poor. Of clinical importance, in the same patients, the proportions of PB CD4 and CD8 T cells expressing ICOS were significantly increased in active phase, compared with those from stable phase. In very active SLE patients the proportions of ICOS-expressing CD45RO cells, especially CD45RO+ CD4 T cells, were markedly increased as compared to those of normal individuals, and had some relationship with the level of serum anti-dsDNA antibodies and the concentration of serum immunoglobulin in patients with SLE. The percentages of ICOS+CD28+ and ICOS+CD28" cells in peripheral blood CD4 and CD8 T cells from SLE patients in different disease-stages were significantly higher than those of RA patients and normal individuals, and had some relationship with the level of serum anti-dsDNA antibodies and the concentration of serum immunoglobulin, which indicated that expression of ICOS might be partly independent on CD28, and that T cells not expressing CD28 might also play an important role in the pathogenesis of SLE. In the mean time, we also compared the proportions of peripheral blood CD4, CD8, CD28, CD45RO, and CD45RA cells in SLE patients and normal individuals. Our results showed that theproportion of CD8 T cells was significantly increased in SLE patients which led to the lower ratio of CD4/CD8. In addition, the proportion of CD45RA cells was markedly decreased while the proportion of CD45RO cells was slightly increased. In active SLE patients, the percentage of IFN-y-secreting T helper (Th) cells significantly decreased compared with stable SLE patients and normal subjects after in vitro PMA/IO stimulation, the percentage of IFN-y-secreting cytotoxic T (Tc) cells significantly reduced compared with normal individuals, but IL-4-secreting Th cells and Tc cells did not vary significantly, which showed that there was a severe immune imbalance in patients with SLE.Through this study we propose that ICOS-ICOSL interaction might play an important immunopathological role(s) in T-cell proliferation and activation, Thl and Th2 cytokine secretion, and T-cell-dependent autoantibody production in patients with SLE which need further study.Part II In vitro study of ICOS-mediated costimulation in the immune regulation of T and B cell interaction in patients with SLEIn this part we firstly evaluated the kinetics of ICOS expression on peripheral blood CD4 and CD8 T cells from patients with SLE and normal individuals after in vitro PMA+IO or anti-CD3 antibody stimulation, then we studied the immunopathological role(s) of ICOS-ICOSL interaction in T-cell and B-cell proliferation and the production of Thl and Th2 cytokines and T-cell-dependent autoantibodies in patients with SLE and normal individuals. Our in vitro experimental results showed that after PMA+IO or anti-CD3 antibody stimulation ICOS expression on peripheral blood CD4 and CD8 T cells from patients with SLE at any time (except 48 hours) was significantly higher than that from normal individuals. ICOS had a similar costimulatory role to CD28 in enhancing the production of Thl and Th2 cytokines from SLE PBMC and control PBMC. When we simultanously blocked ICOS and CD28 costimulation with hICOS-mFc and hCTLA4-hFc soluble proteins, the proliferative responses of PBMC from SLE patients and healthy subjects were further reduced as compared to blockade of either ICOS or CD28 costimulation alone.But ICOS-mediated costimulation failed to enhance T-cell proliferation and IL-2 production from patients with SLE. Most importantly, we also demonstrated that the ICOS-ICOSL interaction markedly enhanced the production of total IgG and pathogenic anti-dsDNA antibodies by B cells from patients with SLE. Human ICOS-mFc fusion protein blocked anti-dsDNA antibody production in patients with SLE stimulated with PWM/ IL-10/ HIgG in a dose dependent manner. When we simultaneously blocked ICOS and CD28 costimulation with hICOS-mFc and hCTLA4-hFc soluble proteins, the production of anti-dsDNA antibodies from active SLE patients was further reduced as compared to blockade of either ICOS or CD28 costimulation alone, which indicated that ICOS-mediated costimulation could enhanced the production of T-cell-dependent autoantibodies through CD28-dependent and CD28-independent way. The study proved the immunopathological roles of ICOS-ICOSL interaction in T-cell and B-cell proliferation and activation, Thl and Th2 cytokines production, and pathogenic autoantibody production in patients with SLE, which may provide with a new strategy in the therapy of autoimmune diseases by blocking ICOS-mediated costimulation.Part III MAP-Kinase signaling cascades in ICOS-mediated costimulatory pathway in peripheral blood T cells from patients with SLETo our knowledge, it was the first time to study MAP-kinase signaling cascades in ICOS-costimulated T cells from patients with SLE and normal individuals. In this part we observed the tyrosine phosphorylation levels of three important MAP-kinase family members, ERK, JNK, and p38 MAPK, during T-cell activation by CD3 induced by costimulation with ICOS and attempted to clarify the role of ERK and p38 MAPK in T-cell proliferation. Our results showed that anti-ICOS antibody strongly enhanced CD3-mediated tyrosine phosphorylation of ERK, JNK, and p38 MAPK in peripheral blood CD4 T cells from patients with SLE and normal individuals to a similar or slightly lower degree as anti-CD28 antibody, but the levels of tyrosine phosphorylation of ERK, JNK, and p38 MAPK, especially ERK, in normal individuals were stronger than those in patients with SLE. Things are more complex...