| Backgroud and objectiveSystemic lupus erythematosus (SLE) remains the classic example of a systemic autoimmune disease. The chronic clinical manifestations of SLE remain a challenge to the most clinician today. SLE is usually a postpubertal disease, with onset of clinical symptoms usually in the 20s to 30s. One of the most striking features of SLE is its female predominance. This disease, given its capacity to encompass every organ system, also can lead to miscarriage, a dead embryo and neonatal lupus erythematosus. The classic pattern, one of exacerbations, or " flares" of disease activity, is now called the " relapsing remitting pattern". The second pattern, one of chronic (or continuous) activity, is equally common. The third pattern is more accurately called " long quiescence". Immunosuppressant including glucocorticoid is the main medical treatment. However, some patients ultimately die of infection, central nervous system damage or renal failure. The causative agents that initiate the disease, and the underlying mechanisms that result in damage, remain largely obscure. The major immunologic alterations characterize individuals with SLE. The diverse collection of autoantibodies that are found in SLE, raised conceptual problems about their origin and the nature of the autoimmunity state in SLE. The antibodies cause injury through their ability to bind to molecules that are or become accessible on the cell membrane, that are normally found in plasma, or that are released into blood or connective tissue from intracellular sites of injured cells. Autoantibodies also may mediate injury either by forming circulating complexes or by binding to released autoantigens that have first bound to components of connective tissue. The autoantibody production in SLE is driven by self antigens and involves both T-cell and B-cell recognition. Despite the numbers of circulating B cells in SLE are not increased, the state of B-cell differentiation is altered, with increased numbers of plasmacytoid B cells characterized by cytoplasmic immunoglobulin (Ig) and the secretion of IgG immunoglobulin, particularly in periods of heightened clinical activity. Autoantigen-specific T-cell help (CD4 positive T cell) involves B-cell activation. Owing to their central role in the humoral response against autoantigens,CD4+ cells are thought to be the primary T lymphocyte subpopulation involved in lupus autoimmune response (1,2). Although some reports of studies in humans suggested that cell cytotoxicity is impaired in SLE (3,4), studies in several rodent models indicate that CD8+ T lymphocytes may also contribute to this response, either directly, as a noxious element of the cellular response, or indirectly, by providing supplies for overcoming mechanisms of tolerance to autoantigens. Mice deprived of CD8+ T cells following deficiency in major histocompatibility complex class I antigen expression are resistant to experimental SLE (5). NZB mice deficient in β 2-microglobulin had a lower incidence and a delayed onset of antierythrocyte autoantibody production compared with that seen in normal NZB mice (6). More recently, NZB mice deficient in type I IFN receptor were shown to have a significant decrease in splenic CD8+ cells and a reduced lupus-like disease (7). Recently Blanco et al also revealed a quantitative and functional increase in CD8+ cytotoxic T lymphocytes that is highly correlated with SLE disease activity and that may be responsible for the increased production of autoantigens (8). T-cell-specific autoimmune recognition is at the center of the pathogenesis of SLE. Following the induction of autoreactive T cells in the induction phase of SLE, the immune system responds by progressively enlarging and strengthening the autoimmune response. The T-cell clone expanding, being driven by the available supply of autoantigen presented by B cells or other antigen-presenting cells, results in the disordered balance of T cell subsets. Active cytotoxic T lymphocytes (CTL) and nature killer (NK) cells mediate death of target cell via granule exocytosis, including perforin. Michael et al have identified at least three subsets of perforin positive T-cells. These are CD8 single positive cytotoxic T-cells, the numbers of perforin positive T-cells increased with the age of the animal and their populations increased after specific antigen stimulation in vitro. So perforin expression can define CD8 positive lymphocyte subsets (9).Ca2+-dependent pore-forming proteins isolated from these granules is named perforin, because it can polymerize into a channel which lead to osmotic target cell lysis in the membrane. The mechanism of cell death involves apoptosis and cytolysis. During physiologic conditions, cellular components degraded by apoptosis are rapidly cleared, without initiating an inflammatory or immune response. During phathologic conditions, cellular components degraded by cytolysis are cleared, with releasing fragments and initiating an inflammatory or immune response. This raises the possibility that dysregulation of the cellular machinery which mediates removal of fragments could be a mechanism whereby increased quantities of autoantigens could be made available for presentation to the immune system in the individual predisposed to developing SLE, perhaps by shunting to dendritic cells where it might serve to initiate or accelerate autoimmunity. That chronic inflammatory response continues in SLE individuals accounts for imperative cytolysis. PF initiates target cells to lysis, while increased apoptosis is observed. This suggests that PF may be involved in the pathogenesis of SLE. Several studies have presented that glucocorticoid can decrease the number of lymphocytes expressing perforin and the mean of perforin density in every lymphocyte in vitro. Blanco et al noted an 3-fold increase in the median percentages of perfori positive CD8+ T cells in the active-disease group compared with the quiescent-disease group. The intracellular expression of perforin in the quiescent-disease group was similar to that in the healthy controls (8). The mechanisms of cytotoxicity operating in murine CTL have been examined extensively using knockout mice, including perforin-deficient mice. The data obtained from the experiments using perforin-deficient mice suggest that the granule exocytosis pathway is dominant in murine CD8+ CTL-mediated cytotoxicity (10). These results identify the role of perforin in SLE pathophysiology. It appears that treatment that can down-regulate the presentation of perforin to a primed immune system may significantly contribute to treat this disease (11). Perforin represents a key target for SLE therapy. Many members of the tumor necrosis factor receptor (TNFR) superfamily can down-regulate the expression of perforin. Muta et al. showed CD30 signals down-regulated the expression of perforin in the large granular lymphoma line YT, quantitative analysis of normalized expression of perforin mRNA after adding 5 mg/ml of the agonistic anti-human CD30 monoclonal antibody (MoAb) revealed a 2-fold suppression from 100% to about 50% of the RNA level for gene products in microchip assays and by RNase protection assay (12). CD134 that belonged to type I transmembrane glycoprotein was a member of TNFR superfamily and its relative molecular mass was about 48,000. The human CD134 gene that was cloned in 1994 was a 1.4kb nucleotide sequence and coded a 277-amino acid sequence of the single transmembrane protein whose relative molecular mass was 27, 777, it could be mapped to chromosome band 1p36 and was, thus, linked to the genes for CD30, 4-1BB, TNFR II and DR3, they had similar function. There are no reports about that CD 134 had similar function with CD30 and inhibit perforin. To study this possibility mechanism, we investigated the expression and activity of perforin in peripheral blood lymphocytes of some patients with active SLE and addressed the mechanism of SLE pathegenesis. We also observed effect of anti-CD134 blocking MoAb on the expression and activity of perforin detected by reverse transcription-polymerase chain reaction (RT-PCR) technique, flow cytometry and cytolysis assay respectively and compared with that of Methylprednisolone (MP), in order that more information about anti-CD134 blocking MoAb used to for SLE therapy. Objects and methods1 ObjectsThirty consecutive SLE patients were included in the present study between February 2005 and February 2006. Patients met at least 4 of the American College of Rheumatology 1997 revised criteria for SLE (13). All clinically and biologically relevant information concerning the patients was documented for statistical analyses.Clinical disease activity was scored using the SLE Disease Activity Index (SLEDAI) (14,15). Two groups of patients were defined. The active-disease group included 15 patients with a flare of disease, defined as a score >9 for patients at diagnosis. The quiescent-disease group included 15 patients with a SLEDAI score (?)9 and with no variations throughout the entire followup period. Healthy individuals from our staff (12 women and 3 men) were studied as a control group. For patients who presented with a disease flare (n=15), the concomitant or the closest biologic variables measured before treatment were considered for statistical analyses. For patients with quiescent disease throughout the entire followup period (n=15), the last biologic variables were used for statistical analyses. All blood samples were obtained after the patients and control subjects had given their informed consent.2 MethodsPerforin mediated cytolysis against rabbit erythrocytes was detected. Perforin and T lymphocyte subsets were marked by monoclonal antibody with fluorescence and detected by flow cytometry.The expression of perform mRNA was measured by using RT-PCR.The intracellular perforin was observed by using immunohistochemistry.The level of NF-κB P65 protein was examined by using western blot.To explore optimal concentration and best time of effect of anti-CD134blocking MoAb, PBMCs were induced with 50μg/mL phytohemagglutinin,by using three different does, 1μg/mL, 5μg/mL or 10μg/mL, anti-CD 134blocking MoAb for four different time, 6 hours, 12hours, 24hours and 48hours. Perforin mediated cytolysis against rabbit erythrocytes was detected.The expression of perforin mRNA was measured by RT-PCR technique. Theexpression of perforin protein was measured by flow cytometry. Totalnumber of groups are 16.To compare with that of MP and analysis treatment of anti-CD134 blockingMoAb in SLE. Human peripheral blood mononuclear cells (PBMCs) wereinduced by using 5 μg/mL anti-CD134 blocking MoAb for 24 hours.Perforin mediated cytolysis against human erythrocytes was detected. Theexpression of perforin mRNA was measured by reversetranscription-polymerase chain reaction (RT-PCR) technique. Theexpression of perforin protein was measured by flow cytometry.ANOVA and Student-Newman-Keuls test were used for comparison of morethan two groups. P values less than 0.05 were considered significant. Weused the Spearman test to determine the correlation between the effect ofanti-CD134 blocking MoAb on perforin and the clinical indices. The testswere carried out using SPSS 13.0 for Windows (SPSS Inc., Michigan,USA).ResuIts1 The expression and activity of perforin in PBMCs of healthy individualsand SLE patients and active PBMCs of healthy individuals: the expressionand activity of cytolysis against human erythrocytes in the control groupwas significantly lower than that in the active-disease group and similar to that in the quiescent-disease group. Phytohemagglutinin (PHA) induced upregulation of the expression and activity of perforin in PBMCs of healthy individuals in a dose-dependent manner. When Concanamycin A (CMA), the specific perforin inhibitor, was added simultaneously, the effect on the activity of perforin mediated cytolysis against rabbit erythrocytes was no significant difference before and after their action in PHA group, it shows that activity of cytolysis against rabbit erythrocytes is mediated by perforin.2 PBMCs were induced with 50 μg/mL PHA, by using 1 μg/mL, 5 μg/mL or 10 μg/mL anti-CD134 blocking MoAb for 6 hours, 12 hours, 24 hours and 48 hours. The expression and activity of Perforin mediated cytolysis against human erythrocytes under anti-CD134 blocking MoAb were detected: Our data showed that the perforin mediated cytolysis in PBMCs was downregulated by various concentrations of anti-CD134 blocking MoAb for different times and reached minimal at 24 hours at any concentration. Anti-CD134 blocking MoAb induced downregulation of perforin mediated cytolysis of PBMC in a dose-dependent manner in the range of 1 μg/mL-5 μg/mL. Perforin mediated cytolysis of PBMCs reached a plateau when the concentration of anti-CD134 blocking MoAb exceeded 5 μg/mL. Anti-CD134 blocking MoAb also induced inhibit the expression of perforin protein and mRNA in PBMCs.3 The expression and activity of Perforin mediated cytolysis against rabbit erythrocytes under anti-CD134 blocking MoAb were detected at 24 hour after anti-CD134 blocking MoAb action: the expression and activity of perforin mediated cytolysis against rabbit erythrocytes in the active-disease group were reduced, but no significant change was observed in the quiescent-disease group or in the control group. When CMA, the specific perforin inhibitor, was added simultaneously, the effect on the activity of perforin mediated cytolysis against rabbit erythrocytes vanished and turned out to be no significant difference before and after their action in every group, it shows that activity of cytolysis against rabbit erythrocytes is mediated by perforin.4 The effect of anti-CD134 blocking MoAb on the peripheral blood lymphocytes: In the control group, the percentage of CD4+CD3+ cells in total PBMCs was higher than that of CD8+CD3+ cells, this ratio was similar to that in the quiescent-disease group. In the active-disease group, the percentage of CD4+CD3+ cells was lower, while that of CD8+CD3+ cells was significantly higher. At 24 hour after anti-CD134 blocking MoAb action, in the active-disease group the percentage of CD4+CD3+ cells didn't change significantly, but that of CD8+CD3+ cells was reduced. While in the control group or in the quiescent-disease group, their proportion didn't change significantly before and after antirCD134 blocking MoAb action.5 Effect of anti-CD134 blocking MoAb on the cell viability of PBMCs: Anti-CD134 blocking MoAb at the highest concentration used (10 μg/mL) did not affect the viability of PBMCs determined by trypan blue exclusion method.6 The effect of anti-CD134 blocking MoAb on the expression of NF-κB P65: NF-κB P65 expression in PBMCs was lower in the control group than that in the active-disease group and similar to that in the quiescent-disease group. At 24 hour after anti-CD134 blocking MoAb action, except for the active-disease group that showed a lower NF-κB P65 expression than before, the other two groups displayed no significant change. In every SLE groups, a positive correlation was observed between NF-κB P65 expression and perforin mRNA expression. When simultaneously adding NF-κB P65 specific inhibitor Pyrrolidine dithiocarbamate (PDTC), the effect of anti-CD134 blocking MoAb vanished, no significant difference was detected in the perforin mRNA expression before and after their action in every group.7 Effect of 5 μg/mL anti-CD134 blocking MoAb is compared with that of MP: the effect of anti-CD 134 blocking MoAb approached to that of MP except for the effect on the peripheral blood lymphocytes. At 24 hour after anti-CD134 blocking MoAb action, in the active-disease group the percentage of CD4+CD3+ cells didn't change significantly, but that of CD8+CD3+ cells was reduced. While in the control group or in the quiescent-disease group, their proportion didn't change significantly before and after anti-CD 134 blocking MoAb action. At 24 hour after MP action, both the percentages of CD4+CD3+ cells and CD8+CD3+cells were decreased in the active-disease group, and also showed a tendency to decrease in the other two groups.8 The correlation between the activity of perforin and antibody titer: In the active-disease group, a positive correlation was noted between the activity of perforin mediated cytolysis against rabbit erythrocytes and anti-double stranded deoxyribonucleic acid (ds-DNA) antibody titer (P=0.040, r=0.535), while in the quiescent-disease group there was no correlation between them. There was no correlation between the activity of perforin mediated cytolysis against rabbit erythrocytes and the other antibodies titer in every SLE group.9 The correlation between the effect of anti-CD134 blocking MoAb on perforin and the clinical indices in SLE patients: In the quiescent-disease group, the inhibition rate of perforin mRNA expression caused by anti-CD134 blocking MoAb showed no correlation with the SLEDAI score and the clinical indices. However, we can see that the inhibition rate of perforin mRNA expression caused by anti-CD134 blocking MoAb in the active-disease group displayed a positive correlation with the SLEDAI score (P<0.01, r=0.755) and a strong positive correlation with quantity of 24hours' urinary protein (P<0.01, r=0.853).Conclusions and significances1 The expression and activity of perforin in PBMCs of healthy individualsand SLE patients and active PBMCs of healthy individuals: the expression and activity of cytolysis against rabbit erythrocytes in the control group was significantly lower than that in the active-disease group and similar to that in the quiescent-disease group. PHA induced upregulation of the expression and activity of perforin in PBMCs of healthy individuals in a dose-dependent manner. When CMA, the specific perforin inhibitor, was added simultaneously, the effect on the activity of perforin mediated cytolysis against rabbit erythrocytes was no significant difference before and after their action in PHA group, it shows that activity of cytolysis against rabbit erythrocytes is mediated by perforin.2 The effect of 5 μg/mL anti-CD 134 blocking MoAb on expression and activity in healthy human PBMCs induced by PHA is optimal at 24 hours, while no effect in healthy human PBMCs without inducing.3 The effect of 5 μg/mL anti-CD134 blocking MoAb on expression and activity in the active-disease group is optimal at 24 hours, while no effect in the quiescent-disease group or the control group. When CMA, the specific perforin inhibitor, was added simultaneously, the effect on the activity of cytolysis againstr rabbit erythrocytes vanished and turned out to be no significant difference before and after their action in every group. It shows that activity of cytolysis against rabbit erythrocytes is mediated by perforin.4 In the control group, the percentage of CD4+CD3+ cells in total PBMCs was higher than that of CD8+CD3+ cells, this ratio was similar to that in the quiescent-disease group. In the active-disease group, the percentage of CD4+CD3+ cells was lower, while that of CD8+CD3+ cells was significantly higher. At 24 hour after anti-CD134 blocking MoAb action, in the active-disease group the percentage of CD4+CD3+ cells didn't change significantly, but that of CD8+CD3+ cells was reduced. While in the control group or in the quiescent-disease group, their proportion didn't change significantly before and after anti-CD134 blocking MoAb action. 5 Anti-CD134 blocking MoAb at the highest concentration used (10 μg/mL) did not affect the viability of PBMCs determined by trypan blue exclusion method, indicating that anti-CD134 blocking MoAb decreases in the expression of perforin were not due to the PBMCs death.6 NF-κB P65 expression in PBMCs was lower in the control group than that in the active-disease group and similar to that in the quiescent-disease group. At 24 hour after anti-CD134 blocking MoAb action, except for the active-disease group that showed a lower NF-κB P65 expression than before, the other two groups displayed no significant change. In every SLE group, a positive correlation was observed between NF-κB P65 expression and perforin mRNA expression. When simultaneously adding NF-κB P65 specific inhibitor PDTC, the effect of anti-CD134 blocking MoAb vanished, no significant difference was detected in the perforin mRNA expression before and after their action in every group. It shows that anti-CD134 blocking MoAb exerts its positive regulatory effect on the perforin expression through NF-κB signaling pathway.7 Effect of 5 μg/mL anti-CD 134 blocking MoAb is compared with that of MP: the effect of anti-CD 134 blocking MoAb approached to that of MP except for the effect on the peripheral blood lymphocytes. Anti-CD 134 blocking MoAb, unlike MP, may not lead to serious immune suppression during the treatment.8 In the active-disease group, a positive correlation was noted between the activity of perforin mediated cytolysis against rabbit erythrocytes and ds-DNA antibody titer (P=0.040, r=0.535), while in the quiescent-disease group there was no correlation between them. There was no correlation between the activity of perforin mediated cytolysis against rabbit erythrocytes and the other antibodies titer in every SLE group. It indicates that the enhancement of the activity of perforin mediated cytolysis against rabbit erythrocytes may result in the increase of antibody production.9 In the quiescent-disease group, the inhibition rate of perforin mRNA expression caused by anti-CD134 blocking MoAb showed no correlation with the SLEDAI score and the clinical indices. However, we can see that the inhibition rate of perforin mRNA expression caused by anti-CD134 blocking MoAb in the active-disease group displayed a positive correlation with the SLEDAI score (P<0.01, r=0.755) and a strong positive correlation with quantity of 24hours' urinary protein (P<0.01, r=0.853). This may be associated with that CD134 is highly expressed in lupus nephritis patients with active disease but non-expressed or hypo-expressed in SLE patients with quiescent and in normal individuals.
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