Birgit Kovacs

B cells from patients with systemic lupus erythematosus display abnormal antigen receptor-mediated early signal transduction events.

To understand the molecular mechanisms that are responsible for the B cell overactivity that is observed in patients with SLE, we have conducted experiments in which the surface immunoglobulin (sIg)-mediated early cell signaling events were studied. The anti-sIgM-mediated free intracytoplasmic calcium ([Ca2+]i) responses were significantly higher in SLE B cells compared with responses of normal individuals and to those of patients with other systemic autoimmune rheumatic diseases. The anti-IgD mAb induced [Ca2+]i responses were also higher in lupus B cells than in controls. The magnitude of anti-sIgM-mediated Ca2+ release from intracellular stores was also increased in B cells from SLE patients compared with normal controls. The amount of inositol phosphate metabolites produced upon crosslinking of sIgM was slightly higher in patients with lupus than in normal controls, although the difference was not statistically significant. In contrast, the degree of anti-sIgM-induced protein tyrosine phosphorylation was obviously increased in lupus patients. Our study demonstrates clearly for the first time that SLE B cells exhibit aberrant early signal transduction events, including augmented calcium responses after crosslinking of the B cell receptor and increased antigen-receptor-mediated phosphorylation of protein tyrosine residues. Because the above abnormalities did not correlate with disease activity or treatment status, we propose that they may have pathogenic significance.

Increased expression of functional Fas-ligand in activated T cells from patients with systemic lupus erythematosus.

The Fas ligand induces apoptosis upon binding to Fas/APO-1 (CD95) bearing target cells. Activation induced cell death (AICD) in T cells is mediated by upregulation of Fas ligand on the cell surface membrane upon crosslinking of the TCR. AICD is considered to be essential for the elimination of autoreactive T cells in the peripheral blood. To elucidate possible abnormalities in the process of AICD in human SLE, we studied the expression and function of Fas ligand in polyclonal T cell lines from patients with SLE, patients with other rheumatic diseases and normal controls. SLE T cells expressed on their surface significantly higher amounts of Fas ligand compared to the two control groups. Stimulation of the cells with anti-CD3 mAb lead to further increase in surface membrane Fas ligand expression in all three groups with SLE expressing the highest amounts. The percentage of increase was though lower in SLE T cells than in normal T cells or disease control cells. The T cells were examined for Fas ligand-mediated cytotoxicity in a 51Cr release assay using Fas-expressing normal T cells as target cells. There was no difference in SLE and control T cells with regard to specific 51Cr lysis, indicating that the Fas ligand expressed by the SLE T cells is functional. Our data show that activated T cells from patients with SLE express high amounts of functional Fas ligand with intact TCR-mediated upregulation. This could account for the high apoptotic rates that have been observed in lymphocytes from patients with SLE.

LYMPHOCYTES, CYTOKINES, INFLAMMATION, AND IMMUNE TRAFFICKING

Studies that were published over the past year have added new knowledge to our understanding of cellular abnormalities in systemic lupus erythematosus (SLE). Antigen-specific and “pathogenic” T cells can be identified and characterized in SLE. Interleukin-10 has been added to the factors that may promote B cell overactivity and autoantibody production. Protein kinase isozyme I was shown to be deficient in patients with SLE, indicating defects in cell signaling events. Aberrant expression of adhesion molecules on the surface membrane of leukocytes and endothelial cells was shown, with important mechanistic and therapeutic implications. Disruption of the lymphokine network (anti-interleukin-10 antibody) and the function of adhesion-costimulatory molecules (CTLA-4-immunoglobulin) were shown to be therapeutically significant in murine SLE.

C1 inhibitor prevents capillary leakage after thermal trauma.

ObjectiveIn burned patients, activation of the complement and clotting systems is suggested to play an important role in the development of the capillary leak syndrome and inflammatory tissue destruction. In an animal model of thermal trauma, the possible protective effect of C1 inhibitor (C1Inh), a major control protein of both the complement and clotting systems, was investigated. DesignProspective, controlled study. SettingAnimal model. SubjectsHealthy pigs weighing 30 kg. InterventionsPigs were scalded for 25 secs with 75°C hot water to achieve a 30% total body surface deep partial-thickness burn. The treatment group (n = 8) received C1Inh concentrate at an initial dose of 100 units/kg body weight immediately after thermal trauma, followed by three further applications every 12 hrs. Two control groups included animals that were either scalded (n = 8) or not scalded (n = 7) and treated with lactated Ringer’s solution. MeasurementsBefore and at various time points after trauma blood samples were analyzed for complement activation (APH50, CH50, SC5b-9, C3). Continuous monitoring of hemodynamic variables was performed and postmortem histologic examination of specimens from lung, heart, liver, kidney, stomach, duodenum, jejunum, ileum, and colon was carried out. Aseptically collected mesenteric lymph nodes were pooled and screened for bacterial translocation. For evaluation of the burn wound, biopsies from defined scalded and not scalded areas were taken daily. As a measure for edema formation, the weight of the animals was recorded every 2 hrs. ResultsAfter C1Inh treatment, which led to a significantly reduced complement activation, the clinical outcome was clearly improved, as indicated by vital signs and as demonstrated by reduced edema formation. Treated animals presented a diminished bacterial translocation. Pathologic alterations were clearly diminished in the burned skin, in shock-related organs, and in the intestines. ConclusionApplication of C1Inh appears to be an effective means to prevent capillary leakage and inflammatory tissue destruction after thermal trauma.

Crosslinking of Fas/CD95 suppresses the CD3-mediated signaling events in Jurkat T cells by inhibiting the association of the T-cell receptor ζ chain with src-protein tyrosine kinases and ZAP70

Crosslinking of Fas (APO-1/CD95) on the surface of T cells initiates a biochemical cascade leading to programmed cell death. We have previously shown that crosslinking of Fas with an apoptosis-inducing IgM anti-Fas mAb results in suppression of the CD3-initiated cell signaling including Ca2+ mobilization and protein tyrosine phosphorylation. We conducted experiments to decipher the mechanisms whereby the cross talk between the Fas- and CD3 signaling pathways occur. We used lysates from Jurkat T and examined the composition of the TCR ζ chain-precipitated immune complexes using immunoblots. While crosslinking of Fas affected the association of p59fyn and p56lck tyrosine kinases with the TCR ζ chain to a limited degree, it dramatically inhibited the association of the protein tyrosine kinase ZAP70 with the ζ chain. In cells that were preincubated with an apoptosis-inducing anti-Fas mAb, the binding of the protein tyrosine phosphatases SHP-1 to the TCR ζ chain was increased. These experiments indicate that crosslinking of Fas interferes with early T cell signaling events by promoting the recruitment of SHP-1 and decreasing the association of protein tyrosine kinases with TCR ζ chain. Therefore, crosslinking of Fas antigen may regulate the antigen-induced T cell response and play an active role in the T cell anergy.