Time between onset of apoptosis and release of nucleosomes from apoptotic cells: putative implications for systemic lupus erythematosus.

OBJECTIVE: To investigate the kinetics of nucleosome leakage from apoptotic cells in an in vitro system and extrapolate the results to autoimmune disease, in particular systemic lupus erythematosus. METHODS: A sensitive nucleosome enzyme linked immunosorbent assay (ELISA) was developed, using a monoclonal antibody (mAb) against histone 3 and an mAb against nucleosomes. Nucleosome release during apoptotic cell death was studied in Jurkat cells. AnnexinV binding (early apoptosis) and propidium iodide positivity (late apoptosis) of the cells were compared with nucleosome release at different times after apoptosis induction. RESULTS: Nucleosomes appeared in culture supernatant of Jurkat cells 24 to 48 hours after apoptosis induction, when the cells had been late apoptotic for more than 12 hours. CONCLUSION: Nucleosomes are released from late apoptotic Jurkat cells, with a 12 hour delay from the appearance of AnnexinV binding cells. This result suggests that in vivo scavenger mechanisms have 12 hours to remove apoptotic material from the circulation.

Soluble CD95 concentrations are increased in patients with severe systemic lupus erythematosus, but not in their first degree relatives.

OBJECTIVE: Plasma concentrations of soluble CD95 (sCD95) are raised in patients with systemic lupus erythematosus (SLE) before clinical relapses become manifest. Increased sCD95 concentrations may therefore be a familial characteristic that is associated with susceptibility to severe disease. To test this, sCD95 concentrations were measured in healthy first degree relatives of patients with severe and non-severe SLE. METHODS: Seventy seven first degree relatives of 26 patients with severe, and 72 relatives of 25 patients with non-severe lupus were studied. Controls were 42 first degree relatives of 17 patients with chronic cutaneous lupus erythematosus (CCLE) and 63 partners of the patients with their first degree relatives. Severe lupus was defined as both multi-organ disease and cyclophosphamide treatment, non-severe lupus as neither. Organ damage was assessed with the SLICC-ACR index, disease activity with SLEDAI. RESULTS: Soluble CD95 concentrations in relatives of patients with severe SLE were similar to those in relatives of patients with non-severe SLE, relatives of patients with CCLE, and controls (median (interquartile range) sCD95 concentration 0.59 (0.52-0.66) v 0.57 (0.50-0.63), 0.56 (0.51-0.71), and 0.55 (0.49-0.61) ng/ml, p=0.25, p=0.94, and p=0.17, respectively). Increased concentrations of sCD95, however, were found in patients with severe SLE compared with those in patients with non-severe SLE, patients with CCLE, and control relatives (0.77 (0.70-0.97) v 0.60 (0.54-0.67), 0.57 (0.54-0.71), and 0.57 (0.52-0.63) ng/ml, respectively, p<0.001). Concentrations of sCD95 were significantly correlated with damage index scores (rs=0.47, p<0.01). Basic and clinical characteristics of patients with SLE, including SLEDAI scores, could not explain these observations. CONCLUSION: Soluble CD95 concentrations are associated with severity of the disease and not with susceptibility for severe SLE.

Drug-induced apoptosis in lung cnacer cells is not mediated by the Fas/FasL (CD95/APO1) signaling pathway.

Anticancer drugs exert at least part of their cytotoxic effect by triggering apoptosis. We previously identified chemotherapy-induced apoptosis in lung cancer cells and suggested a role for p53 alternative or complementary pathways in this process. Recently, a role for the Fas/FasL (CD95/Apo1) signaling system in chemotherapy-induced apoptosis was proposed in some cell types. In the present work, the involvement of the Fas/FasL system in drug-induced apoptosis in lung cancer cells was investigated upon exposure to four cytotoxic drugs (cisplatin, gemcitabine, topotecan, and paclitaxel). We assessed the expression of Fas and FasL and the function of the Fas pathway in six lung cancer cell lines (H460, H322, GLC4, GLC4/ADR, H187, and N417). All lung cancer cell lines expressed Fas and FasL at RNA and protein levels, and apoptosis could be induced in four of six cell lines upon exposure to the Fas agonistic monoclonal antibody (mAb) CLB-CD95/15. Nevertheless, after drug exposure, no significant FasL up-regulation was observed, whereas the Fas expression was increased in the wild-type p53 cell line H460, but not in the other lines, proved to be mutant p53 by direct gene sequencing. Moreover, no correlation was observed in lung cancer cell lines between sensitivity to drugs and to a Fas agonistic mAb, and preincubation of cells with either the Fas-antagonistic mAb CLB-CD95/2 or a FasL-neutralizing mAb did not protect from drug-induced apoptosis. Taken together, these observations strongly argue against a role of the Fas/FasL signaling pathway in drug-induced apoptosis in lung cancer cells. Interestingly, caspase-8 activation was observed upon drug exposure, independently from Fas/FasL signaling.

Interferon (IFN)-beta treatment enhances CD95 and interleukin 10 expression but reduces interferon-gamma producing T cells in MS patients.

Interferon (IFN)-beta has been shown to favorably alter the disease course of relapsing-remitting multiple sclerosis (RRMS) patients. Although its mode of action is still unclear, there is ample evidence from in vitro studies that IFN-beta directly modulates the function of immune cells. We analyzed here the effects of IFN-beta treatment on immune functions in vivo in a group of 25 RRMS patients who received IFN-beta (8 MIU) on alternate days. At baseline and at 1, 3 and 6 months from the start of the treatment, parameters for differentiation and activation states of both monocytes and T lymphocytes were assessed. A transient increase was seen in plasma (p) interleukin (IL)-10 level whereas pIL-12 (p40) was not affected. A similar change was found in the ability of monocytes to secrete these cytokines in vitro. Notably, patients who in vitro readily responded to IFN-beta with enhanced IL-10 production had the highest pIL-10 levels. Concerning T-cell differentiation, flow cytometric analysis of cytokine production showed that treatment with IFN-beta moderately decreased the mean percentages of CD8pos T cells producing IL-2 and IFN-gamma and CD8neg T cells producing IL-4 (p<0.05 for all cytokines), whereas a more significant decline was seen in the mean percentage of CD8neg T cells producing IFN-gamma (p<0.01). This resulted in a significant lower ratio T(HELPER(H))1 vs. T(HELPER(H))2 type cells in the CD8pos T-cell subset (p<0.05), but not in the CD8neg T-cell subset. Finally, IFN-beta treatment resulted in an initial rise in the mean percentage of CD95pos T cells and in a gradual increase in the mean level of soluble CD95 (sCD95) in plasma (p<0.01). Additional in vitro studies showed that IFN-beta indeed rapidly (within 24 h) upregulates CD95 expression on both primed and unprimed T cells and augments the release of sCD95 in culture supernatants. Thus, we confirm here that IFN-beta treatment leads to similar changes in cytokine production of T cells and monocytes as previously described in vitro. Enhanced IL-10 secretion may downmodulate cytokine secretion by activated T cells and in this way dampen newly-induced and/or ongoing immune responses. In addition, we identified a novel effect of IFN-beta treatment, i.e., induction of CD95 expression. The augmentation of CD95 expression may directly interfere with T-cell selection, notably of autoaggressive T cells. Future studies are needed to show whether this increased CD95 expression indeed leads to increased apoptosis of immune cells.

Patients with systemic lupus erythematosus with high plasma levels of sFas risk relapse.

OBJECTIVE: We related soluble Fas (sFas) levels to the Systemic Lupus Erythematosus Disease Activity Index (SLEDAI) in a longitudinal series of plasma samples of patients with SLE to evaluate the relation between excessive production of sFas and disease activity. METHODS: We generated 21 monoclonal antibodies against Fas. Two of these were used to develop and validate a sensitive sandwich ELISA for the longitudinal analysis of sFas levels in plasma of 30 patients and 25 controls. RESULTS: At the start of followup, a significant elevation (p<0.0001) was found in sFas levels in SLE (1167+/-347 pg/ml sFas) compared to controls (618+/-98 pg/ml sFas). Also, at the start of the followup a significant difference (p = 0.0028) existed between patients who were going to have a relapse (1236+/-402 pg/ml sFas) during followup and patients who were not (809+/-276 pg/ml sFas). While sFas did not fluctuate with disease activity in individual patients, we found a strong correlation (r = 0.75, p<0.0001) between sFas and SLEDAI, but only at the time of relapse, when we analyzed the patients as a group. CONCLUSION: In individual patients with SLE, sFas does not fluctuate with disease activity. However, patients with high plasma levels of sFas are at risk of relapse.

Do elevated levels of serum-soluble fas contribute to the persistence of activated lymphocytes in systemic lupus erythematosus?

Systemic lupus erythematosus (SLE) is characterized by generalized immune activation. Part of this might be explained by a decreased rate of apoptosis, possibly related to elevated levels of soluble Fas (sFas) which can inhibit Fas mediated apoptosis of lymphocytes. In order to substantiate the relation between levels of sFas and lymphocyte activation in SLE we monitored sFas levels, lymphocyte activation and disease activity in 25 SLE patients. SLEDAI scores were registered and sera were assayed for sFas levels by an enzyme-linked immunosorbent assay. Flow cytometry was used to monitor the state of activation of lymphocyte subsets. Eighteen healthy, age-matched volunteers served as controls. Soluble Fas levels were elevated in SLE patients (n=25) compared to healthy controls (n=18, P=0.002). Soluble Fas levels correlated with SLEDAI scores (r=0.45, P=0.02). Levels of sFas correlated with the percentages of activated B cells defined as CD20(+)CD38(+) cells (r=0.47, P=0.009). Percentages of CD20(+)CD38(+) cells were increased in quiescent SLE compared to healthy controls (P=0.003). The expression of activation markers on CD4(+) T lymphocytes (IL-2R, P=0.04; HLA-DR, P=0.01) and CD8(+) T lymphocytes (HLA-DR, P=0.007) was also increased in quiescent SLE compared to controls. Activation markers on all lymphocyte subsets tended to increase further during disease activity. No correlation was observed between percentages of activated T lymphocyte subsets and levels of sFas. In conclusion, soluble Fas levels are increased in SLE patients and correlate with disease activity as measured by the SLEDAI score and B and T cell subsets are activated even during quiescent SLE. Serum levels of sFas correlate with percentages of activated B cells but not with that of activated T cells.

Soluble Fas levels in sera of bone marrow transplantation recipients are increased during acute graft-versus-host disease but not during infections.

Graft-versus-host disease (GVHD) and infections are two major complications of allogeneic bone marrow transplantation (BMT). In the course of GVHD, one of the pathways that activated cytotoxic T cells use to execute their killing mechanisms is the Fas/Fas ligand pathway. This killing mechanism might be accompanied by the release of soluble Fas (sFas) in the circulation. To examine the association of serum sFas levels and post-BMT complications, we have analyzed sFas levels in sera of bone marrow recipients with and without GVHD. Postallogeneic BMT sFas levels were significantly increased during clinically relevant acute GVHD (aGVHD; P = .002). However, during infections sFas levels tended to decrease (P = .088). Yet, the simultaneous occurrence of GVHD and infections resulted in extreme high sFas levels. These results suggested that sFas release may be correlated with the amount of tissue damage, because aGVHD induces more damage than infections. The presence of significantly increased sFas levels during aGVHD provides new insights into the GVHD pathogenesis.

Fish immunology: the utility of immortalized lymphoid cells--a mini review.

Long term cell lines can be readily established at high frequency with PBLs from normal channel catfish. Depending upon the mode of stimulation, morphologically and functionally distinct catfish lymphoid cell lines resembling B cells, T cells and monocytes have been developed. These fish cell lines appear unique from their putative mammalian counterparts in that they are immortalized without the need for exogenous factors or overt attempts at transformation.