In vitro apoptosis and expression of apoptosis‐related molecules in lymphocytes from patients with systemic lupus erythematosus and other autoimmune diseases

Abstract

Objective. To analyze factors related to apoptosis in systemic lupus erythematosus (SLE) peripheral blood mononuclear cells (PBMC) and to compare the findings in SLE PBMC with those in normal donor PBMC or PBMC from patients with other autoimmune diseases. Methods. PBMC from normal healthy donors or patients with SLE, mixed connective tissue disease (MCTD), rheumatoid arthritis (RA), or various vasculitides were isolated. The percentage of apoptosis after activation through different signaling pathways was quantified using propidium iodide staining. Protein expression of Fas/APO‐1 or bcl‐2, and messenger RNA (mRNA) expression of bcl‐2, bcl‐xL, bax, bak, Fas/APO‐1, Fas ligand (Fas‐L), c‐myc, mad, or max were determined. Results. We confirmed previous findings of increased numbers of apoptotic cells in SLE PBMC compared with normal donor cells after in vitro incubation. After activation of PBMC with CD28 monoclonal antibody plus phorbol myristate acetate (CD28 MAb/PMA), staphylococcal enterotoxin B (SEB), or phytohemagglutinin (PHA), the percentage of apoptotic cells was unchanged (SEB) or diminished (CD28 MAb/PMA, PHA) in SLE cells, and the difference between normal donor and SLE cells was less pronounced. On the mRNA level, expression of apoptosis‐related gene products did not differ between SLE cells and normal donor cells. Expression of Fas/APO‐1 protein was increased in freshly isolated SLE T lymphocytes compared with normal donor T lymphocytes, whereas bcl‐2 protein was up‐regulated after a 3‐day culture period. Cellular activation further increased bcl‐2 protein levels, eliminating differences between normal donors and SLE patients. In RA cells, the percentage of apoptosis was similar to that in normal donor PBMC, whereas results using cells from patients with other autoimmune diseases (MCTD, Wegener's granulomatosis, Takayasu arteritis, polyarteritis nodosa) were comparable with those found using SLE PBMC. Addition of growth factors such as interleukin‐2 (IL‐2), IL‐4, or IL‐15 to culture medium decreased the percentage of in vitro apoptosis in both normal donor and SLE cells. Conclusion. Based on these data, we conclude that accelerated in vitro apoptosis and increased Fas/APO‐1 and bcl‐2 protein expression in SLE are nonspecific for the disease, and might be explained at least in part by the increased in vivo activation levels of PBMC from patients with SLE, MCTD, or autoimmune vasculitides combined with in vitro incubation under “noninflammatory” conditions and growth factor withdrawal.

Differential Role for IL‐2 and IL‐15 in the Inhibition of Apoptosis in Short Term Activated Human Lymphocytes

“…Importantly, we have found nonselective up-regulation of both pro-apoptotic and anti-apoptotic gene products on mRNA or protein levels after PHA stimulation of PBMC for 6 days. The increase in the expression of those gene products seems to be a non-specific consequence of cellular activation, as we could observe up-regulation of fas/Apo-1, bcl-2 or other apoptosis related gene products after cellular activation independent of the quality of the stimulus [55]. Importantly, however, after stimulation of PHA blasts with IL-2 or IL-15 we could not find a selective up-regulation of anti-apoptotic gene products, yet there was significant and rapid inhibition of apoptosis.…”

Nucleosomes inhibit phagocytosis of apoptotic thymocytes by peritoneal macrophages from MRL+/+ lupus-prone mice

“…Our data show that there does not appear to be any intrinsic functional defect of macrophages from young MRLϩ/ϩ mice to recognize and phagocytose apoptotic thymocytes. It is interesting to note that we found that challenge of macrophages from young MRLϩ/ϩ mice with nucleosomes (as a mimic of in vivo increased cell apoptosis in lupus) [18][19][20][21][22] markedly reduced their ability to phagocytose apoptotic cells. The inhibitory effect elicited by nucleosomes was almost achieved with low concentrations of the autoantigen, and was not a consequence of any cytotoxic effect.…”

“…In these experiments, however, PBMC from SLE patients were cultured over 6 days before the phagocytic assay. In light of our results, it is conceivable that the observed depressed phagocytosis of apoptotic cells may result, at least in part, from an increased SLE peripheral blood mononuclear cell susceptibility to apoptosis [18,20,38], leading to the release of high levels of nucleosomes [18] and, consequently, to an inhibition in uptake of apoptotic cells.…”

“…Increased rates of spontaneous cell apoptosis in vitro have been detected both in human and mouse models of SLE [18][19][20]. Thus, these defects in apoptosis could account for the presence of the elevated concentrations of nucleosomes found in the circulation of lupus patients [21,22].…”

Programmed Cell Death in Rheumatoid Arthritis Peripheral Blood T-Cell Subpopulations Determined by Laser Scanning Cytometry

“…In human RA peripheral blood cells, the percentage of total apoptosis induced with CD28 mAb/PMA has previously been found similar to that in normal donor peripheral blood mononuclear cells (PBMCs); this phenomenon has been explained by activated T-cell migration to the peripheral joints from peripheral vessels (Emlen et al, 1994;Lorenz et al, 1997). Because PBMCs play an important role in the pathogenesis of various systemic autoimmune diseases, such as systemic lupus erythematosus (Bijl et al, 2001;Funauchi et al, 2001;Papo et al, 1998), Sjögren's syndrome (Zeher et al, 1999), and systemic sclerosis, (Stummvoll et al, 2000), we further investigated this phenomenon in RA.…”