IN-VITRO APOPTOSIS AND EXPRESSION OF APOPTOSIS-RELATED MOLECULES IN LYMPHOCYTES FROM PATIENTS WITH SYSTEMIC LUPUS-ERYTHEMATOSUS AND OTHER AUTOIMMUNE-DISEASES

Objective. To analyze factors related to apoptosis in systemic lupus e rythematosus (SLE) peripheral blood mononuclear cells (PBMC) and to co mpare the findings in SLE PBMC with those in normal donor PBMC or PBMC from patients with other autoimmune diseases. Methods. PBMC from norm al healthy donors or patients with SLE, mixed connective tissue diseas e (MCTD), rheumatoid arthritis (RA), or various vasculitides were isol ated. The percentage of apoptosis after activation through different s ignaling pathways was quantified using propidium iodide staining. Prot ein expression of Fas/APO-1 or bcl-2, and messenger RNA (mRNA) express ion of bcl-2, bcl-x(L), bar, 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 nor mal donor cells after in vitro incubation. After activation of PBMC wi th CD28 monoclonal antibody plus phorbol myristate acetate (CD28 MAW P MA), staphylococcal enterotoxin B (SEE), or phytohemagglutinin (PHA), the percentage of apoptotic cells was unchanged (SEE) or diminished (C D28 MAb/PMA, PHA) in SLE cells, and the difference between normal dono r and SLE cells was less pronounced. On the mRNA level, expression of apoptosis-related gene products did not differ between SLE cells and n ormal donor cells. Expression of Fas/APO-1 protein was increased in fr eshly isolated SLE T lymphocytes compared with normal donor T lymphocy tes, whereas bcl-2 protein was up-regulated after a 3-day culture peri od. Cellular activation further increased bcl-2 protein levels, elimin ating differences between normal donors and SLE patients. In Ri cells, the percentage of apoptosis was similar to that in normal donor PBMC, whereas results using cells from patients with other autoimmune disea ses (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 prot ein expression in SLE are nonspecific for the disease, and might be ex plained at least in part by the increased in vivo activation levels of PBMC from patients,vith SLE, MCTD, or autoimmune vasculitides combine d with in vitro incubation under ''noninflammatory'' conditions and gr owth factor withdrawal.