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 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.


CONCLUSIONS
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.
Lorenz
H M
HM
Institute for Clinical Immunology and Rheumatology, University of Erlangen-Nuremberg, Germany.
Grünke
M
M
Hieronymus
T
T
Herrmann
M
M
Kühnel
A
A
Manger
B
B
Kalden
J R
JR
D017209:Apoptosis / Q000235:genetics; D001327:Autoimmune Diseases / Q000097:blood* / Q000209:etiology; D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; D050976:Basic-Leucine Zipper Transcription Factors; D004268:DNA-Binding Proteins / Q000235:genetics; D053222:Fas Ligand Protein; D014890:Granulomatosis with Polyangiitis / Q000097:blood; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000166:cytology; D008180:Lupus Erythematosus, Systemic / Q000097:blood* / Q000209:etiology; D008214:Lymphocytes / Q000502:physiology*; D008562:Membrane Glycoproteins / Q000235:genetics; D008565:Membrane Proteins / Q000235:genetics; D008947:Mixed Connective Tissue Disease / Q000097:blood; D010488:Polyarteritis Nodosa / Q000097:blood; D011506:Proteins / Q000235:genetics; D011518:Proto-Oncogene Proteins / Q000235:genetics; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics; D016271:Proto-Oncogene Proteins c-myc / Q000235:genetics; D012333:RNA, Messenger / Q000378:metabolism; D012097:Repressor Proteins; D013625:Takayasu Arteritis / Q000097:blood; D014157:Transcription Factors; D050998:bcl-2 Homologous Antagonist-Killer Protein; D051028:bcl-2-Associated X Protein
C494772:BAK1 protein, human; C494826:BAX protein, human; D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; D050976:Basic-Leucine Zipper Transcription Factors; D004268:DNA-Binding Proteins; C505661:FASLG protein, human; D053222:Fas Ligand Protein; C499330:MAX protein, human; C079439:MXD1 protein, human; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; C067850:Myc associated factor X; D011506:Proteins; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D016271:Proto-Oncogene Proteins c-myc; D012333:RNA, Messenger; D012097:Repressor Proteins; D014157:Transcription Factors; D050998:bcl-2 Homologous Antagonist-Killer Protein; D051028:bcl-2-Associated X Protein