Regulation of NF-kappa B nuclear activity in peripheral blood mononuclear cells: role of CD28 antigen.
The NF-kappa B/rel family of transcription factors regulates the expression of a number of genes, including interleukin 2 (IL-2), IL-2 receptor alpha chain (Tac), and others, controlling T lymphocyte activation. The CD28 antigen is involved in regulation of T cell activation. To investigate whether CD28 antigen regulates NF-kappa B factors, we analyzed the effect of an anti-CD28 monoclonal antibody (mAb), CLB-CD28/1, on the nuclear activity of NF-kappa B complexes in resting and CD3-activated peripheral blood mononuclear cells (PBMC) of 11 donors. Cells were incubated with or without the anti-CD3 mAb OKT3 and/or the mAb CLB- CD28/1. Then nuclear extracts were obtained and analyzed for their binding to a 32P-labeled oligonucleotide, corresponding to the NF-kappa B 5'-CAACGGCAGGGGAATCTCCCTCTCCTT-3' consensus sequence in electrophoretic mobility shift assays. PBMC incubated with control medium did not appear to contain significant levels of NF-kappa B nuclear activity. The anti-CD28 mAb did not induce any detectable NF-kappa B nuclear activity in PBMC when used alone, except for two cases. However, cells incubated with the anti-CD3 mAb displayed NF-kappa B nuclear activity in 7 of the 11 cases. The addition of anti-CD28 to the anti-CD3 mAb-stimulated cells enhanced the levels of NF-kappa B activity in eight PBMC, while it did not modify PBMC in one sample and partially inhibited the induction of NF-kappa B in the remaining two samples. The stimulatory effect of anti-CD28 mAb on NF-kappa B nuclear activity was detected also on CD3-stimulated purified T lymphocytes. By analysis with antisera recognizing the p50 and p65 components of the NF-kappa B/rel family, NF-kappa B complexes of CD3+CD28-stimulated PBMC were found to contain both p50 and p65 proteins. An enhanced production of IL-2 was detected in cultures of CD3+CD28-stimulated PBMC. Our results indicate that CD28 triggering can modulate the activity on NF-kappa B nuclear complexes in T lymphocytes stimulated via CD3. Such an effect appeared not to require the presence of accessory cells (AC) or AC-derived cytokines.
Dipartimento di Biologia e Patologia Molecolare e Cellulare, University Federico II, Naples, Italy.
M F
MF
Romano
R
R
Bisogni
A
A
Lamberti
M C
MC
Turco
A
A
Petrella
P
P
Tassone
R
R
Van Lier
S
S
Formisano
S
S
Venuta
D000911:Antibodies, Monoclonal / Q000494:pharmacology; D001483:Base Sequence; D018106:CD28 Antigens / Q000097:blood*; D017252:CD3 Complex / Q000097:blood; D016384:Consensus Sequence; D015342:DNA Probes / Q000235:genetics; D015870:Gene Expression; D006801:Humans; D007376:Interleukin-2 / Q000096:biosynthesis; D007963:Leukocytes, Mononuclear / Q000276:immunology* / Q000378:metabolism*; D008969:Molecular Sequence Data; D016328:NF-kappa B / Q000097:blood* / Q000235:genetics
D000911:Antibodies, Monoclonal; D018106:CD28 Antigens; D017252:CD3 Complex; D015342:DNA Probes; D007376:Interleukin-2; D016328:NF-kappa B