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In the present study we have analyzed superinduction of TNF-alpha mRNA and enhancement of TNF-alpha gene transcription by cycloheximide (Chx) in human blood monocytes isolated by continuous Percoll gradient and activated in vitro. In the same monocyte cultures, we have compared the rate of gene transcription of TNF-alpha, IL-1 beta, IL-8, and the P53-antioncogene under the influence of plastic adherence, Staphylococcus aureus Cowan 1 (SAC), and Chx added at different times of monocyte culture. It was shown that the cytokine genes have low or negligible transcriptional activity in freshly isolated monocytes, whereas P53 gene transcription was constant in freshly isolated and in vitro-stimulated cells. Transcription of the IL-1 beta and IL-8 genes was induced by adherence and was not more enhanced by SAC. Transcription of the TNF-alpha gene was not induced by adherence. Chx added at the beginning of the monocyte culture did not block TNF-alpha or IL-1 beta gene transcription. IL-8 gene transcription, however, was abrogated by Chx. Addition of SAC to monocyte culture containing Chx caused significant enhancement of TNF-alpha gene transcription. Addition of Chx after 2.5 or 4 h of SAC activation caused "superinduction" of TNF-alpha mRNA and enhancement of TNF-alpha gene transcription. The data imply that TNF-alpha gene transcription in activated human monocytes might be regulated by both positive and negative regulatory factors that differ in their stability and protein synthesis dependence. In addition, results demonstrate that TNF-alpha, IL-1 beta, IL-8, and p53 genes in human monocytes are differently regulated.

The p53 gene has been associated with malignant transformation as well as "anti-oncogene" activity. In the present report expression of p53 in resting and activated human blood monocytes and lymphocytes is analyzed. It is found that human monocytes freshly isolated by continuous percoll gradient centrifugation contained detectable level of p53 mRNA. Stimulation of monocytes by potent activation inducer Staphylococcus Aureus Cowan I for 3-5 hr caused disappearance of r53 mRNA. In contrast, induction of high level of TNF-alpha mRNA was detected. Addition of cycloheximide had no effect on p53 mRNA content in stimulated monocytes, and caused disappearance of mRNA in resting cells. In lymphocytes cultures p53 mRNA was absent in freshly isolated cells and in resting lymphocytes cultured for 20 hr. Activation of lymphocytes by lectin caused accumulation of p53 mRNA. We suggest that r53 gene regulation and functions might be different in human monocytes and lymphocytes.

Membrane lymphotoxin (LT) complex is a trimer composed of two subunits , LT-alpha and LT-beta of which the latter is a 33-kDa transmembrane protein. The LT-beta gene is expressed in lymphoid cells and organs, but little is known about its inducible regulation. Previously, the surface expression of LT-beta in Jurkat cells has been shown to increase in response to PMA. In this report, we used this model to study the transcriptional control of the human and murine LT-beta genes. PMA strongly induced the expression of LT-beta mRNA, and the level of induction was not changed markedly by cycloheximide (CHX) treatment. The LT-beta promoter region contains conserved Egr-1, nuclear factor (NF)-kappaB, and Ets binding sites, and PMA-inducible factors bound to these sites were detected by the gel-retardation technique (electrophoretic mobility shift assay (EMSA)). To identify sequences involved in transcriptional control, sets of human and mouse promoter-chloramphenicol acetyltransferase (CAT) constructs were generated and assayed by transient transfections. The PMA response was lost after deletion of the distal Ets binding site at -110. Mutations at either the Ets or NF-kappaB sites that prevented factor binding dramatically reduced PMA-inducible promoter activity, suggesting cooperative interaction between corresponding transcription factors in PMA activation. Mutation at the Egr-1 site also resulted in substantial loss of promoter activity, and the residual activity may be attributed to binding of constitutively expressed Sp-1 to the same site. We propose that the interaction between the members of NF-kappaB and Ets families of transcription factors and their cognate sites in the promoter is the major determinant of inducible expression of the LT-beta gene in Jurkat cells.