Interleukin-10 (IL-10), a pleiotropic cytokine that inhibits inflammatory and cell-mediated immune responses, is produced by a wide variety of cell types including T and B cells and monocytes/macrophages. Regulation of pro-and anti-inflammatory cytokines has been suggested to involve distinct signaling pathways. In this study, we investigated the regulation of the human IL-10 (hIL-10) promoter in the human monocytic cell line THP-1 following activation with lipopolysaccharide (LPS). Analysis of hIL-10 promoter sequences revealed that DNA sequences located between base pairs ؊652 and ؊571 are necessary for IL-10 transcription. A computer analysis of the promoter sequence between base pairs ؊652 and ؊571 revealed the existence of consensus sequences for Sp1, PEA1, YY1, and EpsteinBarr virus-specific nuclear antigen-2 (EBNA-2)-like transcription factors. THP-1 cells transfected with a plasmid containing mutant Sp1 abrogated the promoter activity, whereas plasmids containing the sequences for PEA1, YY1, and EBNA-2-like transcription factors did not influence hIL-10 promoter activity. To understand the events upstream of Sp1 activation, we investigated the role of p38 and extracellular signal-regulated kinase mitogenactivated protein kinases by using their specific inhibitors. SB202190 and SB203580, the p38-specific inhibitors, inhibited LPS-induced IL-10 production. In contrast, PD98059, a specific inhibitor of extracellular signal-regulated kinase kinases, failed to modulate IL-10 production. Furthermore, SB203580 inhibited LPS-induced activation of Sp1, as well as the promoter activity in cells transfected with a plasmid containing the Sp1 consensus sequence. These results suggest that p38 mitogen-activated protein kinase regulates LPS-induced activation of Sp1, which in turn regulates transcription of the hIL-10 gene.

The costimulatory molecule B7.2 (CD86) plays a vital role in immune activation and development of Th responses. The molecular mechanisms by which B7.2 expression is regulated are not understood. We investigated the role of mitogen-activated protein kinases (MAPK) in the regulation of B7.2 expression in LPS-stimulated human monocytic cells. LPS stimulation of human monocytes resulted in the down-regulation of B7.2 expression that could be abrogated by anti-IL-10 Abs. Furthermore, SB202190, a specific inhibitor of p38 MAPK, inhibited LPS-induced IL-10 production and reversed B7.2 down-regulation, suggesting that LPS-induced B7.2 down-regulation may be mediated, at least in part, via regulation of IL-10 production by p38 MAPK. In contrast to human promonocytic THP-1 cells that are refractory to the inhibitory effects of IL-10, LPS stimulation enhanced B7.2 expression. This IL-10-independent B7.2 induction was not influenced by specific inhibitors of either p38 or p42/44 MAPK. To ascertain the role of the c-Jun N-terminal kinase (JNK) MAPK, dexamethasone, an inhibitor of JNK activation, was used, which inhibited LPS-induced B7.2 expression. Transfection of THP-1 cells with a plasmid expressing a dominant-negative stress-activated protein/extracellular signal-regulated kinase kinase 1 significantly reduced LPS-induced B7.2 expression, thus confirming the involvement of JNK. To study the signaling events downstream of JNK activation, we show that dexamethasone did not inhibit LPS-induced NF-κB activation in THP-1 cells, suggesting that JNK may not be involved in NF-κB activation leading to B7.2 expression. Taken together, our results reveal the distinct involvement of p38 in IL-10-dependent, and JNK in IL-10-independent regulation of B7.2 expression in LPS-stimulated monocytic cells.

SUMMARYT helper (Th) responses are mediated in part by immunoregulatory cytokines and the signals delivered by the costimulatory CD28-B7 pathway. In this study, we have investigated the relationship between the regulation of B7 isoform expression on antigen-presenting cells from HIV þ individuals and the production of Th cytokines. The level of expression of both B7.1 and B7.2 isoforms as measured by mean channel fluorescence was significantly decreased on freshly isolated monocytes from HIV þ individuals compared with HIV ¹ controls. However, the levels of expression of B7.1 and B7.2 on both B cells and monocytes increased significantly following culture in HIV þ individuals compared with HIV ¹ controls. B7 expression is subject to regulation by immunoregulatory cytokines. Therefore, we analysed the regulation of B7 expression by cytokines, namely IL-10 and tumour necrosis factor-alpha (TNF-a), the production of which is enhanced in HIV infection and have similar inhibitory effects on B7 expression. Two groups of HIV þ individuals were distinguished on the basis of the inhibitory effect of IL-10 and TNF-a on monocyte B7.2 expression. IL-10 inhibited B7.2 expression on monocytes from some HIV þ individuals (termed responders) like the HIV ¹ controls. However, in a subset of HIV þ individuals (non-responders) this inhibitory effect was lost. Loss of inhibition of B7.2 expression by IL-10 was associated with significantly reduced IL-2 production by phytohaemagglutinin (PHA)-stimulated peripheral blood mononuclear cells (PBMC). These observations showing an association of B7 dysregulation on monocytes and B cells with altered production of IL-2 may have implications in HIV immunopathogenesis.

We investigated the expression of membrane-bound CD14 (mCD14) on monocytes and soluble CD14 (sCD14) released into the culture supernatants of peripheral blood lymphocytes (PBMC) from human immunodeficiency virus (HIV)-infected individuals. Monocytes from HIV-positive individuals exhibited both enhanced mCD14 expression and sCD14 production in the PBMC culture supernatants compared to the levels of mCD14 and sCD14 in HIV-negative individuals. This enhanced mCD14 expression and sCD14 production in HIV-infected individuals may be due to the effects of cytokines, the bacterial product lipopolysaccharide (LPS), and/or the HIV regulatory antigens Tat and Nef. Interleukin-10 (IL-10), an immunoregulatory cytokine, as well as LPS enhanced mCD14 expression and the release of sCD14 in the culture supernatants. HIV-Nef, unlike Tat, enhanced mCD14 expression on monocytes but did not induce the release of sCD14 into the culture supernatants. Studies conducted to investigate the mechanism underlying HIV-Nef-induced mCD14 expression revealed that HIV-Nef upregulated mCD14 expression via a mechanism that does not involve endogenously produced IL-10. In contrast, LPS upregulated the expression of mCD14 and increased the release of sCD14 via a mechanism that involves, at least in part, endogenously produced IL-10. Furthermore, dexamethasone, an anti-inflammatory and immunosuppressive agent, inhibited HIV-Nef-induced CD14 expression in an IL-10-independent manner. In contrast, dexamethasone inhibited IL-10-dependent LPS-induced CD14 expression by interfering with IL-10-induced signals but not by blocking IL-10 production. These results suggest that HIV-Nef and IL-10 constitute biologically important modulators of CD14 expression which may influence immunobiological responses to bacterial infections in HIV disease.