Distinct Role of p38 and c-Jun N-Terminal Kinases in IL-10-Dependent and IL-10-Independent Regulation of the Costimulatory Molecule B7.2 in Lipopolysaccharide-Stimulated Human Monocytic Cells

Abstract

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.

STAT-1 Mediates the Stimulatory Effect of IL-10 on CD14 Expression in Human Monocytic Cells

“…IL-10 was also shown to enhance the survival of astrocytes through PI3K activation (26). In addition, there is evidence to suggest that IL-10 can induce the activation of p38 and p42/44 ERK MAPK (23,(27)(28)(29). However, the role of MAPK in IL-10-mediated effects is not clear at present.…”

“…The PBMCs were isolated by density gradient centrifugation over Ficoll Hypaque (Amersham Biosciences), as previously described (28). Briefly, the cell layer containing mononuclear cells was collected and washed three times in PBS.…”

“…Briefly, the cell layer containing mononuclear cells was collected and washed three times in PBS. Purified, nonactivated monocytes were isolated by negative selection by depletion of T cells and B cells using magnetic polystyrene M-450 Dynabeads coated with Abs specific for CD2 (T cells, NK cells) and CD19 (B cells) (Dynal Biotech), as described earlier (28,37). CD2 ϩ CD19 ϩ cells were separated magnetically from the CD2 Ϫ CD19 Ϫ cells.…”

“…CD2 Ϫ CD19 Ϫ cells were incubated at 37°C for 2 h, following which nonadherent cells were removed. The adherent mononuclear cells obtained contained Ͻ1% CD2 ϩ T cells and CD19 ϩ B cells as determined by flow cytometry (28,37).…”

“…The EMSA was performed as described earlier (28,37). Briefly, cells harvested in Tris-EDTA-saline buffer (pH 7.8) were lysed followed by collection of nuclear extracts essentially as described earlier (28,37).…”