1α,25-dihydroxyvitamin D3 (D3) promotes the maturation of myeloid cells and surface expressions of CD14 and CD11b, markers of cell differentiation in response to D3. To examine how these responses are regulated, THP-1 cells were grown in serum-free medium and incubated with D3. This was associated with rapid and transient increases in phosphatidylinositol 3-kinase (PI 3-kinase) activity. Furthermore, induction of CD14 expression in response to D3 was abrogated by (a) the PI 3-kinase inhibitors LY294002 and wortmannin; (b) antisense oligonucleotides to mRNA for the p110 catalytic subunit of PI 3-kinase; and (c) a dominant negative mutant of PI 3-kinase. In THP-1 cells, induction of CD11b expression by D3 was also abrogated by LY294002 and wortmannin. Similarly, LY294002 and wortmannin inhibited D3-induced expression of both CD14 and CD11b in peripheral blood monocytes. In contrast to CD14 and CD11b, hormone-induced expression of the Cdk inhibitor p21 in THP-1 cells was unaffected by either wortmannin or LY294002. These findings suggest that PI 3-kinase selectively regulates D3-induced monocyte differentiation, independent of any effects on p21.

Lipoarabinomannan (LAM) is a putative virulence factor of

The isoform identity of activated protein kinase C (PKC) and its regulation were investigated in bacterial lipopolysaccharide (LPS)-treated human monocytes. Resolution of detergent-soluble lysates prepared from LPS-treated, peripheral blood monocytes using Mono Q anion-exchange chromatography revealed two principal peaks of myelin basic protein kinase activity. Immunoblotting and immunoprecipitation with isoform-specific anti-PKC antibodies showed that the major and latest eluting peak is accounted for by PKC-. In addition to primary monocytes, activation of PKCin response to LPS was also observed in the human promonocytic cell lines, U937 and THP-1. Consistent with its identity as PKC-, the kinase did not depend upon the presence of lipids, Ca 2؉ , or diacylglycerol for activity. In addition, the kinase phosphorylates peptide ⑀ and myelin basic protein with equal efficiency but phosphorylates Kemptide and protamine sulfate poorly. Translocation of PKC-from the cytosolic to the particulate membrane fraction upon exposure of monocytes to LPS provided further evidence for activation of the kinase.Preincubation of monocytes with the phosphatidylinositol 3-kinase (PI 3-kinase) inhibitors, wortmannin or LY294002, abrogated LPS-induced activation of PKC-. Furthermore, activation of PKC-failed to occur in U937 cells transfected with a dominant negative mutant of the p85 subunit of PI 3-kinase. PKC-activity was also observed to be enhanced in vitro by the addition of phosphatidylinositol 3,4,5P 3 . These findings are consistent with a model in which PKC-is activated downstream of PI 3-kinase in monocytes in response to LPS.

Mechanisms regulating lipopolysaccharide (LPS)-induced adherence to intercellular adhesion molecule (ICAM)-1 were examined using THP-1 cells transfected with CD14-cDNA (THP-1wt). THP-1wt adherence to ICAM-1 was LPS dose-related, time-dependent, and inhibited by antibodies to either CD14 or leukocyte function associated antigen (LFA)-1, but was independent of any change in the number of surface expressed LFA-1 molecules. A potential role for phosphatidylinositol (PI) 3-kinase (PI 3-kinase) in LPS-induced adherence was examined using the PI 3-kinase inhibitors LY294002 and Wortmannin. Both inhibitors selectively attenuated LPS-induced, but not phorbol 12-myristate 13-acetateinduced adherence. Inhibition by these agents was unrelated to any changes in either LPS binding to or LFA-1 expression by THP-1wt cells. LPS-induced adherence was also abrogated in U937 cells transfected with a dominant negative mutant of of PI 3-kinase. Toxin B from Clostridium difficile, an inhibitor of the Rho family of GTP-binding proteins, abrogated both PI-3 kinase activation and adherence induced by LPS. Cytohesin-1, a phosphatidylinositol 3,4,5-triphosphate-regulated adaptor molecule for LFA-1 activation, was found to be expressed in THP-1wt cells. In addition, treatment of THP1wt with cytohesin-1 antisense attenuated LPS-induced adherence. These findings suggest a model in which LPS induces adherence through a process of "inside-out" signaling involving CD14, Rho, and PI 3-kinase. This converts low avidity LFA-1 into an active form capable of increased binding to ICAM-1. This change in LFA-1 appears to be cytohesin-1-dependent.Adherence of monocytes to endothelial cells is an essential requirement for the localization of these cells to sites of tissue inflammation (1-3). Several reports have shown that this process is dependent upon the monocyte surface molecule lymphocyte function-associated antigen-1 (LFA-1) 1 (CD11a/CD18; ␣ L ␤ 2 ) (Refs. 3-5 and reviewed in Refs. 6 and 7). Intercellular adhesion molecule-1 (ICAM-1) (CD54) has been identified as a high affinity counter-receptor for LFA-1 (8). Interactions of ICAM-1 with LFA-1 mediate several important functions in the immune system in addition to adherence (6). The basal affinity of LFA-1 for ICAM-1 or its other ligands is low and LFA-1 must be activated to mediate stable adhesion (4, 5). Indeed, in its activated form, the affinity of LFA-1 for ICAM-1 increases 200-fold in comparison with its affinity in the resting state (9). This is consistent with a process of "inside-out" signaling that converts LFA-1 into an activated form capable of mediating increased adhesion. It is important to note that conditions which give rise to increased adherence do not necessarily lead to increased cell surface expression of LFA-1 (5, 10). The signaling events that link cell stimulation to the activation of LFA-1 are incompletely understood. Recently, a regulatory protein that interacts with the cytoplasmic tail of CD18 has been cloned (11). This protein, cytohesin-1, contains a pleckstrin homology...