Mononuclear cells from patients recovered from cutaneous leishmaniasis respond to Leishmania major amastigote class I nuclease with a predominant Th1-like response.

The Leishmania major amastigote class I nuclease (LmaCIN) is a developmentally regulated protein that is highly expressed in the amastigote stage of L. major. This protein is homologous to the P4 nuclease of L. pifanoi, which has been shown to induce protective immune response in a murine model. To evaluate LmaCIN as a potential human vaccine candidate, cellular immune responses to recombinant LmaCIN were examined in individuals recovered from Old World cutaneous leishmaniasis. Peripheral blood mononuclear cells (PBMC) from patients recovered from L. major infection were cultured either with recombinant LmaCIN or autoclaved L. major (ALM) as control. rLmaCIN induced significant proliferation of PBMC from 90% of recovered patients. Phenotypic analysis of proliferating cells showed that CD8(+) cells were the predominant cell type proliferating in response to rLmaC1N. Screening of culture supernatants for cytokines showed that rLmaCIN induced high levels of interferon (IFN)-gamma (mean +/- s.e.m.: 1398 +/- 179 pg/ml) associated with little interleukin (IL)-10 and little or no IL-5 production. These findings show that LmaCIN is immunogenic in humans during L. major infection and that it can elicit immunological responses relevant to immunoprophylaxis of leishmaniasis.

Molecular characterization of a novel amastigote stage specific Class I nuclease from Leishmania major.

Leishmania parasites like other kinetoplastids are unable to synthesize purines de novo and so are reliant on a salvage pathway for recycling ribonucleotides. A stage specific class one nuclease enzyme, 3'-Nucleotidase/nuclease, has been implicated in salvage of preformed purines in Leishmania insect stage promastigote via hydrolysis of 3'-nucleotides and nucleic acids. Although a similar activity is known to exist in amastigotes which reside in infected mammalian cells, the homologous gene and the corresponding protein responsible for carrying out this function have not been well characterized. Using primers specific for conserved regions of trypanosomatid class one nucleases, a gene encoding a novel class one nuclease from amastigotes of Leishmania major (LmaC1N) was cloned and sequenced. The coding sequence consists of 951 bp encoding a 316 amino acid protein with a predicted molecular mass of 35,300 Da. Analysis of the deduced amino acid sequence showed that LmaC1N is highly homologous to other class I nucleases and contains all five conserved regions reported for promastigotes 3'-Nucleotidase/nuclease. Analysis by reverse transcriptase polymerase chain reaction and Western blotting demonstrated that expression of LmaC1N gene is regulated in a stage-specific manner. Whereas the gene appeared to be silenced in promastigotes, high level expression in amastigotes implied an important function in support of parasite survival and multiplication in the mammalian cells.

1alpha,25-Dihydroxyvitamin D3-induced monocyte antimycobacterial activity is regulated by phosphatidylinositol 3-kinase and mediated by the NADPH-dependent phagocyte oxidase.

We investigated the basis for the induction of monocyte antimycobacterial activity by 1alpha,25-dihydroxyvitamin D(3) (D(3)). As expected, incubation of Mycobacterium tuberculosis-infected THP-1 cells or human peripheral blood, monocyte-derived macrophages with hormone resulted in the induction of antimycobacterial activity. This effect was significantly abrogated by pretreatment of cells with either of the phosphatidylinositol 3-kinase (PI 3-K) inhibitors, wortmannin or LY294002, or with antisense oligonucleotides to the p110 subunit of PI 3-Kalpha. Cells infected with M. tuberculosis alone or incubated with D(3) alone produced little or undetectable amounts of superoxide anion (O(2)). In contrast, exposure of M. tuberculosis-infected cells to D(3) led to significant production of O(2), and this response was eliminated by either wortmannin, LY294002, or p110 antisense oligonucleotides. As was observed for PI 3-K inactivation, the reactive oxygen intermediate scavenger, 4-hydroxy-TEMPO, and degradative enzymes, polyethylene glycol coupled to either superoxide dismutase or catalase, also abrogated D(3)-induced antimycobacterial activity. Superoxide production by THP-1 cells in response to D(3) required prior infection with live M. tuberculosis, since exposure of cells to either killed M. tuberculosis or latex beads did not prime for an oxidative burst in response to subsequent hormone treatment. Consistent with these findings, redistribution of the cytosolic oxidase components p47(phox) and p67(phox) to the membrane fraction was observed in cells incubated with live M. tuberculosis and D(3) but not in response to combined treatment with heat-killed M. tuberculosis followed by D(3). Redistribution of p47(phox) and p67(phox) to the membrane fraction in response to live M. tuberculosis and D(3) was also abrogated under conditions where PI 3-K was inactivated. Taken together, these results indicate that D(3)-induced, human monocyte antimycobacterial activity is regulated by PI 3-K and mediated by the NADPH-dependent phagocyte oxidase.

The Salmonella spvB virulence gene encodes an enzyme that ADP-ribosylates actin and destabilizes the cytoskeleton of eukaryotic cells.

ADP-ribosylating enzymes, such as cholera and diphtheria toxins, are key virulence factors for a variety of extracellular bacterial pathogens but have not been implicated previously during intracellular pathogenesis. Salmonella strains are capable of invading epithelial cells and localizing in macrophages during infection. The spvB virulence gene of Salmonella is required for human macrophage cytotoxicity in vitro and for enhancing intracellular bacterial proliferation during infection. Here, we present evidence that spvB encodes an ADP-ribosylating enzyme that uses actin as a substrate and depolymerizes actin filaments when expressed in CHO cells. Furthermore, site-directed mutagenesis demonstrates that the ADP-ribosylating activity of SpvB is essential for Salmonella virulence in mice. As spvB is expressed by Salmonella strains after invasion of epithelial cells or phagocytosis by macrophages, these results suggest that SpvB functions as an intracellular ADP-ribosylating toxin critical for the pathogenesis of Salmonella infections.

Exploitation of host cell signaling machinery: activation of macrophage phosphotyrosine phosphatases as a novel mechanism of molecular microbial pathogenesis.

Intracellular pathogens, particularly those that target host mononuclear phagocytes, have evolved strategies to either evade or inhibit cellular mechanisms of host defense. Mycobacterium tuberculosis and Leishmania donovani exemplify a diverse group of microorganisms that have developed the ability to invade and replicate within host macrophages, leading to disease expression. Recent studies have suggested that the pathogenesis of intracellular infection may involve interference with host cell signaling. Drawing upon examples from in vitro models that focused on M. tuberculosis and L. donovani, we review evidence that activation of host cell phosphotyrosine phosphatases may contribute to pathogenesis. A leading candidate appears to be the Src homology 2 domain containing phosphotyrosine phosphatase SHP-1, the activation of which may contribute to the development of infection and disease progression.

1alpha,25-dihydroxyvitamin D(3)-induced myeloid cell differentiation is regulated by a vitamin D receptor-phosphatidylinositol 3-kinase signaling complex.

1alpha,25-dihydroxyvitamin D(3) (D(3)) promotes the maturation of myeloid cells and surface expressions of CD14 and CD11b, markers of cell differentiation in response to D(3). To examine how these responses are regulated, THP-1 cells were grown in serum-free medium and incubated with D(3). This was associated with rapid and transient increases in phosphatidylinositol 3-kinase (PI 3-kinase) activity. Furthermore, induction of CD14 expression in response to D(3) 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 D(3) was also abrogated by LY294002 and wortmannin. Similarly, LY294002 and wortmannin inhibited D(3)-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 D(3)-induced monocyte differentiation, independent of any effects on p21.

Activation of phosphotyrosine phosphatase activity attenuates mitogen-activated protein kinase signaling and inhibits c-FOS and nitric oxide synthase expression in macrophages infected with Leishmania donovani.

Intracellular protozoan parasites of the genus Leishmania antagonize host defense mechanisms by interfering with cell signaling in macrophages. In this report, the impact of Leishmania donovani on mitogen-activated protein (MAP) kinases and nitric oxide synthase (NOS) expression in the macrophage cell line RAW 264 was investigated. Overnight infection of cells with leishmania led to a significant decrease in phorbol-12-myristate-13-acetate (PMA)-stimulated MAP kinase activity and inhibited PMA-induced phosphorylation of the MAP kinase substrate and transcription factor Elk-1. Simultaneously, leishmania infection markedly attenuated the induction of c-FOS and inducible nitric oxide synthase (iNOS) expression in response to PMA and gamma interferon (IFN-gamma), respectively. These effects correlated with decreased phosphorylation of p44 and p42 MAP kinases on tyrosine residues. Consistent with the latter finding, lysates prepared from leishmania-infected cells contained an activity that dephosphorylated MAP kinase in vitro, suggesting the possibility of a phosphatase acting in vivo. Attenuation of both MAP kinase activity and c-FOS and iNOS expression was reversed by treatment of macrophages with sodium orthovanadate prior to infection. It was also found that the specific activity of the Src homology 2 domain containing tyrosine phosphatase (SHP-1) toward MAP kinase was markedly increased in leishmania-infected cells. These findings indicate that infection with L. donovani attenuates MAP kinase signaling and c-FOS and iNOS expression in macrophages by activating cellular phosphotyrosine phosphatases. This may represent a novel mechanism of macrophage deactivation during intracellular infection.

Monocyte adherence induced by lipopolysaccharide involves CD14, LFA-1, and cytohesin-1. Regulation by Rho and phosphatidylinositol 3-kinase.

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-acetate-induced 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 THP-1wt 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.

Attenuation of HLA-DR expression by mononuclear phagocytes infected with Mycobacterium tuberculosis is related to intracellular sequestration of immature class II heterodimers.

MHC class II expression was examined in macrophages infected with Mycobacterium tuberculosis. IFN-gamma increased the surface expression of class II molecules in THP-1 cells and this was markedly reduced in cells infected with M. tuberculosis. Despite this effect, steady state levels of HLA-DRalpha, HLA-DRbeta, and invariant (Ii) chains were equivalent in control and infected cells. Metabolic labeling combined with pulse-chase experiments and biochemical analysis showed that the majority of class II molecules in infected cells became resistant to endoglycosidase H, consistent with normal Golgi processing. However, results of intracellular staining and dual color confocal microscopy revealed a significant defect in transport of newly synthesized class II molecules through the endocytic compartment. Thus, compared with findings in control cells, class II molecules in infected cells colocalized to a minimal extent with a lysosomal-associated membrane protein-1+ endosomal compartment. In addition, in contrast to control cells, class II molecules in infected cells failed to colocalize with endocytosed BSA under conditions where this marker is known to label late endosomes, lysosomes, and the MHC class II compartment. Consistent with defective transport along the endocytic pathway, the maturation of SDS-stable class II alphabeta dimers--dependent upon removal of Ii chain and peptide loading of class II dimers in the MHC class II compartment--was markedly impaired in M. tuberculosis-infected cells. These findings indicate that defective transport and processing of class II molecules through the endosomal/lysosomal system is responsible for diminished cell surface expression of MHC class II molecules in cells infected with M. tuberculosis.

Phenotypic difference between Bcg(r) and Bcg(s) macrophages is related to differences in protein-kinase-C-dependent signalling.

Mice of diverse genetic backgrounds may be classified as being either resistant or susceptible to infection with Mycobacteria. These phenotypes appear to be determined by a single gene on chromosome 1, the Bcg gene, and are expressed at the level of the macrophage in vitro. When compared to macrophages from mice of the susceptible phenotype (Bcg[s]), macrophages from mice of the resistant phenotype (Bcg[r]) show enhanced functional properties including increased expression of MHC class II molecules, increased nitric oxide production, and greater capacity to inhibit the growth of several intracellular pathogens. The bacteriostatic activity of B10R and B10S macrophages correlated with the amount of nitric oxide produced by the macrophages. Since protein kinase C (PKC) has been shown to be involved in the induction of a range of macrophage functional activities, experiments were conducted to examine the possibility that phenotypic differences between Bcg(r) and Bcg(s) macrophages may be related to differences in PKC-dependent signalling. Macrophage cell lines were derived from mice congenic at the Bcg locus that are either resistant (B10R) or susceptible (B10S) to infection with Mycobacteria. In the basal state, PKC-specific activity was significantly increased in the cytosolic fractions of B10R cells when compared to B10S cells. Following phorbol myristate acetate (PMA) treatment and following the stimulation with Mycobacteria bovis BCG, PKC-specific activity increased significantly in membrane fractions of both B10R and B10S cells, but the absolute level was significantly greater in particulate fractions from B10R macrophages. Furthermore, B10R cells had a superior ability to phosphorylate endogenous substrates compared to B10S macrophages. Scatchard analysis of phorbol ester receptors revealed no differences between B10R and B10S cells. In contrast, the sensitivity of partially purified PKC from B10S cells to activation in vitro by diacylglycerol was decreased by approximately 50% when compared to enzyme from B10R cells. Western-blotting analysis using antibodies specific for PKC isoforms (alpha, beta, delta, epsilon, zeta and eta) showed similar levels of PKC isoforms present in B10R and B10S cells. To examine whether differences in PKC activity of B10R and B10S cells had functional consequences, the induction of c-fos gene expression was compared in the two cell lines. In response either to infection with M. bovis BCG or to stimulation with PMA, c-fos mRNA levels in B10R macrophages were increased 2-4-fold in comparison to B10S macrophages. Since we have previously found that the bacteriostatic activity of B10R and B10S macrophages correlated with the amount of nitric oxide produced by the macrophages, we have tested if the enhancement of PKC activity in these macrophages affects their ability to produce nitric oxide. We have found that interferon-gamma-(IFNgamma)-induced secretion of nitric oxide by B10R macrophages could be augmented a few fold by the activation of PKC whereas, in B10S macrophages stimulated with IFNgamma, nitric oxide release could be augmented by only about 10-20%. These results indicate that the differences in PKC activity between B10R and B10S macrophages may contribute to altered responsiveness to IFNgamma that results in different production of effector molecules crucial for bacteriostatic activity against M. bovis BCG.

Phosphatidylinositol 3-kinase is involved in the induction of macrophage growth by oxidized low density lipoprotein.

Early atherosclerotic lesions are characterized by the presence of cholesterol-rich, macrophage-derived foam cells. It has recently been shown that macrophage proliferation occurs during the development of early lesions and that oxidized low density lipoprotein (LDL) stimulates macrophage growth. Possible mechanisms for this induction of macrophage growth include potentiation of mitogenic signal transduction by a component of oxidized LDL following internalization and degradation, interaction with integral plasma membrane proteins coupled to signaling pathways, or direct or indirect activation of growth factor receptors on the cell surface (e.g. GM-CSF receptor) through an autocrine/paracrine mechanism. The present study was undertaken to characterize some of the early intracellular signaling events by which oxidized LDL mediates macrophage cell growth. Extensively oxidized LDL increased protein-tyrosine phosphorylation and caused a 2-fold increase in phosphatidylinositol (PI) 3-kinase activity in phorbol ester-pretreated THP-1 cells (a human monocyte-like cell line). Similar concentrations of native LDL had no effect. Oxidized LDL also stimulated growth of resident mouse peritoneal macrophages, and this effect was reduced by 40-50% in cells treated with PI 3-kinase inhibitors (100 nM wortmannin or 20 microM LY294002). These results suggest that PI 3-kinase mediates part of the mitogenic effect of oxidized LDL, but parallel pathways involving other receptors and signal transduction pathways are likely also involved.

Lipoarabinomannan of Mycobacterium tuberculosis promotes protein tyrosine dephosphorylation and inhibition of mitogen-activated protein kinase in human mononuclear phagocytes. Role of the Src homology 2 containing tyrosine phosphatase 1.

Lipoarabinomannan (LAM) is a putative virulence factor of Mycobacterium tuberculosis that inhibits monocyte functions, and this may involve antagonism of cell signaling pathways. The effects of LAM on protein tyrosine phosphorylation in cells of the human monocytic cell line THP-1 were examined. LAM promoted tyrosine dephosphorylation of multiple cell proteins and attenuated phorbol 12-myristate 13-acetate-induced activation of mitogen-activated protein kinase. To examine whether these effects of LAM could be related to activation of a phosphatase, fractions from LAM-treated cells were analyzed for dephosphorylation of para-nitrophenol phosphate. The data show that LAM induced increased phosphatase activity associated with the membrane fraction. The Src homology 2 containing tyrosine phosphatase 1 (SHP-1) is important for signal termination and was examined as a potential target of LAM. Exposure of cells to LAM brought about (i) an increase in tyrosine phosphorylation of SHP-1, and (ii) translocation of the phosphatase to the membrane. Phosphatase assay of SHP-1 immunoprecipitated from LAM-treated cells, using phosphorylated mitogen-activated protein kinase as substrate, indicated that LAM promoted increased activity of SHP-1 in vivo. LAM also activated SHP-1 directly in vitro. Exposure of cells to LAM also attenuated the expression of tumor necrosis factor-alpha, interleukin-12, and major histocompatibility class II molecules. These results suggest that one mechanism by which LAM deactivates monocytes involves activation of SHP-1.

Phosphatidylinositol 3-kinase-dependent activation of protein kinase C-zeta in bacterial lipopolysaccharide-treated human monocytes.

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-zeta. In addition to primary monocytes, activation of PKC-zeta in response to LPS was also observed in the human promonocytic cell lines, U937 and THP-1. Consistent with its identity as PKC-zeta, the kinase did not depend upon the presence of lipids, Ca2+, or diacylglycerol for activity. In addition, the kinase phosphorylates peptide epsilon and myelin basic protein with equal efficiency but phosphorylates Kemptide and protamine sulfate poorly. Translocation of PKC-zeta 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-zeta. Furthermore, activation of PKC-zeta failed to occur in U937 cells transfected with a dominant negative mutant of the p85 subunit of PI 3-kinase. PKC-zeta activity was also observed to be enhanced in vitro by the addition of phosphatidylinositol 3,4,5P3. These findings are consistent with a model in which PKC-zeta is activated downstream of PI 3-kinase in monocytes in response to LPS.

Leishmania major: molecular cloning, sequencing, and expression of the heat shock protein 60 gene reveals unique carboxy terminal peptide sequences.

Heat shock proteins (HSP) in the size range of M(r) 60,000 are major targets of the immune response in vivo. The leishmania heat-inducible proteins of M(r) 65-67,000 are expressed at relatively high levels in infected macrophages (Infection and Immunity 1993, 61, 3265-3272) and may be important targets of the host response. To facilitate further studies concerned with these proteins, the HSP60 gene of Leishmania major was cloned, sequenced, and expressed. A lambdaEMBL-3 L. major genomic library was screened with a PCR-generated DNA probe derived from a highly conserved region of the leishmania HSP60 gene. A single clone that hybridized strongly was characterized. Sequence analysis revealed an open reading frame of 1770 bp encoding a putative polypeptide of 589 amino acids with a predicted size of M(r) 64,790 and with the highest degree of amino acid sequence similarity (56%) to HSP60 from Trypanosoma cruzi. Less extensive amino acid sequence similarity (48%) was observed between that leishmania HSP60 and the corresponding human protein. Notably, significant regions of sequence dissimilarity between the leishmania and human proteins were identified principally within the carboxy-terminal regions of the proteins. The entire coding region of the leishmania HSP60 gene was subcloned into the pET-3a vector and expressed in Escherichia coli. Purified recombinant protein was used to examine sera from patients with tegumentary leishmaniasis from Colombia for the presence of antibodies to HSP60. Unlike sera from healthy, uninfected controls, sera from patients reacted strongly with recombinant leishmania HSP60. This recognition had specificity in that these same sera showed little or no reactivity with either recombinant mycobacterial HSP65 or recombinant human HSP60. These findings indicate that patients with tegumentary forms of leishmaniasis have humoral responses to leishmania HSP60. Further studies of this protein will clarify its importance as a target of the immune response and as a potential antigen for serodiagnosis.

TGF-beta attenuates the class II transactivator and reveals an accessory pathway of IFN-gamma action.

In the present report, the induction of the HLA-DRA gene in response to IFN-gamma is shown to be selectively attenuated by TGF-beta. Thus, the accumulation in response to IFN-gamma of mRNA for the DRA gene, but not for the guanylate binding protein-2 gene, is markedly reduced in the presence of TGF-beta. Moreover, the data presented show that the mechanism by which TGF-beta inhibits expression of DRA involves attenuation of the class II transactivator (CIITA) gene. This conclusion is based on the finding that induction of CIITA gene expression in response to IFN-gamma is completely abrogated in TGF-beta-treated cells. In contrast, TGF-beta did not affect IFN-gamma-induced tyrosine phosphorylation of Jak1, Jak2, or the signal transducer and activator of transcription-1 (Stat1). TGF-beta also did not inhibit the appearance of IFN-gamma-activated, Stat1 DNA-binding activity in intact cells. Thus, the effects of TGF-beta on CIITA could not be explained by altered signaling through Jak-Stat1. Potential alternative targets for the inhibitory effects of TGF-beta were identified in renaturation tyrosine kinase assays, which revealed three IFN-gamma-activated protein tyrosine kinases that, in contrast to the Janus kinases, are sensitive to TGF-beta. These findings 1) indicate that inhibition of MHC class II gene expression by TGF-beta involves attenuation of the CIITA gene independently of effects on Janus kinases, 2) provide direct evidence that IFN-gamma activates both Janus and non-Janus protein tyrosine kinases, and 3) identify an accessory pathway of IFN-gamma action involving tyrosine kinases that, unlike the Jak-Stat1 pathway, are impaired by TGF-beta.

Bacterial lipopolysaccharide induces the association and coordinate activation of p53/56lyn and phosphatidylinositol 3-kinase in human monocytes.

p53/56lyn and other src family tyrosine kinases become activated in monocytes treated with LPS. In a variety of systems, phosphatidylinositol 3-kinase (PI 3-kinase) is believed to be a downstream effector of tyrosine kinases, and activation of PI 3-kinase results in increased levels of D3-phosphorylated metabolites of phosphatidylinositol (PtdIns). To examine whether LPS activates PI 3-kinase, freshly isolated human, peripheral blood monocytes were labeled in vitro with [32P]orthophosphate, and inositol phospholipids were detected after extraction and separation of lipids by TLC. Levels of PtdIns 3,4,5-trisphosphate (PtdIns 3,4,5-P3) were elevated within minutes of exposure of cells to LPS. Analysis of 32P-labeled lipid extracts of U937 cells by HPLC confirmed that levels of PtdIns 3,4,5-P3 increased rapidly following LPS treatment. Increased levels of PtdIns 3,4,5-P3 in LPS-treated cells resulted from an increase in the specific activity of PI 3-kinase. Thus, anti-PI 3-kinase immunoprecipitates prepared from unlabeled monocytes and assayed in an in vitro phosphorylation assay, using PtdIns as substrate, showed higher enzymatic activity when these were prepared from lysates of LPS-treated cells as compared with control cells. PI 3-kinase activity in immunoprecipitates was elevated as early as 2 min after LPS exposure and was dose dependent, with increased activity being observed at LPS concentrations as low as 10 pg/ml. Activation of PI 3-kinase involved signaling through the monocyte cell surface molecule CD14, since pretreatment of cells with Abs to CD14 abrogated LPS-induced increases in PtdIns 3,4,5-P3. Immunoprecipitates of p53/56lyn from LPS-treated cells showed a time-dependent and transient increase in PI 3-kinase activity assayed in vitro, coordinate with activation of p53/56lyn, indicating that LPS induces the association and simultaneous activation of these two enzymes in vivo. These findings indicate that monocytes respond to LPS with the rapid activation of PI 3-kinase, resulting in transient increases in levels of PtdIns 3,4,5-P3. This process is CD14 dependent and involves the physical association of PI 3-kinase with activated p53/56lyn.

Attenuation of gamma interferon-induced tyrosine phosphorylation in mononuclear phagocytes infected with Leishmania donovani: selective inhibition of signaling through Janus kinases and Stat1.

The induction of gene transcription in response to gamma interferon is impaired in mononuclear phagocytes infected with Leishmania donovani, and the mechanisms involved are not fully understood. The changes in gene expression brought about by gamma interferon are thought to involve transient increases in the activities of cellular protein tyrosine kinases, including the Janus kinases Jak1 and Jak2, leading to tyrosine phosphorylation of the transcription factor Stat1. To investigate the mechanisms accounting for the impaired responses to gamma interferon, a model system for examining overall changes in protein tyrosine phosphorylation, activation of Jak1 and Jak2 and phosphorylation of Stat1 was developed in phorbol 12-myristate 13-acetate-differentiated U-937 cells. Analysis of whole-cell lysates by antiphosphotyrosine immunoblotting showed that incubation with gamma interferon brought about specific increases in phosphotyrosine labeling of several proteins. Increased labeling of these proteins occurred to similar extents in control cells and in cells that had been infected with L. donovani for 16 h. Jak1, Jak2, and Stat1 were immunoprecipitated from control and interferon-treated cells, and tyrosine phosphorylation of these proteins, detected by antiphosphotyrosine immunoblotting was used to measured their activation. Tyrosine phosphorylation of Jak1, Jak2, and Stat1 increased markedly, in a dose-dependent manner, in U-937 cells incubated with gamma interferon. In contrast, in cells infected with L. donovani, tyrosine phosphorylation of Jak1, Jak2, and Stat1 was markedly impaired. This effect was dependent upon the duration of exposure to L. donovani and was maximal and complete at 16 h. Results similar to those observed with U-937 cells were also obtained with human peripheral blood monocytes. These findings indicate that infection of human mononuclear phagocytes with L. donovani leads to impaired gamma interferon-mediated tyrosine phosphorylation and selective effects on the Jak-Stat1 pathway. Unresponsiveness to gamma interferon for activation of this pathway may explain impaired transcriptional responses in leishmania-infected cells.

CD14-dependent activation of protein kinase C and mitogen-activated protein kinases (p42 and p44) in human monocytes treated with bacterial lipopolysaccharide.

To investigate mechanisms of mononuclear phagocyte cell signaling, the effects of bacterial LPS on protein kinase activities in normal human peripheral blood monocytes were examined. Incubation of intact monocytes with LPS brought about time- and concentration-dependent increases in myelin basic protein (MBP) phosphotransferase activity in high speed supernatants of cell lysates. Anion-exchange chromatography on Mono Q demonstrated that LPS treatment resulted in two principal peaks of stimulated MBP kinase activity. Evidence was obtained to indicate that the first eluted peak of MBP kinase activity is accounted for by p42 and p44 mitogen-activated protein (MAP) kinases. Thus, 1) MBP kinase activity within peak 1 was quantitatively precipitated by anti-MAP kinase Abs, 2) the enzyme effectively phosphorylated a specific peptide substrate, 3) peak 1 contained proteins of subunit size M(r) 42,000 and M(r) 44,000 that reacted specifically with anti-MAP kinase Abs, and that 4) were recognized by anti-phosphotyrosine Abs only after stimulation of cells with LPS. Studies of the second peak of LPS-stimulated MBP kinase activity indicate that it is an isoform of protein kinase C (PKC) because: 1) enzyme activity was quantitatively immunoprecipitated by anti-PKC Abs, 2) the activity of the enzyme was potently and selectively inhibited by a specific peptide modeled on the autoinhibitory domain of PKC, and 3) the presence of a protein of subunit size M(r) 80,000 recognized by anti-PKC Abs. Because the second peak of MBP kinase activity (like the first) was active in the absence of added calcium and in the presence of 2 mM EGTA, it appears to be a type II, calcium-independent isoform of PKC. Abs to CD14 completely abrogated LPS-induced activation of both Mono Q peaks of MBP phosphotransferase activity. These results indicate that LPS coordinately activates both an apparently calcium-independent PKC and MAP kinase in mononuclear phagocytes and these responses appear to be initiated by signaling through the cell surface receptor, CD14.

Altered cell signaling and mononuclear phagocyte deactivation during intracellular infection.

Given the critical antimicrobial properties of mononuclear phagocytes, an important concern in cell biology and immunology has been to understand how intracellular microbes are able to establish states of chronic infection within these cells. Recent studies indicate that mononuclear phagocytes become functionally deactivated during intracellular infection. Here, Neil Reiner considers the experimental evidence to indicate that this is a frequent event that may be accounted for by induced defects in the signaling pathways required to bring cells to an activated state.

Gamma interferon induces rapid and coordinate activation of mitogen-activated protein kinase (extracellular signal-regulated kinase) and calcium-independent protein kinase C in human monocytes.

Gamma interferon plays an important role in regulating the functional properties of mononuclear phagocytes. In the present study, the role of activated protein kinases in the mechanism of action of gamma interferon cell signaling in human peripheral blood monocytes was investigated. Analysis in vitro of 100,000 x g cytosolic fractions from untreated and interferon-treated cells showed that agonist treatment resulted in time- and concentration-dependent increases in phosphotransferase activity when myelin basic protein (MBP) was used as the substrate. Anion-exchange chromatography of high-speed supernatants prepared from detergent extracts of interferon-treated cells revealed two discrete peaks of MBP phosphotransferase activity. Immunoblotting of fractions from these peaks with antiphosphotyrosine antibodies and with antibodies that specifically recognize the family of mitogen-activated protein (MAP) kinases detected a MAP kinase with a subunit M(r) of 42,000 in the earliest-eluting peak (peak 1). Phosphorylation of the 42,000-M(r) protein on tyrosine was observed only after treatment of cells with interferon. The contribution of MAP kinase to the interferon-stimulated activity in peak 1 was confirmed by quantitative immunoprecipitation with anti-MAP kinase and antiphosphotyrosine antibodies. The conclusion that the interferon-activated MBP kinase in peak 1 could be accounted for by an activated MAP kinase was also supported by the finding that fractions from Mono Q peak 1 demonstrated activity towards a MAP kinase-specific substrate. The later-eluting peak of interferon-activated MBP phosphotransferase activity appeared to be accounted for by an activated protein kinase C (PKC). This conclusion is based upon analyses of immunoblotting and immunoprecipitation experiments with antibodies to PKC and was also supported by the observed inhibition of this kinase with a PKC pseudosubstrate peptide. The interferon-stimulated PKC present in Mono Q peak 2 was active in the absence of calcium ions, suggesting that it is a calcium-independent isoform of PKC.