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MCP-1–induced monocyte chemotaxis is a crucial event in inflammation and atherogenesis. Identifying the important signal transduction pathways that control monocyte chemotaxis can unravel potential targets for preventive therapies in inflammatory disease conditions. Previous studies have shown that the focal adhesion kinase Pyk2 plays a critical role in monocyte motility. In this study, we investigated the MCP-1–mediated activation of Pyk2 (particularly by the phosphorylation of Tyr402) in primary human peripheral blood monocytes. We showed that MCP-1 induces Src phosphorylation in a similar time frame and that the MCP-1–induced Pyk2 tyrosine phosphorylation is controlled by the Src family kinase. We also report, in this study, that PKCβ, an isoform of PKC, is required for both Src and Pyk2 activation/phosphorylation in response to MCP-1 stimulation. We identified Lyn as the specific Src kinase isoform that is activated by MCP-1 and acts upstream of Pyk2 in primary monocytes. Furthermore, Lyn is found to be indispensable for monocyte migration in response to MCP-1 stimulation. Moreover, our coimmunoprecipitation studies in monocytes revealed that PKCβ, Pyk2, and Lyn exist constitutively in a molecular complex. To our knowledge, our study has uncovered a novel PKCβ–Lyn–Pyk2 signaling cascade in primary monocytes that regulates MCP-1–induced monocyte adhesion and migration.

Monoamine oxidase A (MAO-A) is a mitochondrial flavoenzyme implicated in the pathogenesis of atherosclerosis and inflammation and also in many neurological disorders. MAO-A also has been reported as a potential therapeutic target in prostate cancer. However, the regulatory mechanisms controlling cytokine-induced MAO-A expression in immune or cancer cells remain to be identified. Here, we show that MAO-A expression is co-induced with 15-lipoxygenase (15-LO) in interleukin 13 (IL-13)-activated primary human monocytes and A549 non-small cell lung carcinoma cells. We present evidence that MAO-A gene expression and activity are regulated by signal transducer and activator of transcription 1, 3, and 6 (STAT1, STAT3, and STAT6), early growth response 1 (EGR1), and cAMP-responsive element–binding protein (CREB), the same transcription factors that control IL-13–dependent 15-LO expression. We further established that in both primary monocytes and in A549 cells, IL-13–stimulated MAO-A expression, activity, and function are directly governed by 15-LO. In contrast, IL-13–driven expression and activity of MAO-A was 15-LO–independent in U937 promonocytic cells. Furthermore, we demonstrate that the 15-LO–dependent transcriptional regulation of MAO-A in response to IL-13 stimulation in monocytes and in A549 cells is mediated by peroxisome proliferator–activated receptor γ (PPARγ) and that signal transducer and activator of transcription 6 (STAT6) plays a crucial role in facilitating the transcriptional activity of PPARγ. We further report that the IL-13–STAT6–15-LO–PPARγ axis is critical for MAO-A expression, activity, and function, including migration and reactive oxygen species generation. Altogether, these results have major implications for the resolution of inflammation and indicate that MAO-A may promote metastatic potential in lung cancer cells.

Injury initiates recruitment of macrophages to support tissue repair; however, excessive macrophage activity may exacerbate tissue damage causing further destruction and subsequent delay in wound repair. Here we show that the peroxisome proliferation–activated receptor-γ agonist, rosiglitazone (Rosi), a medication recently reintroduced as a drug to treat diabetes and with known anti-inflammatory properties, paradoxically generates pro-inflammatory macrophages. This is observed in both IL-6-deficient mice and control wild-type mice experimentally induced to produce high titers of auto-antibodies against IL-6, mimicking IL-6 deficiency in human diseases. IL-6 deficiency when combined with Rosi-mediated upregulation of suppressor of cytokine signaling 3 leads to an altered ratio of nuclear signal transducer and activator of transcription 3/NF-κB that allows hyper-induction of inducible nitric oxide synthase (iNOS). Macrophages activated in this manner cause de novo tissue destruction, recapitulating human chronic wounds, and can be reversed in vivo by recombinant IL-6, blocking macrophage infiltration, or neutralizing iNOS. This study provides insight into an unanticipated paradoxical role of Rosi in mediating hyper-inflammatory macrophage activation significant for diseases associated with IL-6 deficiency.

Macrophage accumulation is a critical step during development of chronic inflammation, initiating progression of many devastating diseases. Leukocyte-specific integrin αDβ2 (CD11d/CD18) is dramatically upregulated on macrophages at inflammatory sites. Previously we found that CD11d overexpression on cell surfaces inhibits in vitro cell migration due to excessive adhesion. In this study, we have investigated how inflammation-mediated CD11d upregulation contributes to macrophage retention at inflammatory sites during atherogenesis. Atherosclerosis was evaluated in CD11d−/−/ApoE−/− mice after 16 wk on a Western diet. CD11d deficiency led to a marked reduction in lipid deposition in aortas and isolated macrophages. Macrophage numbers in aortic sinuses of CD11d−/− mice were reduced without affecting their apoptosis and proliferation. Adoptive transfer of fluorescently labeled wild-type and CD11d−/− monocytes into ApoE−/− mice demonstrated similar recruitment from circulation, but reduced accumulation of CD11d−/− macrophages within the aortas. Furthermore, CD11d expression was significantly upregulated on macrophages in atherosclerotic lesions and M1 macrophages in vitro. Interestingly, expression of the related ligand-sharing integrin CD11b was not altered. This difference defines their distinct roles in the regulation of macrophage migration. CD11d-deficient M1 macrophages demonstrated improved migration in a three-dimensional fibrin matrix and during resolution of peritoneal inflammation, whereas migration of CD11b−/− M1 macrophages was not affected. These results prove the contribution of high densities of CD11d to macrophage arrest during atherogenesis. Because high expression of CD11d was detected in several inflammation-dependent diseases, we suggest that CD11d/CD18 upregulation on proinflammatory macrophages may represent a common mechanism for macrophage retention at inflammatory sites, thereby promoting chronic inflammation and disease development.

Monocyte chemoattractant protein-1 (MCP-1)-induced monocyte chemotaxis is a major event in inflammatory disease. Our prior studies have demonstrated that MCP-1-dependent chemotaxis requires release of arachidonic acid (AA) by activated cytosolic phospholipase A(2) (cPLA(2)). Here we investigated the involvement of AA metabolites in chemotaxis. Neither cyclooxygenase nor lipoxygenase pathways were required, whereas pharmacologic inhibitors of both the cytochrome-P450 (CYP) and the soluble epoxide hydrolase (sEH) pathways blocked monocyte chemotaxis to MCP-1. To verify specificity, we demonstrated that the CYP and sEH products epoxyeiscosatrienoic acids (EETs) and dihydroxyeicosatrienoic acids (DHETs), respectively, restored chemotaxis in the presence of the inhibitors, indicating that sEH-derived products are essential for MCP-1-driven chemotaxis. Importantly, DHETs also rescued chemotaxis in cPLA(2)-deficient monocytes and monocytes with blocked Erk1/2 activity, because Erk controls cPLA(2) activation. The in vitro findings regarding the involvement of CYP/sEH pathways were further validated in vivo using two complementary approaches measuring MCP-1-dependent chemotaxis in mice. These observations reveal the importance of sEH in MCP-1-regulated monocyte chemotaxis and may explain the observed therapeutic value of sEH inhibitors in treatment of inflammatory diseases, cardiovascular diseases, pain, and even carcinogenesis. Their effectiveness, often attributed to increasing EET levels, is probably influenced by the impairment of DHET formation and inhibition of chemotaxis.

IL-13 is a potent stimulator of alternative monocyte/macrophage activation. During alternative activation, the expression of several proteins is induced including 15-lipoxygenase (15-LO), a lipid-peroxidating enzyme and the scavenger receptor CD36. We previously reported that αMβ2 integrin activation or clustering suppresses the expression of both 15-LO and CD36. In this study we focused on exploring the molecular mechanisms that down-regulate CD36 expression and CD36-mediated foam cell formation in IL-13-stimulated monocytes/macrophages after αMβ2 activation. Our studies reveal that αMβ2 integrin activation inhibits the IL-13 activation of several critical pathways that are required for macrophage alternative activation; namely, blocking Jak2 and Tyk2 phosphorylation, which bind to the cytoplasmic tails of the IL-4Rα/IL-13Rα1 complex. This leads to the inhibition of tyrosine phosphorylation of Stats (Stat1, Stat3, and Stat6) and prevents the formation of a signaling complex (containing p38MAPK, PKCδ, and Stat3) that are critical for the expression of both 15-LO and CD36. Jak2-mediated Hck activation is also inhibited, thereby preventing Stats serine phosphorylation, which is essential for downstream Stat-dependent gene transcription. Moreover, inhibition of Jak2, Tyk2, or their downstream target 15-LO with antisense oligonucleotides profoundly inhibits IL-13-induced CD36 expression and CD36-dependent foam cell formation, whereas13(S) Hydroperoxyoctadecadienoic acid (HPODE), a 15-LO product and peroxisome proliferator-activated receptor-γ ligand, completely restores CD36 expression in monocytes treated with 15-LO antisense. αMβ2 integrin activation controls CD36 expression and foam cell formation in alternatively activated monocyte/macrophages by blocking Tyk2/Jak2 phosphorylation via a 15-LO-dependent pathway. The discovery of this mechanism helps our understanding of the potential role of alternatively activated macrophages in atherogenesis and highlights the impact of integrin αMβ2 on this process. Background: CD36, a macrophage receptor critical for oxidized lipid uptake during atherogenesis, is up-regulated after IL-4/IL-13 stimulation. Results: The mechanism whereby integrin αMβ2 inhibits CD36 gene expression and related foam-cell formation is elucidated. Conclusion: Activated integrin αMβ2 suppresses CD36-mediated lipid uptake by blocking Tyk2- and Jak2-mediated signaling pathways. Significance: αMβ2 integrin regulation of CD36 expression and function reveals a new mechanism that may control atherogenesis.

Glioblastoma multiforme (GBM) is one of the most lethal forms of cancer, with a survival rate of only 13% to 27% within 2 years of diagnosis despite optimal medical treatment. We hypothesize that the presence of a unique IL-13Rα2 decoy receptor prevents GBM apoptosis. This receptor has a high affinity for interleukin-13 (IL-13), binds the cytokine, and competitively inhibits the intracellular signaling cascade initiated by IL-13. In cells lacking the IL-13Rα2 decoy receptor, IL-13 initiates the production of 15-lipoxygenase-1 (15-LOX-1), which has been implicated in cellular apoptosis. Our group and others have shown that induction of 15-LOX-1 correlates with tumor cell death in colorectal, pancreatic, and prostate cancer. How 15-LOX-1 induces apoptosis remains unclear. Preliminary evidence in GBM cells implicates an apoptotic process mediated by PPARγ. 15-LOX-1 metabolites can modulate PPARγ and activation of PPARγ can suppress tumor growth. We hypothesize that in GBM, IL-13 can induce 15-LOX-1, which regulates cell apoptosis via signaling through PPARγ and that expression of IL-13Rα2 prevents apoptosis and contributes to tumor growth. Our in vitro and in vivo data support this. Knocking down IL-13Rα2 with short interfering RNA dramatically induces 15-LOX-1 expression, promotes apoptosis, and reduces GBM tumor growth in vivo. These findings identify a mechanism for eliminating the blockade of endogenous IL-13 signaling and for promotion of apoptosis, and characterize a role for 15-LOX-1 in GBM apoptosis. Identifying a mechanistic pathway that can be targeted for pharmacologic intervention will have applied implications to developing novel and effective treatments of GBM. Mol Cancer Ther; 10(7); 1149–60. ©2011 AACR.

Zymosan, a mimic of fungal pathogens, and its opsonized form (ZOP) are potent stimulators of monocyte NADPH oxidase, resulting in the production of O2.–, which is critical for host defense against fungal and bacterial pathogens and efficient immune responses; however, uncontrolled O2.– production may contribute to chronic inflammation and tissue injury. Our laboratory has focused on characterizing the signal transduction pathways that regulate NADPH oxidase activity in primary human monocytes. In this study, we examined the involvement of various pattern recognition receptors and found that Dectin-1 is the primary receptor for zymosan stimulation of O2.– via NADPH oxidase in human monocytes, whereas Dectin-1 and CR3 mediate the activation by ZOP. Further studies identified Syk and Src as important signaling components downstream of Dectin-1 and additionally identified PKCδ as a novel downstream signaling component for zymosan-induced O2.– as well as phagocytosis. Our results show that Syk and Src association with Dectin-1 is dependent on PKCδ activity and expression and demonstrate direct binding between Dectin-1 and PKCδ. Finally, our data show that PKCδ and Syk but not Src are required for Dectin-1-mediated phagocytosis. Taken together, our data identify Dectin-1 as the major PRR for zymosan in primary human monocytes and identify PKCδ as a novel downstream signaling kinase for Dectin-1-mediated regulation of monocyte NADPH oxidase and zymosan phagocytosis.

Rationale: The alternative activation of monocytes by interleukin (IL)-13 and IL-4 is a significant component of the inflammatory response. The consequences of alternative activation in inflammatory diseases remain to be determined. Objective: In this report, we explored how integrins, receptors important for monocyte migration to inflammatory sites, regulate IL-13–mediated monocyte activation. We focused on the analysis of 2 proteins, which are upregulated during the alternative activation and are important for the development of atherosclerosis, an oxidative enzyme 15-lipoxygenase (15-LO) and a scavenger receptor CD36. Methods and Results: We found that adhesion of resting monocytes through β 2 integrins and inside-out activation of β 2 integrins by monocyte chemoattractant protein-1 did not change IL-13–stimulated 15-LO upregulation; however, preincubation of monocytes with the antibody MEM48, which generates full activation of β 2 integrins, significantly inhibited 15-LO mRNA and protein expression. In contrast, activation of β 1 integrins had no effect on 15-LO expression. Analysis of integrin clustering through α M , α L , α X , and α D subunits demonstrated the pivotal role for integrin α M β 2 in inhibiting 15-LO expression. IL-13 treatment upregulates 15-LO–dependent CD36 expression on human monocytes; our studies showed that β 2 integrin activation and α M integrin clustering significantly inhibited IL-13–dependent CD36 mRNA and protein expression, as well as CD36-related foam cell formation. Moreover, IL-13 stimulation of α M -deficient peritoneal macrophages demonstrated an upregulated level of 15-LO induction, CD36 expression, and lipid accumulation as compared with wild-type controls. Conclusions: The adhesion of monocytes/macrophages through activated integrin α M β 2 has a regulatory and potential atheroprotective function during the alternative activation of macrophages.

Monocyte chemoattractant protein-1 (MCP-1) directs migration of blood monocytes to inflamed tissues. Despite the central role of chemotaxis in immune responses, the regulation of chemotaxis by signal transduction pathways and their in vivo significance remain to be thoroughly deciphered. In this study, we examined the intracellular location and functions of two recently identified regulators of chemotaxis, Ca(2+)-independent phospholipase (iPLA(2)beta) and cytosolic phospholipase (cPLA(2)alpha), and substantiate their in vivo importance. These enzymes are cytoplasmic in unstimulated monocytes. Upon MCP-1 stimulation, iPLA(2)beta is recruited to the membrane-enriched pseudopod. In contrast, cPLA(2)alpha is recruited to the endoplasmic reticulum. Although iPLA(2)beta or cPLA(2)alpha antisense oligodeoxyribonucleotide (ODN)-treated monocytes display reduced speed, iPLA(2)beta also regulates directionality and actin polymerization. iPLA(2)beta or cPLA(2)alpha antisense ODN-treated adoptively transferred mouse monocytes display a profound defect in migration to the peritoneum in vivo. These converging observations reveal that iPLA(2)beta and cPLA(2)alpha regulate monocyte migration from different intracellular locations, with iPLA(2)beta acting as a critical regulator of the cellular compass, and identify them as potential targets for antiinflammatory strategies.

We reported previously that interleukin-13 (IL-13) induces tyrosine phosphorylation/activation of Jak2 and Tyk2 kinases and Stats 1, 3, 5, and 6 in primary human monocytes. We recently revealed that p38 MAPK-mediated serine phosphorylation of both Stat1 and Stat3 is required for the induction of 15-lipoxygenase (15-LO) expression by IL-13. In this study, we present data indicating that another serine/threonine kinase, PKCdelta, is also required for IL-13-induced 15-LO expression. PKCdelta, a member of the novel protein kinase C (PKC) subclass, was rapidly phosphorylated and activated upon exposure to IL-13. Treatment of cells with rottlerin, a PKCdelta inhibitor, blocked IL-13-induced 15-LO mRNA and protein expression, whereas Go6976, an inhibitor of the conventional PKC subclass, had no inhibitory effects. Down-regulation of cellular PKCdelta protein levels by PKCdelta-specific antisense oligodeoxyribonucleotides also inhibited 15-LO expression markedly. IL-13-induced 15-LO expression resulted in significant inhibition of synthesis of the potent chemotactic factor leukotriene B4, and that process was reversed by rottlerin, presumably through the blockage of PKCdelta-dependent 15-LO expression. Furthermore, our data demonstrate that IL-13-mediated activation of PKCdelta and p38 MAPK are independent pathways, because inhibition of one kinase activity had no effect on the other, suggesting that the two pathways act in parallel to regulate the downstream targets necessary for 15-LO expression. Inhibition of PKCdelta activation by rottlerin also markedly attenuated IL-13-induced Stat3 DNA binding activity. Our findings indicate that PKCdelta plays an important role in regulating IL-13-induced 15-LO expression in human monocytes and subsequently modulates the inflammatory responses mediated by 15-LO products.

T cells from cancer patients are often functionally impaired, which imposes a barrier to effective immunotherapy. Most pronounced are the alterations characterizing tumor-infiltrating T cells, which in renal cell carcinomas includes defective NF-κB activation and a heightened sensitivity to apoptosis. Coculture experiments revealed that renal tumor cell lines induced a time-dependent decrease in RelA(p65) and p50 protein levels within both Jurkat T cells and peripheral blood T lymphocytes that coincided with the onset of apoptosis. The degradation of RelA/p50 is critical for SK-RC-45-induced apoptosis because overexpression of RelA in Jurkat cells protects against cell death. The loss of RelA/p50 coincided with a decrease in expression of the NF-κB regulated antiapoptotic protein Bcl-xL at both the protein and mRNA level. The disappearance of RelA/p50 protein was mediated by a caspase-dependent pathway because pretreatment of T lymphocytes with a pan caspase inhibitor before coculture with SK-RC-45 blocked RelA and p50 degradation. SK-RC-45 gangliosides appear to mediate this degradative pathway, as blocking ganglioside synthesis in SK-RC-45 cells with the glucosylceramide synthase inhibitor, PPPP, protected T cells from tumor cell-induced RelA degradation and apoptosis. The ability of the Bcl-2 transgene to protect Jurkat cells from RelA degradation, caspase activation, and apoptosis implicates the mitochondria in these SK-RC-45 ganglioside-mediated effects.

CC chemokine receptor 8 (CCR8) has been detected in vitro on type 2 helper and regulatory lymphocytes, which might exert beneficial functions in multiple sclerosis (MS) and on macrophages and microglia, possibly promoting tissue injury in MS lesions. To discriminate the relevant expression pattern in vivo, we defined the cell types that expressed CCR8 in MS lesions and determined the relationship of CCR8 expression and demyelinating activity. CCR8 was not expressed on T cells but was associated with phagocytic macrophages and activated microglia in MS lesions and directly correlated with demyelinating activity. To identify factors associated with CCR8 expression, the study was extended to other central nervous system (CNS) pathologies. CCR8 was consistently expressed on phagocytic macrophages and activated microglia in stroke and progressive multifocal leukoencephalopathy, but not expressed on microglia in pathologies that lacked phagocytic macrophages such as senile change of the Alzheimer’s type. CCR8 was up-regulated by macrophage differentiation and activating stimuli in vitro. In summary CNS CCR8 expression was associated with phagocytic macrophages and activated microglial cells in human CNS diseases, suggesting that CCR8 may be a feasible target for therapeutic intervention in MS. CCR8 expression may also indicate a selective program of mononuclear phagocyte gene expression.

The NADPH oxidase of human monocytes is activated upon exposure to opsonized zymosan and a variety of other stimuli to catalyze the formation of superoxide anion. Assembly of the NADPH oxidase complex is believed to be a highly regulated process, and molecular mechanisms responsible for this regulation have yet to be fully elucidated. We have previously reported that cytosolic phospholipase A2 (cPLA2) expression and activity are essential for superoxide anion production in activated human monocytes. In this study, we investigated the mechanisms involved in cPLA2 regulation of NADPH oxidase activation by evaluating the effects of cPLA2 on translocation and phosphorylation of p67 phox and p47 phox . We report that translocation and phosphorylation of p67 phox , as well as p47 phox , occur upon activation of human monocytes and that decreased cPLA2 protein expression, mediated by antisense oligodeoxyribonucleotides (AS-ODN) specific for cPLA2mRNA, blocked the stimulation-induced translocation of p47 phox and p67 phox from the cytosol to the membrane fraction. Inhibition of translocation of both p47 phox and p67 phox by cPLA2 AS-ODN was above 85%. Arachidonic acid (AA), a product of cPLA2 enzymatic activity, completely restored translocation of both of these oxidase components in the AS-ODN-treated, cPLA2-deficient human monocytes. These results represent the first report that cPLA2activity or AA is required for p67 phox and p47 phox translocation in human monocytes. Although cPLA2 was required for translocation of p47 phox and p67 phox , it did not influence phosphorylation of these components. These results suggest that one mechanism of cPLA2 regulation of NADPH oxidase activity is to control the arachidonate-sensitive assembly of the complete oxidase complex through modulating the translocation of both p47 phox and p67 phox . These studies provide insight into the mechanisms by which activation signals are transduced to allow the induction of superoxide anion production in human monocytes.

Monocytes/macrophages are versatile cells centrally involved in host defense and immunity. Th1 cytokines induce a classical activation program in monocytes/macrophages leading to a proinflammatory M1 macrophage phenotype while Th2 cytokines IL-4 and IL-13 promote monocyte differentiation into an alternatively activated, anti-inflammatory M2 macrophage phenotype. Although monoamine oxidase A (MAO-A) is primarily known for its action in the nervous system, several recent studies have identified MAO-A as a signature marker of alternative activation of monocytes/macrophages. In this brief review we explore the signaling pathways/molecules that regulate MAO-A expression in alternatively activated monocytes/macrophages. We further discuss the contribution of MAO-A to the resolution of inflammation and identify potential therapeutic targets for controlling inflammation. Altogether this review provides deeper insight into the role of MAO-A in alternative activation of monocytes/macrophages and their participation in the inflammatory response.

Mononuclear phagocytes (monocytes, macrophages, and microglia) are considered central to multiple sclerosis (MS) pathogenesis. Molecular cues that mediate mononuclear phagocyte accumulation and activation in the central nervous system (CNS) of MS patients may include chemokines RANTES/CCL5 and macrophage inflammatory protein-1alpha/CCL3. We analyzed expression of CCR1 and CCR5, the monocyte receptors for these chemokines, on circulating and cerebrospinal fluid CD14+ cells, and in MS brain lesions. Approximately 70% of cerebrospinal fluid monocytes were CCR1+/CCR5+, regardless of the presence of CNS pathology, compared to less than 20% of circulating monocytes. In active MS lesions CCR1+/CCR5+ monocytes were found in perivascular cell cuffs and at the demyelinating edges of evolving lesions. Mononuclear phagocytes in early demyelinating stages comprised CCR1+/CCR5+ hematogenous monocytes and CCR1-/CCR5- resident microglial cells. In later stages, phagocytic macrophages were uniformly CCR1-/CCR5+. Cultured in vitro, adherent monocytes/macrophages up-regulated CCR5 and down-regulated CCR1 expression, compared to freshly-isolated monocytes. Taken together, these findings suggest that monocytes competent to enter the CNS compartment derive from a minority CCR1+/CCR5+ population in the circulating pool. In the presence of ligand, these cells will be retained in the CNS. During further activation in lesions, infiltrating monocytes down-regulate CCR1 but not CCR5, whereas microglia up-regulate CCR5.

Monocyte chemoattractant protein 1 (MCP-1) has an important influence on monocyte migration into sites of inflammation. Our understanding of the signal transduction pathways involved in the response of monocytes to MCP-1 is quite limited yet potentially significant for understanding and manipulating the inflammatory response. Prior studies have demonstrated a crucial regulatory role for cytosolic phospholipase A2 (cPLA2) in monocyte chemotaxis to MCP-1. In these studies we investigated the role for another PLA2, calcium-independent PLA2 (iPLA2) in comparison to cPLA2. Pharmacological inhibitors of PLA2 were found to substantially inhibit chemotaxis. Using antisense oligodeoxyribonucleotide treatment we found that iPLA2 expression is required for monocyte migration to MCP-1. Complete blocking of the chemotactic response was observed with inhibition of either iPLA2 or cPLA2 expression by their respective antisense oligodeoxyribonucleotide. In reconstitution experiments, lysophosphatidic acid completely restored MCP-1-stimulated migration in iPLA2-deficient monocytes, whereas lysophosphatidic acid was without effect in restoring migration in cPLA2-deficient monocytes. To the contrary, arachidonic acid fully restored migration of cPLA2-deficient monocytes while having no effect on the iPLA2-deficient monocytes. Additional studies revealed that neither enzyme appears to be upstream of the other indicating that iPLA2 and cPLA2 represent parallel regulatory pathways. These data demonstrate novel and distinct roles for these two phospholipases in this critical step in inflammation.

The Janus kinase, JAK3 plays an important role in interleukin-2 (IL-2)–dependent signal transduction and proliferation of T lymphocytes. Our findings show that prostaglandin E2(PGE2) can inhibit upregulation of JAK3 protein in naive T cells and can downregulate its expression in primed cells. Reduction in JAK3 was selective because expression of other tyrosine kinases (JAK1, p56lck, and p59fyn) and signal transducer and activator of transcription (STAT)5, which are linked to IL-2 receptor (IL-2R) signaling pathway, were not affected. Inhibition of JAK3 may be controlled by intracellular cyclic adenosine monophosphate (cAMP) levels, as forskolin, a direct activator of adenylate cyclase and dibutyryl cAMP (dbcAMP), a membrane permeable analogue of cAMP suppressed JAK3 expression. Moreover, 3-isobutyl-1-methylxanthine (IBMX), an inhibitor of cAMP phosphodiesterase, potentiated PGE2-induced suppression of JAK3. In naive T cells, but not primed T cells, PGE2 and other cAMP elevating agents also caused a modest reduction in surface expression of the common gamma chain (γc) that associates with JAK3. The absence of JAK3, but not IL-2R in T cells correlated with impaired IL-2–dependent signal transduction and proliferation. The alteration in IL-2 signaling included decreased tyrosine phosphorylation and DNA binding activity of STAT5 and poor induction of the c-Myc and c-Jun pathways. In contrast, IL-2–dependent induction of Bcl-2 was unaffected. These findings suggest that suppression of JAK3 levels may represent one mechanism by which PGE2 and other cAMP elevating agents can inhibit T-cell proliferation.

Abstract There is considerable evidence to suggest that cytokines modulate the pathological cellular events that occur in human atherosclerosis. We sought to determine the effects of T-helper-lymphocyte (T H )-1- and T H 2-type cytokines on the ability of human monocytes to oxidize LDL, one of the pathological processes believed to occur in atherosclerosis. The ability of opsonized zymosan (ZOP)-activated human monocytes to oxidize LDL in a 24-hour period was significantly enhanced by pretreatment of the monocytes with the T H 2 cytokines, interleukin (IL)-4, or IL-13 compared with untreated monocytes. In contrast, interferon (IFN)-γ, a T H 1 cytokine, inhibited LDL oxidation by activated monocytes. Treatment with IFN-γ also prevented the IL-4- and IL-13-mediated enhancement of LDL oxidation by ZOP-activated monocytes. Untreated or cytokine-treated unactivated monocytes did not oxidize LDL. The enhancement of LDL oxidation mediated by IL-4 or IL-13 treatment was not due to a mitogenic effect of the cytokines on the monocytes, nor to modulation of superoxide anion (O 2 − ) production. The cytokine regulation of 15-lipoxygenase (LO) in the monocytes was also examined. IL-4 and IL-13 induction of 15-LO mRNA and 15-LO activity in the monocytes was confirmed, as was the previously reported inhibition of induction by IFN-γ. In summary, IL-4 and IL-13 enhance the ability of activated human monocytes to oxidize LDL, whereas IFN-γ inhibits the cell-mediated oxidation. The up- and downregulation of activated monocyte-mediated LDL oxidation by these cytokines correlates with the expression of 15-LO activity. Considerable evidence suggests that the progression of atherosclerosis includes events that are immunologically mediated, lending potential physiological relevance to these in vitro observations.

Our previous studies have shown that monocyte activation and release of O-2 are required for monocyte-mediated low density lipoprotein (LDL) lipid oxidation. We have also found that intracellular Ca2+ levels and protein kinase C activity are requisite participants in this potentially pathogenic process. In these studies, we further investigated the mechanisms involved in the oxidation of LDL lipids by activated human monocytes, particularly the potential contributions of the cytosolic phospholipase A2 (cPLA2) signaling pathway. The most well-studied cPLA2, has a molecular mass of 85 kDa and has been reported to be regulated by both Ca2+ and phosphorylation. We found that cPLA2 protein levels and cPLA2 enzymatic activity were induced upon activation of human monocytes by opsonized zymosan. Pharmacologic inhibition of cPLA2 activity by AACOCF3, which has been reported to be a specific inhibitor of cPLA2 as compared with sPLA2, caused a dose-dependent inhibition of cPLA2 enzymatic activity and LDL lipid oxidation by activated human monocytes, whereas sPLA2 activity was not affected. To corroborate these findings, we used specific antisense oligonucleotides to inhibit cPLA2. We observed that treatment with antisense oligonucleotides caused suppression of both cPLA2 protein expression and enzymatic activity as well as monocyte-mediated LDL lipid oxidation. Furthermore, antisense oligonucleotide treatment caused a substantial inhibition of O-2 production by activated human monocytes. In parallel experimental groups, cPLA2 sense oligonucleotides did not affect cPLA2 protein expression, cPLA2 enzymatic activity, O-2 production, or monocyte-mediated LDL lipid oxidation. These studies support the proposal that cPLA2 activity is required for activated monocytes to oxidize LDL lipids.

Aims Atherosclerosis is a chronic inflammatory disorder of cholesterol deposition in monocyte-derived macrophages (MDM) within the arterial wall leading to impingement on the lumen of the vessel. In atherosclerotic lesions, MDM are the primary source of NADPH oxidase-derived superoxide anion (O2−) inducing low-density lipoprotein (LDL) oxidation leading to their unregulated uptake of oxidized LDL and foam cell formation. We recently discovered that zymosan potently activates monocyte NADPH oxidase via the non-toll pattern recognition receptor (PRR), Dectin-1. Other PRRs bind endogenous human ligands, yet no such ligands have been identified for Dectin-1. Our hypothesis was that inflammation generates endogenous ligands for Dectin-1 that activate O2− production and thereby contributes to atherogenesis. Methods and results Human: anti-zymosan antibodies were used to identify similar, cross-reactive epitopes in human atherosclerotic tissue extracts. Immunoblot analysis revealed consistent antibody reactive protein bands on one- and two-dimensional gel electrophoreses. Vimentin was identified by mass spectrometry in the immunoreactive bands across different tissue samples. Direct binding of vimentin to Dectin-1 was observed using BIACORE. Further data revealed that vimentin induces O2− production by human monocytes. Analysis of human atherosclerotic lesions revealed that vimentin was detected extracellularly in the necrotic core and in areas of active inflammation. Vimentin also co-localized with Dectin-1 in macrophage-rich regions where O2− is produced. Conclusion We conclude that vimentin is an endogenous, activating ligand for Dectin-1. Its presence in areas of artery wall inflammation and O2− production suggests that vimentin activates Dectin-1 and contributes to the oxidation of lipids and cholesterol accumulation in atherosclerosis.

Upon activation, human peripheral blood monocytes and U937 cells oxidized low density lipoprotein (LDL), converting it to a cytotoxin. The oxidized LDL loses its ability to interact specifically with the native LDL (apoB/E) receptor and becomes a ligand for the scavenger receptors and two other receptors, Fc gamma RII (CD32) and CD36. We performed a series of studies to evaluate the potential contribution of each of these receptors to the process of monocyte-mediated LDL oxidation. To assess the participation of the apoB/E receptor, we tested the ability of activated human monocytes to oxidize LDL after up- and down-regulation of apoB/E receptors. Neither up-regulation nor down-regulation of the apoB/E receptor significantly modified the level of LDL lipid oxidation. Acetylated LDL, a ligand for scavenger receptors, was also oxidized by the activated monocytes. Methylated LDL, a chemically modified LDL that is not recognized by the apoB/E or scavenger receptors, was oxidized as well. Thus, LDL does not need to interact with either the apoB/E receptor or scavenger receptors in order to undergo lipid oxidation. Additionally, monoclonal antibodies to CD36 and CD32 were used to block these two receptors that recognize oxidized LDL. Although both antibodies interfered with oxidized LDL binding to these receptors, neither treatment interfered with LDL lipid oxidation mediated by activated human monocytes. Our results suggest that interaction with these receptors is not a requirement for LDL lipid oxidation by activated human monocytes.

Oxidized LDL is present in human atherosclerotic lesions, but the mechanisms responsible for oxidation in vivo have not been definitively demonstrated. Circumstantial evidence has implicated the enzyme 15-lipoxygenase as a contributor to the formation of oxidized lipids in this disease. To assess whether oxidized lipids are indeed formed by the action of 15-lipoxygenase on polyunsaturated fatty acids (PUFAs) in vivo, we have used a sensitive and specific method (chiral phase HPLC) to analyze the lipid oxidation products present in human atherosclerotic lesions. Human 15-lipoxygenase is an omega-6 lipoxygenase that has previously been shown to oxidize esterified PUFA in a stereospecific manner, forming predominantly cholesteryl hydroperoxy-octadecadienoate (13(S)-HPODE) from cholesteryl linoleate substrate in LDL. This property allows its activity to be distinguished from nonenzymatic oxidation, which results in the formation of equal quantities of the S and R stereoisomers of the same oxidation product. A total of 80 specimens of human atherosclerotic plaque were analyzed. Esterified, oxidized linoleate was purified from human atherosclerotic lesions and from LDL oxidized by copper, and the chirality of these oxidation products was compared. There was significantly greater stereospecificity of oxidation in the oxidized linoleate from human atherosclerotic lesions. Even greater stereospecificity was detected in the HPODE derived from cholesteryl ester, purified from human lesions. Cholesteryl HPODE is the primary oxidation product from cholesteryl linoleate, the major esterified PUFA that accumulates in atherosclerotic vessels. Cholesteryl HPODE and its reduced form, cholesteryl hydroxy-octadecadienoate, were detected in all lesions analyzed. Neither the stereospecificity of oxidation nor the percentage of available substrate oxidized to primary oxidation products was correlated with the stage of disease of the lesions examined. We conclude that 15-lipoxygenase contributes to the formation of oxidized lipids in human atherosclerotic lesions.

IL-13 is a potent stimulator of alternative monocyte/macrophage activation. During alternative activation, the expression of several proteins is induced including 15-lipoxygenase (15-LO), a lipid-peroxidating enzyme and the scavenger receptor CD36. We previously reported that α(M)β(2) integrin activation or clustering suppresses the expression of both 15-LO and CD36. In this study we focused on exploring the molecular mechanisms that down-regulate CD36 expression and CD36-mediated foam cell formation in IL-13-stimulated monocytes/macrophages after α(M)β(2) activation. Our studies reveal that α(M)β(2) integrin activation inhibits the IL-13 activation of several critical pathways that are required for macrophage alternative activation; namely, blocking Jak2 and Tyk2 phosphorylation, which bind to the cytoplasmic tails of the IL-4Rα/IL-13Rα1 complex. This leads to the inhibition of tyrosine phosphorylation of Stats (Stat1, Stat3, and Stat6) and prevents the formation of a signaling complex (containing p38MAPK, PKCδ, and Stat3) that are critical for the expression of both 15-LO and CD36. Jak2-mediated Hck activation is also inhibited, thereby preventing Stats serine phosphorylation, which is essential for downstream Stat-dependent gene transcription. Moreover, inhibition of Jak2, Tyk2, or their downstream target 15-LO with antisense oligonucleotides profoundly inhibits IL-13-induced CD36 expression and CD36-dependent foam cell formation, whereas13(S) Hydroperoxyoctadecadienoic acid (HPODE), a 15-LO product and peroxisome proliferator-activated receptor-γ ligand, completely restores CD36 expression in monocytes treated with 15-LO antisense. α(M)β(2) integrin activation controls CD36 expression and foam cell formation in alternatively activated monocyte/macrophages by blocking Tyk2/Jak2 phosphorylation via a 15-LO-dependent pathway. The discovery of this mechanism helps our understanding of the potential role of alternatively activated macrophages in atherogenesis and highlights the impact of integrin α(M)β(2) on this process.

Monocytes/macrophages are innate immune cells that play a crucial role in the resolution of inflammation. In presence of Th2 cytokines interleukin-4 (IL-4) and interleukin-13 (IL-13), they display an anti-inflammatory profile and this activation pathway is known as alternative activation. In this study we compare and differentiate pathways mediated by IL-4 and IL-13 activation of human monocytes/macrophage. Here we report differential regulation of IL-4 and IL-13 signaling in monocytes/macrophages starting from IL-4/IL-13 cytokine receptors to Jak-Stat-mediated signaling pathways that ultimately control expression of several infl1ammatory genes. Our data demonstrate that while the receptor-associated tyrosine kinases Jak2 and Tyk2 are activated after the recruitment of IL-13 to its receptor (containing IL-4Rα and IL-13Rα1), IL-4 stimulates Jak1 activation. We further show that Jak2 is upstream of Stat3 activation and Tyk2 controls Stat1 and Stat6 activation in response to IL-13 stimulation. In contrast, Jak1 regulates Stat3 and Stat6 activation in IL-4-induced monocytes. Our results further reveal that while IL-13 utilizes both IL-4Rα-Jak2-Stat3 and IL-13Rα1-Tyk2-Stat1/Stat6 signaling pathways, IL-4 can only use the IL-4Rα-Jak1-Stat3/Stat6 cascade to regulate the expression of some critical inflammatory genes including 15-lipoxygenase (15-LO), monoamine oxidase A (MAO-A) and scavenger receptor CD36. Moreover, we demonstrate here that IL-13 and IL-4 can uniquely affect the expression of particular genes like dual specificity phosphatase 1 (DUSP1) and tissue inhibitor of metalloprotease-3 (TIMP3) and do so through different Jak kinaes. As evidence of differential regulation of gene function by IL-4 and IL-13, we further report that MAO-A-mediated reactive oxygen species (ROS) generation is influenced by different Jak kinases. Collectively, these results have major implications for understanding the mechanism and function of alternatively activated monocytes/macrophages by IL-4 and IL-13 and add novel insights into the pathogenesis and potential treatment of different inflammatory diseases.

It has come to the attention of the American Heart Association (AHA), in a public manner,1⇓–3 that there are …

A workshop organized by the Society for Leukocyte Biology offered advice to young scientists on how to decipher the grant-submission process of the US National Institutes of Health and compose a clear, compelling and fundable grant.

Low density lipoprotein that was oxidized by activated human monocytes was analyzed to determine the identity of oxidized fatty acids present and the conditions required for their formation. The oxidized lipids were also analyzed under conditions allowing preservation of their oxidation state. Using reversed-phase high performance liquid chromatography (HPLC) analysis of native and saponified lipid extracts of oxidized low density lipoprotein (LDL), we found that the major fatty acid oxidation product was esterified hydroperoxyoctadecadienoic acid (HPODE), the oxidized product of the most abundant polyunsaturated fatty acid in human LDL, linoleic acid. Although some esterified hydroxyoctadecadienoic acid (HODE) was also detected, the reduction of HPODE to HODE did not appear to be monocyte-dependent. Essentially all of the HPODE was found to be esterified with the majority being esterified to cholesterol followed by phospholipids and generally following the abundance of esterified linoleic acid within the lipid classes. The percent of cholesteryl linoleate converted to cholesteryl HPODE and cholesteryl HODE at the end of the 24-h incubation was determined to be approximately 13.5%. The formation of oxidized esterified linoleic acid in the LDL was shown to require immunological activation of the human monocytes, a previously observed requirement for general LDL oxidation in this culture system. The oxidized esterified linoleic acid was present in the supernatant with the LDL and was not cell-associated. HPODE formation on LDL was prevented by including superoxide dismutase (SOD) or eicosatetraynoic acid (ETYA) during the 24-h coincubation of activated monocytes with LDL whereas indomethacin was without effect. The analysis of the lipid oxidation products in oxidized LDL can provide insight into the mechanisms involved in oxidation of LDL by activated human monocytes.

Our previous studies have shown that human monocytes can oxidize native low density lipoprotein (LDL) and transform it to a cytotoxin. We also found that intracellular Ca2+ levels are integrally involved in lipid oxidation of LDL by activated monocytes. In these studies, we investigated the protein kinase C (PKC) signaling pathway for its contribution to the process of monocyte oxidation of LDL lipids. We found substantial protein phosphorylation induced upon monocyte activation. Pharmacologic inhibition of PKC activity with the PKC inhibitors H-7 (1-100 microM), calphostin C (1-10 microM), and GF109203X (0.1-10 microM) caused a dose-dependent inhibition of cellular protein phosphorylation, including that of several previously identified PKC substrates. These inhibitors of PKC activity also substantially inhibited LDL lipid oxidation by activated monocytes. This inhibition was correlated with a profound suppression of superoxide anion production by these cells. In contrast, inhibition of cAMP-dependent protein kinase activity altered neither monocyte-mediated LDL lipid oxidation nor O2- production by activated monocytes. Delaying the addition of PKC inhibitors until after the peak production of O2-, which occurs during the respiratory burst, still resulted in inhibition of LDL lipid oxidation, suggesting roles for PKC in both early and late events. To corroborate these findings using other approaches, we used phorbol 12-myristate 13-acetate to down-regulate PKC activity and also used antisense oligonucleotides as specific PKC inhibitors. Results of both types of studies support the conclusion that PKC activity is required for activated monocytes to oxidize LDL lipids. Thus, PKC activation in this system is essential, one critical pathway regulated by PKC activity is the production of O2-, and continued PKC activity is required for optimal oxidation of LDL lipids.

IL-13 is a Th2 cytokine that promotes alternative activation (M2 polarization) in primary human monocytes. Our studies have characterized the functional IL-13 receptor complex and the downstream signaling events in response to IL-13 stimulation in alternatively activated monocytes/macrophages. In this report, we present evidence that IL-13 induces the activation of a Src family tyrosine kinase, which is required for IL-13 induction of M2 gene expression, including 15-lipoxygenase (15-LO). Our data show that Src kinase activity regulates IL-13-induced p38 MAPK tyrosine phosphorylation via the upstream kinases MKK3 or MKK6. Our findings also reveal that the IL-13 receptor-associated tyrosine kinase Jak2 is required for the activation of both Src kinase as well as p38 MAPK. Further, we found that Src tyrosine kinase-mediated activation of p38 MAPK is required for Stat1 and Stat3 serine 727 phosphorylation in alternatively activated monocytes/macrophages. Additional studies identify Hck as the specific Src family member, stimulated by IL-13 and involved in regulating both p38 MAPK activation and p38 MAPK-mediated 15-LO expression. Finally we show that the Hck regulates the expression of other alternative state (M2)-specific genes (Mannose receptor, MAO-A, and CD36) and therefore conclude that Hck acts as a key regulator controlling gene expression in alternatively activated monocytes/macrophages.

The alternative activation of monocytes by interleukin (IL)-13 and IL-4 is a significant component of the inflammatory response. The consequences of alternative activation in inflammatory diseases remain to be determined.In this report, we explored how integrins, receptors important for monocyte migration to inflammatory sites, regulate IL-13-mediated monocyte activation. We focused on the analysis of 2 proteins, which are upregulated during the alternative activation and are important for the development of atherosclerosis, an oxidative enzyme 15-lipoxygenase (15-LO) and a scavenger receptor CD36.We found that adhesion of resting monocytes through β(2) integrins and inside-out activation of β(2) integrins by monocyte chemoattractant protein-1 did not change IL-13-stimulated 15-LO upregulation; however, preincubation of monocytes with the antibody MEM48, which generates full activation of β(2) integrins, significantly inhibited 15-LO mRNA and protein expression. In contrast, activation of β(1) integrins had no effect on 15-LO expression. Analysis of integrin clustering through α(M), α(L), α(X), and α(D) subunits demonstrated the pivotal role for integrin α(M)β(2) in inhibiting 15-LO expression. IL-13 treatment upregulates 15-LO-dependent CD36 expression on human monocytes; our studies showed that β(2) integrin activation and α(M) integrin clustering significantly inhibited IL-13-dependent CD36 mRNA and protein expression, as well as CD36-related foam cell formation. Moreover, IL-13 stimulation of α(M)-deficient peritoneal macrophages demonstrated an upregulated level of 15-LO induction, CD36 expression, and lipid accumulation as compared with wild-type controls.The adhesion of monocytes/macrophages through activated integrin α(M)β(2) has a regulatory and potential atheroprotective function during the alternative activation of macrophages.

Lipoxygenase (LO) activity has been implicated in the process by which activated human monocytes oxidize normal human low density lipoprotein (LDL) and render it toxic to target cells. Here we examined the role of 5-LO in activated monocyte-mediated LDL modification. Five putative inhibitors of 5-LO (A63162, CGS8515, PF5901, RG6866, and MK886) were used to determine if they prevented activated monocytes from oxidizing LDL. Only RG6866, A63162, and CGS8515 inhibited monocyte-mediated LDL oxidation. Nonspecific effects of these drugs on LDL oxidation by activated monocytes were examined. RG6866 and A63162 were both found to be general antioxidants at their effective concentrations. CGS8515 was toxic at its effective concentration. A63162, CGS8515, and RG6866 also inhibited 15-LO activity in vitro. MK886 and PF5901 did not exhibit the nonspecific effects above and did not inhibit monocyte-mediated LDL oxidation, whereas both MK886 and PF5901 inhibited production of 5-LO metabolites by activated monocytes at concentrations that had no effect on LDL oxidation by the activated monocytes. Since neither of these agents inhibited LDL oxidation, we conclude that 5-LO is not involved in human monocyte oxidation of LDL. The possibility that a cellular 12- or 15-LO is involved in human monocyte-mediated LDL oxidation remains to be evaluated.

IL-13 induces profound expression of 15-lipoxygenase (15-LO) in primary human monocytes. Our studies have defined the functional IL-13R complex, association of Jaks with the receptor components, and the tyrosine phosphorylation of several Stat molecules in response to IL-13. Furthermore, we identified both p38MAPK and protein kinase Cδ as critical regulators of 15-LO expression. In this study, we report an ERK1/2-dependent signaling cascade that regulates IL-13–mediated 15-LO gene expression. We show the rapid phosphorylation/activation of ERK1/2 upon IL-13 exposure. Our results indicate that Tyk2 kinase is required for the activation of ERK1/2, which is independent of the Jak2, p38MAPK, and protein kinase Cδ pathways, suggesting bifurcating parallel regulatory pathways downstream of the receptor. To investigate the signaling mechanisms associated with the ERK1/2-dependent expression of 15-LO, we explored the involvement of transcription factors, with predicted binding sites in the 15-LO promoter, in this process including Elk1, early growth response-1 (Egr-1), and CREB. Our findings indicate that IL-13 induces Egr-1 nuclear accumulation and CREB serine phosphorylation and that both are markedly attenuated by inhibition of ERK1/2 activity. We further show that ERK1/2 activity is required for both Egr-1 and CREB DNA binding to their cognate sequences identified within the 15-LO promoter. Furthermore, by transfecting monocytes with the decoy oligodeoxyribonucleotides specific for Egr-1 and CREB, we discovered that Egr-1 and CREB are directly involved in regulating 15-LO gene expression. These studies characterize an important regulatory role for ERK1/2 in mediating IL-13–induced monocyte 15-LO expression via the transcription factors Egr-1 and CREB.

We have compared various mitogenic stimuli for their ability to induce hydrogen peroxide (H2O2) and superoxide anion (O2-) production by PBMC and the effect of these reactive oxygen species and hydroxyl radical (OH.) has been assessed on proliferation. Our results show that pokeweed mitogen (PWM) stimulated PBMC to release H2O2 which interfered with proliferation since inclusion of catalase enhanced PBMC thymidine uptake. In contrast, phytohemagglutinin (PHA) and monoclonal antibody to CD3 (alpha CD3) did not induce PBMC to generate H2O2. O2- release by PBMC, which is readily induced by phorbol myristate acetate (PMA), did not occur when the cells were stimulated with PWM, PHA, or alpha CD3. In correlation, the O2- scavenger enzyme superoxide dismutase (SOD) had no effect on the proliferative response of the cells to the same mitogens, whereas it impaired the thymidine uptake of PMA-stimulated PBMC. A regulatory role for OH. was implied by studies using a battery of OH scavengers known to inhibit PMA-stimulated PBMC proliferation. OH. scavengers markedly inhibited the lymphoblastic transformation of alpha CD3-stimulated cells but had little or no effect on PHA- and PWM-stimulated PBMC. Thus, one manner by which PBMC proliferation is regulated is through oxygen free radical production which varies depending on the type of mitogenic stimulus.

We have been studying the mechanisms involved in the oxidative modification of low density lipoprotein (LDL) that lead to its transformation to a cytotoxic complex. Here we examine the direct effect-of soybean lipoxygenase (SLO), a 15-lipoxygenase, on normal human LDL. SLO oxidized LDL and rendered it cytotoxic; agents known to interfere with lipoxygenase activity inhibited this reaction. Enhancement of both the SLO-mediated LDL oxidation and the conversion of LDL to a cytotoxin was observed when either superoxide dismutase or copper (II) (3,5,-diisopropylsalicylic acid)2, both of which dismute superoxide anion, were included during the incubation of SLO with LDL. In contrast, catalase inhibited this reaction in the presence or absence of agents that dismute superoxide anion. Thus, purified lipoxygenase can mediate LDL modification and superoxide anion inhibits this reaction, Furthermore, H2O2 is essential for SLO-mediated LDL oxidation and conversion of LDL to a cytotoxin.

Signal-activated phospholipases are a recent focus of the rapidly growing field of lipid signaling. The extent of their impact on the pathways regulating diverse cell functions is beginning to be appreciated. A critical step in inflammation is the attraction of leukocytes to injured or diseased tissue. Chemotaxis of leukocytes, a requisite process for monocyte and neutrophil extravasation from the blood into tissues, is a critical step for initiating and maintaining inflammation in both acute and chronic settings. Recent studies have identified new important and required roles for two signal-activated phospholipases A2 (PLA2) in regulating chemotaxis. The two intracellular phospholipases, cPLA2alpha (Group IVA) and iPLA2beta (Group VIA), act in parallel to provide distinct lipid mediators at different intracellular sites that are both required for leukocytes to migrate toward the chemokine monocyte chemoattractant protein-1. This review will summarize the separate roles of these phospholipases as well as what is currently known about the influence of two other classes of intracellular signal-activated phospholipases, phospholipase C and phospholipase D, in regulating chemotaxis in eukaryotic cells, but particularly in human monocytes. The contributions of these phospholipases to chemotaxis both in vitro and in vivo will be highlighted.

Identification of novel signal transduction pathways regulating monocyte chemotaxis can indicate unique targets for preventive therapies for treatment of chronic inflammatory diseases. To aid in this endeavor we report conditions for optimal transfection of primary human monocytes coupled with a new model system for assessing their chemotactic activity in vivo. This method can be used as a tool to identify the relevant signal transduction pathways regulating human monocyte chemotaxis to MCP-1 in the complex in vivo environment that were previously identified to regulate chemotaxis in vitro. MCP-1-dependent chemotaxis of monocytes is studied in an adoptive transfer model where human monocytes transfected with mutant cDNAs are transferred to mice followed by initiation of peritonitis. Harvesting peritoneal cells at 24 h diminishes the contribution of immunologic responses to the cross-species transfer. Validation of relevant regulatory molecules in vivo is critical for understanding the most relevant therapeutic targets for drug development.

Background—Signal transducer and activator of transcription 1 (Stat1) potently regulates gene expression after stimulation by certain cytokines involved in tumorigenesis and host defenses. The present study investigated a novel role for Stat1 in foam cell formation and atherosclerosis.Methods and Results—Inhibition of Stat1 activity by a Stat1-specific DNA “decoy” oligomer transfected into differentiated human THP-1 cells, and deficiency ofstat1in mouse macrophages significantly inhibited foam cell formation assessed by lipid staining and cholesteryl ester accumulation compared with control cells. The mechanism of Stat1 regulation of foam cell formation was uniquely dependent on the scavenger receptor CD36. Blunted Stat1 activity andstat1deficiency significantly decreased expression of CD36 but not of scavenger receptor-A compared with controls, as assessed by immunoblotting and flow cytometry. Deficiency of CD36 but not scavenger receptor-A in mouse macrophages removed any dependency of foam cell formation on Stat1. In an intraperitoneal model of foam cell formation in which foam cells form in vivo independently of the model ligands used in vitro,stat1deficiency significantly inhibited foam cell formation and CD36 expression. Transplantation of bone marrow fromapolipoprotein e−/−×stat1−/−mice into lethally irradiated, atherosclerosis-susceptibleapolipoprotein e−/−recipients significantly reduced both en face aortic lesion coverage and aortic root lesions compared with recipients of bone marrow from genetically matchedapolipoprotein e−/−mice.Conclusions—Stat1 regulates CD36 expression and foam cell formation in macrophages in vitro; the Stat1 regulation of foam cell formation requires CD36. The regulation of CD36 expression by Stat1 may be important in other pathophysiological CD36-dependent events. Stat1 deficiency reduces atherosclerosis in anapolipoprotein e−/−atherosclerosis-susceptible bone marrow transplantation model.

Our previous studies demonstrated that the IL-13-induced 15-lipoxygenase expression in primary human monocytes is regulated by the activation of both Stat1 and Stat3 and by protein kinase C (PKC)δ. IL-13 stimulated the phosphorylation of Stat3 on both Tyr705 and Ser727. In this study we show that IL-13 induces the association of PKCδ with Stat3, not with Stat1, and is required for Stat3 Ser727 phosphorylation. We found a novel IL-13-dependent cytosolic signaling complex of PKCδ and tyrosine-phosphorylated Stat3. A tyrosine kinase inhibitor blocked PKCδ association with Stat3 as well as Stat3 Ser727 phosphorylation. We therefore hypothesized that tyrosine phosphorylation was required for Stat3 interaction with PKCδ and subsequent PKCδ-dependent phosphorylation of Stat3 Ser727. We developed an efficient transfection protocol for human monocytes. Expression of Stat3 containing a mutation in Tyr705 inhibited the association of PKCδ with Stat3 and blocked Stat3 Ser727 phosphorylation, whereas transfection with wild-type Stat3 did not. Furthermore, by transfecting monocytes with Stat3 containing mutations in Tyr705 or Ser727 or with wild-type Stat3, we demonstrated that both Stat3 tyrosine and serine phosphorylations are required for optimal binding of Stat3 with DNA and maximal expression of 15-lipoxygenase, an important regulator of inflammation and apoptosis.

Phospholipases A(2) (PLA(2)) are potent regulators of the inflammatory response. We have observed that Group IV cPLA(2) activity is required for the production of superoxide anion (O(2)(-)) in human monocytes [Li Q., Cathcart M.K. J. Biol. Chem. 272 (4) (1997) 2404-2411.]. We have previously identified PKCalpha as a kinase pathway required for monocyte O(2)(-) production [Li Q., Cathcart M.K. J. Biol. Chem. 269 (26) (1994) 17508-17515.]. We therefore investigated the potential interaction between PKCalpha and cPLA(2) by evaluating the requirement for specific PKC isoenzymes in the process of activating cPLA(2) enzymatic activity and protein phosphorylation upon monocyte activation. We first showed that general PKC inhibitors and antisense oligodeoxyribonucleotides (ODN) to the cPKC group of PKC enzymes inhibited cPLA(2) activity. To distinguish between PKCalpha and PKCbeta isoenzymes in regulating cPLA(2) protein phosphorylation and enzymatic activity, we employed our previously characterized PKCalpha or PKCbeta isoenzyme-specific antisense ODN [Li Q., Subbulakshmi V., Fields A.P., Murray, N.R., Cathcart M.K., J. Biol. Chem. 274 (6) (1999) 3764-3771]. Suppression of PKCalpha expression, but not PKCbeta expression, inhibited cPLA(2) protein phosphorylation and enzymatic activity. Additional studies ruled out a contribution by Erk1/2 to cPLA(2) phosphorylation and activation. We also found that cPLA(2) co-immunoprecipitated with PKCalpha and vice versa. In vitro studies demonstrated that PKCalpha could directly phosphorylate cPLA(2).and enhance enzymatic activity. Finally, we showed that addition of arachidonic acid restored the production of O(2)(-) in monocytes defective in either PKCalpha or cPLA(2) expression. Taken together, our data suggest that PKCalpha, but not PKCbeta, is the predominant cPKC isoenzyme required for cPLA(2) protein phosphorylation and maximal induction of cPLA(2) enzymatic activity upon activation of human monocytes. Our data also support the concept that the requirements for PKCalpha and cPLA(2) in O(2)(-) generation are solely due to their seminal role in generating arachidonic acid.

Phosphorylation of the reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase components p67phox and p47phox accompanies the assembly and activation of this enzyme complex. We have previously reported that activation of human monocytes with opsonized zymosan (ZOP), a potent stimulator of NADPH oxidase activity, results in the phosphorylation of p67phox and p47phox. In this study, we investigated the regulation of p67phox phosphorylation. Although protein kinase C (PKC)α has previously been shown to regulate NADPH oxidase activity, we found that inhibition of PKCα had no effect on p67phox phosphorylation. Our studies demonstrate that pretreatment of monocytes with antisense oligodeoxyribonucleotides specific for PKCδ or rottlerin, a selective inhibitor for PKCδ, inhibited the phosphorylation of p67phox in monocytes, and Go6976, a specific inhibitor for conventional PKCs, PKCα and PKCβ, had no such inhibitory effect. Additional studies indicate that ZOP stimulation of monocytes induces PKCδ and p67phox to form a complex. We also demonstrate that lysates from activated monocytes as well as PKCδ immunoprecipitates from activated monocytes can phosphorylate p67phox in vitro and that pretreatment of monocytes with rottlerin blocked the phosphorylation in each case. We further show that recombinant PKCδ can phosphorylate p67phox in vitro. Finally, we show that PKCδ-deficient monocytes produce significantly less superoxide anion in response to ZOP stimulation, thus emphasizing the functional significance of the PKCδ regulation of p67phox phosphorylation. Taken together, this is the first report to describe the requirement of PKCδ in regulating the phosphorylation of p67phox and the related NADPH oxidase activity in primary human monocytes.

Our laboratory is interested in understanding the regulation of NADPH oxidase activity in human monocyte/macrophages. Protein kinase C (PKC) is reported to be involved in regulating the phosphorylation of NADPH oxidase components in human neutrophils; however, the regulatory roles of specific isoforms of PKC in phosphorylating particular oxidase components have not been determined. In this study calphostin C, an inhibitor for both novel PKC (including PKCδ, -ε, -θ, and -η) and conventional PKC (including PKCα and -β), inhibited both phosphorylation and translocation of p47phox, an essential component of the monocyte NADPH oxidase. In contrast, GF109203X, a selective inhibitor of classical PKC and PKCε, did not affect the phosphorylation or translocation of p47phox, suggesting that PKCδ, -θ, or -η is required. Furthermore, rottlerin (at doses that inhibit PKCδ activity) inhibited the phosphorylation and translocation of p47phox. Rottlerin also inhibited O⨪2 production at similar doses. In addition to pharmacological inhibitors, PKCδ-specific antisense oligodeoxyribonucleotides were used. PKCδ antisense oligodeoxyribonucleotides inhibited the phosphorylation and translocation of p47phox in activated human monocytes. We also show, using the recombinant p47phox-GST fusion protein, that p47phox can serve as a substrate for PKCδ in vitro. Furthermore, lysate-derived PKCδ from activated monocytes phosphorylated p47phox in a rottlerin-sensitive manner. Together, these data suggest that PKCδ plays a pivotal role in stimulating monocyte NADPH oxidase activity through its regulation of the phosphorylation and translocation of p47phox.