Role of guanine nucleotide regulatory protein in polyphosphoinositide degradation and activation of phagocytic leukocytes by chemoattractants

Leukocyte activation by chemoattractants provides an important model to study the biochemical mechanisms of stimulus-response coupling in these cells. Well-defined chemotactic factors induce readily quantifiable responses in phagocytic leukocytes. These include directed migration and the production and release of toxic substances including oxygen radicals and lysosomal enzymes. The development of radiolabeled synthetic oligopeptides with potent chemotactic activity allowed the demonstration of chemoattractant receptors on polymorphonuclear leukocytes (PMNs) as well as macrophages. In membrane preparations from these cells, these receptors exist in high- and low-affinity states which are regulated by guanosine di- and triphosphates. This suggested that chemoattractant receptors interact with guanine nucleotide regulatory proteins (N or G proteins). Although chemoattractants elicit a rapid but transient increase in intracellular cAMP levels, they neither stimulate nor inhibit membrane-bound adenylate cyclase, suggesting a novel role for N proteins in certain receptor-transduction mechanisms. Stimulation of phagocytes by chemoattractants is also associated with a rapid increase in cytosolic Ca2+ concentrations ([ Ca2+]i) which appears to result from the production of inositol 1,4,5-triphosphate (IP3) as a consequence of the diesteric cleavage of phosphatidylinositol 4,5-bisphosphate (PIP2). Treatment of phagocytes with pertussis toxin (PT), which ADP-ribosylates and thereby inactivates certain N proteins, abolishes the cells' responsiveness to chemoattractants. More direct evidence for a role of a PT-sensitive N protein in leukocyte activation was provided by the demonstration that chemoattractants stimulate the hydrolysis of PIP2 in PMN membranes only in the presence of GTP. This receptor-mediated hydrolysis of PIP2 is not observed in plasma membranes prepared from PT-treated PMNs. Therefore, these studies suggest that occupancy of chemoattractant receptors activates a PT-sensitive N protein. The activated N protein shifts the Ca2+ requirement for phospholipase C activity from supraphysiological levels to ambient cytosolic Ca2+ concentrations. Cleavage of PIP2 results in the formation of the second messenger molecules, IP3 and 1,2-diacylglycerol, which can initiate cellular activation. These messengers also seem to activate responses which feed back to attenuate receptor stimulation of phospholipase.     

Role of a guanine nucleotide regulatory protein in the activation of phospholipase C by different chemoattractants

It is well established that formyl peptide chemoattractants can activate a phospholipase C in leukocytes via a pertussis toxin (PT)-sensitive guanine nucleotide regulatory (G) protein. Whether this pathway is similarly used by chemoattractant receptors as a class has been unclear. We now report that lipid and peptide chemoattractants in direct comparative studies induced similar amounts of initial (less than or equal to 15 sec) inositol trisphosphate (IP3) release in human polymorphonuclear leukocytes, but the response to lipid chemoattractants was more transient. Production of IP3 by all chemotactic factors was inhibited by treatment of the cells with PT, indicating that chemotactic factor receptors as a class are coupled to phospholipase C via a G protein that is a substrate for ADP ribosylation by PT. The peptide and lipid factors had comparable chemotactic activity, which was also inhibitable by PT. However, transient activation of phospholipase C is apparently an insufficient signal for full cellular activation, since the lipid chemotactic factor leukotriene B4 and platelet-activating factor were poor stimuli for O2- production and lysosomal enzyme secretion compared with N-formyl-methionyl-leucyl-phenylalanine (fMet-Leu-Phe). Nonetheless, treatment with PT inhibited O2- production and enzyme secretion in response to all chemoattractants, but as previously noted, did not affect Ca2+ ionophores, lectins, or phorbol myristate acetate. Formyl peptide and lipid chemotactic factors induced similar levels of Ca2+ mobilization when monitored by Quin 2 or chlortetracycline (CTC) fluorescence. Although these responses to fMet-Leu-Phe were blocked by PT, the Quin 2 and initial CTC response to the lipid factors were only partially susceptible. Thus, the lipid factors apparently utilize an additional PT-resistant mechanism for redistributing intracellular Ca2+. This latter process requires extracellular Ca2+ and may be independent of the PT-sensitive G protein.     

A pertussis/choleratoxin-sensitive N protein may mediate chemoattractant receptor signal transduction

Chemoattractant receptors on phagocytic leukocytes utilize a guanine nucleotide regulatory (N) protein to activate phospholipase C and subsequent biological responses. Since pertussis toxin inhibits activation of leukocytes by chemoattractants and ribosylates a ca. 40 kD protein in these cells it had generally been assumed that chemoattractant receptors are coupled to Ni. We now report that human polymorphonuclear leukocytes (PMNs), monocytes, and the myeloid HL-60 and U937 cell lines, but not erythrocytes or bovine brain contain a ca. 40 kD protein which is a substrate for ADP ribosylation by choleratoxin (CT). This N protein, termed Nc for chemotaxis-related N protein, comigrates with the ca. 40 kD PT substrate during one-dimensional gel electrophoresis. In vivo treatment of PMNs with PT or CT reduced high affinity binding of chemoattractants to membrane preparations from the cells, implying that chemoattractant receptors are coupled to an N protein which is a substrate for both PT and CT. We suggest that Nc rather than Ni couples chemoattractant receptors to phospholipase C.     

Cyclic nucleotides regulate the morphologic alterations required for chemotaxis in monocytes

The initial morphologic response of human monocytes to chemoattractants is a change in shape from round to a triangular "motile" configuration (polarization). At doses chemotactic in vitro, chemoattractants induced rapid (t 1/2 = 45 sec), sustained (greater than 40 min) polarization of monocytes in suspension. Extracellular Ca++ was not required for polarization induced by chemoattractants, but in the absence of Ca++ kinetics were slowed (t 1/2 = 6.5 min). Phenylephrine, carbamycholine, serotonin, and ascorbate also caused rapid polarization of monocytes. Unlike chemoattractants, polarization by the pharmacologic agents was unsustained (less than 15 min), absolutely required extracellular Ca++, and affected about 50% of the cells responsive to chemoattractants. Based on relative sensitivities to alpha 1- and alpha 2-adrenergic agonists and antagonists, polarization caused by adrenergic agents was mediated by alpha 2-receptors. Muscarinic and alpha 2-adrenergic agonists, serotonin, and ascorbate enhanced the rate and number of monocytes polarizing to suboptimal doses of chemoattractants. Thus, the initial morphologic changes induced by chemoattractants appear to utilize an activation pathway shared with a variety of agents that enhance cGMP levels and inhibit adenylate cyclase. In contrast, theophylline, histamine, and isoproterenol, all agents that activate adenylate cyclase and elevate cAMP levels, inhibited monocyte polarization to chemoattractants. As in PMN, pharmacologic agents that increase cAMP levels inhibited monocyte chemotaxis in vitro, whereas those that inhibit adenylate cyclase and increase cGMP enhanced monocyte chemotactic responses. Thus, the initial morphologic response of monocytes to chemoattractants as well as the processes required for sustained directional motility are modulated by cyclic nucleotides.     

Cutting edge: inflammatory responses can be triggered by TREM-1, a novel receptor expressed on neutrophils and monocytes

We have identified new activating receptors of the Ig superfamily expressed on human myeloid cells, called TREM (triggering receptor expressed on myeloid cells). TREM-1 is selectively expressed on blood neutrophils and a subset of monocytes and is up-regulated by bacterial LPS. Engagement of TREM-1 triggers secretion of IL-8, monocyte chemotactic protein-1, and TNF-alpha and induces neutrophil degranulation. Intracellularly, TREM-1 induces Ca2+ mobilization and tyrosine phosphorylation of extracellular signal-related kinase 1 (ERK1), ERK2 and phospholipase C-gamma. To mediate activation, TREM-1 associates with the transmembrane adapter molecule DAP12. Thus, TREM-1 mediates activation of neutrophil and monocytes, and may have a predominant role in inflammatory responses.     

Essential role for Ca2+ in regulation of IL-1beta secretion by P2X7 nucleotide receptor in monocytes,macrophages, and HEK-293 cells

Interleukin (IL)-1beta is a proinflammatory cytokine that elicits the majority of its biological activity extracellularly, but the lack of a secretory signal sequence prevents its export via classic secretory pathways. Efficient externalization of IL-1beta in macrophages and monocytes can occur via stimulation of P2X7 nucleotide receptors with extracellular ATP. However, the exact mechanisms by which the activation of these nonselective cation channels facilitates secretion of IL-1beta remain unclear. Here we demonstrate a pivotal role for a sustained increase in cytosolic Ca2+ to potentiate secretion of IL-1beta via the P2X7 receptors. Using HEK-293 cells engineered to coexpress P2X7 receptors with mature IL-1beta (mIL-1beta), we show that activation of P2X7 receptors results in a rapid secretion of mIL-1beta by a process(es) that is dependent on influx of extracellular Ca2+ and a sustained rise in cytosolic Ca2+. Moreover, reduction in extracellular Ca2+ attenuates approximately 90% of P2X7 receptor-mediated IL-1beta secretion but has no effect on enzymatic processing of precursor IL-1beta (proIL-1beta) to mIL-1beta by caspase-1. Similar experiments with THP-1 human monocytes and Bac1.2F5 murine macrophages confirm the unique role of Ca2+ in P2X7 receptor-mediated secretion of IL-1beta. In addition, we report that cell surface expression of P2X7 receptors in the absence of external stimulation also results in enhanced release of IL-1beta and that this can be repressed by inhibitors of P2X7 receptors. We clarify an essential role for Ca2+ in ATP-induced IL-1beta secretion and indicate an additional role of P2X7 receptors as enhancers of the secretory apparatus by which IL-1beta is released.     

Development of receptors for leukotriene B4 on HL-60 cells induced to differentiate by 1 alpha,25-dihydroxyvitamin D3

The incubation of HL-60 human promyelocytic leukemia cells for 7 days with 100 nM 1 alpha,25-dihydroxyvitamin D3 [1,25(OH)2D3] induced differentiation into monocyte-like cells, as assessed by morphologic and biochemical characteristics. Stereospecific receptors for leukotriene B4 (LTB4) developed on the surface of the HL-60 cell-derived monocytes that had the capacity to transduce LTB4 stimulation of a transient increase in the cytosolic concentration of calcium ([Ca+2]in). HL-60 cell-derived monocytes, but not undifferentiated HL-60 cells, expressed a high affinity subset of 6400 +/- 3700 receptors per cell with a dissociation constant (Kd) of 2.3 +/- 1 nM (mean +/- SD, n = 3) and a low affinity subset of approximately 2.2 X 10(6) receptors per cell with an apparent Kd of 680 +/- 410 nM. Derivatives of LTB4 inhibited the binding of [3H]LTB4 to HL-60 cell-derived monocytes with a rank order of potency of LTB4 greater than 20-OH-LTB4 greater than 3-aminopropyl amide-LTB4, which is similar to the order for LTB4 receptors of human blood PMNL. In contrast, leukotrienes C4 and D4 and formyl-methionyl chemotactic peptides did not inhibit the binding of [3H] LTB4, which demonstrates the specificity of these receptors for isomers of 5,12-dihydroxy-eicosatetraenoic acid. LTB4 stimulated an increase in [Ca+2]in in HL-60 cell-derived monocytes which reached 50% of the maximal level at an LTB4 concentration of 0.5 nM (EC50). Preincubation of HL-60 cell-derived monocytes with 10 nM LTB4 resulted in a selective loss of high affinity receptors, as assessed by binding of [3H]LTB4, and a 200-fold increase in the EC50 for stimulation by LTB4 of increases in [Ca+2]in, without alterations in either the low affinity receptors for LTB4 or the responsiveness of [Ca+2]in to formyl-methionyl chemotactic peptides. HL-60 cells that are induced to differentiate into monocytes thus develop stereospecific receptors for LTB4 with binding and transductional characteristics similar to those of human blood PMNL.     

Chemotaxis of rat mast cells toward adenine nucleotides.

Rat mucosal mast cells express P2 purinoceptors, occupation of which mobilizes cytosolic Ca2+ and activates a potassium conductance. The primary function of this P2 system in mast cell biology remains unknown. Here, we show that extracellular ADP causes morphological changes in rat bone marrow-cultured mast cells (BMMC) typical of those occurring in cells stimulated by chemotaxins, and that the nucleotides ADP, ATP, and UTP are effective chemoattractants for rat BMMC. ADP was also a chemotaxin for murine J774 monocytes. The nucleotide selectivity and pertussis toxin sensitivity of the rat BMMC migratory response suggest the involvement of P2U receptors. Poorly hydrolyzable derivatives of ADP and ATP were effective chemotaxins, obviating a role for adenosine receptors. Buffering of external Ca2+ at 100 nM or reduction of the electrical gradient driving Ca2+ entry (by elevating external K+) blocked ADP-driven chemotaxis, suggesting a role for Ca2+ influx in this process. Anaphylatoxin C5a was a potent chemotaxin (EC50 approximately 0.5 nM) for J774 monocytes, but it was inactive on rat BMMC in the presence or absence of laminin. Ca2+ removal or elevated [K+] had modest effects on C5a-driven chemotaxis of J774 cells, implicating markedly different requirements for Ca2+ signaling in C5a- vs ADP-mediated chemotaxis. This is supported by the observation that depletion of Ca2+ stores with thapsigargin completely blocked migration induced by ADP but not C5a. These findings suggest that adenine nucleotides liberated from parasite-infested tissue could participate in the recruitment of mast cells by intestinal mucosa.     

Thrombin-induced Ca2+ mobilization in vascular smooth muscle utilizes a slowly ribosylating pertussis toxin-sensitive G protein. Evidence for the involvement of a G protein in inositol trisphosphate-dependent Ca2+ release.

The role of pertussis toxin (PT)-sensitive and -insensitive guanine nucleotide-binding proteins (G proteins) in the stimulation of Ca2+ mobilization by thrombin was investigated in cultured rat aortic smooth muscle cells. Characterization using immunoblotting with specific antisera indicated the presence in isolated membranes of the G alpha i2, G alpha i3, G alpha s, G beta 35, and G beta 36 protein subunits as well as a lower molecular weight species of unknown identity. To assess the importance of G proteins in the coupling of thrombin receptors to Ca2+ mobilization, we investigated the effect of PT on Ca2+ responses using fluorescence spectroscopy and the Ca2+ indicator dye Fura-2. Pretreatment of cells for 2 h with PT (1 microgram/ml), which produced 91.3% ADP-ribosylation of PT-sensitive G proteins, did not affect the magnitude of thrombin-induced release of Ca2+ from internal stores, suggesting that the residual 8.7% of PT-sensitive G proteins, or PT-insensitive mechanisms, was responsible for Ca2+ release. However, after an 18-h pretreatment with PT, which produced ADP-ribosylation of the total complement of PT-sensitive G proteins, the thrombin-induced peak Ca2+ response was inhibited by approximately 72%, suggesting that the major fraction of the Ca2+ response was mediated by a slowly ribosylating component. The delayed effect of the toxin was not caused by down-regulation of the beta-subunit of G proteins because quantitative immunoblots showed that levels of the beta-subunit remained constant throughout the period of PT pretreatment. It was also not caused by a reduction in the size of the thrombin-releasable Ca2+ pool because Ca2+ release induced by agents that release Ca2+ directly from internal stores, 2,5-di-tert-butylhydroquinone or thapsigargin, was not affected. In addition, the delayed effect of PT could not be explained in terms of differences in thrombin-induced [3H]inositol trisphosphate (IP3) formation because the level of inhibition of IP3 formation after a 2-h PT treatment was similar to that present after an 18-h pretreatment. The results indicate that a slowly ribosylating PT-sensitive species is the major G protein pathway that couples thrombin-receptor activation to Ca2+ mobilization. This G protein appears to be involved not in the mechanisms that generate IP3 but rather possibly in coupling at the level of the intracellular Ca2+ store.     

Receptor-mediated activation of electropermeabilized neutrophils. Evidence for a Ca2+- and protein kinase C-independent signaling pathway

Electrically permeabilized neutrophils were used to study the mechanism of activation of the respiratory burst by the chemotactic agent formyl-methionyl-leucyl-phenylalanine (fMLP). Permeabilization was assessed by flow cytometry, radioisotope trapping, and by the requirement for exogenous NADPH for oxygen consumption. A respiratory burst could be elicited by fMLP, phorbol ester, or diacylglycerol in permeabilized cells suspended in EGTA-buffered medium with 100 nM free Ca2+. The fMLP response persisted even in cells depleted of intracellular Ca2+ stores by pretreatment with ionomycin. Therefore, a change in cytosolic free Ca2+ ([Ca2+]i) is not required for receptor-mediated stimulation of the respiratory burst. The responses induced by phorbol ester and diacylglycerol were largely inhibited by H7, a protein kinase C antagonist. In contrast, the stimulation of oxygen consumption by fMLP was unaffected by H7. These results suggest that a third signaling pathway, distinct from changes in [Ca2+]i and activation of protein kinase C, is involved in the response of neutrophils to chemoattractants.     

Differential inhibition of human neutrophil activation by cyclosporins A, D, and H. Cyclosporin H is a potent and effective inhibitor of formyl peptide-induced superoxide formation.

Cyclosporin (Cs)A but not CsH inhibits activation of human lymphocytes. We studied the effects of CsA, CsD, and CsH on human neutrophil activation induced by chemoattractants and by various substances that circumvent receptor stimulation. CsH inhibited superoxide (O2-) formation induced by the chemotactic peptide, FMLP (30 nM), with a half-maximal effect at 40 nM. O2- formation was abolished by CsH at 1 microM. CsH increased the concentration of FMLP causing half-maximal activation of O2- formation from 30 nM to 0.8 microM and substantially reduced the stimulatory effect of FMLP at supra-maximally effective concentrations. The inhibitory effect of CsH on O2- formation was evident immediately after addition to neutrophils. CsH also markedly inhibited the increase in cytosolic Ca2+ ([Ca2+]i), beta-glucuronidase, and lysozyme release and aggregation stimulated by FMLP. CsA and CsD were considerably less effective than CsH to inhibit FMLP-induced O2- formation. CsA and CsD were without effect on exocytosis, rises in [Ca2+]i, and aggregation induced by the chemotactic peptide. Cyclosporines inhibited FMLP-induced O2- formation in an additive manner, indicating that they acted through a mechanism they had in common. Cyclosporines only slightly inhibited O2- formation and lysozyme release induced by C5a. Aggregation and rises in [Ca2+]i stimulated by C5a were not affected by cyclosporines, and they did not inhibit O2- formation and exocytosis induced by platelet-activating factor and leukotriene B4. Cyclosporines partially inhibited O2- formations induced by NaF and gamma-hexachlorocyclohexane. CsA marginally inhibited PMA-induced O2- formation and lysozyme release. CsA, CsD, and CsH did not inhibit arachidonic acid-induced O2- formation and its potentiation by NaF or stable guanine nucleotides in a cell-free system from DMSO-differentiated HL-60 cells. CsH partially inhibited binding of FML [3H]P to formyl peptide receptors in membranes from DMSO- or dibutyryl cAMP-differentiated HL-60 cells. Our data show that: 1) cyclosporines differentially inhibit activation of human neutrophils; and 2) CsH is, indeed, not immunologically inactive but is a potent and effective inhibitor of FMLP-induced O2- formation. 3) CsH interferes with agonist binding to formyl peptide receptors and in addition, cyclosporines may also act at sites distal to chemoattractant receptors.     

Thioredoxin specifically cross-desensitizes monocytes to MCP-1

Thioredoxin (Trx) is a protein disulfide oxidoreductase which can be secreted and acts as a cytokine. As we recently reported that Trx is chemotactic, we investigated whether it desensitizes monocytes or PMN to other chemokines. Preincubation for 15 min with Trx inhibited the chemotactic response of monocytes to MCP-1, but not to fMLP. This effect was independent of whether Trx was present during the chemotaxis assay or only during the preincubation. Preincubation (5 min) with Trx also inhibited the increase in intracellular Ca(2+) induced by MCP-1 in monocytes, but not that induced by fMLP. Preincubation with Trx did not affect the chemotactic response induced in PMN by IL-8. The inhibition of chemotactic and Ca(2+) responses to MCP-1 in monocytes was not due to a down-regulation of the MCP-1 receptor, as shown by receptor binding studies. The Ca(2+) response to MCP-1 was also inhibited by Trx in a CCR2-transfected cell line. It is suggested that Trx inhibits monocyte responses to chemokines by acting downstream of the chemokine receptors. Since there are high concentrations of circulating Trx in infection and inflammatory diseases, this might act as an inhibitor of monocyte migration in vivo.     

Chemoattractant-elicited alterations of cAMP levels in human polymorphonuclear leukocytes require a Ca2+-dependent mechanism which is independent of transmembrane activation of adenylate cyclase

The affinity of the chemoattractant receptor for N-formyl-methionyl-leucyl-phenylalanine (fMet-Leu-Phe) on human polymorphonuclear leukocytes (PMNs) is regulated by guanine nucleotides, and chemoattractants stimulate increased intracellular cAMP levels in PMNs. Our data, however, indicate that this receptor does not activate membrane-bound adenylate cyclase via direct nucleotide regulatory protein (N) coupling but instead raises cAMP levels indirectly via a mechanism which appears to require Ca2+ mobilization. This conclusion is based on the following data: 1) prostaglandin E1 (PGE1) activated and alpha 2-adrenergic treatment inhibited adenylate cyclase activation in PMN plasma membranes; fMet-Leu-Phe, however, neither activated nor inhibited adenylate cyclase in these membranes; 2) depletion of extracellular Ca2+ had no effect on isoproterenol and PGE1 elicited cAMP responses in intact PMNs while peak fMet-Leu-Phe and A23187-induced responses were reduced by approximately 50 and 80%, respectively; 3) 8-(N,N-diethylamino)-octyl-3,4,5-trimethoxybenzoate, a purported Ca2+ antagonist, caused almost complete inhibition of fMet-Leu-Phe and ionophore-induced cAMP responses in intact cells but had no effect on PGE1 and isoproterenol; 4) alpha 2-adrenergic agonists inhibited PGE1 but not chemoattractant- or A23187-elicited cAMP responses in intact PMNs; and 5) pretreatment of cells with a phosphodiesterase inhibitor (isobutylmethylxanthine) greatly potentiated the PGE1 and isoproterenol cAMP responses but nearly abolished the peak fMet-Leu-Phe response. Thus, chemoattractants appear to utilize a novel mechanism to raise cAMP levels which appear to require Ca2+ mobilization and could be mediated in part through a transient inhibition of phosphodiesterases. We suggest that stimulation of PMN functions by chemoattractants may utilize an N-coupled process to generate a Ca2+ signal which could in turn raise intracellular cAMP levels indirectly and thereby provide negative regulation.     

Differential CCR1-mediated chemotaxis signaling induced by human CC chemokine HCC-4/CCL16 in HOS cells

Human CC chemokine-4 (HCC-4)/CCL16 is a chemoattractant for monocytes and lymphocytes. Although HCC-4 binds to multiple CC chemokine receptors, the receptor-mediated signal transduction pathway induced by HCC-4 has not been characterized. Human osteogenic sarcoma cells stably expressing CCR1 were used to investigate HCC-4-mediated chemotaxis signaling events via CCR1. The chemotactic activity of HCC-4 as well as those of other CCR1-dependent chemokines including MIP-1alpha/CCL3, RANTES/CCL5, and Lkn-1/CCL15 was inhibited by the treatment of pertussis toxin, an inhibitor of Gi/Go protein, U73122, an inhibitor of phospholipase C (PLC), and rottlerin, a specific inhibitor of protein kinase Cdelta (PKCdelta). These results indicate that HCC-4-induced chemotaxis signaling is mediated through Gi/Go protein, PLC, and PKCdelta. SB202190, an inhibitor of p38 mitogen activated protein kinase, only blocked the chemotactic activity of HCC-4, but not those of other CCR1-dependent chemokines. SB202190 inhibited HCC-4-induced chemotaxis in a dose-dependent manner (P < 0.01). HCC-4 induces p38 activation in both a time and dose-dependent manner. However, such p38 activation was not induced by other CCR1-dependent chemokines. To further investigate the differential effect of HCC-4, the Ca2+ mobilization was examined. HCC-4 induced no intracellular Ca2+ flux in contrast to other CCR1-dependent chemokines. These results indicate that HCC-4 transduces signals differently from other CCR1-dependent chemokines and may play different roles in the immune response.     

Histamine as a growth factor and chemoattractant for human carcinoma and melanoma cells: action through Ca2(+)-mobilizing H1 receptors

Histamine receptors are present on the surface of various normal and tumor-derived cell types, where their biological function is incompletely understood. Here we report that histamine not only stimulates cell proliferation under serum-free conditions, but also is chemotactic for human carcinoma (Hela and A431) and melanoma (A875) cells expressing H1 type receptors. Histamine was found to be a potent activator of phospholipase C, leading to polyphosphoinositide hydrolysis and subsequent intracellular Ca2+ mobilization. In addition, histamine also causes the protein kinase C-mediated activation of Na+/H+ exchange, as evidenced by an amiloride-sensitive rise in cytoplasmic pH. All histamine-induced responses, including chemotaxis and DNA synthesis, are completely inhibited by the H1 receptor antagonist pyrilamine, but not by cimetidine, an inhibitor of histamine H2 type receptors. Our results suggest that histamine may have a previously unrecognized role in the migration and proliferation of cells expressing H1 receptors.     

TLR signaling paralyzes monocyte chemotaxis through synergized effects of p38 MAPK and global Rap-1 activation

Toll-like receptors (TLRs) that recognize pathogen associated molecular patterns and chemoattractant receptors (CKRs) that orchestrate leukocyte migration to infected tissue are two arms of host innate immunity. Although TLR signaling induces synthesis and secretion of proinflammatory cytokines and chemokines, which recruit leukocytes, many studies have reported the paradoxical observation that TLR stimulation inhibits leukocyte chemotaxis in vitro and impairs their recruitment to tissues during sepsis. There is consensus that physical loss of chemokine receptor (CKR) at the RNA or protein level or receptor usage switching are the mechanisms underlying this effect. We show here that a brief (<15 min) stimulation with LPS (lipopolysaccharide) at ~0.2 ng/ml inhibited chemotactic response from CCR2, CXCR4 and FPR receptors in monocytes without downmodulation of receptors. A 3 min LPS pre-treatment abolished the polarized accumulation of F-actin, integrins and PIP(3) (phosphatidylinositol-3,4,5-trisphosphate) in response to chemokines in monocytes, but not in polymorphonuclear neutrophils (PMNs). If chemoattractants were added before or simultaneously with LPS, chemotactic polarization was preserved. LPS did not alter the initial G-protein signaling, or endocytosis kinetics of agonist-occupied chemoattractant receptors (CKRs). The chemotaxis arrest did not result from downmodulation of receptors or from inordinate increase in adhesion. LPS induced rapid p38 MAPK activation, global redistribution of activated Rap1 (Ras-proximate-1 or Ras-related protein 1) GTPase and Rap1GEF (guanylate exchange factor) Epac1 (exchange proteins activated by cyclic AMP) and disruption of intracellular gradient. Co-inhibition of p38 MAPK and Rap1 GTPase reversed the LPS induced breakdown of chemotaxis suggesting that LPS effect requires the combined function of p38 MAPK and Rap1 GTPase.     

Calcium transport in the cellular slime mould Dictyostelium discoideum

Transport of Ca2+ into amoebae of Dictyostelium discoideum was studied using 45Ca and a lanthanum stopping technique. Ca2 uptake was found to be rapid and showed saturation kinetics. No difference was found in Ca2+ uptake between vegetative and aggregation competent cells, the V(max) for unstimulated amoebae being approx. 10 nmol/10(7) cells per min. Ca2+ uptake had the characteristics of passive facilitated diffusion using a saturatable carrier and NaN3 and ouabain were not inhibitory. The chemoattractants cAMP and folate, previously reported to stimulate the uptake of Ca2+ into amoebae, did not stimulate the rate of Ca2+ uptake by this carrier but increased the extent of Ca2+ taken up over the period 10-30 s after chemotactic stimulation. The significance of these findings for the function of Ca2+ in chemotactic signalling is discussed.     

S100 protein CP-10 stimulates myeloid cell chemotaxis without activation

Leukocyte recruitment to inflammatory foci is generally associated with cellular activation. Recent evidence suggests that chemotactic agents can be divided into two classes, “classical chemoattractants” such as FMLP, C5a, and IL-8, which stimulate directed migration and activation events and “pure chemoattractants” such as TGF-β1 which influence actin polymerisation and movement but not oxidative burst and associated granular enzyme release. The studies reported here demonstrate that the murine S100 chemoattractant protein, CP-10, belongs to the “non-classical” group. Despite its potent chemotactic activity for neutrophils and monocytes/macrophages, CP-10 failed to increase [Ca²⁺]_i in human or mouse PMN, although chemotaxis was inhibited by pertussis toxin, confirming the suggestion of a novel Ca²⁺-independent G-protein-coupled pathway for post-receptor signal transduction triggered by “pure chemoattractants.” The co-ordinated up-regulation of Mac-1 and down-regulation of L-selectin induced by FMLP on human PMN in vitro was not observed with CP-10. Quantitative changes in immediate (30 s) actin polymerisation occurred with FMLP and CP-10-treated human PMN. The relative F-actin increases induced in WEHI 265 monocytoid cells by FMLP and CP-10 was optimal at 60 s and declined over 120 s. F-actin changes reflected the concentration and potencies of the agonists required to provoke chemotaxis. After 90 min, CP-10 profoundly altered cell shape and increased both cell size and F-actin within pseudopodia. These changes are typical of those mediating leukocyte deformability, and CP-10 may mediate leukocyte retention within microcapillaries and thereby contribute to the initiation of inflammation in vascular beds. © 1996 Wiley-Liss, Inc.     

Chemotactic factor and P15E-related chemotaxis inhibitor in human melanoma cell lines with different macrophage content and tumorigenicity in nude mice

The present study was designed to characterize the production of chemoattractants by human melanoma lines with high (M4Be, M3Da, NTerDa) or low tumorigenic (Doc8, M1Do) potential when heterotransplanted in nude mice. Supernatants from the Doc8 and M1Do cell lines were strongly chemotactic in vitro for mononuclear phagocytes. Chemotactic activity was destroyed by proteolytic enzymes, and upon gel filtration on Sephadex G75, it eluted in the cytochrome c region corresponding to an apparent m.w. of 12,000. Upon chromatofocusing, the Sephadex-separated tumor-derived chemotactic factor (TDCF) showed an isoelectric point of 5.5 to 6. Cell lines with high tumorigenic potential contained low or no detectable chemotactic activity. When culture supernatants of cell lines with modest (M3Da) or no (M4Be) chemotactic activity were exposed to immobilized monoclonal antibodies directed against the retroviral transmembrane protein P15E, appreciable chemotactic activity was detectable (M4Be) or preexisting levels increased (M3Da). The material eluted from Sepharose-bound anti-P15E antibodies inhibited the migration of monocytes in response to chemoattractants. These findings demonstrate the coexistence in some human melanoma cell line supernatants of factors (TDCF and P15E-related inhibitor) with opposite influence on monocyte chemotaxis. That tumor cell products play a pivotal role in regulating the extravasation of monocytes into neoplastic tissues is suggested by the close correlation observed between macrophage levels in melanomas grown in nude mice and levels of chemotactic activity detectable in culture supernatants.     

Co-expression of chemotactic ligand receptors on human peripheral blood monocytes

Directed migration of monocytes is dependent upon interaction of cell surface receptors and specific chemotactic ligands. To determine whether circulating human monocytes express multiple chemotactic ligand receptors or whether subpopulations of monocytes exist with a single receptor specificity, nonoverlapping fluorescent probes for two chemotactic ligands, N-formyl methionyl leucyl phenylalanine (FMLP) and C5a, were developed to simultaneously evaluate the expression of receptors for these ligands on individual monocytes. The subsequent incubation with different fluorochrome labeled C5a and FMLP probes and monoclonal antibodies specific for antigenic determinants on distinct subsets of mononuclear cells followed by analysis with dual parameter flow microfluorometry indicated that cells that express C5a and FMLP receptors are the OKM1, Mac-1, and Fc gamma receptor positive population. Furthermore, it was demonstrated that approximately 90% of peripheral blood monocytes expressed FMLP receptors, and the majority of FMLP+ cells were also C5a receptor positive. In addition, a parallel spectrum of chemotactic ligand receptor density from low to high levels was demonstrated for both C5a and FMLP. Additional analysis revealed that the density of chemotactic ligand receptors on resting peripheral blood monocytes did not correlate with monocyte maturation levels measured by HLA-DR expression. Elucidation of the monocyte chemotactic receptor-ligand interactions that lead to migration and/or activation may provide insight into the regulation of monocyte function in inflammation.