Search

Neutrophil recruitment and directional movement toward chemotactic stimuli are important processes in innate immune responses. This study examines the role of Fer kinase in neutrophil recruitment and chemotaxis to various chemoattractants in vitro and in vivo. Mice targeted with a kinase-inactivating mutation (Fer(DR/DR)) or wild type (WT) were studied using time-lapse intravital microscopy to examine leukocyte recruitment and chemotaxis in vivo. In response to keratinocyte-derived cytokine, no difference in leukocyte chemotaxis was observed between WT and Fer(DR/DR) mice. However, in response to the chemotactic peptide WKYMVm, a selective agonist of the formyl peptide receptor, a 2-fold increase in leukocyte emigration was noted in Fer(DR/DR) mice (p < 0.05). To determine whether these defects were due to Fer signaling in the endothelium or other nonhematopoietic cells, bone marrow chimeras were generated. WKYMVm-induced leukocyte recruitment in chimeric mice (WT bone marrow to Fer(DR/DR) recipients or vice versa) was similar to WT mice, suggesting that Fer kinase signaling in both leukocytes and endothelial cells serves to limit chemotaxis. Purified Fer(DR/DR) neutrophils demonstrated enhanced chemotaxis toward end target chemoattractants (WKYMVm and C5a) compared with WT using an under-agarose gel chemotaxis assay. These defects were not observed in response to intermediate chemoattractants (keratinocyte-derived cytokine, MIP-2, or LTB(4)). Increased WKYMVm-induced chemotaxis of Fer(DR/DR) neutrophils correlated with sustained PI3K activity and reduced reliance on the p38 MAPK pathway compared with WT neutrophils. Together, these data identify Fer as a novel inhibitory kinase for neutrophil chemotaxis toward end target chemoattractants through modulation of PI3K activity.

Chemerin is the ligand of the ChemR23 receptor and a chemoattractant factor for human immature dendritic cells (DCs), macrophages, and NK cells. In this study, we characterized the mouse chemerin/ChemR23 system in terms of pharmacology, structure-function, distribution, and in vivo biological properties. Mouse chemerin is synthesized as an inactive precursor (prochemerin) requiring, as in human, the precise processing of its C terminus for generating an agonist of ChemR23. Mouse ChemR23 is highly expressed in immature plasmacytoid DCs and at lower levels in myeloid DCs, macrophages, and NK cells. Mouse prochemerin is expressed in most epithelial cells acting as barriers for pathogens but not in leukocytes. Chemerin promotes calcium mobilization and chemotaxis on DCs and macrophages and these functional responses were abrogated in ChemR23 knockout mice. In a mouse model of acute lung inflammation induced by LPS, chemerin displayed potent anti-inflammatory properties, reducing neutrophil infiltration and inflammatory cytokine release in a ChemR23-dependent manner. ChemR23 knockout mice were unresponsive to chemerin and displayed an increased neutrophil infiltrate following LPS challenge. Altogether, the mouse chemerin/ChemR23 system is structurally and functionally conserved between human and mouse, and mouse can therefore be considered as a good model for studying the anti-inflammatory role of this system in the regulation of immune responses and inflammatory diseases.

Receptors for IgG Abs (Fcgamma receptors) are capable of triggering diverse cell responses in leukocytes. In neutrophils, two Fcgamma receptors, namely FcgammaRIIA and FcgammaRIIIB, are constitutively expressed. The signaling pathways that regulate FcgammaRIIA-mediated phagocytosis have been relatively well described. However, the different signaling pathways that lead to NF activation after engagement of each Fcgamma receptor have only been partially described. To address this problem, neutrophils were stimulated by cross-linking selectively each type of Fcgamma receptor with specific mAbs, and NF activation was then analyzed. FcgammaRIIIB, but not FcgammaRIIA, promoted a robust increase in phosphorylated ERK in the nucleus, and also efficient phosphorylation of the NF Elk-1. Complete mAb 3G8 (anti-FcgammaRIIIB) induced a higher response than did F(ab')(2) fragments of mAb 3G8, suggesting a possible synergistic effect of both FcgammaR receptors. However, mAb IV.3 (anti-FcgammaRIIA) alone did not cause an increase of phosphorylated ERK in the nucleus. FcgammaRIIIB-induced nuclear phosphorylation of ERK, and of Elk-1, was not affected by Syk, PI3K, or MEK inhibitors. In contrast, FcgammaRIIA- or FcgammaRIIIB-mediated phosphorylation of cytoplasmic ERK depended on Syk, PI3K, and MEK. Also, ERK, but not MEK, was constitutively present in the nucleus, and FcgammaRIIIB cross-linking did not increase the levels of nuclear ERK or MEK. These data clearly show that different neutrophil Fcgamma receptors possess different signaling capabilities. FcgammaRIIIB, but not FcgammaRIIA, activates a unique signaling pathway leading to the nuclear-restricted phosphorylation of ERK and Elk-1, independently of Syk, PI3K, or MEK.

PI3K plays a fundamental role in regulating neutrophil recruitment into sites of inflammation but the role of the different isoforms of PI3K remains unclear. In this study, we evaluated the role of PI3Kgamma and PI3Kdelta for neutrophil influx induced by the exogenous administration or the endogenous generation of the chemokine CXCL1. Administration of CXCL1 in PI3Kgamma(-/-) or wild-type (WT) mice induced similar increases in leukocyte rolling, adhesion, and emigration in the cremaster muscle when examined by intravital microscopy. The induction of neutrophil recruitment into the pleural cavity or the tibia-femoral joint induced by the injection of CXCL1 was not significantly different in PI3Kgamma(-/-) or WT mice. Neutrophil influx was not altered by treatment of WT mice with a specific PI3Kdelta inhibitor, IC87114, or a specific PI3Kgamma inhibitor, AS605240. The administration of IC87114 prevented CXCL1-induced neutrophil recruitment only in presence of the PI3Kgamma inhibitor or in PI3Kgamma(-/-) mice. Ag challenge of immunized mice induced CXCR2-dependent neutrophil recruitment that was inhibited by wortmannin or by blockade of and PI3Kdelta in PI3Kgamma(-/-) mice. Neutrophil recruitment to bronchoalveolar lavage induced by exogenously added or endogenous production of CXCL1 was prevented in PI3Kgamma(-/-) mice. The accumulation of the neutrophils in lung tissues was significantly inhibited only in PI3Kgamma(-/-) mice treated with IC87114. Neutrophil recruitment induced by exogenous administration of C5a or fMLP appeared to rely solely on PI3Kgamma. Altogether, our data demonstrate that there is a tissue- and stimulus-dependent role of PI3Kgamma and PI3Kdelta for neutrophil recruitment induced by different chemoattractants in vivo.

Neutrophils act as the first line of innate immune defense against invading microorganisms during infection and inflammation. The tightly regulated production of reactive oxygen species (ROS) through activation of NADPH oxidase is a major weapon used by neutrophils and other phagocytic leukocytes to combat such pathogens. Cellular adhesion signals play important physiological roles in regulating the activation of NADPH oxidase and subsequent ROS formation. We previously showed that the initial suppression of the oxidase response of chemoattractant-stimulated adherent neutrophils is mediated via inhibition of Vav1-induced activation of the NADPH oxidase regulatory GTPase Rac2 by adhesion signals. In this study we show that prior exposure of neutrophils to a number of cytokines and inflammatory mediators, including TNF-alpha, GM-CSF, and platelet-activating factor, overcomes the adhesion-mediated suppression of ROS formation. Proline-rich tyrosine kinase 2 (pyk2) activity is enhanced under these conditions, correlating with the restoration of Vav1 and Rac2 activities. Both dominant negative pyk2 and a pyk2-selective inhibitor prevented restoration of ROS production induced by TNF-alpha, GM-CSF, and platelet-activating factor, and this loss of pyk2 activity resulted in decreased Vav1 tyrosine phosphorylation and subsequent Rac2 activation. Our studies identify pyk2 as a critical regulatory component and a molecular switch to overcome the suppression of leukocyte oxidant generation by cell adhesion. This activity constitutes a mechanism by which cytokines might lead to rapid elimination of invading pathogens by adherent neutrophils under normal conditions or enhance tissue damage in pathological states.

The antimicrobial and proinflammatory neutrophil granule protein cathepsin G (CaG) has been reported as a chemoattractant for human phagocytic leukocytes by using a putative G protein coupled receptor. In an effort to identify potential CaG receptor(s), we found that CaG-induced phagocyte migration was specifically attenuated by the bacterial chemotactic peptide fMLP, suggesting these two chemoattractants might share a receptor. In fact, CaG chemoattracts rat basophilic leukemia cells (RBL cells) expressing the high affinity human fMLP receptor FPR, but not parental RBL cells or cells transfected with other chemoattractant receptors. In addition, a specific FPR Ab and a defined FPR antagonist, cyclosporin H, abolished the chemotactic response of phagocytes and FPR-transfected cells to CaG. Furthermore, CaG down-regulated the cell surface expression of FPR in association with receptor internalization. Unlike fMLP, CaG did not induce potent Ca(2+) flux and was a relatively weaker activator of MAPKs through FPR. Yet CaG activated an atypical protein kinase C isozyme, protein kinase Czeta, which was essential for FPR to mediate the chemotactic activity of CaG. Thus, our studies identify CaG as a novel, host-derived chemotactic agonist for FPR and expand the functional scope of this receptor in inflammatory and immune responses.

Mucosal mast cells strategically located at the optimal site interact with invading bacteria. Presence of VacA, the virulent Helicobacter pylori cytotoxin, is correlated with the severity of H. pylori-induced gastritis. To examine the mechanisms of inflammation in H. pylori-induced gastritis, we administered VacA to the mice. Inoculation of VacA resulted in epithelium vacuolization and marked infiltrations of mast cells and mononuclear cells into the mucosal epithelium within 24 h. In an in vitro study using bone marrow-derived mast cells, VacA directly bound and showed a chemotactic activity to the mast cell. In addition, VacA induced bone marrow-derived mast cells to produce proinflammatory cytokines, TNF-alpha, macrophage-inflammatory protein-1alpha, IL-1beta, IL-6, IL-10, and IL-13 in a dose-dependent manner without causing degranulation. The present study suggests that early activation of mast cells by VacA may be the host early response to clear the bacteria and also may contribute to the pathogenesis of H. pylori-induced gastritis.

Ecalectin, produced by Ag-stimulated T lymphocytes, is a potent eosinophil-specific chemoattractant in vitro as well as in vivo and thus is implicated in allergic responses. Ecalectin differs structurally from other known eosinophil chemoattractants (ECAs); ecalectin belongs to the galectin family defined by their affinity for beta-galactosides and by their conserved carbohydrate recognition domains. These characteristic features suggest that ecalectin has unique activities associated with allergic inflammation besides ECA activity. Conversely, ecalectin may mediate ECA activity by binding to a receptor of a known ECA via affinity for the beta-galactosides present on this receptor. In this study, we have tested whether ecalectin mediates ECA activity by binding to a receptor of a known ECA, and we have assessed its effects on eosinophils. Ecalectin did not mediate ECA activity by binding to the IL-5R or to CCR3. Also, the ECA activity of ecalectin was mainly chemokinetic. In addition, ecalectin induced concentration-dependent eosinophil aggregation, a marker for eosinophil activation. Ecalectin induced concentration-dependent superoxide production from eosinophils but did not induce degranulation; usually these two events are coupled in eosinophil activation. Moreover, ecalectin directly prolonged eosinophil survival in vitro and did not trigger eosinophils to secrete cytokines that prolong eosinophil survival. These results demonstrate that ecalectin has several unique effects on eosinophils. Therefore, we conclude that ecalectin is a novel eosinophil-activating factor. Presumably, these effects allow ecalectin to play a distinctive role in allergic inflammation.

Receptors for the bacterial chemotactic peptide fMLP are implicated in inflammation and host defense against microbial infection. We investigated the expression and function of fMLPR in microglial cells, which share characteristics of mononuclear phagocytes and play an important role in proinflammatory responses in the CNS. The expression of the genes encoding formyl peptide receptor (FPR)1 and FPR2, the high- and low-affinity fMLPR, was detected in a murine microglial cell line N9, but these cells did not respond to chemotactic agonists known for these receptors. N9 cells incubated with bacterial LPS increased the expression of fMLPR genes and developed a species of specific, but low-affinity, binding sites for fMLP, in association with marked calcium mobilization and chemotaxis responses to fMLP in a concentration range that typically activated the low-affinity receptor FPR2. In addition, LPS-treated N9 cells were chemoattracted by two FPR2-specific agonists, the HIV-1 envelope-derived V3 peptide, and the 42 aa form of the amyloid beta peptide which is a pathogenic agent in Alzheimer's disease. Primary murine microglial cells also expressed FPR1 and FPR2 genes, but similar to N9 cells, exhibited FPR2-mediated activation only after LPS treatment. In contrast to its effect on the function of FPR2, LPS reduced N9 cell binding and biological responses to the chemokine stromal cell-derived factor-1alpha. Thus, LPS selectively modulates the function of chemoattractant receptors in microglia and may promote host response in inflammatory diseases in the CNS.

Cryptococcal capsular Ags induce the production of proinflammatory cytokines in patients with cryptococcal meningitis. Despite this, their cerebrospinal fluid typically contains few neutrophils. Capsular glucuronoxylomannan is generally considered to mediate the inhibition of neutrophil extravasation. In the current study, culture supernatant harvested from the nonglucuronoxylomannan-producing strain CAP67 was found to be as potent as supernatant from wild-type strains in preventing migration. We identified capsular mannoprotein (MP)-4 as the causative agent. Purified MP-4 inhibited migration of neutrophils toward platelet-activating factor, IL-8, and fMLP, probably via a mechanism involving chemoattractant receptor cross-desensitization, as suggested by its direct chemotactic activity. Supporting this hypothesis, MP-4 elicited Ca(2+) transients that were inhibited by preincubation with either fMLP, IL-8, or C5a, but not platelet-activating factor, and vice versa. Moreover, MP-4 strongly decreased the neutrophil surface expression of L-selectin and induced shedding of TNF receptors p55/p75, whereas CD11b/18 increased. Finally, MP-4 was clearly detectable in both serum and cerebrospinal fluid of patients suffering from cryptococcal meningitis. These findings identify MP-4 as a novel capsular Ag prematurely activating neutrophils and desensitizing them toward a chemoattractant challenge.

Thrombin is a procoagulant and proinflammatory molecule in vivo. In vitro, thrombin has been shown to induce endothelial activation, notably IL-8 secretion and adhesion molecule expression. In this study, we showed that thrombin may induce a new cascade leading from acute to chronic inflammation. Thrombin was able to induce the production of both IL-6 and monocyte chemotactic protein-1 (MCP-1) by HUVEC independently of IL-1alphabeta and TNF-alpha. Addition of physiological concentrations of exogenous soluble IL-6Ralpha (sIL-6Ralpha) to thrombin-activated HUVEC was sufficient to increase the amounts of MCP-1 produced, but not those of IL-8. These effects could be blocked by anti-IL-6 or anti-sIL-6Ralpha blocking mAb, demonstrating the existence of an autocrine loop of MCP-1 secretion, involving the IL-6/IL-6Ralpha/gp130 complex on HUVEC. In addition, we identified IL-8-activated neutrophils as a potential source of sIL-6Ralpha because IL-8 induced IL-6Ralpha shedding from the neutrophil membranes and increased in parallel sIL-6Ralpha concentrations in neutrophil supernatants. Furthermore, addition of neutrophils to thrombin-activated HUVEC significantly increased MCP-1 secretion, which could be decreased by blocking IL-6. Thus, thrombin-activated endothelium may induce a cascade of events characterized by IL-8 secretion, neutrophil local infiltration, and the release of IL-6Ralpha from neutrophil membranes. sIL-6Ralpha may then complex with IL-6 and increase the amount of MCP-1 produced by thrombin-activated endothelium, favoring monocyte infiltration, and the transformation of acute into chronic inflammation.

Phosphorothioate cytosine-guanine oligodeoxynucleotides (CpG PS-ODNs) has been reported to induce Th1 immune responses against coadministered Ags more efficiently than phosphodiester CpG ODNs (CpG PO-ODNs). Here, we demonstrated that PS-ODNs, but not PO-ODNs, have a chemotactic effect on primary macrophages, which is independent of the CpG motif. In addition, the conjugation of a hexameric dG run (dG(6) run) at the 3' terminus reduced the concentration required for the optimal chemotactic activity of PS-ODNs by approximately 10-fold. Endosomal maturation blockers, such as monensin and chloroquine, inhibited the chemotactic effect of PS-ODNs. The inhibition of the activities of p38 mitogen-activated protein (MAP) kinase, and extracellular signal-related kinases (ERKs) as well as phosphoinositide 3-kinase with their specific inhibitors also resulted in suppressing the chemotaxis of primary macrophages induced by PS-ODNs. These results indicate that the PS-ODN-mediated chemotaxis requires the activation of ERKs, p38 MAP kinase, and phosphoinositide 3-kinase as well as endosomal maturation. In addition, the phosphorylations of the p38 MAP kinase, ERKs, and protein kinase B, Akt, were induced by PS-ODN, which were further enhanced by the presence of both a dG(6) run and CpG motifs. Our findings suggest that the chemotactic activity of PS-ODNs may be one of the mechanisms by which PS-ODNs exhibit stronger immunomodulatory activities than PO-ODNs in vivo.

Dendritic cells (DC) are highly motile and play a key role in mediating immune responses in various tissues and lymphatic organs. We investigated locomotion of mononuclear cell-derived DC at different maturation stages toward gradients of sensory neuropeptides in vitro. Calcitonin gene-related peptide, vasoactive intestinal polypeptide, secretin, and secretoneurin induced immature DC chemotaxis comparable to the potency of RANTES, whereas substance P and macrophage-inflammatory protein-3beta stimulated immature cell migration only slightly. Checkerboard analyses revealed a true chemotactic response induced by neuropeptides. Upon maturation of DC, neuropeptides inhibited spontaneous, macrophage-inflammatory protein-3beta- and 6Ckine-induced cell migration. Maturation-dependent changes in migratory behavior coincided with distinct neuropeptide-induced signal transduction in DC. Peripheral neuropeptides might guide immature DC to peripheral nerve fibers where high concentrations of these peptides can arrest the meanwhile matured cells. It seems that one function of sensory nerves is to fasten DC at sites of inflammation.

Neutrophils migrate through endothelium using an ordered sequence of adhesive interactions and activating signals. To investigate the consequences of disruption of this sequence, we characterized adhesion and migration of neutrophils perfused over HUVEC that had been treated with TNF-alpha for 4 h and evaluated changes caused by exogenously added chemotactic agents. When HUVEC were treated with 2 U/ml TNF, flowing neutrophils adhered, with the majority rolling and relatively few migrating through the monolayer. If fMLP, IL-8, zymosan-activated plasma (a source of activated complement factor C5a), epithelial cell-derived neutrophil-activating peptide (ENA-78), or growth-regulating oncogene, GRO-alpha, was perfused over these neutrophils, they stopped rolling and rapidly migrated over the monolayer, but did not penetrate it. When HUVEC were treated with 100 U/ml TNF, the majority of adherent neutrophils transmigrated. If neutrophils were treated with fMLP, IL-8, C5a, ENA-78, or GRO-alpha just before perfusion over this HUVEC, transmigration, but not adhesion, was abolished. However, when platelet-activating factor was used to activate neutrophils, migration through HUVEC treated with 100 U/ml TNF was not impaired, and migration through HUVEC treated with 2 U/ml TNF was actually increased. Transmigration required ligation of CXC chemokine receptor-2 on neutrophils, and differential desensitization of this receptor (e.g., by fMLP but not platelet-activating factor) may explain the pattern of disruption of migration. Thus, transmigration may require presentation of the correct activators in the correct sequence, and inappropriate activation (e.g., by systemic activators) could cause pathological accumulation of neutrophils in the vessel lumen.

In this study, we have examined the ability of chemokine receptor antagonists to prevent neutrophil extravasation in the mouse. Two murine CXC chemokines, macrophage-inflammatory protein (MIP)-2 and KC, stimulated the accumulation of leukocytes into s.c. air pouches, although MIP-2 was considerably more potent. The leukocyte infiltrate was almost exclusively neutrophilic in nature. A human CXC chemokine antagonist, growth-related oncogene (GRO)-alpha(8-73), inhibited calcium mobilization induced by MIP-2, but not by platelet-activating factor in leukocytes isolated from the bone marrow, indicating that this antagonist inhibits MIP-2 activity toward murine leukocytes. Pretreatment of mice with GROalpha(8-73) inhibited, in a dose-dependent manner, the MIP-2-induced influx of neutrophils to levels that were not significantly different from control values. Moreover, this antagonist was also effective in inhibiting the leukocyte recruitment induced by TNF-alpha, LPS, and IL-1beta. Leukocyte infiltration into the peritoneal cavity in response to MIP-2 was also inhibited by prior treatment of mice with GROalpha(8-73) or the analogue of platelet factor 4, PF4(9-70). The results of this study indicate 1) that the murine receptor for MIP-2 and KC, muCXCR2, plays a major role in neutrophil recruitment to s.c. tissue and the peritoneal cavity in response to proinflammatory agents and 2) that CXCR2 receptor antagonists prevent acute inflammation in vivo.

Human C5a is a potent chemoattractant for granulocytes, monocytes, and dendritic cells. In mice C5a has been shown to be chemotactic for germinal center (GC) B cells. To date, no information is available on the effects of C5a on human B cell locomotion. Here we demonstrate that rC5a increases polarization and migration of human tonsillar B cells. The locomotory response was due to both chemokinetic and chemotactic activities of rC5a. Moreover, memory and, at a lesser extent, naive B cell fractions from purified tonsillar populations displayed rC5a-enhanced migratory properties, whereas GC cells did not. Flow cytometry revealed C5aR (CD88) on approximately 40% memory and 10% naive cells, respectively, whereas GC cells were negative. Immunohistochemistry showed that a few CD88+ cells were of the B cell lineage and localized in tonsillar subepithelial areas, where the majority of memory B cells settle. Pretreatment of memory B cells with the CD88 mAb abolished their migratory responsiveness to rC5a. Finally, the C5 gene was found to be expressed in naive, GC, and memory B lymphocytes at both the mRNA and the protein level. This study delineates a novel role for C5a as a regulator of the trafficking of human memory and naive B lymphocytes and supports the hypothesis that the B cells themselves may serve as source of C5 in secondary lymphoid tissues.

A recombinant soluble form of human Fas ligand (sFasL) was tested for its chemotactic activity against human and mouse polymorphonuclear neutrophils (PMN) by the Boyden chamber method. sFasL exhibited a potent chemotactic activity against both human and mouse PMN and HL-60 cells when differentiated into neutrophils or monocytes. A neutralizing anti-FasL mAb abolished the chemotactic activity, while control mAb did not. Ligation of Fas by either IgM- or IgG-type anti-Fas mAb also induced PMN migration. PMN derived from lpr mice that express few Fas molecules did not respond to sFasL. In contrast, those derived from lpr(cg) mice that express Fas molecules with a mutated death domain normally responded to sFasL chemotaxis. These results directly indicated a chemotactic activity of sFasL against PMN and suggest a novel signaling function of Fas, which appears to be independent of the death domain-mediated apoptosis.

Melanoma growth stimulating activity (MGSA) and IL-8 are related chemokines that are potent chemoattractants and activators of neutrophils both in vitro and in vivo. Increasing evidence suggests that these molecules play an important role in inflammation; thus, antagonists of their action could be useful therapeutically as antiinflammatory agents. We have generated an MGSA mutant, H19A, that shows a dissociation between receptor binding and biologic activity. The biologic activity of the H19A mutant is between 133-fold and 282-fold less potent than that of wild-type MGSA measured by three independent assays of neutrophil function, i.e., elastase release chemotaxis and the up-regulation of CD18. In addition, pretreatment of cells with the H19A mutant inhibited the ability of MGSA to induce elastase release and chemotaxis and to increase intracellular calcium. However, competition binding studies in cells transfected with the CXCR2 receptor and in neutrophils demonstrate that the receptor affinity of the H19A mutant is only 13-fold less than that of wild-type MGSA. These studies suggest that the mutant MGSA is defective in activating signaling through the receptor and indicate that binding to the receptor is not sufficient to activate a biologic response. The dissociation between receptor binding and activation for this mutant suggests that it should be possible to design antagonists of MGSA that may be of clinical utility.

To further elucidate mechanisms involved in mast cell accumulation at sites of cutaneous inflammation, we have studied the ability of human leukemic mast cells (HMC-1 cells) to express functionally active IL-8 receptors. Expression of mRNA for both types of IL-8 receptors (CXCR1 and CXCR2) was demonstrated by PCR and of both proteins by flow cytometry. Binding and competition studies with 125I-labeled IL-8 and its homologue melanoma growth stimulating activity (125I-labeled MGSA) revealed two specific binding sites for IL-8, K1 = 1.1 x 10(11) M(-1) and K2 = 5 x 10(7) M(-1); and for MGSA, K1 = 2.8 x 10(10) M(-1) and K2 = 5 x 10(7) M(-1). This finding was supported by a dose-dependent rise of cytosolic free calcium concentration ([Ca2+]i) induced by both chemokines and to a lesser extent by the homologue neutrophil-activating peptide-2 (NAP-2). A significant migratory response of human leukemic mast cells (HMC-1) was observed with all three chemokines at a range from 10(-8) M to 10(-9) M. Moreover, the formation of cellular F-actin was induced in a rapid, dose-dependent fashion, with a maximally 1.7-fold increase at 10(-7) M. Using postembedding immunoelectron microscopy, we could show the expression of CXCRI on the cytoplasmatic membrane of isolated human skin mast cells whereas CXCR2 was located in mast cell-specific granules. These findings demonstrate for the first time the functional expression of both types of IL-8 receptors on human mast cells, suggesting a role for their ligands during mast cell activation and recruitment.

IL-8 is one of the major mediators of the transendothelial migration of neutrophils from the circulation to the site of injury and infection. In this work we demonstrate that the CXC or alpha-chemokines, IL-8 and melanoma growth stimulatory activity (MGSA) induce myeloid suppression via direct action on progenitor cells, mediated by activation of the murine homologue of the CXC chemokine receptor-2 (CXCR2) or IL-8R B. We first show that proliferation of the IL-3-dependent murine myeloid progenitor cell line 32D is suppressed by human IL-8 and the functionally and structurally related peptide, MGSA. Second, we show for the first time the high endogenous expression of the murine CXCR2 in 32D cells, as demonstrated by Northern blot analysis, binding to [125I]macrophage inflammatory protein-2, and macrophage inflammatory protein-2-induced calcium responses in 32D cells. Third, we demonstrate that IL-8 and MGSA induce a rise in intracellular calcium in 32D cells. The IL-8-induced Ca2+ response is desensitizing, since a second dose of IL-8 did not trigger a second calcium response. Other chemokines, including neutrophil-activating protein-2, platelet factor-4, RANTES, and macrophage chemotactic protein-1, neither suppressed the proliferation of 32D cells nor induced a rise in intracellular calcium. Finally, the IC50 of IL-8- and MGSA-dependent suppression of proliferation of 32D cells is in good agreement with the EC50 of IL-8- and MGSA-dependent activation of neutrophil Mac-1 up-regulation and chemotaxis. Our studies are consistent with the idea that IL-8 and MGSA suppress the proliferation of 32D cells by activation of murine CXCR2.

Human neutrophils contain a highly specific dehydrogenase that converts 5-hydroxy-6,8,11,14-eicosatetraenoic acid to 5-oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE). 5-Oxo-ETE is a potent stimulator of calcium mobilization, chemotaxis, and aggregation in these cells and has similar effects on eosinophils. The primary objectives of the current study were to determine whether this compound could increase the surface expression of integrins and stimulate actin polymerization in neutrophils. 5-Oxo-ETE stimulated the expression of CD11b and, to a lesser extent, CD11c, on neutrophils, but had no significant effects on the expression of CD11a, CD16 (Fc gammaRIII), or CD32 (Fc gammaRII). Surface expression of CD11b in response to 5-oxo-ETE was maximal after 12 min and remained constant thereafter. The EC50 for this response (50 nM) was lowered to 20 nM by preincubation of neutrophils with PMA. 5-Oxo-ETE (EC50, 10 nM) also rapidly stimulated actin polymerization in neutrophils, with a maximal response at 20 s. This response was blocked by pretreatment of neutrophils with the Gi protein inhibitor, pertussis toxin, and by homologous desensitization due to preincubation with 5-oxo-ETE. However, preincubation with leukotriene B4 or platelet-activating factor had no effect on the response of neutrophils to subsequent addition of 5-oxo-ETE. The adherence of neutrophils to plasma-coated plastic was also stimulated by 5-oxo-ETE with a time course similar to that for the surface expression of CD11b. Low concentrations of PMA (0.3 nM) enhanced this response. These results raise the possibility that 5-oxo-ETE could contribute to the infiltration of neutrophils into inflammatory sites.

At inflammatory sites in vivo, leukocytes may confront multiple, competing chemoattractive signals. We found significant differences between eosinophils and neutrophils in transendothelial chemotaxis to a chemoattractant diffusing from the lower chamber, when a chemoattractant that binds to another receptor is present at uniform concentration. The transendothelial migration of eosinophils to FMLP, C5a, RANTES, or MCP-3 was totally inhibited by the presence of the homologous chemoattractant, and only RANTES and MCP-3 showed mutual inhibition. C5a and to a lesser extent FMLP chemokinetically stimulated migration to RANTES and MCP-3, without stimulating random migration. Results with neutrophils contrasted. The presence of FMLP not only abrogated neutrophil transmigration to FMLP but also strongly decreased chemotaxis to C5a, IL-8, and Gro-alpha. Similarly, C5a inhibited neutrophil chemotaxis to IL-8 and Gro-alpha. IL-8 almost totally abrogated chemotaxis to Gro-alpha, but Gro-alpha only moderately inhibited chemotaxis to IL-8. Neither IL-8 nor Gro-alpha significantly inhibited transmigration to FMLP or C5a. Actin polymerization in eosinophils and neutrophils was desensitized by the same combinations of chemoattractants that desensitized chemotaxis. We conclude that eosinophils have at least three noninterfering receptor-signal transduction pathways for chemotaxis and actin polymerization. In contrast, the signaling pathways for FMLP, C5a, and IL-8/Gro-alpha in neutrophils are heterologously cross-desensitized, with a hierarchy of resistance to competing signals of FMLP > C5a > IL-8 > Gro-alpha, in agreement with previous results in neutrophils on the Ca2+-mobilizing response. These results may have important implications for the behavior of these cell types in inflammatory sites.

Human neutrophils undergo rapid homologous receptor desensitization following repeated stimulation with chemoattractants such as IL-8, C5a, and FMLP. It has also been demonstrated that cross-desensitization among these chemoattractant receptors occurs. We investigated the mechanisms underlying the cross-desensitization of responses to IL-8 induced by pretreatment with FMLP or C5a. In [125I]-labeled IL-8 binding studies we found that the cross-desensitization induced by FMLP or C5a was associated with a subsequent reduction in IL-8 binding to neutrophils. There was no recovery of [125I]-labeled IL-8 binding on removal of the C5a or FMLP pretreatment. FACS analysis using mAbs specific for the two IL-8R subtypes showed differential regulation of IL-8R A and IL-8R B cell surface expression after chemoattractant pretreatment. Homologous desensitization by IL-8 resulted in internalization of IL-8R A and IL-8R B, but only IL-8R A was completely re-expressed after removal of agonist. FMLP stimulation led to a substantial loss of IL-8R B from the cell surface, whereas C5a stimulation induced only a partial loss. In both cases there was no re-expression of IL-8R B on removal of the chemoattractant stimulation. C5a and FMLP did not affect IL-8R A expression. Calcium mobilization studies using melanoma growth stimulatory activity and IL-8 suggest that a sustained loss of IL-8R B may play a part in maintaining FMLP-induced IL-8R cross-desensitization. Chemoattractant-induced cross-desensitization of neutrophils may be of importance in regulating neutrophil accumulation during the inflammatory response in vivo.

To date, the activities of the alpha chemokines for human peripheral B cells from normal subjects (N-B cells) or from HIV-infected subjects (HIV-B cells) are not well established. No report on the IL-8R expression on N-B cells and HIV-B cells has been seen. We report in this work that the alpha chemokines IL-8 and growth-regulatory oncogene-alpha (GRO-alpha) induce a chemotactic migration of N-B cells and HIV-B cells via stimulating the IL-8RB on these cells. The chemotaxis of N-B cells can be inhibited by IFN-gamma and IL-2, and augmented by IL-4 and IL-13, whereas TNF-alpha and IL-10 have no influence. The chemotaxis of HIV-B cells can be inhibited by IFN-gamma and IL-2, and augmented by TNF-alpha, IL-4, and IL-10, whereas IL-13 has no influence. IL-8R are expressed more abundantly on freshly isolated HIV-B cells than N-B cells (51% and 15%, respectively). The IL-8R on N-B cells can be down-regulated by IFN-gamma, IL-2, and TNF-alpha (selectively on IL-8RA), and up-regulated by IL-4 and IL-13, whereas IL-10 has no influence. The IL-8R on HIV-B cells can be down-regulated by IFN-gamma and IL-2, and up-regulated by TNF-alpha, IL-4, and IL-10, whereas IL-13 has no influence. Importantly, N-B cell and HIV-B cell chemotaxis toward IL-8 and GRO-alpha can be blocked by anti-IL-8RB polyclonal Ab, but not by anti-IL-8RA polyclonal Ab. Our results demonstrate that IL-8 and GRO-alpha are important inflammatory mediators that stimulate the directional migration and recruitment of B lymphocytes. The migratory behavior and the expression of IL-8R on HIV-B cells and some of the reactions to Th1- and Th2-like cytokines are modified significantly during HIV infection.

The newly described products of 5-hydroxyeicosanoid dehydrogenase, 5-oxo-6,8,11,14-eicosatetraenoic acid (ETE) and 5-oxo-15(OH)ETE, induced directional migration and actin polymerization of human monocytes in vitro. At peak concentrations, the two eicosanoids had a chemotactic activity of about 40% of that observed in the presence of an optimal concentration of FMLP and twice the activity elicited by the related eicosanoid 5-hydroxy-6,8,11,14-eicosatetraenoic acid (5-HETE). 15-Oxo-ETE showed a very low but detectable chemotactic activity. All of these chemotactic responses were blocked by Bordetella pertussis toxin, but were resistant to LY255283, a leukotriene B4 (LTB4) receptor antagonist. 5-Oxo-ETEs and 5-HETE induced homologous desensitization of chemotactic response, but did not cross-desensitize to other chemotactic agonists (e.g., monocyte chemotactic protein (MCP)-1 and LTB4). 5-Oxo-ETEs increased in a synergistic fashion the monocyte migration to MCP-1 and MCP-3. In the same range of concentrations, 5-oxo-ETE increased MCP-1-induced release of arachidonic acid from labeled monocytes. No synergistic interaction was observed when FMLP was used as chemoattractant. Thus, this study identifies monocytes as cells responsive to 5-oxo-ETEs and shows that monocyte activation by 5-oxo-ETEs occurs through an LTB4 receptor-independent mechanism that associates with pertussis toxin-sensitive G proteins. The synergistic interaction between 5-oxo-ETEs and C-C chemokines, two families of mediators both synthesized by phagocytic cells, may be relevant in vivo for the regulation of monocyte accumulation at sites of allergic and inflammatory reactions.

We have previously reported that cytokines such as IL-9, IL-4, and IL-6 protect murine thymic lymphoma cell lines against dexamethasone-induced apoptosis. A similar activity, which could not be ascribed to any of these factors, was found in a number of human T cell supernatants that enabled mouse BW5147 thymic lymphoma not only to escape apoptosis but also to maintain proliferation. The protein responsible for this activity was purified to homogeneity from the culture medium of activated leukemic T cells and was found to be identical with the I-309 chemokine. Half-maximal anti-apoptotic activity was obtained with approximately 1 ng/ml, a concentration considerably lower than that required for the monocyte chemotactic activity of this molecule, as measured on THP-1 cells. The purified I-309 also improved the survival of two other mouse thymic lymphoma cell lines. This activity was as potent as that of IL-9, which was the strongest anti-apoptotic factor found to date for these cells. Similar results were obtained for BW5147 cells with recombinant I-309 and with T cell activation gene-3, the murine homologue of I-309, but not with other members of the chemokine family, including IL-8, neutrophil-activating peptide-2, granulocyte chemotactic protein-2, macrophage inflammatory protein-1a, RANTES (regulated upon activation, normal T cell expressed and secreted), monocyte chemotactic protein-1 (MCP-1), and MCP-2. MCP-3, however, showed a minor, but significant effect in this model. Unlike that of IL-9, the activity of I-309 was completely inhibited in the presence of pertussis toxin, indicating the involvement of a G protein in this process.

The present study compares the activity of TCA3 with other beta-chemokines (macrophage inflammatory protein (MIP)-1 alpha, MIP-1 beta, and monocyte chemoattractant protein (MCP)-1) on rat vascular smooth muscle cells. TCA3, MIP-1 alpha, and MCP-1 (but not MIP-1 beta) treatment stimulates chemotaxis of vascular smooth muscle cells. TCA3-mediated chemotactic responses are sensitive to treatment with pertussis toxin, suggesting that G alpha-i proteins are involved in TCA3 signaling of smooth muscle. In addition, TCA3, MIP-1 alpha, and MCP-1 increase vascular smooth muscle cell adhesiveness to type III collagen. In contrast, stimulation with TCA3, but not other beta-chemokines, induces proliferation of vascular smooth muscle cells. TCA3 receptors were identified on rat vascular smooth muscle cells by direct binding of radiolabeled ligand. TCA3 binds to this receptor with high affinity (3 nM). Rat vascular smooth muscle cells display approximately 75,000 binding sites/cell. Competitive inhibition studies indicated that murine MIP-1 alpha, murine MCP-1, and human RANTES are weak partial competitors of TCA3 binding, demonstrating the existence of a unique receptor for TCA3. Murine MIP-1 beta, which fails to stimulate any biologic functions in vascular smooth muscle cells, also does not inhibit TCA3 binding. The combined data demonstrate that TCA3 and other beta-chemokines can modulate vascular smooth muscle cell function.

Mast cells are known to accumulate at sites of inflammation, however, the chemotaxins involved are undefined. Since most natural leukocyte secretagogues also induce cell migration, and since the anaphylatoxins C3a and C5a are mast cell secretagogues, we hypothesized that both C3a and C5a are also mast cell chemotaxins. Here we report that C3a and C5a are, in fact, potent chemotaxins for the human mast cell line HMC-1. The optimal concentrations, half-maximal effective concentrations (a measure of agonist potency) and the efficacy (response at the optimal concentration) compared with medium control were, for C3a: 10 nM, 0.5 nM, and 256%, respectively; for C5a: 1 nM, 10 pM and 145%. Chemotaxis of HMC-1 cells to both C3a and C5a was blocked by pertussis toxin, suggesting that Gi-coupled receptors are involved in signal transduction. C3a and C5a also induced transient pertussis toxin-inhibitable increases in [Ca2+]i (ED50 = 1 nM for both) that could be homologously but not heterologously desensitized, suggesting that the receptors for C3a and C5a are distinct. These results make C3a the most effective mast cell chemotaxin identified to date. The chemotactic potency described here for C3a is also 100- to 1000-fold greater than for all of its previously described cellular actions. Direct chemoattraction of mast cells by C3a and C5a may help explain the rapid accumulation of mast cells at sites of inflammation.

Neutrophil and monocyte chemotactic factors were isolated from conditioned media of mouse fibroblasts and epithelial cells. Neutrophil chemotactic activities were purified to homogeneity using a four-step chromatographic procedure, and the corresponding proteins were identified by amino acid sequence analysis. Natural forms of the murine chemokines KC and macrophage inflammatory protein-2 were isolated from virus-infected fibroblasts. However, the major neutrophil chemotactic activity from fibroblasts stimulated with endotoxin plus double-stranded RNA and from PMA-treated epithelial cells resided in other 7- and 8-kDa proteins. Amino acid sequence analysis revealed a novel Cys-Xaa-Cys chemokine structure, characterized by the conservation of four cysteines and the Glu-Leu-Arg motif. Based on the completely identified primary structure of this natural protein, this chemokine must be considered to be the murine homologue of human and bovine granulocyte chemotactic protein-2 (GCP-2; 61 and 64% identical residues, respectively). Due to NH2-terminal cleavage, 11 different forms of mouse GCP-2 were discovered. In contrast to human and bovine GCP-2, functional comparison of long and short NH2-terminal forms of mouse GCP-2 demonstrated that truncated mouse GCP-2 (short form) has a higher specific activity in neutrophil activation (gelatinase B release) and chemotaxis assays. Furthermore, mouse GCP-2 was more potent than human GCP-2 on human neutrophils, and more active than murine KC and macrophage inflammatory protein-2 on mouse neutrophils. In view of the absence of a murine homologue for IL-8, NH2-terminally processed GCP-2 can be considered a major neutrophil chemoattractant in the mouse during the inflammatory response.

A crucial early event in tuberculosis is the ingestion of Mycobacterium tuberculosis (Mtb) by alveolar macrophages. Chemotactic factors released by infected macrophages are likely to initiate a granulomatous response, a key feature of host resistance to tuberculosis. To date, the role of mycobacterial products in regulating the granulomatous response has not been clearly defined. Here we report that the mycobacterial cell wall glycophospholipid lipoarabinomannan (LAM) could specifically induce human peripheral blood T cell chemotaxis in vitro. Both terminally mannosylated LAM isolated from Mtb and LAM lacking the terminal mannosyl units isolated from an avirulent mycobacterium could induce T cell migration in the absence of serum. In contrast, terminally mannosylated LAM isolated from Mycobacterium bovis BCG failed to induce T cell chemotaxis. These observations represent the first report that LAM is capable of directly inducing biologic responses in human T cells. Flow cytometry analysis revealed that CD4+, CD8+, and CD45RO+ lymphocytes were present in the migrating cell populations at ratios similar to those found in nonmigrating cells. The chemotactic response was found to require new protein synthesis, and could be blocked by inhibitors of protein tyrosine kinases at concentrations that did not affect random migration. Acyl groups at the reducing terminus of LAM appear to be required for the chemotactic activity of this mycobacterial glycolipid. Lastly, culture supernatants from human alveolar macrophages infected in vitro with a virulent strain of Mtb could induce T cell migration. Much of the migratory activity present in these supernatants could be blocked using a mAb against LAM, suggesting that LAM is one of the chemotactic factors released by Mtb-infected alveolar macrophages.

The mechanisms by which neutrophils migrate through the alveolar interstitium during acute lung inflammation are unknown. We wished to determine whether platelet-activating factor (PAF) and IL-8, two important mediators in neutrophil transendothelial migration, stimulated neutrophil adherence and motility on lung fibroblasts. Canine fibroblasts grown from lung explants were characterized by light and electron microscopy, and flow cytometry. Unstimulated neutrophils adhered poorly (less than 2%) to cultured fibroblasts. However, neutrophils stimulated with PAF (20-200 nM) showed a dose-dependent increase in adherence that was largely (70%) mediated by the beta 2 (CD11/CD18) integrins; adherence was less dependent (50%) on fibroblast intercellular adhesion molecule-1. Conversely, neutrophils stimulated with canine rIL-8 did not increase their adherence to fibroblasts. PAF-stimulated neutrophils were nonmotile on the surface of the fibroblast, but subsequent addition of rIL-8 (10(-8) M) induced motility that was entirely CD1 8 dependent. Fibroblasts stimulated with human rTNF-alpha or Escherichia coli endotoxin (LPS) were a significant source of IL-8 mRNA. In response to rTNF-alpha (50 U/ml), IL-8 mRNA was detected at 2 h by northern blot analysis; it peaked at 6 h and returned to baseline by 24 h. Fibroblasts stimulated with rTNF-alpha secreted IL-8 protein into the culture medium; secreted IL-8 was chemotactic for neutrophils. These data suggest that fibroblasts can function not only as an adhesive substrate, but also as a source of stimulation for neutrophil migration through the inflamed alveolar interstitium.

The human cytokine growth-regulated oncogene (GRO)-alpha is a small glycoprotein secreted by monocytes, endothelial cells, glycoprotein secreted by monocytes, endothelial cells, fibroblasts, synovial cells, and some tumor cells such as melanoma cells. It is structurally related to IL-8 and can activate neutrophils, whereas it induces chemotaxis, exocytosis, and a respiratory burst in neutrophils. To date, its functions on T lymphocytes have not been well established. We report here that recombinant human (rh)GRO-alpha is a potent chemoattractant for freshly isolated T lymphocytes, but not for anti-CD3 mAb-activated T lymphocytes. It attracts CD4+ and CD8+ T lymphocyte subsets to an equal extent. The migrating T lymphocytes toward rhGRO-alpha are predominantly CD45RO+ memory CD4+ and CD8+ subsets. The chemotactic migration of T lymphocytes toward rhGRO-alpha is stimulated via the IL-8 receptors on the cells. This process can be augmented by IFN-gamma and TNF-alpha, and inhibited by IL-4, IL-10, and IL-13. In addition, we also document that on T lymphocytes there exist IL-8 receptors that can be up-regulated by IFN-gamma, TNF-alpha, and IL-2. Our results demonstrate that rhGRO-alpha gene encodes for an inflammatory mediator that stimulates the directional migration of T lymphocytes. It may thus be another important mediator in the diseases in which T lymphocytes form the major constituent of the cellular infiltration.

Trafficking to tissues and then to lymph nodes is a crucial aspect of the immunobiology of dendritic cells. The present study was designed to identify molecules able to direct the migration of human blood-derived dendritic cells. fMLP (representative of formyl peptides of bacterial origin), C5a, and the C-C chemokines monocyte chemotactic protein (MCP)-3, MIP-1 alpha/LD78, and RANTES elicited chemotactic migration and a rise of intracellular free calcium in dendritic cells. In contrast, the C-X-C chemokines IL-8 and IP-10 and the C-C chemokines MCP-1 and MCP-2 were inactive as chemoattractants. Thus, dendritic cells respond to classical chemotactic signals and to a set of chemokines distinct from that active on monocytes and neutrophils. Chemoattractants are likely to contribute to localization and trafficking of dendritic cells and provide tools to recruit these cells in the design of immunization strategies.

We investigated the regulatory mechanism of the expression of IL-8R, IL-8R type A (IL-8RA), and IL-8R type B (IL-8RB) on human neutrophils by IL-8. The expression of IL-8RA/B was analyzed by flow cytometry using mAb specific for each receptor. IL-8 down-modulated > 90% of IL-8RA and IL-8RB expression within 5 min. A related C-X-C chemokine, melamona growth stimulatory activity, down-modulated IL-8RB but not IL-8RA. It required 7 to 13 times more IL-8 to down-modulate IL-8RA than IL-8RB, as determined by the half-maximal effective concentration of IL-8. Scatchard analysis showed that the affinity of IL-8RA for IL-8 was lower than that of IL-8RB. The possible functions of each IL-8R were explored by comparing 1) the expression levels of IL-8RA/B on migrated neutrophils during in vitro chemotaxis assay and 2) the recovery rate of IL-8RA/B expression after down-modulation by IL-8. Results obtained from the in vitro chemotaxis show that the expression level of IL-8RB, but not IL-8RA, on neutrophils that migrated into the chamber containing low concentrations (< 1 nM) of IL-8 was significantly reduced compared with the control level. This suggests that IL-8RB may play as active role in the initiation of neutrophil migration distant from the site of inflammation, where the concentration of IL-8 is at the picomolar level. After down-modulation by 119 nM IL-8, the expression of IL-8RA fully recovered within 1.5 h, while the recovery rate of IL-8RB expression was slow and never reached more than 40% of the control level during a 3-h culture period. The rapid reexpression of IL-8RA suggests that the low affinity IL-8RA may play a more active role in mediating IL-8 signal at the site of inflammation, where the concentration of IL-8 is high.

Although previous studies have shown that different cells and cell lines of murine origin bind human C1q, suggesting that they display cell surface receptors for C1q, no information is available to indicate whether mouse or human mast cells express C1q receptors. This paper presents the first evidence to show that murine mast cells express specific receptors for C1q. Western blot analysis of cell membrane proteins prepared from a bone marrow-derived mouse cell line using two monospecific polyclonal Abs, one directed against the 60-kDa C1q receptor (C1q-R) that binds to the collagen-like stalk of C1q (cC1q-R) and the other directed against the 33-kDa molecule that binds to the globular "heads" of C1q (gC1q-R), show that both of these receptors are present on these cells. In addition, C1q can induce mast cell migration in a specific and dose-dependent manner. Interestingly, the C1q-induced migratory response was found to be biphasic; the first response peaked at a C1q concentration of 0.1 nM, whereas the second phase peaked at approximately 40 nM. Checkerboard analysis of the mast cell migratory response to C1q showed that the first phase was primarily due to chemotaxis and the second phase was attributable to chemokinesis. Preincubation of C1q with Abs specific for the collagen-like tail of the molecule abolished both its chemotactic and chemokinetic response, whereas heat inactivation of C1q (56 degrees C, 1 h) resulted in 85% abrogation of the chemotactic phase and 42% reduction in the chemokinetic phase. The observed mast cell migratory responses were mediated by cell surface C1q-R(s), as inclusion of a mixture of anti-C1q-R and anti-gC1q-R Abs with the cells inhibited their migratory response toward C1q. However, incubation of cells with various doses of C1q did not result in histamine release. Furthermore, engagement of mast cell C1q-Rs by the ligand C1q induced an antiproliferative response, as coculturing of mast cells with C1q resulted in a specific and dose-dependent decrease in DNA synthesis. These data suggest that C1q-Rs may play a significant role in mast cell function and regulation by providing an important signal through which mast cells can be recruited to inflammatory sites of increased C1q concentration.

IL-8 is a potent proinflammatory cytokine that has a key role in the recruitment and activation of neutrophils during inflammation. IL-8 reacts with neutrophils via two distinct types of IL-8-R. Receptor-specific Abs were raised against peptides derived from the first extracellular domain of each IL-8-R. Anti-IL-8-R1 and anti-IL-8-R2 selectively block 125I-IL-8 binding to rIL-8-R type 1 or 2, respectively. The anti-peptide Abs were used to assess the role of each receptor in the chemotactic response of neutrophils to GRO alpha and to IL-8. Anti-IL-8-R2 blocks GRO alpha-induced chemotaxis of neutrophils. Chemotaxis to GRO alpha is not inhibited by anti-IL-8-R1. Thus GRO alpha stimulates chemotaxis exclusively through IL-8-R2 and independently of IL-8-R1. Surprisingly, anti-IL-8-R1 inhibits the majority (78 +/- 3%) of IL-8-induced neutrophil chemotaxis. Only a minor proportion of IL-8-induced chemotaxis (29 +/- 5%) is inhibited by anti-IL-8-R2. These findings indicate that chemotaxis to IL-8 is mediated predominantly by type 1 IL-8-Rs and suggest that IL-8-R1 is an appropriate target for therapeutic strategies to limit neutrophil influx in diseases where neutrophils contribute to pathophysiology.

It has been shown that CC chemokines activate basophil and eosinophil leukocytes with different selectivities and patterns of activity. The most effective are monocyte chemotactic protein-1 (MCP-1), a potent stimulus of mediator release in basophils without effects on eosinophils, RANTES, a weak stimulus of release and strong chemoattractant for basophils and eosinophils, and MCP-3, which combines the activities of MCP-1 and RANTES. We have now compared MCP-2, which has 62 and 60% of sequence identity with MCP-1 and MCP-3, respectively, with the other CC chemokines. MCP-2 induced mediator release by human basophils with lower efficacy and potency than MCP-1 and MCP-3. It promoted transient changes of cytosolic-free calcium concentration ([Ca2+]i) and chemotactic responses in both basophils and eosinophils, however somewhat less efficiently than MCP-3 and RANTES. Desensitization studies indicate that MCP-2 interacts with receptors recognizing MCP-1 as well as RANTES. These results demonstrate that MCP-2 and MCP-3 exert qualitatively similar biologic activities on basophils and eosinophils. In basophils that had not been treated with IL-3, MCP-2 induced minimal exocytosis only, but desensitized the cells toward MCP-1 and MCP-3, suggesting that MCP-2 may act as a functional inhibitor of CC chemokine actions. The results of this study further indicate that MCP analogues display partially distinct, partially overlapping bioactivities toward eosinophils and basophils, and may thus regulate inflammatory processes involving these effector cell types.

We have determined that several chemokines induce mast cell migration in vitro. This directed migration is dependent on the presence of particular extracellular matrix proteins and the activation status of the cells. Mast cell haptotactic responses were observed in response to various chemokines on vitronectin-, laminin-, and fibronectin-coated filters. Unstimulated mast cells were chemoattracted only by monocyte chemotactic protein-1 and RANTES on vitronectin-coated and, to a lesser extent, laminin-coated filters, whereas IgE-activated mast cells migrated in response to monocyte chemotactic protein-1, regulated on activation normal T expressed and secreted, platelet factor-4, and macrophage inflammatory protein-1 alpha on all three matrix proteins. No significant migration was observed on collagen type IV-coated or uncoated filters. Mast cell migration in response to chemokines on extracellular matrices and its enhancement by IgE-dependent activation provide a mechanism by which cells may be drawn to sites of inflammation. Chemokine-induced mast cell recruitment may be particularly relevant in host defense responses to parasitic infections, allergic reactions, Jones-Mote reactions, and in wound healing.

Expression of mRNA for the neutrophil (PMN) chemokine, KC, in rat models of lung injury suggests a role for this chemokine in pulmonary inflammation. We addressed this hypothesis at the protein level by functionally characterizing recombinant rat KC (rKC) in vitro and in vivo. In vitro, rKC induced PMN chemotaxis and increased the expression of CD11b/CD18 on PMNs. Recombinant KC also induced a respiratory burst (quantitated by flow cytometry) in rat PMNs, similar to that caused by its human structural homologue, gro/melanoma growth-stimulating activity, on human PMNs, but less than that caused by IL-8 on human PMNs. Intratracheal instillation of rKC induced dose-dependent PMN influx into airspaces (average PMNs in bronchoalveolar lavage: vehicle = 1.5%, n = 4; rKC (1 microgram) = 11.5%, n = 2; rKC (10 micrograms) = 77.3%, n = 2). A neutralizing anti-KC Ab reduced the chemotactic activity of rat bronchoalveolar lavage fluid collected after the intratracheal administration of LPS (48.3 +/- 8% of control, n = 4). Anti-KC neutralizing Ab markedly inhibited PMN accumulation (71 +/- 6%) within the lungs in response to an intratracheal challenge of LPS. We conclude that rat KC is a major but not exclusive mediator of PMN activation and recruitment during LPS-induced pulmonary inflammation.

It remains unknown which factor(s) control mast cell recruitment in chronic immune reactions. Although TGF-beta has been shown to function as a potent chemotactic factor for monocytes, fibroblasts, and neutrophils, its effect on mast cells has not been previously determined. In this study, TGF-beta 1 was shown to cause directed migration of cultured mouse mast cells at femtomolar concentrations, with a maximal chemotactic response observed at 25 fM. Moreover, chemotaxis to TGF-beta was also seen using freshly isolated rat peritoneal mast cells. Addition of neutralizing Ab to TGF-beta abrogated its chemotactic activity for both freshly isolated rat peritoneal mast cells and cultured mouse mast cells, whereas an irrelevant species-matched control Ab had no effect. Checkerboard analysis confirmed the mast cell chemotactic activity after exposure to concentration gradients of TGF-beta. Mast cells were observed to undergo rapid and extensive shape changes on exposure to TGF-beta, assuming a polarized morphology in preparation for migration. Other known mast cell chemoattractants including laminin, c-kit ligand, and IL-3 were found to be considerably less potent on a molar basis in inducing directed migration. Affinity cross-linking studies identified TGF-beta binding proteins with M(r) at 70 and 288 kDa, consistent with types I and III TGF-beta receptors on the mast cells. In summary, TGF-beta is the most potent chemoattractant described for mast cells and conceivably relevant, because pathologic processes mediated by TGF-beta are often associated with mast cell accumulation.

Human neutrophils were exposed to the chemotactic factors C5a, FMLP, IL-8, leukotriene B4, and PAF, for 30 s, and subsequently analyzed for protein tyrosine phosphorylation by immunoblotting whole cell lysates with a polyclonal antiphosphotyrosine Ab. All chemotactic factors caused the rapid de novo tyrosine phosphorylation of a broad band of approximately 120 kDa and increased the phosphotyrosine content of several other proteins, including two with molecular masses of 60 and 56 kDa that were present in the unstimulated neutrophil. Tyrosine phosphorylation was evident as early as 10 s after stimulation and was maintained for 1 to 3 min before dephosphorylation occurred. The extent of tyrosine phosphorylation was dependent on the concentration of chemotactic factor, with stimulation observed at concentrations as low as 10 to 100 nM. To investigate the pathway used by chemotactic factors to transduce this signal, neutrophils were treated with PMA. PMA also stimulated tyrosine phosphorylation in the neutrophil but with a slower response time and a different pattern of affected proteins. Additional experiments suggested that tyrosine phosphorylation is involved in the regulation of the neutrophil respiratory burst because the tyrosine kinase inhibitor, herbimycin A, inhibited C5a-induced protein tyrosine phosphorylation and also prevented C5a- and FMLP-induced superoxide anion production. Herbimycin A also inhibited PMA-induced tyrosine phosphorylation and superoxide anion production. To confirm that the ability to stimulate tyrosine phosphorylation was intrinsic to the C5a receptor, tyrosine phosphorylation was examined in both undifferentiated U937 cells (C5a receptor negative) and cAMP differentiated U937 cells (C5a receptor positive). C5a induced tyrosine phosphorylation only in differentiated U937 cells. Analysis of the C5a receptor mRNA using the PCR confirmed its presence in differentiated and its absence in undifferentiated U937 cells. Therefore, C5a stimulates tyrosine phosphorylation via a receptor-mediated mechanism and U937 cells provide a system in which G-coupled receptor-mediated tyrosine phosphorylation can be investigated.

The murine S100 chemotactic protein of m.w. 10,000 termed (CP-10), has potent chemotactic activity for murine and human myeloid cells. We examined the ability of a synthetic CP-10 hinge region peptide CP-10(42-55) and rCP-10 to act as chemotactic agents and induce expression of the adhesion molecule Mac-1 (CD 11b/CD 18) in vivo. Maximal neutrophil (PMN) accumulation occurred between 2 to 8 h after mouse footpad injection of rCP-10 (10(-7) M) or CP-10 peptide (10(-6) M). The infiltrating PMN expressed high levels of Mac-1, and low levels of the murine L-selectin Mel-14. Injection of CP-10 peptide i.p. also induced infiltration of PMNs that expressed high levels of Mac-1. Cell suspensions obtained after i.p. injection of CP-10 peptide could be significantly inhibited from adhering to fibrinogen-coated plates when incubated with anti-Mac-1 antibody. The chemotactic activity of CP-10 peptide toward murine inflammatory PMN in vitro was also inhibited by anti-Mac-1 antibody. Neither CP-10 analogue stimulated or primed murine inflammatory or human blood neutrophils for superoxide production or granular enzyme release. The localization of CP-10 in vivo was examined using murine footpads injected with LPS and was found to be concentrated around the endothelial cells of the small blood vessels. This distribution suggests that the accumulated CP-10 the may contribute to the generation of a chemotactic gradient.

In recent years, there has been a growing body of evidence suggesting that IL-8 and granulocyte-macrophage CSF (GM-CSF) play an important role in inflammatory processes. We show that after GM-CSF treatment, the exposure of human neutrophils to IL-8 results in the synthesis of leukotriene (LT)B4 and platelet-activating factor. In GM-CSF-treated cells, IL-8 induced a concentration-dependent synthesis of both lipid mediators, with a threshold at 10 to 30 nM, suggesting that IL-8 could stimulate phospholipase A2 activity, an enzyme essential for both syntheses. Accordingly, IL-8 induced a substantial release of 3H-arachidonic acid in GM-CSF-treated PMN. It was also found that IL-8 activates the neutrophil 5-lipoxygenase (5-LO), the other key enzyme in LT biosynthesis. IL-8 induced 5-LO activation in a time- and concentration-dependent manner, with a threshold at 1 nM, and prior treatment of neutrophils with GM-CSF enhanced this effect of IL-8 over the 1 to 300 nM range. Neutrophil-activating peptide-2 and the melanoma growth-stimulatory activity, two peptides that are closely related to IL-8, also had the ability to activate the 5-LO and stimulate LT synthesis, albeit less potently than IL-8. Finally, pertussis toxin and the 5-LO translocation inhibitor, MK-886, both blocked the IL-8-elicited 5-LO activation. Taken together, our results raise the possibility that the combined presence of IL-8 and of GM-CSF at inflammatory foci could result in the synthesis of platelet-activating factor and LTB4 by neutrophils, thereby contributing to the amplification of the inflammatory response.

Human IL-10 (hIL-10) is a newly described cytokine that was originally identified as a cytokine synthesis inhibitory factor regulating the production of several pro-inflammatory cytokines such as IL-1 alpha, IL-1 beta, IL-6, IL-8, TNF-alpha, and IFN-gamma. Additionally, hIL-10 also inhibits the macrophage-dependent proliferative response of CD4+ T lymphocytes to Ag stimulation, and it down-regulates the constitutive class II MHC expression on human monocytes. We report hIL-10 to be a potent and specific chemotaxin for human T lymphocytes with optimum activity in the range between 10 and 100 U/ml. Checkerboard analysis shows the activity to be chemotactic and not chemokinetic. The chemotactic activity is directed toward CD8+ T lymphocytes and not towards CD(4+)-enriched cells. Also, hIL-10 lacks chemotactic activity toward human monocytes or neutrophil granulocytes. Further, we found that hIL-10 inhibits the chemotactic response of CD4+, but not CD8+, T cells toward IL-8. Because hIL-10 can be produced by several cells including CD4+ T cells of the Th2 type, our results suggest that hIL-10 participates in a complex regulatory circuit between CD4+ and CD8+ T cells with implications for the control of lymphocyte-mediated inflammatory responses.

Chemotactic factors regulate the recruitment of monocytes-macrophages to inflammatory sites and neoplastic tissues. The purpose of this study was to determine whether MCAF also influences the activation of C3H/HeN macrophages to become tumoricidal. Several metastatic and nonmetastatic clones of the K-1735 murine melanoma cells syngeneic to C3H/HeN mice were transfected with expression vectors containing the human MCAF gene or control DNA. Tumor cells producing high levels of MCAF were significantly lysed by macrophages treated with LPS, whereas parental or control transfected cells were not. Control-activated macrophages incubated with both IFN-gamma and LPS lysed all the melanoma cells regardless of MCAF production. Pretreatment of macrophages with MCAF significantly enhanced their response to low concentrations of LPS, muramyl tripeptide, and a synthetic bacterial LPP, as measured by lysis of murine melanoma cells. These data suggest that in addition to being a chemotactic factor, MCAF can prime macrophages to respond to endotoxins and other bacterial products and therefore may regulate several levels of macrophage-tumor interactions in situ.

Monocyte chemoattractant protein-1 (MCP-1) cDNA was cloned from guinea pig spleen cells stimulated with Con A. The cDNA comprised 647 bp with an open reading frame that encoded a 120 amino acid protein. The sequence similarity of the first 99 amino acids to human MCP-1 is 56%. Recombinant guinea pig MCP-1 was expressed in COS-7 cells, then purified by a three step procedure with orange A-agarose, carboxymethyl-HPLC, and reversed phase-HPLC. The purified protein was found around 25 kDa as a broad band on a polyacrylamide gel under reducing conditions. Guinea pig MCP-1 attracted about 34% of input human monocytes at 5 x 10(-9) M. Guinea pig peritoneal exudate macrophages migrated toward guinea pig MCP-1 dose dependently, but only 1% of input cells responded to guinea pig MCP-1 at its optimal concentration of 5 x 10(-9) M. Human MCP-1 attracted 1% of input guinea pig peritoneal exudate macrophages at its highest concentration of 2.5 x 10(-8) M. Neither human nor guinea pig MCP-1 attracted guinea pig peritoneal resident macrophages. These results suggest that monocytes lose responsiveness to MCP-1 after differentiating to macrophages. Finally, intradermal injection of the recombinant protein into guinea pigs caused marked macrophage infiltration. Cloned and expressed guinea pig MCP-1 will help in studying the role of MCP-1 in vivo.

Macrophage inflammatory protein (MIP)-1 alpha, part of a family termed chemokines, has been implicated in suppression of hemopoietic stem and progenitor cell proliferation. The chemokine family has been organized into two subgroups with MIP-1 alpha, MIP-1 beta, macrophage chemotactic and activating factor (MCAF) and RANTES belonging to one subgroup, and GRO-alpha, MIP-2 alpha (GRO-beta), MIP-2 beta (GRO-gamma), platelet factor 4 (PF4), IL-8, and neutrophil activating peptide (NAP)-2 belonging to the other. These molecules were evaluated for effects on colony formation by human bone marrow multipotential (CFU-GEMM), erythroid (BFU-E) and granulocyte-macrophage (CFU-GM) progenitor cells. None of the chemokines stimulated colony formation in the absence of CSF, or influenced colony formation stimulated by a single growth factor such as granulocyte-macrophage-CSF or erythropoietin. However, MIP-1 alpha, MIP-2 alpha, PF4, IL-8, and MCAF suppressed in dose-response fashion colony formation of immature subsets of myeloid progenitor cells stimulated by GM-CSF plus steel factor. Effects were apparent on low density and CD34 HLA-DR(+)-sorted marrow cells in which up to 88.4% of the cells were composed of progenitor cells, suggesting direct effects on the progenitors themselves. Up to 2500-fold less of each chemokine could be used to demonstrate synergistic suppression when any two of these five chemokines were used together at low concentrations, effects also apparently directly on the progenitors. In contrast, MIP-1 beta, MIP-2 beta, GRO-alpha, NAP-2, and RANTES were not suppressive nor did they synergize with MIP-1 alpha, MIP-2 alpha, PF4, IL-8, or MCAF to suppress. However, a fivefold excess of MIP-1 beta blocked the suppressive effects of MIP-1 alpha. Similarly, a fivefold excess of either MIP-2 beta or GRO-alpha blocked the suppressive effects of IL-8 and PF4. These suppressing, synergizing and blocking effects may be of relevance to blood cell regulation.

RANTES is a member of the 8-kDa cytokine family that has been shown to possess chemotactic activity for monocytes and CD4 T cells. In this study, we investigated whether RANTES could affect eosinophil chemotaxis and function. Peripheral blood eosinophils from blood donors were isolated on Percoll gradients to > 98% purity and then used for chemotaxis, flow cytometry, eosinophil cationic protein release assay, and survival assay. We found that RANTES is chemotactic for eosinophils at 10(-9) to 10(-8) M concentrations. RANTES elicited 65% of the chemotactic response to 10(-7) M platelet-activating factor in all experiments. The mechanism of chemotaxis was investigated by studying the expression of adhesion molecules on eosinophils by flow cytometry. We found that RANTES up-regulated the expression of CD11b/CD18 on eosinophils in a dose-dependent manner. In another set of experiments, purified eosinophils incubated with various concentrations of RANTES released eosinophil cationic protein as measured by a RIA. We also investigated the effect of RANTES on eosinophil density. Leukocytes were incubated in the presence or absence of RANTES, and the distribution of eosinophils on discontinuous Percoll gradients was then examined. We found that eosinophils became hypodense (< 1.085) when incubated in RANTES. However, unlike IL-3, RANTES did not affect the survival of eosinophils in a 4-day culture system. Thus, we established that RANTES is a chemotactic and activating factor for eosinophils.