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Chemokines were originally characterized by their ability to direct migration and induce activation of selected leukocyte populations. The beta-chemokines MIP-1 alpha, MIP-beta, and RANTES have been implicated in the suppression of viral replication by CD8+ T cells from HIV-infected individuals. The present study was undertaken to evaluate the effect of beta-chemokines on HIV replication in cocultures of dendritic cells (DCs) and CD4+ T cells, and an in vitro model of the lymphoid microenvironment. In the acute infection system, where DCs from uninfected individuals are pulsed with HIV and cocultured with autologous CD4+ T cells, no inhibition of replication of monocytotropic or T cell tropic viral isolates by MIP-1 alpha, MIP-1 beta, and RANTES, alone or in combination, was observed. In contrast, in an endogenous infection system, where the DCs and CD4+ T cells were obtained from HIV-infected subjects, addition of recombinant beta-chemokines suppressed HIV replication. However, neutralizing antibodies to beta-chemokines did not affect the suppressive activity of CD8+ T cells from HIV-infected donors in either system, suggesting that CD8+ T cell-mediated suppression is not due exclusively to beta-chemokines. Furthermore, no significant differences in secretion of MIP-1 alpha, MIP-1 beta, and RANTES by purified CD8+ T cells were noted in uninfected versus HIV-infected donors, regardless of the stage of disease. These results indicate that HIV suppression by CD8+ T cells derived from HIV-infected donors is a multifactorial phenomenon and not limited to the action of MIP-1 alpha, MIP-1 beta, and RANTES.

Interleukin-13 (IL-13) is a recently described lymphokine. Like IL-4, IL-13 induces the production of IL-1 receptor antagonists and suppresses the production of inflammatory cytokines by macrophages and monocytes. In this study, we investigated the effects of IL-13 on the migration and proliferation of human umbilical vein endothelial cells (HUVECs) and human dermal microvascular endothelial cells (HMVECs). We examined the effect of IL-13 on endothelial chemotaxis under two conditions: first, endothelial cells were exposed to a combination of IL-13 and a known chemotaxin, basic fibroblast growth factor (bFGF); second, endothelial cells were exposed to IL-13 alone. The effects of IL-13 on endothelial proliferation were also examined. IL-13 showed no inhibitory effects on bFGF-induced chemotactic activity on either HUVECs or HMVECs. IL-13 demonstrated chemotactic activity for HUVECs and HMVECs. For both cell types, peak chemotactic activity was at or above that of bFGF (60 nM). By varying concentrations of IL-13 in the upper and lower wells of the chemotaxis chambers, we found IL-13 to be chemotactic, and not chemokinetic, for HUVECs and HMVECs. IL-13 did not induce mitogenesis of either HUVECs or HMVECs. Although IL-13 has been shown to have many anti-inflammatory actions, these results suggest a novel role for IL-13 as a proinflammatory proangiogenic cytokine.

In studies of disease processes, increasing knowledge leads to an increased awareness of the complexity of the underlying mechanisms. The intense research activity in the chemokine field has made this acutely manifest. Numerous chemokines have been discovered through the use of (1) bioassay of in vitro cell culture supernatants and in vivo exudates from animal models of inflammation and (2) molecular biology techniques. Any one chemokine can often be produced by a number of different cell types and exert its effects on different target cells. This has been interpreted by some as implying a high degree of redundancy. Although this is understandable, in disease processes parallel and sequential mechanisms are possible, and potentially important therapeutic targets have emerged. There is compelling evidence from animal and clinical studies that eosinophils are important effector cells in asthma, but this relationship is as yet unproven in the human disease. Two possible targets to prevent eosinophil recruitment to the lung are IL-5 and its receptor, which are important in several aspects of eosinophil biology, and eotaxin and its receptor, CCR3. The eotaxin receptor is particularly attractive as a target as it is expressed in high numbers on eosinophils, but not other leukocytes, and appears to be the major detector of the eosinophil for eotaxin and other chemokines such as MCP-4. Eotaxin and CCR3 knockout mice are being developed, and animal models will continue to be invaluable when antagonists are available. In the shape of receptor antagonists, the chemokine field may yet provide the final proof of concept for the long-established eosinophil theory of asthma in humans.

A key rate-limiting step in the adaptive immune response at peripheral challenge sites is the transmission of antigen signals to T cells in regional lymph nodes. Recent evidence suggests that specialized dendritic cells (DC) fulfill this surveillance function in the resting state, but their relatively slow turnover in most peripheral tissues brings into question their effectiveness in signaling the arrival of highly pathogenic sources of antigen which require immediate mobilization of the full range of host defenses for maintenance of homeostasis. However, the present report demonstrates that recruitment of a wave of DC into the respiratory tract mucosa is a universal feature of the acute cellular response to local challenge with bacterial, viral, and soluble protein antigens. Consistent with this finding, we also demonstrate that freshly isolated respiratory mucosal DC respond in vitro to a variety of CC chemokines as well as complementary cleavage products and N-formyl-methionyl-leucine-phenylalanine. This suggests that rapid amplification of specific antigen surveillance at peripheral challenge sites is an integral feature of the innate immune response at mucosal surfaces, and serves as an "early warning system" to alert the adaptive immune system to incoming pathogens.

Expansion of mature neutrophils has been observed in mice lacking the murine interleukin (IL) 8 receptor homolog [mIL-8Rh(-/-)], and human (hu) IL-8 suppresses proliferation of primitive myeloid cells in vitro and in vivo. To evaluate involvement and relevance of murine IL-8 receptor homolog (mIL-8Rh) in negative regulation of myelopoiesis, we studied mIL-8Rh(-/-) and (+/+) mice raised in a normal or germ-free environment. Immature myeloid progenitors from mIL-8Rh(+/+) mice bred under normal or germ-free conditions were significantly suppressed in vitro by recombinant huIL-8, macrophage inflammatory protein (MIP)-1 alpha, platelet factor (PF) 4, interferon inducible protein (IP) 10, monocyte chemotactic peptide (MCP) 1, and H-ferritin. In contrast, progenitors from mIL-8Rh(-/-) mice were insensitive to inhibition by IL-8, but not to these other chemokines and H-ferritin. Mouse MIP-2, a ligand for mIL-8Rh, suppressed progenitors from normal but not mIL-8Rh(-/-) mice. Under normal environmental conditions, enhanced numbers of myeloid progenitors were found in femur, spleen, and blood of mIL-8Rh(-/-) compared with mIL-8Rh(+/+) mice. Numbers of myeloid progenitors were greatly decreased in mIL-8Rh(-/-)and (+/+) mice in germ-free conditions, and were either not significantly enhanced in mIL-8Rh(-/-) mice compared with (+/+) mice or were only moderately so. Differences in progenitors/organ between a germ-free and normal environment were greater for the mIL-8Rh(-/-) mice. These results document selective insensitivity of myeloid progenitor cells from mIL-8Rh(-/-) mice to inhibition by huIL-8 and mouse MIP-2 and a large expansion of myeloid progenitors in these mice, the latter effect being environmentally inducible. This provides strong support for a negative myeloid regulatory role played by the mIL-8Rh in vivo, whose active ligand may be MIP-2.

We have developed a nude mouse model in which tumor regression is reproducibly induced by coinjection with or intratumor inoculation of EBV-immortalized B cells. A wide spectrum of tumor-derived human cell lines can be established as subcutaneous tumors in sublethally irradiated athymic mice. Most of these tumors are induced to regress by human B cells immortalized with EBV. The tumor regression process is characterized by superficial necrosis and scarring that progressively extends itself to involve all or most of the tumor. Microscopically, tumor regression is characterized by tumor tissue necrosis, evidence of vascular damage with intimal thickening and capillary thrombosis, and macrophage, but not lymphocyte or neutrophil, infiltration. Profiles of cytokine expression differed between progressive and regressive Burkitt's tumors in that regressive tumors expressed larger levels of TNF-alpha, IL-6, IFN-gamma, IP-10, Mig, and IL-12 p35, but not other chemokines/cytokines. IP-10 and IL-12 were found to act as potent inhibitors of angiogenesis in vivo. IL-12 is an inducer of IFN-gamma and indirectly of IP-10, an IFN-gamma-inducible protein, raising the possibility that the antiangiogenic effect of IL-12 is mediated by IP-10. Previous studies have demonstrated that IL-12 has potent antitumor activity in vivo. Much of this activity was dependent on the presence of IFN-gamma and of immune T cells. The observation, made in our studies, that IP-10 is an inhibitor of angiogenesis raises the possibility that IP-10 might contribute to the antitumor effects of IL-12 by inhibiting angiogenesis. Inhibition of angiogenesis by IP-10 and IL-12 is T-cell independent, suggesting that IL-12 targets at least two compartments, T cells and capillaries, each capable of mediating antitumor effects.

Effects of the human C-C chemokines eotaxin, MIP-1 alpha, and RANTES on human eosinophil or neutrophil adhesion to human lung microvascular endothelial cells (LMVEC) were investigated. Basal adhesion of unstimulated eosinophils to LMVEC was increased following pretreatment of LMVEC with TNF alpha (10ng/ml) for 6h. Stimulation of eosinophils with eotaxin (30 and 100ng/ml) resulted in increased adhesion to LMVEC pretreated with TNF alpha but not culture medium. Neutrophil adhesion was not increased by eotaxin under similar conditions. Neither MIP-1 alpha (3-100 ng/ml) nor RANTES (3-100 ng/ml) increased eosinophil or neutrophil adhesion to LMVEC pretreated for 6 h with either TNF alpha (10 ng/ml) or culture medium. Monoclonal antibodies (mAb) against eosinophil adhesion molecule VLA-4 (2B4; 30 micrograms/ml) but not CD18 (6.5E; 10 micrograms/ml) inhibited eotaxin-induced eosinophil adhesion to TNF alpha-activated LMVEC. 2B4 in combination with 6.5E reduced adhesion to basal levels. These data show that eotaxin, but not MIP-1 alpha or RANTES, stimulates eosinophil adhesion to LMVEC and that this effect can be abolished by anti-VLA-4 and CD18 mAb in combination. These results suggest that eotaxin may facilitate eosinophil migration from blood vessels in the lung by increasing eosinophil adhesion to endothelial cells.

The effect of anticoagulant (heparin vs EDTA) on chemokine induced CD11b upregulation on neutrophils, eosinophils, and monocytes in human whole blood was determined. For most of the chemokines (IL-8, GRO-alpha, MCP-1, MIP-1 alpha) the difference in the response of leukocytes in EDTA anticoagulated blood vs those in heparinized blood was the degree of their maximal response, with a slightly higher maximal increase in CD11b expression usually seen in cells from EDTA anticoagulated blood. Two chemokines were exceptions to this: RANTES and MIP-1 beta. RANTES is considered to be a stimulator of monocytes and eosinophils and not of neutrophils. As expected, neutrophils in heparinized whole blood did not respond to RANTES; however, neutrophils in EDTA anticoagulated blood had a significant increase in CD11b when exposed to high concentrations (1 microM) of RANTES. RANTES-induced CD11b expression on monocytes and eosinophils in these samples were the same in either heparin or EDTA. In EDTA anticoagulated blood, MIP-1 beta did not elicit a response in either monocytes, eosinophils or neutrophils; however, in heparinized blood, all three cell types increased CD11b expression upon exposure to 1 microM MIP-1 beta.

Chemokines are a family of chemotactic cytokines which attract different types of leukocytes. This property, combined with some additional inflammatory and growth-regulatory activities, demonstrate their crucial role in the immune system. Chemokines are low molecular weight proteins and possess a typical positioning of four conserved cysteines. This family is further subdivided in two subfamilies depending on whether the first two cysteines are adjacent or not (CC and CXC chemokines, respectively). The CXC chemokines (including interleukin-8) predominantly attract neutrophils, whereas CC chemokines induce migration of monocytes, as well as other leukocyte cell types. In this article, the general characteristics of chemokines are reviewed. Furthermore, the murine CC chemokines, JE/MCP-1, MCP-3/MARC, MIP-1 alpha, MIP-1 beta, RANTES, TCA3, C10/MRP-1, MRP-2, and eotaxin, are discussed more in detail.

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.

Chemotactic factors are postulated to direct emigration of lymphocytes from the blood stream into sites of inflammation. Members of a family of chemotactic cytokines, termed chemokines, have been shown to attract lymphocytes but efficacy, i.e., the maximal percentage of attracted cells, has been low. We have identified a highly efficacious lymphocyte chemotactic activity in the supernatants of the murine bone marrow stroma cell line MS-5 which attracts 10-fold more lymphocytes in vitro than currently described lymphocyte chemoattractants. Purification of this chemotactic activity revealed identity to stromal cell-derived factor 1 (SDF-1). SDF-1 acts on lymphocytes and monocytes but not neutrophils in vitro and is both a highly efficacious and highly potent mononuclear cell attractant in vivo. In addition, SDF-1 induces intracellular actin polymerization in lymphocytes, a process that is thought to be a prerequisite for cell motility. Since SDF-1 is expressed constitutively in a broad range of tissues it may have a role in immune surveillance and in basal extravasation of lymphocytes and monocytes rather than in inflammation.

Because dendritic cells (DC) are the most potent antigen-presenting cells involved in many pathophysiological responses, we investigated the effect of chemokines on the migration of these cells in an effort to determine whether chemokines may contribute to the initiation of immune responses. CD34+ progenitor cells isolated from umbilical cord blood were grown in suspension cultures with cytokines and expanded 50- to 100-fold. A variable proportion of the cells expressed markers consistent with DC. The proportion of CD1a+ DC was increased when the cells were cultured with interleukin-4 (IL-4). These cells expressed specific binding sites for C-C and C-X-C chemokines. Cells cultured with or without IL-4 had similar binding profiles. All C-C chemokines tested, including monocyte chemotactic protein (MCP)-1, MCP-2, MCP-3, macrophage inflammatory protein-1 alpha (MIP1 alpha), MIP-1 beta, and RANTES, induced migration of DC-enriched cells cultured with or without IL-4 with MCP-3 being the most potent chemoattractant. Phenotypic analysis of cell migrating in response to C-C chemokines showed that CD1a+ cells were indeed attracted across the polycarbonate filters, and there was no preferential attraction of contaminating CD14+ monocytes by C-C chemokines. DC-enriched cells also expressed specific binding sites for IL-8 and NAP2, which failed to induce cell migration. Our results suggest that C-C chemokines may participate in the recruitment of DC to amplify host defense.

Following the intracranial injection of lipopolysaccharide or during acute neuronal degeneration, there is a paucity of polymorphonuclear leukocyte recruitment to the brain parenchyma and a delay in monocyte recruitment. The present study investigates whether the injection of specific leukocyte chemoattractants into the murine central nervous system can override this intrinsic resistance. Recombinant alpha-(IL-8/NAP-1 MIP-2, IP-10) and beta-chemokines (MCP-1, RANTES) were injected into the murine hippocampus and leukocyte recruitment was assessed histologically. Injections were also made into the dermis of the hind flank for comparison. At doses of 1 microgram, MCP-1 was found to be the most potent monocyte chemoattractant in the brain parenchyma and skin with IP-10 and RANTES producing minimal monocyte recruitment to both sites. In contrast IL-8, and MIP-2 provoked dramatic polymorphonuclear leukocyte recruitment in both the central nervous system and skin. The polymorphonuclear leukocyte recruitment was associated with a breaching of the blood brain barrier that was particularly severe after MIP-2. Both L-8 and MIP-2 induced blood brain barrier breakdown could be attenuated by prior depletion of the circulating leukocytes. The regulation of polymorphonuclear leukocyte chemoattractants in the brain parenchyma during injury and infection is an important area for future studies.

Chemokines are chemotactic cytokines that activate and direct the migration of leukocytes. There are two subfamilies, the CXC and the CC chemokines. We recently found that the CXC-chemokine stromal cell-derived factor-1 (SDF-1) is a highly efficacious lymphocyte chemoattractant. Chemokines act on responsive leukocyte subsets through G-protein-coupled seven-transmembrane receptors, which are also used by distinct strains of HIV-1 as cofactors for viral entry. Laboratory-adapted and some T-cell-line-tropic (T-tropic) primary viruses use the orphan chemokine receptor LESTR/fusin (also known as fusin), whereas macrophage-tropic primary HIV-1 isolates use CCR-5 and CCR-3 (refs 7-11), which are receptors for known CC chemokines. Testing of potential receptors demonstrated that SDF-1 signalled through, and hence 'adopted', the orphan receptor LESTR, which we therefore designate CXC-chemokine receptor-4 (CXCR-4). SDF-1 induced an increase in intracellular free Ca2+ and chemotaxis in CXCR-4-transfected cells. Because SDF-1 is a biological ligand for the HIV-1 entry cofactor LESTR, we tested whether it inhibited HIV-1. SDF-1 inhibited infection by T-tropic HIV-1 of HeLa-CD4 cells, CXCR-4 transfectants, and peripheral blood mononuclear cells (PBMCs), but did not affect CCR-5-mediated infection by macrophage-tropic (M-tropic) and dual-tropic primary HIV-1.

A putative chemokine receptor that we previously cloned and termed LESTR has recently been shown to function as a co-receptor (termed fusin) for lymphocyte-tropic HIV-1 strains. Cells expressing CD4 became permissive to infection with T-cell-line-adapted HIV-1 strains of the syncytium-inducing phenotype after transfection with LESTR/fusin complementary DNA. We report here the indentification of a human chemokine of the CXC type, stromal cell-derived factor 1 (SDF-1), as the natural ligand for LESTR/fusin, and we propose the term CXCR-4 for this receptor, in keeping with the new chemokine-receptor nomenclature. SDF-1 activates Chinese hamster ovary (CHO) cells transfected with CXCR-4 cDNA as well as blood leukocytes and lymphocytes. In cell lines expressing CXCR-4 and CD4, and in blood lymphocytes, SDF-1 is a powerful inhibitor of infection by lymphocyte-tropic HIV-1 strains, whereas the CC chemokines RANTES, MIP-1 alpha and MIP-1 beta, which were shown previously to prevent infection with primary, monocyte-tropic viruses, are inactive. In combination with CC chemokines, which block the infection with monocyte/macrophage-tropic viruses, SDF-1 could help to decrease virus load and prevent the emergence of the syncytium-inducing viruses which are characteristic of the late stages of AIDS.

An immunogenic murine fibrosarcoma cell line was genetically modified to express and produce the human RANTES chemokine stably. In in vitro chemotaxis assays purified recombinant human RANTES as well as human RANTES secreted by the modified murine tumor cells were strongly chemoattractant for mouse CD8+ /Thy-1+ tumor-infiltrating lymphocytes (TIL). RANTES production did not alter the growth of these cytokine gene-modified tumor cells in vitro, but injection of RANTES-secreting cells resulted in the abolition of the ability of those cells to form solid tumors in vivo. The growth of tumors could be restored by co-administration of monoclonal antibodies that inhibit the function of various subsets of immune cells. For example, depletion of CD8+ T cells by antibody administration resulted in complete restoration of solid tumor formation by RANTES-secreting cells, whereas depletion of the CD4+ T cell population resulted in a partial restoration of tumor formation. Additionally, administration of an anti-CR3 monoclonal antibody known to inhibit the in vivo migration of macrophages also completely restored the tumorigenicity of RANTES-secreting fibrosarcoma cells. Thus, the human RANTES chemokine can abolish tumorigenicity of an immunogenic fibrosarcoma in an in vivo murine model, and this process is mediated by various subpopulations of immune effector cells.

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.

Leukocyte emigration possibly requires dynamic regulation of integrin adhesiveness for endothelial and extracellular matrix ligands. Adhesion assays on purified vascular cell adhension molecule (VCAM)-1, fibronectin, and fibronectin fragments revealed distinct kinetic patterns for the regulation of very late antigen (VLA)-4 (alpha 4 beta 1) and VLA-5 (alpha 5 beta 1) avidity by the CC chemokines monocyte inflammatory protein (MIP)-1 alpha, RANTES (regulated on activation, normal T expressed and secreted), or monocyte chemoattractant protein (MCP)-1 in monocytes. CC chemokines induced early activation and subsequent deactivation of VLA-4, whereas upregulation of VLA-5 avidity occurred later and persisted. Controlled detachment assays in shear flow suggested that adhesive strength of VLA-4 for VCAM-1 or the 40-kD fragment of fibronectin (FN40) is more rapidly increased and subsequently reduced by MCP-1 than by MIP-1 alpha, and confirmed late and sustained activation of the adhesive strength of VLA-5 for the 120-kD fragment of fibronectin (FN120). Mn2+ or the stimulating beta 1 mAb TS2/16 strongly and stably enhanced monocyte binding to VCAM-1 or fibronectin, and locked beta 1 integrins in a high avidity state, which was not further modulated by CC chemokines. Mn2+ and mAb TS2/16 inhibited CC chemokine-induced transendothelial migration, particularly chemotaxis across stimulated endothelium that involved VLA-4 and VCAM-1. VLA-4 on Jurkat cells is of constitutively high avidity and interfered with migration across barriers expressing VCAM-1. Low but not high site densities of VCAM-1 or FN40 promoted, while FN120 impaired, beta 1 integrin-dependent monocyte chemotaxis to MCP-1 across filters coated with these substrates. Thus, we show that CC chemokines can differentially and selectively regulate avidity of integrins sharing common beta subunits. Transient activation and deactivation of VLA-4 may serve to facilitate transendothelial diapedesis, whereas late and prolonged activation of VLA-5 may mediate subsequent interactions with the basement membrane and extracellular matrix.

Aims/background: The subcutaneous injection of bacterial endotoxin in Lewis rats produces an acute intraocular inflammation evolving over a 24 hour period. This endotoxin induced uveitis (EIU) is characterised by a biphasic protein exudation and a cellular infiltrate composed of macrophages and polymorphonuclear neutrophils (PMNs). This model was used to study the mechanism of cellular infiltration in ocular inflammation., Methods: EIU was induced by a subcutaneous injection of lipopolysaccharide (LPS) (S typhimurium) at 350 micrograms/kg. The levels of cytokine induced neutrophil chemoattractant (CINC) were measured every 2 hours in the serum and in the aqueous humour by ELISA. The intraocular inflammation was quantified by protein measurement and leucocyte counting., Results: The kinetics of CINC production in the systemic circulation showed a rapid rise, peaking 2 hours after LPS injection, followed by a progressive decline over the next 8 hours. In the eye, the CINC levels increased above the serum levels 10 hours after EIU induction corresponding to the time of cellular infiltration. When leucocyte entry in the eye was inhibited by 56% and 64% with an antiadhesion molecule antibody, there was only a slight reduction in the aqueous humour CINC levels of 9% and 16%, respectively, indicating that CINC was produced by ocular tissue cells. The specific effect of CINC in the eye was confirmed when a direct intraocular injection of 250 ng of purified CINC was followed by significant PMN infiltration, in the absence of protein exudation., Conclusion: The data indicate that the production of the CINC chemotactic factor by ocular tissue participates in the inflammatory reaction in EIU.

Human immunodeficiency virus-type 1 (HIV-1) entry requires fusion cofactors on the CD4+ target cell. Fusin, a heterotrimeric GTP-binding protein (G protein)-coupled receptor, serves as a cofactor for T cell line-tropic isolates. The chemokines RANTES, MIP-1alpha, and MIP-1beta, which suppress infection by macrophage-tropic isolates, selectively inhibited cell fusion mediated by the corresponding envelope glycoproteins (Envs). Recombinant CC CKR5, a G protein-coupled receptor for these chemokines, rendered CD4-expressing nonhuman cells fusion-competent preferentially with macrophage-tropic Envs. CC CKR5 messenger RNA was detected selectively in cell types susceptible to macrophage-tropic isolates. CC CKR5 is thus a fusion cofactor for macrophage-tropic HIV-1 strains.

The past few years have seen significant growth in the number of known chemokines. Along with expansion in this field has come an appreciation of a role for chemokines beyond the chemotaxis of leukocytes. In particular, it appears that these molecules may represent major products of activated lymphocytes and that, in addition to chemotaxis, some of them may also regulate the growth, activation, and differentiation of lymphocytes.

Recently we have found that only cytokine-induced neutrophil chemoattractant (CINC)-3 induces an increase in intracellular [Ca2+] in rat neutrophils stimulated first with CINC-1, CINC-2 alpha or CINC-2 beta, and hypothesized that rat neutrophils have another receptor specific for CINC-3 in addition to a common receptor for all CINCs [Shibata F., et al., Eur. J. Biochem., 231, 306 (1995)]. To evaluate this hypothesis, chemotactic activity of these CINCs in vitro was determined by using rat neutrophils together with each CINC. In the presence of CINC-1, migration of neutrophils toward CINC-1, CINC-2 alpha and CINC-2 beta was inhibited, but that toward CINC-3 was not. Similar results were obtained when CINC-2 alpha or CINC-2 beta was added to neutrophils. However, addition of CINC-3 to neutrophil suspension in the upper chamber resulted in the complete inhibition of the neutrophil migration toward each CINC. The results support our hypothesis and indicate both the putative receptors can mediate chemotaxis of rat neutrophils.

Although there is a mounting body of evidence that eosinophils are recruited to sites of allergic inflammation by a number of beta-chemokines, particularly eotaxin and RANTES, the receptor that mediates these actions has not been identified. We have now cloned a G protein-coupled receptor, CC CKR3, from human eosinophils which, when stably expressed in AML14.3D10 cells bound eotaxin, MCP-3 and RANTES with Kds of 0.1, 2.7 and 3.1 nM, respectively. CC CKR3 also bound MCP-1 with lower affinity, but did not bind MIP-1 alpha or MIP-1 beta. Eotaxin, RANTES, and to a lessor extent MCP-3, but not the other chemokines, activated CC CKR3 as determined by their ability to stimulate a Ca(2+) -flux. Competition binding studies on primary eosinophils gave binding affinities for the different chemokines which were indistinguishable from those measured with CC CKR3. Since CC CKR3 is prominently expressed in eosinophils we conclude that CC CKR3 is the eosinophil eotaxin receptor. Eosinophils also express a much lower level of a second chemokine receptor, CC CKR1, which appears to be responsible for the effects of MIP-1 alpha.

A novel human CC chemokine complementary DNA was identified in a library constructed from human fetal RNA, cloned into a baculovirus vector, and expressed in Sf9 insect cells. The mature recombinant protein that was released had the NH2-terminal sequence pyro-QPDALNVPSTC...and consisted of 75 amino acids. Minor amounts of two variants of 77 and 82 residues (NH2 termini: LAQPDA...and FNPQGLAQPDA...) were released as well. The novel chemokine was designated monocyte chemotactic protein 4 (MCP-4) and the variants were designated (LA)MCP-4 and (FNPQGLA)MCP-4. MCP-4 shares the pyroglutamic acidproline NH2-terminal motif and 56-61% sequence identity with the three known monocyte chemotactic proteins and is 60% identical to eotaxin. It has marked functional similarities to MCP-3 and eotaxin. Like MCP-3, MCP-4 is a chemoattractant of high efficacy for monocytes and T lymphocytes. On these cells, it binds to receptors that recognize MCP-1, MCP-3, and RANTES. On eosinophils, MCP-4 has similar efficacy and potency as MCP-3, RANTES, and cotaxin. It shares receptors with eotaxin and shows full cross-desensitization with this cosinophil-selective chemokine. Of the two variants, only (LA)MCP-4 could be purified in sufficient quantities for testing and was found to be at least 30-fold less potent than MCP-4 itself. This suggests that the 75-residue form with the characteristic NH2 terminus of an MCP is the biologically relevant species.

The proteolytic cleavage product of complement component 3, (C3a), is like C4a and C5a, is a potent anaphylatoxin and induces the production of inflammatory mediators in phagocytes. Notably, mast cells respond to C3a with the release of vasoactive substances, including histamine. We have examined the function and receptor binding of C3a in a human leukemic mast cell line, HMC-1. Similar to chemoattractant agonists in leukocytes, C3a induced rapid cytosolic free calcium concentration increases in HMC-1 cells. EGTA did not diminish this response, indicating that mobilizable Ca2+ was from intracellular stores. Receptors of C3a in HMC-1 cells couple in part to Bordetella pertussis toxin-sensitive G-proteins and, therefore, appear to belong to the family of serpentine receptors that require G-proteins for signal transduction. HMC-1 cells express two types of C3a receptors, C3aR1 and C3aR2, that were shown to bind 125I-C3a with high-(Kd1 = 2.1-4.8 nM) or low-affinity (Kd2 = 30-150 nM), and both receptors are expressed at high level: 3 x 10(5)-6 x 10(5) C3aR1/cell and 5 x 10(5)-2.3 x 10(6) C3aR2/cell. Results from cross-linking experiments with 125I-C3a fully agree with the presence of two different classes of C3a receptors in HMC-1 cells. Two membrane proteins with apparent molecular masses of 54-61 kDa (p57) and 86-107 kDa (p97) could be covalently modified with 125I-C3a, and this cross-linking was inhibited with an excess of unlabeled C3a. Many of the known agonists for leukocytes including 13 chemokines (IL-8, NAP-2, GRO alpha, ENA-78, IP10, PF4, MCP-1, 2 and 3, RANTES, MIP-1 alpha, MIP-1 beta and I309), three neuropeptides (neuropeptide Y, somatostatin and calcitonin), as well as C5a, did not activate HMC-1 cells, indicating that C3a is one of a few protein ligands for which this cell line expresses specific receptors. The apparent selectivity for C3a and the abundant expression of C3a receptors make the HMC-1 cell line an excellent choice for the cloning of the receptor genes.

The CC chemokine eotaxin is a selective chemoattractant for guinea pig eosinophils, first purified from bronchoalveolar lavage fluid in a guinea pig model of allergic airway inflammation. We have now isolated the gene and cDNA for a human counterpart of eotaxin. The gene maps to chromosome 17 and is expressed constitutively at high levels in small intestine and colon, and at lower levels in various other tissues. The deduced mature protein sequence is 66% identical to human monocyte chemoattractant protein-1, and 60% identical to guinea pig eotaxin. Recombinant human eotaxin produced in insect cells induced a calcium flux response in normal human eosinophils, but not in neutrophils or monocytes. The response could not be desensitized by pretreatment of eosinophils with other CC chemokines, suggesting a unique receptor. In this regard, we show that human eotaxin is a potent and highly specific agonist for CC chemokine receptor 3, a G protein-coupled receptor selectively expressed in human eosinophils. Thus eotaxin and CC chemokine receptor 3 may be host factors highly specialized for eosinophil recruitment in inflammation, and may be good targets for the development of selective drugs for inflammatory diseases where eosinophils contribute to pathogenesis, such as asthma.

While chemokines primarily promote chemotaxis, it is apparent that these cytokines also modulate a number of other biologic activities, including adhesion, degranulation, cytotoxicity, and enzyme release. We demonstrate here that the beta chemokines, recombinant human macrophage inflammatory protein-1 alpha and -1 beta, RANTES (regulated upon activation, normally T cell expressed and secreted), and macrophage chemotactic peptide-1, are capable of directly costimulating purified human T cell and human T cell clone proliferation and IL-2 production in the presence of anti-CD3 mAb, but not phorbol esters, in vitro. This costimulatory activity was dose and donor dependent and required the presence of free extracellular calcium as well as endogenously produced IL-2. Chemokine treatment of human T cells in vitro increased the level of cell surface CD25 and soluble CD25. In addition, these chemokines enhanced both Ag- and alloantigen-specific T cell and T cell clone proliferation. This activity was further augmented in the presence of the CD28 ligand, B7-1. Neutralization analyses using chemokine-specific Abs revealed that endogenously produced chemokines are important costimulatory mediators in human T cell activation. Together, these results suggest that chemokines not only play an important role in lymphocyte recruitment to inflammatory sites, but also participate in T cell activation.

Previous studies from this laboratory have demonstrated that the chemokines RANTES (recombinant human regulated upon activation, normally T cell expressed and presumably secreted), macrophage chemotactic peptide-1, recombinant human macrophage inflammatory protein-1 alpha (rhMIP-1 alpha) IL-8, and IP-10 are capable of inducing human T cell infiltration into the injection site of severe combined immunodeficiency (SCID) mice reconstituted with human PBL. However, the ability of these chemokines to facilitate T cell homing into various lymphoid tissues has not been examined. Initial studies focused on the ability of rhMIP-1 beta to induce human T cell infiltration into injection sites in human PBL-SCID mice. SCID mice received s.c. injections of rhMIP-1 beta or PBS (1 microgram/injection) in the hindflank for 4 h or sequential injections for 3 days. Biopsies of the MIP-1 beta injection site revealed the presence of significant mononuclear cell accumulation 72 h after injection. Immunohistologic evaluation determined that significant numbers of human CD3+ T cells were recruited in response to MIP-1 beta injections, and this infiltration could be specifically blocked by co-administration of anti-MIP-1 beta antiserum. We subsequently examined these chemokine-injected mice for the effect of trafficking of human T cells to peripheral lymphoid organs. Flow cytometric analysis of the thymus in human PBL-SCID mice revealed that treatment with rhMIP-1 beta or rhRANTES, but not platelet factor-4, resulted in improved thymic homing of the human T cells after 72 h. This trafficking effect was shown to be direct, as pretreatment of the human T cells with the chemokines in vitro also improved peripheral lymphoid trafficking of the human cells. In addition, co-injection of rhMIP-1 beta with anti-1 beta antiserum abrogated the increase in T cell homing to the thymus. These data demonstrate that MIP-1 beta and RANTES directly augment human T cell trafficking to peripheral murine lymphoid tissues. Chemokines may, therefore, under either isogeneic or xenogeneic conditions, play a role in normal lymphocyte recirculation and homing, and may be of potential clinical use in promoting immune cell trafficking and function.

The chemokines interleukin-8 (IL-8) and GRO alpha bind in neutrophils to the interleukin-8 receptor alpha and beta (IL-8R alpha and beta) triggering reorganization of the actin cytoskeleton and activation of phospholipase C (PLC). Reconstitution of chemokine-induced activation of PLC indicated coupling of IL-8R alpha and beta to pertussis toxin-insensitive G alpha 16-proteins as well as to pertussis toxin-sensitive G alpha i2- or G alpha i3-proteins. To identify the signal transduction mechanisms of chemokine-induced actin response, undifferentiated human leukemia cells (HL-60 cells) constitutively expressing G alpha 16-, G alpha i2- and G alpha i3-proteins were chosen for reconstitution studies. Expression of recombinant receptors after transfection of the cells with the cDNA of IL-8R alpha and beta was confirmed by binding studies with radiolabeled ligands. IL-8R alpha bound IL-8 with high affinity (Kd approximately 1 nM) and GRO alpha with low affinity (Kd approximately 1 microM), whereas IL-8R beta bound both IL-8 and GRO alpha with high affinity (Kd approximately 1nM). Flow cytometric actin measurements indicated that high affinity ligand-receptor interactions in both receptor transfectants displayed inducible responses. Pretreatment of transfectants with pertussis toxin caused ADP-ribosylation of G-proteins and blocked chemokine-induced polymerization, indicating involvement of G alpha i2- or G alpha i3-proteins, but not G alpha 16-proteins in this response.

Interleukin 5 (IL-5) is a T-cell derived cytokine that induces eosinophil growth and differentiation in both mouse and human bone marrow cultures. Elevated levels of IL-5 as well as eosinophils have been detected in the sputum and Bronchoalveolar lavage (BAL) fluids of asthmatics. Since the recruitment of inflammatory cells to tissues requires the participation of adhesion molecules, we have developed a rapid and sensitive assay to examine the effect of IL-5 and other activation stimuli on eosinophil adhesion to recombinant intercellular adhesion molecule-1 (ICAM-1), and vascular cell adhesion molecule-1 (VCAM-1). Human recombinant IL-5, granulocyte-macrophage colony stimulating factor (GM-CSF), interleukin 3 (IL-3), tumour necrosis factor alpha (TNF-alpha), RANTES, MCP-3, C5a, PAF, fMLP, PMA and ConA all induced adhesion of purified eosinophils obtained from normal donors to ICAM-1 and VCAM-1 in a dose and time dependent manner. Adhesion was rapid, within 15 minutes of culture at 37 degrees C, and plateaued within 30 minutes. Activated eosinophils also adhered rapidly to immobilized IgG via the type II Fc gamma receptor (CD32). Analysis of the effect of IL-5 on surface molecule expression by FACS analysis revealed increased expression of CD11b molecules and decreased expression of L-selectin, but no change in the expression of CD11a, CD18, CD29, CD49d and CD32. We also show that Mac-i plays an important role in the regulation of eosinophil activation, since antibodies to CD11b can block IL-5 induced adhesion to IgG and IL-5 induced degranulation.

The human cytokine, interferon-inducible protein-10 (IP-10), is a small glycoprotein secreted by activated monocytes, T cells, keratinocytes, astrocytes, and endothelial cells and is structurally related to the alpha subfamily of chemotactic cytokines called chemokines (Taub and Oppenheim, Cytokine 5:175, 1993). However, in contrast to other alpha chemokines that induce neutrophil migration, IP-10 has been shown to chemoattract monocytes and T lymphocytes in vitro, suggesting a role in T-cell-mediated immune responses. We therefore examined the effects of human IP-10 after in vivo administration. IP-10 induces significant mononuclear cell infiltration after subcutaneous injections in normal mice. In an effort to study the in vivo effects of IP-10 on human leukocyte migration, we then examined the ability of recombinant human IP-10 (rhIP-10) to induce human-T-cell infiltration using a human/severe combined immune deficiency (SCID) mouse model. SCID mice received an intraperitoneal injection of human peripheral blood lymphocytes (10(8) cells), followed by a subcutaneous injection of rhIP-10 (1 micrograms/injection) in the hind flank for 4 hours or sequential injections for 3 days. The skin and underlying tissue from the rhIP-10 injection site were then biopsied and examined for the extent of mononuclear cell infiltration. rhIP-10 again induced significant mononuclear cell accumulation 72 hours after injection. Immunohistologic evaluation determined that a significant number of human CD3+ T cells were recruited in response to rhIP-10 injections. These results show that rhIP-10 is capable of inducing human T-cell migration in vivo and may play an important role in monocyte and lymphocyte recruitment into inflammatory sites.

We describe here that members of the CC chemokines exhibit biological activities other than chemotaxis. Macrophage inflammatory protein (MIP)-1 alpha, MIP-1 beta, monocyte chemoattractant protein-1 and RANTES, but not interleukin (IL)-8, induce the generation of cytolytic cells, designated here as CHAK (CC chemokine-activated killer) cells to distinguish them from IL-2-activated (LAK) cells. Like IL-2, CC chemokines can induce the proliferation and activation of killer cells. While incubating CC chemokines with CD4+ or CD8+ cells did not generate CHAK activity, all CC chemokines were capable of inducing CHAK activity upon incubating with CD56+ cells, suggesting that the primary effectors are NK cells. However, the presence of other cell types, such as CD4+ or CD8+, are necessary to induce the proliferation of CD56+ cells. Confirming the involvement of T cell-derived factors in inducing the proliferation of these cells, anti-IL-2 and anti-interferon-gamma, but not anti-IL-1 beta, anti-tumor necrosis factor-alpha, anti-IL-8, or anti-granulocyte/monocyte-colony-stimulating factor inhibited RANTES-induced proliferation of nylon wool column-nonadherent cells. Our results may have important clinical applications for the utilization of CHAK cells in the treatment of cancer and immunodeficient patients.

Whether the chemokines macrophage inflammatory protein-1 beta (MIP-1 beta) and regulated on activation normal T expressed and secreted (RANTES), which interact specifically with glycosaminoglycans and thus mediate the recruitment, attachment, and migration of leukocytes to vascular endothelia and extracellular matrix, are also involved in interactions between CD4+ murine T lymphocytes and keratinocytes was examined. We have previously observed that depending on the local pH, a mammalian extracellular matrix-degrading enzyme, endo-beta-D glucuronidase (heparanase), which cleaves heparin sulfate proteoglycans, can function wither as an enzyme or as an adhesion molecule for CD4+ T lymphocytes. Herein, the involvement of heparanase in T cell-keratinocyte interactions was also probed. At 37 degree C and pH 7.2, radioactively labeled MIP-1 beta, RANTES, and heparanase bound to confluent layers of resting keratinocytes in a saturable and an heparan sulfate- or heparin-dependent manner, and thereby induced the adhesion of resting CD4+ T cells to keratinocytes. At a relatively acidic pH characteristic of inflammatory milieu, enzymatically active heparanase did not bind to the keratinocytes but, rather, inhibited the binding of MIP-1beta, RANTES, and the enzymatically quiescent heparanase to keratinocytes. These results suggest that certain chemokines and heparanase may function to restrict passing leukocytes, notable T lymphocytes, in the cutaneous micro-environment, a site which is continuously challenged with antigens. These keratinocyte-bound lymphocytes can serve as a reservoir of immediate responders to immunological stimuli.

In the mammalian host, Leishmania are obligate intracellular parasites and invade macrophages and Langerhans cells. The accessory functions of both types of host cells are important for regulation of the specific cellular immune response and involve the following activities: infiltration into the site of infection, initiation of a T cell response, maintenance of immunity and the effector mechanisms that control intracellular parasite replication.

Chemokines are a family of structurally related, low m.w. proteins that regulate leukocyte migration both in vitro and in vivo. By virtue of their target cell specificity, chemokines have the potential to selectively recruit leukocyte subpopulations into sites of inflammation during the genesis of an immune response. Chemokines have been shown to induce leukocyte adhesion to endothelium, to facilitate trans-endothelial passage, and to direct cell migration along a protein gradient (chemotaxis). The chemokines (macrophage inflammatory protein-1 alpha, macrophage inflammatory protein-1 beta, RANTES, and IFN-inducible protein-10) have recently been reported to be chemotactic for T cells. We have investigated the potential activity of these proteins in regulation of T cell adhesion. These chemokines induce T cell adhesion to purified, recombinant human adhesion molecules (rhICAM-1, rhVCAM-1) and to ECM proteins: fibronectin, collagen, and laminin. The chemokine-induced adhesion process occurs rapidly, is dose-dependent, and appears to be mediated via beta 1 and beta 2 integrins. The enhanced T cell adhesion is not associated with an increased surface expression of adhesion proteins, suggesting that chemokines stimulate the development of a high affinity state in the integrin molecules. Our findings provide in vitro evidence of a critical role for chemokines in T cell adhesion to endothelial adhesion molecules and ECM proteins, thereby promoting haptotactic migration of T cells to sites of inflammation in vivo.

The responses of cloned human NK cells (ERNK57) to seven CC chemokines (monocyte chemotactic protein-1 (MCP-1), MCP-2, MCP-3, RANTES (regulated on activation, normal T cell expressed and secreted), macrophage inflammatory protein-1 alpha (MIP-1 alpha), MIP-1 beta, and 1309) and two CXC chemokines (IL-8 and IP-10) were studied. Except for 1309, all CC chemokines induced chemotaxis of the NK cells in vitro, whereas the CXC chemokines were inactive. Maximal activity was obtained at 1 nM for MCP-1 and 10 to 100 nM for the other CC chemokines. The response showed a typically bimodal concentration dependence in all cases, except for RANTES, which induced a linear increase of migration over the concentration range of 0.1 to 1000 nM. A transient rise of the cytosolic-free Ca2+ concentration ([Ca2+]i), which is characteristic for chemokine-stimulated leukocytes, was observed in NK cells after stimulation with all six active chemokines. Since granule exocytosis is required for NK cell-dependent target killing, the effect of CC chemokines on exocytosis was tested. All CC chemokines that induced chemotaxis and [Ca2+]i changes also induced the release of granzyme A and N-acetyl-beta-D-glucosaminidase from cloned and blood NK cells, as well as CD8+ T cells after pretreatment with cytochalasin B. Maximum release was obtained from NK cells, and amounted to 35% and 13% of the total content of granzyme A and N-acetyl-beta-D-glucosaminidase, respectively. The capacity of cloned NK cells and CD8+ T cells to respond to chemokines depended on the time in culture after stimulation with PHA in the presence of irradiated feeder cells, and maximum responses were observed after 10 to 16 days. Our results demonstrate that CC chemokines activate NK cells, and are, therefore, not only attractants for monocytes, T lymphocytes, and eosinophil and basophil granulocytes.

The release of mediators from mast cells and basophils represents the central event in the development of immediate hypersensitivity reactions with release of substances such as histamine, Leukotrienes C4/D4, Platelet Activating Factor (PAF), and Prostaglandin D2. Cytokines such as IL-1, TNF alpha, and IL-4 may also be secreted. Histamine Releasing Factors (HRF) are cytokine-like molecules that interact with basophils and/or mast cells to cause cell activation and secretion of mediators. Histamine release is the best characterized of these and has been used as the assay for HRFs, but a wide variety of inflammatory mediators can potentially be secreted. We believe this type of cell to cell communication to be important in tissue inflammation, in which infiltrating cells may produce HRFs in proximity to infiltrating basophils and/or mast cells and cause them to degranulate. Such a reaction appears to be independent of IgE antibody, may no longer require the presence of any inciting antigen, and appears to be pertinent to the allergic late phase reaction as it occurs in the nose, lungs, and skin. It is thought that an ongoing antigenic stimulus, as seen in seasonal or perennial allergic rhinitis and asthma, or in certain types of urticaria, or in atopic dermatitis, leads to a perpetuating inflammatory reaction that persists for many weeks or months. A chronic inflammatory reaction of this sort appears to be required for an allergic reaction (IgE mediated) to manifest as an allergic disease. The relationship of HRF to the chemokine group of cytokine-like molecules and the importance of HRF in the pathogenesis of bronchial hyperreactivity and asthma has been reviewed in this paper.

We report here the ability of the beta chemokines MIP-1 alpha, MIP-1 beta, RANTES, and MCP-1 to enhance some lymphocyte effector functions. Initial studies focused on the effects of chemokines on human and mouse cytotoxic T lymphocyte (CTL)- and natural killer (NK) cell-specific cytolytic responses. The results demonstrate that beta chemokines are capable of augmenting mouse and human CTL and human NK- but not lymphokine-activated killer cell- or antibody-dependent cell cytotoxicity-specific cytolytic responses. Neutralization analysis utilizing integrin-specific antibodies revealed that CTL/NK tumor cell conjugate formation is required for chemokine-induced killing. In addition, both CTLs and NK cells incubated with various beta chemokines were induced to degranulate and release granule-derived serine esterases, suggesting that chemokines may be important costimulators of CTL and NK cell degranulation and may thus augment local target cell destruction. Chemokines also modulate antigen-driven T cell proliferative responses as well as effects on lymphokine production. Many of the beta chemokines were found to potentiate human and mouse antigen-specific Th1 and Th2 clone activation promoting cellular proliferation and the release of various lymphokines. This chemokine-mediated T cell proliferation was chemokine and antigen dose dependent as well as clone dependent. Chemokine pretreatment analyses with T cells and antigen-presenting cells (APCs) revealed that chemokines up-regulate both T cells and antigen- presenting cells (APCs) revealed that chemokines up-regulate both T cell and APC functions. Costimulation assays using immobilized antiCD3 monoclonal antibody-coated plates and purified human and mouse T cells and T cell clones in the presence of various chemokines also exhibited enhanced proliferation and lymphokine secretion. This costimulation was interleukin-2 dependent and required the presence of free extracellular calcium. Examination of chemokine-treated APCs revealed that the T cell costimulatory molecule B7-1 was induced by various beta chemokines. Neutralization of endogenously produced chemokines, with specific antibodies during an antigen-specific T cell response blocked cellular proliferation, suggesting that the chemokines have an autocrine role in antigen-induced T cell proliferative responses. Together, these results suggest that chemokines play a significant role in the activation of polyclonal as well as antigen-specific helper and cytotoxic T cells during the genesis of an immune response.

A novel CC chemokine, HCC-1, was isolated from the hemofiltrate of patients with chronic renal failure. HCC-1 has a relative molecular mass of 8,673 and consists of 74 amino acids including four cysteines linked to disulfide bonds. HCC-1 cDNA was cloned from human bone marrow and shown to code for the mature protein plus a putative 19-residue leader sequence. Mature HCC-1 has sequence identity of 46% with macrophage inflammatory protein (MIP)-1 alpha and MIP-1 beta, and 29-37% with the other human CC chemokines. Unlike MIP-1 alpha and the other CC chemokines, HCC-1 is expressed constitutively in several normal tissues (spleen, liver, skeletal and heart muscle, gut, and bone marrow), and is present at high concentrations (1-80 nM) in plasma. HCC-1 has weak activities on human monocytes and acts via receptors that also recognize MIP-1 alpha. It induced intracellular Ca2+ changes and enzyme release, but no chemotaxis, at concentrations of 100-1,000 nM, and was inactive on T lymphocytes, neutrophils, and eosinophil leukocytes. In addition, HCC-1 enhanced the proliferation of CD34+ myeloid progenitor cells. It was as effective as MIP-1 alpha, but about 100-fold less potent.

The CC chemokine monocyte chemoattractant protein-3 (MCP-3) activates human monocytes, lymphocytes, basophils, and eosinophils. MCP-3 has been reported to induce [Ca2+]i changes in cells transfected with the monocyte-selective MCP-1 receptor 2B (CC CKR2B) and competes for 125I-MCP-1 binding on CC CKR2B, suggesting that it may mediate monocyte responses to MCP-3. However, we now show that MCP-3 is a ligand and potent agonist for the macrophage inflammatory protein-1 alpha (MIP-1 alpha)/regulated on activation, normal T expressed, and secreted protein (RANTES) receptor CC CKR1 (rank order for [Ca2+]i changes = MIP-1 alpha > MCP-3 > RANTES), which is expressed in monocytes > neutrophils > eosinophils. 125I-MCP-3 bound directly to CC CKR1 and CC CKR2B (Ki = 8 and 7 nM, respectively). Binding to CC CKR1 was competed by all CC chemokines tested except MCP-1. In contrast, binding to CC CKR2B was competed only by MCP-3 and MCP-1. Both MCP-1 and MCP-3 were equipotent agonists (EC50 = 10 nM for [Ca2+]i changes). Thus, MCP-3 is a functional ligand for both CC CKR1 and CC CKR2B, which otherwise have distinct selectivities for CC chemokines. These data suggest that monocyte responses to MCP-3 could be mediated by both CC CKR2B and CC CKR1, whereas eosinophil responses to MCP-3 could be mediated by CC CKR1.

Eight chemokines were tested for ability to elicit transendothelial chemotaxis of unstimulated peripheral blood T lymphocytes. The C-C chemokines monocyte chemotactic protein (MCP)-2, MCP-3, RANTES, macrophage inflammatory protein (MIP)-1 alpha, MIP-1 beta, and, as previously described, MCP-1 induced significant, dose-dependent transendothelial chemotaxis of CD3+ T lymphocytes. In contrast, the C-X-C chemokines interleukin-8 (IL-8) and interferon-gamma inducible protein-10 (IP-10) failed to induce transendothelial chemotaxis of CD3+ T lymphocytes or T lymphocyte subsets. RANTES, MIP-1 alpha, and MIP-1 beta induced significant transendothelial chemotaxis of CD4+, CD8+, and CD45R0+ T lymphocyte subsets. Phenotyping of mononuclear cells that underwent transendothelial migration to MCP-2, MCP-3, RANTES, or MIP-1 alpha showed both monocytes and activated (CD26 high), memory-type (CD45R0+) T cells. Both CD4+ and CD8+ T lymphocytes were recruited, but not natural killer cells or significant numbers of B cells. MCP-2 was the only C-C chemokine tested that attracted a significant number of naive-type (CD45RA+) T lymphocytes. In the absence of endothelium, IL-8 but not IP-10 promoted modest but significant chemotoxis of CD3+ T lymphocytes. Our data support the hypothesis that C-C, not the C-X-C chemokines IL-8 or IP-10, promote transendothelial chemotaxis of T lymphocytes.

We have found that two chemokines, recombinant gro-alpha and gro-beta, specifically inhibit growth factor-stimulated proliferation of capillary endothelial cells in a dose-dependent manner, whereas gro-gamma has no inhibitory effect. In vivo, gro-beta inhibits blood vessel formation in the chicken chorioallantoic membrane assay. It is sufficiently potent to effectively suppress basic fibroblast growth factor-induced corneal neovascularization after systemic administration in mice. Further, gro-beta significantly inhibits the growth of murine Lewis lung carcinoma in syngeneic C57B16/J and immunodeficient nude mice without toxicity. In vitro, Lewis lung carcinoma cells are completely insensitive to recombinant gro-beta at high concentrations that significantly inhibit endothelial cell proliferation. This finding supports the conclusion that gro-beta inhibits Lewis lung tumor growth by suppression of tumor-induced neovascularization.