Cloning and characterization of a novel murine beta chemokine receptor, D6. Comparison to three other related macrophage inflammatory protein-1alpha receptors, CCR-1, CCR-3, and CCR-5

The beta-chemokine macrophage inflammatory protein-1alpha (MIP-1alpha) is chemotactic for many hemopoietic cell types and can inhibit hemopoietic stem cell (HSC) proliferation, effects mediated through G-protein coupled heptahelical receptors. We have isolated cDNAs for seven chemokine receptors, CCR-1 to -5, MIP-1alphaRL1, and a novel cDNA, D6. Chinese hamster ovary cells expressing CCR-1, -3, -5, and D6 bound 125I-murine MIP-1alpha: the order of affinity was D6 > CCR-5 > CCR-1 > CCR-3. Each bound a distinct subset of other beta-chemokines: the order of competition for 125I-murine MIP-1alpha on D6 was murine MIP-1alpha > human and murine MIP-1beta > human RANTES approximately JE > human MCP-3 > human MCP-1. Human MIP-1alpha and the alpha-chemokines did not compete. Like other chemokine receptors, D6 induced transient increases in [Ca2+] in HEK 293 cells upon ligand binding. D6 mRNA was abundant in lung and detectable in many other tissues. Bone marrow cell fractionation demonstrated T-cell and macrophage/monocyte expression of D6, and CCR-1, -3, and -5. Moreover, we could detect expression of CCR-3, CCR-5, and to a greater extent D6 in a cell population enriched for HSCs. Thus, we have characterized four murine beta chemokine receptors that are likely involved in mediating the pro-inflammatory functions of MIP-1alpha and other chemokines, and we present D6, CCR-3, and CCR-5 as candidate receptors in MIP-1alpha-induced HSC inhibition.     

Induction of the chemokine beta peptides, MIP-1 alpha and MIP-1 beta, by lipopolysaccharide is differentially regulated by immunomodulatory cytokines gamma-IFN, IL-10, IL-4, and TGF-beta

The macrophage occupies a central role in the host response to invasion, exerting its control over the developing inflammatory response largely through the elaboration of an assortment of endogenous mediators including many cytokines. The beta chemokine peptides, macrophage inflammatory protein [MIP]-1 alpha and MIP-1 beta, are two such effectors markedly up-regulated in macrophages following exposure to bacterial lipopolysaccharide (LPS). These highly homologous peptides, like the other members of the beta chemokine family, exhibit diverse but partially overlapping biological activity profiles, suggesting that the cellular participants and intensity of an inflammatory response may in part be regulated by selective expression of these chemokines. Studies reported here demonstrate that, in contrast to the "balanced" MIP-1 alpha/MIP-1 beta chemokine responses of LPS-stimulated macrophage cultures in vitro, circulating levels of MIP-1 beta are significantly higher than those of MIP-1 alpha following LPS administration in vivo. Further studies have revealed that several immunomodulatory cytokines known to be up-regulated in vivo as a consequence of exposure to an invasive stimulus (gamma-IFN, IL-10, IL-4, and transforming growth factor [TGF]-beta) down-regulated the LPS-induced release of MIP-1 alpha by macrophages in vitro, but spared the MIP-1 beta response. This altered pattern of secretion may explain, at least in part, the high circulating levels of MIP-1 beta relative to MIP-1 alpha observed in vivo in response to LPS challenge.     

Bacterial superantigens induce down-modulation of CC chemokine responsiveness in human monocytes via an alternative chemokine ligand-independent mechanism

Staphylococcal superantigens (SAgs) are very potent T cell mitogens, but they can also activate monocytes by binding directly to MHC class II molecules in a manner independent of TCR coengagement. Induction of proinflammatory cytokines and chemokine expression in monocytes by superantigens has recently been reported. Here we report that superantigen stimulation of human peripheral blood monocytes results in a rapid, dose-dependent, and specific down-regulation of chemokine (macrophage inflammatory protein-1alpha (MIP-1alpha), monocyte chemotactic protein-1 and MIP-1beta) binding sites (e.g., CCR1, CCR2, and CCR5), which correlates with a concomitant hyporesponsiveness of human monocytes to these CC chemokine ligands. This down-regulation occurs 15-30 min following superantigen stimulation and is specific to chemokine receptors, in that binding and responsiveness of monocytes to the chemoattractant formyl-tripeptide FMLP are not affected. We further demonstrate that SAg-induced down-modulation of chemokine binding and monocyte hyporesponsiveness to the chemokines MIP-1alpha, monocyte chemotactic protein-1, and MIP-1beta is mediated through cellular protein tyrosine kinases, and the down-modulation can be mimicked by an MHC class II-specific mAb. Additionally, our observations indicate that SAg-induced loss of chemokine binding and monocyte responsiveness is probably mediated by secreted serine proteinases. Bacterial SAg-induced down-modulation of chemokine responsiveness represents a previously unrecognized strategy by some bacteria to subvert immune responses by affecting the intricate balance between chemokine and chemokine receptor expression and function.     

STRL22 is a receptor for the CC chemokine MIP-3alpha

STRL22 is a human seven transmembrane domain orphan receptor related to known chemokine receptors and expressed in peripheral blood lymphocytes, tumor infiltrating lymphocytes and lymphoid tissues. MIP-3alpha/LARC/Exodus is a CC chemokine that is chemotactic for lymphocytes and that is expressed in activated cells, including monocytes, T cells, endothelial cells, and fibroblasts, and in liver, lung, and some lymphoid tissues. We report here that STRL22-transfected human embryonic kidney 293 cells demonstrated specific binding for MIP-3alpha and that MIP-3alpha, but no other chemokines, produced a calcium flux in the STRL22-transfected cells. We show that MIP-3alpha, unlike other chemokines, produced a calcium flux in freshly-isolated peripheral blood lymphocytes and we show that MIP-3alpha also produced a signal in tumor infiltrating lymphocytes that express STRL22. Since STRL22 is the sixth functional CC chemokine receptor identified, it should be re-named CCR6.     

Dendritic cell chemotaxis and transendothelial migration are induced by distinct chemokines and are regulated on maturation

The capacity of dendritic cells (DC) to initiate immune responses is dependent on their specialized migratory and tissue homing properties. Chemotaxis and transendothelial migration (TEM) of DC were studied in vitro. Immature DC were generated by culture of human monocytes in granulocyte-macrophage colony-stimulating factor and IL-4. These cells exhibited potent chemotaxis and TEM responses to the CC chemokines macrophage inflammatory protein (MIP)-1alpha, MIP-1beta, RANTES, and monocyte chemotactic protein-3, and weak responses to the CC chemokine MIP-3beta and the CXC chemokine stromal cell-derived factor (SDF)-1alpha. Maturation of DC induced by culture in lipopolysaccharide, TNF-alpha or IL-1beta reduced or abolished responses to the former CC chemokines but markedly enhanced responses to MIP-3beta and SDF-1alpha. This correlated with changes in chemokine receptor expression: CCR5 expression was reduced while CXCR4 expression was enhanced. These findings suggest two stages for regulation of DC migration in which one set of chemokines may regulate recruitment into or within tissues, and another egress from the tissues.     

The T cell-directed CC chemokine TARC is a highly specific biological ligand for CC chemokine receptor 4

Thymus and activation-regulated chemokine (TARC) is a recently identified CC chemokine that is expressed constitutively in thymus and transiently in stimulated peripheral blood mononuclear cells. TARC functions as a selective chemoattractant for T cells that express a class of receptors binding TARC with high affinity and specificity. To identify the receptor for TARC, we produced TARC as a fusion protein with secreted alkaline phosphatase (SEAP) and used it for specific binding. By stably transfecting five orphan receptors and five known CC chemokine receptors (CCR1 to -5) into K562 cells, we found that TARC-SEAP bound selectively to cells expressing CCR4. TARC-SEAP also bound to K562 cells stably expressing CCR4 with a high affinity (Kd = 0.5 nM). Only TARC and not five other CC chemokines (MCP-1 (monocyte chemoattractant protein-1), RANTES (regulated upon activation, normal T cells expressed and secreted), MIP-1alpha (macrophage inflammatory protein-1alpha), MIP-1beta, and LARC (liver and activation-regulated chemokine)) competed with TARC-SEAP for binding to CCR4. TARC but not RANTES or MIP-1alpha induced migration and calcium mobilization in 293/EBNA-1 cells stably expressing CCR4. K562 cells stably expressing CCR4 also responded to TARC in a calcium mobilization assay. Northern blot analysis revealed that CCR4 mRNA was expressed strongly in human T cell lines and peripheral blood T cells but not in B cells, natural killer cells, monocytes, or granulocytes. Taken together, TARC is a specific functional ligand for CCR4, and CCR4 is the specific receptor for TARC selectively expressed on T cells.     

EBI1/CCR7 is a new member of dendritic cell chemokine receptor that is up-regulated upon maturation

Dendritic cells (DC) that are stimulated with inflammatory mediators can maturate and migrate from nonlymphoid tissues to lymphoid organs to initiate T cell-mediated immune responses. This migratory step is closely related to the maturation of the DC. In an attempt to identify chemokine receptors that might influence migration and are selectively expressed in mature DC, we have discovered that the chemokine receptor, EBI1/CCR7, is strikingly up-regulated upon maturation in three distinct culture systems: 1) mouse bone marrow-derived DC, 2) mouse epidermal Langerhans cells, and 3) human monocyte-derived DC. The EBI1/CCR7 expressed in mature DC is functional because ELC/MIP-3beta, recently identified as a ligand of EBI1/CCR7, induces a rise in intracellular free calcium concentrations and directional migration of human monocyte-derived mature DC (HLA-DRhigh, CD1a(low), CD14-, CD25+, CD83+, and CD86high) in a dose-dependent manner, but not of immature DC (HLA-DRlow, CD1a(high), CD14-, CD25-, CD83-, and CD86-). In contrast, macrophage inflammatory protein-1alpha (MIP-1alpha), monocyte chemotactic protein-3 (MCP-3), and RANTES are active on immature DC but not on mature DC. Thus, it seems likely that MIP-1alpha, MCP-3, and RANTES can mediate the migration of immature DC located in peripheral sites, whereas ELC/MIP-3beta can direct the migration of Ag-carrying DC from peripheral inflammatory sites, where DC are stimulated to up-regulate the expression of EBI1/CCR7, to lymphoid organs. It is postulated that different chemokines and chemokine receptors are involved in DC migration in vivo, depending on the maturation state of DC.     

The N-terminal extracellular segments of the chemokine receptors CCR1 and CCR3 are determinants for MIP-1alpha and eotaxin binding, respectively, but a second domain is essential for efficient receptor activation

CCR1 and CCR3 are seven-transmembrane domain G protein-coupled receptors specific for members of the CC chemokine subgroup of leukocyte chemoattractants. Both have been implicated in the inflammatory response, and CCR3, through its expression on eosinophils, basophils, and Th2 lymphocytes, may be especially important in allergic inflammation. CCR1 and CCR3 are 54% identical in amino acid sequence and share some ligands but not others. In particular, macrophage inflammatory protein 1alpha (MIP-1alpha) is a ligand for CCR1 but not CCR3, and eotaxin is a ligand for CCR3 but not CCR1. To map ligand selectivity determinants and to guide rational antagonist design, we analyzed CCR1:CCR3 chimeric receptors. When expressed in mouse pre-B cells, chimeras in which the N-terminal extracellular segments were switched were both able to bind both MIP-1alpha and eotaxin, but in each case, binding occurred via separate sites. Nevertheless, neither MIP-1alpha nor eotaxin were effective agonists at either chimeric receptor in either calcium flux or chemotaxis assays. These data are consistent with a multi-site model for chemokine-chemokine receptor interaction in which one or more subsites determine chemokine selectivity, but others are needed for receptor activation. Agents that bind to the N-terminal segments of CCR1 and CCR3 may be useful in blocking receptor function.     

Macrophage-derived chemokine is a functional ligand for the CC chemokine receptor 4

Macrophage-derived chemokine (MDC) is a recently identified member of the CC chemokine family. MDC is not closely related to other chemokines, sharing most similarity with thymus- and activation-regulated chemokine (TARC), which contains 37% identical amino acids. Both chemokines are highly expressed in the thymus, with little expression seen in other tissues. In addition, the genes for MDC and TARC are encoded by human chromosome 16. To explore this relationship in greater detail, we have more precisely localized the MDC gene to chromosome 16q13, the same position reported for the TARC gene. We have also examined the interaction of MDC with CC chemokine receptor 4 (CCR4), recently shown to be a receptor for TARC. Using a fusion protein of MDC with secreted alkaline phosphatase, we observed high affinity binding of MDC-secreted alkaline phosphatase to CCR4-transfected L1.2 cells (Kd = 0.18 nM). MDC and TARC competed for binding to CCR4, while no binding competition was observed for six other chemokines (MCP-1, MCP-3, MCP-4, RANTES (regulated on activation normal T cell expressed and secreted), macrophage inflammatory protein-1 alpha, macrophage inflammatory protein-1 beta). MDC was tested for calcium mobilization in L1.2 cells tranfected with seven different CC chemokine receptors. MDC induced a calcium flux in CCR4-transfected cells, but other receptors did not respond to MDC. TARC, which also induced calcium mobilization in CCR4 transfectants, was unable to desensitize the response to MDC. In contrast, MDC fully desensitized a subsequent response to TARC. Both MDC and TARC functioned as chemoattractants for CCR4 transfectants, confirming that MDC is also a functional ligand for CCR4. Since MDC and TARC are both expressed in the thymus, one role for these chemokines may be to attract CCR4-bearing thymocytes in the process of T cell education and differentiation.     

Selective G protein coupling by C-C chemokine receptors

The C-C chemokines are major mediators of chemotaxis of monocytes and some T cells in inflammatory reactions. The pathways by which the C-C chemokine receptors activate phospholipase C (PLC) were investigated in cotransfected COS-7 cells. The C-C chemokine receptor-1 (CKR-1), the MCP-1 receptor-A (MCP-1Ra), and MCP-1Rb can reconstitute ligand-induced accumulation of inositol phosphates with PLC beta2 in a pertussis toxin-sensitive manner, presumably through G beta gamma released from the Gi proteins. However, these three receptors demonstrated different specificity in coupling to the alpha subunits of the Gq class. While none of the receptors can couple to Galphaq/11, MCP-1Rb can couple to both Galpha14 and Galpha16, but its splicing variant, MCP-1Rb, cannot. Since MCP-1Ra and -b differ only in their C-terminal intracellular domains, the C-terminal ends of MCP-1Rs determine G protein coupling specificity. CKR-1 can couple to Galpha14 but not to Galpha16, suggesting some of the C-C chemokine receptors, unlike the C-X-C chemokine receptors, discriminate against Galpha16, a hematopoietic-specific Galpha subunit. The intriguing specificity in coupling of the Gq class of G proteins implies that the chemokines may be involved in some distinct functions in vivo. The commonality of the chemokine receptors in coupling to the Gi-Gbetagamma-PLC beta2 pathway provides a potential target for developing broad spectrum anti-inflammatory drugs.     

Selective chemokine mRNA expression following brain injury

Injury in non-neuronal tissues stimulates chemokine expression leading to recruitment of inflammatory cells responsible for orchestration of repair processes. The signals involved in directing repair of damage to the brain are less well understood. We hypothesized that following brain injury, chemokines are expressed and regulate the rate and pattern of inflammatory cell accumulation. The two chemokine subfamilies are alpha(alpha)-chemokines, which primarily function as neutrophil chemoattractants, and the beta(beta)-chemokines, which function primarily as monocyte chemoattractants. We assessed alpha and beta chemokine mRNA expression patterns and leukocyte accumulation following a cerebral cortical lesion. Cortical lesions were produced with and without addition of endotoxin, Escherichia coli lipopolysaccharide (LPS), which stimulates cytokine expression. We studied the expression of the beta-chemokines: monocyte chemoattractant protein (gene product JE; MCP-1/JE), macrophage inflammatory protein-1 alpha and beta (MIP-1alpha and MIP-1beta), and the regulated upon activation normal T expressed and secreted chemokine (RANTES) as well as the alpha-chemokines: interferon-gamma-inducible protein (IP-10) and N51/KC (KC; a murine homologue of MIP-2). Changes in gene expression were analyzed by Northern analysis at different time points following injury. Leukocyte and macrophage densities were analyzed by immunohistochemistry at the same time intervals. All chemokines were elevated following cortical injury/endotoxin. MCP-1 and MIP-1alpha were elevated at 2 h and peaked 6 h, MIP-1beta peaked at 6 h, but declined more rapidly than MCP-1 or MIP-1alpha, and IP-10 peaked at 6 h and showed the most rapid decline. KC was elevated at 1 h, and peaked at 6 h following LPS. RANTES was elevated at 1 h and achieved a plateau level between 6 and 18 h, then declined. In contrast, sterile injuries produced in the absence of endotoxin only induced the mRNA of the beta-chemokine MCP-1, and its expression was delayed compared to the cortical injury/endotoxin group. The presence of chemokine message as early as 1 h indicates that expression of this class of molecules is an early response in the repair process following traumatic brain injury. Macrophage/microglia accumulation occurred more rapidly, activated microglia further from the lesion border, and more cells accumulated in cortical injury/endotoxin than in cortical lesions produced under sterile conditions. Thus, there was a positive correlation between beta-chemokine expression and the number of beta-chemokine responsive cells (i.e. microglia) accumulating in injury sites. This is the first comprehensive study using a panel of chemokine probes and specific marcophage/microglial markers to study in vivo activation of the brain following injury. Our data show that the brain is capable of expression of multiple chemokine genes upon appropriate stimulation (e.g. LPS-treatment). The gradient of microglial activation is consistent with physical damage stimulating release of chemokines that diffuse from the injury site. These data strongly suggest that chemokines are instrumental in the initiation of repair processes following brain injury.     

Effect of N-terminal truncation and solution conditions on chemokine dimer stability: nuclear magnetic resonance structural analysis of macrophage inflammatory protein 1 beta mutants

Chemokines (chemotactic cytokines) are a family of immune system proteins, several of which have been shown to block human immunodeficiency virus (HIV) infection in various cell types. While the solved structures of most chemokines reveal protein dimers, evidence has accumulated for the biological activity of individual chemokine monomers, and a debate has arisen regarding the biological role of the chemokine dimer. Concurrent with this debate, several N-terminal truncations and modifications in the CC subfamily of chemokines have been shown to have functional significance, in many cases antagonizing their respective receptors and in some cases retaining the ability to block HIV entry to the cell. As the dimer interface of CC chemokines is located at their N-terminus, a structural study of N-terminally truncated chemokines will address the effect that this type of mutation has on the dimer-monomer equilibrium. We have studied the structural consequences of N-terminal truncation in macrophage inflammatory protein 1 beta (MIP-1 beta), a CC chemokine that has been shown to block HIV infection. Examination of nuclear magnetic resonance (NMR) spectra of a series of N-terminally truncated MIP-1 beta variants reveals that these proteins possess a range of ability to dimerize. A mutant beginning at amino acid Asp6 [termed MIP(6)] has near wild-type dimer properties, while further truncation results in weakened dimer affinity. The mutant MIP(9) (beginning with amino acid Thr9) has been found to exist solely as a folded monomer. Relaxation measurements yield a rotational correlation time of 8.6 +/- 0.1 ns for wild-type MIP-1 beta and 4.5 +/- 0.1 ns for the MIP(9) mutant, consistent with a wild-type dimer and a fully monomeric MIP(9) variant. The presence of physiological salt concentration drastically changes the monomer-dimer equilibrium for both wild-type and most mutant proteins, heavily favoring the dimeric form of the protein. These results have implications for structure-function analysis of existing chemokine mutants as well as for the larger debate regarding the biological existence and activity of the chemokine dimer.     

Macrophage-inflammatory protein-3 beta/EBI1-ligand chemokine/CK beta-11, a CC chemokine, is a chemoattractant with a specificity for macrophage progenitors among myeloid progenitor cells

Chemoattractants are potential factors influencing cell migration. Stromal cell-derived factor-1, a CXC chemokine, is the only chemokine reported to have chemotactic activity for hemopoietic progenitor cells (HPC). We report in this work another chemokine of the CC subfamily, which is chemotactic for HPC. Macrophage-inflammatory protein (MIP)-3 beta/EBI1-ligand chemokine/CK beta-11 attracted bone marrow and cord blood CD34+ cells. In contrast to stromal cell-derived factor-1, which attracts multiple types of HPC, MIP-3beta attracted mainly CFU granulocyte macrophage, but not other HPC such as burst-forming unit erythrocyte or CFU granulocyte, erythrocyte, macrophage, and megakaryocyte. Chemoattracted CD34+ cells formed CFU granulocyte macrophage-like colonies, which were morphologically determined as large macrophages. These progenitors were selectively responsive to stimulation by macrophage CSF, demonstrating that MIP-3 beta attracts macrophage progenitors. Expression of CCR7, the receptor for MIP-3 beta, was detected at a mRNA level in the attracted CD34+ cells as well as input CD34+HPC. Expression of MIP-3 beta mRNA was not constitutive, but was inducible in bone marrow stromal cells by inflammatory agents such as bacterial LPS, IFN-gamma, and TNF-alpha. Taken together, our findings suggest that MIP-3 beta is expressed in the bone marrow environment after induction with certain inflammatory cytokines and LPS, and may play a role in trafficking of macrophage progenitors in and out of the bone marrow in inflammatory conditions.     

CK beta-11/macrophage inflammatory protein-3 beta/EBI1-ligand chemokine is an efficacious chemoattractant for T and B cells

We examined the functional properties of CK beta-11/MIP-3 beta/ELC, a recently reported CC chemokine that specifically binds to a chemokine receptor, EBI1/BLR2/CCR7. CK beta-11/MIP-3 beta/ELC is distantly related to other CC and CXC chemokines in primary amino acid sequence structure. Recombinant human CK beta-11/MIP-3 beta/ELC expressed from a mammalian cell system showed potent chemotactic activity for T cells and B cells but not for granulocytes and monocytes. An optimal concentration of CK beta-11/MIP-3 beta/ELC attracted most input T cells within 3 h, a chemotactic activity comparable with that of stromal cell derived factor 1 (SDF-1), a highly efficacious CXC chemokine. CK beta-11/MIP-3 beta/ELC equally attracted naive CD45RA+ and memory type CD45RO+ T cells. CK beta-11/MIP-3 beta/ELC also strongly attracted both CD4+ and CD8+ T cells, but the attraction for CD4+ T cells was greater. CK beta-11/MIP-3 beta/ELC was also a more efficacious chemoattractant for B cells than MIP-1 alpha, a known B cell chemoattractant. CK beta-11/MIP-3 beta/ELC induced actin polymerization in lymphocytes, and chemotaxis was completely blocked by pertussis toxin showing its receptor, most likely EBI1/BLR2/CCR7, is coupled to a G(alpha i) protein. CK beta-11/MIP-3 beta/ELC induced calcium mobilization in lymphocytes, which could be desensitized by SDF-1, suggesting possible cross-regulation in their signaling. Human CK beta-11/MIP-3 beta/ELC attracted murine splenocytes suggesting functional conservation of CK beta-11/MIP-3 beta/ELC between human and mouse. The efficacy of chemoattraction by CK beta-11/MIP-3 beta/ELC and tissue expression of its mRNA suggest that CK beta-11/MIP-3 beta/ELC may be important in trafficking of T cells in thymus, and T cell and B cell migration to secondary lymphoid organs.     

Generation of CD8 suppressor factor and beta chemokines, induced by xenogeneic immunization, in the prevention of simian immunodeficiency virus infection in macaques

Previous xenogeneic immunization experiments in rhesus macaques with simian immunodeficiency virus (SIV) grown in human CD4(+) T cells consistently elicited protection from challenge with live SIV. However, the mechanism of protection has not been established. We present evidence that xenogeneic immunization induced significant CD8 suppressor factor, RANTES (regulated upon activation, normal T cell expressed and secreted), macrophage inflammatory protein (MIP) 1alpha, and MIP-1beta (P < 0.001 - P < 0.02). The concentrations of these increased significantly in protected as compared with infected macaques (P < 0.001). Xenogeneic stimulation in vitro also up-regulated CD8 suppressor factors (SF; P < 0.001) and the beta chemokines which were neutralized by antibodies to the 3 beta chemokines. Recombinant human RANTES, MIP-1alpha and MIP-1beta which bind to simian CCR5, suppressed SIV replication in a dose-dependent manner, with RANTES being more effective than the other two chemokines. The results suggest that immunization with SIV grown in human CD4(+) T cells induces CD8-suppressor factor, RANTES, MIP-1alpha and MIP-1beta which may block CCR5 receptors and prevent the virus from binding and fusion to CD4(+) cells.     

An important role for the chemokine macrophage inflammatory protein-1 alpha in the pathogenesis of the T cell-mediated autoimmune disease, experimental autoimmune encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE) is a CD4+ T cell-mediated, inflammatory demyelinating disease of the central nervous system (CNS) that serves as a model for the human demyelinating disease, multiple sclerosis. A critical event in the pathogenesis of EAE is the entry of both Ag-specific T lymphocytes and Ag-nonspecific mononuclear cells into the CNS. In the present report we investigated the role of two C-C chemokines (macrophage inflammatory protein-1 alpha (MIP-1 alpha) and monocyte chemotactic protein-1) and a C-x-C chemokine (MIP-2) in the pathogenesis of EAE. Production in the CNS of MIP-1 alpha, but not that of MIP-2, a rodent homologue of IL-8, or monocyte chemotactic protein-1, correlated with development of severe clinical disease. Administration of anti-MIP-1 alpha, but not that of anti-monocyte chemotactic protein-1, prevented the development of both acute and relapsing paralytic disease as well as infiltration of mononuclear cells into the CNS initiated by the transfer of neuroantigen peptide-activated T cells. Ab therapy could also be used to ameliorate the severity of ongoing clinical disease. Anti-MIP-1 alpha did not affect the activation of encepahlitogenic T cells as measured by cytokine secretion, surface marker expression, and ability to adoptively transfer EAE. These results demonstrate that MIP-1 alpha plays an important role in directing the chemoattraction of mononuclear inflammatory cells in the T cell-mediated autoimmune disease, EAE.