Reduced chemokine and chemokine receptor expression in spinal cords of TCR BV8S2 transgenic mice protected against experimental autoimmune encephalomyelitis with BV8S2 protein

The perivascular transmigration and accumulation of macrophages and T lymphocytes in the CNS of mice with experimental autoimmune encephalomyelitis (EAE) may be partly regulated by low m.w. chemotactic cytokines. Using the RNase protection assay and ELISA, we quantified expression of chemokines and chemokine receptors in the spinal cord (SC), brain, and lymph nodes of BV8S2 transgenic mice that developed or were protected from EAE by vaccination with BV8S2 protein. In paralyzed control mice, the SC had increased cellular infiltration and strong expression of the chemokines RANTES, IFN-inducible 10-kDa protein, and monocyte chemoattractant protein-1 and the cognate chemokine receptors CCR1, CCR2, and CCR5, with lower expression of macrophage-inflammatory protein (MIP)-1alpha, MIP-1beta, and MIP-2; whereas brain had less infiltration and a lower expression of a different pattern of chemokines and receptors. In TCR-protected mice, there was a decrease in the number of inflammatory cells in both SC and brain. In SC, the reduced cellular infiltrate afforded by TCR vaccination was commensurate with profoundly reduced expression of chemokines and their cognate chemokine receptors. In brain, however, TCR vaccination did not produce significant changes in chemokine expression but resulted in an increased expression of CCR3 and CCR4 usually associated with Th2 cells. In contrast to CNS, lymph nodes of protected mice had a significant increase in expression of MIP-2 and MIP-1beta but no change in expression of chemokine receptors. These results demonstrate that TCR vaccination results in selective reduction of inflammatory chemokines and chemokine receptors in SC, the target organ most affected during EAE.     

A role for macrophage inflammatory protein-3 alpha/CC chemokine ligand 20 in immune priming during T cell-mediated inflammation of the central nervous system

Chemokines are a family of cytokines that exhibit selective chemoattractant properties for target leukocytes and play a significant role in leukocyte migration. In this study, we have investigated the role of the C-C chemokine, macrophage inflammatory protein (MIP)-3alpha/CC chemokine ligand 20, in the pathogenesis of experimental autoimmune encephalomyelitis (EAE), a model of T cell-dependent inflammation. Expression in the CNS of MIP-3alpha, as determined by RT-PCR, increased in a time-dependent manner such that peak expression correlated with peak clinical disease. Similarly, levels of immunoreactive MIP-3alpha in the draining lymph nodes increased up to 10-fold 9 days postimmunization and remained elevated for up to 21 days postimmunization. The increased production of MIP-3alpha coincided with onset of clinical disease. Treatment of mice with specific neutralizing anti-MIP-3alpha Abs significantly reduced the severity of both clinical EAE and neuroinflammation by inhibiting the sensitization of lymphocytes to the specific Ag and release of lymphocytes from the draining lymph nodes. In contrast, adoptive transfer experiments indicated that MIP-3alpha was not essential for the effector phase of EAE. Together, these data demonstrate that MIP-3alpha plays a critical role in the sensitization phase of EAE.     

Regulation of macrophage chemokine expression by lipopolysaccharide in vitro and in vivo.

The host response to Gram-negative LPS is characterized by an influx of inflammatory cells into host tissues, which is mediated, in part, by localized production of chemokines. The expression and function of chemokines in vivo appears to be highly selective, though the molecular mechanisms responsible are not well understood. All CXC (IFN-gamma-inducible protein (IP-10), macrophage inflammatory protein (MIP)-2, and KC) and CC (JE/monocyte chemoattractant protein (MCP)-1, MCP-5, MIP-1alpha, MIP-1beta, and RANTES) chemokine genes evaluated were sensitive to stimulation by LPS in vitro and in vivo. While IL-10 suppressed the expression of all LPS-induced chemokine genes evaluated in vitro, treatment with IFN-gamma selectively induced IP-10 and MCP-5 mRNAs, but inhibited LPS-induced MIP-2, KC, JE/MCP-1, MIP-1alpha, and MIP-1beta mRNA and/or protein. Like the response to IFN-gamma, LPS-mediated induction of IP-10 and MCP-5 was Stat1 dependent. Interestingly, only the IFN-gamma-mediated suppression of LPS-induced KC gene expression was IFN regulatory factor-2 dependent. Treatment of mice with LPS in vivo also induced high levels of chemokine mRNA in the liver and lung, with a concomitant increase in circulating protein. Hepatic expression of MIP-1alpha, MIP-1beta, RANTES, and MCP-5 mRNAs were dramatically reduced in Kupffer cell-depleted mice, while IP-10, KC, MIP-2, and MCP-1 were unaffected or enhanced. These findings indicate that selective regulation of chemokine expression in vivo may result from differential response of macrophages to pro- and antiinflammatory stimuli and to cell type-specific patterns of stimulus sensitivity. Moreover, the data suggest that individual chemokine genes are differentially regulated in response to LPS, suggesting unique roles during the sepsis cascade.     

Induction of β-family chemokines mRNA in human embryonic astrocytes by inflammatory cytokines

The cellular infiltration found during CNS inflammation consists of monocytes and activated T cells, suggesting the presence of cell-specific chemotactic signals during inflammatory responses. Astrocyte chemokine expression might contribute to site-specific leukocyte infiltration within the CNS. To investigate the factors that regulate astrocyte chemokine expression, we examined the ability of human fetal astrocytes to induce β-family chemokine mRNA. Astrocyte-derived monocyte chemoattractant protein-1 (MCP-1), RANTES, macrophage inflammatory protein-1α (MIP-1α), and MIP-1β mRNA were easily induced by lipopolysaccharide and/or the proinflammatory cytokines (IFNγ and/or TNF-α), respectively. Addition of both IFNγ and TNF-α together did not lead to an additive effect but resulted in the inhibition of MCP-1 and MIP-1β mRNA expression, indicating that interaction between chemokines and cytokines may play a key role in regulating the local immune response of resident and infiltrating cells at the site of lesion. Interestingly, ultraviolet light-inactivated measles virus, but not cytomegalovirus, strongly induced expression of MCP-1, RANTES, MIP-1α, and MIP-1β mRNA in human embryonic astrocytes, especially MCP-1 and MIP-1β. An association occurs between the β-family chemokine expression in astrocytes and inflammatory factors/virus, suggesting a possible role for β-family chemokines in the pathogenesis of CNS inflammatory disease.     

Chemokine receptor expression and function in CD4+ T lymphocytes with regulatory activity

We have investigated the chemokine receptor expression and migratory behavior of a new subset of nickel-specific skin-homing regulatory CD4(+) T cells (Th(IL-10)) releasing high levels of IL-10, low IFN-gamma, and undetectable IL-4. These cells inhibit in a IL-10-dependent manner the capacity of dendritic cells to activate nickel-specific Tc1 and Th1 lymphocytes. RNase protection assay and FACS analysis revealed the expression of a vast repertoire of chemokine receptors on resting Th(IL-10), including the Th1-associated CXCR3 and CCR5, and the Th2-associated CCR3, CCR4, and CCR8, the latter at higher levels compared with Th2 cells. The most active chemokines for resting Th(IL-10), in terms of calcium mobilization and in vitro migration, were in order of potency: CCL2 (monocyte chemoattractant protein-1, CCR2 ligand), CCL4 (macrophage-inflammatory protein-1beta, CCR5 ligand), CCL3 (macrophage-inflammatory protein-1alpha, CCR1/5 ligand), CCL17 (thymus and activation-regulated chemokine, CCR4 ligand), CCL1 (I-309, CCR8 ligand), CXCL12 (stromal-derived factor-1, CXCR4), and CCL11 (eotaxin, CCR3 ligand). Consistent with receptor expression down-regulation, activated Th(IL-10) exhibited a reduced or absent response to most chemokines, but retained a significant migratory capacity to I-309, monocyte chemoattractant protein-1, and thymus and activation-regulated chemokine. I-309, which was ineffective on Th1 lymphocytes, attracted more efficiently Th(IL-10) than Th2 cells. I-309 and CCR8 mRNAs were not detected in unaffected skin and were up-regulated at the skin site of nickel-allergic reaction, with an earlier expression kinetics compared with IL-10 and IL-4. Results indicate that skin-homing regulatory Th(IL-10) lymphocytes coexpress functional Th1- and Th2-associated chemokine receptors, and that CCR8/I-309-driven recruitment of both resting and activated Th(IL-10) cells may be critically involved in the regulation of Th1-mediated skin allergic disorders.     

Fibrinogen stimulates macrophage chemokine secretion through toll-like receptor 4

Extravascular fibrin deposition is an early and persistent hallmark of inflammatory responses. Fibrin is generated from plasma-derived fibrinogen, which escapes the vasculature in response to endothelial cell retraction at sites of inflammation. Our ongoing efforts to define the physiologic functions of extravasated fibrin(ogen) have led to the discovery, reported here, that fibrinogen stimulates macrophage chemokine secretion. Differential mRNA expression analysis and RNase protection assays revealed that macrophage inflammatory protein-1alpha (MIP-1alpha), MIP-1beta, MIP-2, and monocyte chemoattractant protein-1 are fibrinogen inducible in the RAW264.7 mouse macrophage-like cell line, and ELISA confirmed that both RAW264.7 cells and primary murine thioglycolate-elicited peritoneal macrophages up-regulate the secretion of monocyte chemoattractant protein-1 >100-fold upon exposure to fibrinogen. Human U937 and THP-1 precursor-1 (THP-1) monocytic cell lines also secreted chemokines in response to fibrinogen, upon activation with IFN-gamma and differentiation with vitamin D(3), respectively. LPS contamination could not account for our observations, as fibrinogen-induced chemokine secretion was sensitive to heat denaturation and was unaffected by the pharmacologic LPS antagonist polymyxin B. Nevertheless, fibrinogen- and LPS-induced chemokine secretion both apparently required expression of functional Toll-like receptor 4, as each was diminished in macrophages derived from C3H/HeJ mice. Thus, innate responses to fibrinogen and bacterial endotoxin may converge at the evolutionarily conserved Toll-like recognition molecules. Our data suggest that extravascular fibrin(ogen) induces macrophage chemokine expression, thereby promoting immune surveillance at sites of inflammation.     

Differential role of CCR2 in islet and heart allograft rejection: tissue specificity of chemokine/chemokine receptor function in vivo

Chemokines have a pivotal role in the mobilization and activation of specific leukocyte subsets in acute allograft rejection. However, the role of specific chemokines and chemokine receptors in islet allograft rejection has not been fully elucidated. We now show that islet allograft rejection is associated with a steady increase in intragraft expression of the chemokines CCL8 (monocyte chemoattractant protein-2), CCL9 (monocyte chemoattractant protein-5), CCL5 (RANTES), CXCL-10 (IFN-gamma-inducible protein-10), and CXCL9 (monokine induced by IFN-gamma) and their corresponding chemokine receptors CCR2, CCR5, CCR1, and CXCR3. Because CCR2 was found to be highly induced, we tested the specific role of CCR2 in islet allograft rejection by transplanting fully MHC mismatched islets from BALB/c mice into C57BL/6 wild-type (WT) and CCR2-deficient mice (CCR2-/-). A significant prolongation of islet allograft survival was noted in CCR2-/- recipients, with median survival time of 24 and 12 days for CCR2-/- and WT recipients, respectively (p < 0.0001). This was associated with reduction in the generation of CD8+, but not CD4+ effector alloreactive T cells (CD62L(low)CD44(high)) in CCR2-/- compared with WT recipients. In addition, CCR2-/- recipients had a reduced Th1 and increased Th2 alloresponse in the periphery (by ELISPOT analysis) as well as in the grafts (by RT-PCR). However, these changes were only transient in CCR2-/- recipients that ultimately rejected their grafts. Furthermore, in contrast to the islet transplants, CCR2 deficiency offered only marginal prolongation of heart allograft survival. This study demonstrates the important role for CCR2 in early islet allograft rejection and highlights the tissue specificity of the chemokine/chemokine receptor system in vivo in regulating allograft rejection.     

Differential expression of CC chemokines and the CCR5 receptor in the pancreas is associated with progression to type I diabetes

We investigated the biological role of CC chemokines in the Th1-mediated pathogenesis of spontaneous type I diabetes in nonobese diabetic (NOD) mice. Whereas an elevated ratio of macrophage inflammatory protein-1alpha (MIP-1alpha):MIP-1beta in the pancreas correlated with destructive insulitis and progression to diabetes in NOD mice, a decreased intrapancreatic MIP-1alpha:MIP-1beta ratio was observed in nonobese diabetes-resistant (NOR) mice. IL-4 treatment, which prevents diabetes in NOD mice by polarizing intraislet Th2 responses, decreased CCR5 expression in islets and potentiated a high ratio of MIP-1beta and monocyte chemotactic protein-1 (MCP-1): MIP-1alpha in the pancreas. Furthermore, NOD.MIP-1alpha-/- mice exhibited reduced destructive insulitis and were protected from diabetes. Neutralization of MIP-1alpha with specific Abs following transfer of diabetogenic T cells delayed the onset of diabetes in NOD.Scid recipients. These studies illustrate that the temporal expression of certain CC chemokines, particularly MIP-1alpha, and the CCR5 chemokine receptor in the pancreas is associated with the development of insulitis and spontaneous type I diabetes.     

Lack of CCR2 results in increased mortality and impaired leukocyte activation and trafficking following infection of the central nervous system with a neurotropic coronavirus

In the present study, we evaluated the role of CCR2 in a model of viral-induced neurologic disease. An orchestrated expression of chemokines, including the CCR2 ligands monocyte chemoattractant protein-1/CCL2 and monocyte chemoattractant protein-3/CCL7, occurs within the CNS following infection with mouse hepatitis virus (MHV). Infection of mice lacking CCR2 (CCR2(-/-)) with MHV resulted in increased mortality and enhanced viral recovery from the brain that correlated with reduced (p < or = 0.04) T cell and macrophage/microglial (determined by F4/80 Ag expression, p < or = 0.004) infiltration into the CNS. Moreover, MHV-infected CCR2(-/-) mice displayed a significant decrease in Th1-associated factors IFN-gamma (p < or = 0.001) and RANTES/CCL5 (p < or = 0.002) within the CNS as compared with CCR2(+/+) mice. Further, peripheral CD4(+) and CD8(+) T cells from immunized CCR2(-/-) mice displayed a marked reduction in IFN-gamma production in response to viral Ag and did not migrate into the CNS of MHV-infected recombination-activating gene (RAG)1(-/-) mice following adoptive transfer. In addition, macrophage/microglial infiltration into the CNS of RAG1(-/-) mice receiving CCR2(-/-) splenocytes was reduced (p < or = 0.05), which correlated with a reduction in the severity of demyelination (p < or = 0.001) as compared with RAG1(-/-) mice receiving splenocytes from CCR2(+/+) mice. Collectively, these results indicate an important role for CCR2 in host defense and disease by regulating leukocyte activation and trafficking.     

Gamma delta T cell regulation of IFN-gamma production by central nervous system-infiltrating encephalitogenic T cells: correlation with recovery from experimental autoimmune encephalomyelitis

Interferon-gamma has been shown to be important for the resolution of inflammation associated with CNS autoimmunity. Because one of the roles of gamma delta T cells is the regulation of inflammation, we asked whether gamma delta T cells were able to regulate CNS inflammation using the autoimmune disease mouse model experimental autoimmune encephalomyelitis (EAE). We show that the presence of gamma delta T cells was needed to promote the production of IFN-gamma by both CD4 and CD8 T cells in the CNS before the onset of EAE. This regulation was shown to be independent of the ability of gamma delta T cells to produce IFN-gamma, and was specific to T cells in the CNS, as no alterations in IFN-gamma production were detectable in gamma delta T cell-deficient mice in the spleen and lymph nodes of mice with EAE or following immunization. Analysis of TCR gamma delta gene usage in the CNS showed that the only TCR delta V gene families present in the CNS before EAE onset are from the DV7s6 and DV105s1 gene families. We also show that the primary IFN-gamma-producing cells in the CNS are the encephalitogenic T cells, and that gamma delta T cell-deficient mice are unable to resolve EAE disease symptoms like control mice, thus exhibiting a long-term chronic disease course similar to that observed in IFN-gamma-deficient mice. These data suggest that CNS resident gamma delta T cells promote the production of IFN-gamma by encephalitogenic T cells in the CNS, which is ultimately required for the recovery from EAE.     

The chemokine receptor CCR5 is not a necessary inflammatory mediator in kainic acid-induced hippocampal injury: evidence for a compensatory effect by increased CCR2 and CCR3

Chemokines and their receptors have been strongly implicated in the inflammatory process. However, their roles in excitotoxic brain injury are largely unknown. In this study we used C-C chemokine receptor 5 (CCR5) knockout (KO) mice to investigate the role of CCR5 in neurodegeneration induced by intranasal administration of the excitotoxin kainic acid (KA). Although KA treatment resulted in an increased CCR5 mRNA level in the hippocampi of wild-type mice, a CCR5 deficiency in KO mice did not affect either the clinical and pathological changes in vivo or the neuronal susceptibilities to KA insult in vitro. KA treatment stimulated mRNA expression of the monocyte chemoattractant protein-2 (MCP-2) in both the wild-type and KO mice. KA treatment did not affect mRNA levels for the macrophage inflammatory protein-1alpha (MIP-1alpha) or the regulated upon activation normal T cells expressed and secreted protein (RANTES) in either wild-type or CCR5 KO mice. CCR2 mRNA expression was undetectable in the hippocampi of wild-type mice regardless of KA treatment. In contrast, CCR5 KO mice showed CCR2 mRNA expression that was remarkably increased after KA treatment. KA treatment did not affect CCR3 mRNA expression in the wild-type mice, whereas KO mice showed both a higher basal level of CCR3 mRNA expression as well as a strong upregulation following KA treatment. These results indicate that CCR5 is not a necessary inflammatory mediator in KA induced neurodegeneration. The roles of CCR5 in excitotoxic injury in CCR5 deficient mice are compensated by increased CCR2 and CCR3 expression, which share the common MCP-2 ligand with CCR5.     

IFN-inducible protein 10/CXC chemokine ligand 10-independent induction of experimental autoimmune encephalomyelitis

In multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE), autoaggressive T cells traffic into the CNS and induce disease. Infiltration of these pathogenic T cells into the CNS has been correlated with the expression of the chemokine IFN-inducible protein (IP)10/CXC chemokine ligand (CXCL)10, a chemoattractant for activated T cells, and its receptor CXCR3, in the CNS of both MS patients and mice with EAE. In the present study, we report that targeted deletion of IP-10 did not diminish the expression, severity, or histopathology of EAE induced by active immunization with 100 micro g of myelin oligodendrocyte glycoprotein peptide (MOG)p35-55. However, we found that IP-10-deficient mice had a lower threshold for expression of disease compared with wild-type littermates. EAE induced by immunization with 5 micro g of MOGp35-55 resulted in more severe disease characterized by a greater number of CNS lesions and infiltrating mononuclear cells in IP-10-deficient mice compared with wild-type controls. IP-10-deficient mice immunized with MOGp35-55 demonstrated increased levels of IFN-inducible T cell alpha-chemokine/CXCL11 mRNA in the CNS and decreased levels of monokine induced by IFN-gamma/CXCL9 mRNA in draining lymph nodes, suggesting differential compensation for loss of IP-10 in lymphoid vs parenchymal tissue compartments. EAE in IP-10-deficient mice induced by low-dose immunization was associated with enhanced Ag-specific Th1 responses in the draining lymph node, which corresponded with diminished lymph node TGF-beta1 expression. Our data demonstrated that IP-10 was not required for the trafficking of pathogenic T cells into the CNS in EAE but played an unexpected role in determining the threshold of disease susceptibility in the periphery.     

T cell chemokine receptor expression in aging

Changes in chemokine receptor expression are important in determining T cell migration and the subsequent immune response. To better understand the contribution of the chemokine system in immune senescence we determined the effect of aging on CD4(+) T cell chemokine receptor function using microarray, RNase protection assays, Western blot, and in vitro chemokine transmigration assays. Freshly isolated CD4(+) cells from aged (20-22 mo) mice were found to express a higher level of CCR1, 2, 4, 5, 6, and 8 and CXCR2-5, and a lower level of CCR7 and 9 than those from young (3-4 mo) animals. Caloric restriction partially or completely restored the aging effects on CCR1, 7, and 8 and CXCR2, 4, and 5. The aging-associated differences in chemokine receptor expression cannot be adequately explained by the age-associated shift in the naive/memory or Th1/Th2 profile. CD4(+) cells from aged animals have increased chemotactic response to stromal cell-derived factor-1 and macrophage-inflammatory protein-1alpha, suggesting that the observed chemokine receptor changes have important functional consequences. We propose that the aging-associated changes in T cell chemokine receptor expression may contribute to the different clinical outcome in T cell chemokine receptor-dependent diseases in the elderly.     

Distinct temporal patterns of macrophage-inflammatory protein-2 and KC chemokine gene expression in surgical injury

In the present study the regulation of CXC chemokine expression was evaluated in full-thickness abdominal wounds in mice. During the first 24 h after injury, IL-1alphabeta, KC, macrophage-inflammatory protein (MIP)-2, and monocyte chemoattractant protein-1 were the predominant cytokines and chemokines produced; TNF-alpha was not detected. Chemokine mRNA expression and protein secretion occurred in two temporal stages. The first, which reached a maximum at 6 h, was associated with high levels of IL-1alpha and KC and low levels of MIP-2. This stage could be reproduced by intradermal injection of IL-1alpha or IL-1beta and was partially blocked by injection of neutralizing Ab against IL-1alpha but not IL-1beta. In animals depleted of circulating neutrophils, chemokine expression was reduced by nearly 70% during this stage. In the second stage, which peaked at 24 h after injury, modest but significant levels of IL-1beta were detected in association with low levels of KC and high levels of MIP-2. This pattern of chemokine expression could not be mimicked by injection of IL-1alpha or IL-1beta (even with prolonged exposure), although MIP-2 expression could be partially inhibited by intradermal injection of neutralizing Ab against IL-1beta. Surprisingly, neutrophil depletion before injury resulted in sustained high levels of both KC and MIP-2 expression. These observations demonstrate that these two closely related chemokines are under distinct regulatory controls in vivo that are likely to reflect the temporally ordered participation of different cell types and/or extracellular stimuli and inhibitors.     

Characterization of binding sites for chemokines MCP-1 and MIP-1alpha on human brain microvessels

The presence of binding sites for the beta chemokines monocyte chemoattractant protein-1 (MCP-1) and macrophage inflammatory protein-1alpha (MIP-1alpha) has recently been identified on human brain microvessels. We extend these findings in this report to reveal that such sites exemplify characteristics of the recognized major receptors for MCP-1 and MIP-1alpha: CCR2, and CCR1 and CCR5, respectively. Specifically, labeled MCP-1 binding to isolated brain microvessels was inhibited by unlabeled MCP-1 and MCP-3, the latter another CCR2 ligand, but not by MIP-1alpha. Inhibition of labeled MIP-1alpha binding was achieved with unlabeled MIP-1alpha and RANTES, the latter a beta chemokine that binds to both CCR1 and CCR5, but not by MCP-1. Labeled MIP-1alpha binding was also antagonized by unlabeled MCP-3, which is also recognized by CCR1, and MIP-1beta, which is a ligand for CCR5. Labeled MCP-1 and MIP-1alpha were further observed to be internalized within the endothelial cells of brain microvessels, following their binding to the microvascular surface at 37 degrees C. Additionally, exposure of microvessels to unlabeled MCP-1 or MIP-1alpha was accompanied by the initial loss and subsequent recovery of surface binding sites for these chemokines, which occurred on a time scale consistent with ligand-induced endocytosis and recycling. These collective features bear striking similarity to those that characterize interactions of MCP-1 and MIP-1alpha with their receptors on leukocytes and underscore the concept of cognate chemokine receptors on brain microvascular endothelium.     

The Toll-like receptor 5 stimulus bacterial flagellin induces maturation and chemokine production in human dendritic cells

Toll-like receptors (TLRs) are pattern recognition receptors that serve an important function in detecting pathogens and initiating inflammatory responses. Upon encounter with foreign Ag, dendritic cells (DCs) go through a maturation process characterized by an increase in surface expression of MHC class II and costimulatory molecules, which leads to initiation of an effective immune response in naive T cells. The innate immune response to bacterial flagellin is mediated by TLR5, which is expressed on human DCs. Therefore, we sought to investigate whether flagellin could induce DC maturation. Immature DCs were cultured in the absence or presence of flagellin and monitored for expression of cell surface maturation markers. Stimulation with flagellin induced increased surface expression of CD83, CD80, CD86, MHC class II, and the lymph node-homing chemokine receptor CCR7. Flagellin stimulated the expression of chemokines active on neutrophils (IL-8/CXC chemokine ligand (CXCL)8, GRO-alpha/CXCL1, GRO-beta/CXCL2, GRO-gamma/CXCL3), monocytes (monocyte chemoattractant protein-1/CC chemokine ligand (CCL)2), and immature DCs (macrophage-inflammatory protein-1 alpha/CCL3, macrophage-inflammatory protein-1 beta/CCL4), but not chemokines active on effector T cells (IFN-inducible protein-10 kDa/CXCL10, monokine induced by IFN-gamma/CXCL9, IFN-inducible T cell alpha chemoattractant/CXCL11). However, stimulating DCs with both flagellin and IFN-inducible protein-10 kDa, monokine induced by IFN-gamma, and IFN-inducible T cell alpha chemoattractant expression, whereas stimulation with IFN-beta or flagellin alone failed to induce these chemokines. In functional assays, flagellin-matured DCs displayed enhanced T cell stimulatory activity with a concomitant decrease in endocytic activity. Finally, DCs isolated from mouse spleens or bone marrows were shown to not express TLR5 and were not responsive to flagellin stimulation. These results demonstrate that flagellin can directly stimulate human but not murine DC maturation, providing an additional mechanism by which motile bacteria can initiate an acquired immune response.