Mast cells contribute importantly to both protective and pathological IgE-dependent immune responses. We show that the mast cell–expressed orphan serpentine receptor mCCRL2 is not required for expression of IgE-mediated mast cell–dependent passive cutaneous anaphylaxis but can enhance the tissue swelling and leukocyte infiltrates associated with such reactions in mice. We further identify chemerin as a natural nonsignaling protein ligand for both human and mouse CCRL2. In contrast to other “silent” or professional chemokine interreceptors, chemerin binding does not trigger ligand internalization. Rather, CCRL2 is able to bind the chemoattractant and increase local concentrations of bioactive chemerin, thus providing a link between CCRL2 expression and inflammation via the cell-signaling chemerin receptor CMKLR1.
Chemokine-like receptor 1 (CMKLR1), also known as ChemR23, and chemokine (C-C motif) receptor-like 2 (CCRL2) are 7-transmembrane receptors that were cloned in the late 1990’s based on their homology to known G-protein-coupled receptors. They were previously orphan receptors without any known biological roles; however, recent studies identified ligands for these receptors and their functions have begun to be unveiled. The plasma protein-derived chemoattractant chemerin is a ligand for CMKLR1 and activation of CMKLR1 with chemerin induces the migration of macrophages and dendritic cells (DCs) in vitro, suggesting a pro-inflammatory role. However, in vivo studies using CMKLR-deficient mice suggest an anti-inflammatory role for this receptor, possibly due to the recruitment of tolerogenic plasmacytoid DCs. Chemerin/CMKLR1 interaction also promotes adipogenesis and angiogenesis. The anti-inflammatory lipid mediator, resolving E1, is another CMKLR1 ligand and it inhibits leukocyte infiltration and proinflammatory gene expression. These divergent results suggest that CMKLR1 is a multifunctional receptor.
The chemokine CCL5 and CCL19 are reported to bind to CCRL2. Like Duffy antigen for chemokine receptor (DARC), D6 and CCX-CKR, CCRL2 does not signal, but it constitutively recycles, potentially reducing local concentration of CCL5 and CCL19 and subsequent immune responses. Surprisingly, chemerin, a ligand for CMKLR1, is a ligand for CCRL2. CCRL2 binds chemerin and increases local chemerin concentration to efficiently present it to CMKLR1 on nearby cells, providing a link between CCRL2 and CMKLR1. Although these findings suggest an anti-inflammatory role, a recent study using CCRL2-deficient mice indicates a proinflammatory role; thus, CCRL2 may also be multifunctional. Further studies using CMKLR1- or CCRL2-deficient mice are needed to further define the role of these receptors in immune responses and other cellular processes.
chemokine receptor; G-protein-coupled receptor; inflammation; leukocyte trafficking
Human CCRL1 belongs to the family of silent chemokine receptors. This transmembrane protein plays a role in blunting function
of chemokines through binding to them. This will attenuate immune responses. Interaction between CCRL1 and CCL21 determines
this immune extinction. Thus inhibiting the action of this atypical chemokine seems to stimulate immune responses especially in
the case of suppressed and immune deficient conditions. In this study we predicted 3D structure of CCRL1 using comparative
modeling and Hiddebn Markov Model algorithm. Final predicted model optimized by Modeller v9.8 and minimized regarding
energy level using UCSF chimera candidate version1.5.3. ClasPro webserver was used to find interacting residues between CCRL1
and CCL21. Interacting residues were used as target for chemical inhibitors by simulated docking study. For finding potential
inhibitors, library of KEGG compounds screened and 97 obtained chemicals docked against interacting residues between CCRL1-
CCL21 and MolDock was used as docking scoring function. Results indicated that Hexadecanal is a potential inhibitor of CCRL1-
CCL21 interaction. Inhibition of this interaction will increase intercellular level of CCl21 and interaction between CCL21 and CCR7
causes immune potentiaiton.
CCRL1; CCL21; CCR7; Docking; MolDock; Hexadecanal
Rheumatoid arthritis (RA) is characterized by the recruitment of leukocytes and the accumulation of inflammatory mediators within the synovial compartment. Release of the chemokine CCL18 has been widely attributed to antigen-presenting cells, including macrophages and dendritic cells. This study investigates the production of CCL18 in polymorphonuclear neutrophils (PMN), the predominant cell type recruited into synovial fluid (SF). Microarray analysis, semiquantitative and quantitative reverse transcriptase polymerase chain reaction identified SF PMN from patients with RA as a novel source for CCL18 in diseased joints. Highly upregulated expression of other chemokine genes was observed for CCL3, CXCL8 and CXCL10, whereas CCL21 was downregulated. The chemokine receptor genes were differentially expressed, with upregulation of CXCR4, CCRL2 and CCR5 and downregulation of CXCR1 and CXCR2. In cell culture experiments, expression of CCL18 mRNA in blood PMN was induced by tumor necrosis factor α, whereas synthesis of CCL18 protein required additional stimulation with a combination of IL-10 and vitamin D3. In comparison, recruited SF PMN from patients with RA were sensitized for CCL18 production, because IL-10 alone was sufficient to induce CCL18 release. These results suggest a release of the T cell-attracting CCL18 by PMN when recruited to diseased joints. However, its production is tightly regulated at the levels of mRNA expression and protein synthesis.
The non-signalling chemokine receptors, including receptors DARC, D6 and CCX-CKR, have recently been shown to be involved in chemokine clearance and activity regulation. The human chemokine receptor CRAM (also known as HCR or CCRL2) is the most recently identified member of this atypical group. CRAM is expressed on B cells in a maturation-stage dependent manner and absent on T cells. We have recently shown that it competitively binds CCL19. CCL19 and its signalling receptor CCR7 are critical components involved in cell recruitment to secondary lymphoid organs and in maturation. B cell Chronic Lymphocytic Leukemia (B-CLL) is a low-grade lymphoma characterized by proliferative centres (or pseudofollicles). Proliferative centres develop due to abnormal cellular localisation and they are involved in the development of malignant cells. CCR7 is highly expressed on B cells from CLL patients and mediates migration towards its ligands CCL19 and CCL21, while CRAM expression and potential interferences with CCR7 are yet to be characterized.
In this study, we show that B cells from patients with B-CLL present highly variable degrees of CRAM expression in contrast to more consistently high levels of CCR7. We investigated the hypothesis that, similar to the atypical receptor DARC, CRAM can modulate chemokine availability and/or efficacy, resulting in the regulation of cellular activation. We found that a high level of CRAM expression was detrimental to efficient chemotaxis with CCL19. MAP-kinase phosphorylation and intracellular calcium release induced by CCL19 were also altered by CRAM expression. In addition, we demonstrate that CRAM-induced regulation of CCL19 signalling is maintained over time.
We postulate that CRAM is a factor involved in the fine tuning/control of CCR7/CCL19 mediated responses. This regulation could be critical to the pivotal role of CCL19 induced formation of proliferation centres supporting the T/B cells encounter as well as disease progression in B-CLL.
Recovery from acute hepatitis B virus (HBV) infection requires a broad, vigorous T-cell response, which is enhanced in mice when chemokine receptor 5 (CCR5) is missing. To test the hypothesis that production of a nonfunctional CCR5 (CCR5Δ32 [a functionally null allele containing a 32-bp deletion]) increases the likelihood of recovery from hepatitis B in humans, we studied 526 persons from three cohorts in which one person with HBV persistence was matched to two persons who recovered from an HBV infection. Recovery or persistence was determined prior to availability of lamivudine. We determined genotypes for CCR5Δ32 and for polymorphisms in the CCR5 promoter and in coding regions of the neighboring genes, chemokine receptor 2 (CCR2) and chemokine receptor-like 2 (CCRL2). Allele and haplotype frequencies were compared among the 190 persons with viral recovery and the 336 with persistence by use of conditional logistic regression. CCR5Δ32 reduced the risk of developing a persistent HBV infection by nearly half (odds ratio [OR], 0.53; 95% confidence interval [CI], 0.33 to 0.83; P = 0.006). This association was virtually identical in persons with and without a concomitant human immunodeficiency virus infection. Of the nine individuals who were homozygous for the deletion, eight recovered from infection (OR, 0.25; 95% CI, 0.03 to 1.99; P = 0.19). None of the other neighboring polymorphisms examined were associated with HBV outcome. These data demonstrate a protective effect of CCR5Δ32 in recovery from an HBV infection, provide genetic epidemiological evidence for a role of CCR5 in the immune response to HBV, and suggest a potential therapeutic treatment for patients persistently infected with HBV.
Chemokine signals and their cell-surface receptors are important modulators of HIV-1 disease and cancer. To aid future case/control association studies, aim to further characterise the haplotype structure of variation in chemokine and chemokine receptor genes. To perform haplotype analysis in a population-based association study, haplotypes must be determined by estimation, in the absence of family information or laboratory methods to establish phase. Here, test the accuracy of estimates of haplotype frequency and linkage disequilibrium by comparing estimated haplotypes generated with the expectation maximisation (EM) algorithm to haplotypes determined from Centre d'Etude Polymorphisme Humain (CEPH) pedigree data. To do this, they have characterised haplotypes comprising alleles at 11 biallelic loci in four chemokine receptor genes (CCR3, CCR2, CCR5 and CCRL2), which span 150 kb on chromosome 3p21, and haplotyes of nine biallelic loci in six chemokine genes [MCP-1(CCL2), Eotaxin(CCL11), RANTES(CCL5), MPIF-1(CCL23), PARC(CCL18) and MIP-1α(CCL3) ] on chromosome 17q11-12. Forty multi-generation CEPH families, totalling 489 individuals, were genotyped by the TaqMan 5'-nuclease assay. Phased haplotypes and haplotypes estimated from unphased genotypes were compared in 103 grandparents who were assumed to have mated at random.
For the 3p21 single nucleotide polymorphism (SNP) data, haplotypes determined by pedigree analysis and haplotypes generated by the EM algorithm were nearly identical. Linkage disequilibrium, measured by the D' statistic, was nearly maximal across the 150 kb region, with complete disequilibrium maintained at the extremes between CCR3-Y17Y and CCRL2-1243V. D'-values calculated from estimated haplotypes on 3p21 had high concordance with pairwise comparisons between pedigree-phased chromosomes. Conversely, there was less agreement between analyses of haplotype frequencies and linkage disequilibrium using estimated haplotypes when compared with pedigree-phased haplotypes of SNPs on chromosome 17q11-12. These results suggest that, while estimations of haplotype frequency and linkage disequilibrium may be relatively simple in the 3p21 chemokine receptor cluster in population samples, the more complex environment on chromosome 17q11-12 will require a higher resolution haplotype analysis.
chemokine; SNP; haplotype estimation; pedigree analysis; linkage disequilibrium
Chromosome 3p21–22 harbors two clusters of chemokine receptor genes, several of which serve as major or minor coreceptors of HIV-1. Although the genetic association of CCR5 and CCR2 variants with HIV-1 pathogenesis is well known, the role of variation in other nearby chemokine receptor genes remain unresolved. We genotyped exonic single nucleotide polymorphisms (SNPs) in chemokine receptor genes: CCR3, CCRL2, and CXCR6 (at 3p21) and CCR8 and CX3CR1 (at 3p22), the majority of which were non-synonymous. The individual SNPs were tested for their effects on disease progression and outcomes in five treatment-naïve HIV-1/AIDS natural history cohorts. In addition to the known CCR5 and CCR2 associations, significant associations were identified for CCR3, CCR8, and CCRL2 on progression to AIDS. A multivariate survival analysis pointed to a previously undetected association of a non-conservative amino acid change F167Y in CCRL2 with AIDS progression: 167F is associated with accelerated progression to AIDS (RH = 1.90, P = 0.002, corrected). Further analysis indicated that CCRL2-167F was specifically associated with more rapid development of pneumocystis pneumonia (PCP) (RH = 2.84, 95% CI 1.28–6.31) among four major AIDS–defining conditions. Considering the newly defined role of CCRL2 in lung dendritic cell trafficking, this atypical chemokine receptor may affect PCP through immune regulation and inducing inflammation.
Human chemokine receptors are cell surface proteins that may be utilized by HIV-1 for entry into host cells. DNA variation in the HIV-1 major coreceptor CCR5 affects HIV-1 infection and progression. This study comprehensively assesses the role of genetic variation of multiple chemokine receptor genes clustered in the chromosome 3p21 and 3p22 on HIV-1 disease outcomes in HIV-1 natural history cohorts. The multivariate survival analyses identified functional variants that altered disease progression rate in CCRL2, CCR3, and CCR8. CCRL2-F167Y affects the rate to AIDS development through a specific protection against pneumocystis pneumonia (PCP), a common AIDS–defining condition. Our study identified this atypical chemokine receptor CCRL2 as a key factor involved in PCP, possibly through inducing inflammation in the lung.
During pulmonary mycobacterial infection, there is increased trafficking of dendritic cells from the lungs to the draining lymph nodes. We hypothesized that ongoing mycobacterial infection would modulate recruitment and activation of antigen-specific naive CD4+ T cells after airway antigen challenge. BALB/c mice were infected by aerosol with Mycobacterium bovis BCG. At peak bacterial burden in the lungs (4 to 6 weeks postinfection), carboxy-fluorescein diacetate succinimidyl ester-labeled naive ovalbumin-specific DO11.10 T cells were adoptively transferred into infected and uninfected mice. Recipient mice were challenged intranasally with soluble ovalbumin (OVA), and OVA-specific T-cell responses were measured in the lungs, draining mediastinal lymph nodes (MLN), and spleens. OVA challenge resulted in increased activation and proliferation of OVA-specific T cells in the draining MLN of both infected and uninfected mice. However, only BCG-infected mice had prominent OVA-specific T-cell activation, proliferation, and Th1 differentiation in the lungs. BCG infection caused greater distribution of airway OVA to pulmonary dendritic cells and enhanced presentation of OVA peptide by lung CD11c+ cells. Together, these data suggest that an existing pulmonary mycobacterial infection alters the phenotype of lung dendritic cells so that they can activate antigen-specific naive CD4+ T cells in the lungs in response to airway antigen challenge.
Chemokines are a class of inflammatory mediators which main function is to direct leukocyte migration through the binding to G protein-coupled receptors (GPCRs). In addition to these functional, signal-transducing chemokine receptors other types of receptors belonging to the chemokine GPCR family were identified. They are called atypical or decoy chemokine receptors because they bind and degrade chemokines but do not transduce signals or activate cell migration. Here there is the summary of two recent papers that identified other nonchemotactic chemokine receptors: the Duffy antigen receptor for chemokines (DARC) that mediates trancytosis of chemokines from tissue to vascular lumen promoting chemokine-mediated leukocyte transmigration and chemokine (CC motif) receptor-like 2 (CCRL2) that neither internalizes its ligands nor transduces signals but presents bound ligands to functional signaling receptors improving their activity. Collectively these nonchemotactic chemokine receptors do not directly induce cell migration, but appear nonetheless to play a nonredundant role in leukocyte recruitment by shaping the chemoattractant gradient, either by removing, transporting or concentrating their cognate ligands.
Chemokine; chemokine receptor; leukocyte recruitment; chemotaxis; transcytosis
Salmonella enterica subspecies enterica serovar Typhimurium, commonly called S. Typhimurium, can cause intestinal infections in humans and various animal species such as swine. To analyze the host response to Salmonella infection in the pig we used an in vivo gut loop model, which allows the analysis of multiple immune responses within the same animal. Four jejunal gut-loops were each inoculated with 3×108 cfu of S. Typhimurium in 3 one-month-old piglets and mRNA expressions of various cytokines, chemokines, transcription factors, antimicrobial peptides, toll like and chemokine receptors were assessed by quantitative real-time PCR in the Peyer’s patch and the gut wall after 24 h. Several genes such as the newly cloned CCRL1/CCX-CKR were assessed for the first time in the pig at the mRNA level. Pro-inflammatory and T-helper type-1 (Th1) cytokine mRNA were expressed at higher levels in infected compared to non-infected control loops. Similarly, some B cell activation genes, NOD2 and toll like receptor 2 and 4 transcripts were more expressed in both tissues while TLR5 mRNA was down-regulated. Interestingly, CCL25 mRNA expression as well as the mRNA expressions of its receptors CCR9 and CCRL1 were decreased both in the Peyer’s patch and gut wall suggesting a potential Salmonella strategy to reduce lymphocyte homing to the intestine. In conclusion, these results provide insight into the porcine innate mucosal immune response to infection with entero-invasive microorganisms such as S. Typhimurium. In the future, this knowledge should help in the development of improved prophylactic and therapeutic approaches against porcine intestinal S. Typhimurium infections.
Salmonella; Th1 cytokines; pig; CCRL1; pattern recognition receptor
Chemerin is a proinflammatory plasma protein that binds to the GPCR ChemR23/CMKLR1 on macrophages and plasmacytoid dendritic cells, and promotes chemotaxis. An orphan GPCR, CCRL2, has now been identified as an additional receptor for chemerin, providing a unique mechanism by which chemerin enhances inflammation. Furthermore, because recent data shows that chemerin-derived peptides possess antiinflammatory properties, chemerin may be involved in both the initiation and resolution of inflammation.
Initiation of an adaptive cellular immune response depends on intimate interactions with antigen-presenting cells and naive T lymphocytes. We have previously reported that activation of naive Mycobacterium tuberculosis-specific CD4+ T cells depends on dendritic cell (DC) transport of live bacteria from the lungs to the mediastinal lymph node (MDLN). Since the migratory paths of DCs are largely governed by the chemokine receptor CCR7, which is expressed on DCs upon maturation by proinflammatory stimuli, we examined the quantitative contribution of CCR7-dependent DC migration in the context of tuberculosis, and found that early trafficking of DCs from the lungs to the MDLN depended on CCR7-mediated signaling, but alternative mechanism(s) are employed later in infection. Impaired migration of DCs in CCR7−/− mice resulted in delayed dissemination of bacteria to MDLN and spleen, and in delayed kinetics of activation of adoptively-transferred Ag85B-specific CD4+ T cells. Furthermore, in contrast to control mice, we found that naive Ag85B-specific CD4+ T cells are activated to proliferate in the lungs of CCR7−/− mice and, when infected with higher doses of bacteria, resistance to M. tuberculosis infection in CCR7−/− mice is compromised compared to wild type mice.
dendritic cells; bacterial; chemokines; cell activation; cell trafficking
The aim of this study was to investigate whether dendritic cells (DCs) can induce sensitization to aeroallergen in a mouse model of allergic asthma. Ovalbumin-pulsed (OVA-pulsed) or unpulsed myeloid DCs that were injected into the airways of naive mice migrated into the mediastinal lymph nodes. When challenged 2 weeks later with an aerosol of OVA, activated CD4 and CD8 lymphocytes, eosinophils, and neutrophils were recruited to the lungs of actively immunized mice. These CD4+ lymphocytes produced predominantly IL-4 and IL-5 but also IFN-γ, whereas CD8+ lymphocytes produced predominantly IFN-γ. Histological analysis revealed perivascular and peribronchial eosinophilic infiltrates and goblet cell hyperplasia. Studies in IL-4–/– and CD28–/– mice revealed that production of IL-4 by host cells and provision of costimulation to T cells by DCs were critical for inducing the response. Lung CD4+ T cells strongly expressed the Th2 marker T1/ST2, and signaling through this molecule via a ligand expressed on DCs was essential for the establishment of airway eosinophilia. These data demonstrate that DCs in the airways induce sensitization to inhaled antigen and that molecules expressed on the surface of these cells are critical for the development of Th2-dependent airway eosinophilia.
This study reveals a function of endogenous galectin-3, an animal lectin recognizing β-galactosides, in regulating dendritic cell motility both in vitroand in vivo,which to our knowledge is unreported. First, galectin-3-deficient (gal3−/−) bone marrow-derived dendritic cells exhibited defective chemotaxis compared to gal3+/+ cells. Second, cutaneous dendritic cells in gal3−/− mice displayed reduced migration to draining lymph nodes upon hapten stimulation compared to gal3+/+ mice. Moreover, gal3−/− mice were impaired in the development of contact hypersensitivity relative to gal3+/+ mice in response to a hapten, a process in which dendritic cell trafficking to lymph nodes is critical. In addition, defective signaling was detected in gal3−/− cells upon chemokine receptor activation. By immunofluorescence microscopy, we observed that galectin-3 is localized in membrane ruffles and lamellipodia in stimulated dendritic cells and macrophages. Furthermore, galectin-3 was enriched in lipid raft domains under these conditions. Finally, we determined that ruffles on gal3−/− cells contained structures with lower complexity compared to gal3+/+ cells. In view of the participation of membrane ruffles in signal transduction and cell motility, we conclude that galectin-3 regulates cell migration by functioning at these structures.
Eosinophil accumulation is a distinctive feature of lung allergic inflammation. Here, we have used a mouse model of OVA (ovalbumin)-induced pulmonary eosinophilia to study the cellular and molecular mechanisms for this selective recruitment of eosinophils to the airways. In this model there was an early accumulation of infiltrating monocytes/macrophages in the lung during the OVA treatment, whereas the increase in infiltrating T-lymphocytes paralleled the accumulation of eosinophils. The kinetics of accumulation of these three leukocyte subtypes correlated with the levels of mRNA expression of the chemokines monocyte chemotactic peptide-1/JE, eotaxin, and RANTES (regulated upon activation in normal T cells expressed and secreted), suggesting their involvement in the recruitment of these leukocytes. Furthermore, blockade of eotaxin with specific antibodies in vivo reduced the accumulation of eosinophils in the lung in response to OVA by half. Mature CD4+ T-lymphocytes were absolutely required for OVA-induced eosinophil accumulation since lung eosinophilia was prevented in CD4+-deficient mice. However, these cells were neither the main producers of the major eosinophilic chemokines eotaxin, RANTES, or MIP-1alpha, nor did they regulate the expression of these chemokines. Rather, the presence of CD4+ T cells was necessary for enhancement of VCAM-1 (vascular cell adhesion molecule-1) expression in the lung during allergic inflammation induced by the OVA treatment. In support of this, mice genetically deficient for VCAM-1 and intercellular adhesion molecule-1 failed to develop pulmonary eosinophilia. Selective eosinophilic recruitment during lung allergic inflammation results from a sequential accumulation of certain leukocyte types, particularly T cells, and relies on the presence of both eosinophilic chemoattractants and adhesion receptors.
We previously established that the inhibitory receptor LILRB4 mitigates LPS-induced, neutrophil-dependent pathologic effector mechanisms in inflammation. We now report that LILRB4 on dendritic cells (DCs) counterregulates development of an adaptive Th2 immune response and ensuing inflammation in a model of allergic pulmonary inflammation initiated by inhalation sensitization with OVA and LPS followed by airway challenge with OVA. We found that Lilrb4−/− mice had significantly exacerbated eosinophilic pulmonary inflammation as assessed in bronchoalveolar lavage and lung tissue, as well as elevated levels of OVA-specific IgE and Th2 cytokines produced by OVA-restimulated lymph node cells. LILRB4 was preferentially expressed on MHC class IIhighCD86high OVA-bearing DCs in lung-draining lymph nodes after sensitization or challenge. Moreover, the lymph nodes of Lilrb4−/− mice had significantly more of these mature DCs after challenge with OVA, which was accompanied by significantly more IL-4-producing lymphocytes, compared with Lilrb4+/+ mice. Sensitization of naïve Lilrb4+/+ mice by transfer of OVA-LPS-pulsed Lilrb4−/− bone marrow-derived DCs (BMDCs) was sufficient to confer exacerbated allergic lung pathology upon challenge with OVA, compared with mice that received Lilrb4+/+ BMDCs. Our findings establish that maturation and migration of pulmonary DCs to lymph nodes in response to Ag and an innate immune stimulus is associated with upregulated expression of LILRB4. In addition, this receptor attenuates the number of these mature DCs and attendant IL-4-producing lymphocytes in the lymph nodes, and accordingly, the ability of DCs to elicit pathologic Th2 pulmonary inflammation.
Dendritic Cells; Inflammation; Allergy; Lung
Aspergillus fumigatus, a ubiquitous fungus, causes invasive disease in immunocompromised humans. Although monocytes and antigen-specific CD4 T cells contribute to defense against inhaled fungal spores, how these cells interact during infection remains undefined. Investigating the role of inflammatory monocytes and monocyte-derived dendritic cells during fungal infection, we find that A. fumigatus infection induces an influx of chemokine receptor CCR2- and Ly6C-expressing inflammatory monocytes into lungs and draining lymph nodes. Depletion of CCR2+ cells reduced A. fumigatus conidial transport from lungs to draining lymph nodes, abolished CD4 T cell priming following respiratory challenge, and impaired pulmonary fungal clearance. In contrast, depletion of CCR2+Ly6Chi monocytes during systemic fungal infection did not prevent CD4 T cell priming in the spleen. Our findings demonstrate that pulmonary CD4 T cell responses to inhaled spores require CCR2+Ly6Chi monocytes and their derivatives, revealing a compartmentally restricted function for these cells in adaptive respiratory immune responses.
Chemokine receptor (CCR) 5 is expressed on dendritic cells, macrophages, CD8 cells, memory CD4 T cells, and stromal cells, and is frequently used as a marker of T helper type 1 cells. Interventions that abrogate CCR5 or interfere with its ligand binding have been shown to alter T helper type 2–induced inflammatory responses. The role of CCR5 on allergic airway responses is not defined. CCR5-deficient (CCR5−/−) and wild-type (CCR5+/+) mice were sensitized and challenged with ovalbumin (OVA) and allergic airway responses were monitored 48 hours after the last OVA challenge. Cytokine levels in lung cell culture supernatants were also assessed. CCR5−/− mice showed significantly lower airway hyperresponsiveness (AHR) and lower numbers of total cells, eosinophils, and lymphocytes in bronchoalveolar lavage (BAL) fluid compared with CCR5+/+ mice after sensitization and challenge. The levels of IL-4 and IL-13 in BAL fluid of CCR5−/− mice were lower than in CCR5+/+ mice. Decreased numbers of lung T cells were also detected in CCR5−/− mice after sensitization and challenge. Transfer of OVA-sensitized T cells from CCR5+/+, but not transfer of CCR5−/− cells, into CCR5−/− mice restored AHR and numbers of eosinophils in BAL fluid after OVA challenge. Accordingly, the numbers of airway-infiltrating donor T cells were significantly higher in the recipients of CCR5+/+ T cells. Taken together, these data suggest that CCR5 plays a pivotal role in allergen-induced AHR and airway inflammation, and that CCR5 expression on T cells is essential to the accumulation of these cells in the airways.
rodent; T cells; cytokines; chemokines; lung
There is growing evidence that chemokines and their receptors regulate the movement and interaction of antigen-presenting cells such as dendritic cells (DCs) and T cells. We tested the hypothesis that the CC chemokine receptor (CCR)2 and CCR5 and the chemokine macrophage inflammatory protein (MIP)-1α, a ligand for CCR5, influence DC migration and localization. We found that deficiency of CCR2 but not CCR5 or MIP-1α led to distinct defects in DC biology. Langerhans cell (skin DC) density in CCR2-null mice was normal, and their ability to migrate into the dermis was intact; however, their migration to the draining lymph nodes was markedly impaired. CCR2-null mice had lower numbers of DCs in the spleen, and this was primarily due to a reduction in the CD8α1 T helper cell type 1 (Th1)-inducing subset of DCs. Additionally, there was a block in the Leishmania major infection–induced relocalization of splenic DCs from the marginal zone to the T cell areas. We propose that these DC defects, in conjunction with increased expression of B lymphocyte chemoattractant, a B cell–specific chemokine, may collectively contribute to the striking B cell outgrowth and Th2 cytokine–biased nonhealing phenotype that we observed in CCR2-deficient mice infected with L. major. This disease phenotype in mice with an L. major–resistant genetic background but lacking CCR2 is strikingly reminiscent of that observed typically in mice with an L. major–susceptible genetic background. Thus, CCR2 is an important determinant of not only DC migration and localization but also the development of protective cell-mediated immune responses to L. major.
infectious immunity; chemokine; cytokine; knockout; T helper cell
The CC chemokine receptor 3 (CCR3) is expressed by eosinophils, mast cells, and Th2 cells. We used CCR3–/– mice to assess the role of CCR3 in a murine model of allergic skin inflammation induced by repeated epicutaneous sensitization with ovalbumin (OVA), and characterized by eosinophil skin infiltration, local expression of Th2 cytokines, and airway hyperresponsiveness (AHR) to inhaled antigen. Eosinophils and the eosinophil product major basic protein were absent from the skin of sham and OVA-sensitized CCR3–/– mice. Mast cell numbers and expression of IL-4 mRNA were normal in skin of CCR3–/– mice, suggesting that CCR3 is not important for infiltration of the skin by mast cells and Th2 cells. CCR3–/– mice produced normal levels of OVA-specific IgE, and their splenocytes secreted normal amounts of IL-4 and IL-5 following in vitro stimulation with OVA, indicating effective generation of systemic Th2 helper responses. Recruitment of eosinophils to lung parenchyma and bronchoalveolar lavage (BAL) fluid was severely impaired in CCR3–/– mice, which failed to develop AHR to methacholine following antigen inhalation. These results suggest that CCR3 plays an essential role in eosinophil recruitment to the skin and the lung and in the development of AHR.
Dendritic cells are potent antigen-presenting cells involved in the initiation of immune responses. The trafficking of these cells to tissues and lymph nodes is mediated by members of the chemokine family. Recently, a novel CC chemokine known as MIP-3α or liver and activation-regulated chemokine has been identified from the EMBL/GenBank/DDBJ expressed sequence tag database. In the present study, we have shown that the messenger RNA for MIP-3α is expressed predominantly in inflamed and mucosal tissues. MIP-3α produced either synthetically or by human embryonic kidney 293 cells is chemotactic for CD34+-derived dendritic cells and T cells, but is inactive on monocytes and neutrophils. MIP-3α was unable to displace the binding of specific CC or CXC chemokines to stable cell lines expressing their respective high affinity receptors, namely CCR1–5 and CXCR1 and CXCR2, suggesting that MIP-3α acts through a novel CC chemokine receptor. Therefore, we used degenerate oligonucleotide-based reverse transcriptase PCR to identify candidate MIP-3α receptors in lung dendritic cells. Our results show that the orphan receptor known as GCY-4, CKRL-3, or STRL-22 is a specific receptor for MIP-3α, and that its activation leads to pertussis toxin–sensitive and phospholipase C–dependent intracellular Ca2+ mobilization when it is expressed in HEK 293 cells.
Trafficking of corneal antigen-presenting cells (APC) to draining lymph nodes (LN) is critical in triggering immune responses. However, very little is known about the molecular regulation of this pathway. We investigated the expression and function of the chemokine receptor CCR7 in mediating corneal APC migration in inflammation.
Expression of CCR7 and its ligands, CCL21 and CCL19, in the normal and inflamed corneas was analyzed by RT-PCR and immunofluorescence staining. The phenotype of CCR7-expressing cells was identified by double-staining with different cell surface markers. To trace the trafficking of APC to draining LN, we injected corneal grafts with Alexa488-conjugated ovalbumin (OVA) and transplanted to syngeneic recipients. CCR7 expression on the Alexa488-conjugated OVA+ cells in the ipsilateral draining LN was analyzed by flow cytometry. To determine the functional role of CCR7, we injected anti-CCL21 neutralizing antibody subconjunctivally after corneal transplantation and analyzed changes in numbers of OVA+ cells in the draining LN. Each experiment was repeated at least three times.
Both CCR7 and its ligand CCL21 were significantly upregulated in inflamed corneas as measured by RT-PCR and immunofluorescence staining. CCR7+ cells were detected especially in the corneal periphery near LYVE-1+ lymphatic vessels. CCR7+ cells were universally CD11b+CD11c+, and a majority were major histocompatibility complex class II positive, suggesting a monocytic dendritic cell lineage and a relative state of maturation. Forty-eight h after syngeneic transplantation with OVA-loaded grafts, CCR7 expression was detected on the OVA+ cells in both the host corneal beds and the draining LN. Local administration of anti-CCL21 led to a significant suppression in the flow of OVA+CD11c+ cells to the draining LN.
These data suggest that in inflammation, APC expressing CCR7 on their cell surface interact with CCL21 to facilitate their migration from the cornea to draining LN via afferent lymphatics.
Patients with atopic dermatitis (AD) often suffer from food allergy and develop flares upon skin contact with food allergens. However, it is unclear whether T cells sensitized to allergens in the gut promote this skin inflammation. To address this question, we orally immunized WT mice and mice lacking the skin-homing chemokine receptor Ccr4 (Ccr4–/– mice) with OVA and then challenged them epicutaneously with antigen. Allergic skin inflammation developed in the WT mice but not in the mutants and was characterized by epidermal thickening, dermal infiltration by eosinophils and CD4+ T cells, and upregulation of Th2 cytokines. T cells purified from mesenteric lymph nodes (MLNs) of orally immunized WT mice transferred allergic skin inflammation to naive recipients cutaneously challenged with antigen, but this effect was lost in T cells purified from Ccr4–/– mice. In addition, the ability of adoptively transferred OVA-activated T cells to home to the skin following cutaneous OVA challenge was ablated in mice that lacked lymph nodes. These results indicate that cutaneous exposure to food antigens can reprogram gut-homing effector T cells in LNs to express skin-homing receptors, eliciting skin lesions upon food allergen contact in orally sensitized AD patients.
Dendritic cells (DC) migrate from sites of inflammation to lymph nodes to initiate primary immune responses, but the molecular mechanisms by which DC are replenished in the lungs during ongoing pulmonary inflammation are unknown. To address this question, we analyzed the secondary pulmonary immune response of Ag-primed mice to intratracheal challenge with the particulate T cell-dependent Ag sheep erythrocytes (SRBC). We studied wild-type C57BL/6 mice and syngeneic gene-targeted mice lacking either both endothelial selectins (CD62E and CD62P), or the chemokine receptors CCR2 or CCR6. DC, defined as non-autofluorescent, MHC class II+CD11cmod cells, were detected in blood, enzyme-digested minced lung, and bronchoalveolar lavage fluid using flow cytometry and immunohistology. Compared with control mice, Ag challenge increased the frequency and absolute numbers of DC, peaking at day 1 in peripheral blood (6.5-fold increase in frequency), day 3 in lung mince (20-fold increase in total DC), and day 4 in bronchoalveolar lavage fluid (55-fold increase in total DC). Most lung DC expressed CD11c, CD11b, and low levels of MHC class II, CD40, CD80, and CD86, consistent with an immature myeloid phenotype. DC accumulation depended in part upon CCR2 and CCR6, but not endothelial selectins. Thus, during lung inflammation, immature myeloid DC from the bloodstream replace emigrating immature DC and transiently increase total intrapulmonary APC numbers. Early DC recruitment depends in part on CCR2 to traverse vascular endothelium, plus CCR6 to traverse alveolar epithelium. The recruitment of circulating immature DC represents a potential therapeutic step at which to modulate immunological lung diseases.