CCL17 and CCL22 induce CCR4 receptor expression and promote cytokine-induced killer cells migration.

Recently, cytokine-induced killer (CIK) cells have been shown to possess effective cytotoxic activity against some tumor cells both in vitro and in clinical research. Furthermore, dendritic cell-activated CIK (DC-CIK) cells display significantly increased antitumor activity compared to unstimulated CIK cells. Study findings indicate DC cells can secrete chemokine C-C motif ligand 17 (CCL17) and chemokine C-C motif ligand 22 (CCL22) with a common receptor molecule, C-C chemokine receptor type-4(CCR4). CCL17 and CCL22 levels were measured by ELISA from CIK cell culture supernatants and the expression of CCR4 on CIK and DC-CIK cells was analyzed by flow cytometry. Through Migration and Killing assays, further analyzed the effects of the altered expression levels of CCR4 on the chemotactic ability and the tumor-killing efficiency of CIK cells. We found markedly increased CCL17 and CCL22 in supernatants of DC-CIK co-cultures. Similarly, the expression of CCR4 was also increased on CIK cells in these co-cultures. Further, the stimulation of CCL17 and CCL22 increased expression of the CCR4 and enhanced the migratory capacity and antitumor efficacy of CIK cells. Simultaneously, similar effects had achieved by transfecting the CCR4 gene into CIK cells. DC cells may promote the expression of CCR4 on CIK cells by secreting CCL17 and CCL22, thereby promoting infiltration of DC-CIK cells into the tumor microenvironment, and exerting stronger antitumor activity than CIK cells.

Epstein-Barr virus-positive pyothorax-associated lymphoma expresses CCL17 and CCL22 chemokines that attract CCR4-expressing regulatory T cells.

Epstein-Barr virus (EBV)-positive diffuse large B-cell lymphomas associated with chronic inflammation (DLBCL-CI) develop in patients with chronic inflammation but without any predisposing immunodeficiency. Given the expression of the EBV latent genes, DLBCL-CI should have mechanisms for evasion of host antitumor immunity. EBV-positive pyothorax-associated lymphoma (PAL) is a prototype of DLBCL-CI and may provide a valuable model for the study of immune evasion by DLBCL-CI. This study demonstrates that PAL cell lines express and secrete CCL17 and/or CCL22 chemokines, the ligands of C-C motif chemokine receptor 4 (CCR4), in contrast to EBV-negative DLBCL cell lines. Accordingly, culture supernatants of PAL cell lines efficiently attracted CCR4-positive regulatory T (Treg) cells in human peripheral blood mononuclear cells. PAL cells injected into mice also attracted CCR4-expressing Treg cells. Furthermore, this study confirmed that CCR4-expressing Treg cells were abundantly present in primary PAL tissues. Collectively, these findings provide new insight into the mechanisms of immune evasion by PAL, and further studies are warranted on whether such mechanisms eventually lead to the development of DLBCL-CI.

CCR4 Expression in Tumor-Infiltrating Regulatory T Cells in Patients with Squamous Cell Carcinoma of the Lung: A Prognostic Factor for Relapse and Survival.

To clarify the prognostic impact of tumor-infiltrating effector regulatory T cells (eTregs) in non-small cell lung cancer (NSCLC), eTregs were evaluated by immunohistochemical detection of CCR4 and Foxp3 in 108 consecutive surgical NSCLC tumors. Multivariate analysis showed that a high ratio of CCR4+ eTregs to total Tregs (≥40%) was the only independent risk factor for relapse-free survival (odds ratio [OR]: 6.54, 95% confidence interval: 1.67-25.7, p = .007) and overall survival (OR: 3.76, p = .037) in lung squamous cell carcinoma (SqCC). These results highlight the prognostic importance of the balance of tumor-infiltrating Tregs in resected lung SqCC.

Complement-Opsonized HIV-1 Alters Cross Talk Between Dendritic Cells and Natural Killer (NK) Cells to Inhibit NK Killing and to Upregulate PD-1, CXCR3, and CCR4 on T Cells.

Dendritic cells (DCs), natural killer (NK) cells, and T cells play critical roles during primary HIV-1 exposure at the mucosa, where the viral particles become coated with complement fragments and mucosa-associated antibodies. The microenvironment together with subsequent interactions between these cells and HIV at the mucosal site of infection will determine the quality of immune response that ensues adaptive activation. Here, we investigated how complement and immunoglobulin opsonization influences the responses triggered in DCs and NK cells, how this affects their cross talk, and what T cell phenotypes are induced to expand following the interaction. Our results showed that DCs exposed to complement-opsonized HIV (C-HIV) were less mature and had a poor ability to trigger IFN-driven NK cell activation. In addition, when the DCs were exposed to C-HIV, the cytotolytic potentials of both NK cells and CD8 T cells were markedly suppressed. The expression of PD-1 as well as co-expression of negative immune checkpoints TIM-3 and LAG-3 on PD-1 positive cells were increased on both CD4 as well as CD8 T cells upon interaction with and priming by NK-DC cross talk cultures exposed to C-HIV. In addition, stimulation by NK-DC cross talk cultures exposed to C-HIV led to the upregulation of CD38, CXCR3, and CCR4 on T cells. Together, the immune modulation induced during the presence of complement on viral surfaces is likely to favor HIV establishment, dissemination, and viral pathogenesis.

CCR4 promotes metastasis via ERK/NF-κB/MMP13 pathway and acts downstream of TNF-α in colorectal cancer.

Chemokines and chemokine receptors are causally involved in the metastasis of human malignancies. As a crucial chemokine receptor for mediating immune homeostasis, however, the role of CCR4 in colorectal cancer (CRC) remains unknown. In this study, we found that high expression of CCR4 in CRC tissues was correlated with shorter overall survival and disease free survival. In vitro and in vivo experiments revealed that silencing CCR4 attenuated the invasion and metastasis of CRC cells, whereas ectopic overexpression of CCR4 contributed to the forced metastasis of these cells. We further demonstrated that matrix metalloproteinase 13 (MMP13) played an important role in CCR4-mediated cancer cell invasion, which is up-regulated by ERK/NF-κB signaling. Positive correlation between CCR4 and MMP13 expression was also observed in CRC tissues. Moreover, our investigations showed that the level of CCR4 could be induced by TNF-α dependent of NF-κB activation in CRC cells. CCR4 might be implicated in TNF-α-regulated cancer cells metastasis. Combination of CCR4 and TNF-α is a more powerful prognostic marker for CRC patients. These findings suggest that CCR4 facilitates metastasis through ERK/NF-κB/MMP13 signaling and acts as a downstream target of TNF-α. CCR4 inhibition may be a promising therapeutic option for suppressing CRC metastasis.

Versatility of using major histocompatibility complex class II dextramers for derivation and characterization of antigen-specific, autoreactive T cell hybridomas.

Antigen-specific, T cell hybridomas are useful to study the cellular, molecular and functional events, but their generation is a lengthy process. Thus, there is a need to develop robust methods to generate the hybridoma clones rapidly in a short period of time. To this end, we have demonstrated a novel approach using major histocompatibility complex (MHC) class II dextramers to generate T cell hybridomas for an autoantigen, proteolipid protein (PLP) 139-151. Using MHC class II dextramers assembled with PLP 139-151 as screening and sorting tools, we successfully obtained mono antigen-specific clones within seven to eight weeks. In conjunction with other T cell markers, dextramers permitted phenotypic characterization of hybridoma clones for their antigen specificity in a single step by flow cytometry. Importantly, we achieved successful fusions using dextramer(+) cells sorted by flow cytometry as a starting population, resulting in direct identification of multiple antigen-specific clones. Characterization of selected clones led us to identify chemokine receptor, CCR4(+) to be expressed consistently, but their cytokine-producing ability was variable. Our work provides a proof-of principle that the antigen-specific, CD4 T cell hybridoma clones can be generated directly using MHC class II dextramers. The availability of hybridoma clones that bind dextramers may serve as useful tools for various in vitro and in vivo applications.

Increase in activated Treg in TIL in lung cancer and in vitro depletion of Treg by ADCC using an antihuman CCR4 mAb (KM2760).

INTRODUCTION: Tregs infiltrate tumors and inhibit immune responses against them. METHODS: We investigated subpopulations of Foxp3 CD4 T cells previously defined by Miyara et al. (Immunity 30, 899-911, 2009) in peripheral blood mononuclear cells (PBMCs) and tumor infiltrating lymphocytes (TILs) in lung cancer. We also showed that Tregs in healthy donors that express CCR4 could be efficiently eliminated in vitro by cotreatment with antihuman (h) CCR4 mAb (KM2760) and NK cells. RESULTS: In lung cancer, the number of activated/effector Tregs and non-Tregs, but not resting/naive Tregs, was increased in TILs compared with the number of those cells in PBMCs. The non-Treg population contained Th2 and Th17. CCR4 expression on activated/effector Tregs and non-Tregs in TILs was down-regulated compared with that on those cells in PBMCs. Chemokinetic migration of CD25 CD4 T cells containing the Treg population sorted from the PBMCs of healthy donors to CCL22/MDC was abrogated by pretreatment with anti-hCCR4 mAb (KM2760). The inhibitory activity of CD25 CD127 CD4 Tregs on the proliferative response of CD4 and CD8 T cells stimulated with anti-CD3/CD28 coated beads was abrogated by adding an anti-hCCR4 mAb (KM2760) and CD56 NK cells to the culture. CONCLUSIONS: The findings suggested the CCR4 on activated/effector Tregs and non-Tregs was functionally involved in the chemokinetic migration and accumulation of those cells to the tumor site. In vitro findings of efficient elimination of Tregs may give the basis for implementation of a clinical trial to investigate Treg depletion by administration of an anti-hCCR4 mAb to solid cancer patients.

A mutation in cnot8, component of the Ccr4-not complex regulating transcript stability, affects expression levels of developmental regulators and reveals a role of Fgf3 in development of caudal hypothalamic dopaminergic neurons.

While regulation of the activity of developmental control genes at the transcriptional level as well as by specific miRNA-based degradation are intensively studied, little is known whether general cellular mechanisms controlling mRNA decay may contribute to differential stability of mRNAs of developmental control genes. Here, we investigate whether a mutation in the deadenylation dependent mRNA decay pathway may reveal differential effects on developmental mechanisms, using dopaminergic differentiation in the zebrafish brain as model system. In a zebrafish genetic screen aimed at identifying genes controlling dopaminergic neuron development we isolated the m1061 mutation that selectively caused increased dopaminergic differentiation in the caudal hypothalamus, while other dopaminergic groups were not affected. Positional cloning revealed that m1061 causes a premature stop codon in the cnot8 open reading frame. Cnot8 is a component of the Ccr4-Not complex and displays deadenylase activity, which is required for removal of the poly (A) tail in bulk mRNA turnover. Analyses of expression of developmental regulators indicate that loss of Cnot8 activity results in increased mRNA in situ hybridization signal levels for a subset of developmental control genes. We show that in the area of caudal hypothalamic dopaminergic differentiation, mRNA levels for several components of the FGF signaling pathway, including Fgf3, FGF receptors, and FGF target genes, are increased. Pharmacological inhibition of FGF signaling or a mutation in the fgf3 gene can compensate the gain of caudal hypothalamic dopaminergic neurons in cnot8m1061 mutants, indicating a role for Fgf3 in control of development of this dopaminergic population. The cnot8m1061 mutant phenotype provides an in vivo system to study roles of the Cnot8 deadenylase component of the mRNA decay pathway in vertebrate development. Our data indicate that attenuation of Cnot8 activity differentially affects mRNA levels of developmental control genes.

Transcriptional profile of tuberculosis antigen-specific T cells reveals novel multifunctional features.

In latent tuberculosis infection (LTBI) spread of the bacteria is contained by a persistent immune response, which includes CD4(+) T cells as important contributors. In this study we show that TB-specific CD4(+) T cells have a characteristic chemokine expression signature (CCR6(+)CXCR3(+)CCR4(-)), and that the overall number of these cells is significantly increased in LTBI donors compared with healthy subjects. We have comprehensively characterized the transcriptional signature of CCR6(+)CXCR3(+)CCR4(-) cells and found significant differences to conventional Th1, Th17, and Th2 cells, but no major changes between healthy and LTBI donors. CCR6(+)CXCR3(+)CCR4(-) cells display lineage-specific signatures of both Th1 and Th17 cells, but also have a unique gene expression program, including genes associated with susceptibility to TB, enhanced T cell activation, enhanced cell survival, and induction of a cytotoxic program akin to CTL cells. Overall, the gene expression signature of CCR6(+)CXCR3(+)CCR4(-) cells reveals characteristics important for controlling latent TB infections.

Higher serum CCL17 may be a promising predictor of acute exacerbations in chronic hypersensitivity pneumonitis.

BACKGROUND: Recent research has suggested that the Th1 and Th2 chemokine/cytokine axis contributes to the development of chronic hypersensitivity pneumonitis (HP). Acute exacerbations (AE) are significant factors in the prognosis of chronic HP. Little is known, however, about these biomarkers in association with AE in chronic HP patients. METHODS: Fifty-six patients with chronic HP were evaluated, including 14 patients during episodes of AE. Th1 mediators (C-X-C chemokine ligand [CXCL]10 and interferon [IFN]-γ), Th2 mediators (C-C chemokine ligand [CCL]17, interleukin-4, and interleukin-13), and pro-fibrotic mediator (transforming growth factor [TGF]-ß) were measured to evaluate the mediators as predictors of AE. C-C chemokine receptor (CCR)4 (receptor for CCL17)-positive lymphocytes were quantified in lung specimens. RESULTS: Serum CCL17 levels at baseline independently predicted the first episode of AE (HR, 72.0; 95% CI, 5.03-1030.23; p = 0.002). AE was significantly more frequent in the higher-CCL17 group (≥285 pg/ml) than in the lower-CCL17 group (<285 pg/ml) (log-rank test, p = 0.0006; 1-year incidence: higher CCL17 vs. lower CCL17, 14.3% vs. 0.0%). Serum CCL17 levels and CCR4-positive cells during episodes of AE were increased from the baseline (p = 0.01 and 0.031). CONCLUSIONS: Higher serum concentrations of CCL17 at baseline may be predictive of AE in patients with chronic HP, and CCL17 may contribute to the pathology of AE by inducing the accumulation of CCR4-positive lymphocytes in the lungs.

Bath-PUVA therapy decreases infiltrating CCR4-expressing tumor cells and regulatory T cells in patients with mycosis Fungoides.

BACKGROUND: Mycosis fungoides (MF) is a malignant lymphoma characterized by expansion of CD4(+) memory T-cell clones. Infiltrating cells express CCR4, which is attracted to CC chemokine ligands 17 and 22 (thymus and activation-regulated chemokine [TARC]/CCL17 and TARC/CCL22). Bath-psoralen plus ultraviolet A (PUVA) is effective against MF. In patients with psoriasis, bath-PUVA induces circulating regulatory T cells (Tregs), which suppress effector T cells. To understand the mechanisms in MF, we analyzed lesion-infiltrating cells before and after bath-PUVA therapy. PATIENTS AND METHODS: Thirteen patients with MF (12 stage IB, 1 stage III; mean age 69.2 years, range 35-87 years; 6 men, 7 women) were recruited. RESULTS: Immunohistochemical analysis revealed that lesion CCR4-positive (CCR4(+)) cells and Tregs significantly decreased from 105.1 ± 164.8 cells/10(-2) mm(2) to 31.4 ± 39.0 cells/10(-2) mm(2) and from 78.1 ± 67.8 cells/10(-2) mm(2) to 24.7 ± 25.0 cells/10(-2) mm(2), respectively. Serum TARC levels significantly correlated with infiltrating CD3(+) (r = 0.997), CCR4(+) (r = 0.991), and forkhead box P3-positive (Foxp3(+)) cells (r = 0.843). Circulating Tregs before bath-PUVA therapy were not significantly different from those in healthy volunteers. Bath-PUVA did not significantly change the percentage of circulating Tregs. CONCLUSIONS: Bath-PUVA decreased CCR4(+) cells and Tregs in MF lesions but did not induce circulating Tregs, which might suppress effector T cells. Direct effects through skin lesions might eliminate both pathogenetically relevant cells and Tregs. Systemic immunosuppression was not induced.

Human leukocyte antigen (HLA)-B*57:01-restricted activation of drug-specific T cells provides the immunological basis for flucloxacillin-induced liver injury.

UNLABELLED: The role of the adaptive immune system in adverse drug reactions that target the liver has not been defined. For flucloxacillin, a delay in the reaction onset and identification of human leukocyte antigen (HLA)-B*57:01 as a susceptibility factor are indicative of an immune pathogenesis. Thus, we characterize flucloxacillin-responsive CD4+ and CD8+ T cells from patients with liver injury and show that naive CD45RA+CD8+ T cells from volunteers expressing HLA-B*57:01 are activated with flucloxacillin when dendritic cells present the drug antigen. T-cell clones expressing CCR4 and CCR9 migrated toward CCL17 and CCL 25, and secreted interferon-gamma (IFN-γ), T helper (Th)2 cytokines, perforin, granzyme B, and FasL following drug stimulation. Flucloxacillin bound covalently to selective lysine residues on albumin in a time-dependent manner and the level of binding correlated directly with the stimulation of clones. Activation of CD8+ clones with flucloxacillin was processing-dependent and restricted by HLA-B*57:01 and the closely related HLA-B*58:01. Clones displayed additional reactivity against ß-lactam antibiotics including oxacillin, cloxacillin, and dicloxacillin, but not abacavir or nitroso sulfamethoxazole. CONCLUSION: This work defines the immune basis for flucloxacillin-induced liver injury and links the genetic association to the iatrogenic disease.

High-dose vitamin D3 supplementation is a requisite for modulation of skin-homing markers on regulatory T cells in HIV-infected patients.

Vitamin D(3) is known to have an effect on the immune function. We investigated the immunomodulatory capability of vitamin D(3) in HIV-infected patients and studied the expression of chemokine receptors on regulatory T cells (Treg). Vitamin D(3)-deficient HIV-1-seropositive subjects were treated with cholecalciferol (vitamin D(3)) at a dose of 800 IU daily for 3 months (n=9) or 25,000 IU weekly for 2 months (n=7). Peripheral blood mononuclear cells (PBMCs) were isolated and analyzed for skin-homing (CCR4 and CCR10) and gut-homing (CCR9 and integrin α(4)ß(7)) marker expression on Treg, by flow cytometry, before and after supplementation. Serum 25(OH)D(3) and parathyroid hormone (PTH) levels were determined at baseline and after the treatment period. Weekly doses of 25,000 IU cholecalciferol effectively achieved the optimal target serum 25(OH)D(3) concentration of >75 nmol/liter (30 ng/ml) in HIV-infected patients. High-dose cholecalciferol supplementation differentially influenced skin-homing markers on Treg with an increased level of CCR10 expression and while a reduction in CCR4 expression level was observed together with a lower percentage of Treg expressing CCR4. For both dosing regimens, there were no significant differences in the expression of gut-homing markers, CCR9, and integrin α(4)ß(7). High-dose vitamin D(3) supplementation is needed to reverse vitamin D(3) deficiency in HIV-infected individuals and this results in modulation of skin-homing markers but not gut-homing markers expression on Treg. At a standard dose of 800 IU/day, vitamin D(3) is not effective in achieving an optimal 25(OH)D(3) concentration in patients with an underlying T cell dysfunction and is unable to exert any immunomodulatory effects.

CCR4 expressing cells cultured adherently on a capillary wall and formaldehyde fixed as the stationary phase for ligand screening by ACE.

We have developed an on-line screening method for CC chemokine receptor 4 (CCR4) ligands, in which the whole cells expressed with CCR4 were cultured adherently and immobilized on the inner wall of the capillary as the stationary phase for the first time. Moreover, in this method it is unnecessary to isolate and purify the target receptors from cell membranes. Therefore, it is possible to almost completely preserve the native conformation of the target receptors. The binding activities of the immobilized CCR4 did not change. A known antagonist of CCR4, compound A, was employed to validate the bioactivity of the cell layer and stability of this method. The intraday, interday, and batch-to-batch reproducibilities were investigated (RSD ≤ 13.9%). Nonlinear chromatography was used to calculate the binding constant between the compound A and CCR4 (6.4 × 10(4)/M, RSD = 4.96%). Using this method, the qualitative and quantitative characterizations of 23 computer-aided drug design compounds were achieved and the kinetic parameters (K, k(a), k(d), and k') were obtained by nonlinear chromatography. Three active compounds were screened out, which also showed activity in chemotaxis inhibition assay. The experimental results show that this method is simple, sensitive, and efficient for drug screening. Moreover, it offered a novel way to detect the nonspecific interactions between ligands and cell membrane.

Differing requirements for CCR4, E-selectin, and α4ß1 for the migration of memory CD4 and activated T cells to dermal inflammation.

CCR4 on T cells is suggested to mediate skin homing in mice. Our objective was to determine the interaction of CCR4, E-selectin ligand (ESL), and α(4)ß(1) on memory and activated T cells in recruitment to dermal inflammation. mAbs to rat CCR4 were developed. CCR4 was on 5-21% of memory CD4 cells, and 20% were also ESL(+). Anti-TCR-activated CD4 and CD8 cells were 40-55% CCR4(+), and ∼75% of both CCR4(+) and CCR4(-) cells were ESL(+). CCR4(+) memory CD4 cells migrated 4- to 7-fold more to dermal inflammation induced by IFN-γ, TNF, TLR agonists, and delayed-type hypersensitivity than CCR4(-) cells. CCR4(+) activated CD4 cells migrated only 5-50% more than CCR4(-) cells to these sites. E-selectin blockade inhibited ∼60% of CCR4(+) activated CD4 cell migration but was less effective on memory cells where α(4)ß(1) was more important. Anti-α(4)ß(1) also inhibited CCR4(-) activated CD4 cells more than CCR4(+) cells. Anti-E-selectin reduced activated CD8 more than CD4 cell migration. These findings modify our understanding of CCR4, ESL, α(4)ß(1), and dermal tropism. There is no strict relationship between CCR4 and ESL for skin homing of CD4 cells, because the activation state and inflammatory stimulus are critical determinants. Dermal homing memory CD4 cells express CCR4 and depend more on α(4)ß(1) than ESL. Activated CD4 cells do not require CCR4, but CCR4(+) cells are more dependent on ESL than on α(4)ß(1), and CCR4(-) cells preferentially use α(4)ß(1). The differentiation from activated to memory CD4 cells increases the dependence on CCR4 for skin homing and decreases the requirement for ESL.

Increased frequency of CCR4+ and CCR6+ memory T-cells including CCR7+CD45RAmed very early memory cells in granulomatosis with polyangiitis (Wegener's).

INTRODUCTION: Chemokine receptors play an important role in mediating the recruitment of T cells to inflammatory sites. Previously, small proportions of circulating Th1-type CCR5+ and Th2-type CCR3+ cells have been shown in granulomatosis with polyangiitis (GPA). Wondering to what extent CCR4 and CCR6 expression could also be implicated in T cell recruitment to inflamed sites in GPA, we investigated the expression of CCR4 and CCR6 on T cells and its association with T cell diversity and polarization. METHODS: Multicolor flow cytometry was used to analyze CCR4, CCR6, and intracellular cytokine expression of T cells from whole blood of GPA-patients (n = 26) and healthy controls (n = 20). CCR7 and CD45RA were included for phenotypic characterization. RESULTS: We found a significant increase in the percentages of circulating CCR4+ and CCR6+ cells within the total CD4+ T cell population in GPA. In contrast, there was no difference in the percentages of CD8+CCR4+ and CD8+CCR6+ T cells between GPA and healthy controls. CCR4 and CCR6 expression was largely confined to central (TCM) and effector memory T cells (TEM, TEMRA). A significant increase in the frequency of CCR4+ and CCR6+ TEMRA and CCR6+ TCM was shown in GPA. Of note, we could dissect CCR4 and CCR6 expressing CCR7+CD45RAmed very early memory T cells (TVEM) from genuine CCR7+CD45RAhigh naïve T cells lacking CCR4 and CCR6 expression for peripheral tissue-migration within the CCR7+CD45RA+ compartment. The frequencies of CCR4+ and CCR6+ TVEM were also significantly increased in GPA. An increased percentage of IL-17+ and IL-22+ cells was detected in the CCR6+ cell subsets and IL-4+ cells in the CRR4+ cell subset when compared with CD4+ cells lacking CCR4 and CCR6 expression. CONCLUSIONS: Increased frequencies of circulating CCR4+ and CCR6+ memory T cell subsets including hitherto unreported TVEM suggest persistent T cell activation with the accumulation of CCR4+ and CCR6+ cells in GPA. CCR4 and CCR6 could be involved in the recruitment of T cells including cytokine-producing subsets to inflamed sites in GPA.

Proinflammatory chemokines in the intestinal lumen contribute to intestinal dysfunction during endotoxemia.

Intestinal failure is common in patients with septic shock, with dysfunction of the gut often manifesting as both a cause and consequence of their critical illness. Most studies investigating the pathogenesis of intestinal failure focus on the systemic aspect, although few data examine the inflammatory signaling in the intestinal lumen. Having previously demonstrated apical/luminal chemokine secretion in an in vitro model of intestinal inflammation, we hypothesized that endotoxemia would induce secretion of proinflammatory chemokines into the intestinal lumen. In addition, we examined the contribution of these mediators to intestinal dysmotility. C57/BL6 male mice were injected intraperitoneally with LPS. Serum, intestinal tissue, and intestinal luminal contents were harvested for cytokine analysis. For intestinal motility studies, a transit assay was performed after oral gavage of chemokines. Caco-2 cells grown on Transwell culture inserts were used to examine the role of the intestinal epithelium in chemokine secretion. Monocyte chemoattractant protein 1 (MCP-1/CCL2) and macrophage-derived chemokine (MDC/CCL22) were secreted into the lumen of multiple segments of the gut during endotoxemia in mice. In vitro work showed that the intestinal epithelium participates in monocyte chemoattractant protein 1 and MDC secretion and expresses the CCR2 and CCR4 receptors for these chemokines. Intestinal transit studies show that oral gavage of MDC results in impaired gut motility. This study demonstrates that the intestinal lumen is an active compartment in the gut's inflammatory response. Proinflammatory chemokines are secreted into the intestinal lumen during endotoxemia. These intraluminal chemokines contribute to intestinal dysmotility, complicating intestinal failure.

Percutaneous application of peptidoglycan from Staphylococcus aureus induces infiltration of CCR4+ cells into mouse skin.

BACKGROUND: The lesional skin of patients with atopic dermatitis has an increased number of type 2 helper T (TH2) cells in the dermis and is superficially colonized by Staphylococcus aureus. The purpose of this study was to determine the effects of peptidoglycan (PEG) from S aureus on TH2 cell induction in murine skin. METHODS: Mice were sensitized with house dust mite antigen (MA) by topical application to barrier-disrupted abdominal skin. Seven days after sensitization, PEG was applied to the barrier-disrupted dorsal skin. After a further 3 days, C-C chemokine receptor type 4-positive (CCR4+) cells were counted in the PEG-treated skin.The production of chemokine (C-C) motif ligand 17 (CCL17) (thymus- and activation-regulated chemokine) and CCL22 (macrophage-derived chemokine) in the skin was investigated using reverse transcriptase polymerase chain reaction and immunohistological analysis. RESULTS: Application of PEG to the dorsal skin of MA-sensitized mice led to a significant increase in the number of cells expressing CCR4 in the dermis. The skin of PEG-treated mice showed an increased level of CCL17 mRNA expression, which coincided with TH2 cytokine mRNA expression. Immunohistological analysis demonstrated that levels of CCL17 transcripts corresponded to those of protein synthesis in the epidermis. CCL17 production was induced mainly by Langerhans cells stimulated with PEG. Furthermore, intraperitoneal injection of anti-CCL17 antibody abrogated the induction of CCR4+ cells in the skin. CONCLUSION: These results suggest that PEG may induce TH2 cells in the skin through the production of CCL17 by Langerhans cells and would explain the role of colonization by S aureus in patients with atopic dermatitis.

Lung transplantation affects expression of the chemokine receptor type 4 on specific T cell subsets.

Alloreactive T cells that infiltrate the graft after lung transplantation (LTx) play a role in chronic rejection. Chemokines such as thymus and activation-regulated chemokine (TARC), macrophage-derived chemokine (MDC) and monocyte chemotactic protein-1 (MCP-1) are produced locally in the lung and attract T cells via chemokine receptor 4 (CCR4). In a TARC gradient, cells expressing CCR4(++) migrate more efficiently than CCR4(+) -expressing cells. In this study, we compared the CCR4 expression of T cells in blood from 20 lung transplant recipients to healthy controls. We then examined whether CCR4 expression is associated with the occurrence of chronic rejection. The CCR4(++) expression was decreased on CD4 T cells from LTx patients (P < 0·0001) when compared to healthy controls. The analysis of CD4 T cell subsets showed that this decrease was present on central memory, effector memory and terminally differentiated T cells (P = 0·0007, P < 0·0001 and P = 0·05, respectively), while a trend was found for naive CD4 T cells (P = 0·06). Also, the expression of CCR4(+) on regulatory T cells (T(regs) ) was decreased in LTx patients when compared to healthy controls (P = 0·02). Interestingly, the CCR4(++) expression on CD4 effector memory T cells was decreased in patients developing chronic rejection sometimes more than a year before the clinical diagnosis when compared to patients who did not (P = 0·04). The analysis of CD8 T cell subsets only showed the CCR4(+) expression to be increased significantly on effector memory and terminally differentiated CD8 T cells (P = 0·02, P = 0·03, respectively) in LTx patients, but no relation was found in chronic rejection. In conclusion, the expression of CCR4 on T cell subsets was altered after LTx and appears to be related to chronic rejection.