Nakanishi T, etal., Cancer Immunol Immunother. 2006 Nov;55(11):1320-9. Epub 2006 Feb 2.
BACKGROUND: Ligands for CXCR3 chemokines [IFN-gamma-inducible protein of 10 kD (IP-10/CXCL10), monokine induced by IFN-gamma (Mig/CXCL9), IFN-inducible T cell alpha chemoattractant (I-TAC/CXCL11)] and those for CCR4 [macrophage-derived chemokine (MDC/CCL22), thy
mus- and activation-regulated chemokine (TARC/CCL17)] have been shown to play the central roles for T helper-cell recruitment into the tissues. To examine the role of these chemokines in tumor progression of lung cancer, we investigated their expression in human lung cancer tissues to determine the possible relationship between their expression and the prognosis of patients. METHODS: Total RNA was prepared from lung cancer tissues of 40 patients (24 adenocarcinoma and 16 squamous cell carcinoma). We measured gene expression levels of chemokines (IP-10, Mig, I-TAC, MDC and TARC) by real-time quantitative RT-PCR. RESULTS: Higher gene expression of MDC in lung cancer was significantly correlated with longer disease-free survival time and lower risk of recurrence after tumor resection. We could not find any significant relationship of IP-10, Mig, I-TAC and TARC gene expression with disease-free survival or lower risk of recurrence after surgery. CONCLUSIONS: These results suggest that increased gene expression of MDC in tumor tissues may be a predictive marker for improving the prognosis of lung cancer.
Inoue T, etal., Eur Respir J. 2004 Jul;24(1):49-56.
Pulmonary fibrosis is caused by various known and unknown aetiologies, but the key pathogenic mechanisms are still ill-defined. Chemokines are a large family of chemotactic cytokines that play pivotal roles in various inflammatory diseases. In the present study, the roles of chemokines in a rat mode
l of radiation pneumonitis/ pulmonary fibrosis were examined. Accumulation of inflammatory cells and pneumonitis were observed on day 28, and diffuse alveolar wall thickening with extensive fibrosis was observed on day 56. In addition to the previously reported CCL2 (macrophage chemoattractant protein-1) induction, selective upregulation of CCL22 (macrophage-derived chemokine) and CCL17 (thymus and activation-regulated chemokine) were demonstrated for the first time in the irradiated lung tissues. Immunohistochemically, it was demonstrated that CCL22 and CCL17 were localised primarily to alveolar macrophages, whereas their receptor CC chemokine receptor 4 (CCR4) was detected on alveolar lymphocytes and macrophages. On further analysis of bronchoalveolar lavage fluid from patients with idiopathic pulmonary fibrosis and sarcoidosis, elevated levels of CCL22, but not of CCL17, were observed in the idiopathic pulmonary fibrosis patients. Since these two chemokines play pivotal roles in various type-2 T-helper cell-dominant diseases, it was speculated that CCL22, and probably CCL17, are involved in the pathophysiology of radiation pneumonitis/pulmonary fibrosis and idiopathic pulmonary fibrosis through the recruitment of CC chemokine receptor 4-positive type-2 T-helper cells and alveolar macrophages.
Wu C, etal., Respirology. 2010 Apr;15(3):522-9. Epub 2010 Mar 19.
BACKGROUND AND OBJECTIVE: CD4(+)CD25(high) regulatory T cells are increased in tuberculous pleural effusions (TPE). However, the mechanism by which CD4(+)CD25(high) T cells infiltrate into the pleural cavity is unknown. The aim of this study was to investigate whether the chemokines CCL22
t-weight:700;'>CCL22 and CCL17 are present in TPE, and the chemoattractant activity of these chemokines for infiltration of CD4(+)CD25(high) T cells into the pleural space. METHODS: The concentrations of CCL22 and CCL17 were measured in pleural effusions from 33 patients with tuberculous pleurisy, 21 patients with pleural bacterial infections and 18 patients with transudative pleural effusions. T lymphocyte subsets in pleural effusions were assessed by flow cytometry. Pleural effusion cells were analysed for the expression of CCL22. The chemoattractant activity of CCL22 for CD4(+)CD25(high) T cells was assessed in vitro. RESULTS: The frequency of CD4(+)CD25(high) T cells was significantly higher in TPE than in blood. High concentrations of CCL22 were detected in tuberculous effusions, but not in bacterial effusions or transudates. Macrophages and T cells in TPE expressed CCL22. Tuberculous pleural fluid was chemotactic for CD4(+)CD25(high) T cells in vitro, and anti-CCL22 antibody partly inhibited this chemotactic activity. CONCLUSIONS: CCL22 appeared to be increased in TPE compared with bacterial pleural effusions or transudates. CCL22 may be responsible for the infiltration of CD4(+)CD25(high) T cells into the pleural space of patients with tuberculous pleurisy.
BACKGROUND: The omentum is one of the initial sites for peritoneal metastasis because it possesses milky spots that provide a microenvironment for cancer cells to readily migrate and grow into micrometastases. This study investigated the role of the CCL22-CCR4 a
xis in gastric cancer cells selectively infiltrating into milky spots. METHODS: Gastric cancer MFC cells labelled with DiI were injected intraperitoneally into strain 615 mice. The mice were euthanised at specified intervals and the omentum was excised for immunohistochemistry. The effects of CCL22 on the proliferation and migration of MFCs were assessed by MTT and trans-well assays. RT-PCR and Western blot analysis detected CCR4 mRNA and protein expression levels in MFCs. Immunohistochemistry was used to analyse CCL22 and CCR4 expression in the milky spot micrometastases. RESULTS: Two weeks after intraperitoneal injection, the milky spot areas were completely occupied by proliferating gastric cancer cells and cell cluster-type micrometastases were observed. In contrast, cancer cells formed single cell-type micrometastases in the non-milky spot areas. MFCs expressed CCR4, which was localised on the cell surface and or in the cytoplasm. Different concentrations of CCL22 significantly increased the proliferation ability of MFCs. Additionally, concentrations of CCL22 between 10-100 ng/ml significantly increased the migration of MFCs. Within omental milky spots, CCL22 was localised mainly on the cell surface and or in the cytoplasm. Within sections of omental milky spot micrometastases, CCR4 was recognised on or in gastric cancer cells, constituent cells milky spots, blood cells and blood endothelial cells. CONCLUSIONS: Omental milky spots are a congenial microenvironment for peritoneal free gastric cancer cells to migrate, survive, and establish cell cluster-type metastases. The CCL22-CCR4 axis contributes to this selective infiltration process.
CCL22 inactivation in vivo occurs by cleavage at the N-terminus; however, it is unclear whether this encompasses the entire site of CCR4 interaction. CCL17 also binds CCR4 and its function requires binding via two discrete binding sites. Using monoclonal antibo
dies (MAbs), we report that there are two separate sites on CCL22 that are required for CCR4-mediated function. The CCL22-specific antibodies bind with affinities of 632 +/- 297 pM (MC2B7) and 308 +/- 43 pM (MAB4391) and neither exhibited detectable binding to CCL17. Both antibodies are comparable in their ability to inhibit CCL22-mediated calcium mobilization; however, competition binding studies demonstrate that MC2B7 and MAB4391 bind to distinct epitopes on CCL22. Both antibodies inhibit function through CCR4, which is demonstrated by loss of beta-arrestin recruitment in a reporter cell line. In both assays, blocking either site independently abolished CCL22 function, suggesting that concurrent engagement of both sites with CCR4 is necessary for function. This is the first demonstration that CCL22 has two distinct binding sites that are required for CCR4 function. These antibodies are valuable tools for better understanding the interaction and function of CCL22 and CCR4 and will potentially help further understanding of the differential outcomes of CCL17 and CCL22 interaction with CCR4.
Pemphigus Vulgaris (PV), a relatively common autoimmune blistering disease in India, primarily mediated by anti-Desmoglein 3 (anti-Dsg3) autoantibodies. T-helper 17 (Th17) and T-regulatory (Treg) cells play significant role in regulating immune homeostasis in autoimmune disorders. To understand immu
nopathogenesis of PV, it is crucial to unfold the phenotypic expression and functional characteristics of these cells along with their specific homing chemokine receptor-ligand. This proposed study aims to unravel the functional expression of Th17 and Treg cells along with their specific homing chemokine receptor-ligand, transcription factors and cytokine levels to better understand the immunopathogenesis of PV. The Flow cytometry results showed decreased frequency of Treg cells and high number of Th17 cells (p<0.001) indicating immune dysregulation in PV. A significant increase (p<0.001) in the serum levels of Th17 associated molecules (IL-17A, CCL-20) and relative expression of RORgammat, CCR6 and CCL20 was found in patients. For Treg cells, transcription factor FOXp3 was significantly lowered along with defective CCR4-CCL22 (p<0.05) that might be playing an ambiguous role in Treg generated immune regulation, leading to homing defect at lesional sites. This maiden study revealed the role of defective receptor-ligand interface that might have failed to suppress inflammatory milieu produced by Th17 cells thus promoting inflammation and contributing to immunopathogenesis of PV. This chemokine receptor-ligand can further be explored as potential target for development of novel therapies in PV.
Ritter M, etal., Biochem Biophys Res Commun. 2005 Aug 19;334(1):254-62.
TARC (CCL17) and MDC (CCL22) are well-known chemoattractants for Th2 cells. Here, we evaluated the role of both chemokines for cigarette smoke-induced airway inflammation. The expression profiles of MDC, TARC, and their receptor CCR4 were analyzed in models of a
cute and chronic cigarette smoke-induced airway inflammation that is characterized by a Th1 immune response. The results were compared to the expression of both chemokines in models of idiopathic pulmonary fibrosis and acute asthma, which are associated with a Th2 immune response. The expression of MDC and TARC was found to be elevated in all lung inflammation models. In contrast to the findings in the asthma and lung fibrosis models, the increased expression of MDC and TARC in the cigarette-smoke model was not associated with an increased infiltration of Th2 cells into smoke-treated lungs. Our data indicate that instead of Th2 cells, airway epithelial cells expressing CCR4 might be the principal targets for MDC and TARC released from alveolar macrophages during cigarette smoke-induced airway inflammation.
PROBLEM: Regulatory T cells (Treg) are a T-cell subpopulation with suppressive capacities, specifically attracted by CCL22. We aimed to investigate whether CCL22 is expressed in human placentas and whether its presence, toge
ther with Treg infiltration, is associated with miscarriage conditions. METHOD OF STUDY: Paraffin samples were stained for CCL22 and for the Treg-specific transcription factor FoxP3. Expression levels were evaluated in a semi-quantitative manner. Double immunofluorescence was used for the identification of CCL22-producing cells. RESULTS: In all placentas, trophoblasts expressed CCL22. Interestingly, expression in the decidua was only observed in miscarriage conditions. Maternal stromal cells expressed CCL22. Correlation with a higher presence of Treg in the decidua of abortive tissues was observed. CONCLUSION: Our results demonstrate that CCL22 is expressed in human placenta. Decidual expression was only observed in miscarriage conditions and correlates with Treg infiltration. Thus, CCL22 plays a role in human pregnancy and may occur as a negative feedback response to pro-inflammatory events during miscarriage conditions.
Flytlie HA, etal., Cytokine. 2010 Jan;49(1):24-9. doi: 10.1016/j.cyto.2009.10.005. Epub 2009 Nov 25.
The pathogenesis of rheumatoid arthritis (RA) and psoriatic arthritis (PsA) involves an abnormal chemokine regulation. The chemokine receptor CCR4 is necessary for T cell migration to the skin. We, therefore, studied if CCR4 and its ligand macrophage-derived chemokine (MDC/CCL22
00;'>CCL22) could participate in spreading the disease between skin and joints by examining RA, PsA and osteoarthritis (OA) patients. In synovial fluid from RA and PsA patients we observed a significantly higher MDC/CCL22 level compared to OA patients. Additionally, the MDC/CCL22 protein was found to be elevated in RA and PsA plasma compared to OA and healthy volunteers. Flow cytometry revealed that most CD4(+)CCR4(+) lymphocytes also co-expressed CD45RO. Neither the MDC/CCL22 level nor the expression of CCR4 correlated to CRP. Immunohistochemistry of the RA and OA synovial membrane demonstrated CCR4 to be expressed by mononuclear cells and endothelial cells. Our results show that MDC/CCL22 is present within the synovial membrane of RA and OA patients and in high amount in the synovial fluid of patients with RA and PsA. This will enable migration of CCR4 expressing memory cells supporting that MDC/CCR4 could play a role in attracting skin specific memory T cells to the joints.
Inflammatory bone resorption is a hallmark of periodontitis, and Tregs and Th2 cells are independently associated with disease progression attenuation. In this study, we employed an infection-triggered inflammatory osteolysis model to investigate the mechanisms underlying Treg and Th2 cell migratio
n and the impact on disease outcome. Aggregatibacter actinomycetemcomitans-infected C57Bl/6 (wild-type [WT]) mice develop an intense inflammatory reaction and alveolar bone resorption, and Treg and Th2 cell migration is temporally associated with disease progression attenuation. Tregs extracted from the lesions preferentially express CCR4 and CCR8, whereas Th2 cells express CCR3, CCR4, and CCR8. The absence of CCR5 and CCR8 did not significantly impact the migration of Tregs and Th2 cells or affect the disease outcome. CCR4KO mice presented a minor reduction in Th2 cells in parallel with major impairment of Treg migration, which was associated with increased inflammatory bone loss and higher proinflammatory and osteoclastogenic cytokine levels. The blockade of the CCR4 ligand CCL22 in WT mice resulted in an increased inflammatory bone loss phenotype similar to that in the CCR4KO strain. Adoptive transfer of CCR4(+) Tregs to the CCR4KO strain revert the increased disease phenotype to WT mice-like levels; also, the in situ production of CCL22 in the lesions is mandatory for Tregs migration and the consequent bone loss arrest. The local release of exogenous CCL22 provided by poly(lactic-co-glycolic acid) (PLGA) microparticles promotes migration of Tregs and disease arrest in the absence of endogenous CCL22 in the IL-4KO strain, characterized by the lack of endogenous CCL22 production, defective migration of Tregs, and exacerbated bone loss. In summary, our results show that the IL-4/CCL22/CCR4 axis is involved in the migration of Tregs to osteolytic lesion sites, and attenuates development of lesions by inhibiting inflammatory migration and the production of proinflammatory and osteoclastogenic mediators.
BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a chronically progressive interstitial lung disease of unknown etiology. Previously, we have demonstrated the selective upregulation of the macrophage-derived chemokine CCL22 and the thymus activation-regulated
chemokine CCL17 among chemokines, in a rat model of radiation pneumonitis/pulmonary fibrosis and preliminarily observed an increase in bronchoalveolar (BAL) fluid CCL22 levels of IPF patients. METHODS: We examined the expression of CCR4, a specific receptor for CCL22 and CCL17, in bronchoalveolar lavage (BAL) fluid cells, as well as the levels of CCL22 and CCL17, to elucidate their pathophysiological roles in pulmonary fibrosis. We also studied their immunohistochemical localization. RESULTS: BAL fluid CCL22 and CCL17 levels were significantly higher in patients with IPF than those with collagen vascular diseases and healthy volunteers, and there was a significant correlation between the levels of CCL22 and CCL17 in patients with IPF. CCL22 levels in the BAL fluid did not correlate with the total cell numbers, alveolar lymphocytes, or macrophages in BAL fluid. However, the CCL22 levels significantly correlated with the numbers of CCR4-expressing alveolar macrophages. By immunohistochemical and immunofluorescence analysis, localization of CCL22 and CCR4 to CD68-positive alveolar macrophages as well as that of CCL17 to hyperplastic epithelial cells were shown. Clinically, CCL22 BAL fluid levels inversely correlated with DLco/VA values in IPF patients. CONCLUSION: We speculated that locally overexpressed CCL22 may induce lung dysfunction through recruitment and activation of CCR4-positive alveolar macrophages.
Jakubzick C, etal., Am J Pathol. 2004 Oct;165(4):1211-21.
Controversy persists pertaining to the role of CCR4 ligands, namely CCL17 (or thymus and activation regulated chemokine; TARC) and CCL22 (or macrophage-derived chemokine; MDC), in Th2-type cytokine-dominated responses in the lung. Accordingly, the present study
addressed the relative role of each of these CC chemokines during an evolving pulmonary granulomatous response elicited by the intrapulmonary embolization of live Schistosoma mansoni eggs into S. mansoni-sensitized mice. CCL22 protein expression peaked at day 4, but CCL17 levels were not increased significantly at any time after egg challenge. CCR4 transcript and protein expression were highest at day 8 after egg embolization and CCR4 protein was prominently expressed in macrophages surrounding S. mansoni eggs. Systemic immunoneutralization of CCL22 from the time of egg injection into S. mansoni-sensitized mice for 8 days significantly decreased CCR4 protein expression, the eosinophil content, the overall size of the egg granuloma, and its hydroxyproline content. Whole lung levels of interferon-gamma were also significantly increased at day 8 in anti-CCL22-treated mice. The systemic immunoneutralization of CCL17 had a lesser effect on all of the granuloma parameters listed above, but this antibody treatment significantly decreased granuloma hydroxyproline content to a greater extent than the anti-CCL22 antibody treatment. In addition, the immunoneutralization of CCL17 significantly increased whole lung levels of interleukin (IL)-4, IL-5, IL-13, transforming growth factor-beta, IL-12, and tumor necrosis factor-alpha at day 8 after egg infusion. Thus, these studies demonstrate a major role for CCL22 and a lesser role for CCL17 during an evolving S. mansoni egg granuloma in the lung.
S100B is a Ca2+ binding protein and is typically associated with brain and CNS disorders. However, the role of S100B in an inflammatory situation is not clear. The aim of the study was to determine whether S100B is likely to influence inflammation through its effect on macrophages. A murine macroph
age cell line (RAW 264.7) and primary bone marrow derived macrophages were used for in vitro studies and a model of retinal inflammatory disease in which pathogenesis is highly dependent on macrophage infiltration, Experimental Autoimmune Uveoretinitis, for in vitro study. Experimental Autoimmune Uveoretinitis is a model for the human disease posterior endogenous uveoretinitis, a potentially blinding condition, with an autoimmune aetiology, that mainly affects the working age group. To date the involvement of S100B in autoimmune uveoretinitis has not been investigated. Real-time PCR array analysis on RAW 246.7 cells indicated up-regulation of gene expression for various cytokines/chemokines in response to S100B, IL-1beta and CCL22 in particular and this was confirmed by real-time PCR. In addition flow cytometry and ELISA confirmed up-regulation of protein production in response to S100B for pro-IL-1beta and CCL22 respectively. This was the case for both RAW 264.7 cells and bone marrow derived macrophages. Induction of EAU with retinal antigen in mice in which S100B had been deleted resulted in a significantly reduced level of disease compared to wild-type mice, as determined by topical endoscopic fundus imaging and histology grading. Macrophage infiltration was also significantly reduced in S100B deleted mice. Real-time PCR analysis indicated that this was associated with reduction in CCL22 and IL-1beta in retinas from S100B knock-out mice. In conclusion S100B augments the inflammatory response in uveoretinitis and this is likely to be, at least in part, via a direct effect on macrophages.
