Characterising the expression and function of CCL28 and its corresponding receptor, CCR10, in RA pathogenesis.

OBJECTIVE: This study was conducted to determine the expression pattern, regulation and function of CCL28 and CCR10 in rheumatoid arthritis (RA) pathogenesis. METHODS: Expression of CCL28 and CCR10 was assessed in RA compared with other arthritis synovial tissues (STs) or fluids (SFs) by histology or ELISA. The factors modulating CCL28 and CCR10 expression were identified in RA myeloid and endothelial cells by ELISA, FACS and Western blotting. The mechanism by which CCL28 ligation promotes RA angiogenesis was examined in control and CCR10-knockdown endothelial cell chemotaxis and capillary formation. RESULTS: CCL28 and/or CCR10 expression levels were accentuated in STs and SFs of patients with joint disease compared with normal controls and they were predominately coexpressed in RA myeloid and endothelial cells. We show that protein expression of CCL28 and CCR10 was modulated by tumour necrosis factor (TNF)-α and toll-like receptor 4 ligation in RA monocytes and endothelial cells and by interleukin (IL)-6 stimulation in RA macrophages. Neutralisation of CCL28 in RA SF or blockade of CCR10 on human endothelial progenitor cells (EPCs) significantly reduced SF-induced endothelial migration and capillary formation, demonstrating that ligation of joint CCL28 to endothelial CCR10+ cells is involved in RA angiogenesis. We discovered that angiogenesis driven by ligation of CCL28 to CCR10 is linked to the extracellular signal regulated kinase (ERK) cascade, as CCR10-knockdown cells exhibit dysfunctional CCL28-induced ERK signalling, chemotaxis and capillary formation. CONCLUSIONS: The overexpression of CCL28 and CCR10 in RA ST and their contribution to EPC migration into RA joints support the CCL28/CCR10 cascade as a potential therapeutic target for RA.

Chemokine scavenger D6 is expressed by trophoblasts and aids the survival of mouse embryos transferred into allogeneic recipients.

Proinflammatory CC chemokines are thought to drive recruitment of maternal leukocytes into gestational tissues and regulate extravillous trophoblast migration. The atypical chemokine receptor D6 binds many of these chemokines and is highly expressed by the human placenta. D6 is thought to act as a chemokine scavenger because, when ectopically expressed in cell lines in vitro, it efficiently internalizes proinflammatory CC chemokines and targets them for destruction in the absence of detectable chemokine-induced signaling. Moreover, D6 suppresses inflammation in many mouse tissues, and notably, D6-deficient fetuses in D6-deficient female mice show increased susceptibility to inflammation-driven resorption. In this paper, we report strong anti-D6 immunoreactivity, with specific intracellular distribution patterns, in trophoblast-derived cells in human placenta, decidua, and gestational membranes throughout pregnancy and in trophoblast disease states of hydatidiform mole and choriocarcinoma. We show, for the first time, that endogenous D6 in a human choriocarcinoma-derived cell line can mediate progressive chemokine scavenging and that the D6 ligand CCL2 can specifically associate with human syncytiotrophoblasts in term placenta in situ. Moreover, despite strong chemokine production by gestational tissues, levels of D6-binding chemokines in maternal plasma decrease during pregnancy, even in women with pre-eclampsia, a disease associated with increased maternal inflammation. In mice, D6 is not required for syngeneic or semiallogeneic fetal survival in unchallenged mice, but interestingly, it does suppress fetal resorption after embryo transfer into fully allogeneic recipients. These data support the view that trophoblast D6 scavenges maternal chemokines at the fetomaternal interface and that, in some circumstances, this can help to ensure fetal survival.

The lymphatic system controls intestinal inflammation and inflammation-associated Colon Cancer through the chemokine decoy receptor D6.

BACKGROUND AND AIMS: Inflammatory CC chemokines have long been associated with cancer, but unequivocal evidence of a role in clinically relevant models of carcinogenesis is lacking. D6, a promiscuous decoy receptor that scavenges inflammatory CC chemokines, plays a non-redundant role in reducing the inflammatory response in various organs. As inflammation is a key player in the development of inflammatory bowel disease (IBD) and IBD-associated colorectal cancer, we investigated D6 expression in human colitis and colon cancer, and its role in experimental colitis and inflammation-associated colon cancer. RESULTS: In humans, D6 was mainly expressed by lymphatic vessels and leukocytes in the mucosa of individuals with IBD and colon cancer, as well as the mucosa of control individuals. Mice lacking expression of D6 were significantly more susceptible to experimental colitis than wild-type mice and failed to resolve colitis, with significantly higher levels of several pro-inflammatory chemokines. In bone marrow chimeric mice, the ability of D6 to regulate colitis was tracked to the stromal/lymphatic compartment, with no contribution of haemopoietic cells. Finally, after administration of the carcinogen azoxymethane, D6(-/-) mice showed increased susceptibility to colitis-associated cancer in the distal segment of the colon compared with wild-type mice. CONCLUSIONS: D6 expressed on lymphatic vessels plays a key role in the control of intestinal inflammation and the development of inflammation-associated colon cancer. Our results reveal a new unexpected role for the lymphatic system in the pathogenesis of IBD and intestinal cancer, and candidate chemokines as novel players in tumour promotion and progression.

The chemokine scavenging receptor D6 limits acute toxic liver injury in vivo.

The chemokine decoy receptor D6 is a promiscuous chemokine receptor lacking classical signaling functions. It negatively regulates inflammation by targeting CC chemokines to cellular internalization and degradation. Here we analyze the function of D6 in acute CCl(4)-induced liver damage in constitutive D6(-/-) and wild-type mice. The degree of liver injury was assessed by liver histology, serum transaminases, IL-6, and TNFalpha mRNA expression. Protein levels of D6 ligands (CCL2, CCL3, CCL5) and the non-D6-ligand CXCL9 within the livers were determined by ELISAs. The intrahepatic infiltration of immune cells was characterized by FACS. Genetic deletion of D6 led to prolonged liver damage after acute CCl(4) administration. The augmented liver damage in D6(-/-) mice was associated with increased protein levels of intrahepatic inflammatory chemokines CCL2, CCL3, and CCL5 after 48 h, whereas CXCL9 was not different between knockout and wild-type mice. Functionally, increased intra-hepatic CC chemokine concentrations led to increased infiltration of CD45(+) leukocytes, which were mainly identified as T and NK cells. In conclusion, the chemokine scavenger receptor D6 has a non-redundant role in acute toxic liver injury in vivo. These results support the importance of post-translational chemokine regulation and describe a new mechanism of immune modulation within the liver.

The atypical chemokine receptor D6 contributes to the development of experimental colitis.

Proinflammatory CC chemokines control leukocyte recruitment and function during inflammation by engaging chemokine receptors expressed on circulating leukocytes. The D6 chemokine receptor can bind several of these chemokines, but appears unable to couple to signal transduction pathways or direct cell migration. Instead, D6 has been proposed to act as a chemokine scavenger, removing proinflammatory chemokines to dampen leukocyte responses. In this study, we have examined the role of D6 in the colon using the dextran sodium sulfate-induced model of colitis. We show that D6 is expressed in the resting colon, predominantly by stromal cells and B cells, and is up-regulated during colitis. Unexpectedly, D6-deficient mice showed reduced susceptibility to colitis and had less pronounced clinical symptoms associated with this model. D6 deletion had no impact on the level of proinflammatory CC chemokines released from cultured colon explants, or on the balance of leukocyte subsets recruited to the inflamed colon. However, late in colitis, inflamed D6-deficient colons showed enhanced production of several proinflammatory cytokines, including IFN-gamma and IL-17A, and there was a marked increase in IL-17A-secreting gammadelta T cells in the lamina propria. Moreover, Ab-mediated neutralization of IL-17A worsened the clinical symptoms of colitis at these later stages of the response in D6-deficient, but not wild-type, mice. Thus, D6 can contribute to the development of colitis by regulating IL-17A secretion by gammadelta T cells in the inflamed colon.

An indispensable role for the chemokine receptor CCR10 in IgA antibody-secreting cell accumulation.

The differential expression of chemokines and chemokine receptors, by tissues and leukocytes, respectively, contributes to the specific accumulation of leukocyte subsets to different tissues. CCR10/CCL28 interactions are thought to contribute to the accumulation of IgA Ab-secreting cells (ASC) to mucosal surfaces, such as the gastrointestinal tract and the lactating mammary gland. Although the role of CCL28 in lymphocyte homing is well established, direct in vivo evidence for CCR10 involvement in this process has not been previously shown. In this study, we describe the generation of a CCR10-deficient mouse model. Using this model, we demonstrate that CCR10 is critical for efficient localization and accumulation of IgA ASC to the lactating mammary gland. Surprisingly, IgA ASC accumulation to the gastrointestinal tract is minimally impacted in CCR10-deficient mice. These results provide the first direct evidence of CCR10 involvement in lymphocyte homing and accumulation in vivo, and demonstrate that reliance on CCR10-mediated recruitment of IgA ASC varies dramatically within mucosal tissues.