CCL20/macrophage inflammatory protein 3alpha and tumor necrosis factor alpha production by primary uterine epithelial cells in response to treatment with lipopolysaccharide or Pam3Cys.
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Having previously shown that CCL20/macrophage inflammatory protein 3alpha and tumor necrosis factor alpha (TNF-alpha) are released by polarized primary rat uterine epithelial cells (UEC) in response to Escherichia coli but not to Lactobacillus rhamnosus, we sought to determine if epithelial cells are responsive to pathogen-associated molecular patterns (PAMP), including lipopolysaccharide (LPS), lipoteichoic acid (LTA), and Pam(3)Cys, a bacterial lipoprotein analog. Epithelial cells were grown to confluence on Nunc cell culture inserts prior to apical treatment with PAMPs. In response to LPS, LTA, and Pam(3)Cys (EMC Microcollection GmbH, Tubingen, Germany), CCL20 levels increased (4- to 10-fold) while PAMPs caused increased TNF-alpha (1- to 4-fold) in the medium collected after 24 h of incubation. Both apical and basolateral secretion of CCL20 and TNF-alpha increased in response to PAMPs, but treatments had no effect on cell viability and integrity, as measured by transepithelial resistance. Time course studies of CCL20 and TNF-alpha release in response to Pam(3)Cys and LPS indicated that CCL20 release peaked between 2 and 4 h after treatment, whereas TNF-alpha release was gradual over the length of the incubation. Freeze-thaw and cell lysis experiments, along with actinomycin D studies, suggested that CCL20 and TNF-alpha are synthesized in response to PAMP stimulation. Taken together, these studies demonstrate that E. coli and selected PAMPs have direct effects on the production of CCL20 and TNF-alpha without affecting cell integrity. Since CCL20 is known to be both chemotactic and antimicrobial, the increase in apical and basolateral release by UEC in response to PAMPs suggests a new mechanism of innate immune protection in the female reproductive tract. (+info)
Increased lymphocyte trafficking to colonic microvessels is dependent on MAdCAM-1 and C-C chemokine mLARC/CCL20 in DSS-induced mice colitis.
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Although enhanced lymphocyte trafficking is associated with colitis formation, little information about its regulation is available. The aim of this study was to examine how the murine liver and activation-regulated chemokine (mLARC/CCL20) contributes to lymphocyte recruitment in concert with vascular adhesion molecules in murine chronic experimental colitis. T and B lymphocytes isolated from the spleen were fluorescence-labelled and administered to recipient mice. Lymphocyte adhesion to microvessels of the colonic mucosa and submucosa was observed with an intravital microscope. To induce colitis, the mice received two cycles of treatment with 2% dextran sodium sulphate (DSS). In some of the experiments antibodies against the adhesion molecules or anti-mLARC/CCL20 were administered, or CC chemokine receptor 6 (CCR6) of the lymphocytes was desensitized with excess amounts of mLARC/CCL20. Significant increases in T and B cell adhesion to the microvessels of the DSS-treated mucosa and submucosa were observed. In chronic colitis, the accumulation of lymphocytes was significantly inhibited by anti-mucosal addressin cell adhesion molecule (MAdCAM)-1 mAb, but not by anti-vascular cell adhesion molecule-1. In DSS-treated colonic tissue, the expression of mLARC/CCL20 was significantly increased, the blocking of mLARC/CCL20 by monoclonal antibody or the desensitization of CCR6 with mLARC/CCL20 significantly attenuated the DSS-induced T and B cell accumulation. However, the combination of blocking CCR6 with MAdCAM-1 did not further inhibit these accumulations. These results suggest that in chronic DSS-induced colitis, both MAdCAM-1 and mLARC/CCL20 may play important roles in T and B lymphocyte adhesion in the inflamed colon under flow conditions. (+info)
Characterization of CCL20 secretion by human epithelial vaginal cells: involvement in Langerhans cell precursor attraction.
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Mucosa represents the main site of pathogen/cell interactions. The two main types of cells forming the epithelial structure [epithelial cells and Langerhans cells (LC)] coordinate the first defense responses to avoid infection. To evaluate the involvement of epithelial cells in the early steps leading to a specific adaptive immune response, we have studied the interactions between vaginal epithelial and LC through the establishment of a human vaginal epithelial mucosa. We demonstrate that normal human vaginal epithelial cells constitutively secrete the chemokine macrophage inflammatory protein 3alpha/CC chemokine ligand 20 (CCL20), known to recruit LC precursors (LCps) selectively via its cognate CC chemokine receptor 6 (CCR6). This secretion is up-regulated by the proinflammatory cytokine interleukin-1beta through the nuclear factor-kappaB pathway. Similar results were obtained with the human vaginal epithelial cell line SiHa, which displays numerous homologies with normal vaginal cells. The chemotactic activity of the secreted CCL20 was demonstrated by its ability to attract LCp CCR6+. Moreover, the use of neutralizing polyclonal antibodies directed against the CCL20 molecule abolished this migration completely, suggesting that CCL20 is the main attracting factor for LCps, which is produced by the vaginal cells. These data indicate that vaginal epithelial cells play an important role in the immunological defense by attracting immune cells to the site of epithelial/pathogen contact. (+info)
Effects of estradiol on lipopolysaccharide and Pam3Cys stimulation of CCL20/macrophage inflammatory protein 3 alpha and tumor necrosis factor alpha production by uterine epithelial cells in culture.
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We have previously demonstrated that rat uterine epithelial cells (UEC) produce CCL20/macrophage inflammatory protein 3 alpha (MIP3alpha) and tumor necrosis factor alpha (TNF-alpha) in response to live and heat-killed Escherichia coli and to the pathogen-associated molecular patterns (PAMP) lipopolysaccharide (LPS) and Pam3Cys. To determine whether estradiol (E2) modulates PAMP-induced CCL20/MIP3alpha and TNF-alpha secretion, primary cultures of rat UEC were incubated with E2 for 24 h and then treated with LPS or Pam3Cys or not treated for an additional 12 h. E2 inhibited the constitutive secretion of TNF-alpha and CCL20/MIP3alpha into culture media. Interestingly, E2 pretreatment enhanced CCL20/MIP3alpha secretion due to LPS and Pam3Cys administration. In contrast, and at the same time, E2 lowered the TNF-alpha response to both PAMP. To determine whether estrogen receptors (ER) mediated the effects of E2, epithelial cells were incubated with E2 and/or ICI 182,780, a known ER antagonist. ICI 182,780 had no effect on E2 inhibition of constitutive TNF-alpha and CCL20/MIP3alpha secretion. In contrast, ICI 182,780 reversed the stimulatory effect of E2 on LPS- and/or Pam3Cys-induced CCL20/MIP3alpha secretion as well as partially reversed the inhibitory effect of E2 on TNF-alpha production by epithelial cells. Overall, these results indicate that E2 regulates the production of TNF-alpha and CCL20/MIP3alpha by UEC in the absence as well as presence of PAMP. Since CCL20/MIP3alpha has antimicrobial activity and is chemotactic for immune cells, these studies suggest that regulation of CCL20/MIP3alpha and TNF-alpha by E2 and PAMP may have profound effects on innate and adaptive immune responses to microbial challenge in the female reproductive tract. (+info)
HIV type 1 glycoprotein 120 inhibits human B cell chemotaxis to CXC chemokine ligand (CXCL) 12, CC chemokine ligand (CCL)20, and CCL21.
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We analyzed the modulation of human B cell chemotaxis by the gp120 proteins of various HIV-1 strains. X4 and X4/R5 gp120 inhibited B cell chemotaxis toward CXCL12, CCL20, and CCL21 by 40-50%, whereas R5 gp120 decreased inhibition by 20%. This gp120-induced inhibition was strictly dependent on CXCR4 or CCR5 and lipid rafts but not on CD4 or V(H)3-expressing BCR. Inhibition did not impair the expression or ligand-induced internalization of CCR6 and CCR7. Our data suggest that gp120/CXCR4 and gp120/CCR5 interactions lead to the cross-desensitization of CCR6 and CCR7 because gp120 does not bind CCR6 and CCR7. Unlike CXCL12, gp120 did not induce the activation of phospholipase Cbeta3 and PI3K downstream from CXCR4, whereas p38 MAPK activation was observed. Similar results were obtained if gp120-treated cells were triggered by CCL21 and CCL20. Our results are consistent with a blockade restricted to signaling pathways using phosphatidylinositol-4,5-bisphosphate as a substrate. X4 and X4/R5 gp120 induced the cleavage of CD62 ligand by a mechanism dependent on matrix metalloproteinase 1 and 3, CD4, CXCR4, Galpha(i), and p38 MAPK, whereas R5 gp120 did not. X4 and X4/R5 gp120 also induced the relocalization of cytoplasmic CD95 to the membrane and a 23% increase in CD95-mediated apoptosis. No such effects were observed with R5 gp120. The gp120-induced decrease in B cell chemotaxis and CD62 ligand expression, and increase in CD95-mediated B cell apoptosis probably have major deleterious effects on B cell responsiveness during HIV infection and in vaccination trials. (+info)
Monocyte-derived dendritic cells activated by bacteria or by bacteria-stimulated epithelial cells are functionally different.
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Dendritic cells (DCs) are able to open the tight junctions between adjacent epithelial cells (ECs) and to take up both invasive and noninvasive bacteria directly from the intestinal lumen. In this study, we describe a tight cross talk between ECs and human monocyte-derived DCs (MoDCs) in bacterial handling across epithelial monolayers. We show that the release of proinflammatory mediators by ECs in response to bacteria is dependent on bacterial invasiveness and on the presence of flagella. This correlates with the capacity of EC-derived factors to modulate MoDC function. MoDCs incubated with supernatants of bacteria-treated ECs are "noninflammatory" as they release interleukin-10 (IL-10) but not IL-12 and can drive only T helper (Th)-2 type T cells. Moreover, noninflammatory MoDCs release chemokines aimed at recruiting Th2 and T-regulatory cells. In contrast, when MoDCs are incubated with ECs and bacteria in a transwell coculture system, and can contact directly the bacteria across stimulated EC monolayers, they are more inflammatory as they release IL-12 and IL-10 and induce both Th1 and Th2 responses. These results suggest that ECs are not simply a barrier to bacteria entering via the oral route, but they actively influence the activating properties of DCs. (+info)
Physiological role of macrophage inflammatory protein-3 alpha induction during maturation of intestinal macrophages.
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Intestinal macrophages (IMAC) are a central component in the defense of the intestinal mucosa against luminal microbes. In normal mucosa, monocytes differentiate to immunologically tolerant IMAC with a typical phenotype lacking activation markers such as CD14 and TLRs 2 and 4. CD33+ IMAC were isolated from normal intestinal mucosa by immunomagnetic beads. A subtractive hybridization subtracting mRNA from normal IMAC from those of in vitro differentiated macrophages was performed. IMAC differentiation was studied in multicellular spheroids (MCS). Functional assays on migration of CD45R0+ T cells were performed in MCS coculture models. Of 76 clones, 3 obtained by subtractive mRNA hybridization showed >99% homology to mRNA of MIP-3alpha, indicating that this chemokine is induced in IMAC compared with in vitro differentiated macrophages. MIP-3alpha protein expression was confirmed in cryostat sections of normal intestinal mucosa by immunohistochemistry. IMAC in the lamina propria stained positive for MIP-3alpha. FACS of purified IMAC clearly indicated expression of MIP-3alpha in these cells. In the MCS-in vitro differentiation model for IMAC, MIP-3alpha protein expression was absent on day 1 but detectable on day 7 of coculture, demonstrating the induction of MIP-3alpha during differentiation of IMAC. IMAC attracted CD45R0+ T cells to migrate into an MCS coculture model. In human mucosa, a close contact between IMAC and CD45R0+ T cells could be demonstrated. MIP-3alpha is induced during the differentiation of monocytes into IMAC. Our data suggest that MIP-3alpha expression could be involved in the recruitment of CD45R0+ cells into the lamina propria. (+info)
CXCL12 and CXCR4 expression by human gingival fibroblasts in periodontal disease.
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CXCL12 is a CXC chemokine that is related to lymphocyte infiltration and angiogenesis in inflammatory sites such as arthritis. However, the expression and roles of CXCL12 in periodontal disease are uncertain. The aim of this study was to assess the expression of CXCL12 and its receptor, CXCR4, in periodontal tissue and to investigate the properties of CXCL12 and CXCR4 expression by human gingival fibroblasts (HGF). RT-PCR analysis revealed that CXCL12 and CXCR4 mRNA were expressed in both normal gingival tissues and periodontal diseased tissues. Immunohistochemistry disclosed that CXCL12 was expressed and CXCR4 positive cells were found in both normal and periodontal diseased gingival tissues. Our in vitro experiments elucidated that HGF constitutively produced CXCL12, and the levels were enhanced by stimulation with tumour necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma), transforming growth factor-beta (TGF-beta), regulated upon activation normal T cell expressed and secreted (RANTES) and macrophage inflammatory protein 3(alpha) (MIP-3(alpha)). On the other hand, heat killed Porphyromonas gingivalis (P. gingivalis) and P. gingivalis LPS reduced the CXCL12 production by HGF. Flow cytometry analysis clarified that CXCR4 was highly expressed on HGF, and CXCR4 expression was abrogated by TNF-alpha, IFN-gamma and P. gingivalis LPS. Moreover, CXCL12 induced vascular endothelial growth factor (VEGF) production by HGF. Our results demonstrated that CXCL12 might be related to CXCR4+ cells infiltration and angiogenesis both in normal periodontal tissues and periodontal diseased tissue. P. gingivalis, a known periodontal pathogen, inhibits the production of CXCL12 and the expression of CXCR4 by HGF. This fact means that P. gingivalis may inhibit CXCR4+ cells infiltration and neovascularization in periodontal tissue and escape from the immune response. (+info)