Proinflammatory cytokines induce liver and activation-regulated chemokine/macrophage inflammatory protein-3alpha/CCL20 in mucosal epithelial cells through NF-kappaB [correction of NK-kappaB].

Liver and activation-regulated chemokine (LARC)/CCL20 is expressed by surface-lining epithelial and epidermal cells, and is likely to link innate and acquired immunity by attracting immature dendritic cells, effector memory T cells and B cells via CCR6. Here we examined the mechanism of LARC expression in epithelial-type cells. Either IL-1beta or tumor necrosis factor (TNF)-alpha strongly induced LARC mRNA in intestinal cell lines Caco-2 and T84, while both were effective on HEK 293T cells. Induction of LARC was also demonstrated in the intestinal epithelium of BALB/c mice upon treatment with IL-1alpha or TNF-alpha. Transient transfection assays using murine LARC promoter-reporter constructs identified a region essential for IL-1beta- or TNF-alpha-induced promoter activation in Caco-2 and 293T cells. Using site-directed mutagenesis, we demonstrated that an NF-kappaB site located between -96 and -87 bp upstream from the transcriptional start site was both necessary and sufficient for IL-1beta- or TNF-alpha-induced promoter activation in Caco-2 and 293T cells. Electrophoretic mobility shift assays demonstrated that p50/p65 heterodimer and p65 homodimer of NF-kappaB bound to this site in 293T cells upon treatment with IL-1beta and TNF-alpha, and p50/p65 heterodimer bound to this site in Caco-2 cells upon treatment with IL-1beta. Co-expression of constitutively active p65 strongly activated the promoter construct carrying the intact NF-kappaB site in 293T and Caco-2 cells. Collectively, LARC expression in intestinal epithelial-type cells is induced by proinflammatory cytokines such as IL-1 and TNF-alpha primarily through activation of NF-kappaB.

Inducible expression of a CC chemokine liver- and activation-regulated chemokine (LARC)/macrophage inflammatory protein (MIP)-3 alpha/CCL20 by epidermal keratinocytes and its role in atopic dermatitis.

Liver-and activation-regulated chemokine (LARC)/macrophage inflammatory protein (MIP)-3alpha/CCL20 is a CC chemokine which is constitutively expressed by follicle-associated epithelial cells in the mucosa, and attracts cells expressing CCR6 such as immature dendritic cells and alpha(4)beta(7)(high) intestine-seeking memory T cells. Here, we examine LARC/CCL20 expression in the skin. LARC/CCL20 mRNA and protein were induced in primary human keratinocytes upon stimulation with proinflammatory cytokines such as IL-1alpha and tumor necrosis factor (TNF)-alpha. In mice, intradermal injection of IL-1alpha and TNF-alpha rapidly induced a local accumulation of transcripts for LARC/CCL20 and its receptor CCR6 with a lag of several hours in the latter. In humans, immunostaining of LARC/CCL20 was weak if any in normal skin tissues but strongly augmented in lesional skin tissues with atopic dermatitis. Furthermore, massive infiltration of cells with markers such as CD1a, CD3 or HLA-DR was present in atopic skin lesions. Many infiltrating cells were also found to be CCR6(+) by a newly generated monoclonal anti-CCR6. However, Langerhans cells residing within the epidermis were hardly stained by anti-CCR6 in normal and atopic skin tissues. Furthermore, plasma levels of LARC/CCL20 were found to be elevated in patients with atopic dermatitis. Collectively, our results suggest that epidermal keratinocytes produce LARC/CCL20 upon stimulation with proinflammatory cytokines such as IL-1alpha and TNF-alpha, and attract CCR6-expressing immature dendritic cells and memory/effector T cells into the dermis of inflamed skin such as atopic dermatitis. LARC/CCL20 may not, however, play a major role in homeostatic migration of Langerhans cells into the skin.

Expression profile of active genes in mouse lymph node high endothelial cells.

High endothelial venules (HEV) allow rapid and selective lymphocyte trafficking from the blood into secondary lymphoid tissues. Here we report the expression profile of active genes in mouse high endothelial cells (HEC). HEC were first purified from mouse lymph nodes (LN) by magnetic cell sorting with MECA-79 mAb and a 3'-directed cDNA library that faithfully represents the composition of mRNA was constructed. A total of 1495 cDNA sequences were obtained from randomly selected clones. Based on their sequence identity, they were grouped into 754 different species [gene signatures (GS)] of which 335 GS were identified in GenBank. Among the previously identified genes, expression of several endothelial cell surface molecules including endoglin and ICAM-1 was detected in HEC. Comparison of the gene expression profile with that of purified CD31(+) flat endothelial cells identified several molecules, such as KC chemokine and Duffy antigen/receptor for chemokines, that are known to be selectively expressed in activated endothelial cells or post-capillary venules. Interestingly, mac25/TAF, which is known to be expressed specifically in tumor vessels and implicated in the regulation of cell adhesion, was highly and selectively expressed in HEC in mouse LN, suggesting that it may participate in regulating HEC-specific functions. Comparison with the expression profiles obtained from 35 different cell types showed at least 22 GS that were apparently specific to HEC. Our results illustrate the expression differences between HEC and CD31(+) flat endothelial cells, and will be useful for the identification and characterization of genes specific for HEC.

Molecular cloning of a novel CC chemokine, interleukin-11 receptor alpha-locus chemokine (ILC), which is located on chromosome 9p13 and a potential homologue of a CC chemokine encoded by molluscum contagiosum virus.

Molluscum contagiosum virus (MCV) encodes a CC chemokine MC148R which is likely to have been acquired from the host. By a homology search employing MC148R as a probe, we have identified a novel CC chemokine whose gene exists next to the IL-11 receptor alpha (IL-11Ralpha) gene in both humans and mice. Thus, this chemokine maps to chromosome 9p13 in humans where IL-11Ralpha has been assigned. We term this novel chemokine IL-11Ralpha-locus chemokine (ILC). ILC has the highest homology to MC148R among the known human CC chemokines. Furthermore, ILC is strongly and selectively expressed in the skin where infection of MCV also takes place. Thus, ILC is likely to be the original chemokine of MC148R.

Molecular cloning of mXCR1, the murine SCM-1/lymphotactin receptor.

Single C motif-1 (SCM-1)/lymphotactin is a C-type member of the chemokine superfamily. Previously, we identified its specific receptor XCR1. Here we isolated the murine homologue of XCR1 (mXCR1). To demonstrate its biological activity, we produced recombinant mouse SCM-1 by the baculovirus expression system. B300-19 murine pre-B cells expressing mXCR1 responded to mSCM-1 in chemotactic and calcium-mobilization assays. mXCR1 mRNA was weakly expressed in spleen and lung of normal C57BL/6 mice. In spleen, CD8+ cells and NK1.1+ cells were found to express mXCR1. Identification of mXCR1 will now allow us to study the role of this unique cytokine system in the mouse models of inflammation and immunity.

A novel, high endothelial venule-specific sulfotransferase expresses 6-sulfo sialyl Lewis(x), an L-selectin ligand displayed by CD34.

L-selectin mediates lymphocyte homing by facilitating lymphocyte adhesion to unique carbohydrate ligands, sulfated sialyl Lewis(x), which are expressed on high endothelial venules (HEV) in secondary lymphoid organs. The nature of the sulfotransferase(s) that contribute to sulfation of such L-selectin counterreceptors has been uncertain. We herein describe a novel L-selectin ligand sulfotransferase, termed LSST, that directs the synthesis of the 6-sulfo sialyl Lewis(x) on L-selectin counterreceptors CD34, GlyCAM-1, and MAdCAM-1. LSST is predominantly expressed in HEV and exhibits striking catalytic preference for core 2-branched mucin-type O-glycans as found in natural L-selectin counterreceptors. LSST enhances L-selectin-mediated adhesion under shear compared to nonsulfated controls. LSST therefore corresponds to an HEV-specific sulfotransferase that contributes to the biosynthesis of L-selectin ligands required for lymphocyte homing.

Constitutive expression of GlyCAM-1 core protein in the rat cochlea.

Glycosylation-dependent cell adhesion molecule-1 (GlyCAM-1) is a mucin-like glycoprotein previously identified on high endothelial venules (HEV) of lymph nodes and also in lactating mammary glands. A specifically glycosilated form of GlyCAM-1 on HEV has been shown to be a ligand for a leukocyte L-selectin, which plays an important role in leukocyte rolling along the inflamed endothelium. Here we report that GlyCAM-1 is also expressed in the cochlea. Immunohistochemistry revealed the lateral wall of the cochlea, tectorial membrane, modiolus, organ of corti, and spiral modiolar vein (SMV) to be strongly stained with polyclonal anti-GlyCAM-1 antibody. Moreover, RT-PCR of the cochlear tissue by the use of specific oligonucleotide primers for rat GlyCAM-1 generated a 378 bp product which was then verified by nucleotide sequencing to represent GlyCAM-1. Electron microscopic investigation revealed the presence of GlyCAM-1 over the entire lumenal surface of the vessels, and the basolateral infoldings in stria vascularis. However, soluble L-selectin or mAb MECA-79 which recognizes a carbohydrate epitope on functional L-selectin ligands bound only to the spiral ligament, tectorial membrane and modiolus. These observations suggest that GlyCAM-1 expressed in the cochlear region is heterogenous in terms of its glycosylation.

RNase P RNA of Mycoplasma capricolum.

There are at least six small stable RNAs in Mycoplasma capricolum cells besides tRNAs and rRNAs. One of them, MCS5 RNA, is a homolog of RNase P RNA. The predicted secondary structure of this RNA is essentially the same as that of other eubacterial RNase P RNAs. MCS5 RNA is more similar to the RNase P RNA of B. Subtilis than to that of E. coli. This is consistent with previous conclusions that mycoplasmas are phylogenetically related to the low G + C Gram-positive bacterial group. The major substrates for MCS5 RNA must be the precursors of tRNAs. The precursor of MCS6 RNA, which is a homolog of the E. coli 10Sa RNA, may also be a substrate for the MCS5 RNA because this RNA has a tRNA-like structure at its 5' and 3' ends.