Identification of a novel adhesion molecule in human leukocytes by monoclonal antibody LB-2

Monoclonal antibody LB-2 to a surface antigen on human B cells, lymphoblast, monocytes and vascular endothelial cells largely inhibited adhesion among Epstein Barr virus-immortalized normal B cells (EBV-B) and concanavalin A-stimulated blood mononuclear cells (Con A-BMC) before and after phorbol ester treatment. The antibody inhibited to a lesser extent phorbol ester-induced aggregation of monocytes, U937 cells and fresh BMC and had virtually no inhibitory effect on the adhesion among enriched T cells and granulocytes. A surface glycoprotein band of 84 kDa was obtained from EBV-B cells by immunoprecipitation and gel electrophoresis. Immunological and biochemical studies clearly distinguished this molecule from gp90 and associated glycoproteins which also mediate leukocyte adhesion.     

Coxsackie- and adenovirus receptor (CAR) is expressed in lymphatic vessels in human skin and affects lymphatic endothelial cell function in vitro

Lymphatic vessels play an important role in tissue fluid homeostasis, intestinal fat absorption and immunosurveillance. Furthermore, they are involved in pathologic conditions, such as tumor cell metastasis and chronic inflammation. In comparison to blood vessels, the molecular phenotype of lymphatic vessels is less well characterized. Performing comparative gene expression analysis we have recently found that coxsackie- and adenovirus receptor (CAR) is significantly more highly expressed in cultured human, skin-derived lymphatic endothelial cells (LECs), as compared to blood vascular endothelial cells. Here, we have confirmed these results at the protein level, using Western blot and FACS analysis. Immunofluorescence performed on human skin confirmed that CAR is expressed at detectable levels in lymphatic vessels, but not in blood vessels. To address the functional significance of CAR expression, we modulated CAR expression levels in cultured LECs in vitro by siRNA- and vector-based transfection approaches. Functional assays performed with the transfected cells revealed that CAR is involved in distinct cellular processes in LECs, such as cell adhesion, migration, tube formation and the control of vascular permeability. In contrast, no effect of CAR on LEC proliferation was observed. Overall, our data suggest that CAR stabilizes LEC-LEC interactions in the skin and may contribute to lymphatic vessel integrity.     

The murine CD99-related molecule CD99-like 2 (CD99L2) is an adhesion molecule involved in the inflammatory response

CD99, a glycoprotein found on the surfaces of leukocytes and concentrated at the borders of endothelial cells, plays a major role in the migration of leukocytes across endothelial cells into sites of inflammation, and has other roles in thymocyte development. The human and mouse genomes encode only two proteins related to CD99. One of these, XGA, is a red blood cell surface antigen. The function of the other, CD99-like 2 (CD99L2), is not known. We cloned mouse CD99L2 and used CD99L2 isolated from transfected cells to raise specific antibodies. Similar to human CD99, CD99L2 was expressed at the borders between transfected cells as well as on mouse leukocytes and vascular endothelial cells in situ. Transfection of L cell fibroblasts with CD99L2 imparted to them the ability to adhere to each other in a divalent cation-dependent, homophilic manner. Anti-CD99L2 antibody blocked influx of neutrophils and monocytes into a site of inflammation in vivo.     

Glycoconjugate and adhesion molecule changes during the cell cycle in a human embryonic epithelial cell line

The changes in the expression of glycoconjugates and adhesion molecules were studied by selective lectin binding and immunocytochemical reactions in a human embryonic epithelial cell line (EUE cells), synchronized in the cell cycle phases. The results can be summarized as follows: most of the tested lectins display a more diffuse binding for the cytoplasm in G₁ than S and G₂ phases; in the S and particularly in G₂ phases the cytoplasm glycoconjugates are rearranged around the nucleus; cells in mitosis always show a strong binding towards all tested lectins. Cellular fibronectin and its receptor β₁ integrin are well expressed in G₁, but the strongest reaction is observed in the S phase. The immunoreactions for laminin and uvomorulin (L-CAM) are poorly positive in all cell cycle phases. The immunocytochemical reaction for heparan sulfate is positive, with a stronger reaction in S and G₂ than in G₁; on the contrary a diffuse staining with the anti-dermatan sulfate proteoglycan antibody appears unchanged during the cell cycle.     

Curcumin decreases toll-like receptor-2 gene expression and function in human monocytes and neutrophils

Toll-like receptor-2 (TLR2) is a pattern recognition receptor that senses many types of bacterial components and activates signaling pathways that induce inflammatory cytokines. A hyperresponsiveness to pathogens caused by increased expression of TLR2 triggers exaggeration of some inflammatory diseases. Here, we showed that curcumin, a well-known anti-inflammatory agent derived from the curry spice turmeric, inhibits TLR2 expression in various TLR2-expressing innate immune cell lines such as monocytic THP-1 cells, neutrophilic-differentiated HL-60 cells. Strong suppression of TLR2 gene expression was specifically observed at concentrations of curcumin in the range 40-100 μM. Consistent with decreased expression of TLR2 mRNA, protein expression and ligand-responsiveness of TLR2 were markedly reduced by curcumin treatment. Moreover, curcumin-dependent down-regulation of TLR2 expression and function was also observed in primary peripheral blood monocytes (MC) and polymorphonuclear neutrophils (PMN). Finally, we determined the importance of curcumin-dependent radical generation for the suppressive effect of curcumin on TLR2 expression. Thus, our data demonstrate that curcumin inhibits TLR2 gene expression and function possibly via an oxidative process.     

Polymorphisms in vitamin D receptor,toll-like receptor 2 and Toll-Like receptor 4 genes links with Dengue susceptibility

Host genetic factors are known to determine disease susceptibility in dengue virus infection. Therefore, in this study association of gene polymorphisms of Vitamin D Receptor [rs731236 (Taq) and rs7975232 (Apa1)], Toll-like receptor 2 [rs5743708 (Arg735Gln) and rs5743704 (Pro631His)] and Toll-like receptor 4 [rs4986790A/G(Asp299Gly13843) and rs4986791 C/T(Thr399Ile)] were studied in cases with dengue as compared to controls. Total 98 cases of confirmed dengue virus infection and 98 age, sex and geographically matched healthy controls were enrolled and their genetic polymorphisms for the above mentioned regions were studied by Sanger sequencing. Mutant genotypes CC of VDR rs731236 (Taq1) [(OR 3.808, p value =0.02, CI 1.160-12.498)], GG of VDR rs7975232 (Apa1) [(OR 3.485, p value =0.02, CI 1.162-10.45)] and heterozygous genotypes of TLR4 rs4986790 A/G Asp299Gly [OR 2.40, p value= 0.02, CI 1.12-5.14], TLR4 rs4986791 C/T Thr399Ile [OR 2.09, p value=0.02, CI 1.12-5.14] were found to be significantly more in cases with dengue virus infection as compared to the controls. Also, at these positions mutant alleles were observed in significantly higher number of cases than controls. The values for C allele at VDR rs731236 (Taq1) were OR 1.86, p value 0.009, CI 1.162-3.001; for allele G at rs7975232( Apa1) were OR 2.71, p value 0.006, CI 1.196-2.98 for allele G at TLR4s rs4986790 A/G Asp299Gly were OR 2.35, p value 0.009, CI 1.23-4.50 and for allele T at rs4986791 C/T Thr399Ile were OR 2.36, p value=0.006, CI 1.28-4.38. VDR and TLR4 but not TLR2 gene polymorphisms were found to be associated with dengue susceptibility in Indian population.     

Protein kinase D1 and D2 are involved in chemokine release induced by toll-like receptors 2, 4, and 5

The protein kinase D (PKD) family consists of three serine-threonine kinases involved in cellular proliferation, motility, and apoptosis. We previously reported that human toll-like receptor 5 (TLR5) contains a consensus PKD phosphorylation site. Flagellin stimulation of cells activated PKD1, and inhibition of PKD1 reduced flagellin-induced interleukin-8 (IL-8) production in epithelial cells. In the current work, we examined PKD1 and PKD2 involvement downstream of TLR5, TLR4 and TLR2. We found that inhibition of either kinase with shRNA reduced IL-8 and CCL20 release due to TLR4 and TLR2 agonists to a similar extent as previously reported for TLR5. PKD1 and PKD2 inhibition reduced NF-κB activity but not MAPK activation. These results demonstrate that both PKD1 and PKD2 are required for inflammatory responses following TLR2, TLR4, or TLR5 activation, although PKD1 is more strongly involved. These kinases likely act downstream of the TLRs themselves to facilitate NF-κB activation but not MAP kinase phosphorylation.     

Detection of intracellular cytokines during antioxidant supplementation in corticoid-dependent asthmatics and modulation of adhesion molecule expression on cultured endothelial cells

In this study, we report on the interferon-γ (IFN-γ) and interleukin-4 (IL-4) cytokine responses to phorbol myristate acetate (PMA)+ionomycin-stimulated CD3+ lymphocytes in asthmatic subjects when compared with normal donors. There was a significantly lower production of intracellular IFN-γ in asthmatic patients. No difference was found for IL-4 production between these two groups. After administration of a multivitamin-mineral supplement containing selenium, zinc, vitamin A, vitamin B₆, vitamin C, and vitamin E for 6 mo, a significant increase in the percentage of CD3+/IL-4 positive cells (p<0.05) was found. The induction of endothelial cell adhesion molecule (CAM) expression in cultured human umbilical vein endothelial cells (HUVEC) and whole-blood mixture was studied using flow cytometry. The ICAM-1 and VCAM-1 expressions were higher in the patients than in control donors (p<0.05). There is a correlation between the increased percentage of CD3+/IFN-γ positive cells and reduced endothelial ICAM-1 and VCAM-1 expression after 6 mo of intervention period. No apparent effect of supplementation on CAM expression was found, suggesting that these changes do not arise from an antioxidant mechanism. This newly developed whole-blood technique for the assessment of CAM expression can be of use for monitoring therapy in inflammatory diseases.     

Human immunodeficiency virus type 1 Nef in human monocyte-like cell line THP-1 expands treg cells via toll-like receptor 2

CD4(+) CD25(+) regulatory T cells (Tregs) represent a unique T-cell lineage that is endowed with the ability to actively suppress immune responses in order to inhibit pathogenic damage resulting from over activation of the immune system. In human immunodeficiency virus-1 (HIV-1) infection, suppression of the immune response by Tregs appears to play an opposing role that promotes chronic viral infection. Treg expansion is known as a marker of the severity of HIV infection and as a potential prognostic marker of disease progression. HIV-1 Nef is one of the earliest expressed viral regulatory genes whose expression may play an important role in regulating Treg cells. We established a THP-1 cell line stably expressing HIV-1 Nef and showed that Nef protein was a potent factor for increasing Treg numbers in vitro. We further found that TLR2 plays a critical role in the increase in Treg cells induced by Nef using TLR2-specific siRNA. Our results suggest new strategies for therapeutic and preventive interventions of HIV infection.     

Activation of BV2 microglia by lipopolysaccharide triggers an inflammatory reaction in PC12 cell apoptosis through a toll-like receptor 4-dependent pathway

Microglia play an important role in neuronal protection and damage. However, the molecular and cellular relationship between microglia and neurons is unclear. We carried out a prospective study to detect that activation of BV2 microglia induced PC12 cell apoptosis in vitro through the TLR4/adapter protein myeloid differentiation factor 88 (MyD88)/nuclear factor-κB (NF-κB) signaling pathway. BV2 microglia were treated with different concentrations of LPS for 24 h. Western blot was utilized to detect the expression of TLR4 and the downstream signaling pathway. The level of inflammatory mediator was quantified using a specific ELISA kit. The supernatant of 10 μg/ml LPS-treated BV2 cells was used as conditioned medium (CM). PC12 cells were co-culture with CM for 24 h. Cell viability was determined by MTT assay and cell apoptosis was tested by flow cytometry. BV2 microglia were treated with 10, 20, or 30 μg/ml LPS for 24 h. The expression of TLR4, MyD88, and NF-κB significantly increased. When PC12 cells were co-cultured with CM for 24 h, cell viability decreased. CM up-regulated the Bax level and down-regulated the Bcl-2 protein level in PC12 cells. PC12 cells pretreated with interleukin-1 receptor antagonist (IL-1RA) for 30 min, significantly alleviated CM-induced PC12 cell apoptosis. These results suggest that BV2 microglia activated by LPS triggered TLR4/MyD88/NF-κB signaling pathway that induced the release of IL-1β and could participate in the PC12 cells injury.     

Thrombin-stimulated Pyk2 phosphorylation in human endothelium is dependent on intracellular calcium and independent of protein kinase C and Src kinases

G-protein-coupled receptor agonists (GPCAs) cause functional responses in endothelial cells including secretion, proliferation, and altering monolayer permeability. These events are mediated in part by activation of the p42/44 mitogen-activated protein kinase (MAPK) cascade. The cytosolic tyrosine kinase Pyk2 is postulated to link GPCA-induced changes in intracellular calcium to activation of the MAP kinase cascade. We have investigated the regulation of Pyk2 in human umbilical vein endothelial cells in response to GPCAs and show that (1) thrombin, a PAR-1 peptide, and histamine cause rapid concentration- and time-dependent phosphorylation on tyrosines 402 (Src kinase binding site), 881 (Grb2 binding site), and 580 (an autophosphorylation site), (2) thrombin-stimulated phosphorylation is dependent on intracellular calcium and independent of PKC and PI-3 kinase, and (3) inhibition of Src kinases has no significant effect on thrombin-stimulated phosphorylation, implying that tyrosine phosphorylation of Pyk2 is independent of Src binding.     

Effects of a 50 Hz sinusoidal magnetic field on cell adhesion molecule expression in two human osteosarcoma cell lines (MG-63 and Saos-2)

The possibility that a sinusoidal 50 Hz magnetic field with a magnetic flux density of 0.5 mT can induce variations in the expression of cell adhesion molecules (CAMs) in two human osteosarcoma cell lines (MG-63 and Saos-2) was investigated. In particular, the expression of two important integrins, VLA-2, the receptor for collagen, and VLA-5, the receptor for fibronectin, as well as CD44, were examined in both cell lines after these had been exposed for 7 and 14 days to a 50 Hz, 0.5 mT field. Cell surface morphology (scanning electron microscopy), cell growth characteristics (growth curves and cell cycle phase distribution), and cell death (necrosis and apoptosis) were also examined. The results demonstrate that no variations in surface morphology and cell death occurred between control and exposed cells in both MG-63 and Saos-2 cells, while significant changes were noted in cell growth and fibronectin and CD44 expression in MG-63 cells. The results are discussed in view of the important role that CAMs play in controlling various cancer cell functions, particularly proliferation and metastasis.     

Distributional characteristics of lymphatic vessels in normal human nasal mucosa and sinus mucosa

An immunohistochemical staining technique with the D2-40 antibody was undertaken to examine the functional and morphological features of lymphatic networks in tissue sections and whole-mount preparations of normal nasal mucosa and ethmoid sinus mucosa. In normal nasal mucosa, most lymphatic vessels were found in the superficial mucosa beneath the epithelial layer. Some of these vessels were dilated, whereas others were compressed and had a slit-like lumen. Whole-mount preparations revealed the extent of lymphatic vessels in normal ethmoid sinus mucosa. A network of lymphatic vessels was mainly found in the subepithelial layer, where lymphatic vessels represented rich networks, possessing antler-like branches and typical blind ends. However, these lymphatic networks were not arranged in the form of lymphangion chains, with each lymphangion consisting of a contractile compartment and valve. Thus, recognition of the distinctive features of the lymphatic network in normal nasal and sinus mucosa might aid investigations of lymphatic involvement in sinonasal diseases, such as rhinitis, sinusitis, and malignancy. This work was supported by a Grant-in-Aid for Scientific Research from the Communication Disorders Center, Korea University, Korea.     

The cell-cell adhesion molecule carcinoembryonic antigen-related cellular adhesion molecule 1 inhibits IL-2 production and proliferation in human T cells by association with Src homology protein-1 and down-regulates IL-2 receptor

The cell adhesion molecule, carcinoembryonic Ag-related cellular adhesion molecule 1, shown by others to both activate and inhibit T cell proliferation, exhibits a reciprocal relationship to IL-2R expression over the time course of activation of PBMCs, and upon Ab ligation, inhibits both the production of IL-2 and cell proliferation. Carcinoembryonic Ag-related cellular adhesion molecule 1 associates with CD3 and is found in lipid rafts of PBMCs, is phosphorylated on the immunoreceptor tyrosine-based inhibitory motifs (ITIMs) of the -4L isoform, and associates with Src homology protein-1, providing an explanation for its inhibitory activity. When the ITIM-containing -4L and non-ITIM-containing -4S isoforms are transfected into Jurkat cells that produce, but do not depend on IL-2 for growth, both IL-2 production and cell proliferation are differentially inhibited, demonstrating that the two isoforms signal via different pathways. When the two isoforms are transfected into Kit-225 cells that depend on IL-2 for growth, IL-2Rbeta and gamma, but not alpha subunits are down-regulated, and the -4L, but not the -4S isoform inhibits cell proliferation by 6-fold in an IL-2 dose-response study.     

ShcA regulates neurite outgrowth stimulated by neural cell adhesion molecule but not by fibroblast growth factor 2: evidence for a distinct fibroblast growth factor receptor response to neural cell adhesion molecule activation

Homophilic binding in trans of the neural cell adhesion molecule (NCAM) mediates adhesion between cells and leads, via activation of intracellular signaling cascades, to neurite outgrowth in primary neurons as well as in the neuronal cell line PC12. NCAM mediates neurite extension in PC12 cells by two principal routes of signaling: NCAM/Fyn and NCAM/fibroblast growth factor receptor (FGFR), respectively. Previous studies have shown that activation of mitogen-activated protein kinases is a pivotal point of convergence in NCAM signaling, but the mechanisms behind this activation are not clear. Here, we investigated the involvement of adaptor proteins in NCAM and fibroblast growth factor 2 (FGF2)-mediated neurite outgrowth in the PC12-E2 cell line. We found that both FGFR substrate-2 and Grb2 play important roles in NCAM as well as in FGF2-stimulated events. In contrast, the docking protein ShcA was pivotal to neurite outgrowth induced by NCAM, but not by FGF2, in PC12 cells. Moreover, in rat cerebellar granule neurons, phosphorylation of ShcA was stimulated by an NCAM mimicking peptide, but not by FGF2. This activation was blocked by inhibitors of both FGFR and Fyn, indicating that NCAM activates FGFR signaling in a manner distinct from FGF2 stimulation, and regulates ShcA phosphorylation by the concerted efforts of the NCAM/FGFR as well as the NCAM/Fyn signaling pathway.     

Single-bulb garlic oil regulates toll-like receptors and Nrf2 cross-talk and IL-17 production in mice fed with high-fat diet

The present study aimed to evaluate the effect of single-bulb garlic oil (SGO) on toll-like receptors 3 and 4 (TLR3 and TLR 4) and nuclear erythroid factor-like 2 (Nrf2) signaling pathway resulted from a high-fat diet and its underlying mechanism. Twenty-four Balb/c mice allocated into six groups: 1) N: mice fed with standard chow; 2) HFD: mice fed a high-fat diet for 45 days without any treatment; 3) HFD + Simv: mice fed a high-fat diet for 45 days and treated with simvastatin; 4–6) HFD + SGO 100, 200, 400 (mice fed a high-fat diet for 45 days and treated with single-bulb garlic oil at dose: 100, 200, and 400 mg/kg body weight for 30 days), respectively. At the end of treatment, spleen and hepar were isolated. The flow cytometry analysis was performed to analyze the relative number of nrf2, superoxide dismutase (SOD), malondialdehyde (MDA), TLR3, TLR4 and interleukin (IL-17). The results showed that HFD induction significantly reduced Nrf-2 and antioxidant enzyme levels. Furthermore, HFD induction increased TLR3 and TLR4 signaling and IL-17 production. Interestingly, 200 mg/kg BW of SGO increased the relative number Nrf-2 followed by SOD and HO-1 elevation at a dose of 100 mg/kg BW. SGO100 notably decrease the relative number of TLR3 (CD11b⁺TLR3⁺) and TLR4 (CD11b⁺TLR4⁺). The production of IL-17 by CD4 and CD8 were also reduced after receiving SGO at 200 mg/kg BW. This study suggests that the protective effect of SGO treatment on HFD mice was achieved by modulating TLR-Nrf2 cross-talks and decreasing IL-17 production. Our findings support a potential beneficial role of SGO for treating metabolic disease caused by a high-fat diet.     

Roxithromycin downmodulates Th2 chemokine production by keratinocytes and chemokine receptor expression on Th2 cells: its dual inhibitory effects on the ligands and the receptors

Roxithromycin (RXM), an anti-bacterial macrolide, has various immunomodulatory activities. To investigate the ability of RXM to downregulate skin-infiltration of T-lymphocytes, we examined the effects of RXM on keratinocyte production of chemokines and T cell expression of chemokine receptors. Normal human and HaCaT keratinocytes were cultured with RXM and stimulants. RXM at 1 or 10 microM significantly suppressed the production/expression of Th2 chemokines MDC and TARC in these keratinocytes, but the production of IP-10 was not affected. The effect of RXM on T-cell expression of the corresponding chemokine receptors was also tested in Th2-rich peripheral blood lymphocytes. The IL-2-enhanced expression level of Th2 chemokine receptor CCR4 was decreased by RXM at 10 microM, whereas the expression of CXCR3 was unchanged. Thus, RXM downmodulates both the production and receptor expression of Th2 but not Th1 chemokines involved in cutaneous immunity, suggesting its beneficial therapeutic effects on Th2-mediated or allergic skin disorders.