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S W Caughman

Publications and source records attributed to S W Caughman.

At least 37 records · Page 2Linked to original sources
PubMed1997

Ionizing radiation induces, via generation of reactive oxygen intermediates, intercellular adhesion molecule-1 (ICAM-1) gene transcription and NF kappa B-like binding activity in the ICAM-1 transcriptional regulatory region.

Ionizing radiation produces reactive oxygen intermediates in mammalian tissues and may serve as a model system for the investigation of the biologic effects of free radicals. We have previously shown that the adhesion molecule ICAM-1 is induced by ionizing radiation, and here we have investigated the molecular mechanisms responsible. ICAM-1 mRNA and cell surface expression was induced in HeLa and HaCaT cells after exposure to ionizing radiation. This induction was blocked by preincubation with the antioxidants PDTC and N-acetyl cysteine. ICAM-1 promoter activity was assessed by transiently transfecting HeLa cells with CAT-reporter gene constructs containing sequential ICAM-1 5' deletions. ICAM-1 5' fragments -1162/+1 (relative to the transcription start site) and -277/+1 displayed increased promoter activity when cells were exposed to ionizing radiation, but no induction was seen in a -182/+1 construct associating positions -277 to around -182 with inducibility by ionizing radiation. Nuclear extracts from HaCaT cells were tested in mobility shift assays using an NF kappa B-like binding site of the ICAM-1 5' region (positions -186/-177). There was marked enhancement of DNA-protein complex forming in extracts from irradiated versus untreated cells. Incubation of cells with antioxidants prior to irradiation prevented the radiation-dependent increase in complex formation. We conclude that reactive oxygen intermediates are involved in ICAM-1 induction by ionizing radiation. The ionizing radiation-induced, antioxidant-inhibitable binding at the ICAM-1 NF kappa B-like binding site is consistent with the view that NF kappa B is a pro-oxidant transcription factor.

Binding Sites↗
PubMed1996

Differential regulation of vascular cell adhesion molecule-1 gene transcription by tumor necrosis factor alpha and interleukin-1 alpha in dermal microvascular endothelial cells.

As part of the inflammatory response, the localization of leukocytes depends to an important degree on cytokine-induced expression of vascular cell adhesion molecule-1 (VCAM-1) on endothelial cells (EC). We have previously shown that VCAM-1 expression is induced on human umbilical vein EC (HUVEC) by both tumor necrosis factor alpha (TNF-alpha) and interleukin-1 alpha (IL-1 alpha), whereas on human dermal microvascular EC (HDMEC) only TNF alpha results in VCAM-1 expression. To explore molecular mechanisms responsible for these contrasting patterns of VCAM-1 induction in HUVEC versus HDMEC, we performed transcriptional activation studies with VCAM-1-based reporter constructs and in vitro binding assays using two adjacent NF-kappa B binding sequences of the VCAM-1 promoter as a DNA probe. Previous studies have established that these NF-kappa B motifs are required for cytokine-induced VCAM-1 transcription, and may further mediate cell-specific VCAM-1 gene activation by cytokines. The findings reported here demonstrate a significant HDMEC-specific attenuation of VCAM-1 gene transcription in response to IL-1 alpha, but not TNF alpha. An upstream VCAM-1 gene regulatory region distinct from the NF-kappa B sites appears to function as an IL-1 alpha-mediated transcriptional repressor within HDMEC. This repressor region conveys IL-1 alpha-dependent, but not TNF alpha-dependent, inhibition of transcription driven by a heterologous cytokine response element and promoter.

Base Sequence↗
PubMed1996

Anti-metastatic prostacyclins inhibit the adhesion of colon carcinoma to endothelial cells by blocking E-selectin expression.

Prostacyclins have long been shown to have anti-metastatic activity. One hypothesis is their modulation of cell adhesion molecule (CAM) expression by target organ endothelial cells. We have postulated that prostacyclin, its analogs, and mechanistic mimics decrease colon carcinoma adhesion to cytokine-stimulated endothelial cells by blocking endothelial expression of the adhesion molecule E-selectin, but not the vascular cell adhesion molecule-1 (VCAM-1). Cultured human microvascular endothelial cells (HDMEC) were pre-incubated with prostacyclin (PGI2), dibutyrl-cAMP (dbcAMP), forskolin (FOR), and/or iso-methylbutylxanthine (IBMX) for 15 min, then co-incubated with the cytokine tumor necrosis factor (TNF) for 4 h. HDMEC surface expression of E-selectin and VCAM-1 was evaluated by flow cytometry and ELISA. Adherence of 51Cr-labeled colon carcinoma cells to HDMEC monolayers was then determined. In parallel assays, HDMECs were incubated with anti-E-selectin and anti-VCAM-1 monoclonal antibody (1:100) prior to the addition of tumor cells. Prostacyclins, its analogs, and mimics significantly reduced E-selectin expression by HDMEC, while the reduction of VCAM-1 expression was much less pronounced. Prostacyclins also significantly decreased colon carcinoma adherence to stimulated HDMECs. The inhibition of E-selectin expression, but not VCAM-1 expression, corresponded to the reduction of tumor cell adherence. Prostacyclin's effects on tumor adhesion were nullified by pre-incubation with E-selectin antibody. The inhibition of colon carcinoma adherence to cytokine-stimulated endothelial cells treated with prostacyclin, its analogs, and mimics appears to result from blocking endothelial E-selectin, but not VCAM-1, expression. These data support the hypothesis that prostacyclins may exert their anti-metastatic effect, in part, by inhibiting CAM-mediated adherence of colon carcinoma to endothelial cells in metastatic target organs.

1-Methyl-3-isobutylxanthine↗
PubMed1995

Transcriptional regulation of intercellular adhesion molecule-1: PMA-induction is mediated by NF kappa B.

The surface glycoprotein intercellular adhesion molecule-1 (ICAM-1) mediates important immunologic cell interactions during cutaneous inflammatory processes by binding to the leukocyte integrin lymphocyte function-associated antigen-1. The expression of ICAM-1 is induced in epidermal keratinocytes by certain pro-inflammatory stimuli, and this modulation is transcriptionally regulated. To identify the molecular mechanisms involved in the regulation of ICAM-1 gene expression, we have previously cloned the transcriptional regulatory region of the human ICAM-1-gene and have characterized a functional promoter. Here we have used the phorbol ester phorbol-12-myristate-13-acetate (PMA) to further evaluate the transcriptional mechanisms of ICAM-1 gene induction in A431 cells. Exposure to PMA induced ICAM-1 both at the mRNA and cell surface level. Promoter activity and PMA-enhanced effects were assessed by transiently transfecting A431 cells with chloramphenicol acetyl transferase reporter gene constructs containing a series of sequential ICAM-1 5' deletions. Constructs containing ICAM-1 5' fragments from -1162/+1 (relative to the transcription start site) to -277/+1 displayed a threefold increase in promoter activity when cells were stimulated with PMA. Inducibility dropped below 1.5-fold in chloramphenicol acetyl transferase construct -182/+1. Using electrophoretic mobility shift assays, a PMA-inducible binding site was identified for an NF kappa B-like complex within positions -186/-177. A -199/-170 fragment containing this NF kappa B-like element conferred PMA responsiveness when cloned into a thymidine kinase-driven chloramphenicol acetyl transferase vector, indicating that the region containing this NF kappa B-like element is not only necessary but also sufficient for PMA induction of ICAM-1.

Base Sequence↗
PubMed1995

Polarized expression and basic fibroblast growth factor-induced down-regulation of the alpha 6 beta 4 integrin complex on human microvascular endothelial cells.

Endothelial cells rest on a basement membrane that anchors them to the vessel wall. The alpha 6 beta 4 integrin complex has been described on epithelial cells, frequently localizes to basement-membrane structures, and appears to play a role in binding epithelial cells to laminin. We have determined that human microvascular endothelial cells express the beta 4 integrin chain in vivo and that it preferentially localizes to the endothelial basement membrane. Human microvascular endothelial cells and human umbilical vein endothelial cells also express cell-surface beta 4 in vitro. In addition, the expression of beta 4 appears to be polarized to the undersurface of endothelial cell monolayers in vitro, mimicking its in vivo localization. Stimulation of microvascular endothelial cells with basic fibroblast growth factor or phorbol 12-myristate 13-acetate, agents previously shown to induce endothelial cell migration in vitro, resulted in a marked decrease in cell-surface expression of the beta 4 integrin chain, associated with a decrease in beta 4 mRNA. These data demonstrate that human endothelial cells express the beta 4 integrin chain in vivo and in vitro, the expression of this integrin chain is polarized, and its expression is regulated on microvascular endothelial cells by factors important in wound healing and vascular regeneration.

Antigens, Surface↗
PubMed1994

A factor in human plasma permits persistent expression of E-selectin by human endothelial cells.

E-selectin is an inducible endothelial cell adhesion protein that is a critical element in the binding of leukocytes to activated endothelial cells. It is induced by a variety of pro-inflammatory soluble substances including interleukin-1 (IL-1), tumor necrosis factor (TNF), or bacterial lipopolysaccharide (LPS). In vitro studies of a large vessel endothelial cells demonstrate that stimulation with TNF or IL-1 leads to a rapid, but transient, induction of E-selectin expression that disappears within 24 hours. However, in vivo studies have shown that microvascular endothelial cells persistently express E-selectin in chronic inflammatory states, particularly in the skin where it serves as a homing receptor for memory T cells. Stimulation of dermal-derived microvascular endothelial cells (HDMECs) with single doses of IL-1 alpha, TNF alpha, or LPS resulted in transient but slightly more persistent expression of E-selectin than seen after stimulation of large vessel derived umbilical vein endothelial cells (HUVECs). However, stimulation of either HDMECs or HUVECs with repetitive doses of IL-1 alpha, TNF alpha, or LPS in the presence of human serum or plasma resulted in persistent E-selectin expression in vitro. The persistent E-selectin cell surface expression was associated with persistent E-selectin mRNA expression and correlated with E-selectin-mediated HL-60 binding to endothelial cell monolayers. The effect of human plasma or serum was dose dependent, and fractionation of human plasma by gel filtration demonstrated that the E-selectin persistence activity resolved into high and low molecular peaks. These data demonstrate that human endothelial cells are capable of persistent E-selectin expression in vitro and that factors in human serum or plasma are critical in preventing cytokine refractoriness and loss of E-selectin expression. This study provides a basis to resolve the apparent discrepancies between previous in vivo and in vitro dynamics of E-selectin expression.

Blotting, Northern↗
PubMed1994

Selective upregulation of intercellular adhesion molecule (ICAM-1) by ultraviolet B in human dermal microvascular endothelial cells.

Although a portion of ultraviolet light (UV) penetrates into the dermis, histologic changes that occur within the dermal microvasculature have largely been attributed to the elaboration of biologic substances, such as interleukin 1 (IL-1), from other constitutive cells of the skin, as opposed to a direct effect of UV on the endothelial cell. As a potential model for understanding early molecular events occurring in UV-induced cutaneous inflammation, we have examined the direct effects of UVB, as well as cytokine-positive controls, upon human dermal microvascular endothelial cells (HDMEC) cell adhesion molecule (CAM) gene expression. Cultured HDMEC were exposed to varying dosages of UVB, and examined for cell surface and mRNA expression of the CAMs intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and E-selectin (formerly ELAM-1). Following UVB exposure, dose-dependent increases in baseline cell surface expression of ICAM-1 were demonstrated by fluorescence-activated cell sorter analysis with concomitant increases in ICAM-1 mRNA, as shown by Northern blot analysis; there was no induction of either E-selectin or VCAM-1. The UVB-induced ICAM-1 upregulation could not be blocked by antibodies to IL-1 or tumor necrosis factor alpha (TNF-alpha). In fact, ICAM-1 gene regulatory region based CAT reporter gene plasmids, including constructs containing IL-1- and TNF-alpha-responsive elements, did not display increased CAT expression after transfection into HDMEC followed by UVB exposure, though control cytokine-treated transfectants did. Thus, UVB selectively upregulates ICAM-1, but not E-selectin or VCAM-1, mRNA and cell surface expression in HDMEC, and this upregulation is not dependent upon the autologous secretion and activity of either IL-1 or TNF-alpha.

Antibodies↗
PubMed1994

Basic fibroblast growth factor increases expression of the alpha v beta 3 integrin complex on human microvascular endothelial cells.

Modulation of the expression of the alpha v beta 3 complex on human dermal microvascular endothelial cells (HDMEC) may be crucial in wound healing and angiogenesis. Therefore, we examined the influence of basic fibroblast growth factor (bFGF), transforming growth factor beta, and interferon-gamma (IFN-gamma) on the expression of this complex. Stimulation of HDMEC with bFGF increased cell surface expression of both alpha v and beta 3 in a dose- and time-dependent manner associated with the development of a spindled, elongated cell morphology. Northern blot analysis of HDMEC stimulated with bFGF demonstrated a marked increase in beta 3 but not alpha v mRNA expression. Incubation of HDMEC with transforming growth factor-beta or interferon-gamma alone resulted in modest decreases in cell surface alpha v beta 3, and co-incubation of HDMEC with bFGF and transforming growth factor-beta or interferon-gamma inhibited bFGF-induced changes in cell morphology, increases in cell surface alpha v beta 3 expression, and increases in beta 3 mRNA. These data demonstrate that both growth factors and pro-inflammatory cytokines alter the expression of alpha v beta 3 on microvascular endothelial cells and that these alterations correlate with changes in cell morphology.

Blotting, Northern↗
PubMed1994

Ionizing radiation induces human intercellular adhesion molecule-1 in vitro.

Intercellular adhesion molecule-1 (ICAM-1) plays a central role in various inflammatory reactions and its expression is readily induced by inflammatory stimuli such as cytokines or ultraviolet irradiation. We have investigated the effect of ionizing radiation (IR) on human ICAM-1 expression in human cell lines and skin cultures. ICAM-1 mRNA levels in HL60, HaCaT, and HeLa cells were elevated at 3-6 h after irradiation and increased with doses from 10-40 Gy. The rapid induction of ICAM-1 occurred at the level of transcription, was independent of de novo protein synthesis, and did not involve autocrine stimuli including tumor necrosis factor-alpha and interleukin-1. IR also induced ICAM-1 cell surface expression within 24 h. Immunohistologic analysis of cultured human split skin revealed ICAM-1 upregulation on epidermal keratinocytes and dermal microvascular endothelial cells 24 h after exposure to 6 Gy. In conclusion, we propose ICAM-1 as an important radiation-induced enhancer of immunologic cell adhesion, which contributes to inflammatory reactions after local and total body irradiation.

Antibodies↗
PubMed1993

A 5' portion of the ICAM-1 gene confers tissue-specific differential expression levels and cytokine responsiveness.

Intercellular adhesion molecule-1 (ICAM-1), a cell-adhesion molecule critically involved in leukocyte trafficking and adherence, displays tissue-specific and cytokine-specific expression profiles. Although human dermal microvascular endothelial cells (HDMEC) constitutively express ICAM-1, keratinocytes (HK) do not. Interleukin-1 (IL-1) upregulates ICAM-1 expression in HDMEC, but fails to do so in either HK or A431, a human squamous carcinoma cell line, even though both have IL-1 receptors and express ICAM-1 on exposure to other cytokines. We have previously characterized a human ICAM-1 genomic clone that contains the 5' flanking transcriptional regulatory region. To test the hypothesis that tissue- and cytokine-specific ICAM-1 gene expression results from the interaction of constitutive and inducible tissue-specific trans-acting factors with distinct cis-elements of the ICAM-1 gene, various ICAM-1-based reporter gene (CAT) plasmids were constructed. Transcriptional activity of these various constructs was assessed after transient transfection into HDMEC and A431. A critical ICAM-1 region was identified that conferred enhanced expression of CAT in HDMEC and suppressed expression of CAT in A431. This same region further enhanced CAT expression in transfected HDMEC treated with IL-1 alpha, yet no such enhancement was seen with IL-1 treatment of identically transfected A431. However, treatment of A431 transfectants with IFN gamma did result in enhanced CAT expression, demonstrating reversal of A431 cell context suppression of the ICAM-1-based reporter gene construct. These data implicate the existence of both tissue- and cytokine-specific responsive elements in the 5' flanking region of the ICAM-1 gene and demonstrate that regulatory effects directed by such elements are dependent upon their cellular context. Moreover, they provide the basis for identification of specific cis-acting genetic elements, the trans-acting factors with which they interact, and the molecular mechanisms by which they regulate transcription of the ICAM-1 gene.

Base Sequence↗
PubMed1992

Regulation of vascular cell adhesion molecule 1 on human dermal microvascular endothelial cells.

Vascular endothelial cell adhesion molecule 1 (VCAM-1) is an adherence molecule that is induced on endothelial cells by cytokine stimulation and can mediate binding of lymphocytes or tumor cells to endothelium. Because these interactions often occur at the level of the microvasculature, we have examined the regulation of expression of VCAM-1 in human dermal microvascular endothelial cells (HDMEC) and compared it to the regulation of VCAM-1 in large vessel human umbilical vein endothelial cells (HUVEC). Both cell populations were judged pure as assessed by expression of von Willebrand factor and uptake of acetylated low density lipoprotein. Expression of VCAM-1 was not detectable on either unstimulated HDMEC or HUVEC when assessed by ELISA or flow cytometry. Stimulation of either HDMEC or HUVEC with TNF-alpha resulted in a time- and dose-dependent induction of VCAM-1. However, although TNF-alpha-induced cell surface and mRNA expression of VCAM-1 in HDMEC was transient, peaking after 16 h of stimulation, TNF stimulation led to persistently elevated cell surface expression of VCAM-1 on HUVEC. IL-1 alpha also induced cell surface expression of VCAM-1 on HUVEC in a time- and dose-dependent manner, but stimulation of HDMEC with IL-1 alpha at doses up to 1000 U/ml failed to induce significant cell surface expression. However, IL-1 alpha induced time- and dose-dependent increases in ICAM-1 on HDMEC. Similarly, IL-4 induced VCAM-1 expression and augmented TNF-alpha-induced expression on HUVEC but did not affect VCAM-1 expression on HDMEC. Binding of Ramos cells to cytokine-stimulated endothelial cell monolayers correlated with VCAM-1 induction. Increased binding was seen after stimulation of HDMEC with TNF-alpha, which was blocked by anti-VCAM-1 mAb, but no increases in binding were noted after stimulation of HDMEC monolayers with IL-1 alpha. These data provide additional evidence for the existence of endothelial cell heterogeneity and differences in cell adhesion molecule regulation on endothelial cells derived from different vascular beds.

Cell Adhesion Molecules↗
PubMed1992

Human intercellular adhesion molecule-1 gene and its expression in the skin.

Cell adhesion molecules are cell-surface proteins that allow specific cell-cell interactions among leukocytes, as well as between leukocytes and other cells. Recent studies have shown that the differential expression of selected cell-adhesion molecules plays a critical role in cutaneous inflammation, immunologic responses, and wound repair. Intercellular adhesion molecule-1 (ICAM-1) is a cell-adhesion molecule that is constitutively expressed on human dermal microvascular endothelial cells (HDMEC) and is inducible on human keratinocytes (HK). Its regulated expression is vital to the initiation and evolution of localized inflammatory processes in the skin. ICAM-1 serves as a specific ligand for lymphocyte function-associated antigen-1 (LFA-1), a cell-surface protein expressed on all leukocytes. The regulated expression of ICAM-1 allows leukocytes to bind to endothelial cells at sites of inflammation and, after exiting into the tissue, to interact with specific target cells, such as HK. Furthermore, specific cytokines are capable of differentially regulating ICAM-1 expression on HDMEC, HK, and other cells. The biologic relevance of ICAM-1 expression in cutaneous inflammation is further supported by functional studies demonstrating the critical role of ICAM-1/LFA-1 interactions in mediating the binding of peripheral blood leukocytes to HDMEC and to HK--cells known to be participants and targets in specific cutaneous immunologic responses. Thus, the delineation of precise molecular mechanisms that regulate the tissue-specific and cytokine-specific expression if ICAM-1 is important to both our understanding of the biology of localized inflammation and to the development of directed anti-inflammatory therapeutic strategies. Current evidence indicates that ICAM-1 expression is regulated at the level of gene transcription. Recently our laboratory has isolated and characterized a human genomic clone that contains the 5' regulatory region of the ICAM-1 gene. In the current studies, we further describe the genomic ICAM-1 clones isolated to date and demonstrate the presence of consensus regulatory elements located within the 5' flanking region of the ICAM-1 gene that are potentially involved in regulating ICAM-1 gene transcription.

Animals↗
PubMed1992

Human keratinocytes and A-431 cells synthesize and secrete factor B, the major zymogen protease of the alternative complement pathway.

Biosynthetic radiolabeling studies demonstrate that human keratinocytes and A-431 cells, a human epidermoid carcinoma cell line, synthesize and secrete factor B as a monomeric 105-kD protein. Epithelial cell-derived factor B comigrates in SDS-PAGE with that produced by HepG2 cells, a human hepatoma cell line traditionally employed in studies of complement component biosynthesis. Comparative pulse-chase studies in A-431 and HepG2 cells show that this alternative pathway complement component is produced as co-migrating 100-kD intracellular proteins that are processed in both cell types to 105-kD extracellular factor B. Quantitatively, immunoprecipitable factor B accounts for 0.05% of radiolabeled proteins in A-431 cell culture media. Treatment of biosynthetically radiolabeled A-431 cell culture media with cobra venom factor and factor D for 60 min at 37 degrees C produces the specific factor B cleavage products Ba and Bb. These fragments are not identifiable in control culture media subjected to similar treatment in the absence of alternative pathway activators. Northern blot analysis of total cellular RNA from human keratinocytes, A-431 cells, and HepG2 cells reveals qualitative identity of a 2.8-kb factor B mRNA species in these three cell types. The relative level of factor B mRNA expression in these cells parallels their level of factor B protein synthesis (i.e., HepG2 cells greater than A-431 cells greater than human keratinocytes). Epithelial cell-derived factor B may play an important role in local inflammatory reactions and also directly interact with epithelial cell derived C3--a key classical and alternative pathway complement component recently shown to be produced by human keratinocytes.

Animals↗