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M J Holtzman

Publications and source records attributed to M J Holtzman.

At least 19 recordsLinked to original sources
PubMed1996

Molecular cloning, functional expression, and selective regulation of ovine prostaglandin H synthase-2.

Structural characterization for ovine prostaglandin H synthase-1 (PGHS-1) is extensive, but the corresponding structure for the homologous ovine PGHS-2 isoform is undefined. Accordingly, we isolated a full-length (3.4 kb) ovine PGHS-2 cDNA from a primary-culture cell model (ovine tracheal epithelial cells) originally described as containing both PGHS isoforms. Analysis of ovine PGHS-2 cDNA sequence indicated conservation of critical amino acid residues, but differences in other hydrophilic regions allowed for the development of an anti-peptide antibody highly selective for PGHS-2. Enzymatic activities of the recombinant ovine PGHS isozymes indicated significant differences in response to aspirin-acetylation consistent with the characteristics of endogenous cellular PGHS activities under basal and serum-induced conditions. The results fully account for previous evidence of two distinct PGHS activities in cultured airway epithelial cells and provide for additional definition of PGHS structure-function relationships.

Amino Acid Sequence↗
PubMed1995

Stat1 depends on transcriptional synergy with Sp1.

STAT (signal transducer and activator of transcription) proteins combine with cytokine receptors and receptor-associated kinases in distinct protein/protein interactions that are critical for STAT-dependent signal transduction events, but the nature of any subsequent STAT interactions with DNA-binding proteins in the nucleus is less certain. Based on assays of DNA/protein binding and activity of transfected reporter plasmids, we determined that occupation of contiguous DNA-binding sites for Stat1 (the first member of the STAT family) and the transcriptional activator Sp1 are both required for full activation of the intercellular adhesion molecule-1 gene by interferon-gamma. Thus, Stat1 binding to DNA cannot by itself be equated with biologic actions of Stat1. In co-immunoprecipitation experiments, we also obtained evidence of direct and selective Stat1/Sp1 interaction (in primary culture cells without overexpression), further indicating that Stat1/Sp1 synergy confers an element of specificity in the pathway leading to cytokine-activated transcription and cytokine-dependent immunity and inflammation.

Animals↗
PubMed1995

A hierarchy for integrin expression and adhesiveness among T cell subsets that is linked to TCR gene usage and emphasizes V delta 1+ gamma delta T cell adherence and tissue retention.

To define the relationship between T cell phenotype and adhesiveness, we examined T cell adhesion to endothelial cell, fibroblast, and epithelial cell monolayers as well as extracellular matrix proteins (collagen and fibronectin) using a three-color flow cytometry-based adherence assay that minimizes basal adhesion levels and facilitates quantitative lymphocyte subtyping. Regardless of monolayer type, monolayer stimulation conditions, or T cell activation status, we found that the gamma delta-TCR-bearing T cells adhered more efficiently than alpha beta T cells. The difference was based predominantly on increased levels of activatable LFA-1 (and to a lesser degree VLA-4) because: 1) it correlated precisely with inhibitability by anti-LFA-1 (and VLA-4) mAbs and the levels of LFA-1 (and VLA-4) on the cell surface, and 2) it persisted after maximal LFA-1 (and VLA-4) activation with phorbol dibutyrate. In contrast to most cases of alpha beta T cell behavior, gamma delta T cell adhesion to cell monolayers was not linked to memory status, i.e., there was no difference between naive V delta 1+ and memory V delta 2+ populations in levels of LFA-1 (or VLA-4) expression or LFA-1- (or VLA-4-) dependent adhesion to cell monolayers. However, V delta 1+ cells exhibited higher levels of VLA-5 that correlated with an increased adhesiveness to fibronectin and to a 120-kDa fibronectin fragment (FN-120) that contains only the VLA-5-binding domain but not to type I collagen or to a fibronectin fragment (FN-40) that binds only VLA-4. Taken together, the results define a hierarchy for integrin (LFA-1, VLA-4, and VLA-5) expression and consequent adhesion among T cell subsets that is linked to TCR gene usage (but not necessarily linked to memory status) and may thereby help to explain the accumulation and retention of V delta 1+ gamma delta T cells in epithelial and connective tissues.

Antibodies, Monoclonal↗
PubMed1994

Prostaglandin H synthase and lipoxygenase gene families in the epithelial cell barrier.

Epithelial barrier cells (in skin, gut, and airway) are both active modulators and important targets of the inflammatory response, and some of these cellular events may be regulated at a molecular level by products of phospholipid-arachidonic acid metabolism. Accordingly, we have defined some of the characteristics of gene expression and enzyme regulation for distinct members of the PGH synthase and lipoxygenase gene families in normal and inflamed epithelial tissues and in epithelial cells isolated from mucosal and epidermal tissue (Table 1). A unifying scheme for our findings includes the following enzymatic systems: (i) a PGH synthase-1/PG isomerase pathway responsible for constitutive generation of prostaglandins (e.g., PGE2) and maintenance of physiologic epithelial function; (ii) a PGH synthase-2/PG isomerase and synthase pathway capable of producing additional prostaglandins (e.g., excess PGE2 and/or PGF2 alpha and PGD2) especially after stimulation by growth factors and cytokines; and (iii) a family of arachidonate 12- and 15-lipoxygenases that may serve to generate hydroxy acids (e.g., 12- and 15-HETE) as mediators of basal epithelial function and that (after overexpression and oxidant activation) may also catalyze membrane peroxidation that contributes to epithelial damage during inflammation. The regulatory mechanisms inherent in the control of this scheme provide a biochemical rationale for balancing constitutive and inducible oxygenation activities and maintaining epithelial barrier function.

Animals↗
PubMed1994

Selective interaction of a subset of interferon-gamma response element-binding proteins with the intercellular adhesion molecule-1 (ICAM-1) gene promoter controls the pattern of expression on epithelial cells.

Intercellular adhesion molecule-1 (ICAM-1) modulates epithelial and endothelial leukocyte adherence, but epithelial cell ICAM-1 levels (unlike endothelial cell levels) are selectively sensitive to interferon-gamma (IFN-gamma). Nuclear run-off assays indicated that IFN-gamma regulation of epithelial ICAM-1 levels occurs at a transcriptional level, so the basis for selective cytokine control of ICAM-1 expression was investigated using the ICAM-1 gene promoter region. A plasmid construct containing 5.0 kilobases of ICAM-1 gene 5'-flanking region fused to a reporter gene was selectively responsive to IFN-gamma in (human tracheal) epithelial cells but not in (human umbilical vein) endothelial cells, indicating that this region is sufficient to mediate proper cell-specific expression. An IFN-gamma response element (IRE) was localized to a DNA segment (nucleotides -130 to -94) in the ICAM-1 gene by comparisons of nested 5'-deletional constructs and by demonstrating that this segment confers IFN-gamma responsiveness on heterologous promoters. This same IRE formed a single major binding complex (IRE-BC) in gel retardation assays with nuclear proteins from IFN-gamma-stimulated but not unstimulated epithelial cells, and mutation of the IRE consensus motif (TTTCCGGGAAA at -116 to -106) resulted in loss of IRE-protein binding and abolished IFN-gamma responsiveness, indicating that this sequence is required for ICAM-1 IRE function. Comparison of the ICAM-1 IRE to DNA elements that confer IFN responsiveness in other human genes indicated similarity only to response regions in the Fc gamma RI and IRF-1 genes. The findings provide evidence for a distinct IRE subset that combines a DNA element common to all IFN-responsive genes (GAAA) with a distinct flanking sequence (the inverted repeat GAAA) in order to fine-tune IFN responses and activate a subset of immune response genes (ICAM-1, Fc gamma RI, and IRF-1).

Base Sequence↗
PubMed1994

Epidermis contains platelet-type 12-lipoxygenase that is overexpressed in germinal layer keratinocytes in psoriasis.

Human epidermal cells exhibited none of the cytosolic lipoxygenase activity that is prominent in mucosal epithelial cells, but instead contained a microsomal activity that converted arachidonic acid to 12-hydroxyeicosatetraenoic acid (12-HETE). Identification of the extractable 12-HETE-forming activity as a 12-lipoxygenase (distinct from cytochrome P-450) included (S)-12-stereospecificity of product formation, trapping of 12-hydroperoxyeicosatetraenoic acid as an intermediate reaction product, and lack of NADPH dependence for activity. Epidermal cell poly(A)+ RNA contained high levels of a 2.3-kb mRNA that selectively hybridized with human platelet 12-lipoxygenase cDNA, and partial cDNA sequence of this mRNA indicated identity to platelet 12-lipoxygenase. The epidermal 12-lipoxygenase was not recognized by antibodies against the leukocyte-type 12- and 15-lipoxygenases (found in leukocytes, reticulocytes, and mucosal epithelial cells) but was detected by an antiplatelet 12-lipoxygenase antibody. The epidermal 12-lipoxygenase antigen was selectively expressed in germinal layer keratinocytes in healthy and psoriatic skin, and these layers exhibited hyperplasia and increased immunostaining in inflamed psoriatic skin. Together with previous results, these observations indicate that 1) epidermis generates 12-HETE by either cytochrome P-450 or lipoxygenase-based mechanisms depending on reaction conditions, and 2) 12-lipoxygenases (originally described in hematopoietic cell types) may be expressed in at least two distinct isoforms in epithelial barriers in humans, and in the case of the skin, a microsomal (platelet-type) 12-lipoxygenase is selectively overexpressed in germinal layer keratinocytes during psoriatic inflammation.

12-Hydroxy-5,8,10,14-eicosatetraenoic Acid↗
PubMed1994

Selective differences in vascular endothelial- vs. airway epithelial-T cell adhesion mechanisms.

The basis for T cell adhesion to airway epithelial and vascular endothelial cells was studied using a quantitative flow cytometry-based assay that avoids extensive leukocyte purification and labeling. Compared with standard cell-labeling methods, the flow cytometry-based assay yielded a lower level of constitutive T cell adhesion, despite a similar level of stimulated adhesion (after T cell activation with phorbol dibutyrate) using endothelial or epithelial cell monolayers. Endothelial T cell adhesion was further increased by monolayer treatment with tumor necrosis factor-alpha (less so with interleukin-1 beta and least with interferon-gamma), whereas epithelial T cell adhesion was most sensitive to interferon-gamma. Cytokine stimulation of adhesion was invariably concentration dependent and closely matched to the cellular levels of intracellular adhesion molecule-1 (ICAM-1). Accordingly, stimulated T cell adhesion was markedly inhibited by anti-ICAM-1 or anti-beta 2-integrin antibody (95-97% inhibition for epithelial cells and 57-67% inhibition for endothelial cells) directed against ICAM-1 interaction with lymphocyte function-associated antigen-1 (LFA-1; alpha L beta 2-integrin). Residual endothelial T cell adhesion that correlated with endothelial vascular cell adhesion molecule-1 (VCAM-1) levels was blocked by an anti-alpha 4-integrin antibody directed against VCAM-1 interaction with very late activation antigen-4 (VLA-4; alpha 4 beta 1-integrin). The results suggest that 1) peripheral blood T cells without exogenous activation exhibit little LFA-1- or VLA-4-dependent adherence except to endothelial or epithelial cells expressing high levels of ICAM-1 and/or VCAM-1; and 2) differences in endothelial vs. epithelial cell mechanisms to bind activated and unactivated T cells (e.g., dependence on a mixed- vs. a single-ligand system and distinct cytokine-responsiveness of ligand levels) may help to coordinate T cell traffic to epithelial barriers.

Cell Adhesion Molecules↗
PubMed1993

A cryptic, microsomal-type arachidonate 12-lipoxygenase is tonically inactivated by oxidation-reduction conditions in cultured epithelial cells.

Cultured ovine tracheal epithelial cells converted arachidonic acid to prostaglandin E2 (PGE2), but microsome-containing subcellular fractions prepared from these cells under calcium-free conditions converted arachidonic acid to PGE2 and to 12-hydroxyeicosatetraenoic acid (12-HETE) at a high rate (2-4 nmol/mg of protein/15 min). Identification of the membrane-bound 12-HETE-forming activity as a 12-lipoxygenase included 12S-stereospecificity of product formation and trapping of 12-hydroperoxyeicosatetraenoic acid as a reaction product. The 12-lipoxygenase activity was extracted from cell membranes only with detergent (1% Triton X-100), and the activity (membrane-bound or detergent-solubilized) was completely inactivated by mixing with the cytosol-containing subcellular fraction. The inhibitory effect of the cytosolic fraction was reversed by treating the cytosol with GSH-depleting agents (2-cyclohexene-1-one or N-ethylmaleimide) or by mixing it with lipid hydroperoxide (13-hydroperoxyoctadecadienoic acid) at a concentration that had little direct effect on enzyme activity. Inhibition of 12-lipoxygenase activity could also be achieved by treatment of enzyme preparations with GSH at levels (0.1-10 mM) found in epithelial cell cytosol. In addition, treatment of cultured epithelial cells with a GSH-depleting agent (buthionine sulfoximine) and lipid hydroperoxide restored cellular 12-lipoxygenase activity. Little or no detectable 12-lipoxygenase activity was found in freshly isolated ovine tracheal epithelial cells, but the cytosolic 12-lipoxygenase found in freshly isolated bovine tracheal epithelial cells was relatively insensitive to regulation by GSH or lipid hydroperoxide. These observations indicate that a 12-lipoxygenase is expressed in a cryptic, microsomal-type form in primary-culture epithelial cells and that this form of the enzyme may be selectively regulated by changes in cellular oxidation-reduction conditions dependent on cytosolic levels of GSH versus lipid hydroperoxide.

12-Hydroxy-5,8,10,14-eicosatetraenoic Acid↗
PubMed1993

Induction of epithelial arachidonate 12-lipoxygenase at active sites of inflammatory bowel disease.

To determine if epithelial lipoxygenases are regulated by mucosal inflammation, we examined the distribution of arachidonate 12-lipoxygenase in healthy colonic tissue and in involved and uninvolved sections of colon with inflammatory bowel disease. Immunohistochemistry of formaldehyde-fixed, paraffin-embedded intestinal tissue using two anti-12-lipoxygenase antibodies and indirect biotin-avidin-peroxidase detection demonstrated that, in contrast to tissue from normal colon (n = 8), in which 12-lipoxygenase antigen was undetectable in mucosal epithelial cells, the mucosal and glandular epithelium of inflamed colon in ulcerative colitis (n = 4) or Crohn's disease (n = 4) exhibited markedly positive immunostaining with anti-12-lipoxygenase antibodies. Immunoblotting of whole cell extracts with anti-12-lipoxygenase antibodies showed immunoperoxidase staining of a single protein band that comigrated with purified 12-lipoxygenase (in relative molecular weight: M(r) = 72,000) in mucosal samples from inflamed colon from subjects with ulcerative colitis or Crohn's disease but no comparable band in samples from uninvolved sections of the same colons or from colons of subjects without inflammatory bowel disease. Assays of 12-lipoxygenase activity indicated a corresponding increase in enzymatic activity in the same mucosal samples. The increased levels of 12-lipoxygenase antigen in the mucosal and glandular epithelial cells in regions of colon affected by inflammatory bowel disease, the corresponding increases in 12-lipoxygenase activity, and the absence of detectable 12-lipoxygenase antigen or enzymatic activity in the same cell types in noninflamed colonic tissue all suggest that epithelial cell 12-lipoxygenase is induced by local mediators of colonic inflammation.

Adult↗
PubMed1993

Histochemical evidence for induction of arachidonate 15-lipoxygenase in airway disease.

We have previously described the distribution of the arachidonate 15-lipoxygenase in lung tissue obtained from healthy human subjects. In the present study, we have utilized the same immunohistochemical methodology to examine the expression of 15-lipoxygenase in bronchial biopsy tissue from subjects with airway disease. Immunohistochemistry of bronchial tissue using two antibodies against distinct epitopes of the 15-lipoxygenase and indirect biotin-avidin-peroxidase detection demonstrated that, in contrast to airway tissue from normal subjects (n = 10) in which 15-lipoxygenase antigen was confined to the uppermost airways (nose and trachea) and was almost undetectable in bronchi, the bronchial tissue obtained from subjects with asthma (n = 7) or chronic bronchitis (n = 7) exhibited markedly positive immunostaining of mucosal epithelial cells with both anti-15-lipoxygenase antibodies. Specificity of 15-lipoxygenase immunostaining was verified by antigen competition experiments and by the lack of immunostaining with preimmune serum or control anti-5-lipoxygenase antibodies. The increased levels of 15-lipoxygenase antigen in the bronchial epithelial cells of asthmatic and bronchitic subjects compared with the same cell population in normal subjects coupled with the previous findings of increased 15-lipoxygenase activity in asthmatic airways suggest that epithelial 15-lipoxygenase is induced by airway inflammatory disease.

Adolescent↗
PubMed1992

Identification of a pharmacologically distinct prostaglandin H synthase in cultured epithelial cells.

Nonsteroidal anti-inflammatory drugs inhibit the action of prostaglandin H synthase (PGH synthase), and this effect may constitute the basis for therapeutic and idiosyncratic responses to these agents. We found that aspirin treatment of cultured ovine tracheal epithelial cells blocked PGH synthase-catalyzed formation of PG as expected but also caused a dose-dependent increase in 15-hydroxyeicosatetraenoic acid (15-HETE) production from arachidonic acid. In contrast, aspirin caused only inhibition of PG production without enhancing 15-HETE formation in ovine seminal vesicle and other tissues. The 15-HETE formed by aspirin-treated ovine tracheal epithelial cells was generated by a PGH synthase-dependent mechanism because: (i) the 15-HETE forming activity was just as sensitive as PG forming activity to selective inhibition by indomethacin; (ii) both 15-HETE and PG forming activities were quantitatively immunoprecipitated (depleted from supernatants and recovered in immune complex pellets) by a specific anti-PGH synthase antiserum. Additional immunoprecipitation experiments indicated that anti-PGH synthase monoclonal antibodies (cyo-1 and cyo-5) raised against the aspirin-inhibited form of the enzyme (contained in seminal vesicle) did not recognize the aspirin-stimulated 15-HETE-forming PGH synthase (contained in cultured epithelial cells). Thus, sequential immunoprecipitation of cultured epithelial cell material first with excess cyo-1 followed by anti-PGH synthase antiserum indicated that two isoforms of PGH synthase were expressed in these cells. SDS-polyacrylamide gel electrophoresis of immunoprecipitated PGH synthase from cultured epithelial cells revealed distinct protein bands for each form of the enzyme (M(r) = 70,000 and 72,000). The identification of a distinct PGH synthase which may be modified by aspirin so that selective oxygenation of fatty acid substrate is enhanced (while PG formation is inhibited) indicates that isozymes of PGH synthase exist which are pharmacologically distinct.

Amino Acid Sequence↗
PubMed1992

Arachidonic acid metabolism in airway epithelial cells.

Airway epithelial cells carry out their physiologic role in part by activating phospholipase-fatty acid oxygenation pathways. Recent discoveries include the facts that (a) airway epithelial cells contain abundant stores of fatty acid substrates, including arachidonic acid, for oxygenation, (b) the cells release arachidonic acid upon activation of specific phospholipases, (c) the cells contain novel cyclooxygenases and lipoxygenases at high levels relative to other cell types, and (d) some of the arachidonate metabolites have potent biologic effects on airway end organs such as smooth muscle, nerves, mucus glands, and epithelial cells themselves. Studies of arachidonate metabolism in airway epithelial cells have often been done on a heterogeneous cell population of basal, ciliated, and goblet cells, so information on individual cell types and alterations during cellular differentiation is still poorly defined. Potential cell-cell interaction via transcellular synthesis of eicosanoids also requires further study. Each of these aims would be aided by the use of cultured airway epithelial cells, but the culture system has proven problematic in preserving the oxygenation phenotype of the original tissue. The same access of the epithelial cells to inhaled agents will permit lipid-modifying drugs to be delivered to them, and much of this therapeutic potential is still unexplored. Therefore, determining the factors that regulate arachidonic acid metabolism in airway epithelial cells is still a fundamental goal for unraveling the role of arachidonate products in airway function and for altering eicosanoid production in the airway.

Animals↗
PubMed1992

Selective expression of an arachidonate 12-lipoxygenase by pancreatic islet beta-cells.

The immunohistochemical distribution of arachidonate lipoxygenases in rat pancreas was characterized with specific polyclonal anti-5-lipoxygenase and anti-12-lipoxygenase antibodies. Immunohistochemical analysis of formaldehyde-fixed paraffin-embedded rat pancreas using anti-12-lipoxygenase antibody and biotin-avidin-peroxidase detection demonstrated specific staining of islets and no staining of pancreatic exocrine tissue. Less intense staining of pancreatic vascular myocytes and endothelial cells was also observed. Immunoblotting of isolated pancreatic islet extracts with the anti-12-lipoxygenase antibody demonstrated immunoperoxidase staining of a single protein band which comigrated with purified 12-lipoxygenase (relative molecular weight = 72,000) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. Dispersed cells prepared from isolated islets and then subjected to fluorescence-activated cell sorting and immunostaining exhibited 12-lipoxygenase antigen in beta-cell populations but not in non-beta-cell (predominantly alpha-cell) populations. Assays of enzymatic activity confirmed that the 12-lipoxygenase-catalyzed conversion of arachidonic acid to 12-hydroxyeicosatetraenoic acid methyl ester occurred only with purified beta-cells and not with islet non-beta-cells. No evidence of 5-lipoxygenase antigen or enzymatic activity was found in purified beta-cells or in islet non-beta-cells. We conclude that rat pancreatic islet beta-cells contain an arachidonate 12-lipoxygenase which shares antigenic epitopes with the homologous enzyme contained in tissues from other species. In addition, the selective localization of the 12-lipoxygenase to pancreatic beta-cells and its absence in pancreatic acinar cells and in islet non-beta-cells support observations suggesting that 12-lipoxygenase products may participate in glucose-induced insulin secretion from beta-cells.

12-Hydroxy-5,8,10,14-eicosatetraenoic Acid↗
PubMed1992

Selective induction of intercellular adhesion molecule-1 by interferon-gamma in human airway epithelial cells.

To evaluate the factors controlling migration of leukocytes into pulmonary airway epithelium, we determined the biochemical mechanisms responsible for the regulation of intercellular adhesion molecule-1 (ICAM-1) expression on cultured monolayers of human tracheal epithelial cells (HTECs) or SV40 virus-transformed human bronchial epithelial cells (BEAS-2B). Validation experiments with human umbilical vein endothelial cells (HUVECs) demonstrated little detectable ICAM-1 expression on unstimulated cells or on cells incubated with interferon-gamma (IFN-gamma), but HUVEC monolayers responded to interleukin-1 beta (IL-1 beta) or tumor necrosis factor-alpha (TNF-alpha) with significant increases in ICAM-1 and ICAM-1-dependent adherence of polymorphonuclear leukocytes (PMNs). HTEC monolayers also exhibited no significant basal ICAM-1 expression but, in contrast to HUVEC monolayers, had marked increases in ICAM-1 expression and ICAM-1-dependent PMN adherence only after incubation with IFN-gamma (and not after IL-1 beta or TNF-alpha) treatment. BEAS-2B cells also exhibited relatively selective IFN-gamma stimulation of ICAM-1 expression and ICAM-1-dependent PMN adherence but (like late passage HTEC) showed significant basal ICAM-1 expression. Differences in IFN-gamma effect on ICAM-1 levels between HUVEC and HTEC monolayers were not due to differences in number or responsiveness of IFN-gamma receptors, because both cell types exhibited a similar number of receptors and other IFN-gamma-dependent responses of HUVECs remained active. In all analyses, ICAM-1 mRNA levels correlated closely with detection of ICAM-1 on the cell surface.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell Adhesion↗