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Biomedical subjects

T M McIntyre

Publications and source records attributed to T M McIntyre.

At least 55 records · Page 3Linked to original sources
PubMed1998

Human endothelial cells regulate polymorphonuclear leukocyte degranulation.

Neutrophil degranulation is an important event in inflammatory responses. We examined the regulation of neutrophil (PMN) degranulation by resting and activated human endothelial cells. Whereas PMNs adherent to endothelial cells that were stimulated to express P-selectin and platelet-activating factor did not release the specific granule marker lactoferrin or the primary granule enzyme, elastase, PMNs adherent to endothelial cells stimulated with interleukin-1 (IL-1) or tumor necrosis factor secreted both. PMN degranulation was dependent on the time of incubation of endothelial cells with the cytokine, its concentration, and the time of incubation of the PMNs with endothelial cells. Degranulation of PMNs and their adhesion to stimulated endothelial cells are correlated events, but they could be dissociated by blocking the tethering molecules used by the endothelial cells and neutrophils under these conditions. This suggested that paracrine signaling molecules that induce PMN degranulation are produced by cytokine-stimulated endothelial cells. We found that endothelial cells stimulated with IL-1 release newly synthesized degranulating factors that require transcription and translation. IL-8 was synthesized, released, and signaled granular secretion by PMNs. However, experiments with blocking antibodies indicated the presence of an additional degranulating factor not accounted for by IL-8. These experiments demonstrate that human endothelial cells regulate degranulation of neutrophils by generating signaling factors that are expressed differentially depending on the endothelial agonist and other features. Active modification of neutrophil granular secretion by endothelial cells can influence physiologic acute inflammatory responses but may also contribute to pathologic vascular and tissue damage.

Cell Adhesion↗
PubMed1997

Retrograde inflammatory signaling from neutrophils to endothelial cells by soluble interleukin-6 receptor alpha.

Endothelial cells initiate the inflammatory response by recruiting and activating leukocytes. IL-6 is not an agonist for this, but we found soluble IL-6 receptor alpha-subunit (IL-6Ralpha), with their constitutive IL-6 synthesis, stimulated endothelial cells to synthesize E-selectin, intracellular adhesion molecule-1, vascular cellular adhesion molecule-1, IL-6, and IL-8, and to bind neutrophils. Neutrophils express significant amounts of IL-6Ralpha and upon stimulation shed it: this material activates endothelial cells through a newly constituted IL-6 receptor. Retrograde signaling from PMN activated in the extravascular compartment to surrounding endothelial cells will recruit more and a wider variety of leukocytes. The limiting signal is a soluble receptor, not a cytokine.

Animals↗
PubMed1997

Alpha toxin from Clostridium perfringens induces proinflammatory changes in endothelial cells.

Alpha toxin from Clostridium perfringens type A, a phospholipase C, has been implicated in many of the localized and systemic features of gas gangrene. We demonstrated that human endothelial cells synthesize two vasoactive lipids, platelet-activating factor (PAF) and prostacyclin, in response to alpha toxin treatment. The stimulated synthesis of PAF required the enzymatic activity of the toxin and subsequent protein kinase C activation. Alpha toxin-treated endothelial cells accumulated the products of the phospholipase C reaction, diacylglycerol and ceramide, and exhibited a decrease in the enzymatic precursors phosphatidylcholine and sphingomyelin. Furthermore, the temporal accumulation of PAF depended on the concentration of the toxin in the overlying medium and was blocked in the presence of a neutralizing antibody. The cultured endothelial cells also exhibited enhanced neutrophil adhesion in response to alpha toxin which was mediated through the PAF receptor and P-selectin. P-selectin expression by endothelial cells and extravascular neutrophil accumulation were also observed in tissue sections from alpha toxin-injected Sprague-Dawley rats. These endothelial cell-mediated processes are important in maintaining vascular homeostasis and, when activated in a dysregulated manner by C. perfringens alpha toxin, may contribute to localized and systemic manifestations of gas gangrene including enhanced vascular permeability, localized neutrophil accumulation, and myocardial dysfunction.

Animals↗
PubMed1997

Oncostatin M is a proinflammatory mediator. In vivo effects correlate with endothelial cell expression of inflammatory cytokines and adhesion molecules.

Oncostatin M is a member of the IL-6 family of cytokines that is primarily known for its effects on cell growth. Endothelial cells have an abundance of receptors for oncostatin M, and may be its primary target. We determined if oncostatin M induces a key endothelial cell function, initiation of the inflammatory response. We found that subcutaneous injection of oncostatin M in mice caused an acute inflammatory reaction. Oncostatin M in vitro stimulated: (a) polymorphonuclear leukocyte (PMN) transmigration through confluent monolayers of primary human endothelial cells; (b) biphasic PMN adhesion through rapid P-selectin expression, and delayed adhesion mediated by E-selectin synthesis; (c) intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 accumulation; and (d) the expression of PMN activators IL-6, epithelial neutrophil activating peptide-78, growth-related cytokine alpha and growth-related cytokine beta without concomitant IL-8 synthesis. The nature of the response to oncostatin M varied with concentration, suggesting high and low affinity oncostatin M receptors independently stimulated specific responses. Immunohistochemistry showed that macrophage-like cells infiltrating human aortic aneurysms expressed oncostatin M, so it is present during a chronic inflammatory reaction. Therefore, oncostatin M, but not other IL-6 family members, fulfills Koch's postulates as an inflammatory mediator. Since its effects on endothelial cells differ significantly from established mediators like TNFalpha, it may uniquely contribute to the inflammatory cycle.

Animals↗
PubMed1997

Alternative splicing of the human diacylglycerol kinase zeta gene in muscle.

Diacylglycerol can function as a second messenger, and one mechanism for the attenuation of this signal is its conversion to phosphatidic acid, which is catalyzed by diacylglycerol kinase (DGK). We screened a cDNA library from human skeletal muscle and isolated two DGKzeta cDNAs that differed from the 3.5-kb clone originally identified in endothelial cells. One transcript, which was 3.4 kb long, was shown to be nonfunctional; it had a 77-bp deletion that included the translation initiation site. The other was 4.1 kb long with a unique 5' sequence of 853 bp. We also isolated a genomic clone of DGKzeta and determined its organization and location; it contains 32 exons, spans approximately 50 kb of genomic sequence, and maps to chromosome 11p11.2. The protein encoded by the 4.1-kb transcript contains two cysteine-rich regions, a catalytic domain, and ankyrin repeats like the endothelial form of DGKzeta, as well as a unique N-terminal domain. The coding sequence was shown to be derived from alternative splicing of the DGKzeta gene. In cells transfected with the 4.1-kb clone, we detected a 130-kDa protein with an antibody to DGKzeta and demonstrated that it was localized predominantly in the nucleus. We conclude that alternative splicing generates tissue-specific variants of DGKzeta that share some properties but may have unique ones as well.

Alternative Splicing↗
PubMed1997

Vitamin C blocks inflammatory platelet-activating factor mimetics created by cigarette smoking.

Cigarette smoking within minutes induces leukocyte adhesion to the vascular wall and formation of intravascular leukocyte-platelet aggregates. We find this is inhibited by platelet-activating factor (PAF) receptor antagonists, and correlates with the accumulation of PAF-like mediators in the blood of cigarette smoke-exposed hamsters. These mediators were PAF-like lipids, formed by nonenzymatic oxidative modification of existing phospholipids, that were distinct from biosynthetic PAF. These PAF-like lipids induced isolated human monocytes and platelets to aggregate, which greatly increased their secretion of IL-8 and macrophage inflammatory protein-1alpha. Both events were blocked by a PAF receptor antagonist. Similarly, blocking the PAF receptor in vivo blocked smoke-induced leukocyte aggregation and pavementing along the vascular wall. Dietary supplementation with the antioxidant vitamin C prevented the accumulation of PAF-like lipids, and it prevented cigarette smoke-induced leukocyte adhesion to the vascular wall and formation of leukocyte-platelet aggregates. This is the first in vivo demonstration of inflammatory phospholipid oxidation products and it suggests a molecular mechanism coupling cigarette smoke with rapid inflammatory changes. Inhibition of PAF-like lipid formation and their intravascular sequela by vitamin C suggests a simple dietary means to reduce smoking-related cardiovascular disease.

Animals↗
PubMed1997

Human endothelial cells synthesize ENA-78: relationship to IL-8 and to signaling of PMN adhesion.

The interaction of endothelial cells and polymorphonuclear leukocytes (PMNs, neutrophils) is a critical determinant of the acute inflammatory response, and mirrors cell-cell interactions in other biologic systems. Adhesion molecules that tether the two cells together, and signaling factors that bind to receptors on the leukocytes and mediate their spatially-localized activation, govern PMN responses as they adhere to and traverse stimulated endothelial cells. Here we show that cultured human endothelial cells express two members of the C-X-C family of chemokines, epithelial neutrophil activating peptide-78 (ENA-78) and interleukin (IL)-8, when stimulated by IL-1 or certain other agonists. ENA-78, previously thought to be exclusively a product of epithelium, is expressed by in situ endothelium in inflamed human lung and other tissues as well as by cultured endothelial cells. The regulation of ENA-78 and IL-8 share certain features in common and they have overlapping biologic activities, including the ability to induce PMN adhesiveness. This was demonstrated in experiments in which we found that ENA-78 induces inside-out signaling of beta2 integrins on the PMN surface, as does IL-8. Antibody blocking experiments demonstrated that ENA-78 contributes to the proadhesive activity for neutrophils that is released by endothelial cells stimulated with IL-1 for a prolonged period and that it acts in concert with IL-8, which provides the major component of the signal for adhesion under this condition. We also show, however, that the temporal expression and secretion of ENA-78 and other characteristics of its handling by stimulated endothelial cells vary from the expression of IL-8, indicating that differential regulation of the two signaling chemokines occurs in this cell type.

Adult↗
PubMed1996

Borrelia burgdorferi outer membrane protein A induces nuclear translocation of nuclear factor-kappa B and inflammatory activation in human endothelial cells.

Lyme disease is caused by infection with Borrelia burgdorferi, and is characterized by bacterial persistence and inflammation in a number of host tissues. B. burgdorferi outer surface lipoproteins possess cytokine stimulatory properties that may be responsible for localized inflammation. B. burgdorferi presence is correlated with severity of disease, and the pathology of many tissues, particularly the arthritic joint, is consistent with localized cytokine production. Spirochete invasion of tissues requires interaction with and penetration of vascular endothelium, suggesting endothelial cells may participate in the inflammation of Lyme disease. In this study, outer surface protein A (OspA), a model B. burgdorferi lipoprotein, was found to be a potent stimulant of nuclear factor-kappa B (NF-kappa B) nuclear translocation in human endothelial cells, resulting in nuclear levels similar to those seen in response to known inflammatory mediators. Only the lipid-modified OspA had activity, and activity was not due to contamination with LPS. Nuclear NF-kappa B was detectable within 15 min, suggesting that OspA directly mediates NF-kappa B nuclear translocation. OspA also rapidly up-regulated endothelial cell production of several proteins whose transcription is dependent on NF-kappa B: the cytokine IL-6; the chemokine IL-8; and the adhesion molecules E-selectin, VCAM-1, and ICAM-1. The adhesion molecules were functional, as demonstrated by enhanced binding of neutrophils to OspA-stimulated endothelial monolayers. These data suggest that OspA may initiate synthesis of many proteins essential for localized inflammation via the direct activation of NF-kappa B-dependent transcription. These observations suggest that the interaction of B. burgdorferi lipoproteins with the endothelium may directly induce the inflammation responsible for the symptoms of Lyme disease.

Antigens, Surface↗
PubMed1996

Platelet-activating factor acetylhydrolase deficiency. A missense mutation near the active site of an anti-inflammatory phospholipase.

Deficiency of plasma platelet-activating factor (PAF) acetylhydrolase is an autosomal recessive syndrome that has been associated with severe asthma in Japanese children. Acquired deficiency has been described in several human diseases usually associated with severe inflammation. PAF acetylhydrolase catalyzes the degradation of PAF and related phospholipids, which have proinflammatory, allergic, and prothrombotic properties. Thus, a deficiency in the degradation of these lipids should increase the susceptibility to inflammatory and allergic disorders. Miwa et al. reported that PAF acetylhydrolase activity is absent in 4% of the Japanese population, which suggests that it could be a common factor in such disorders, but the molecular basis of the defect is unknown. We show that inherited deficiency of PAF acetylhydrolase is the result of a point mutation in exon 9 and that this mutation completely abolishes enzymatic activity. This mutation is the cause of the lack of enzymatic activity as expression in E. coli of a construct harboring the mutation results in an inactive protein. This mutation as a heterozygous trait is present in 27% in the Japanese population. This finding will allow rapid identification of subjects predisposed to severe asthma and other PAF-mediated disorders.

1-Alkyl-2-acetylglycerophosphocholine Esterase↗
PubMed1996

Endothelial cell inflammatory responses to tumor necrosis factor alpha. Ceramide-dependent and -independent mitogen-activated protein kinase cascades.

Ceramide generation by stimulated sphingomyelinase activity has been implicated in tumor necrosis factor alpha (TNF) signaling of apoptosis and differentiation. We examined the role of ceramide in a major action of TNF: the initiation of inflammatory events. Sphingomyelinase C at high levels induced inflammatory protein expression in endothelial cells resulting in leukocyte adhesion, but the pattern of induction of adhesion molecules (E-selectin, ICAM-1, VCAM-1) and cytokines (interleukins 6 and 8) differed from that induced by TNF. TNF induced only a small increase in ceramide: using lower doses of sphingomyelinase to mimic this we found that small amounts of ceramide did not induce protein expression, but still rapidly activated Raf-1, mitogen-activated protein/extracellular regulated kinase (ERK) kinase (MEK) and ERKs. TNF additionally caused rapid p38 and JNK-1 mitogen-activated protein kinase activation and efficient NF-kappaB translocation, which could not be achieved even by high levels of ceramide. Thus activation of the ERK cascade alone is an incomplete endothelial cell stimulus, and the TNF receptor generates at least two signals: Raf-1 activation, which could be ceramide-dependent; and ceramide-independent efficient NF-kappaB translocation and activation of p38 and JNK-1 mitogen-activated kinases.

Biological Transport↗
PubMed1996

Prostaglandin H synthase 2 is expressed abnormally in human colon cancer: evidence for a transcriptional effect.

Evidence from epidemiological studies, clinical trials, and animal experiments indicates that inhibitors of prostaglandin synthesis lower the risk of colon cancer. We tested the hypothesis that abnormal expression of prostaglandin H synthase 2 (PHS-2), which can be induced by oncogenes and tumor promoters, occurs during colon carcinogenesis by examining its level in colon tumors. Human colon cancers were found to have an increased expression of PHS-2 mRNA compared with normal colon specimens from the same patient (n = 5). In situ hybridization showed that the neoplastic colonocytes had increased expression of PHS-2 (n = 4). Additionally, five colon cancer cell lines were shown to express high levels of PHS-2 mRNA even in the absence of a known inducer of PHS-2. To study the basis for this increased gene expression, we transfected a colon cancer cell line, HCT-116, with a reporter gene containing 2.0 kb of the 5' regulatory sequence of the PHS-2 gene. Constitutive transcription of the reporter gene was observed, whereas normal control cell lines transcribed the reporter only in response to an exogenous agonist. We conclude that PHS-2 is transcribed abnormally in human colon cancers and that this may be one mechanism by which prostaglandins or related compounds that support carcinogenesis are generated.

Adenocarcinoma↗
PubMed1996

Molecular cloning and characterization of a novel human diacylglycerol kinase zeta.

Diacylglycerol (DAG) occupies a central position in the synthesis of complex lipids and also has important signaling roles. For example, DAG is an allosteric regulator of protein kinase C, and the cellular levels of DAG may influence a variety of processes including growth and differentiation. We previously demonstrated that human endothelial cells derived from umbilical vein express growth-dependent changes in their basal levels of diacylglycerol and diacylglycerol kinase activity (Whatley, R. E., Stroud, E. D., Bunting, M., Zimmerman, G. A., McIntyre, T. M., and Prescott, S. M. (1993) J. Biol. Chem. 268, 16130-16138). To further explore the role of diacylglycerol metabolism in endothelial responses, we used a degenerate reverse transcription-polymerase chain reaction method to identify diacylglycerol kinase isozymes expressed by human endothelial cells. We report the isolation of a 3.5-kilobase cDNA encoding a novel diacylglycerol kinase (hDGKzeta) with a predicted molecular mass of 103.9 kDa. Human DGK zeta contains two zinc fingers, an ATP binding site, and four ankyrin repeats near the carboxyl terminus. A unique feature, as compared with other diacylglycerol kinases, is the presence of a sequence homologous to the MARCKS phosphorylation site domain. From Northern blot analysis of multiple tissues, we observed that hDGKzeta mRNA is expressed at highest levels in brain. COS-7 cells transfected with the hDGKzeta cDNA express 117-kDa and 114-kDa proteins that react specifically with an antibody to a peptide derived from a unique sequence in hDGK zeta. The transfected cells also express increased diacylglycerol kinase activity, which is not altered in the presence of R59949, an inhibitor of human platelet DGK activity. The hDGKzeta displays stereoselectivity for 1,2-diacylglycerol species in comparison to 1,3-diacylglycerol, but does not exhibit any specificity for molecular species of long chain diacylglycerols.

Amino Acid Sequence↗