Biomedical subjects

M E Gerritsen

Publications and source records attributed to M E Gerritsen.

At least 37 records · Page 2Linked to original sources

PubMed1997

Lymphocytes mediate TNF-alpha-induced endothelial cell adhesion molecule expression: studies on SCID and RAG-1 mutant mice.

TNF-alpha is known to elicit a rapid increase in the expression of specific endothelial cell adhesion molecules (ECAMs) within different vascular beds. The aim of this study was to determine whether lymphocytes contribute to the increased ECAM expression elicited by TNF-alpha. A dual radiolabeled mAb technique was used to quantify constitutive and TNF-alpha-induced expression of ICAM-1, VCAM-1, E-selectin, and P-selectin in different vascular beds (lung, heart, stomach, mesentery, small intestine, large intestine, and muscle) in wild-type and SCID mice. In reconstitution experiments, either whole splenocytes, T cell-enriched splenocytes, or B cell-enriched splenocytes were injected into SCID mice 48 h before TNF-alpha administration. Although the constitutive expression of ECAMs differed only slightly between wild-type and SCID mice, TNF-alpha-induced ECAM expression was markedly blunted in SCID mice compared with wild-type mice. This blunted response to TNF-alpha was also demonstrated for VCAM-1 in recombination activating gene (RAG)-1 mutant mice. Reconstitution studies revealed that administration of 50 x 10(6) splenocytes in SCID mice at 48 h before cytokine treatment restored the TNF-alpha-induced expression of VCAM-1 to levels normally observed in wild-type mice. Reconstitution with T cell- but not B cell-enriched splenocytes, also restored the TNF-alpha-induced expression of VCAM-1 in SCID mice to wild-type levels. These results implicate circulating T lymphocytes as modulators of the increased ECAM expression elicited by TNF-alpha.

Animals↗

PubMed1997

Novel inhibitors of cytokine-induced IkappaBalpha phosphorylation and endothelial cell adhesion molecule expression show anti-inflammatory effects in vivo.

We have identified two compounds that inhibit the expression of endothelial-leukocyte adhesion molecules intercellular adhesion molecule-1, vascular cell adhesion molecule-1, and E-selectin. These compounds act by inhibiting tumor necrosis factor-alpha-induced phosphorylation of IkappaB-alpha, resulting in decreased nuclear factor-kappaB and decreased expression of adhesion molecules. The effects on both IkappaB-alpha phosphorylation and surface expression of E-selectin were irreversible and occurred at an IC50 of approximately 10 microM. These agents selectively and irreversibly inhibited the tumor necrosis factor-alpha-inducible phosphorylation of IkappaB-alpha without affecting the constitutive IkappaB-alpha phosphorylation. Although these compounds exhibited other activities, including stimulation of the stress-activated protein kinases, p38 and JNK-1, and activation of tyrosine phosphorylation of a 130-140-kDa protein, these effects are probably distinct from the effects on adhesion molecule expression since they were reversible. One compound was evaluated in vivo and shown to be a potent anti-inflammatory drug in two animal models of inflammation. The compound reduced edema formation in a dose-dependent manner in the rat carrageenan paw edema assay and reduced paw swelling in a rat adjuvant arthritis model. These studies suggest that inhibitors of cytokine-inducible IkappaBalpha phosphorylation exert anti-inflammatory activity in vivo.

Animals↗

PubMed1997

Endothelial-dependent mechanisms regulate leukocyte transmigration: a process involving the proteasome and disruption of the vascular endothelial-cadherin complex at endothelial cell-to-cell junctions.

Although several adhesion molecules expressed on leukocytes (beta1 and beta2 integrins, platelet endothelial cell adhesion molecule 1 [PECAM-1], and CD47) and on endothelium (intercellular adhesion molecule 1, PECAM-1) have been implicated in leukocyte transendothelial migration, less is known about the role of endothelial lateral junctions during this process. We have shown previously (Read, M.A., A.S. Neish, F.W. Luscinskas, V.J. Palambella, T. Maniatis, and T. Collins. 1995. Immunity. 2:493-506) that inhibitors of the proteasome reduce lymphocyte and neutrophil adhesion and transmigration across TNF-alpha-activated human umbilical vein endothelial cell (EC) monolayers in an in vitro flow model. The current study examined EC lateral junction proteins, principally the vascular endothelial (VE)-cadherin complex and the effects of proteasome inhibitors (MG132 and lactacystin) on lateral junctions during leukocyte adhesion, to gain a better understanding of the role of EC junctions in leukocyte transmigration. Both biochemical and indirect immunofluorescence analyses of the adherens junction zone of EC monolayers revealed that neutrophil adhesion, not transmigration, induced disruption of the VE-cadherin complex and loss of its lateral junction localization. In contrast, PECAM-1, which is located at lateral junctions and is implicated in neutrophil transmigration, was not altered. These findings identify new and interrelated endothelial-dependent mechanisms for leukocyte transmigration that involve alterations in lateral junction structure and a proteasome-dependent event(s).

Acetylcysteine↗

PubMed1997

CREB-binding protein/p300 are transcriptional coactivators of p65.

CBP (CREB-binding protein) and p300 are versatile coactivators that link transcriptional activators to the basal transcriptional apparatus. In the present study, we identify CBP and p300 as coactivators of the nuclear factor-kappaB (NF-kappaB) component p65 (RelA). Consistent with their role as coactivators, both CBP and p300 potentiated p65-activated transcription of E-selectin and VCAM-1-CAT reporter constructs. The N- and C-terminal domains of both CBP/p300 functionally interact with a region of p65 containing the transcriptional activation domain as demonstrated by mammalian two-hybrid assays. Direct physical interactions of CBP/p300 with p65 were demonstrated by glutathione S-transferase fusion protein binding, and coimmunoprecipitation/Western blot studies. The adenovirus E1A 12S protein, which complexes with CBP and p300, inhibited p65-dependent gene expression. Reporter gene expression could be rescued from E1A inhibition by overexpression of CBP or p300. CBP and p300 act as coactivators of p65-driven gene activation and may play an important role in the cytokine-induced expression of various immune and inflammatory genes.

Adenovirus E1A Proteins↗

PubMed1997

Integrin activation protects pulmonary endothelial cells from the genotoxic effects of bleomycin.

Integrin activation promotes the survival of endothelial cells undergoing diverse forms of stress. Here we determined the ability of integrins to inhibit DNA strand breakage by bleomycin (BLM), a DNA-cleaving antitumor antibiotic that causes acute endothelial injury and subsequent pulmonary fibrosis. We found that BLM produced DNA breakage in cultured murine lung endothelial cells (MLEC) within 45 min of treatment as measured by DNA sedimentation and in situ labeling of 3'-OH by nick translation (ISNT). Two hours after the removal of BLM, we found a marked but incomplete reduction in DNA strand breakage as measured by ISNT, indicating that the damage was reversible. DNA sedimentation and ISNT demonstrated that strand breakage due to BLM was inhibited in MLEC cultured on fibronectin, and no evidence of breakage was found 2 h after removal of the drug in ISNT experiments. Gelatin, type IV collagen, laminin, and the integrin ligand peptide Gly-Arg-Gly-Asp-Ser-Pro, but not the inactive Gly-Arg-Ala-Asp-Ser-Pro peptide, also inhibited DNA strand breakage. Activation of integrins, either by coating surfaces with antibodies to alpha 5-, beta 1-, or beta 3-integrin subunits or by receptor clustering with the soluble antibodies, inhibited BLM-induced DNA breakage. Inhibition of BLM-induced DNA strand breakage by soluble beta 1-integrin antibody increased with increasing antibody concentration and duration of receptor clustering before BLM treatment. Thus integrin activation protects pulmonary endothelial cells from the genotoxic effects of BLM.

Animals↗

PubMed1997

Low-density lipoprotein receptor knockout mice exhibit exaggerated microvascular responses to inflammatory stimuli.

The objective of this study was to determine whether genetically induced hypercholesterolemia affects leukocyte-endothelial cell interactions in postcapillary venules of the mouse cremaster muscle. Leukocyte adhesion, emigration, and other microvascular parameters were assessed in venules of normal (wild-type) and low-density lipoprotein receptor-deficient (LDLr-/-) mice maintained on either normal rodent chow or on a high cholesterol diet (HCD). Measurements were obtained under control conditions and after administration of either leukotriene B4 (LTB4), platelet-activating factor (PAF), or tumor necrosis factor-alpha (TNF-alpha). Elevated numbers of adherent and emigrated leukocytes were observed in venules of LDLr-/- (compared with wild-type) mice on HCD, both under baseline conditions and after exposure to either LTB4, PAF, or TNF-alpha. Plasma TNF-alpha levels were also elevated in LDLr-/- versus wild-type mice. Administration of blocking monoclonal antibodies demonstrated that intercellular adhesion molecule-1, but not vascular cell adhesion molecule-1, mediates the exaggerated leukocyte-endothelial cell adhesion observed in LDLr-/- mice. The results of these studies indicate that chronic hypercholesterolemia predisposes the microvasculature to intense leukocyte-endothelial cell adhesion in response to different inflammatory stimuli.

Animals↗

PubMed1997

Differential up-regulation of circulating soluble and endothelial cell intercellular adhesion molecule-1 in mice.

Although circulating levels of soluble intercellular adhesion molecule-1 (sICAM-1) are frequently used as an indicator of the severity of different immune, inflammatory, or neoplastic diseases, little is known about the factors that govern plasma sICAM-1 concentration and its relationship to the membranous form of ICAM-1 (mICAM-1) expressed on vascular endothelial cells. Plasma sICAM-1 concentration (measured by enzyme-linked immunosorbent assay) and mICAM-1 expression (measured using the dual radiolabeled monoclonal antibody technique) in different vascular beds (eg, lung, small intestine, and spleen) were monitored in wild-type (C57BL) and ICAM-1-deficient mice, before and after administration of tumor necrosis factor (TNF)-alpha. In wild-type mice, TNF-alpha elicited time-dependent increases in lung and intestine mICAM-1 (plateau achieved at 12 hours), with a corresponding increase in plasma sICAM-1 (peaked at 5 hours and then declined). The initial increases in mICAM-1 and pulmonary leukocyte sequestration (measured as lung myeloperoxidase activity) induced by TNF-alpha preceded any detectable elevation in sICAM-1. In ICAM-1-deficient mice, plasma sICAM-1 was reduced by approximately 70%, with > 95% reductions of mICAM-1 in lung and intestine, and > 75% reduction in splenic accumulation of anti-ICAM-1 antibody. Although TNF-alpha doubled plasma sICAM-1 in ICAM-1-deficient mice, mICAM-1 was unaffected in all tissues. Either splenectomy or pretreatment with cycloheximide resulted in an attenuated TNF-induced increase in sICAM-1, without affecting mICAM-1 expression. These findings indicate that plasma sICAM-1 concentration does not accurately reflect the level of ICAM-1 expression on endothelial cells in different vascular beds.

Animals↗

PubMed1996

Brain endothelium lack one of two pathways of P-selectin-mediated neutrophil adhesion.

P-selectin, an endothelial leukocyte adhesion receptor, is rapidly translocated to the cell surface upon release from storage granules called Weibel-Palade bodies and is also transcriptionally upregulated upon cytokine stimulation of endothelial cells (ECs). These two pathways of surface expression are coincident with the rapid and cytokine-inducible pathway of neutrophil adhesion to ECs. Constitutive P-selectin expression is largely absent in cultured murine brain microvascular EC (BMEC) monolayers, but interleukin-1beta and tumor necrosis factor-alpha stimulation for 4 hours leads to dramatic P-selectin upregulation. The functional relevance of differential P-selectin expression in these cells was examined by studying BMECs derived from wild-type mice and P-selectin-deficient mice. We show that P-selectin deficiency does not affect Weibel-Palade body formation or their release in response to short-acting agonists. However, in the absence of P-selectin, the brain endothelium is unable to support neutrophil adhesion after stimulation with these agonists, which may contribute to the immune privilege status of the brain. We show that P-selectin does play a major role in supporting neutrophil adhesion in the cytokine-induced pathway in BMECs in the context of other cytokine-inducible endothelial-leukocyte adhesion receptors, E-selectin, ICAM-1, and VCAM-1.

Animals↗

PubMed1996

Postinduction transcriptional repression of E-selectin and vascular cell adhesion molecule-1.

TNF-alpha induction of the E-selectin and vascular cell adhesion molecule-1 (VCAM-1) genes leads to transient accumulation of high levels of mRNA in endothelial cells. The increase in these mRNAs after induction is due to an increase in the rate of gene transcription, which is maintained for several hours in the continuous presence of cytokine. Cytokine-induced transcriptional activation of these genes requires the transcription factor, nuclear factor-kappaB. Following removal of TNF-alpha, there is rapid postinduction transcriptional repression common to both of these genes. The repression is protein synthesis dependent and correlates with protein synthesis-dependent loss of both the p50 and p65 subunits of nuclear factor-kappaB from the nucleus. IkappaBalpha is capable of specifically displacing endothelial-derived heterodimeric p50/p65 from the E-selectin and VCAM-1 kappaB elements, while having no effect on binding of p50 homodimer. In the presence of agents that block proteasomal degradation of IkappaBalpha, endogenous IkappaBalpha can be visualized in the nucleus of both resting and TNF-alpha-activated endothelial cells. Endogenous IkappaBalpha is readily detected in the nucleus of HeLa cells, and its nuclear localization is increased following removal of TNF-alpha. Repression of E-selectin and VCAM-1 transcription following cytokine removal requires the loss of nuclear p50 and p65, and involves IkappaBalpha. This postinduction transcription repression mechanism may be one component of a program that prevents inappropriate and prolonged expression of adhesion molecules.

Animals↗

PubMed1996

L- and P-selectins, but not CD49d (VLA-4) integrins, mediate monocyte initial attachment to TNF-alpha-activated vascular endothelium under flow in vitro.

Monocyte adhesion to the vascular endothelium is a pivotal step during their egress to tissues at sites of inflammation and immune reactions, and during atherogenesis. In this study, an in vitro flow model and blocking mAb were used to define the role of adhesion molecules in monocyte interactions with activated HUVEC under flow conditions. By videomicroscopy, freely flowing monocytes abruptly halted (initial attachment) on 6-h TNF-alpha-activated HUVEC under flow via L- and P-selectin, whereas E-selectin was not involved. CD49d/CD29 integrin (VLA-4), which can mediate initial attachment of certain T cells to VCAM-1 under flow, did not support monocyte initial attachment. Once initially attached, a small number of monocytes began rolling at 9 microns/s through a mechanism involving L-selectin, as well as CD49d and CD11/CD18 integrins, while the remaining monocytes became firmly adherent, or released to the flow stream. Monocyte stable arrest and subsequent transendothelial migration occurred rapidly and efficiently through either CD49d or CD18 integrin adhesion pathways. Transendothelial passage was also dependent on PECAM-1 (CD31). These data reveal monocytes initially attach to activated endothelium via an L-selectin-dependent mechanism, with a smaller contribution from P-selectin and no contribution by CD49d. Subsequent monocyte rolling, arrest, and transmigration require overlapping functions between multiple members of the selectin, integrin, and Ig gene families.

Animals↗

PubMed1996

Integrins inhibit LPS-induced DNA strand breakage in cultured lung endothelial cells.

Collagen inhibits acute DNA strand breakage and apoptosis in sheep pulmonary artery endothelial cells (SPAEC) treated with lipopolysaccharide (LPS). Here we tested the ability of major basement membrane components, type IV collagen, laminin and fibronectin, and integrin ligands and anti-integrin antibodies to inhibit DNA breakage caused by LPS in SPAEC and BALB/c murine lung endothelial cells (MLEC). In situ labeling of DNA strand breaks with terminal deoxynucleotidyl transferase revealed similar DNA breakage in attached SPAEC and MLEC within 2 h after incubation with 1 microgram LPS/ml. Acute DNA strand breakage was reduced in cells plated on gelatin, type IV collagen, laminin, cellular fibronectin, or plasma fibronectin. DNA breakage was also suppressed by plating cells on surfaces coated with the integrin ligand hexapeptide, GRGDSP (40 micrograms/cm2), but not with GRADSP. LPS-induced DNA strand breakage was inhibited in MLEC plated on surfaces coated with antibodies to murine alpha 5-, beta 1, or beta 3-integrin subunits. Addition of anti-integrin antibodies, but not GRGDSP, to the medium above cell monolayers inhibited strand breakage. Despite similar acute DNA breakage, MLEC exhibited less detachment and apoptosis than SPAEC, consistent with a difference in the sensing or processing systems for apoptosis in these two cell types. These results demonstrate that extracellular matrices and integrin activation can inhibit the genotoxicity of LPS.

Animals↗

PubMed1996

Apigenin inhibits tumor necrosis factor-induced intercellular adhesion molecule-1 upregulation in vivo.

OBJECTIVE: Apigenin is a flavonoid that effectively blocks intercellular adhesion molecule-1 (ICAM-1) upregulation and leukocyte adhesion in response to cytokines in vitro. In the present study, we characterized the effects of tumor necrosis factor (TNF) on ICAM-1 expression in different tissues of the rat. We then assessed whether apigenin alters this response. METHODS: ICAM-1 expression was measured under baseline conditions or 5 h after treatment with rTNF. We used 125I-labeled anti-rat ICAM-1 monoclonal antibody (mAb) and an isotype-matched control mAb labeled with 131I to correct for nonspecific accumulation of the binding mAb. Animals were pretreated with either placebo, apigenin, narigenin (a flavonoid without inhibitory effect in vitro), or vehicle. Additional groups of animals were treated with either allopurinol, glutathione, dimethyl-thiourea, or an anti-CD18 monoclonal antibody in order to assess possible actions of flavonoids that were mediated via free radical scavenging or through interference with neutrophil function. RESULTS: Treatment with rTNF resulted in a marked increase in ICAM-1 expression in all organs studied. The magnitude of the response varied in different organs and increases ranged from onefold (lung) to threefold (muscle). Treatment with apigenin blocked ICAM-1 upregulation in organs with low to intermediate responses to rTNF and it significantly attenuated the increased ICAM-1 expression in organs that normally exhibit more marked upregulation. Treatment with narigenin or vehicle did not affect rTNF-induced ICAM-1 upregulation in all tissues studied. Pretreatment with either allopurinol, free radical scavengers, or anti-CD18 monoclonal antibody did not affect the ICAM-1 upregulatory response to rTNF. CONCLUSIONS: TNF-induced ICAM-1 upregulation in vivo effectively is blocked by apigenin through a mechanism that is unrelated to free radical scavenging or leukocyte function.

Animals↗

PubMed1996

Cytokeratin expression and hyaluronic acid production in cultures of human synovial microvascular endothelial cells: influence of cytokines and growth factors.

OBJECTIVES: To isolate and characterize human synovial endothelial cells and to determine the effects of cytokines and fibroblast growth factor on human synovial endothelial (HSE) cell hyaluronic acid production. METHODS: Endothelial cells were isolated from primary cultures of human synovial cells by fluorescent activated cell sorting based on the incorporation of a fluorescent derivative of acetylated low-density lipoprotein (DiI-Ac-LDL). Identity of endothelial cells was confirmed by positive immunostaining for von Willebrand factor (vWf), cytokeratins, endoglin, and reactivity with the lectin ulex europeans agglutinin (UEA). Hyaluronic acid production was measured by a radioligand-binding assay. RESULTS: HSE cells were isolated and maintained in long-term culture. The identity of the cultured cells as endothelial was based on uniform uptake of a (DiI-Ac-LDL), immunoreactivity for vWf, and endoglin and the binding of the lectin UEA. In addition, small blood vessels in the synovium were stained selectively with anticytokeratin antibodies K462 (cytokeratin 19 specific) and K8.13 (reactive for cytokines 1, 5, 6, 7, 8, 10, 11, and 18). Isolated HSE cells also demonstrated immunoreactivity with these cytokeratin antibodies. The cytokeratins identified by the monoclonal antibody clone K8.13 demonstrated a diffuse, fibrillar staining pattern. The cytokeratin distribution revealed with monoclonal antibody K4.62 (cytokeratin 19) was also fibrillar; however, the majority of cells also demonstrated numerous punctuate cytoplasmic vesicular structures. Treatment of HSE cells with interleukin-1 alpha (IL-1 alpha) or acidic fibroblasts growth factor (aFGF), but not tumor necrosis factor (TNF alpha), dramatically reduced the vesicular structures staining with the K4.62 antibody. HSE cells produced hyaluronic acid (HA) at a constitutive rate of 200-800 ng/10(5) cells/24 h, which could be upregulated when the cells were incubated with either IL-1 alpha or aFGF. HA production was not significantly increased when HSE cells were incubated with TNF alpha, IL-4 or interferon-gamma. CONCLUSIONS: Synovial microvascular endothelial cells produce and secrete HA and endothelial HA secretion is upregulated by IL-1 and aFGF. IL-1 and aFGF also reduce the number of vesicular-like structures immunoreactive with a monoclonal antibody to cytokeratin 19. These studies suggest that cytokine stimulation of local endothelial secretion and/or accumulation of HA may influence leukocyte adhesion to the synovial endothelium.

Adult↗

PubMed1996

Direct stimulation of limbal microvessel endothelial cell proliferation and capillary formation in vitro by a corneal-derived eicosanoid.

12(R)-Hydroxyeicosatrienoic acid (12(R)-HETrE), a corneal epithelial derived inflammatory eicosanoid, elicits blood vessel growth into the avascular cornea in the classical corneal micropocket bioassay. Using an in vivo stimulated angiogenesis assay and 12(R)-HETrE as the angiogenic stimulus, we isolated a homogeneous population of rabbit limbal microvessel endothelial cells, the target for angiogenic factors in the anterior surface of ocular tissues, and analyzed the mitogenic and angiogenic potential of this eicosanoid. 12(R)-HETrE stereospecifically increased cell number by approximately 45%, an effect comparable to that of basic fibroblast growth factor (0.6 nmol/L; 10 ng/ml). This potent mitogenic response was maximal at 0.1 nmol/L. An additive effect (approximately 90% above control) on cell proliferation was observed when 12(R)-HETrE (0.1 nmol/L) and basic fibroblast growth factor (0.6 nmol/L) were added to quiescent cultures of rabbit limbal microvessel endothelial cells. We also show that 12(R)-HETrE, but not 12(S)-HETrE, induces cultured rabbit limbal microvessel endothelial cells to organize themselves as a network of branching cords reminiscent of capillaries. This effect was evident within 48 hours, maximal by 5 days of culture, and paralleled the effect observed with basic fibroblast growth factor. This study describes a novel method for testing site-directed angiogenesis in vitro and further strengthens the angiogenic properties of 12(R)-HETrE by demonstrating a direct effect on limbal microvessel endothelial cells.

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

PubMed1996

Microvascular endothelial cells from E-selectin-deficient mice form tubes in vitro.

Studies of capillary morphogenesis and angiogenesis in vitro have suggested a role for E-selectin (CD62E) in the process of differentiation into tube-like structures. Recent studies by our group and others have demonstrated that mice lacking E-selectin because of germline inactivation of the E-selectin gene by gene targeting are viable and fertile, without apparent deficiencies in vascular development. Murine lung endothelial cells from wild-type and E-selectin-deficient animals were isolated using an activation-dependent sterile sorting method, and differentiation into tube-like structures on sparse fibronectin, Matrigel, and collagen gels was compared. Both types of murine lung endothelial cells spontaneously organized to form multicellular tubes and extensive anastomotic networks. There were no major differences in either the time course of development or the general appearance of the multicellular cords or tube-like structures formed by murine lung endothelial cells from wild-type or E-selectin-deficient mice, although different patterns were observed on different extracellular matrices. These studies, thus, demonstrate that E-selectin is not required for morphogenesis of 3-dimensional vascular structures in vitro.

Animals↗

PubMed1996

IL-4 induced leucocyte trafficking in cynomolgus monkeys: correlation with expression of adhesion molecules and chemokine generation.

BACKGROUND: Interleukin-4 (IL-4) is an immunoregulatory cytokine which has a wide variety of effects on immune cell function. In addition, recent studies suggest that IL-4 may have effects on other cells including endothelial cells in terms of the regulation of adhesion molecule expression and leucocyte extravasation from the vascular space to sites of tissue inflammation. Consequently, IL-4 may have an important role in the pathogenesis of allergic inflammation and disease. OBJECTIVE: The purpose of this study was to learn more about the potential role of IL-4 in inflammatory disease, specifically in regard to the potential of IL-4 to induce the expression of adhesion molecules on vascular endothelial cells and promote the adherence and transmigration of circulating leucocytes to sites of tissue inflammation. METHODS: Single subcutaneous injections of human IL-4 were administered to cynomolgus monkeys and tissue biopsy samples were obtained and analysed for adhesion molecule expression on vascular endothelium and inflammatory cell infiltrates. In another series of experiments, multiple subcutaneous injections of human IL-4 were administered (bid on four consecutive days) and the effects on peripheral blood leucocytes and plasma levels of various cytokines and chemokines were examined. RESULTS: Intradermal injection of IL-4 induced the expression of vascular cell adhesion molecule-1 (VCAM-1) on cutaneous vascular endothelium that was present at 8 hr and persisted out to 24 h post injection. The expression of VCAM-1 was associated with an inflammatory cell infiltrate comprised of granulocytes and mononuclear cells. Multiple injections of IL-4 resulted in a dose-related decrease in the relative percentage and total number of circulating lymphocytes and an increase in circulating neutrophils (4.6 +/- 1-2.1 +/- 0.2 x 10(6)/mL and 1.7 +/- 0.3-7.0 +/- 1 x 10(6)/mL, respectively). Analysis of cell surface markers by flow cytometry revealed a transient decrease in the number of CD4+T lymphocytes and a sustained decrease in CD16+ cells. In addition, IL-4 administration resulted in a large increase in plasma MCP-1 concentration. CONCLUSION: This is the first study to demonstrate an acute effect of IL-4 consistent with lymphocyte trafficking out of the vascular space, the induction of VCAM-1 expression on vascular endothelium and increases in plasma levels of MCP-1 in vivo. We suggest that IL-4 may be involved in the early recruitment of mononuclear cells to sites of tissue inflammation by the upregulation of VCAM-1 expression on vascular endothelium and the generation and release of potent chemoattractants.

Animals↗

PubMed1996

Physiological and pathophysiological roles of eicosanoids in the microcirculation.

Eicosanoids are important mediators of both physiological and pathophysiological responses of the microcirculation. These biologically active lipid mediators can be produced locally by the cellular elements within the microvasculature (endothelium, pericytes, smooth muscle cells) as well as interstitial cells (e.g. fibroblasts, mast cells) and by various cells in the vascular compartment (e.g. leukocytes, platelets). Transcellular metabolism of intermediates produced by one cell type in close contact with another also has the potential to generate potent metabolites. This review integrates some of the older literature with recent developments in eicosanoid synthesis and release, the molecular advances in the enzymology and receptor specificity of the eicosanoids and the regulation by autocoids and inflammatory mediators. The potential roles of eicosanoids in regulation of local blood flow, vascular reactivity, angiogenesis and leukocyte adhesion and transmigration are discussed.

Animals↗

PubMed1995

Transfection of the human heme oxygenase gene into rabbit coronary microvessel endothelial cells: protective effect against heme and hemoglobin toxicity.

Heme oxygenase (HO) is a stress protein and has been suggested to participate in defense mechanisms against agents that may induce oxidative injury such as metals, endotoxin, heme/hemoglobin, and various cytokines. Overexpression of HO in cells might therefore protect against oxidative stress produced by certain of these agents, specifically heme and hemoglobin, by catalyzing their degradation to bilirubin, which itself has antioxidant properties. We report here the successful in vitro transfection of rabbit coronary microvessel endothelial cells with a functioning gene encoding the human HO enzyme. A plasmid containing the cytomegalovirus promoter and the human HO cDNA complexed to cationic liposomes (Lipofectin) was used to transfect rabbit endothelial cells. Cells transfected with human HO exhibited an approximately 3.0-fold increase in enzyme activity and expressed a severalfold induction of human HO mRNA as compared with endogenous rabbit HO mRNA. Transfected and nontransfected cells expressed factor VIII antigen and exhibited similar acetylated low-density lipoprotein uptake (two important features that characterize endothelial cells) with > 85% of cells staining positive for each marker. Moreover, cells transfected with the human HO gene acquired substantial resistance to toxicity produced by exposure to recombinant hemoglobin and heme as compared with nontransfected cells. The protective effect of HO overexpression against heme/hemoglobin toxicity in endothelial cells shown in these studies provides direct evidence that the inductive response of human HO to such injurious stimuli represents an important tissue adaptive mechanism for moderating the severity of cell damage produced by these blood components.

Animals↗