Biomedical subjects

R M Marks

Publications and source records attributed to R M Marks.

At least 55 records · Page 3Linked to original sources

PubMed1989

Monokine-induced neutrophil chemotactic factor gene expression in human fibroblasts.

Although fibroblasts are important in providing a structural framework for most tissues, they also appear to be active participants in the inflammatory process via the production of specific mediators. The production of inflammatory mediators by fibroblasts is especially important in relation to their strategic location within connective tissue as they may act as a cellular communication bridge between the interstitium and vasculature. In this paper, we demonstrate that fibroblasts may participate in these inflammatory reactions by the production of a neutrophil chemotactic factor (NCF) with characteristics similar to a recently isolated and cloned monocyte-derived NCF. Either tumor necrosis factor-alpha-, interleukin-1 alpha-, or interleukin-1 beta-stimulated fibroblasts showed both a time- and dose-dependent increase in steady-state levels of NCF mRNA and secretion of chemotactic activity. In contrast, lipopolysaccharide and interleukin-6 failed to induce fibroblast-derived NCF. The expression of fibroblast-derived NCF mRNA was first detectable by 30 min poststimulation, whereas chemotactic activity was significantly observed 3-4 h postchallenge. Heat-inactivated monokine (100 degrees C) failed to induce NCF mRNA expression, suggesting that only the active proteins are capable of inducing NCF. Gel filtration analysis using high pressure liquid chromatography indicated peak chemotactic activity with an approximate molecular mass of 8000 daltons. This peak of NCF activity was found to be relatively stable to both heat and trypsin inactivation. Specificity of the fibroblast-derived neutrophil chemotactic activity was demonstrated with inhibition of chemotaxis by the addition of neutralizing antibody directed against recombinant human neutrophil chemotactic factor. These data provide evidence that monokine-treated fibroblasts can synthesize a potent chemotactic agent with molecular and physicochemical characteristics similar to monocyte-derived NCF and that this factor may contribute to neutrophil-mediated disease processes.

Base Sequence↗

PubMed1989

Rapid induction of neutrophil-endothelial adhesion by endothelial complement fixation.

The adhesion of neutrophils to vascular endothelium is an early event in their recruitment into acute inflammatory lesions. In evaluating potential neutrophil-endothelial adhesive mechanisms in acute inflammation, important considerations are that adhesion in vivo may occur very rapidly following injury and that the specificity of the reaction resides in altered endothelium. That is, neutrophils adhere only to altered endothelium adjacent to an inflammatory focus, rather than at random as would be expected if activation of neutrophils were the initiator of adhesion. We have explored a possible bridging role for complement in causing early neutrophil-endothelial cell adhesion. The complement system is involved in inflammatory processes, is capable of rapid amplification, and endothelial complement fixation at sites of inflammation could generate an endothelium-restricted signal for neutrophil adhesion. We have now developed a model in which this can be investigated without complicating factors such as immunoglobulin deposition, by constructing a novel molecule, a hybrid of the endothelial binding lectin Ulex europaeus I and of the complement activator cobra venom factor. This molecule has the capacity to cause fixation of complement on human umbilical vein endothelial cells. We show that complement fixation is a potent and rapid stimulus for neutrophil adhesion. Neutrophil adhesion requires only endothelial deposition of C3, and is mediated through the type 3 complement receptor.

Cell Adhesion↗

PubMed1989

Endothelial cell gene expression of a neutrophil chemotactic factor by TNF-alpha, LPS, and IL-1 beta.

Human endothelial cells produced a neutrophil chemotactic factor (NCF) upon stimulation with tumor necrosis factor-alpha (TNF-alpha), interleukin-1 beta (IL-1 beta), or lipopolysaccharide (LPS). The expression of endothelial cell-derived NCF messenger RNA and biological activity was both time- and concentration-dependent. Maximal NCF mRNA expression occurred at 10 and at 2 nanograms per milliliter for TNF and IL-1 beta, respectively; mRNA expression was first observed 1 hour after stimulation and was maintained for at least 24 hours. In situ hybridization analysis showed that NCF mRNA peaked in treated cells by 24 hours, whereas unstimulated cells were negative. These studies demonstrated that endothelial cells may participate in neutrophil-mediated inflammation by synthesizing a chemotactic factor in response to specific monokines and LPS.

Blotting, Northern↗

PubMed1989

The acute inflammatory reaction.

A major theme in current developments in this field has been the realization that the target tissue, in particular the endothelium, has an active role in initiating and participating in inflammatory reactions and is not a passive target, as was previously thought. Attempts to delineate the mechanisms involved have benefited immeasurably from the application of the modern techniques of molecular and cellular biology, and will continue to do so. It is axiomatic that a fuller understanding of these mechanisms will result in the development of new strategies designed to block the acute inflammatory response more specifically and effectively.

Acute-Phase Reaction↗

PubMed1989

Isolation and propagation of endothelial cells derived from rheumatoid synovial microvasculature.

Synovial angiogenesis may play an important part in the destruction of articular cartilage in patients with rheumatoid arthritis (RA). As an important first step towards developing in vitro models of synovial angiogenesis, microvascular endothelial cells have been isolated, purified, and cultured from operative synovial specimens obtained from adult patients with RA.

Arthritis, Rheumatoid↗

PubMed1989

Cytotoxicity of tumor necrosis factor-alpha for human umbilical vein endothelial cells.

Human umbilical vein endothelial cells were examined for sensitivity to killing by human recombinant tumor necrosis factor-alpha (TNF-alpha). Treatment of the cells with concentrations of TNF-alpha up to 50 ng/ml for 18 hours did not produce evidence of cytotoxicity. However, a marked cytotoxic effect was found when TNF-alpha pretreated cells were incubated in Hanks' balanced salt solution for a further 4 hours. Exposure of the cells to heat-inactivated or antibody-neutralized TNF-alpha did not result in cytotoxicity. Human recombinant interleukin-1 also lysed endothelial cells under the same conditions, whereas human recombinant macrophage-colony stimulating factor did not. Inclusion of superoxide dismutase, catalase, or soybean trypsin inhibitor in the culture medium during the time of endothelial cell exposure to TNF-alpha had no protective effects. Likewise, allopurinol (a xanthine oxidase inhibitor) and nordihydro-guaiaretic acid (a lipoxygenase inhibitor) were not protective under the same conditions. In contrast, the ferric iron chelator deferoxamine mesylate and three different cyclooxygenase inhibitors provided significant protection against TNF-alpha induced cytotoxicity. When human dermal fibroblasts and human squamous epithelial cells were used in place of the umbilical vein endothelial cells, these cells were resistant to TNF-alpha mediated killing. These findings demonstrate that under the experimental conditions employed, TNF-alpha is cytotoxic for human umbilical vein endothelial cells. This may have implications in a number of in vivo situations in which TNF-alpha is thought to play a role.

Cell Survival↗

PubMed1988

Monokine-induced gene expression of a human endothelial cell-derived neutrophil chemotactic factor.

Monokines have been increasingly recognized as communication signals that interact with both immune and non-immune cells during inflammation. Specifically, interleukin-1 alpha (IL-1 alpha), interleukin-1 beta (IL-1 beta) and tumor necrosis factor-alpha (TNF-alpha) possess potent effector activities on various cell types. We present novel data demonstrating that human endothelial cells are a major source of a neutrophil chemotactic factor (NCF) synthesized upon stimulation with either IL-1 alpha, IL-1 beta, or TNF-alpha; but not with interleukin-6 (IL-6). Northern blot analysis demonstrated that 20 ng/ml of either IL-1 or TNF-alpha could induce endothelial cells to express significant levels of NCF mRNA, while IL-6 was not active in this system. These data demonstrate that monokines play an important role in mediating acute inflammation via induction of an endothelial cell-derived NCF.

Autoradiography↗

PubMed1988

The effects of scleroderma serum on human microvascular endothelial cells. Induction of antibody-dependent cellular cytotoxicity.

To investigate the vascular immunopathology of systemic sclerosis, we developed a model consisting of human microvascular endothelial cells, leukocytes, and serum. Sera from 19% of the patients studied mediated antibody-dependent cellular cytotoxicity against endothelial cells. Some sera also mediated cytotoxicity against aortic endothelium and fibroblasts. K lymphocytes, the cells that mediate antibody-dependent cellular cytotoxicity, were identified in the skin of some patients. The sera alone were not cytotoxic or growth inhibitory, and did not affect endothelial prostacyclin production.

Antibody-Dependent Cell Cytotoxicity↗

PubMed1986

Use of diathermy for weeding heterogeneous tissue cultures.

Cultures generated from tissues consisting of multiple types of cells are often heterogeneous. Unless the cell type of interest has or can be given some selective growth advantage it may be overgrown by other cells. While developing techniques for the tissue culture of microvascular endothelial cells we evaluated an electrosurgical generator (diathermy) to selectively kill nonendothelial cells. Primary cell cultures were observed at X 100 magnification under phase contrast microscopy and a needle electrode apposed to the cell to be destroyed. A return electrode was constructed by placing a sterile clip in contact with the culture medium. The diathermy power setting controlled the area of lysis. Use of this technique allowed weeding of unwanted cells without damage to endothelial cells, which were able to grow to confluence in pure culture.

Capillaries↗

PubMed1986

Killing of human dermal capillary endothelial cells by antibody-dependent cellular cytotoxicity.

Damage to the microvasculature occurs commonly in autoimmune diseases affecting the skin. There has been little investigation of immune mechanisms other than immune complex deposition as a major cause of microvascular damage. We have investigated the potential of antibody-dependent cellular cytotoxicity (ADCC) to cause vascular destruction in an in vitro model consisting of human dermal microvascular endothelial cells to which were added antibody and leukocytes. Severe damage was induced by antibody and lymphocytes. Monocytes or neutrophils were not able to mediate cytotoxicity although activated neutrophils caused endothelial detachment. The cytotoxic cells were OKT3-, Leu 11+ and were identified as K cells. ADCC was not inhibited by human serum or aggregated IgG. These results imply that ADCC may have a role in causing the vascular destruction observed in some human autoimmune skin diseases.

Antibody-Dependent Cell Cytotoxicity↗

PubMed1986

Mast cell granules cause proliferation of human microvascular endothelial cells.

To investigate the possible role of mast cells in blood vessel formation, rat mast cell granules were studied for their proliferative effect on human microvascular endothelial cells. It was found that granules had a marked proliferative effect and that most of this activity was restricted to a dialyzable fraction. The dialyzable mast cell granule constituent histamine was found to be mitogenic, an effect that was shown with the use of specific agonists and antagonists to be mediated through an H1 receptor. H1 antagonists reduced the proliferation caused by the untreated mast cell granules to the level of proliferation caused by dialyzed granules, suggesting that all the dialyzable mitogenic activity was due to histamine. Histamine was also shown to cause proliferation of cells that were growth arrested by serum deprivation, suggesting that it is an endothelial growth factor. The compound responsible for the undialyzable mitogenic activity could not be identified but was shown not to be mast cell heparin. This demonstration of mast cell granule-induced endothelial proliferation suggests that the mast cell may be of importance in the process of angiogenesis.

Animals↗

PubMed1985

Human dermal microvascular endothelial cells: an improved method for tissue culture and a description of some singular properties in culture.

Tissue culture of human large vessel endothelium is now routine in many laboratories but tissue culture of human microvascular endothelium remains a difficult procedure, preventing study of features of endothelial function that may be peculiar to the microvasculature. This report describes an improved method for tissue culture of human dermal microvascular endothelium derived from foreskin. The method is rapid, reproducible, avoids contamination with nonendothelial cells, and does not require the use of a tumor-conditioned medium. The major modifications over existing techniques are the use of a Percoll density gradient to remove the majority of nonendothelial cells followed by a simplified weeding procedure that removes residual nonendothelial cells and leaves large numbers of endothelial cells to grow rapidly to confluence. The cells are identified as endothelial by their morphology and by positive immunofluorescence for Factor VIII. Proliferation experiments demonstrate their requirement for an exogenous matrix and for a high concentration of human serum. Whole serum was required as platelet-poor plasma serum had poor growth stimulatory activity. Proliferation could be enhanced by dibutyryl cyclic AMP or endothelial cell growth substance and was maximal with the combination of endothelial cell growth substance and heparin. However, the use of these agents did not remove the requirement for an exogenous matrix. Fibroblast growth factor, platelet-derived growth factor, epidermal growth factor, nerve growth factor, and thrombin did not increase proliferation.

Animals↗

PubMed1976

Lymphokine-like activity in the serum of patients with haemopoietic malignancies.

Serum from patients with haemopoietic malignancies was tested for the presence of migration inhibition factor (MIF) and leucocyte inhibition factor (LIF) activity by standard assays. There was no correlation between the specific disease state and positivity of the sera for MIF or LIF activity. MIF activity in the serum of one patient with myeloma was present in the isolated IgG cryoglobulin fraction.

Animals↗

PubMed1975

Salmonella enteritidis infection in rats: antigens involved in cell-mediated immunity.

When rats are sub-lethally infected with S. entertidis by the intravenous route, there is a period of at least 14 days in which peritoneal exudate cells are unable to migrate from capillary tubes on to glass surfaces; thereafter they migrate as do macrophages from normal animals. The migration of peritoneal cells from rats infected 18-21 days previously is inhibited by lipopolysaccharide (LPS) prepared from homologous and heterologous strains of Salmonella. The apparent non-specificity of LPS activity in the macrophage migration inhibition test has been resolved by demonstrating that lipid A, containing less than 0.01% protein amino acids, is also capable of eliciting this test of cell-mediated immunity.

Animals↗

PubMed1973