Biomedical subjects

D F Gibbs

Publications and source records attributed to D F Gibbs.

At least 19 recordsLinked to original sources

PubMed1992

Mesangial cell killing by leukocytes: role of leukocyte oxidants and proteolytic enzymes.

Mesangial cells from human and rat kidney were examined for sensitivity to killing by neutrophils. Cells from both species were sensitive to killing by phorbol myristate acetate-stimulated neutrophils. Catalase was highly protective while superoxide dismutase was less protective and a number of protease inhibitors were not protective. Strong protection was also observed with the iron chelators, deferoxamine and phenanthroline, and with the hydroxyl radical scavengers, dimethylthiourea and 5,5-dimethyl-1-pyrroline N-oxide. Pretreatment of the mesangial cells with deferoxamine followed by washing also provided protection. Mesangial cells were also killed by reagent hydrogen peroxide (H2O2) but were much less sensitive to injury by direct application of proteolytic enzymes. The ability of H2O2 to injure mesangial cells was prevented by pre-incubation of the H2O2 with human leukocyte myeloperoxidase. These data suggest that killing is due primarily to the generation of H2O2 by the stimulated neutrophils and its further reduction in an iron-catalyzed reaction. The hydroxyl radical may be the reduction product that actually mediates lethal injury but lack of scavenger specificity prevents definitively concluding this. Mesangial cell killing by activated neutrophils could be significantly inhibited by monoclonal antibodies to CD11/CD18 molecules, suggesting that close contact between the target and effector cells is required for cytotoxicity. Although qualitatively similar to endothelial cells, the mesangial cells appeared to be quantitatively more oxidant sensitive than previously examined human and rat endothelial cells. Taken together, these data show that mesangial cells from rat and human are sensitive to leukocyte-induced injury and that injury results via an oxidant pathway.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗

PubMed1992

Modulation of Ca2+ levels in keratinocytes by all-trans retinoic acid.

Human epidermal keratinocytes, that have been growth-arrested by removal of epidermal growth factor from the culture medium, are stimulated to proliferate by all-trans retinoic acid (RA). The same treatment inhibits the onset of differentiated features and reduces cell-substrate adhesion. In the present study we show that the same treatment results in a decrease in total cell-associated Ca2+ as measured by changes in the amount of 45Ca2+ bound to cells at equilibrium following RA treatment and by a decrease in intracellular free Ca2+ levels as measured with the Ca(2+)-sensitive dye, Indo-1. The alterations in Ca2+ levels were evident within an hour after RA treatment, were in the range of 30-35% and occurred over the same RA concentration range that stimulated proliferation (i.e., 0.25-1.0 micrograms/ml). When the extracellular Ca2+ concentration was elevated from the normal level of 0.15-1.4 mM, intracellular free Ca2+ increased by a factor of 2 while total cell-associated Ca2+ increased approximately 6-fold. Even under conditions of high extracellular Ca2+, RA was able to reduce cell-associated and intracellular free Ca2+. These data indicate that RA has the capacity to lower Ca2+ levels in keratinocytes concomitantly with its effects on biological behavior.

Calcium↗

PubMed1992

Human umbilical vein endothelial cell killing by activated neutrophils. Loss of sensitivity to injury is accompanied by decreased iron content during in vitro culture and is restored with exogenous iron.

First passage human umbilical vein endothelial cells (HUVECs) were sensitive to killing by activated neutrophils and reagent hydrogen peroxide (H2O2). Catalase and deferoxamine prevented killing whereas soybean trypsin inhibitor and superoxide dismutase did not. In these regards, HUVECs are similar to previously characterized endothelial cells from bovine and rat. Although first passage HUVECs were killed by activated neutrophils, sensitivity fell off rapidly as the cells were maintained in culture. At passage 2 (four population doublings), and beyond, HUVECs were highly resistant. The cells also became resistant to killing by reagent H2O2. The acquisition of resistance to killing was not accompanied by a failure to up-regulate neutrophil adhesion molecules or to support neutrophil adhesion. Levels of intracellular anti-oxidants (total thiols, though not glutathione, glutathione peroxidase or catalase activity) increased as a function of passage in culture. However, levels of glutathione and total thiols in late passage (resistant) HUVECs were similar to levels in late passage rat pulmonary artery endothelial cells, that were sensitive to killing by activated neutrophils. Cell-associated iron in HUVECs fell as a function of time in culture. By passage 2, the amount of total iron measurable with the Ferrozine reagent was only about 30% of the amount recovered from first passage HUVECs. The loss of iron from the cells may underlie much of the concomitant resistance to killing because when the cells were pretreated with iron under conditions in which it could be taken up, sensitivity to killing by activated neutrophils and by H2O2 was restored.

Cells, Cultured↗

PubMed1991

Hydrogen peroxide-induced cell and tissue injury: protective effects of Mn2+.

Recent evidence indicates that under in vitro conditions, superoxide anion and hydrogen peroxide (H2O2) are unstable in the presence of manganese ion (Mn2+). The current studies show that in the presence of Mn2+, H2O2-mediated injury of endothelial cells is greatly attenuated. A source of bicarbonate ion and amino acid is required for Mn2+ to exert its protective effects. Injury by phorbol ester-activated neutrophils is also attenuated under the same conditions. EDTA reverses the protective effects. Acute lung injury produced in vivo in rats by intratracheal instillation of glucose-glucose oxidase is almost completely blocked in rats treated with Mn2+ and glycine. Conversely, treatment of rats with EDTA, a chelator of Mn2+, markedly accentuates lung injury caused by glucose-glucose oxidase. These data are consistent with the findings of others that Mn2+ can facilitate direct oxidation of amino acids with concomitant H2O2 disproportionation. This could form the basis of a new therapeutic approach against oxygen radical-mediated tissue injury.

Animals↗

PubMed1991

Effects of all-trans retinoic acid on neutrophil-mediated endothelial cell injury in vitro and immune complex injury in rats.

All-trans retinoic acid (RA) has beneficial effects when used in a variety of inflammatory skin conditions. In this study, the authors found that RA inhibited superoxide anion production and proteolytic enzyme release by human and rat neutrophils. Concomitantly, the authors found that RA-treated neutrophils were less able than untreated neutrophils to injure endothelial cells in culture even though the adhesion of the RA-treated neutrophils to endothelial cell monolayers was not diminished. Inhibition of cytotoxicity occurred over the same range of concentrations that inhibited oxygen radical formation and protease release. In additional studies, it was observed that pretreatment of endothelial cells with RA-induced resistance to subsequent injury by activated neutrophils. Finally, in vivo studies showed that pretreatment of rats for 3 days with RA (1-10 mg/day, IP) reduced the degree of injury in the lungs and skin sites after treatment with bovine serum albumin and antibodies to bovine serum albumin in the reverse-passive Arthus reaction. Thus, RA can modulate neutrophil-mediated endothelial cell injury by an effect on both the neutrophils and their target cells. Together, these effects may underlie the reduction in immune complex-mediated injury seen in experimental animals. The beneficial effects that retinoids have in a variety of inflammatory skin diseases may likewise be a reflection of their effects on the physiology of both neutrophils and endothelial cells.

Animals↗

PubMed1991

Inhibition of epithelial cell adhesion by retinoic acid. Relationship to reduced extracellular matrix production and alterations in Ca2+ levels.

Human squamous epithelial cells maintained in growth factor-deficient medium were examined for sensitivity to all-trans retinoic acid (retinoic acid). Under conditions of low external Ca2+ (0.15 mmol/l [millimolar]), or high external Ca2+ (1.4 mmol/l), retinoic acid stimulated proliferation. Concomitantly, cell-substrate adhesion was decreased. Enzyme-linked immunosorbent assays were used to assess production of two extracellular matrix components, ie, fibronectin and thrombospondin. In the presence of retinoic acid, production of both was decreased. Because both fibronectin and thrombospondin serve as epithelial cell adhesion factors, the decreased production of these moieties could contribute to reduced adhesion. Using 45Ca2+ to measure total cell-associated Ca2+ and the Ca2(+)-sensitive dye Indo-1 to measure intracellular free Ca2+, it was found that concentrations of retinoic acid that altered cell-substrate adhesion in the squamous epithelial cells had no effect on total, cell-associated Ca2+, but reduced intracellular free Ca2+ by 50% to 60%. Because Ca2+ is a regulator of adhesion, the ability of retinoic acid to modulate Ca2+ levels in the squamous epithelial cells may explain, in part, how retinoic acid influences their adhesiveness.

Calcium↗

PubMed1990

H2O2-mediated cytotoxicity of rat pulmonary endothelial cells. Changes in adenosine triphosphate and purine products and effects of protective interventions.

H2O2-mediated cytotoxicity (as measured by 51Cr-release) of rat pulmonary artery endothelial cells was time-dependent and related to the concentration of H2O2 employed. The cytotoxic effects of H2O2 were, as expected, prevented by catalase and the degree of protection was directly related to its time of addition. Endothelial cells were incubated with [14C]adenosine to achieve intracellular labeling of ATP, after which the cells were exposed to H2O2. Based on analysis of cell extracts by high-performance liquid chromatography, there was a time-dependent loss of intracellular radioactivity and ATP with the simultaneous appearance of purine degradation products including xanthine/hypoxanthine. Approximately 50% of the intracellular ATP was lost after 15 minutes of exposure and up to 80% was lost by 30 minutes. The extracellular fluid of cells exposed to H2O2 contained significant amounts of xanthine/hypoxanthine. The ferric iron chelator deferoxamine provided almost complete protection against H2O2-mediated cytotoxicity. Two inhibitors of xanthine oxidase, allopurinol and oxypurinol, were also protective as was deoxycoformycin, an inhibitor of adenosine deaminase. Remarkably, cells protected by these agents showed the same loss of intracellular ATP as unprotected, H2O2-treated cells. These findings demonstrate the dissociation between ATP loss per se and oxidant injury of endothelial cells. ATP breakdown may be an important event leading to cellular injury in that this results in the formation of substrate for xanthine oxidase.

Adenosine Triphosphate↗

PubMed1989

Interaction of mammalian cells with polymorphonuclear leukocytes: relative sensitivity to monolayer disruption and killing.

Monolayers of murine fibrosarcoma cells that had been treated either with histone-opsonized streptococci, histone-opsonized Candida globerata, or lipoteichoic acid-anti-lipoteichoic acid complexes underwent disruption when incubated with human polymorphonuclear leukocytes (PMNs). Although the architecture of the monolayers was destroyed, the target cells were not killed. The destruction of the monolayers was totally inhibited by proteinase inhibitors, suggesting that the detachment of the cells from the monolayers and aggregation in suspension were induced by proteinases releases from the activated PMNs. Monolayers of normal endothelial cells and fibroblasts were much resistant to the monolayer-disrupting effects of the PMNs than were the fibrosarcoma cells. Although the fibrosarcoma cells were resistant to killing by PMNs, killing was promoted by the addition of sodium azide (a catalase inhibitor). This suggests that the failure of the PMNs to kill the target cells was due to catalase inhibition of the hydrogen peroxide produced by the activated PMNs. Target cell killing that occurred in the presence of sodium azide was reduced by the addition of a "cocktail" containing methionine, histidine, and deferoxamine mesylate, suggesting that hydroxyl radicals but not myeloperoxidase-catalyzed products were responsible for cell killing. The relative ease with which the murine fibrosarcoma cells can be released from their substratum by the action of PMNs, coupled with their insensitivity to PMN-mediated killing, may explain why the presence of large numbers of PMNs at the site of tumors produced in experimental animals by the fibrosarcoma cells is associated with an unfavorable outcome.

Animals↗

PubMed1989

Vascular endothelial cell killing by combinations of membrane-active agents and hydrogen peroxide.

Previous studies have demonstrated that a number of membrane-active agents are capable of binding to the surface of polymorphonuclear leukocytes (PMN) resulting in an augmentation of superoxide anion and hydrogen peroxide (H2O2) production in response to soluble stimuli. It is now demonstrated that these same membrane-active agents can bind to the surface of endothelial cells and enhance their susceptibility to killing by H2O2. Membrane-active agents which are capable of synergizing with H2O2 include cationic proteins, cationic poly-amino acids, lysophosphatides and enzymes which are capable of degrading membrane phospholipids (e.g., phospholipase C, phospholipase A2 and streptolysin S). In each case, treatment of the target cells with the membrane-active agent and H2O2 produces greater damage than the sum of the damage produced by either agent separately. Since inflammatory lesions, particularly sites of bacterial infection, may contain a rich mixture of cationic substances, phospholipases and phospholipid breakdown products, these substances may contribute to the tissue damage observed at sites of inflammation by enhancing endothelial cell sensitivity to PMN-generated H2O2 as well as by augmenting the generation of H2O2 by PMNs.

Animals↗

PubMed1989

Endothelial cell killing by neutrophils. Synergistic interaction of oxygen products and proteases.

Killing of rat pulmonary artery endothelial cells by activated polymorphonuclear leukocytes (PMNs), as measured at 4 hours, is catalase sensitive, iron dependent, and unaffected by addition of protease inhibitors. If the time course for exposure of endothelial cells to activated PMNs is extended to 18 hours, progressive injury occurs. Endothelial cell injury resulting at 18 hours is partially inhibited by catalase and partially inhibited by soybean trypsin inhibitor. Together, these two inhibitors function synergistically to protect the cells from injury. Exposure of endothelial cells to reagent H2O2 and purified proteolytic enzymes (trypsin, chymotrypsin, elastase, and cathepsin G) mimics the effects of activated PMNs: H2O2 alone is cytotoxic with maximal killing achieved by 4 hours; proteolytic enzymes produce cytotoxicity only at high concentrations and only after prolonged incubation (longer than 8 hours); and, in combination, H2O2 and proteolytic enzymes act synergistically. These data provide compelling evidence that PMN-mediated injury of endothelial cells involves interaction between oxygen products and proteases.

Adult↗

PubMed1975