Charles F. Moldow

Oxygen radicals mediate endothelial cell damage by complement-stimulated granulocytes. An in vitro model of immune vascular damage.

During hemodialysis, alternative pathway complement activation leads to pulmonary sequestration of granulocytes, with loss of pulmonary vascular endothelial integrity and, at times, protein-rich pulmonary edema. An in vitro model of this phenomenon was constructed utilizing 51Cr-labeled human umbilical vein endothelial cell cultures. In this system, granulocytes, when exposed to activated complement (C), induce endothelial damage; this injury is mediated primarily by oxygen radicals produced by the granulocytes. C5a appears to be the C component responsible for granulocyte-induced cytotoxicity; studies with cytochalasin B-treated granulocytes suggest that close approximation of the granulocytes and endothelial cells is necessary for maximal cell injury.

Complement-induced granulocyte aggregation: an unsuspected mechanism of disease.

The capacity of blood cells to aggregate, best exemplified by the response of platelets to vascular injury, is generally thought to be beneficial. However, if aggregation occurs inappropriately—tha...

Abnormal Adherence of Sickle Erythrocytes to Cultured Vascular Endothelium: POSSIBLE MECHANISM FOR MICROVASCULAR OCCLUSION IN SICKLE CELL DISEASE

The abnormal shape and poor deformability of the sickled erythrocyte (RBC) have generally been held responsible for the microvascular occlusions of sickle cell disease. However, there is no correlation between the clinical severity of this disease and the presence of sickled RBC. In searching for additional factors that might contribute to the pathophysiology of sickle cell disease, we have investigated the possibility that sickle RBC might be less than normally repulsive of the vascular endothelium. After RBC suspensions are allowed to settle onto plates of cultured human endothelial cells, normal RBC are completely removed by as few as six washes. In contrast, sickle RBC remain adherent despite multiple washes. On subconfluent culture plates, normal RBC are distributed randomly, whereas sickle RBC cluster around endothelial cells. Sickle RBC adherence is not enhanced by deoxygenation but does increase with increasing RBC density. The enzymatic removal of membrane sialic acid greatly diminishes the adherence of sickle RBC to endothelial cells, suggesting that sialic acid participates in this abnormal cell-cell interaction. Although net negative charge appears normal, sickle RBC mainfest an abnormal clumping of negative surface charge as demonstrated by localization of cationized ferritin. These abnormalities are reproduced in normal RBC loaded with nonechinocytogenic amounts of calcium. We conclude that sickle RBC adhere to vascular endothelial cells in vitro, perhaps caused by a calcium-induced aberration of membrane topography. This adherence may be a pathogenetic factor in the microvascular occlusions characteristic of sickle cell disease.

Plasma Exchange in the Treatment of Immune Disease

Plasma exchange was investigated as an alternative to the use of toxic drugs to remove unwanted antibody. Studies in rabbits immunized with bovine serum albumin demonstrate that exchange transfusion after a primary immunization results in a rebound of antibody to above preexchange levels. However, exchange transfusion seven, 11, or 18 days after secondary immunization results in permanent lowering of antibody levels.

Deleterious effects of endotoxin on cultured endothelial cells: an in vitro model of vascular injury.

The effects of endotoxin-triggered granulocytes on the viability of endothelial cells in vitro was investigated. Endotoxin or its lipid A component caused granulocytes to adhere to and significantly damage cultured endothelial cells. Fresh serum is not necessary but does amplify both adherence and endothelial injury. Much of the endothelial injury was inhibited by free-radical scavengers or by blocking granulocyte adhesion to endothelial cells and appears to result from free radical production by the stimulated granulocyte. Studies in this model suggest a pathogenic role for the endotoxin-triggered granulocyte in the Shwartzman reaction and perhaps related clinical disorders.

Endothelial Cell Heterogeneity: Antioxidant Profiles Determine Vulnerability to Oxidant Injury

Abstract Human umbilical vein endothelial cells were more sensitive to hydrogen peroxide lysis than cow pulmonary artery endothelial cells. Conversely, activated neutrophils which utilize hydrogen peroxide-mediated cell cytotoxicity cell mechanisms were more toxic to the cow pulmonary artery cells. This discordance was not related to neutrophil adhesion to either cell type or cell passage number. The antioxidant profiles of the endothelial cells revealed that cow pulmonary artery cells were rich in catalase to consume bolus hydrogen peroxide presented to them, while human umbilical vein endothelial cells utilize glutathione peroxidase-linked mechanisms to detoxify a slower more sustained release of hydrogen peroxide generated by neutrophils. Endothelial cells from different species and sites may utilize diversified antioxidant protective mechanisms.

Folate-Induced Remission in Aplastic Anemia with Familial Defect of Cellular Folate Uptake

Severe aplastic anemia developed in a young man with an extensive family history of leukemia, pancytopenia, and neutropenia. Megaloblastic changes became evident, and treatment with high doses of folic acid resulted in striking clinical improvement. However, red-cell folate levels remained persistently low despite high serum folate levels. A defect in cellular folate uptake was suspected, and, indeed, uptake of 5-14CH3-H4-folate by stimulated lymphocytes and by bone-marrow cells from the patient was significantly reduced (P less than 0.05 as compared to normal cells. Further characterization of folate metabolism showed that intestinal absorption of the vitamin, membrane transport of 5-14CH3-H4-folate by mature red cells, folate utilization in the conversion of deoxyuridylate to thymidylate and polyglutamate formation were all normal. At least five other family members manifest decreased uptake of 5-14CH3-H4-folate by stimulated lymphocytes. These studies suggest that a genetically induced abnormality of folate uptake contributed to this patients severe, but reversible, aplasia.

THERAPEUTIC RAMIFICATIONS OF THE INTERACTION OF COMPLEMENT, GRANULOCYTES, AND PLATELETS IN THE PRODUCTION OF ACUTE LUNG INJURY*

Evidence is reviewed that activation of complement through its ability to cause adhesion of granulocytes to the pulmonary endothelium (margination) and aggregation and embolization of these cells to the lung microvasculature is an important cause of pulmonary injury. Agents capable of diminishing such stimulated adhesiveness might be rational therapeutic adjuncts in vascular-damaging syndromes and are being investigated. To date, corticosteroids, such as methylprednisolone (in enormous doses), and certain nonsteroidal anti-inflammatory drugs, such as ibuprofen, have been shown to diminish granulocyte stickiness in vitro and diminish complement-mediated tissue damage in vivo. Moreover, platelets amplify complement/granulocyte-induced endothelial damage by releasing serotonin, which, in turn, potentiates granulocyte adhesion to endothelium. This potentiation is abrogated by the serotonin antagonists imiprimine and methysergide. Since these diverse agents inhibit granulocyte-adhesion phenomena by different mechanisms, they might predictably be synergistic in their beneficial effect. This prediction is validated for methylprednisolone and ibuprofen, for which we report a threefold synergism in preventing complement-mediated granulocyte aggregation in vitro. Preliminary studies indicate utility of this synergism in vivo as well, in that the degree of myocardial infarction [a complement-activating phenomenon) is diminished in coronary-ligated animals in which rather small doses of the two drugs administered together.

[48] Endothelial culture, neutrophil or enzymic generation of free radicals: In vitro methods for the study of endothelial injury

Publisher Summary In vitro culture of endothelial cells from a variety of anatomic sites and various species permits the study of the inflammatory response at a cellular level. The interactions of endothelial cells with polymorphonuclear leukocytes (PMN), particularly those activated by inflammatory mediators, such as certain complement components, seem critical to the vasodilatation and permeability alterations that characterize acute inflammation. The metabolic products of activated PMN, especially reactive oxygen species (ROS) and lysosomal proteases, contribute to the inflammatory response and have been implicated in many clinical syndromes. This chapter describes a method for determining toxic effects of PMN products upon endothelial cells in vitro. It uses cultured human umbilical vein endothelial cells that are mixed with activated PMN to induce release of endothelial-bound radiolabel and measures both cellular injury and detachment. Specific inhibitors may be used to determine the contribution of ROS (or other substances) produced by the neutrophil while PMN production of these products can be measured directly and correlated with the degree of endothelial injury. ROS may also be generated enzymically in the absence of PMN to further explore their effects upon the endothelial cells.

Familial Hodgkin's disease and the major histocompatibility complex.

Abstract. Two sisters who developed nodular sclerosis Hodgkins disease (H.D., N.S.) within 3 months of their 25th birthdays were HLA and MLC identical. All family members possessed the HLA‐B7 antigen. The propositi, a third HLA/MLC identical sister, and the mother, shared the maternal B7 and lacked Dw2. The father and a fourth sibling with the paternal B7 were heterozygous for Dw2. In six unrelated patients with H.D., N.S., the presence of B7 and Dw2 correlated precisely. In 69 consecutive patients with H.D., N.S., the frequencies of 23 HLA antigens did not differ significantly from the frequencies in 245 control subjects.