cytochalasin-b and Sepsis

We have identified a patient with a number of neutrophil dysfunctions. The patient was a female baby who lived for 8 months. During her life, she developed severe bacterial infections and showed omphalitis, impaired wound healing, and a pronounced leukocytosis. She was not a patient with leukocyte adhesion deficiency, because all leukocyte CD18 complex proteins were expressed at normal levels. Yet, neutrophil polarization and chemotaxis to platelet-activating factor, leukotriene B4, or formyl-methionyl-leucyl-phenylalanine (FMLP) were completely absent. We found a strong defect in actin polymerization in response to chemotactic stimuli, but only a retarded or even normal reaction with other stimuli. This indicates that the cellular dysfunctions were not due to an intrinsic defect in actin metabolism. Instead, the regulation of actin polymerization with chemotactic stimuli seemed to be defective. We concentrated on FMLP-induced responses in the patient's neutrophils. Functions dependent on activation of complement receptor type 3, such as aggregation or adherence to endothelial cells, were normally induced. Binding to serum-coated coverslips was normal in cell number; however, spreading was not observed. Exocytosis from the specific granules was readily induced. In contrast, FMLP failed to induce a respiratory burst activity or degranulation of the azurophil granules. FMLP induced a normal increase in free intracellular Ca2+, but a decreased formation of diglycerides (especially the 1-O-alkyl,2-acyl compounds). Thus, we have described a patient whose neutrophils show a severe defect in functional activation via chemotaxin receptors, resulting in a selective absence of NADPH oxidase activity, exocytosis from the azurophil granules, and actin polymerization. Our findings show that actin polymerization for neutrophil spreading and locomotion is regulated differently from that for phagocytosis. Also, the release of azurophil and specific granule contents is clearly shown to be regulated in a different way.

Topics: Actins; Antigens, CD; Calcimycin; Calcium; CD18 Antigens; CD4 Antigens; CD8 Antigens; Cell Adhesion; Cell Aggregation; Chemotaxis, Leukocyte; Cytochalasin B; Endothelium, Vascular; Female; Humans; Immunologic Deficiency Syndromes; In Vitro Techniques; Infant, Newborn; Kinetics; Leukocyte Count; Leukotriene B4; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils; Oxygen Consumption; Platelet Activating Factor; Reference Values; Sepsis; Syndrome; T-Lymphocyte Subsets

This study examined whether alterations in cellular Ca2+ regulation contribute to previously observed changes in skeletal muscle sugar transport during bacteremia. Fasted male rats received saline (control) or bacteria (4 X 10(10) Escherichia coli/kg) intraperitoneally. Twelve hours later, basal and insulin-mediated 3-O-methylglucose (3MG) transport was measured in isolated soleus muscles. Measurements of 3MG transport in the presence of cytochalasin b or at a low temperature (0.5 degree C) indicated that altered sugar transport in bacteremic rat muscles was not due to nonspecific membrane permeability changes. To determine the role of Ca2+ in the pathogenesis of altered sugar transport during bacteremia, rats were treated with the Ca2+ antagonist diltiazem (DZ, 0.6-2.4 mg/kg) at various times (0, 0 + 7.5, 10 h) after saline or bacterial injection. In bacteremic rats given 2.4 mg/kg DZ at 10 h, basal and insulin-mediated transport were similar to control values. This dose of DZ had little effect on control muscles. The addition of 20 microM DZ to the incubation media did not affect basal or insulin-mediated 3MG transport in bacteremic rat muscles. Addition of the Ca2+ agonist BAY K 8644 to the incubation media had no effect on sugar transport in bacteremic rat muscles but caused alterations in control rat muscles that were comparable to those observed in bacteremia. These results suggest that alterations in Ca2+ regulation could contribute to the previously observed changes in sugar transport in skeletal muscles from bacteremic rats.

Topics: 3-O-Methylglucose; Animals; Biological Transport, Active; Carbon Radioisotopes; Cytochalasin B; Diltiazem; Escherichia coli Infections; Insulin; Kinetics; Male; Methylglucosides; Methylglycosides; Monosaccharide Transport Proteins; Muscles; Radioisotope Dilution Technique; Rats; Reference Values; Sepsis; Thermodynamics

There is a growing body of data to suggest that marginated granulocytes mediate much of the pulmonary damage observed during endotoxemia. The mechanism(s) by which endotoxemia initiates neutrophil margination and cytotoxicity remain either controversial or unknown. The objectives of this study were 1) to determine the temporal relationship between endotoxin-induced decreases in mean arterial pressure and circulating neutrophils, 2) to monitor neutrophil activation in vivo by measuring myeloperoxidase (MPO) activity in the plasma and lymph, and 3) to assess the interaction between endotoxin and complement in activation of neutrophilic oxidative metabolism in vitro. We found that a bolus injection of endotoxin causes a concurrent decrease in both mean arterial pressure and circulating neutrophils at 2 min postinfusion. Blood pressure recovered to approximately 70% of control values by 180 min, whereas circulating neutrophils remain depressed at 20% of control values for the entire experimental period. Using MPO as a marker for neutrophil activation, we found that infusion of endotoxin produces a dramatic increase in plasma and lymph MPO activity, suggesting activation of neutrophilic metabolism in vivo. In vitro data showed that both endotoxin and plasma were required for optimal neutrophilic degranulation and superoxide formation. We conclude that 1) the appearance of MPO in the plasma (or lymph) may be a useful neutrophil marker for neutrophil activation in vivo and may prove useful in following the course of neutrophil-mediated tissue injury during endotoxemia, and 2) endotoxin-activated complement (C5a) activates neutrophils to produce cytotoxic oxidants.

Topics: Animals; Blood Pressure; Complement C5; Complement C5a; Cytochalasin B; Dogs; Endotoxins; Female; Leukocyte Count; Male; Neutropenia; Neutrophils; Peroxidase; Sepsis; Superoxides