Neutrophil apoptosis in the lung after hemorrhage or endotoxemia: apoptosis and migration are independent of IL-1beta.

Hemorrhage and endotoxemia are associated with neutrophil accumulation in the lungs and the development of acute inflammatory lung injury. Because alterations in the rate of apoptosis may affect the number and function of neutrophils in the lungs, we determined the percentage of neutrophils undergoing apoptosis in the lungs of control, hemorrhaged, or endotoxemic mice. In control mice, 18.5 +/- 1.2% of pulmonary neutrophils were apoptotic. The proportion of apoptotic neutrophils in the lungs was significantly decreased 1 h after hemorrhage (6.5 +/- 1.6%, P < 0.01 compared to control) or endotoxemia (7.0 +/- 0.9%, P < 0.01 compared to control). Between 1 and 24 h after endotoxemia or hemorrhage, the proportion of apoptotic neutrophils in the lung remained significantly depressed compared to that in control, unmanipulated mice. By 48 h, the proportion of apoptotic neutrophils returned to baseline levels in the lungs of hemorrhaged (21.4 +/- 1.4%) or endotoxemic (16.4 +/- 1. 6%) mice. Lung neutrophil IL-1beta mRNA was significantly increased from that of control mice [i.e., 0.12 +/- 0.06 relative absorbance units (RAU)] 1 h after hemorrhage (5.19 +/- 0.068 RAU, P < 0.05 compared to control) or endotoxemia (8.90 +/- 1.53 RAU, P < 0.01 compared to control). In IL-1beta-deficient mice, there was no significant difference in lung neutrophil apoptosis or neutrophil entry into the lung after hemorrhage or endotoxemia compared to wild-type mice. Our results show that apoptosis among lung neutrophils is decreased for more than 24 h after hemorrhage or endotoxemia. Although IL-1beta expression is increased in lung neutrophils under these conditions, IL-1beta is not responsible for either the influx of neutrophils into the lung or the reduction of apoptosis in neutrophil populations after hemorrhage or endotoxemia.

Neutrophils are major contributors to intraparenchymal lung IL-1 beta expression after hemorrhage and endotoxemia.

Acute lung injury and the acute respiratory distress syndrome (ARDS) are significant causes of morbidity and mortality following sepsis and hemorrhage. Increased IL-1beta production in the lung is important in the development of acute inflammatory lung injury. Although neutrophils are an important component of the inflammatory response that characterizes acute lung injury, there is little information to suggest that they are capable of initiating cytokine-mediated immune responses in the lung. To explore the role of neutrophils in the early stages of acute lung injury, we examined IL-1beta production by mouse lung neutrophils after hemorrhage and endotoxemia. There was a significant increase in IL-1beta expression among intraparenchymal pulmonary neutrophil/mononuclear cells (IPNMC) 1 h after hemorrhage or endotoxemia. IL-1beta was detected only in a neutrophil-rich fraction of the IPNMC, but not in T and B lymphocytes positively selected from the IPNMC. Cyclophosphamide (CTX)-treated neutropenic mice expressed significantly less IL-1beta in IPNMC after hemorrhage or endotoxemia compared with CTX-untreated controls. Immunohistochemical analysis of lung sections from mice after hemorrhage or endotoxemia revealed IL-1beta expression in infiltrating neutrophils. These data indicate that IL-1beta-producing neutrophils traffic to the lungs rapidly in response to hemorrhage or endotoxemia and support the concept that proinflammatory cytokine production by lung neutrophils may contribute to the development of lung injury after blood loss and sepsis.

Actin polymerization and pseudopod reorganization accompany anti-CD3-induced growth arrest in Jurkat T cells.

T cell activation via CD3/Ti linked pathways results in the polymerization and reorganization of actin. However, little is known about the morphology and temporal appearance of filamentous actin (F-actin) after activation. Similarly, little is known about the relationship between F-actin and changes in cell shape or other parameters of activation, such as the appearance of proteins newly phosphorylated on tyrosine, that occur after stimulation via the CD3/Ti complex. Accordingly, we have characterized changes in cell shape and F-actin morphology occurring in the Jurkat T cell leukemia attached to the surface of culture vessels by immobilized anti-CD3 antibodies (OKT3, UCHT-1, SPV-T3b). These antibodies induced activation within 30 min as measured by increased protein tyrosine kinase activity and conversion of the proto-oncogene product, lck, from 56 kDa to 60 kDa (p56lck conversion), and after 12 to 96 h as measured by growth arrest and, in some experiments, IL-2 production. Activation was not seen when cells were attached to the substrates using antibodies directed to other cell surface proteins including CD71 (transferrin receptor), CD7, and CD11a (LFA-1), demonstrating the specificity of activation for immobilized anti-CD3 antibodies. Temporal changes in cell shape and F-actin morphology were characterized in Jurkat cells attached by immobilized anti-CD3 antibodies (stimulatory antibodies) and compared with the patterns obtained obtained in Jurkat cells attached by antibodies specific for the other markers (nonstimulatory antibodies). In these experiments, Jurkat cells were incubated with antibody-coated substrates for 1 to 30 min at 37 degrees C and actin rearrangements were visualized on fixed, detergent-permeabilized cells using rhodamine-conjugated phalloidin. Analysis of cell shape and F-actin morphology during the first 30 min of activation revealed a unique pattern that was observed only when cells were stimulated with anti-CD3 antibodies. Jurkat cells attached by either stimulatory or nonstimulatory antibodies reorganized their actin similarly after the first minute of culture, as characterized by the formation of small, F-actin rich pseudopods at the sites of attachment. After 5 min of culture in cells attached by stimulatory antibodies, the actin was polymerized into a dense collar rimming the inner edge of the cell. From 15 to 60 min, this collar was replaced by numerous F-actin rich, branched pseudopods. These branched pseudopods were larger and had longer microfilament bundles than their earlier counterparts. By contrast, in cells attached by nonstimulatory antibodies, the initial configuration was maintained for at least 60 min, except that a decrease in microfilament bundle length was noted.(ABSTRACT TRUNCATED AT 400 WORDS)