Mononuclear phagocytes are known to be long-lived cells, capable of synthesis of new cellular constituents and of major alterations in metabolic activities and morphology (1). In contrast, the neutrophil generally has been considered an end-stage cell, incapable of significant functional or metabolic modulation. However, Granelli-Piperno et ai. (2, 3) have reported that human neutrophils actively synthesize protein and RNA and secrete plasminogen activator when placed in culture, and that these activities can be stimulated by incubation of the cells with concanavalin A and inhibited by incubation with glucocorticoids. These findings, and the capacity of small amounts of bacterial endotoxin (lipopolysaccharide, LPS) ~ to prime mononuclear phagocytes for enhanced stimulated release of superoxide anion (O~) (4, 5), suggested to us that neutrophil function might be modified by exposure to LPS. We report here that incubation of human neutrophils for 30-60 min with LPS primes these cells for enhanced release of O~ or hydrogen peroxide (H~O~) upon subsequent contact with a variety of stimuli. This increase in oxidative metabolism might permit increased effectiveness of the neutrophil in host defense. These results also raise the possibility that exposure of neutrophils to bacterial LPS could enhance the capacity of these cells to damage tissue, which might occur in conditions such as endotoxic shock or infection-induced adult respiratory distress syndrome. Materials and MethodsPreparation of Neutrophils. Human neutrophils were isolated from venous blood anticoagulated with 10% sodium citrate (3.8% solution in water; Fisher Scientific Co., Pittsburgh, PA). 3 vol blood were added to 1 vol 6% dextran (70,000 tool we) in 0.9% sodium chloride solution (Cutter Laboratories, Berkeley, CA) and allowed to stand at This work was supported by grants AI 14148, HL 21565, and GM 24834 from the National Institutes of Health. L. C. M.'s present address is: Dept. of Biochemistry Bowman Gray School of Medicine, Winston-Salem, NC 27103. Reprint requests should be addressed to R. B. J.l Abbreviations used in this paper: BSA, bovine serum albumin; FMLP, formyl-methionyl-leucylphenylalanine; KRPD, Krebs-Ringer phosphate buffer with dextrose, 2 mg/ml; LPS, lipopolysaccharide; OL superoxide anion; PBS, phosphate-buffered saline, pH 7.4; PMA, phorbol myristate acetate; TCA, trichloroacetic acid. 1656J. ExP. MEB.

Granulocytes undergoing apoptosis are recognized and removed by phagocytes before their lysis. The release of their formidable arsenal of proteases and other toxic intracellular contents into tissues can create significant damage, prolonging the inflammatory response. Binding and/or uptake of apoptotic cells by macrophages inhibits release of proinflammatory cytokines by mechanisms that involve anti-inflammatory mediators, including TGF-β. To model the direct effects of necrotic cells on macrophage cytokine production, we added lysed or apoptotic neutrophils and lymphocytes to mouse and human macrophages in the absence of serum to avoid complement activation. The results confirmed the ability of lysed neutrophils, but not lymphocytes, to significantly stimulate production of macrophage-inflammatory protein 2 or IL-8, TNF-α, and IL-10. Concomitantly, induction of TGF-β1 by lysed neutrophils was significantly lower than that observed for apoptotic cells. The addition of selected serine protease inhibitors and anti-human elastase Ab markedly reduced the proinflammatory effects, the lysed neutrophils then behaving as an anti-inflammatory stimulus similar to intact apoptotic cells. Separation of lysed neutrophils into membrane and soluble fractions showed that the neutrophil membranes behaved like apoptotic cells. Thus, the cytokine response seen when macrophages were exposed to lysed neutrophils was largely due to liberated proteases. Therefore, we suggest that anti-inflammatory signals can be given by PtdSer-containing cell membranes, whether from early apoptotic, late apoptotic, or lysed cells, but can be overcome by proteases liberated during lysis. Therefore, the outcome of an inflammatory reaction and the potential immunogenicity of Ags within the damaged cell will be determined by which signals predominate.

Phosphatidylserine (PS), ordinarily sequestered in the plasma membrane inner leaflet, appears in the outer leaflet during apoptosis, where it triggers non-inflammatory phagocytic recognition of the apoptotic cell. The mechanism of PS appearance during apoptosis is not well understood but has been associated with loss of aminophospholipid translocase activity and nonspecific flip-flop of phospholipids of various classes. The human leukemic cell line HL-60, the T cell line Jurkat, and peripheral blood neutrophils, undergoing apoptosis induced either with UV irradiation or anti-Fas antibody, were probed in the cytofluorograph for (i) surface PS using fluorescein isothiocyanate-labeled annexin V, (ii) PS uptake by the aminophospholipid translocase using [6-[(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino] caproyl] (NBD)-labeled PS, (iii) nonspecific uptake of phospholipids (as a measure of transbilayer flip-flop) using NBDlabeled phosphatidylcholine, and (iv) the appearance of hypodiploid DNA. In all three types of cells undergoing apoptosis, the appearance of PS followed loss of aminophospholipid translocase and was accompanied by nonspecific phospholipid flip-flop. Importantly, however, in the absence of extracellular calcium, the appearance of PS was completely inhibited despite DNA fragmentation and loss of aminophospholipid translocase activity, the latter demonstrating that loss of the translocase is insufficient for PS appearance during apoptosis. Furthermore, while both the appearance of PS and nonspecific phospholipid uptake demonstrated identical extracellular calcium requirements with an ED 50 of nearly 100 M, the magnitude of PS appearance depended on the level of aminophospholipid translocase activity. Taken together, the data strongly suggest that while nonspecific flip-flop is the driving event for PS appearance in the plasma membrane outer leaflet, aminophospholipid translocase activity ultimately modulates its appearance.The appearance of phosphatidylserine in the outer leaflet of the plasma membrane appears to be a universal phenomenon in cells undergoing apoptosis, or programmed cell death (1). Importantly, outer leaflet PS likely serves as a signal in tissues for the noninflammatory engulfment of apoptotic cells (Ref. 2 and see "Discussion"), a distinctly different response than the pro-inflammatory events following tissue necrosis. While phosphatidylserine (PS) 1 is actively transported from the outer to the inner leaflet by the aminophospholipid translocase (3-5), the mechanism(s) by which PS appears in apoptosis is not well understood. Verhoven et al. (3) reported that PS appearance was accompanied by both loss of aminophospholipid translocase activity and enhanced nonspecific transbilayer movement of phospholipids, perhaps due to activation of a "scramblase." Hence, they proposed that PS may become detectable in the outer leaflet due to the combination of these two events. However, to date, the relative contribution of these two events has not been clarified. Additionally, it is not known what role...

The neutrophil has been implicated as an important mediator of vascular injury, especially after endotoxemia. This study examines neutrophil-mediated injury to human microvascular endothelial cells in vitro. We found that neutrophils stimulated by formyl-methionyl-leucyl-phenylalanine (FMLP), the complement fragment C5a, or lipopolysaccharide (LPS) (1-1,000 ng/ ml) alone produced minimal endothelial injury over a 4-h assay. In contrast, neutrophils incubated with endothelial cells in the presence of low concentrations of LPS (1-10 ng/ml) could then be stimulated by FMLP or C5a to produce marked endothelial injury. Injury was maximal at concentrations of 100 ng/ml LPS and 1o-7 M FMLP. Pretreatment of neutrophils with LPS resulted in a similar degree of injury, suggesting that LPS effects were largely on the neutrophil. Endothelial cell injury produced by LPS-exposed, FMLP-stimulated neutrophils had a time course similar to that induced by the addition of purified human neutrophil elastase, and different from that induced by hydrogen peroxide (H202). Further, neutrophil-mediated injury was not inhibited by scavengers of a variety of oxygen radical species, and occurred with neutrophils from a patient with chronic granulomatous disease, which produced no H202. In contrast, the specific serine elastase inhibitor methoxy-succinyl-alanyl-alanylprolyl-valyl-chloromethyl ketone inhibited 63% of the neutrophilmediated injury and 64% of the neutrophil elastase-induced injury. However, neutrophil-mediated injury was not inhibited significantly by 50% serum, 50% plasma, or purified a, proteinase inhibitor. These results suggest that, in this system, chemotactic factor-stimulated human neutrophil injury of microvascular endothelial cells is enhanced by small amounts of LPS and may be mediated in large part by the action of neutrophil elastase.

Polymorphonuclear neutrophils and monocytes emigrate from the bloodstream to areas of inflammation and play an essential role there in a variety of physiological and pathological events, including defense against infection. Although the biochemical basis for these cells' microbicidal activity is not completely understood, studies primarily with neutrophils indicate that the killing of most organisms depends upon phagocytosis-associated oxidative metabolism. During phagocytosis neutrophils remove oxygen from the surrounding medium and convert it, probably first, to superoxide anion (O2") (1, 2) and then to hydrogen peroxide (H202) (3). H202 and O~. may interact to form the potent oxidant, hydroxyl radical (-OH) (2). Oxidation of glucose through the hexose monophosphate (HMP) shunt is stimulated, perhaps as a result of increased H202 generation (4). The spontaneous dismutation of O~. is believed to result in singlet oxygen formation (reviewed in 2), and the occurrence of this reaction in neutrophils appears to be the most likely explanation for the luminescence that occurs with phagocytosis (2, 5).Like neutrophils, monocytes undergo increased HMP shunt activation (6-8), oxygen consumption (7, 8), and H202 generation (7) during ingestion. Macrophages from mice and guinea pigs generate O~. (9). We report here that human monocytes elaborate O~. and generate chemiluminescence during phagocytosis, on stimulation by a surface-active chemical agent, and on contact with fLxed aggregated IgG. Materials and MethodsPreparation of Cells. Human neutrophils were separated from other leukocytes in 96-99% purity by centrifugation of defibrinated or heparinized venous blood through a Ficoll-Hypaque mixture (2) at 310 g for 35 min at room temperature. Erythrocytes were removed by dextran sedimentation and hypotonic lysis (2), and neutrophils were washed and suspended in KrebsRinger phosphate buffer, pH 7.35, containing 0.2% glucose and 0.2% bovine serum albumin. Monocyte-lymphocyte layers from the same preparations were diluted with 4 vol of buffer and centrifuged at 370 g, 4°C for 10 min. Erythrocytes were removed by hypotenic lysis, and the * Supported by U. S.