R. D'Amico

Increased PMN CD11b/CD18 expression following post-traumatic ARDS

We investigated the role of polymorphonuclear leukocytes (PMN) in the pathogenesis of post-traumatic adult respiratory distress syndrome (ARDS). Two groups of patients were studied: Group I (n = 29) represents trauma patients studied within 24 hr of admission to the SICU, and Group II (n = 10) represents a subset of Group I patients who subsequently developed ARDS. Circulating pulmonary artery PMN were then assayed for CD11b/CD18 expression, MTT-Formazan production, intracellular H2O2 production, and superoxide anion release. PMN from Group II patients were upregulated with regard to all of the PMN functions assayed within 24 hr of the diagnosis of ARDS being made. Subsequently, longitudinal assays were performed on 17 patients at risk for the development of ARDS. In 6 of 7 patients prior to the clinical recognition of ARDS, CD11b/CD18 expression and MTT-Formazan production increased significantly over baseline. These results suggest that: (i) ARDS coincides with increased CD11b/CD18 expression on PMN cell surfaces, (ii) PMN oxidative metabolism increases at the onset of ARDS, and (iii) changes in circulating pulmonary artery PMN may provide markers for the development of ARDS in the traumatized patient.

Endogenous PMN-derived reactive oxygen intermediates provide feedback regulation on respiratory burst signal transduction

The role of polymorphonuclear leukocytes (PMN) in stemming systemic infection is executed mainly by the utilization of molecular O2 leading to the production of reactive oxygen intermediates (ROI). PMN‐derived ROI also serve as intra‐ and extracellular second messengers providing both positive and negative feedback on cellular autoregulation. We investigated the effect of endogenous ROI on two signal transducing pathways: the receptor (R)‐G‐protein‐phospholipase D (PLD) and receptor (R)‐G‐protein‐phospholipase C pathways responsible for the subsequent interleukin‐8 (IL‐8)‐induced PMN respiratory burst. Purified human PMN were primed with LPS adhered to plastic surfaces and stimulated with IL‐8 with or without the presence of each of five different selective ROI scavengers/antioxidants: DMSO, NaN3, L‐alanine, catalase, or superoxide dismutase. Total IL‐8 surface receptor expression was assessed by 125I‐IL‐8 125I‐labeled mAbs against IL‐8R type A and B binding assays; PLD activation was assessed by measuring formation of phosphatidyl ethanol (PEt) in the presence of ethanol; PLC activation was measured by quantitative conversion of [32P]ATP‐labeled phosphatidic acid (PA) into diacylglycerol (DAG); expression of Gα‐inhibitory subunit was assessed by SDS‐PAGE and immunoblotting with polyclonal Abs against this subunit. Production of O2 –, H2O2, HClO, and myeloperoxidase (MPO) in the experimental model was confirmed in a separate set of experiments. The overall impact of antioxidants on each component of the transducing tripartite complex was stimulatory; however, NaN3 and SOD exhibited the most ubiquitous effect with consistent upregulation by NaN3 of IL‐8R expression, whereas even trace amounts of externally added authentic MPO significantly down‐regulated the functional activity of both effector enzymes. These results demonstrate a multiple site‐specific targeting of the signal‐transducing complex by endogenous PMN‐derived ROI and an overall protective effect of ROI scavengers/antioxidants. J. Leukoc. Biol. 62: 268–276; 1997.

Polymorphonuclear leucocyte (PMN)-derived inflammatory cytokines--regulation by oxygen tension and extracellular matrix.

The kinetics of IL‐8, tumour necrosis factor‐alpha (TNF‐α) and IL‐1β release by PMN adhered to fibronectin, laminin or plastic for 24h in response to continuousstimulation with lipopolysaccharide (LPS; 50ng/ml), n‐formyl‐Met‐Leu‐Phe (fMLP; 100mm), or phorbol myristate acetate (PMA; 10ng/ml), was investigated under altered oxygen tension conditions. Cell supernatants were sampled for cytokine content every 6h and measured by ELISA. IL‐8 was the most abundant cytokine, produced in a range of up to 5.4ng/ml; TNF‐α and IL‐1β were produced in a range of up to 1ng/ml. During normoxia, LPS was the most potent stimulus, inducing the release of each cytokine, while fMLP showed a less pronounced effect on IL‐8 and IL‐1β production and markedly inhibited TNF‐α production. PMA markedly suppressed IL‐8 and IL‐1β release and failed to induce any release of TNF‐α. Hypoxia had an overall inhibitory effect on cytokine release except for PMA‐induced IL‐1β release, and hypoxia/reoxygenation had a significant up‐regulating effect except for a further inhibition of fMLP‐induced release of TNF‐α. Integrin–matrix protein ligation differentiated both spontaneous and externally induced cytokine release and its sensitivity to alteration in oxygen tension. Thus the process of PMN elaboration of inflammatory cytokines is controlled on multiple levels of signal transduction, differentiated by integrin–extracellular matrix interactions, and is sensitive to alterations in microenvironmental oxygen tension.

Lipopolysaccharide induces intracytoplasmic migration of the polymorphonuclear leukocyte CD11b/CD18 receptor.

We investigated the effects of lipopolysaccharide (LPS) on the subcellular location of the integrin receptor CD11b/CD18 (Mac-1). Cytoplast and subcellular fractionation experiments were performed to distinguish between receptor shedding and intracellular receptor transport as mechanisms involved for the effects of LPS on CD11b/CD18 expression. Cytoplast preparations demonstrated the same percentage of cell-associated receptors +/- LPS. Subcellular fractionation experiments demonstrated a shift from primarily plasma membrane fractions to azurophilic granules. Protein tyrosine kinase inhibition with genistein (50 microM) inhibited the LPS-induced translocation of CD11b/CD18 receptors to azurophilic granules. The effects of LPS (10 ng/mL) alone were reproduced with LPS (.1 ng/mL) plus heat-inactivated pooled normal human serum. Preincubation of PMN with anti-CD14 monoclonal antibodies prevented the effects of LPS+serum on the translocation of CD11b/CD18 receptors. These results demonstrate that LPS regulates CD11b/CD18 expression by inducing intracytoplasmic translocation of this receptor to azurophilic granules. This process involves activation of protein tyrosine kinase, and endosomal acidification contributes to the degradation of these receptors within azurophilic granules.

IL-1β stimulation induces paracrine regulation of PMN function and apoptosis

Polymorphonuclear leukocytes (PMN) play a crucial role in the primary immunological defense against infectious agents. PMN activation and function is influenced in a paracrine manner by cytokines and bacterial products. While cell-cell communication has been demonstrated between PMN and other cell types, little data is available addressing PMN-PMN communication. Therefore, the aim of this study was to determine whether PMN were able to affect PMN function in vitro in a cell-contact independent manner, and whether IL-1beta influenced this effect. Conditioned medias (CM) were prepared by incubating PMN in HBSS +/- IL-1beta for 1-4 h. Incubation of fresh PMN in these conditioned medias had little or no effect on the expression of cell surface FcgammaR expression or oxidative metabolism. However, incubation of PMN in CM-IL1beta, but not control CM, increased phagocytotic activity and suppressed apoptosis. Additionally, CM-IL1beta, but not control CM, slowed the changes in Mac-1 and CR1 cell surface expression that occurred in HBSS within 2 h of incubation. Finally, control CM down-regulated the cell surface expression of PSGL-1; an effect that was not observed with CM-IL1beta. In conclusion, we demonstrate that PMN are able to communicate with and influence the immunological function of other PMN independent of cell-cell contact, and that this influence is regulated by cytokines such as IL-1beta. The major impact of this paracrine regulation is to down-regulate PMN apoptosis with the potential for an upregulated inflammatory response.

Regulation of IL-8RA (CXCR1) expression in polymorphonuclear leukocytes by hypoxia/reoxygenation.

Interleukin‐8 (IL‐8) is an important mediator of neutrophil (PMN) function and the type A IL‐8 receptor (IL‐8RA) mediates these pro‐inflammatory signals. Hypoxia or hypoxia/reoxygenation (H/R) affects the production of IL‐8, but no data is available regarding its effect on IL‐8RA expression. The purpose of this study was to determine the effects of hypoxia and/or H/R on the expression of IL‐8RA in PMN. We demonstrated that IL‐8RA mRNA levels were similar under normoxic and hypoxic conditions but H/R resulted in a significant reduction in mRNA expression between 30 and 60 min. IL‐8RA protein also decreased with reoxygenation of whole blood, which was altered by the addition of specific antioxidants. Therefore, H/R appears to attenuate the effect of IL‐8 by down‐regulating IL‐8RA in PMN. These data show that changes in oxygen tension within the wound site not only affect the expression of inflammatory cytokines, but also control their actions by regulating their receptors. J. Leukoc. Biol. 65: 171–178; 1999.

THE ROLE OF NEUTROPHIL-DERIVED OXIDANTS AS SECOND MESSENGERS IN INTERLEUKIN 1β-STIMULATED CELLS

ABSTRACT The role of the inflammatory cytokine interleukin 1β (IL-1β) as potent agonist of the PMN respiratory burst signal transduction cascade has been described. We hypothesized that this phenomenon is self-limiting and that polymorphonuclear leukocyte (PMN)-derived reactive oxygen intermediates (ROI) might provide feedback regulation on the IL-1β surface receptor (IL-1βR)-G-protein-effector enzyme transducing tripartite complex that ultimately leads to NADPH oxidase activation. Therefore, we separately assessed either baseline or IL-β-induced activation of each member of the IL-1βR-G-protein-phospholipase D (PLD) or IL-1βR-G-protein-phospholipase C (PLC) signaling systems in the presence or absence of one of several specific ROI scavengers/antioxidants. Purified human PMN were lipopolysaccharide primed, adhered for 2 h, and stimulated with 100 ng/mL IL-1β with or without 1% v/v dimethyl sulfoxide, 10 mM NaN3, 30 mM L-alanine, 200 U catalase, or 300 U superoxide dismutase (SOD). To validate the use of these antioxidants, the production of O2−, H2O2, hypochlorous acid, or myeloperoxidase (MPO) in the employed experimental model was confirmed in a separate set of experiments. The expression of IL-1βR type I or II was assessed by binding with corresponding 125I-labeled monoclonal antibodies and corrected for nonspecific binding. PLD activation was assessed by measuring phosphatidyl ethanol formation in the presence of ethanol. PLC activation was determined by quantitative measurement of diacylglycerol. The level of Gα stimulatory and inhibitory subunits was assessed by Western blotting. IL-1βR type I expression was significantly up-regulated in the presence of catalase and SOD. PLD activation was increased by dimethyl sulfoxide and NaN3, and PLC activation was up-regulated by NaN3, l-alanine, SOD, and catalase. After 5 min of stimulation with IL-1β, Giα expression was significantly down-regulated by NaN3 and SOD, whereas SOD had an up-regulating effect on the expression of Gsα. Increasing concentrations of externally added authentic MPO progressively down-regulated both PLD and PLC activity. Thus, PMN-derived ROI, in addition to their role as antibacterial/fungal agents, serve as second messengers in IL-1β signal transduction, with MPO having the most ubiquitous role as a modulator of PMN second messenger pathways.

Polymorphonuclear leukocyte opsonic receptor expression after hypoxia/reoxygenation

We investigated the effects of hypoxia/reoxygenation (H/R) and subsequent stimulation of polymorphonuclear leukocytes (PMNs) with either formyl-methionyl-leucyl-phenylalanine (FMLP) or phorbol myristate acetate (PMA) on CD32, CD16, CD35, and CD11b/CD18 expression and on degranulation and superoxide anion production. H/R primed both adherent and fluid-phase PMNs for subsequent up-regulation of CD32 and CD16 (Fcgamma receptors) when stimulated with FMLP and primed both Fcgamma and complement (CD35, CD11b/CD18) receptors when stimulated with PMA. Kinetics assays demonstrated maximal up-regulation of CD32 and CD16 induced by H/R plus FMLP after 30 minutes of reoxygenation, whereas maximal receptor stimulation by H/R plus PMA occurred within 15 minutes of reoxygenation. Neither actinomycin D nor cycloheximide abrogated the effect of H/R with subsequent stimulation of PMNs on receptor expression; however, 10(-5) to 10(-8) mol/L concentrations of either taxol or phalloidin completely abrogated the effect of H/R plus FMLP or PMA on opsonic receptor expression. The effect of H/R plus FMLP on CD32 and CD16 expression was blocked by pertussis toxin, whereas staurosporine, H-7, H-9, and genistein had no effect. Conversely, the effect of H/R plus PMA on CD32, CD16, CD35, and CD11b/CD18 expression was blocked by staurosporine and H-7 but not by H-9, pertussis toxin, or genistein. The up-regulation of CD32, CD16, CD35, and CD11b/CD18 induced by H/R plus FMLP or PMA in the presence or absence of matrix proteins resulted in the increased rosetting of E-anti-CD32, E anti-CD16, E-Con A, EC3b, and EC3bi, respectively. Reduced nicotinamide adenine dinucleotide phosphate oxidase inhibition with diphenyleneiodonium blocked the effect of H/R on receptor expression, degranulation, and superoxide anion production. These results demonstrate that H/R primes PMNs for subsequent receptor up-regulation by divergent intracellular signal transduction pathways and that the receptors induced to the cell surface are biologically active.

Intraabdominal sepsis: Effects on polymorphonuclear leukocyte Fc receptor-mediated phagocytosis

In vitro studies have shown that phagocytic cells are capable of undergoing activation in response to inflammatory signals and that the activation process is quite complex. A relationship between polymorphonuclear leukocyte (PMN) Fc receptor-mediated phagocytosis and oxidative metabolism has been seen in humans. We have sequentially examined circulating polymorphonuclear leukocytes (PMNs) from a total of 13 postoperative swine with either no sepsis, untreated intraabdominal sepsis, or treated intraabdominal sepsis to determine phagocytic activity over 8 postoperative days (POD). Products of the oxidative burst (i.e., myeloperoxidase) reduced the phagocytic activity of nonseptic swine PMN. Phagocytic activity was augmented by inhibiting the nonseptic swine oxidative burst with 10 mM sodium azide (an inhibitor of myeloperoxidase). In swine with untreated intraabdominal sepsis, PMN Fc receptor-mediated phagocytosis exhibited a biphasic response. An initial (between POD1 and POD4) increase in PMN function was followed by a subsequent (between POD4 and POD8) decrease in PMN function. Partial preservation of phagocytic capability was seen when swine were reexplored on POD4 and had their intraabdominal sepsis treated. These results indicate that (1) as in humans, nonseptic swine PMN Fc receptor-mediated phagocytosis is augmented by inhibition of the PMN respiratory burst; (2) untreated intraabdominal sepsis produces an initial increase and subsequent decrease in PMN Fc receptor-mediated phagocytosis; (3) early treatment of intraabdominal sepsis results in partial restoration of PMN Fc receptor-mediated phagocytosis.

ALTERED OXYGEN TENSION MODULATES CYTOKINE-INDUCED SIGNAL TRANSDUCTION IN POLYMORPHONUCLEAR LEUKOCYTES: REGULATION OF THE GPLD PATHWAY

We investigated the effect of alterations in buffer oxygen tensions from normoxia (Po2 = 180–200 mmHg) to hypoxia (Po2 < 30 mmHg) and then reoxygenation (Po2 > 140 mmHg) on the GPLD-pathway by measuring phosphatidylethanol formation in the presence of ethanol and subsequent NADPH oxidase activation and O-2 production in polymorphonuclear leukocytes (PMN). Experiments were performed with PMN stimulated with either interleukin (IL)-8, tumor necrosis factor (TNF)-α, or IL-1β in the presence or absence of fibronectin. Hypoxia exerted a downregulating effect on this pathway and reoxygenation restored GPLD activation to levels seen during normoxia; however, supraphysiological concentrations of cytokines were able to reverse this pattern. Changes in GPLD activation correlated best with changes in O-2 production during the hypoxia to hypoxia/reoxygenation transition induced by TNF-α-Fn and IL-1β ± Fn. Thus, changes in oxygen tension can directly modulate the extent of the PMN response to stimulation by IL-8, TNF-α, or IL-1β, and activation of the GPLD-pathway appears to be highly sensitive to hypoxia and hypoxia/reoxygenation.