The phosphatidylinositol 3-kinase pathway selectively controls sIL-1RA not interleukin-1beta production in the septic leukocytes.

Microbial components such as bacterial endotoxin lipopolysaccharide (LPS) can trigger highly lethal septic shock. The cardinal features of septic leukocytes include the repressed production of inflammatory cytokines, such as interleukin-1 beta (IL-1beta), and elevated production of anti-inflammatory cytokines, such as secretory interleukin-1 receptor antagonist (sIL-1RA). Pro- and anti-inflammatory cytokine gene transcriptions are equally repressed in septic leukocytes due to disruption of the LPS signaling pathway at the level of interleukin-1 receptor-associated kinase. The selective elevation of sIL-1RA protein in septic blood is caused by efficient translation of residual sIL-1RA message. In this study, we report that the LPS-inducible phosphatidylinositol 3-kinase (PI3-kinase)-dependent signaling pathway contributes to the elevated translation of sIL-1RA in septic/LPS-adapted leukocytes. We also observe that this pathway is gene specific and does not affect the production of proinflammatory IL-1beta protein.

Endotoxin-adapted septic shock leukocytes selectively alter production of sIL-1RA and IL-1beta.

During septic shock, circulating levels of anti-inflammatory mediators are increased relative to those of pro-inflammatory. The reduced capacity of septic shock blood leukocytes in expressing pro-inflammatory genes in response to bacterial lipopolysaccharide endotoxin (LPS) may contribute to reductions in these mediators, but the reasons for persistent increases in circulating anti-inflammatory mediators are unknown. We determined whether septic shock leukocytes that have adapted to LPS induction of the IL-1beta gene could continue to express sIL-1RA in response to LPS. Septic shock whole-blood leukocytes and neutrophils (PMNs) selectively maintained production of sIL-1RA after treatment with LPS while limiting that of IL-1beta. Repressed transcription of IL-1beta and rapid decay of IL-1beta mRNA in septic shock neutrophils correlated with reductions in levels of IL-1beta after stimulation with LPS. Transcription of sIL-1RA mRNA was also suppressed, but the ability of LPS to stimulate events that lead to efficient translation of a stable sIL-1RA mRNA appeared responsible for maintaining sIL-1RA production. We conclude that LPS adaptation of septic shock leukocytes selectively influences signaling pathways that regulate transcription, mRNA processing, and translation, leading to changes in the balance of production of pro- and anti-inflammatory mediators.

Characterization of interleukin-1 receptor-associated kinase in normal and endotoxin-tolerant cells.

Interleukin-1 receptor-associated kinase (IRAK), a signal transducer for interleukin-1, has also been suggested to participate in the Toll-like receptor-mediated innate immune response to bacterial endotoxin lipopolysaccharide (LPS). Using the human promonocytic THP-1 cell line, we demonstrated that the endogenous IRAK is quickly activated in response to bacterial LPS stimulation, as measured by its in vitro kinase activity toward myelin basic protein. LPS also triggers the association of IRAK with MyD88, the adaptor protein linking IRAK to the Toll-like receptor/interleukin-1beta receptor intracellular domain. Macrophage cells with prolonged LPS treatment become tolerant to additional dose of LPS and no longer express inflammatory cytokines. Endotoxin tolerance is a common phenomenon observed in blood from sepsis patients. We observed for the first time that the quantity of IRAK is greatly reduced in LPS-tolerant THP-1 cells, and its activity no longer responds to further LPS challenge. In addition, IRAK does not associate with MyD88 in the tolerant cells. Furthermore, application of AG126, a putative tyrosine kinase inhibitor, can substantially alleviate the LPS-induced cytokine gene expression and can also decrease IRAK level and activity. Our study indicates that IRAK is essential for LPS-mediated signaling and that cells may develop endotoxin tolerance by down-regulating IRAK.

mRNA and protein stability regulate the differential expression of pro- and anti-inflammatory genes in endotoxin-tolerant THP-1 cells.

The products of proinflammatory genes such as interleukin-1beta (IL-1beta) and cyclooxygenase-2 (COX-2) initiate many of the events associated with sepsis. Transcription of these genes is subsequently down-regulated, whereas expression of anti-inflammatory genes such as secretory interleukin-1 receptor antagonist (sIL-1 RA) is maintained. Differential expression is associated with endotoxin tolerance, a cellular phenomenon common to sepsis and characterized by reduced proinflammatory gene expression after repeated exposure to lipopolysaccharide. As a model for endotoxin tolerance, we examined the expression of COX-2 and sIL-1 RA in a human promonocyte cell line, THP-1. We observed a 5-fold decrease in COX-2 protein in endotoxin-tolerant cells relative to control cells. In contrast, sIL-1 RA protein increased 5-fold in control and tolerant cells and remained elevated. Decreased COX-2 production is due to repressed transcription and not enhanced mRNA degradation. In addition, COX-2 protein is turned over rapidly. Transcription of sIL-1 RA is also repressed during tolerance. However, sIL-1 RA mRNA is degraded more slowly than COX-2 mRNA, allowing continued synthesis of sIL-1 RA protein that is very stable. These results indicate that differential expression during endotoxin tolerance occurs by transcriptional repression of COX-2 and by protein and mRNA stabilization of sIL-1 RA.

Endotoxin inducible transcription is repressed in endotoxin tolerant cells.

Stimulation of the human promonocytic cell line, THP-1, with endotoxin results in a rapid and transient increase in interleukin 1beta expression. Endotoxin pretreatment of THP-1 cells results in tolerance, characterized by decreased levels of endotoxin-induced interleukin 1beta expression due to decreased transcription of the interleukin 1beta gene. We hypothesized that tolerant cells could not activate transcription factors necessary to express the interleukin 1beta gene. This hypothesis was tested in tolerant THP-1 cells by using stable and transiently transfected reporter genes containing the interleukin 1beta promoter. We found decreased endotoxin-induced transcription of all reporter genes tested; however, individual transcription factors, such as NFkappaB, retain normal, CD14-dependent, nuclear translocation and DNA binding. Tolerance is specific for endotoxin, because phorbol ester is still able to activate transcription of the endogenous interleukin 1beta gene and transfected reporter genes. A constitutively active reporter gene that is not inducible by endotoxin is unaffected. We further show that nuclear extracts of tolerant cells show transcription inhibitor activity that is specific for promoter sequences of the interleukin 1beta gene. These results support a mechanism of endotoxin tolerance that is independent of transcription factor DNA binding and appears to be associated with the inability of DNA-bound transcription factors to activate transcription, perhaps through the activity of a repressor.

Human neutrophils express the prostaglandin G/H synthase 2 gene when stimulated with bacterial lipopolysaccharide.

Human blood neutrophils (PMN) rapidly release arachidonic acid (AA) from cellular phospholipids when stimulated in vitro with a variety of inflammatory agonists. Free AA is then metabolized via 5'-lipoxygenase to produce bioactive mediators such as leukotriene B4 and 5-hydroxyeicosatetraenoate. Arachidonic acid can also be metabolized via the cyclooxygenase or prostaglandin G/H synthase (PGHS) pathway to form prostaglandins and thromboxane. We show here that human blood PMN express the PGHS 2 gene when stimulated with bacterial lipopolysaccharide (LPS). PGHS 2 mRNA increases within 30 min after LPS stimulation and PGHS 2 immunoreactive protein is detectable by 5 h. Although PGHS 1 mRNA is detectable in PMN, no immunoreactive protein is observed in either resting or LPS-stimulated cells. Following stimulation with LPS and expression of PGHS 2, PMN increase secretion of prostaglandin E2. This phenotypic change in PMN could be an important mechanism for regulating inflammation.

Interleukin-1beta expression after inhibition of protein phosphatases in endotoxin-tolerant cells.

Endotoxin (lipopolysaccharide [LPS]) is a potent activator of a number of inflammatory genes in blood leukocytes, including interleukin-1 (IL-1). Blood leukocytes isolated from patients with septic shock fail to produce IL-1 in response to LPS, a phenomenon known as endotoxin tolerance. To study the regulation of IL-1 expression in endotoxin-tolerant cells, the protein phosphatase inhibitor okadaic acid was used to examine the effects of protein phosphorylation on IL-1beta gene expression. We found that endotoxin-tolerant cells produced normal levels of IL-1beta when protein phosphatases were inhibited. In the human pro-monocytic cell line THP-1, okadaic acid increased mRNA accumulation and synthesis of IL-1beta protein. Normal and endotoxin-tolerant THP-1 cells accumulated IL-1beta mRNA and protein with similar delayed kinetics. Okadaic acid stabilization of IL-1beta mRNA appears to be the primary mechanism through which endotoxin-tolerant cells accumulate IL-1beta mRNA and protein. Endotoxin-tolerant cells were unable to activate transcription in response to okadaic acid. However, the transcription factor NF-kappaB, which is known to be involved in IL-1beta expression, was translocated to the nucleus in both normal and endotoxin-tolerant cells after treatment with okadaic acid. These studies revealed that protein phosphorylation can affect gene expression on at least two distinct levels, transcription factor activation and mRNA stability. Endotoxin-tolerant cells have decreased transcription activation potential, while IL-1beta mRNA stability remains responsive to protein phosphorylation.

Protein-tyrosine kinase activation is required for lipopolysaccharide induction of interleukin 1beta and NFkappaB activation, but not NFkappaB nuclear translocation.

In human monocytes, interleukin 1beta protein production and steady state mRNA levels are increased in response to lipopolysaccharide, predominantly as a result of increased transcription of the interleukin 1beta gene. Expression of interleukin 1beta and other cytokines, such as interleukin 6 and tumor necrosis factor alpha, has been shown to be dependent on the activation of the transcription factor, NFkappaB. Since recent studies have shown that lipopolysaccharide-induced tyrosine kinase activation is not required for NFkappaB nuclear translocation, we sought to determine whether NFkappaB translocated in the absence of tyrosine kinase activity was active in stimulating transcription. We have found that, in the human pro-monocytic cell line, THP-1, the lipopolysaccharide-induced expression of interleukin 1beta is dependent on tyrosine kinase activation. Tyrosine kinases are not required for lipopolysaccharide-mediated nuclear translocation of NFkappaB. However, in the absence of tyrosine kinase activity, the ability of NFkappaB to stimulate transcription is impaired. This inhibition of transcription is specific for NFkappaB; in the absence of tyrosine kinase activity, AP-1-dependent transcription is enhanced. These results suggest that, while lipopolysaccharide-induced expression of inflammatory mediators requires tyrosine kinase activity, tyrosine kinase activity is not obligatory for lipopolysaccharide signal transduction.

Elevated levels of shed type II IL-1 receptor in sepsis. Potential role for type II receptor in regulation of IL-1 responses.

Two types of cellular IL-1Rs have been characterized and cloned from both human and murine sources. The type II IL-1R has a very short cytoplasmic domain and does not seem to participate in IL-1 signaling. We demonstrate that type II IL-1Rs are released from the surface of neutrophils in response to treatment with TNF or endotoxin. In addition, serum from patients with sepsis syndrome contains elevated levels of soluble type II IL-1Rs. Neutrophils isolated from patients with sepsis have greatly enhanced expression of type II IL-1R mRNA and cell surface receptors and are therefore a likely source for the shed receptors in serum. Of the three forms of IL-1, soluble type II IL-1R binds IL-1 beta with highest affinity and also selectively inhibits IL-1 beta activity. We propose that increased cell surface expression and rapid release of preformed type II IL-1R from neutrophils, as a soluble IL-1 beta binding protein, represents a mechanism that has evolved for regulating IL-1 activity in sepsis.

A labile transcriptional repressor modulates endotoxin tolerance.

Tolerance to bacterial lipopolysaccharide (LPS, endotoxin) is an adaptive cellular process whereby exposure to endotoxin induces a subsequent hyporesponsive state characterized by decreased levels of LPS-induced cytokine mRNA and protein. We demonstrate, in a human promonocytic cell line, THP-1, that endotoxin tolerance is manifested by decreased LPS-induced interleukin 1 beta (IL-1 beta) transcription. Inhibition of protein synthesis reverses the tolerant phenotype by inducing transcription of IL-1 beta in the absence of a second stimulus. These results indicate that a labile protein contributes to the endotoxin-tolerant phenotype, and that this factor acts in a dominant repressive manner to inhibit the activity of existing transcription factors. We provide further data that cellular expression of I kappa B-alpha correlates with downregulated IL-1 beta gene expression during endotoxin tolerance, implicating I kappa B-alpha as a potential candidate for the labile repressor identified herein.

Phosphatidic acid and diacylglycerol synergize in a cell-free system for activation of NADPH oxidase from human neutrophils.

NADPH oxidase, the respiratory burst enzyme of human neutrophils, is a multi-component complex that is assembled and activated during stimulation of the cells by inflammatory or phagocytic stimuli. The signal mechanisms leading to activation of the enzyme are unclear, but it is likely that phospholipases are involved. Recent work has shown that phosphatidic acid, the initial product of phospholipase D activation, is a weak activator of NADPH oxidase in a cell-free system. We now show that diacylglycerol enhances the cell-free activation of NADPH oxidase activation by phosphatidic acid. 1,2-Didecanoyl phosphatidic acid (10:0-PA) and 1,2-dioctanoylglycerol (8:0-DG) each increased levels of NADPH oxidase activity in mixtures of membrane and cytosolic fractions about 2-fold. The combination of both lipids increased NADPH oxidase activity approximately 12-fold, indicative of a synergistic response. Fatty acid and neutral lipid metabolites of 10:0-PA or 8:0-DG were ineffective, suggesting activation is directly mediated by phosphatidic acid and diacylglycerol. Activation was time- and concentration-dependent with maximum activation at 30-60 min and a sharp peak of maximal activity at 10 microM 10:0-PA and 30 microM 8:0-DG. In lipid specificity studies, activity of PA or DG decreased with increasing acyl chain length but was restored by introducing unsaturation in the acyl chain. Natural forms of PA stimulated levels of activity comparable to that seen with 10:0-PA. Synthetic and natural phosphatidylserines, but not other phospholipids, could replace phosphatidic acid in the synergistic response. These studies provide direct evidence for a synergistic interaction between phosphatidic acid and diacylglycerol in mediating a cellular function: the assembly and activation of NADPH oxidase. Our results support the concept that the generation of second messenger lipids by phospholipase D is a key step in activation of the respiratory burst enzyme.

Phospholipases and activation of the NADPH oxidase.

The signal transductional mechanisms regulating the activation of NADPH oxidase, the respiratory burst enzyme in phagocytic cells, are not completely understood. Receptors for most physiologic stimuli trigger the activation of various phospholipases, including phospholipases A2, C, and D. The lipid mediators formed (arachidonic acid, 1,2-diacylglycerol, and phosphatidic acid) have been implicated as second messengers in the induction of the respiratory burst. In intact cells, we have correlated phospholipase D activation and the production of phosphatidic acid with the activation of NADPH oxidase, using the drug propranolol. Phosphatidic acid activated NADPH oxidase in a cell-free system, but the level of activation was low. 1,2-Diacylglycerol markedly enhanced NADPH oxidase activation by phosphatidic acid. The synergistic effect required the diacyl species, since mono- or tri-acylglycerols were ineffective. Phosphatidic acid could be replaced by either lysophosphatidic acid or phosphatidylserine, but not by phosphatidylcholine, phosphatidylethanolamine, or phosphatidylinositol, suggesting specificity for an anionic phospholipid. Since other cell-free activators of NADPH oxidase (arachidonic acid, sodium dodecyl sulfate) are also anionic amphiphiles, phosphatidic acid may directly interact with an enzyme component(s). The targets for phosphatidic acid and diacylglycerol in the cell-free system are currently under investigation. These results emphasize the critical importance of phospholipases, particularly phospholipase D, in the regulation of the respiratory burst.

Lipopolysaccharide-induced expression of prostaglandin H synthase-2 in alveolar macrophages is inhibited by dexamethasone but not by aspirin.

Alveolar macrophages cultured with lipopolysaccharide release markedly increased amounts of prostanoids upon subsequent stimulation, an effect that is due to induction of prostaglandin H synthase-2 (J. Biol. Chem., (1992), 267, 14545-14550, and Biochem. Biophys. Res. Comm., (1992), 187, 1123-1127). The effects of dexamethasone and aspirin on this enhanced formation of thromboxane by stimulated lipopolysaccharide-primed alveolar macrophages were investigated. Under conditions of maximum inhibition, dexamethasone and aspirin decreased the formation of thromboxane by approximately 50% and 80%, respectively. Expression of lipopolysaccharide-induced prostaglandin H synthase-2 in dexamethasone-treated macrophages was similarly inhibited by about 50%, as determined by Northern blot and immunoprecipitation. In contrast, levels of lipopolysaccharide-induced prostaglandin H synthase-2 mRNA and protein were not reduced in aspirin-treated macrophages. We conclude that inhibition of prostaglandin H synthase-2 expression represents a mechanism by which dexamethasone, but not aspirin, may inhibit prostanoid formation by alveolar macrophages.

Tolerance to endotoxin-induced expression of the interleukin-1 beta gene in blood neutrophils of humans with the sepsis syndrome.

The induction of genes of host cells stimulated by microbial products such as endotoxin and the tolerance of cells to endotoxin excitation play critical roles in the pathogenesis of microbial-induced acute disseminated inflammation with multiorgan failure (the sepsis syndrome). One gene that is induced in phagocytic cells by endotoxin and that appears to play an essential role in the pathogenesis of the sepsis syndrome is IL-1 beta. We report here that blood neutrophils (PMN) of patients with the sepsis syndrome (sepsis PMN) are consistently tolerant to endotoxin-induced expression of the IL-1 beta gene, as determined by decreased synthesis of the IL-1 beta protein and reductions in IL-1 beta mRNA. This down-regulation of the IL-1 beta gene in sepsis PMN occurs concomitant with an upregulation in the constitutive expression of the type 2 IL-1 receptor (IL-1R2). These phenotypic changes do not persist in PMN of patients recovering from the sepsis syndrome. Tolerance has stimulus and response specificity since sepsis PMN tolerant to endotoxin can respond normally to Staphylococcus aureus stimulation of IL-1 beta production and they normally secrete elastase. Uninfected patients with severe trauma or shock from causes are not tolerant to endotoxin and tolerance is not limited to patients infected with Gram-negative bacteria. The mechanism responsible for tolerance involves pretranslational events and is not due to loss of the CD14 surface protein, a receptor required for endotoxin induction of IL-1 beta in PMN. The physiological significance of the tolerance to endotoxin and increased expression of IL-1R2 on sepsis PMN is unknown, but may represent an attempt by the host to protect itself from the deleterious effects of disseminated inflammation.

Propranolol, a phosphatidate phosphohydrolase inhibitor, also inhibits protein kinase C.

Propranolol, a beta-adrenergic receptor antagonist, also inhibits phosphatidate phosphohydrolase, the enzyme that converts phosphatidic acid into diacylglycerol. This latter effect has prompted recent use of propranolol in studies examining the importance of diacylglycerol and phosphatidic acid in cellular signalling events. Here, we show that propranolol is also an inhibitor of protein kinase C. At concentrations greater than or equal to 20 microM, propranolol reduced [3H]phorbol dibutyrate binding (IC50 = 200 microM) and phorbol myristate acetate-stimulated superoxide anion release (IC50 = 130 microM) in human neutrophils. Scatchard analysis showed that propranolol lowers the number of phorbol diester binding sites without significantly affecting their affinity. In vitro kinetic analysis, performed in a mixed micellar assay with protein kinase C purified from human neutrophils, suggested a competitive inhibition of propranolol with the cofactor phosphatidylserine. Complex kinetic patterns were observed with respect to diacylglycerol and ATP, approximating competitive and noncompetitive inhibition, respectively. Taken together, these results suggest that the drug interacts at the level of the regulatory domain of the enzyme. Fifty % inhibition occurred at approximately 150 microM propranolol. Similar levels of inhibition were obtained using exogenous (histone) and endogenous (p47-phox, a NADPH oxidase component) substrates. Protein kinase C-alpha and protein kinase C-beta, two protein kinase C isozymes present in human neutrophils, were inhibited by propranolol in a comparable manner. In the range of concentrations tested (30-1000 microM), neither cAMP-dependent protein kinase nor neutrophil protein tyrosine kinases were affected. The racemic form of propranolol and the (+) and the (-) stereoisomers were equally active, and other beta-adrenergic receptor antagonists (pindolol) and agonists (isoproterenol) were inactive. This suggests that the inhibitory action of propranolol on protein kinase C is related to the amphipathic nature of the drug rather than to its beta-adrenergic receptor blocking ability. Analogs of propranolol were synthesized and found to be more potent protein kinase C inhibitors, with IC50 values in the 10-20 microM range. We conclude that the ability of propranolol to inhibit both protein kinase C and PA phosphohydrolase complicates interpretation of results when this drug is used in signal transduction studies. In addition, propranolol may be a useful prototype for the synthesis of new protein kinase C inhibitors.

Comparison of diglyceride production from choline-containing phosphoglycerides in human neutrophils stimulated with N-formylmethionyl-leucylphenylalanine, ionophore A23187 or phorbol 12-myristate 13-acetate.

The turnover of choline-containing phosphoglycerides (PC) in response to agonist stimulation is well documented in human neutrophils. We have now compared the enzymic pathways of N-formylmethionyl-leucylphenylalanine (fMLP)-, A23187- and phorbol-12-myristate 13-acetate (PMA)-induced diglyceride (DG) and phosphatidic acid (PA) generation in these cells. In order to distinguish between phospholipase C- and D-mediated PC breakdown, human neutrophils were radiolabelled with 1-O-[3H]alkyl-2-acyl-glycero-3-phosphocholine and stimulated in the presence of ethanol or propranolol. The addition of 0.5% ethanol to the incubation mixture resulted in the production of phosphatidylethanol, indicative of phospholipase D activation, in response to all three stimuli. Concomitant with phosphatidylethanol formation was a partial block of PA production. The production of DG was also partially blocked by addition of ethanol. Propranolol (200 microM) was also used to assess the contributions of phospholipases C and D toward DG generation. Inhibition of PA phosphohydrolase by propranolol resulted in the complete abolition of DG generation when neutrophils were stimulated with fMLP. In contrast, propranolol only partially inhibited DG generation in response to A23187 and PMA. These results suggested that DG production in response to fMLP stimulation is mediated via the activation of phospholipase D, whereas A23187- or PMA-induced DG generation may involve more than one pathway. However, examination of the water-soluble choline metabolites produced indicated that phospholipase D was responsible for the production of PA and DG in response to all three stimuli.

Lipopolysaccharide induces prostaglandin H synthase-2 in alveolar macrophages.

Prostaglandin H synthase is a key enzyme in the formation of prostaglandins and thromboxane from arachidonic acid. The recent cloning of a second prostaglandin H synthase gene, prostaglandin H synthase-2, which is distinct from the classic prostaglandin H synthase-1 gene, may dramatically alter our concept of how cells regulate prostanoid formation. We have recently shown that the enhanced production of prostanoids by lipopolysaccharide-primed alveolar macrophages involves the induction of a novel prostaglandin H synthase (J. Biol. Chem., (1992), 267, 14547-14550). We report here that the novel PGH synthase induced by lipopolysaccharide in alveolar macrophages is prostaglandin H synthase-2.

Lipopolysaccharide priming of alveolar macrophages for enhanced synthesis of prostanoids involves induction of a novel prostaglandin H synthase.

We report here that lipopolysaccharide (LPS) priming of rabbit alveolar macrophages leads to amplified synthesis of prostanoids, at least in part, by induction of a novel prostaglandin H synthase (PGH synthase). Rabbit alveolar macrophages were cultured with or without added LPS derived from Escherichia coli 0111:B4 for 4 h and then stimulated with opsonized zymosan (OPZ). LPS priming of alveolar macrophages resulted in enhanced release of thromboxane (TX) upon stimulation with OPZ, when compared to stimulated non-LPS controls. Addition of exogenous arachidonic acid to LPS-primed alveolar macrophages also resulted in increased production of TX. The LPS-induced increase in TX formation, in response to OPZ or arachidonic acid, was abolished by the addition of actinomycin D or cycloheximide during the priming period. Gas chromatography/mass spectrometry analysis indicated that levels of prostaglandins D2, E2, and F2 alpha, along with TX, were augmented in stimulated LPS-primed alveolar macrophages, implicating PGH synthase in the priming process. PGH synthase enzymatic activity, as determined by addition of arachidonic acid to macrophage sonicates, was markedly enhanced in LPS-primed alveolar macrophages. This correlated with increased PGH synthase levels detected by immunoprecipitation of 35S-labeled proteins and by Western blot analysis. Finally, Northern blot analysis using a cDNA probe to the recently described mitogen-inducible mouse PGH synthase revealed strong induction of approximately 4.3-kilobase mRNA in LPS-primed alveolar macrophages. Taken together, these results reveal that induction of a novel PGH synthase, probably the rabbit homologue of PGH synthase-2, plays a role in the enhanced synthesis of prostanoids by LPS-primed alveolar macrophages.

Increased expression of the interleukin 1 receptor on blood neutrophils of humans with the sepsis syndrome.

Because of the potential importance of interleukin 1 (IL-1) in modulating inflammation and the observations that human blood neutrophils (PMN) express IL-1 receptors (IL-1R) and synthesize IL-1 alpha and IL-1 beta, we studied the IL-1R on blood PMN from a group of patients with the sepsis syndrome. We report a marked enhancement in the sites per cell of IL-1R expressed on sepsis-PMN of 25 consecutively studied patients compared to 20 controls (patient mean = 9,329 +/- 2,212 SE; control mean = 716 +/- 42 SE, respectively). There was no demonstrable difference in the Kd of IL-1R on sepsis-PMN (approximately 1 nM) as determined by saturation curves of 125I-IL-1 alpha binding and the IL-1R on sepsis-PMN had an apparent Mr approximately 68,000, a value like that of normal PMN. Cytofluorographic analysis indicated that the sepsis-PMN phenotype is a single homogeneous population with respect to IL-1R expression. In contrast, expression of the membrane complement receptor CR3 is not increased on sepsis-PMN. Similar increases in expression of IL-1R were not observed in various other inflammatory processes, including acute disseminated inflammation and organ failure not caused by infection, acute infection without organ failure, and immunopathologies such as active systemic lupus erythematosus and rheumatoid arthritis. Enhanced expression of IL-1R was not related simply to the state of myeloid stimulation. Increased expression of IL-1R on normal PMN was induced in vitro by incubating cells with recombinant human granulocyte-macrophage/colony-stimulating factor for 18 h and this response was inhibited by cycloheximide, suggesting the possibility that de novo synthesis of IL-1R might occur in PMN during the sepsis syndrome.

Phosphatidic acid as a second messenger in human polymorphonuclear leukocytes. Effects on activation of NADPH oxidase.

Receptor-mediated agonists, such as FMLP, induce an early, phospholipase D (PLD)-mediated accumulation of phosphatidic acid (PA) which may play a role in the activation of NADPH oxidase in human PMN. We have determined the effect of changes in PA production on O2 consumption in intact PMN and the level of NADPH oxidase activity measured in a cell-free assay. Pretreatment of cells with various concentrations of propranolol enhanced (less than or equal to 200 microM) or inhibited (greater than 300 microM) PLD-induced production of PA (mass and radiolabel) in a manner that correlated with enhancement or inhibition of O2 consumption in PMN stimulated with 1 microM FMLP in the absence of cytochalasin B. The concentration-dependent effects of propranolol on FMLP-induced NADPH oxidase activation was confirmed by direct assay of the enzyme in subcellular fractions. In PA extracted from cells pretreated with 200 microM propranolol before stimulation with 1 microM FMLP, phospholipase A1 (PLA1)-digestion for 90 min, followed by quantitation of residual PA, showed that a minimum of 44% of PA in control (undigested) sample was diacyl-PA; alkylacyl-PA remained undigested by PLA1. Propranolol was also observed to have a concentration-dependent enhancement of mass of 1,2-DG formed in PMN stimulated with FMLP. DG levels reached a maximum at 300 microM propranolol and remained unchanged up to 500 microM propranolol. However, in contrast to PA levels, the level of DG produced did not correlate with NADPH oxidase activation. Exogenously added didecanoyl-PA activated NADPH oxidase in a concentration-dependent manner (1-300 microM) in a reconstitution assay using membrane and cytosolic fractions from unstimulated PMN. In addition, PA synergized with SDS for oxidase activation. Taken together, these results indicate that PA plays a second messenger role in the activation of NADPH oxidase in human PMN and that regulation of phospholipase D is a key step in the activation pathway.