IL-1 and related cytokines enhance thrombin-stimulated PGI2 production in cultured endothelial cells without affecting thrombin-stimulated von Willebrand factor secretion or platelet-activating factor biosynthesis

We examined the effects of various cytokines on alpha-thrombin-stimulated prostaglandin (PG) I2 production, von Willebrand factor (vWF) secretion, and platelet-activating factor (PAF) synthesis in cultured human umbilical vein endothelial cells (HUVEC). A 24-h pretreatment with IL-1 beta doubled the low level of constitutive PGI2 production. In contrast, alpha-thrombin increased PGI2 production fivefold in untreated HUVEC. The most striking increase in PGI2 production was observed in IL-1 beta-treated HUVEC that were subsequently stimulated with thrombin. PGI2 production was two to three times greater than in untreated, thrombin-stimulated HUVEC and nearly eightfold greater than in IL-1 beta-treated but unstimulated HUVEC. Enhanced thrombin-stimulated PGI2 production was also observed in HUVEC pretreated with the related cytokines IL-1 alpha, TNF, or lymphotoxin. This cytokine effect was selective for PGI2 production because none of these cytokines altered either constitutive or thrombin-stimulated vWF secretion or PAF biosynthesis. IL-1 beta enhancement of thrombin-stimulated PGI2 production was concentration and time dependent and required protein synthesis. IL-1 beta pretreatment also enhanced PGI2 production in response to another agonist, histamine, and to exogenously added substrates, arachidonic acid or PGH2. Our results indicate that activation by IL-1 and related cytokines selectively primes endothelial cells for enhanced PGI2 production, but not vWF secretion or PAF synthesis, in response to thrombin and histamine. The evidence suggests that this effect is mediated through specific induction of biosynthetic enzymes for PGI2.     

Biphasic platelet-activating factor synthesis by human monocytes stimulated with IL-1-beta, tumor necrosis factor, or IFN-gamma

The capacity of IL-1-beta, TNF, and IFN-gamma to stimulate platelet-activating factor (PAF) synthesis by human monocytes is examined in our report. All three cytokines induced PAF synthesis in a novel biphasic pattern with peaks of PAF synthesis 1 to 2 and 6 to 8 h after stimulation of the monocytes. In contrast, calcium ionophore A23187 elicited a single peak of early PAF synthesis. PAF in the early peak was largely retained intracellularly whereas PAF in the late peak was largely released into culture fluids. Combinations of cytokines were subadditive or antagonistic in inducing PAF synthesis. Cycloheximide inhibited the late peak of PAF synthesis indicating that protein synthesis is required for synthesis of the phospholipid PAF. Specific antibodies to TNF or IL-1-beta inhibited the late peak of PAF synthesis induced by IFN-gamma indicating that late PAF synthesis is dependent on cytokine synthesis. The quantities of PAF produced by cytokine-activated monocytes are sufficient to activate human monocytes. Thus, these studies suggest that PAF may mediate in part monocyte activation by cytokines.     

Synthesis of platelet-activating factor by polymorphonuclear neutrophils stimulated with interleukin-8.

Human interleukin-8 (IL-8) was evaluated for its capability to induce the synthesis and release of platelet-activating factor (PAF) from human polymorphonuclear neutrophils (PMN). IL-8 promotes in a dose-dependent fashion (1-100 ng/ml) a rapid synthesis of PAF, which is only partially released. The synthesis of PAF is preceded by the activation of acetyl-CoA: 1-alkyl-2-lyso-sn-glycero-3-phosphocholine acetyl-transferase, suggesting that IL-8 activates the "remodeling pathway" of PAF synthesis. By thin layer chromatography and reverse-phase high pressure liquid chromatography, we demonstrated that PAF synthesized by human PMN stimulated with IL-8 is heterogeneous: the 2-acetylated phospholipids having the biological and physicochemical characteristics of PAF include the 1-O-alkyl form, which is produced in large extent (51%), and the 1-acyl form (20%). The analysis of the individual molecular species of radyl chain indicated nine peaks, 16:0 and 18:0 being the predominant forms. These results identify PAF as a direct product of IL-8 stimulation in PMN.     

Platelet-activating factor induces mediator release by human basophils primed with IL-3, granulocyte-macrophage colony-stimulating factor, or IL-5.

The phospholipid platelet-activating factor (PAF) is a potent cell-derived bioactive molecule thought to be involved in diverse inflammatory processes. It has been shown that PAF can activate different leukocyte types and platelets, particularly in synergy with other agonists. In this study we examined the effect of PAF upon the release of histamine and leukotriene (LT) C4 by basophils when added alone and in combination with different agonists and cytokines. PAF by itself did neither induce histamine release nor the generation of LTC4 by basophils. However, basophils primed by the hematopoietic growth factors (hGF) IL-3, granulocyte-macrophage (GM)-CSF, or IL-5 (10 ng/ml) released preformed and de novo synthesized mediators in response to PAF at 10 to 100 nM concentrations. The extent of mediator release by hGF primed basophils in response to PAF was similar to that induced by an optimal concentration of monoclonal anti-IgE. Thus, similar to NAP-1/IL-8 and C3a, PAF efficiently stimulates mediator release in hGF-primed basophils only. However, PAF was clearly a more potent trigger of LTC4 formation in IL-3-primed cells than NAP-1/IL-8 or C3a. When PAF was used as a second trigger, the priming effect of IL-5 was less than that of IL-3 or GM-CSF, whereas the response for other IgE-independent agonists (i.e., C5a or FMLP) was augmented equally by all three hGF. IL-1 beta-pretreated basophils released minimal amounts of histamine in response to PAF. Neither TNF-alpha nor PAF, nor the combination thereof, was able to induce basophil mediator release. The efficiency of the different cytokines to prime for PAF responsiveness was strikingly similar to their capacity to enhance anti-IgE-induced mediator release. Similar to other IgE-independent agonists, the kinetic of mediator release in response to PAF was very rapid. PAF pretreatment of basophils did not enhance mediator release in response to diverse agonists, such as C5a and FMLP, in contrast to the capacity of PAF to augment the response of other leukocyte types to appropriate stimuli. Thus, depending on the presence of IL-3, GM-CSF, or IL-5, PAF is a potent basophil agonist capable of inducing histamine release as well as de novo synthesis of LTC4.     

Synthesis of platelet-activating factor by endothelial cells. The role of G proteins

Production of the potent lipid autacoid, platelet-activating factor (PAF), is a stimulated response of the endothelium which has important physiologic consequences including mediating adherence of inflammatory cells to the endothelium. Consequently, an understanding of the mechanisms that regulate PAF synthesis by the endothelium is important. To this end, we investigated the role of G proteins as a component of the signal transduction pathway that couples hormonal stimuli to PAF production. The addition of aluminum fluoride (AlF-4) to endothelial cells resulted in production of PAF with a maximal effect at 20 mM fluoride and within 20-60 min of exposure. Alf-4 also augmented the production of PAF which occurs in response to hormonal agonists. In addition, submaximal concentrations of AlF-4 converted an ineffective hormonal agonist (thrombin in bovine cells) to a maximally effective agonist. The adherence of neutrophils to endothelial cells that had been exposed previously to AlF-4 was increased in a manner that paralleled PAF production. PAF production in response to AlF-4 was not consistently affected by pertussis or cholera toxin. Introduction of guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) into permeabilized endothelial cells also resulted in PAF production, with reversal by guanosine 5'-O-(2-thiodiphosphate) (GDP beta S), consistent with an effect mediated by a G protein. G protein activation with AlF-4 or GTP gamma S resulted in entry of extracellular Ca2+ as determined using 45Ca2+ flux studies and Indo-1 spectrofluorometry. Our data are consistent with the hypothesis that G proteins couple hormone-receptor binding to opening of a membrane calcium channel, a key step in the initiation of PAF production in endothelial cells.     

Tumor necrosis factor inhibits a polymorphonuclear leukocyte-dependent airway edema in guinea pigs

Intravenously administered endotoxin inhibits the polymorphonuclear leukocyte (PMN)-dependent airway edema produced in guinea pigs exposed to toluene diisocyanate (TDI). Tumor necrosis factor (TNF) is produced in vivo by peripheral blood monocytes and tissue macrophages stimulated with endotoxin and has been shown to activate PMN's and vascular endothelial cells. To determine whether the inhibition of airway edema is mediated by TNF, guinea pigs were treated with intravenous saline or 75,000 U/kg recombinant human TNF 1.5 h before exposure to air or 3 ppm TDI for 1 h. Animals were then injected intravenously with 50 mg/kg Evans blue dye as a marker of protein extravasation. Saline-treated animals exposed to TDI had a significant increase in tracheal Evans blue dye extravasation (85 +/- 6.5 micrograms dye/g trachea, mean +/- SE) compared with saline-treated animals exposed to air (31.3 +/- 2.5, P less than 0.001). The tracheal extravasation of Evans blue dye was significantly inhibited (P less than 0.05) in TDI-exposed animals treated with TNF (64.7 +/- 7.5). Neither heat-inactivated TNF (104.9 +/- 9.5) nor TNF neutralized with a monoclonal antibody against TNF (99.7 +/- 17.9) inhibited TDI-induced airway edema. In addition, treatment with 15,000 U/kg (99.9 +/- 21.3) or 150,000 U/kg (103.2 +/- 17.6) interleukin 1, a monokine also produced in response to endotoxin, did not prevent airway edema. These results suggest that TNF released in response to endotoxin mediates endotoxin's inhibition of a PMN-dependent airway edema.     

Membrane interleukin 1 induction on human endothelial cells and dermal fibroblasts

Human endothelial cells and dermal fibroblasts both expressed a membrane-associated interleukin 1 (IL-1) activity when stimulated with either recombinant tumor necrosis factor (TNF) or recombinant lymphotoxin but stimulated endothelial cells expressed significantly more membrane IL-1 per cell than did fibroblasts. Lipopolysaccharide induced membrane IL-1 activity on endothelial cells but not fibroblasts. Interferon-gamma treatment of endothelial cells and fibroblasts had no direct effect on membrane IL-1 expression and little effect when used as a pretreatment for TNF or lipopolysaccharide stimulation. Endothelial cell membrane IL-1 activity was induced within 24 hr of culture with TNF or lipopolysaccharide, and increased up to 72 hr of incubation. Antibodies raised against human monocyte-derived IL-1 species neutralized the membrane IL-1 activity of TNF-stimulated endothelial cells. Both absorption studies and neutralization with specific sera indicated that endothelial cell membrane IL-1 is structurally related to IL-1 alpha. Endothelial cells expressed both IL-1 beta mRNA in response to TNF, lymphotoxin, and recombinant IL-1 species, as detected by Northern blot analysis. These studies demonstrate that endothelial cells can be activated to express a cell-surface IL-1 activity which is structurally, as well as functionally, related to the secreted form of IL-1.     

Endothelial cell-associated platelet-activating factor: a novel mechanism for signaling intercellular adhesion

The binding of neutrophils (polymorphonuclear leukocytes [PMNs]) to endothelial cells (ECs) presents special requirements in the regulation of intercellular adhesion. ECs that are stimulated by certain agonists, including thrombin and cytokines (tumor necrosis factor alpha, interleukin-1), generate molecular signals that induce the adhesion of PMNs (endothelial cell-dependent neutrophil adhesion). Our experiments demonstrate that the mechanism of binding induced by thrombin is distinct from that induced by the cytokines based on the time courses, the requirement for protein synthesis, and differential binding of HL60 promyelocytic leukemia cells to ECs activated by the two classes of agonists. The rapid EC-dependent PMN adhesion (initiated in minutes) that occurs when the ECs are stimulated by thrombin is temporally coupled with the accumulation of platelet-activating factor, a biologically active phosphoglyceride that remains associated with ECs and that activates PMNs by binding to a cell surface receptor. A portion of the newly synthesized platelet-activating factor (PAF) is on the EC surface, as demonstrated by experiments in which the rate of hydrolysis of PAF synthesized by activated ECs was accelerated by extracellular PAF acetylhydrolase. When ECs were treated with exogenous PAF they became adhesive for PMNs; the PMN binding was prevented by incubating the ECs with PAF acetylhydrolase or by treating the PMNs with competitive PAF receptor antagonists. Thus PAF associated with the EC plasma membrane induces PMN binding, an observation supported by experiments in which PAF in model membranes (liposomes) stimulated rapid PMN adhesion to ECs and to cell-free surfaces. In addition, competitive antagonists of the PAF receptor inhibited the binding of PMNs to ECs activated by thrombin and other rapidly acting agonists, but not to ECs activated by tumor necrosis factor alpha, indicating that PAF that is endogenously synthesized by ECs can mediate neutrophil adhesion. These experiments demonstrate a novel mechanism by which a cell-associated phospholipid, PAF, can serve as a signal for an intercellular adhesive event.     

Two distinct monokines, interleukin 1 and tumor necrosis factor, each independently induce biosynthesis and transient expression of the same antigen on the surface of cultured human vascular endothelial cells

A murine monoclonal antibody (H4/18) raised against cultured human endothelial cells (HEC) prestimulated by the monokine interleukin 1 (IL 1) recognizes a cell surface molecule inducible by IL 1 or by the distinct monokine tumor necrosis factor (TNF) in primary or serially passaged HEC. H4/18 binding is not basally expressed or inducible by IL 1 in an SV-40 transformed HEC line, in human dermal fibroblasts, or in blood leukocytes. Expression of this molecule by HEC in response to IL 1 can be blocked by protein and RNA synthesis inhibitors but not by cyclooxygenase inhibitors. In addition, H4/18 can immunoprecipitate two biosynthetically labeled polypeptides (Mr 100,000 and 120,000) from HEC stimulated with IL 1 but not from control HEC. Thus, the H4/18 binding site appears to be an inducible surface protein specific for HEC. The majority of HEC in a culture can be induced to express the H4/18 binding protein, but expression is transient (peak 4 to 6 hr) and over the next 24 hr declines to near basal levels either in the continued presence of or upon removal of IL 1. The magnitude of the peak response depends upon IL 1 concentration (peak 5 to 10 U/ml), and the response is optimized by the continued presence of IL 1 during the initial 4- to 6-hr induction period. The time of peak H4/18 binding does not appear to be a function of IL 1 concentration. The decline of H4/18 binding from peak levels is prevented by cycloheximide, a protein synthesis inhibitor. HEC maintained in the presence of IL 1 for 24 hr become refractory to restimulation by IL 1; however, IL 1-stimulated cells rested in the absence of IL 1 for 20 hr can be stimulated by fresh IL 1. HEC expression of the H4/18 binding protein is not induced by interleukin 2 or by interferon-alpha, -beta, or -gamma. Induction of H4/18 binding by TNF is also concentration dependent, transient, and dependent upon protein and RNA synthesis. Several observations suggest that IL1 and TNF act independently on HEC. Our TNF is a recombinant protein, expressed from a cloned cDNA and thus free of IL 1 contamination; it also has no activity in a highly sensitive IL 1 assay. Our standard IL 1 preparation is affinity purified and lacks TNF activity on L929 cells. Thus, our monokine preparations are not cross-contaminated. Most interestingly, HEC incubated with IL 1 and refractory to IL1 restimulation can be restimulated by TNF to express H4/18 binding and vice versa.(ABSTRACT TRUNCATED AT 400 WORDS)     

IL-1-induced adhesion of polymorphonuclear leukocytes to cultured human endothelial cells. Role of platelet-activating factor

In early studies we found that IL-1 stimulated endothelial cells (EC) to produce platelet-activating factor (PAF, 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine). Inasmuch as this phospholipid has a wide range of biologic activities, including polymorphonuclear leukocytes (PMN) aggregation and chemotaxis, we investigated whether EC-associated PAF could contribute to IL-1-induced PMN adhesion to EC. When four selective PAF antagonists were added to IL-1-stimulated EC during the PMN adhesion assay, adhesion was reduced in a concentration-related way. Similarly, pre-treatment of PMN with PAF before the adhesion assay to induce desensitization to this phospholipid reduced PMN adhesion to IL-1-treated EC. However, comparing the time course and the concentration response curve of IL-1-induced EC adhesivity and PAF synthesis, we found that increased EC adhesivity to PMN required a shorter incubation time and lower concentration of IL-1 to become apparent than PAF production. When acetyl-coenzyme A was added to EC cultures at a concentration that raised PAF synthesis by 60%, no significant increase in PMN adhesion was observed. In addition, after 9 to 10 doublings, the EC ability to synthesize PAF decreased by 85 to 90%, whereas IL-1-induced EC adhesivity to PMN was only slightly diminished. When IL-1-alpha and -beta were tested on EC, we observed that both were equally active in promoting PMN adhesion to EC but only the alpha-form was able to stimulate PAF production. When PMN were seeded on IL-1-treated EC, increased amounts of PAF were detected even when EC were fixed; in addition, the inhibitory effect of a PAF antagonist was evident also in these conditions. Overall these results indicate that IL-1-induced PAF production by EC does not significantly contribute to PMN adhesion to them. We hypothesize that the observed inhibitory effect of PAF antagonists and PAF desensitization of PMN might be directed at PAF produced by PMN themselves during adhesion to IL-1-treated EC.     

Platelet-activating factor mediates CD40-dependent angiogenesis and endothelial-smooth muscle cell interaction

The aim of the present study was to investigate whether stimulation of CD40 expressed by endothelial or smooth muscle cells triggers the synthesis of platelet-activating factor (PAF), an inflammatory mediator with angiogenic properties, and whether PAF contributes to CD40-induced neoangiogenesis. The results obtained indicate that the interaction of CD40 with soluble CD154 or with CD154 expressed on the membrane of leukocytes (CD154-transfected J558 cells) or of activated platelets, stimulated the synthesis of PAF by endothelial cells but not by smooth cells. The synthesis of PAF triggered by activated platelets was inhibited by a soluble CD40-murine Ig fusion protein that prevents the interaction between membrane CD40 and CD154. Studies with specific inhibitors and evaluation of protein phosphorylation indicated the involvement in PAF synthesis of two intracellular signaling pathways leading to cytosolic phospholipase A(2) activation: a phospholipase Cgamma-protein kinase C-Raf-p42/p44-mitogen-activated protein kinase (MAPK) and a MAPK kinase-3/6-dependent activation of p38 MAPK. PAF synthesized by endothelial cells after CD40 stimulation was instrumental in the in vitro migration and vessel-like organization of endothelial cells, and in the interaction between endothelial cells and smooth muscle cells, as inferred by the inhibitory effect of two different PAF receptor antagonists, WEB2170 and CV3988. In vivo, blockade of PAF receptors prevented the angiogenic effect triggered by CD40 stimulation in a murine model of s.c. Matrigel implantation. In conclusion, these observations indicate that PAF synthesis induced by stimulation of endothelial CD40 contributes to the formation and organization of new vessels. This may be relevant in the vascular remodeling associated with tumor and inflammatory neoangiogenesis.     

Cytokine gene regulation by PGE(2), LTB(4) and PAF

The initial response of the host to noxious stimuli produces a nonspecific inflammatory response. A more specific immune response is believed to be modulated by two classes of molecules: lipid mediators (PG, LT and PAF) and cytokines, synthesized by phagocytes and parenchyreal cells. In this review we discuss the increasing evidence of the interrelationship between eicosanoids, PAF and cytokines: IL-1 and TNF induce PG synthesis in various cells and PG, in turn, modulate cytokine production. We focused on the regulatory effects of LTB(4), PGE(2) and PAF on cytokine gene expression.     

Activation of the acetyl-coenzyme A:lysoplatelet-activating factor acetyltransferase regulates platelet-activating factor synthesis in human endothelial cells.

Platelet-activating factor (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine; PAF) is a phospholipid with many physiological actions. It is synthesized by endothelial cells and a variety of others in response to stimulation with receptor-mediated agonists. In endothelial cells it remains associated with the surface of the cell and serves as a signal for adhesive interactions with leukocytes. Thus, its synthesis must be precisely regulated. In previous work we have shown that PAF synthesis is regulated at the initiating step, a phospholipase A2. Here we demonstrate that the subsequent step of PAF synthesis, the acetyl-CoA:lyso-PAF acetyltransferase, is rapidly activated when cells are exposed to thrombin or other agonists. We found that the activity increased from basal values (5 nmol/mg/min) to approximately 3-fold higher within 1 min following the addition of agonists. The enzyme activity returned to basal levels within 10 min. The pattern of activation and inactivation suggested covalent modification of the enzyme. This was supported in experiments in which we showed that homogenates had stable enhanced activity and that there was no evidence for an activator or inhibitor. Pretreatment of the cells with vanadate, an inhibitor of protein phosphatases, markedly prolonged the activation state. In subsequent studies we pretreated intact cells with vanadate to block inactivation of the enzyme and then measured the accumulation of PAF in response to thrombin. We found that it was markedly augmented and prolonged. From this we conclude that the synthesis of PAF in intact cells is regulated by the activity of the acetyltransferase. We characterized requirements for activation of acetyltransferase and found that it was not dependent on the influx of intracellular calcium but that calcium entry did influence the length of time for which the enzyme was activated. The acetyltransferase in endothelial cells was shown to be a specific enzyme that did not catalyze the transfer of long chain acyl groups from acyl-CoA to lysophospholipids and demonstrated modest specificity for the acceptor lysophospholipids. These results suggest that activation of the acetyltransferase is a crucial determinant of the amount of PAF synthesized in activated endothelial cells.     

Downregulation of tumor necrosis factor receptors of macrophages by interferons and interleukin-1. Role of protein kinase C activation

Freshly harvested murine peritoneal macrophages and a line of transformed murine macrophages (RAW) were used in experiments designed to investigate the effect of different interferons (IFN) and interleukin-1 (IL-1) on tumor necrosis factor (TNF) receptors. Low concentrations of IFN-gamma or somewhat higher concentrations of IFN-alpha drastically downregulated the TNF receptors of RAW cells. A similar, but less pronounced, downregulation of TNF receptors was observed in peritoneal macrophages treated with these IFNs. This downregulation could not be accounted for by an induction of TNF secretion. Furthermore, IFN-alpha and gamma interacted synergistically in downregulating TNF receptors of RAW cells. IL-1 also downregulated TNF receptors. When RAW cells were treated with inhibitors of protein kinase C, the downregulation of TNF receptors by IFNs or IL-1 was reversed, and TNF binding increased up to 2-fold over that of untreated cells. Such increase was also observed in RAW cells treated only with the inhibitor of protein kinase C, staurosporine. However, TNF receptors decreased in peritoneal macrophages treated with staurosporine. This finding was explained by activation of macrophages by staurosporine, which induced secretion of TNF. These findings indicate that protein kinase C activity regulates TNF receptors in macrophages.     

Down regulation of the receptors for tumor necrosis factor by interleukin 1 and 4 beta-phorbol-12-myristate-13-acetate

Binding of radiolabeled tumor necrosis factor (TNF) to cell surface receptors was markedly reduced in human foreskin fibroblasts and cells from SV-80 and HeLa cell lines subsequent to treatment with interleukin 1 (IL-1) or 4 beta-phorbol-12-myristate-13-acetate (PMA). The decrease in TNF binding was initiated within minutes of application of IL-1 or PMA and could not be blocked by cycloheximide, suggesting that it is independent of protein synthesis. Scatchard plot analysis of TNF binding to the SV-80 cells indicated that its decrease in response to IL-1 and PMA reflects a reduced amount of TNF receptors, with no change in their affinity. IL-1 and PMA together had an additive effect on TNF binding. Treatment with TNF did not result in decreased binding of IL-1 to its receptors nor did TNF and IL-1 compete directly for their respective receptors. Human U937 cells on which receptors for IL-1 were below detectable levels exhibited no decrease in TNF binding when treated with IL-1, but did so in response to PMA. In addition to a decrease in TNF receptors, cells treated with IL-1 or PMA exhibited a lesser vulnerability to the cytolytic effect of TNF. The two kinds of changes were not completely correlated. A particularly notable dissimilarity was evident when comparing the rate of their reversal: the TNF receptor level was fully recovered within a few hours of removal of IL-1 or of the water-soluble analogue of PMA, 4 beta-phorbol-12,13-dibutyrate, from pretreated SV-80 cells; yet at that time resistance to the cytotoxicity of TNF was still prominent. These findings indicate that IL-1 as well as tumor-promoting phorbol diesters can down regulate cellular response to TNF by inducing a decrease in the number of receptors for TNF, and apparently through some other effect(s) as well.     

Hypoxia increases stimulus-induced PAF production and release from human umbilical vein endothelial cells.

Hypoxia alters endothelial cell function and metabolism. Since PAF is synthesized by endothelial cells and capable of modulating endothelial cell responses, we investigated the effect of hypoxia on synthesis and release of PAF from endothelial cells. We found: (1) Approx. 90% of the radylPAF derivative in stimulated endothelial cells is acylPAF. (2) Acute hypoxic (15 min-1 h) priming increased ionophore- and thrombin-induced radylPAF accumulation. (3) Long-term hypoxic exposure increased radylPAF accumulation at 24 and 48 h in the presence of ionophore. (4) Bioactive PAF was released into media and hypoxia and ionophore synergistically increased PAF release. (5) Hypoxia and ionophore stimulation increased phospholipase A2 activity and decreased acetylhydrolase activity in endothelial cells. We conclude that hypoxia and ionophore increase PAF synthesis and release from endothelial cells.     

Enhanced synthesis of factor B induced by tumor necrosis factor in human skin fibroblasts is decreased by IL-4

IL-4 is a T cell-derived lymphokine that has multiple biologic functions, affecting B cells, T cells, mast cells, monocytes, macrophages, and hematopoietic progenitor cells. We report that IL-4 also affects human skin fibroblasts. These effects were primarily in modulating the effects of other mediators of inflammation on these cells, with IL-4 producing little or no effects by itself. Synthesis of C proteins in human skin fibroblasts is modulated by TNF and other mediators of inflammation. TNF increased synthesis of factor B by 18.6-fold; IL-4 reduced the TNF effect on factor B synthesis by 83%. This effect started with concentrations of IL-4 as low as 0.1 ng/ml. The effect of IL-4 on the TNF-induced increase in synthesis of factor B occurred after 2 h incubation, the time when the effects of TNF alone were first observed. IL-4 also abrogated the effects of IL-1 and LPS on factor B synthesis, but did not affect the increase in synthesis of factor B induced by IFN-gamma. In contrast to the pattern for effect of IL-4 on factor B synthesis, the TNF-induced rate of synthesis of C3 was augmented by IL-4. The specificity of the IL-4 effect was also apparent when comparing the effect of IL-4 on the TNF-induced synthesis of factor B to the effects on total protein synthesis (increased 1.5-fold) and the TNF-induced increases in synthesis in C1r, C1s, C1 inhibitor, C2, and factor H (no changes). The IL-4-induced decrease in synthesis of factor B was mediated at a pretranslational level and was dependent on synthesis of a new protein. This interaction between IL-4 and mediators of inflammation can potentially modulate the inflammatory response in the setting of activation of Th cells.     

Stimulation of platelet-activating factor synthesis by a nonmetabolizable bioactive analog of platelet-activating factor and influence of arachidonic acid metabolites

Platelet-activating factor (PAF) is a potent neutrophil agonist operating through specific receptors located on the cell surface. Binding of PAF to its receptor may also stimulate further PAF synthesis, thus providing a means of amplifying the PAF signal for the cell of origin and/or other responsive cells. In this report we demonstrate that 1-O-alkyl-2-N-methylcarbamyl-sn-glycero-3-phosphocholine (C-PAF), a nonmetabolizable bioactive analog of PAF, stimulates human neutrophils to synthesize PAF, as detected by [3H]acetate incorporation into PAF. This approach allowed us to conclude that [3H]acetate-labeled PAF was formed from endogenous precursor rather than mere turnover of the stimulatory dose of PAF. PAF's ability to initiate further PAF synthesis was confirmed by measuring the PAF-stimulated conversion of 1-O-[3H]alkyl-2-acylglycerophosphocholine to 1-O-[3H]alkyl-2-acetylglycerophosphocholine by prelabeled human neutrophils and by determining the molecular species of 1-O-alkyl-2-[3H]acetylglycerophosphocholine produced by cells stimulated with a single molecular species of PAF (C15:0). Degradation of exogenously added [3H]PAF was not inhibited by C-PAF/5-hydroxyeicosatetraenoic acid treatment. Thus, inhibition of PAF degradation was ruled out as the mechanism accounting for the appearance of labeled PAF in the stimulated cells. Synthesis of PAF in response to C-PAF was not dependent on cytochalasin B pretreatment but was dramatically potentiated by 5-hydroxyeicosatetraenoic acid, which alone was without effect. Additionally, we have demonstrated that another major arachidonate metabolite of neutrophils, leukotriene B4, stimulates PAF production. Thus, at least three products of activated neutrophils, including PAF itself, can promote PAF synthesis by these cells. This positive feedback effect may amplify autacoid production and the final cellular response.     

Modulation of platelet-activating factor (PAF) synthesis and release from human polymorphonuclear leukocytes (PMN): role of extracellular Ca2+

Extracellular Ca2+ regulated the synthesis and release of platelet-activating factor (PAF) from human polymorphonuclear leukocytes (PMN) stimulated with N'-formyl-methionyl-leucyl-phenylalanine (FMLP) in the presence of cytochalasin B. Maximum PAF synthesis and release required the presence of 0.14 mM Ca2+ whereas 1.4 mM Ca2+ was necessary for maximum lysosomal enzyme secretion. The synthesis of PAF occurred within 2.5 min after PMN stimulation in the presence of 1.4 mM Ca2+; however, PAF release did not occur until 5 min after stimulation. Peak PAF release occurred by 7.5 min but accounted for only 30-40% of the total amount of PAF synthesized, the remainder being retained on or within the PMN. Stimulation of PMN in the presence of 0.01 M EDTA or EGTA decreased PAF synthesis and release by greater than 95%. In the absence of extracellular Ca2+, stimulated PMN synthesized PAF in amounts that were 10-30% of maximum, but there was no release of the newly synthesized PAF. At Ca2+ concentrations greater than 0.01 mM, there was a dose-dependent (up to 0.14 mM) increase in PAF synthesis that was associated with the initiation and concomitant increase in the amount of PAF released. These data suggest the presence of a PAF synthesis-release coupling mechanism in which the extracellular Ca2+-dependent release of PAF stimulates additional PAF synthesis.     

Tumor necrosis factor combines with IL-4 or IFN-gamma to selectively enhance endothelial cell adhesiveness for T cells. The contribution of vascular cell adhesion molecule-1-dependent and -independent binding mechanisms.

The adhesion of lymphocytes to vascular endothelium is the first step in their passage from the blood into inflammatory tissues. By modulating endothelial cell (EC) adhesiveness for lymphocytes, cytokines may regulate lymphocyte accumulation and hence the nature and progression of inflammatory responses. IL-1, TNF, IFN-gamma, and IL-4 each increase EC adhesiveness for T cells when used alone in adhesion assays in vitro. As cytokines are more likely to act in combination at sites of inflammation in vivo, we have studied the stimulating effect of different combinations of cytokines on EC adhesiveness for T cells and polymorphonuclear leukocytes (PMN). Acting alone IL-1, TNF, IFN-gamma, and IL-4 each significantly enhanced EC adhesiveness for T cells (p less than 0.005), whereas only IL-1 (p less than 0.005) and TNF (p less than 0.005) but not IFN-gamma or IL-4 significantly enhanced adhesiveness for PMN. When EC were stimulated with optimal concentrations of TNF in combination with IL-4 or IFN-gamma, there was a significant further increase in adhesiveness for T cells (p less than 0.003), but not PMN, over that seen with TNF alone. The additive effect of TNF and IL-4 was more marked than that of TNF and IFN-gamma. Although approximately equal proportions of T cells and PMN bound to TNF-stimulated EC, nearly double the proportion of T cells compared with PMN bound EC preincubated with TNF and IL-4 together. A similar interaction with IL-4 or IFN-gamma was exhibited by lymphotoxin. mAb-inhibition studies indicated that the extra increase in binding caused by stimulating EC with TNF and IL-4 in combination was mediated by VCAM-1 whereas that caused by stimulating with TNF and IFN-gamma in combination was substantially mediated through leukocyte function-associated Ag-1- and VCAM-1-independent mechanisms. These observations suggest that whereas IL-1 and TNF alone are unselective in terms of leukocyte adhesion to EC, the combination of TNF (or LT) with IL-4 or IFN-gamma may be of key importance in determining the recruitment of a lymphocyte-predominant infiltrate in immune mediated inflammation, and in initiating the transition from acute to chronic inflammation.