Synthesis and release of platelet-activating factor by stimulated human mononuclear phagocytes

Platelet-activating factor (PAF) is a potent phospholipid mediator that may participate in inflammatory responses by virtue of its ability to activate platelets, leukocytes, and vascular cells. We examined the synthesis and release of PAF by human peripheral blood monocytes (PBM) isolated by countercurrent elutriation. PAF was produced after stimulation by calcium ionophore A23187 (IoA), opsonized zymosan (OpsZ), and PMA with a relative order of potency IoA much greater than OpsZ greater than PMA. The portion of PAF subsequently released from the cell was dependent on the specific agonist, the time of incubation, and the presence of albumin. Under optimal conditions, PBM released 67, 49 and 32% of the total PAF produced in response to IoA, OpsZ, and PMA, respectively. Changes in PAF metabolism were observed in PBM that were examined after short term adherence or differentiation into macrophages. Adherent PBM accumulated and released less PAF than suspended monocytes, and monocyte-derived macrophages produced less PAF than the parent PBM. The ability of monocytes to release significant amounts of newly synthesized PAF from the cell is unusual among human cell types, which in general retain the vast majority of the lipid, and may be of particular pathophysiologic importance.     

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.     

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

Human neutrophilic polymorphonuclear leukocytes (PMN) stimulated with N'-formyl-methionyl-leucyl-phenylalanine (FMLP) in the presence of cytochalasin B but in the absence of human serum albumin (HSA) synthesized only small amounts of platelet-activating factor (PAF) that attained maximum levels within 60-120 s after stimulation; in addition, no release of PAF occurred. However, in the presence of 2.5 mg HSA/ml, there was a threefold increase in PAF synthesis, 30-40% of which was released within 5 min after FMLP stimulation. In the presence of 50 mg HSA/ml there was at least a fourfold increase in PAF synthesis and release, with maximal synthesis occurring 10-20 min after stimulation. Thus, the presence of HSA during PMN stimulation not only induced an albumin dose-dependent increase in PAF release but significantly augmented the synthesis of PAF. In contrast to PAF synthesis and release, the presence or absence of HSA had no effect upon lysosomal enzyme secretion from FMLP-stimulated PMN, which was maximal within 30-60s after stimulation. These results demonstrate that HSA plays an essential role in vitro in the synthesis and release of PAF from human PMN, and support the hypothesis that there is a cyclic PAF synthesis-release coupling mechanism in the stimulated human PMN.     

Endothelial cell prostacyclin production induced by activated neutrophils

A bovine aortic endothelial cell (EC) line released prostacyclin (greater than 1 pmol/10(+5) EC cells) when incubated with fMet-Leu-Phe (FMLP)-stimulated rat and human neutrophils (PMNs). This prostaglandin (PG) I2 was shown to come from the ECs and not from the PMNs by radioactive, high-performance liquid chromatography, and immunochemical criteria. Both FMLP-stimulated rat peritoneal and human peripheral PMNs as well as their stimulated cell-free supernatants and unstimulated sonicates could elicit the release of PGI2 from ECs. Since phorbol myristate acetate stimulated PMN adherence but elicited little PGI2 release from ECs, the PGI2 stimulation in ECs is unrelated to PMN adhesion. The addition of catalase and superoxide dismutase to FMLP-stimulated PMNs enhanced rather than reduced PGI2 formation, indicating that activated oxygen products of the PMN are not responsible for the induction of PGI2. Incubation of ECs with leukotriene (LT) B4, LTC4, or LTD4 did not trigger PGI2 release nor did aspirin pretreatment of the PMNs reduce the PGI2 induction. These data suggest that arachidonic acid metabolites of the PMNs were not responsible for the PGI2 induction. Available data indicates that the PMN factor that stimulates PGI2 from ECs is either released concomitantly with the azurophilic granules or is closely related to this event.     

Tumor necrosis factor-alpha priming of phospholipase A2 activation in human neutrophils. An alternative mechanism of priming.

The cytokine, TNF-alpha, interacts with human neutrophils (PMN) via specific membrane receptors and primes leukotriene B4 (LTB4) production in PMN for subsequent stimulation by calcium ionophores. We have further examined the effects of TNF-alpha on arachidonic acid (AA) release, LTB4 production, and platelet-activating factor (PAF) formation in PMN by prelabeling cells with either [3H]AA or [3H]lyso-PAF, priming with human rTNF-alpha, and then stimulating with the chemotactic peptide, FMLP. TNF-alpha, alone, had little effect; minimal AA release, LTB4 or PAF production occurred after PMN were incubated with 0 to 1000 U/ml TNF-alpha. However, when PMN were first preincubated with 100 U/ml TNF-alpha for 30 min and subsequently challenged with 1 microM FMLP, both [3H] AA release and LTB4 production were elevated two- to threefold over control values. Measurement of AA mass by gas chromatography and LTB4 production by RIA confirmed the radiolabeled results. TNF-alpha priming also increased PAF formation after FMLP stimulation. These results demonstrate that TNF-alpha priming before stimulation with a physiologic agonist can enhance activation of phospholipase A2 (PLA2) resulting in increased AA release and can facilitate the activities of 5-lipoxygenase (LTB4 production) and acetyltransferase (PAF formation). Reports in the literature have hypothesized that the priming mechanism involves either production of PLA2 metabolites, increased diglyceride (DG) levels, or enhanced cytosolic calcium levels induced by the priming agent. We investigated these possibilities in TNF-alpha priming of PMN and report that TNF-alpha had no direct effect on PLA2 activation or metabolite formation. Treatment of PMN with TNF-alpha did not induce DG formation and, in the absence of cytochalasin B, no increased DG production (measured by both radiolabel techniques and mass determinations) occurred after TNF-alpha priming followed by FMLP stimulation. TNF-alpha also had no effect on basal cytosolic calcium and did not enhance intracellular calcium levels after FMLP stimulation. These results suggest that an alternative, as yet undefined, mechanism is active in TNF-alpha priming of human PMN.     

Calcium is necessary but not sufficient for the platelet-activating factor release in human neutrophils stimulated by physiological stimuli. Role of G-proteins

Platelet-activating factor (1-O-alkyl-2-acetyl-sn-glycerol-3-phosphocholine; PAF) enhances the release of newly synthesized PAF as measured by [3H]acetate incorporation into PAF in human neutrophils. The response was dose-dependent, rapid, transient, and inhibitable by the PAF antagonist BN-52021. The non-metabolizable bioactive PAF analogue (C-PAF) but not lyso-PAF enhances the release of newly synthesized PAF. Newly synthesized PAF was also released after stimulation of these cells with fMet-Leu-Phe. The human granulocyte-macrophage colony-stimulating factor potentiates the stimulated release of PAF. The intracellular calcium chelator BAPTA inhibits the rise of [Ca2+]i and the release of PAF but not the Na+/H+ antiport activity. PAF release, but not the rise in the intracellular concentration of free calcium, was inhibited in pertussis toxin-treated neutrophils stimulated with PAF. The release of PAF in pertussis toxin-treated cells was also inhibited in cells stimulated with fMet-Leu-Phe or opsonized zymosan. These results suggest that functional pertussis toxin-sensitive guanine nucleotide regulatory protein and/or one or more of the changes produced by phospholipase C activation are necessary for PAF release produced by physiological stimuli. It appears that PAF release requires a coordinated action of receptor-coupled G-proteins, calcium, and other parameters.     

Effect of an inhibitor of protein kinase C on human polymorphonuclear leukocyte degranulation

The protein kinase C inhibitor C-I reduced superoxide production by human neutrophils in response to phorbol myristate acetate by greater than 50%. In contrast to its effects in oxidative metabolism, 100 microM C-I caused minimal inhibition (5-18%) of lysozyme release in response to phorbol myristate acetate. Enzyme release produced by the formylated oligopeptide FMLP was enhanced by 23-54% in neutrophils pretreated with 100 microM C-I. These findings suggest that protein kinase C activation is not required for phorbol myristate acetate induced enzyme release. Enhancement of FMLP stimulated degranulation by C-I suggests that protein kinase C activation may have inhibitory effects on the release of granule enzymes by human neutrophils.     

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.     

Superoxide responses to formyl-methionyl-leucyl-phenylalanine in primed neutrophils. Role of intracellular and extracellular calcium.

For superoxide (O2-) responses of human neutrophils stimulated by FMLP, experiments were designed to assess the requirement of extracellular calcium [( Ca2+]o) for priming of O2- responses by platelet-activating factor (PAF), PMA, or ionomycin. Although priming by PMA did not require [Ca2+]o, there was, as expected, a requirement for [Ca2+]o for the optimal priming effects of PAF and ionomycin. The ED50 value for [Ca2+]o in the priming function of PAF was 105 microM. The [Ca2+]o-dependent priming with ionomycin was bimodal with two ED50 values for [Ca2+]o of 90 microM and 3.2 mM. Optimal priming by PAF required at least 4-min exposure of cells to [Ca2+]o. Cells primed by PAF exhibited faster initial rates of O2-production after addition of FMLP, but the duration of O2- production was not prolonged. Whereas PAF-primed responses to FMLP are usually associated with increases in intracellular calcium [( Ca2+]i) after addition of FMLP, two conditions were found in which O2- responses to FMLP in PAF-primed cells occurred in the absence of any detectable increase in [Ca2+]i. When cells were loaded with the calcium chelator, bis-(O-aminophenoxy)-ethane-H,N,N',N'-tetraacetic acid, and then primed with PAF, normal amounts of inositol 1,4,5-trisphosphate were formed, but no increase in [Ca2+]i occurred after addition of FMLP even though the cells exhibited a fully primed O2- response; in Ca2(+)-depleted and ionomycin-permeabilized cells that were primed with PAF and then stimulated with FMLP, O2- was generated in amounts comparable to reference control (primed) cells, but there was suppressed production of inositol 1,4,5-trisphosphate and no increase in [Ca2+]i after addition of FMLP to PAF-primed cells. These data confirm the requirement of [Ca2+]o for optimal priming of neutrophils by PAF and ionomycin (but not cells primed by PMA) and indicate that, under certain conditions, generation of O2- in response to FMLP in PAF-primed neutrophils can occur independent of any increase in [Ca2+]i.     

The cell surface glycoprotein Mac-1 (CD11b/CD18) mediates neutrophil adhesion and modulates degranulation independently of its quantitative cell surface expression

It has previously been shown that during degranulation Mac-1 (CD11b/CD18)--a glycoprotein that plays a central role in neutrophil adhesion-is up-regulated on PMN surfaces. It has been assumed that this quantitative change in adhesion Ag expression on the cell surface would in turn lead to increased cellular adhesiveness. In contrast, we found that at an incubation temperature of 16 degrees C, stimulated neutrophil adhesion to plastic tissue culture dishes in the presence of FMLP (2.5 x 10(-6) M), TNF (10 ng/ml), or PAF (1 x 10(-4) M) occurred without cellular degranulation or Mac-1 surface up-regulation as measured cytofluorometrically. As shown by functional inhibition studies employing monoclonal antibodies 60.3 (anti-CD18) and 60.1 (anti-CD11b), adhesion at 16 degrees C, where no CD11b/CD18 up-regulation was seen, is mediated by CD11b/CD18 just as it is at 37 degrees C, where degranulation and CD11b/CD18 up-regulation could be demonstrated. The physiologic importance of these findings was underscored by experiments done on endothelial monolayers, which showed that PMN association with endothelial cells is absolutely independent from the quantitative up-regulation of Mac-1 on PMN surfaces. When neutrophils were stimulated at 37 degrees C by endotoxin, an agent that does not induce aggregation (a form of intercellular adhesion), Mac-1 surface expression increased only after cells had become adherent, whereas cells held in suspension to prevent cell-substrate adhesion neither degranulated nor up-regulated their Mac-1 surface expression. Thus, not only is adherence independent of degranulation and Mac-1 cell surface up-regulation, but both degranulation and Mac-1 surface up-regulation appear to depend on the process of adhesion. Correspondingly, incubation of neutrophils with antibodies 60.1 and 60.3 inhibited not only adhesion of cells stimulated with FMLP at 37 degrees C but degranulation as well. These results indicate that Mac-1 influences degranulation as well as it controls adhesion not by its mere quantity on the cell surface, but rather by an yet undefined molecular modulation.     

Granulocyte-macrophage colony-stimulating factor is a stimulant of platelet-activating factor and superoxide anion generation by human neutrophils.

Human granulocyte-macrophage colony-stimulating factor (GM-CSF) was studied for its ability to stimulate the synthesis and release of the inflammatory mediator platelet-activating factor (PAF) from human neutrophils as measured by bioassay and incorporation of [3H]acetate into PAF. GM-CSF stimulated the synthesis but not the release of PAF from neutrophils. PAF synthesis took place in a time- and concentration-dependent manner, was dependent on a pertussis toxin-sensitive G protein and could be inhibited by antibodies to GM-CSF. On the other hand, pre-incubation of neutrophils with GM-CSF followed by stimulation with the bacterial tripeptide formylmethionylleucylphenylalanine caused PAF synthesis and release. The effect of GM-CSF was qualitative and not simply the result of larger amounts of PAF being synthesized since similar amounts were generated in response to the calcium ionophore A23187 but no released PAF could be detected. In functional studies GM-CSF stimulated superoxide anion generation from neutrophils with a time and dose relationship that paralleled PAF synthesis. In addition, the serine protease inhibitor L-1-tosylamide-2-phenylethyl chloromethyl ketone, which inhibits PAF synthesis, reduced PAF accumulation as well as superoxide generation, raising the possibility of a causal relationship between cell-associated PAF and cell activation. These results identify PAF as a direct product of GM-CSF stimulation in neutrophils where it may play a role in signal transduction and demonstrate that PAF is released only after subsequent neutrophil stimulation. The selective release of PAF may play a role in regulating and amplifying the inflammatory response.     

Regulation of platelet-activating factor synthesis in human neutrophils by MAP kinases

Human neutrophils (PMN) are potentially a major source of platelet-activating factor (PAF) produced during inflammatory responses. The stimulated synthesis of PAF in PMN is carried out by a phospholipid remodeling pathway involving three enzymes: acetyl-CoA:lyso-PAF acetyltransferase (acetyltransferase), type IV phospholipase A(2) (cPLA(2)) and CoA-independent transacylase (CoA-IT). However, the coordinated actions and the regulatory mechanisms of these enzymes in PAF synthesis are poorly defined. A23187 has been widely used to activate the remodeling pathway, but it has not been shown how closely its actions mimic those of physiological stimuli. Here we address this important problem and compare responses of the three remodeling enzymes and PAF synthesis by intact cells. In both A23187- and N-formyl-methionyl-leucyl-phenylalanine (fMLP)-stimulated PMN, acetyltransferase activation is blocked by SB 203580, a p38 MAP kinase inhibitor, but not by PD 98059, which blocks activation of the ERKs. In contrast, either agent attenuated cPLA(2) activation. Correlating with these results, SB 203580 decreased stimulated PAF formation by 60%, whereas PD 98059 had little effect. However, the combination of both inhibitors decreased PAF formation to control levels. Although a role for CoA-IT in PAF synthesis is recognized, we did not detect activation of the enzyme in stimulated PMN. CoA-IT thus appears to exhibit full activity in resting as well as stimulated cells. We conclude that the calcium ionophore A23187 and the receptor agonist fMLP both act through common pathways to stimulate PAF synthesis, with p38 MAP kinase regulating acetyltransferase and supplementing ERK activation of cPLA(2).     

Generation and release of platelet-activating factor (PAF) from enriched preparations of rabbit basophils; failure of human basophils to release PAF

Rabbit mononuclear cells containing up to 20% basophils and uncontaminated by neutrophils release PAF when stimulated with goat antiserum to rabbit IgE. The amount of PAF detected was a function of basophil concentration but decreased on a per cell basis at high basophil or high total cell concentrations. Calcium ionophore A23187, but not protein A, C5a, or FMLP, initiated rabbit basophil degranulation and PAF release. By contrast, extensive studies using a variety of human leukocyte preparations failed to demonstrate the release of significant levels of PAF from human basophils by IgE-dependent or -independent mechanisms. These results suggest that cells other than the peripheral blood basophil (e.g., the neutrophil) may act as the primary site of PAF production in man.     

Release of platelet-activating factor and the metabolism of leukotriene B4 by the human neutrophil when studied in a cell superfusion model

Stimulated human neutrophils are known to synthesize large quantities of 1-0-alkyl-2-acetyl-sn-glycero-3-phosphocholine (PAF) and 5,12-dihydroxy-6,14-cis-8,10-trans-transeicosatetraenoic acid (LTB4). However, in an isolated cell suspension the majority of synthesized PAF appears to remain cell associated. In addition, LTB4 is rapidly metabolized to an omega-oxidation product (20-OH-LTB4). Experiments were designed to test the hypothesis that the degree of association of PAF with the neutrophils and the metabolism of LTB4 by the neutrophils is a result of the in vitro condition used during cell activation. Here we have compared in paired experiments ionophore A23187-induced production of PAF and LTB4 by human neutrophils in a concentrated cell suspension, a diluted cell suspension and in a system in which the cells are placed on a matrix and superfused with buffer at a constant flow rate (dynamic system). There was little difference in the amount of PAF synthesized in the concentrated cell suspension and the dynamic system. However, less PAF was produced by neutrophils in the dilution system. The percent of PAF released was consistently greater in the dynamic and dilution systems than in the concentrated cell suspension. For example, more than 40% of PAF measured by incorporation of [3H]acetate or gas chromatography/mass spectrometry was released in the dynamic system and dilution systems. In contrast, less than 15% of the PAF synthesized was released from the cells in the concentrated cell suspension. 1-0-Hexadecyl-2-acetyl-3-GPC was primarily released from the neutrophils. By contrast both 1-0-hexadecyl and 1-0-octadecyl linked species of PAF were found within the cells. Exogenous PAF added to neutrophils in the dynamic or dilution systems was taken up and metabolized at a significantly lower rate than that added to cells in the concentrated cell suspension. Most of the leukotrienes synthesized by the neutrophil during A23187 stimulation were released from the cells. However, studies of LTB4 metabolism revealed differences between the dynamic and concentrated cell suspension designs. By 20 min, most of the LTB4 was recovered as 20-OH-LTB4 in the concentrated cell suspension, whereas in the dynamic system little 20-OH-LTB4 was found in the superfusate over 20 min. These experiments suggest that a large proportion of PAF synthesized by neutrophils may be released. They also suggest that the omega-hydroxylation of LTB4 by neutrophils occurs after synthesized LTB4 is released and taken back up by the cell.     

Enhancement of phorbol ester-induced protein kinase activity in human neutrophils by platelet-activating factor

We have shown that platelet-activating factor (PAF), a weak primary stimulus for neutrophil superoxide generation, synergistically enhances neutrophil oxidative responses to the tumor promoter phorbol myristate acetate (PMA). Since PMA is known to cause cytosol-to-membrane shift of calcium-activated, phospholipid-dependent protein kinase (protein kinase c, PKC) in human neutrophils, we investigated the role of PAF in modifying PMA-induced PKC activation/translocation. Protein kinase activity was measured as the incorporation of 32P from gamma-32P-ATP into histone H1 induced by enzyme in cytosolic and particulate fractions from sonicated human neutrophils. PAF did not alter the sharp decrease in cytosolic PKC activity induced by PMA. However, in the presence of PAF and PMA, total particulate protein kinase activity increased markedly over that detected in the presence of PMA alone (144 +/- 9 pmoles 32P/10(7)PMN/minute in cells treated with 20 ng/ml PMA compared to 267 +/- 24 pmoles 32P in cells exposed to PMA and 10(-6)M PAF). The increase in total particulate protein kinase activity was synergistic for the two stimuli, required the presence of cytochalasin B during stimulation, and occurred at PAF concentrations of 10(-7) M and above. Both PKC and calcium-, phospholipid-independent protein kinase activities in whole particulate fractions were augmented by PAF as were both activities in detergent-extractable particulate subfractions. PAF did not directly activate PKC obtained from control or PMA-treated neutrophils. However, the PKC-enhancing effect of PAF was inhibited in the absence of calcium during cellular stimulation. PAF also increased particulate protein kinase activity in cells simultaneously exposed to FMLP but the effect was additive for these stimuli. These results suggest that PAF enhances PMA-induced particulate PKC activity by a calcium-dependent mechanism. The enhancing effect of PAF may be directly involved in the mechanism whereby the phospholipid "primes" neutrophils for augmented oxidative responses to PMA.     

Neutrophil-mediated biodegradation of medical implant materials

During the acute inflammatory response to implanted medical devices, human neutrophils (PMN) release oxidative and hydrolytic activities which may ultimately contribute to the degradation of the biomaterial. In this study, the biological activities secreted by live PMNs which may contribute to biodegradation were investigated using a 14C label in the monomer unit of a poly(ester-urea-urethane) (PEUU) substrate. By using specific inhibitors, it was possible to propose a mechanism for PMN-mediated biodegradation. PMN, labeled with 3H-arachidonic acid, released significantly more 3H when adherent to PEUU than when adherent to tissue culture grade polystyrene (P<0.05). The phospholipase A2 (PLA2) inhibitors, aristolochic acid (ARIST) and quinacrine (QUIN), decreased the release of 3H and inhibited PEUU biodegradation (>50%, P<0.05). ARIST had no effect on cell viability, whereas QUIN significantly decreased it. The serine protease inhibitor, phenylmethylsulfonylfluoride inhibited biodegradation, but did not decrease cell survival. There is evidence to suggest that activation via the PLA2 pathway caused the release of hydrolytic activities which were able to elicit 14C release from PEUU. The role of oxidative compounds which were released via activation by phorbol myristate acetate (PMA), was not apparent, since PMA inhibited biodegradation and cell survival (>40%, P<0.05). This study has shown that it is possible to find out the differences in PMN activation through the PLA2 pathway when exposed to different material surfaces, making this a model system worthy of further investigation.     

Platelet-activating factor production in human neutrophils by sequential stimulation with granulocyte-macrophage colony-stimulating factor and the chemotactic factors C5A or formyl-methionyl-leucyl-phenylalanine

Besides its function as a growth factor, the cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF) "primes" polymorphonuclear leukocytes (PMN) for enhanced biologic responses to a number of secondary stimuli. We examined the effect of priming PMN with GM-CSF on the production of [3H] platelet-activating factor (PAF) from [3H]acetate upon stimulation with the chemotactic factors FMLP and C5a. In PMN stimulated with the individual peptide mediators alone [3H]PAF levels were close to controls, whereas considerable amounts of [3H]PAF are formed after stimulation of PMN which have been preexposed to GM-CSF. The priming effect was concentration and time dependent. It was optimal after a preincubation period of 2 h. A maximum of [3H]PAF accumulation is reached within 2.5 min (C5a) and 5.0 min (FMLP) after activation of GM-CSF-primed PMN. In addition, we show that PAF isolated from PMN preincubated with GM-CSF and triggered with chemotactic factors is able to enhance the respiratory burst in PMN. PAF formed by sequentially activated PMN could contribute to the enhanced oxygen radical production and cytotoxicity in effector cells and play a role in modulating and amplifying inflammatory reactions.     

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.     

The intracellular retention of newly synthesized platelet-activating factor

The ether phospholipid platelet-activating factor (PAF) has been generally assumed to be released into the extracellular environment by the cells of origin, whereupon it effects its well-known mediator functions. However, during the generation of PAF by human neutrophils, it was noted that the majority of the measurable PAF remained associated with the cells. Accordingly, the intracellular and extracellular distribution of PAF was examined in neutrophils and several other cell types. No PAF was detected in association with unstimulated neutrophils. However, in stimulated neutrophils, PAF was produced and the majority of this material remained in association with the cells independent of the type of stimulus, dose of stimulus, or method of cell isolation used. This pattern of cell association of PAF was seen in all but one of the cell types tested. The retention of PAF by stimulated neutrophils was not due to spurious underestimates of the extracellular levels due to extracellular metabolism and inactivation of released PAF, nor to release followed by readsorption or binding of PAF to the cells. The retention of PAF also occurred in the presence of plasma and appears to be a common phenomenon. Thus, the majority of newly synthesized PAF appears to be retained within the cell and not released.     

Neutrophil degranulation: evidence pertaining to its mediation by the combined effects of leukotriene B4, platelet-activating factor, and 5-HETE

Stimulus-activated polymorphonuclear neutrophils (PMN) produce leukotriene B4 (LTB4), 5-hydroxyeicosatetraenoate (5-HETE), and platelet-activating factor (PAF). Each of these lipids promotes PMN degranulation; in combination they have additive and potentiating effects that result in prominent degranulation responses at relatively low concentrations. Thus, the combined interactions of LTB4, 5-HETE, and PAF may mediate responses in PMN activated by other stimuli. This possibility was examined by measuring the responses of PMN made insensitive to one or more of these lipids. Cells were pretreated with LTB4, 5-HETE, and/or PAF for 8 min; exposed for 2 min to cytochalasin B (which is required for lipid-induced degranulation); and then challenged. PMN challenged with only buffer released minimal amounts of granule-bound enzymes. Furthermore, the lipid-pretreated cells were hyporesponsive to challenge with 1) various combinations of these same lipids or 2) ionophore A23187. The relative potencies of the lipids in producing hyporesponsiveness to themselves or A23187 were: 5-HETE less than PAF less than or equal to LTB4 less than PAF + LTB4 less than PAF + LTB4 + 5-HETE. For both types of challenge, reduced responsiveness occurred in cells pretreated with greater than 0.1 nM LTB4 and/or greater than 0.2 nM PAF, persisted in cells washed after lipid pretreatment, and did not develop in cells pretreated with various combinations of bioinactive structural analogues of the lipids. Thus, PAF, LTB4, and 5-HETE interacted to desensitize PMN, and the degranulating actions of A23187 required cells that were fully responsive to each of the three lipids. This supports the concept that the lipids act together in mediating certain of the ionophore's effects. However, lipid-desensitized PMN degranulated fully when challenged with C5a, a formylated oligopeptide, or phorbol myristate acetate. Degranulation responses, therefore, may proceed through various pathways, only some of which involve the lipid products studied here.