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Paf-acether (paf) is a phospholipid cytokine alloted with potent inflammatory and immunoregulatory properties. Recent reports indicated that in human B cell lines, paf modulated both early and late activation events. In our study, we showed that four of six human B cell lines specifically bound [3H]paf irrespective of the stage of differentiation, the presence of EBV genome or cell surface phenotype. Binding was saturated and fit a one receptor model with a dissociation constant ranging from 1 to 6 nM and a number of sites per cell ranging from approximately equal to 4000 in Rjc13 to approximately equal to 30,000 in Raji or IM9. In addition, our data indicate that 1) maximal expression occurred during the log phase growth; 2) paf itself (10-100 nM) or rIL-4 (100 U/ml) up-regulated by two- to threefold the number of paf binding sites without affecting the affinity. Finally, we found that activated normal B lymphocytes exhibited a higher capacity than resting B cells to incorporate and metabolize [3H]paf at 37 degrees C. Resting B lymphocytes lacked specific binding capacity for paf, yet specific paf receptors were induced upon stimulation via Staphylococcus aureus Cowan I or phorbol 12,13 dibutyrate plus ionomycin. These results suggest that B cell activation is a critical event for paf receptor expression and modulation.

In the present report, we further explored the mechanisms by which 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (paf-acether), a phospholipid mediator of inflammation inhibited PHA-induced CD4+ cell proliferation. Evidence was obtained that CD4+ cells stimulated with either PHA or immobilized OKT3 in the presence of paf at concentrations that block CD4+ cell proliferation, exhibited a marked decrease in high affinity IL-2R expression. Importantly, paf did not prevent the binding of IL-2 to its receptor. Scatchard analysis of the binding data indicated that paf caused more than 50% decrease in the number of IL-2 high affinity sites per cell, whereas the receptor ligand affinity remained essentially constant. Moreover, the down-regulation of high affinity IL-2R was also accompanied by a loss of IL-2-dependent proliferative capacity. Together these data suggest that decreased expression of high affinity IL-2R may contribute to the diminished proliferative activity observed in CD4+ cells stimulated with PHA or immobilized OKT3 in the presence of paf. They further emphasize the potential role of lipid proinflammatory mediators such as paf in the regulation of T cell activation.

We tested the hypothesis that protein kinase C might play a role in the biosynthesis of platelet-activating factor (paf-acether) in human neutrophils. PMA but not its inactive analog 4-alpha-phorbol-12,13-didecanoate induced lyso paf-acether production, followed by acetyltransferase activation, leading to paf-acether synthesis and release. Moreover, PMA was twice as powerful compared to opsonized zymosan (OPZ). 1-Oleoyl-2-acetyl-glycerol also induced acetyltransferase activation and paf and lyso paf production. The paf-acether formed by PMA or OPZ stimulation was composed of alkyl chains C16:0 (84.3 +/- 5% and 80.7 +/- 3.5%, respectively, and C18:0 (15.7 +/- 5% and 19.3 +/- 3.5%, respectively, means +/- SEM) as assessed by gas chromatography-electron capture detection. The inhibitor of protein kinase C, D-sphingosine, markedly decreased paf and lyso paf production and acetyltransferase activation in PMA- as well as OPZ-stimulated neutrophils. These results strongly suggest the involvement of protein kinase C in signal transduction during cell stimulation, leading to the paf biosynthesis.

Rat adherent macrophages (M phi) isolated from 2.5 x 10(6) peritoneal cells release 5.7 +/- 1.3 ng/ml platelet-activating factor (PAF-acether: 1-O-alkyl-2-acetyl-sn-glyceryl-3-phosphorylcholine) when stimulated by zymosan (Z). The yield of PAF-acether was increased to 13.8 +/- 1.8 ng/ml when stimulation occurred in the presence of 0.1 mM acetyl-CoA. That M phi used the acetyl moiety of acetyl-CoA to increase PAF-acether biosynthesis was demonstrated by the following criteria 1) as opposed to sodium acetate, malonyl-CoA, butyryl-CoA, palmitoyl-CoA, and CoA were inefficient in increasing the yield of PAF-acether; 2) radiolabeled PAF-acether was obtained after exposure of Z-stimulated M phi to 3H acetyl-CoA; actually, both radioactivity and biologic activity were eluted from HPLC with the same retention time as synthetic or natural PAF-acether; 3) after treatment with phospholipase A2, both radioactivity and biologic activity disappeared from these fractions. The yield of PAF-acether was further increased to 23.6 +/- 2.9 ng/ml by adding 0.5 microM totally synthetic 2-lyso PAF-acether (1-O-octadecyl-sn-glyceryl-3-phosphorylcholine) to Z-stimulated M phi cultured in the presence of acetyl-CoA. Again, using 3H 2-lyso PAF-acether, the formation of radiolabeled PAF-acether could be demonstrated. These results indicate that an acetyl-transferase is capable of synthesizing PAF-acether from 2-lyso PAF-acether and acetyl-CoA in intact M phi.

Human polymorphonuclear neutrophils, monocytes, and lymphocytes were studied for their ability to synthesize Paf-acether when stimulated with the ionophore A 23187 (Io) or with specific secretagogues. When stimulated with Io, neutrophils produced 100 +/- 8.5 pmol Paf-acether 1 X 10(6) cells (mean +/- 1 SD, n = 5); monocytes were less efficient (44 +/- 3.3 pmol Paf-acether/1 X 10(6) cells), whereas lymphocytes were practically unable to form this mediator (1.0 +/- 0.4 pmol Paf-acether/1 X 10(6) cells). Neutrophils and monocytes released in the extracellular medium 49 and 37% of Paf-acether that they formed, respectively. We attempted to correlate the amount of Paf-acether produced by the various cell types with that of its precursors, 1-O-alkyl-2-acyl-sn-glycero-3-phosphocholine and 1-O-alkyl-sn-glycero-3-phosphocholine (2-lyso Paf-acether). In the three cell types, the amount of 1-O-alkyl-2-acyl-sn-glycero-3-phosphocholine was sufficient to ensure the formation of 2-lyso Paf-acether and consequently that of Paf-acether. The quantity of 2-lyso Paf-acether formed appeared to be the limiting factor only in the case of the neutrophils. These cells increased their synthesis of Paf-acether in the presence of exogenous 2-lyso Paf-acether. To investigate the failure of lymphocytes to produce the mediator, the acetylating step of Paf-acether formation was studied, and we found a very weak activity (0.5 +/- 0.1 nmol Paf-acether/10 min/mg protein) in this cell type as opposed to monocytes (4.0 +/- 2.3 nmol Paf-acether/10 min/mg protein) and neutrophils (17.8 +/- 5.3 nmol Paf-acether/10 min/mg protein). These activities were doubled in Io-stimulated cells. Thus, the modulation of acetyl-transferase activity appears to be a key step in the regulation of Paf-acether biosynthesis. Also, the availability of 2-lyso Paf-acether could regulate Paf-acether synthesis in human neutrophils.

Activated peritoneal macrophages (M phi) from mice injected with Bacilli Calmette-Guérin, trehalose dimycolate, a defined immunostimulant derived from Mycobacterium tuberculosis, or streptococci C74 (St), synthesized two to three times less paf-acether (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) in response to a zymosan challenge than did (0.7 +/- 0.2 nmol/mg protein) resident-M phi (R-M phi). To assess at which step the paf-acether biosynthetic pathway was impaired, the content in phospholipid paf-acether precursors was evaluated. The alkyl-acyl-glycerophosphocholine content was comparable in R-M phi (50.4 +/- 18.6 nmol/mg protein) and activated-M phi (40.0 +/- 6.5 to 63.9 +/- 7.7 nmol/mg protein), as well as the lyso paf-acether content (0.85 +/- 0.18 nmol/mg protein for R-M phi vs 0.63 +/- 0.16 to 1.23 +/- 0.21 nmol/mg protein for activated macrophages). The nonlimiting rate of the phospholipid substrate was strengthened by experiments showing that incubation of the various populations with lyso paf-acether did not yield increased amounts of paf-acether. Similarly, incubation of R-M phi or St-M phi with sodium acetate increased paf-acether production to the same extent in both populations, ruling out the acetate substrate deficiency as the cause of the impaired production. The level of acetyltransferase activity, the enzyme that transfers an acetate moiety of acetyl coenzyme A (acetyl-CoA) onto lyso paf-acether, was very low in thioglycolate (TG)-elicited M phi but high in R-M phi and activated ones. In all cases, it was increased by two to three times upon zymosan challenge. This suggested that increased paf-acether catabolism and not impaired anabolism could be responsible for the marked reduced formation noted in activated macrophages. The addition of acetyl-CoA (200 microM) to the various macrophage monolayers restored paf-acether formation by activated cells to R-M phi values but with delayed kinetics as compared with paf-acether formation induced by zymosan. The enhancing effect of acetyl-CoA on paf-acether production was inhibited upon oleyl-CoA addition, suggesting that acetyl-CoA may increase paf-acether production by preventing the reacylation of lyso paf-acether resulting from paf-acether degradation. In conclusion, the paf-acether output in some inflammatory macrophages may be regulated by the level of acetyltransferase activity, because it is observed in TG-M phi. However, we present the first evidence for another mechanism of regulation, most probably related to the deacylation/reacylation of paf-acether precursors and metabolites.

Paf-acether or platelet-activating factor (1-0-alkyl-2-acetyl-sn-glycero-3-phosphocholine) is a phospholipid mediator of inflammation initially described as a potent platelet-aggregating compound. It is newly formed by a variety of cells including monocytes and is now recognized as a major mediator of cell-cell interactions. The present studies were undertaken to determine whether paf-acether could modulate T cell function. We found that addition of paf-acether to CD4+ cells cultured with phytohemagglutinin markedly inhibited the proliferative response in a dose-dependent manner. Maximal inhibition occurred when paf-acether was present during the first 24 hr of cell culture and the presence of paf-acether did not alter the kinetics of CD4+ cell proliferation. Importantly, the mechanism by which paf-acether inhibited the proliferative response was not related to inhibition of interleukin 2 (IL-2) secretion since the amount of IL-2 in cultures was not altered and addition of exogenous IL-2 failed to restore the CD4+ cell proliferative response. Further, as judged by indirect immunofluorescence, paf-acether did not inhibit IL-2 receptor expression. Taken together, these data indicate that paf-acether interferes with some processes leading to CD4+ cell proliferation. This new role for the chemically defined monokine paf-acether emphasizes the potential role of inflammatory lipid mediators in the regulation of T cell response.

Mouse bone marrow-derived mast cells passively sensitized with monoclonal IgE released paf-acether (platelet-activating factor) and beta-hexosaminidase when challenged with the specific antigen. The formation and the release of paf-acether followed an early increase in the activity of the acetyltransferase, the main enzyme in paf-acether biosynthesis. The antigen-induced activation of the acetyltransferase was dependent on physiologic temperature and on the presence of Ca2+. By using microsomal fractions from unchallenged and challenged mast cells, the Vmax values were 3.5 and 12.0 nmol/min/mg of protein, respectively, whereas in both cases a Km value for acetyl-coenzyme A of 172 microM was measured. The stimulation of acetyltransferase could be mimicked in vitro under experimental conditions which favor phosphorylation, i.e. adding ATP and Mg2+ to lysates from unchallenged mast cells. In contrast, ATP and Mg2+ were uneffective on lysates from challenged cells that exhibited high level of acetyltransferase activity, suggesting that phosphorylation of the enzyme already took place at the time of cell stimulation. Moreover, addition of alkaline phosphatase to microsomal fraction obtained from either antigen-challenged mouse bone marrow-derived mast cells or unchallenged cells, resulted in 52% and 43% loss of acetyltransferase activity, respectively. Phorbol myristate acetate treatment of cells doubled the enzyme activity supporting the phosphorylation hypothesis. Thus, we report on the immunologic activation of a key enzyme for paf-acether synthesis and on the mechanism of this activation in a pure mast cell population. A link between bridging of IgE receptors and the activation of an enzyme critical to the formation of a lipid mediator is thereby evidenced.

Addition of 1 microM dexamethasone (DM) to bone marrow-derived mast cells (BMMC) induced a time-dependent increase in cell histamine content. The latter reached a plateau of 2.5 micrograms/1 x 10(6) cells after 11 days in culture, compared with 100 ng/1 x 10(6) for untreated BMMC. Steroids, such as beta-estradiol, androsterone, and testosterone (1 microM), did not alter the histamine content of BMMC, whereas progesterone (1 microM) induced a moderate increase. Other glucocorticosteroids also enhanced histamine content, suggesting that the observed increase was specific for glucocorticosteroid. Treatment of BMMC with 1 microM DM for 14 days inhibited the Ag-induced, IgE-mediated release of histamine, beta-hexosaminidase, platelet-activating factor-acether, LTB4, and LTC4 by 65 +/- 3%, 66 +/- 1%, 93 +/- 3%, 66 +/- 2%, and 74 +/- 10%, respectively (mean +/- 1 SD, n = 3). In contrast with untreated cells which produce less than 2 ng/1 x 10(6) cells PGD2 after Ag challenge, DM-treated BMMC generated 16.8 +/- 0.3 ng/1 x 10(6) cells PGD2. Moreover, most of DM-treated BMMC became Alcian blue+/safranin+ and by ultrastructure, exhibited numerous cytoplasmic granules filled with abundant and uniform electron-dense matrix. The present results indicate that DM-treated BMMC exhibit biochemical and functional properties different from immature untreated cells, suggesting that a maturation-like process occurred in vitro during DM treatment.

The study of immunoglobulin levels of intact, immunized and/or contaminated axenic mice showed that: 1) axenic mice had lower levels of serum IgG2 and IgG1 than did holoxenic mice, but both immunoglobulin levels rose sharply after immunization or contamination of the former. In contrast, serum IgA remained undetectable in axenic mice after immunization and at 21 days after contamination; 2) serum IgA appeared about 2 months after massive contamination of axenic mice or 6 days after administration of prednisolone to them. The effect of prednisolone on serum IgA seemed to be related to passage of IgA from the intestinal system into the blood without true activation of the serum system.