The tyrosine kinase Hck is an inhibitor of HIV-1 replication counteracted by the viral vif protein.

The virus infectivity factor (Vif) protein facilitates the replication of human immunodeficiency virus type 1 (HIV-1) in primary lymphocytes and macrophages. Its action is strongly dependent on the cellular environment, and it has been proposed that the Vif protein counteracts cellular activities that would otherwise limit HIV-1 replication. Using a glutathione S-transferase pull-down assay, we identified that Vif binds specifically to the Src homology 3 domain of Hck, a tyrosine kinase from the Src family. The interaction between Vif and the full-length Hck was further assessed by co-precipitation assays in vitro and in human cells. The Vif protein repressed the kinase activity of Hck and was not itself a substrate for Hck phosphorylation. Within one single replication cycle of HIV-1, Hck was able to inhibit the production and the infectivity of vif-deleted virus but not that of wild-type virus. Accordingly, HIV-1 vif- replication was delayed in Jurkat T cell clones stably expressing Hck. Our data demonstrate that Hck controls negatively HIV-1 replication and that this inhibition is suppressed by the expression of Vif. Hck, which is present in monocyte-macrophage cells, represents the first identified cellular inhibitor of HIV-1 replication overcome by Vif.

Characterization of human immunodeficiency virus type 1 vif gene in long-term asymptomatic individuals.

We have determined the sequence of the human immunodeficiency virus type 1 (HIV-1) vif genes from a cohort of 42 long-term nonprogressors (LTNP) and compared these sequences to those of 8 late progressors. The coding potential of the vif open reading frame directly derived by nested PCR from uncultured peripheral blood mononuclear cell DNA was conserved in all 50 individuals. The nucleotide distances between vif sequences were not significantly different between LTNP and late progressors, indicating similar selections of viruses within both types of long-term HIV-1-infected subjects. However, a statistically significant correlation between an amino acid signature at position 132 of Vif and the viral load was found within LTNP. Namely, amino acid Ser was associated with low viral load and amino acid Arg with high viral load. This signature was also observed when LTNP with low viral load were compared to progressors. The Ser132 signature was introduced in place of Arg132 present in the HIV-1 YU-2 Vif prototype into chimeric viruses to assess the impact of Vif signature on the virus. While the replication properties in the SupT1 cell line were unmodified, the mutagenized virus revealed a fivefold decreased replication in activated PBMC, suggesting a possible role of this Vif signature for viral production in vivo.

A large subset of neutrophils expressing membrane proteinase 3 is a risk factor for vasculitis and rheumatoid arthritis.

It has been shown previously that proteinase 3 (PR3), a neutrophil intracellular protease that is the main antigen of antineutrophil cytoplasm (ANCA) autoantibodies, is present on the plasma membrane of a subset of freshly isolated neutrophils. This study shows that the size of this subset of membrane PR3-positive (mPR3+) neutrophils is a stable feature of a given individual, most likely genetically controlled. It ranges from 0 to 100% of neutrophils and allows us to define a new polymorphism in the healthy population, with three discrete phenotypes corresponding respectively to less than 20% mPR3 + neutrophils (mPR3low) or to a mean percentage of 47% (mPR3intermediate) and 71.5% (mPR3high) mPR3+ neutrophils. The frequency of the mPR3high phenotype was significantly increased in patients with ANCA-associated vasculitis (85% versus 55% in healthy subjects). The percentage of mPR3+ neutrophils was not affected by disease activity, relapses, or therapy, and did not reflect in vivo cell activation. In addition, mPR3+ phenotypes were normally distributed in cystic fibrosis patients, indicating that infection and/or inflammation per se do not lead to a high percentage of mPR3+ neutrophils. The frequency of the mPR3high phenotype was not related to anti-PR3 autoimmunization, since it was increased in vasculitic patients regardless of the ANCA specificity (anti-PR3, anti-myeloperoxidase, or unknown). Interestingly, the frequency of the mPR3high phenotype was also increased in patients with rheumatoid arthritis. It was normal in type I-diabetes, a T cell-dependent autoimmune disease. It is proposed here that a high proportion of membrane PR3-positive neutrophils could favor the occurrence or the progression of chronic inflammatory diseases.

Priming of blood neutrophils in children with cystic fibrosis: correlation between functional and phenotypic expression of opsonin receptors before and after platelet-activating factor priming.

Blood phagocyte opsonin receptor CR1 (CD35) and CR3 (CD11b) functions were examined in cystic fibrosis (CF) patients with endobronchial Staphylococcus aureus or Pseudomonas aeruginosa chronic infection, CF patients without infection, heterozygous, non-CF patients with chronic pulmonary infection, and healthy controls. Circulating and platelet-activating factor (PAF)-primed phagocyte luminol luminescence responses to complement-opsonized zymosan were increased in both groups of infected CF and non-CF children relative to uninfected CF children and healthy control children and adults. The ratio between circulating and PAF-primed phagocyte responses was significantly elevated in all children with CF, and in these, the ratio could serve as an indicator of response to antibiotic treatment. The ratios of circulating and PAF-primed phenotypic expression for CR1, CR3, and FcgammaRIII (CD16), but not FcgammaRII (CD32), correlated with the functional ratios. Phagocyte opsonin receptor response capacity might be used for evaluation of inflammation and infection in CF patients.

Role of alpha v integrins in mesangial cell adhesion to vitronectin and von Willebrand factor.

This study demonstrates (by flow cytometry and immunoprecipitation after cell surface radiolabeling and by using monoclonal antibodies to alpha v, beta 3, and alpha v beta 3 and alpha v beta 5 complexes) that alpha v beta 3, the vitronectin receptor, and alpha v beta 5 are expressed in vitro on cultured human mesangial cells (HMC) of the 5th to 8th passages. Antibodies to alpha v, beta 3 and alpha v beta 3 respectively precipitated an alpha beta heterodimer with molecular weights of 140 and 97 kDa. We analyzed the role of the various integrins in HMC interactions with vitronectin, and with fibronectin and von Willebrand factor (vWf), which are synthetized respectively by mesangial and endothelial cells. Cell adhesion increased in a dose dependent manner with the concentration of plastic-coated matrix protein and vWf. Inhibition of cell attachment with monoclonal antibodies to integrins indicated that HMC adhesion to vWf primarily involves alpha v beta 3, and that alpha v beta 5 may also contribute to cell binding to vWf. Adhesion to vitronectin involves both alpha v beta 3 and alpha v beta 5 complexes. In contrast, adhesion to fibronectin was not affected by monoclonal antibodies to alpha v beta 3 and alpha v beta 5 complexes. We propose that integrins alpha v beta 3 and alpha v beta 5, present on HMC, could mediate an interaction between mesangial and endothelial cells by binding to vWf, released at the basal site of endothelial cells.

Disturbed myeloperoxidase-dependent activity of neutrophils in cystic fibrosis homozygotes and heterozygotes, and its correction by amiloride.

The present study addresses the question of a possible linkage between the cystic fibrosis (CF) genetic autosomal recessive disorder and disturbance in neutrophil function. Neutrophil-dominated chronic airway inflammation is present at an early age in children with CF, even in the absence of detectable infection. As evidenced by extracellular superoxide anion release (measured by lucigenin luminescence) or intracellular hydrogen peroxide production (measured by 2',7'-dichlorofluorescein (DCF) fluorescence), no significant difference in the nicotinamide-adenine dinucleotide phosphate (NADPH) oxidase activity of isolated neutrophils was observed in noninfected CF children (homozygotes), their mothers or fathers (CF heterozygotes), and controls. In contrast, both myeloperoxidase (MPO)-dependent oxygenation activity (measured by luminol luminescence) and chloramine release were increased significantly in both CF homozygotes and heterozygotes as compared with controls. In the presence of either amiloride (a sodium channel inhibitor and sodium/proton antiport blocker) or EIPA (5-ethyl-N-isopropyl-amiloride, a specific inhibitor of the antiport), or choline buffer, intracellular MPO activity was decreased significantly in controls and in the CF homozygotes and heterozygotes, thus bringing intracellular MPO-dependent activity in CF subjects back to the level of controls. Extracellular release of MPO, measured by an ELISA to provide an activity-independent assessment of the enzyme, was increased only in CF homozygotes, and was decreased by amiloride and choline buffer, but not by EIPA. We conclude that a modification of intracellular pH and/or ionic concentrations may be related to the altered MPO enzymatic activity observed in CF neutrophils.

Inhibition of neutrophil-endothelial cell adhension by a neutrophil product, cathepsin G.

In the present study we investigated the modulation of the polymorphonuclear neutrophil (PMN)-endothelial cell adhesion process by the two main proteinases released from activated PMN during their adhesion to endothelium. Our results showed that, in contrast with elastase, cathepsin G was a powerful inhibitor of PAIN adhesion to interleukin-1 (IL-1)-treated human umbilical vein endothelial cells. This inhibitory effect was linked to the enzymatic activity of the proteinase and was selectively directed against PMN. Because the viability and the reactivity of PMN were not modified by cathepsin G, we looked for a possible effect on adhesion molecules. L-selectin was not cleaved by cathepsin G, whereas it was by chymotrypsin, a closely related proteinase. Cathepsin G blocked PMN adhesion to activated endothelial cells, but also to serum- or fibrinogen-coated plates, three adhesion processes mediated by CD11b/CD18. However, by FACScan analysis or by immunoprecipitation, we failed to find evidence of modifications of CD11b/CD18 expression. Although the precise molecular target(s) of cathepsin G remain(s) to be defined, these data indicate that this proteinase, which is known as an inflammatory mediator, can also be considered as a potential down-regulator of adhesion reactions involved in the inflammatory process.

Potential effect of metabolic acidosis on beta 2-microglobulin generation: in vivo and in vitro studies.

Beta 2-microglobulin (beta 2M) is responsible for dialysis-associated amyloidosis. Level of beta 2M in plasma increase during chronic renal failure; however, retention does not appear to be the sole mechanism responsible. The effect of metabolic acidosis on beta 2M production was examined. Thirty-six patients with stable chronic renal insufficiency, 12 uremic patients before their first dialysis, 8 hemodialysis patients who were assigned to acetate or bicarbonate dialysate and then crossed over to the alternative regimen, and 6 normal subjects given NH4Cl to initiate metabolic acidosis were studied. In vitro studies in the human myeloid cell line U 937 were also performed. beta 2M protein was measured with ELISA, beta 2M mRNA was measured with reverse transcription polymerase chain reaction, and the U 937 cells were studied at two pH levels with FACScan flow cytometry. The cells were exposed in vitro up to 60 min in a buffered incubation medium to either pH 5.10 or pH 7.34. An inverse correlation was found between beta 2M and bicarbonate concentrations in plasma in the stable chronic renal failure patients (r = -0.54; P < 0.05) and in the uremic patients before their first dialysis (r = -0.72; P < 0.05). In hemodialysis patients, blood pH and plasma bicarbonate values were lower (P < 0.05) and beta 2M concentrations in plasma were higher (P < 0.05) with acetate than with bicarbonate dialysate. In normal men, NH4Cl resulted in an increase (P < 0.05) in beta 2M mRNA expression in lymphocytes by an average factor of 1.5 (range, 1.1 to 1.8). In U 937 cells, the cell surface expression of beta 2M and HLA Class I heavy chain assembled with beta 2M decreased at low pH compared with normal pH. Concomitantly, an increase in beta 2M release into the supernatant was observed, possibly as the result of beta 2M dissociation from cell surface HLA Class I complex. The results suggest that metabolic acidosis may enhance cellular beta 2M generation and release.

Bimodal distribution of proteinase 3 (PR3) surface expression reflects a constitutive heterogeneity in the polymorphonuclear neutrophil pool.

Proteinase 3, which is known as an intracellular serine protease of neutrophils, was detected at the surface of a subpopulation of freshly isolated PMN. The proportion of PR3-positive and -negative PMN, observed by flow cytometry with anti-PR3 mAbs or ANCA autoantibodies, varies among individuals but is extremely stable for each individual over prolonged time periods. After PMN degranulation by FMLP with cyt. B, membrane PR3 expression increases but the proportion of low and high PR3-expressing cells remains stable. The existence of a subset of PMN which spontaneously expresses PR3 and varies among individuals, may be relevant to the pathogenesis of anti-PR3 ANCA autoantibody-related vasculitis.

Neutrophil expression of tumour necrosis factor receptors (TNF-R) and of activation markers (CD11b, CD43, CD63) in rheumatoid arthritis.

In vitro analysis of polymorphonuclear neutrophils (PMN) has allowed various stages of cell activation to be distinguished, characterized by the expression level of specific membrane markers and of functional receptors. Among those, TNF-alpha receptors (TNF-R) are modulated by various PMN activators, a mechanism which may be important to control cell responses to TNF in inflammatory reactions such as rheumatoid arthritis (RA). PMN, isolated from the blood of 36 RA patients and from the synovial fluid of 23 of them, were analysed for membrane expression of the two TNF-R (p55 and p75). Soluble p55 and p75 (sTNF-R) and TNF concentrations were measured in the plasma and synovial fluid by specific ELISA assays. Our results show that PMN from the blood of RA patients bear a normal number of TNF-R, with a normal p55/p75 ratio, compared with PMN from normal controls. Soluble TNF-R levels were similar in patients and normal plasma. In spite of high endogenous TNF concentration, patients' circulating PMN were not activated, as shown by a CD11b/CD18 expression similar to that of control resting cells. In contrast with blood neutrophils, PMN from RA patients' synovial fluids had an activated phenotype, characterized by increased expression of CD11b, decreased expression of leukosialin, CD43, and the appearance on the plasma membrane of an azurophil granule protein, CD63. High levels of soluble TNF-R were measured in RA synovial fluids. Nevertheless, membrane TNF-R levels and p55 and p75 proportions were similar to those of PMN from normal blood. These results suggest the existence of regulatory mechanisms which maintain a stable neutrophil expression of TNF-R as well as a balance between both types of receptors in inflammatory situations where neutrophils are strongly activated.

Human neutrophils release their major membrane sialoprotein, leukosialin (CD43), during cell activation.

Leukosialin (CD43) is a sialic acid-rich molecule with a relative molecular mass (M(r)) of 140,000 highly represented on polymorphonuclear neutrophils (PMN) and on most leukocytes. One of its functions may be to prevent nonspecific cell-to-cell interactions through negative charge repulsions. As tested by immunofluorescence, neutrophil CD43 membrane expression was shown to decrease by up to 80% upon cell activation by phorbol myristate acetate (10 ng/ml) or by N-formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP; 10(-6) M) in the presence of cytochalasin B. The kinetic of this decrease paralleled that of CD11b up-regulation. FMLP alone, tumor necrosis factor (TNF-alpha), lipopolysaccharide and granulocyte macrophage colony-stimulating factor had moderate or insignificant effects, while inducing striking CD11b up-regulation. Cell priming with TNF-alpha followed by FMLP stimulation resulted in up to 40% decrease of CD43 expression. Anti-CD43 mAb immunoprecipitated three fragments of M(r) 130,000, 49,000 and 34,000 from the cell-free supernatant of activated neutrophils, suggesting that CD43 is released from the membrane by proteolysis. Indeed, the decrease in CD43 expression was inhibited by phenylmethanesulfonylfluoride (PMSF). Homotypic aggregation of activated PMN was also inhibited by PMSF and could result, at least in part, from the shedding of CD43. The shedding of such a strongly anionic and major membrane protein should drastically modify PMN surface charge and may allow previously hindered interactions by exposing new adhesion molecules.

Biosynthesis of paf-acether. XVI. Acetyltransferase specificity determines composition of paf-acether molecular species in human polymorphonuclear neutrophils.

In human neutrophils, the velocity of the lyso paf-acether:acetyl-CoA acetyltransferase reaction was almost 2-fold higher in the presence of lyso paf-acether bearing a 16:0 alkyl chain at the sn-1 position of glycerol than in that of its 18:0 analog. The paf-acether produced from an equimolar mixture of the two substrates was a 5:1 mixture, respectively, of the 16:0 and 18:0 species. The ratio of 16:0/18:0 lyso paf-acether in microsomal fractions, as analyzed by gas chromatography, was close to 1, whereas the paf-acether formed in these fractions from endogenous phospholipids was nearly exclusively of the 16:0 form. We conclude that acetyltransferase possesses a higher affinity for 16:0 than for 18:0 lyso-PAF and thus might control the molecular composition of paf-acether synthesized by stimulated human polymorphonuclear neutrophils.

Biosynthesis of paf-acether. Activators of protein kinase C stimulate cultured mast cell acetyltransferase without stimulating paf-acether synthesis.

Antigen stimulation of cultured bone-marrow-derived mast cells sensitized with specific monoclonal IgE induced cell degranulation and paf-acether (paf; 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) biosynthesis via the deacylation/acetylation (remodelling) pathway. Phorbol myristate acetate (PMA; 20-100 ng/ml) triggered only acetyltransferase activation, without concomitant lyso-paf (1-O-alkyl-sn-glycero-3-phosphocholine) and paf formation. A low concentration of PMA (5 ng/ml) potentiated antigen-induced degranulation, acetyltransferase activation and paf formation by about 30% but did not change the level of lyso-paf formation. Stimulation of mast cells with antigen increased intracellular Ca2+ from 61 to 269 nM, whereas no modification of Ca2+ influx was observed when cells were pretreated with PMA (5 ng/ml) before antigen challenge. Gas chromatography coupled to electron capture detection revealed that the composition of paf formed by cells stimulated by antigen alone was similar to that of paf formed by PMA-primed antigen-stimulated cells; 84 +/- 8% and 79 +/- 2% (means +/- S.E.M., n = 3) of molecules respectively bore the C16:0 alkyl chain moiety, with the remainder bearing essentially C18:0 molecules. Overnight treatment of mast cells with PMA (200 ng/ml) caused disappearance of protein kinase C (PKC) from both cytosol and membranes. When such cells were stimulated further with antigen, they failed to degranulate, and acetyltransferase activation, paf production and lyso-paf production were decreased by 33 +/- 11%, 57 +/- 4% and 96 +/- 3% respectively (n = 3 or 5). The PKC inhibitors chlorpromazine and staurosporine inhibited to a significant extent both cell degranulation and all steps leading to paf biosynthesis. Our data suggest that PKC-dependent mechanisms are operational during cell degranulation and contribute only in part to paf biosynthesis. The PKC-dependent signal directly generated by PMA or diacylglycerol is not sufficient to trigger the full cell response, which is obtained only through receptor-operated antigen challenge.

Quantitation of paf-acether by release of endogenous platelet serotonin assessed by liquid chromatography with electrochemical detection.

A new method to quantitate paf-acether (paf) was developed. It is based on the measurement of serotonin released from washed rabbit platelets challenged with paf. Platelets (1 X 10(8)/ml) were exposed with or without stirring to various concentrations of paf (26-130 pM) at 37 degrees C or at room temperature. Supernatants were submitted to a 4-min liquid chromatography run and serotonin was measured by electrochemical detection. We quantitated paf from three different biological sources, human neutrophils, mouse peritoneal macrophages, and cultured mast cells, comparing a classical method, i.e., platelet aggregation with the electrochemical detection of endogenous serotonin. We found similar results since, when compared with the aggregation method, the results differed by 12 to 47%. The sensitivity of both methods was 26 pM. The between-day variation coefficient was 23 and 14% (n = 12) for the aggregation method and the serotonin release, respectively, whereas the within-day variation coefficient for serotonin quantitation was less than 5% (n = 12). The superiority of the new method lies in its simplicity, the economy of platelets, and its possibility of automation. It can be applied to any agonist or any mechanism capable of releasing serotonin from platelets and more generally when a simple and fast method for measuring serotonin is desirable.

Biosynthesis of paf-acether. X. Phorbol myristate acetate-induced paf-acether biosynthesis and acetyltransferase activation in human neutrophils.

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.

Biosynthesis of paf-acether. XI. Regulation of acetyltransferase by enzyme-substrate imbalance.

Acetyl-CoA:1-O-alkyl-sn-glycero-3-phosphocholine acetyltransferase is the key enzyme in paf-acether (paf) biosynthesis, since it yields the active mediator from its nonacetylated precursor, lyso-paf. In microsomal fractions obtained from the ionophore A23187-stimulated human polymorphonuclear neutrophils, the optimal conditions allowing the full acetylation of lyso-paf were: 2-2.5 mg.ml-1 bovine serum albumin, 40 microM lyso-paf, 200 microM acetyl-CoA and acetyltransferase of high specific activity, at least 18 nmol.min-1.mg protein- -1. The reaction frequently stopped before the substrate was consumed due to spontaneous decay of the enzyme activity at 37 degrees C and inhibition of the enzyme by the paf formed in the reaction. However, low concentrations of acetyltransferase substrates (lyso-paf or lysophosphatidylcholine) and the antioxidant dithiothreitol, but not the inhibitors of proteinases or phosphatases, protected the enzyme against decay. In contrast, high concentrations of those lyso substrates inhibited the enzyme activity in the assay. This inhibition as well as that due to paf was overcome by raising the concentration of the enzyme contained in the microsomal fraction or the bovine serum albumin in the assay. These results suggest that the biosynthesis of paf in cell-free assay and most probably in intact cells might be controlled to a larger extent by the acetyltransferase concentration rather than by that of its substrates.

Ketotifen inhibits paf-acether biosynthesis and beta-hexosaminidase release in mouse mast cells stimulated with antigen.

Acetyl-CoA acetyltransferase (1-O-alkyl-sn-glycero-3-phosphocholine) is a key enzyme in paf-acether biosynthesis. Its immunological activation as related to paf-acether formation was investigated in mast cells derived from mouse bone marrow. The action of ketotifen, a prophylactic anti-asthma drug, on the antigen-induced activation of acetyltransferase and on the release of paf-acether and beta-hexosaminidase was studied in mast cells. Mast cells were sensitized with dinitrophenyl-specific monoclonal IgE and preincubated for 15 min at 37 degrees C with various concentrations of ketotifen or vehicle prior to challenge with dinitrophenyl coupled to bovine serum albumin (40 ng/ml). Acetyltransferase activity and mediator formation and release were measured. Ketotifen inhibited dose dependently the antigen-induced paf-acether formation and release, beta-hexosaminidase release and acetyltransferase stimulation. The IC50 values were 20.0 +/- 4.4, 11.8 +/- 6.2, 8.8 +/- 3.8 and 20.5 +/- 3.4 microM (mean +/- S.E.M., n = 3) respectively. Mast cells were preincubated with 50 microM ketotifen for 15 min at 37 degrees C then washed prior to antigen challenge. The release of paf-acether and beta-hexosaminidase and the stimulation of acetyltransferase were inhibited by 90.0 +/- 15.0, 91.0 +/- 15.0 and 88.0 +/- 11.0% (n = 3) respectively. In addition, Ca2+ entry was inhibited by 100% as assessed from Quin-2 fluorescence. Thus, the release of a preformed granular enzyme beta-hexosaminidase is inhibited by ketotifen together with the enzymatic formation of a newly formed mediator.(ABSTRACT TRUNCATED AT 250 WORDS)

Biosynthesis of paf-acether. IX. Role for a phosphorylation-dependent activation of acetyltransferase in antigen-stimulated mouse mast cells.

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.

Decrease in IgE Fc receptor expression on mouse bone marrow-derived mast cells and inhibition of paf-acether formation and of beta-hexosaminidase release by dexamethasone.

The effect of dexamethasone (DM) on the immunologic and nonimmunologic release of paf-acether and of the granule marker beta-hexosaminidase (BHEX) from mouse bone marrow-derived mast cells (BMMC) was studied. BMMC (1 X 10(6] in a modified Tyrode's solution containing 0.25% bovine serum albumin (BSA) were sensitized with an optimal dose of dinitrophenyl (DNP)-specific monoclonal IgE, and were washed before challenge with 40 ng/ml of DNP coupled to BSA. Preincubation of BMMC for 24 hr with 1 nM to 1 microM DM inhibited in a dose-dependent fashion the immunologic release of paf-acether and of BHEX as compared with control cells, with a half-maximal effect at 20 nM and 4 nM respectively. By contrast, the ionophore A23187 (1 microM)-induced release of paf-acether and of BHEX was unaffected by DM pretreatment. Finally, the antigen-induced increase in acetyltransferase activity, used as an index of cellular activation, was inhibited by 37 +/- 16% in 1 microM DM-treated BMMC as compared with untreated cells. Preincubation of BMMC with DM for 24 hr caused a dose-dependent inhibition of 125I-IgE binding to the cells, with a half-maximal effect at 14 nM. As determined by Scatchard analysis, the number of IgE Fc receptors was decreased by 55% in 1 microM DM-treated BMMC as compared with untreated cells, although the dissociation constants were comparable (control: 12.6 +/- 4.1 nM; DM-treated cells: 14.1 +/- 6.7 nM; mean +/- 1 SD; n = 3). Cytofluorometer analysis of BMMC sensitized with a saturating amount of purified monoclonal IgE, followed by addition of a fluoresceinated anti-mouse IgG (heavy and light chains), revealed a single cellular population for both DM-treated and untreated BMMC. This demonstrates that the DM-induced decrease in IgE Fc receptor expression was exhibited by every BMMC. The possible link between the decreased sensitization of the cells consequent to the reduction in IgE Fc receptor expression and the alteration of the secretory response and acetyltransferase activity was investigated. BMMC were incubated with IgE under experimental conditions giving half-sensitization of the cells. Upon antigen challenge, a 10.5 +/- 3.7% decrease in acetyltransferase activity and a 29.2 +/- 3.5% decrease in paf-acether release were observed with half-sensitized cells as compared with cells sensitized with a saturating amount of IgE.(ABSTRACT TRUNCATED AT 400 WORDS)