Influence of tyrosine phosphorylation on protein interaction with FcgammaRIIa.

The cytoplasmic tail of Fc(gamma)RIIa present on human neutrophils shares with other antigen receptors a common amino acid sequence called ITAM (Immunoreceptor Tyrosine-based Activation Motif). After receptor ligation, the tyrosine residues within this motif become phosphorylated. We prepared a recombinant fusion protein of the cytoplasmic tail of Fc(gamma)RIIa (containing the ITAM) with glutathione-S-Transferase (GST-CT) to characterize the phosphorylation of Fc(gamma)RIIa and its ability to interact with other proteins involved in signal transduction. The GST-CT became phosphorylated in the presence of Lyn, Hck and Syk (immunoprecipitated from human neutrophils), but not in the presence of Fgr. Of the active kinases, only Lyn (mainly present in the membrane fraction) was found to associate with the GST-CT in the absence of ATP. This association was also observed in immunoprecipitates of Fc(gamma)RIIa from resting neutrophils, suggesting that Lyn might be the kinase responsible for the initial Fc(gamma)RIIa phosphorylation. Moreover, we observed specific association of Syk and the p85 subunit of PI 3-kinase after incubation of the GST-CT with neutrophil cytosol. This interaction was dependent on tyrosine phosphorylation of the GST-CT. Substitution of 269Tyr by Phe almost completely abolished tyrosine phosphorylation of the fusion protein. Substitution of either 253Tyr or 269Tyr eliminated Syk binding, but only 253Tyr appeared to be essential for p85 binding. We hypothesize that, upon activation, the membrane-associated Lyn is responsible for the initial tyrosine phosphorylation of Fc(gamma)RIIa, thus creating a docking site for Syk and PI 3-kinase.

The anti-Fc gamma RIII mAb 3G8 induces neutrophil activation via a cooperative actin of Fc gamma RIIIb and Fc gamma RIIa.

Human neutrophils express two types of Fc gamma receptors, the transmembrane Fc gamma RIIa and the glycan-phosphatidylinositol-anchored Fc gamma RIIIb, that show synergism in provoking a cellular response. To analyse further the requirements for this synergism to occur we used the monoclonal antibody 3G8, directed against Fc gamma RIII. This antibody is able to induce neutrophil activation, as measured by an increase in the intracellular free Ca2+ concentration and homotypic neutrophil aggregation, but only when the Fc part of the antibody is able to interact with Fc gamma RIIa. We observed that binding of the Fab parts of 3G8 mAb to two Fc gamma RIIIb molecules and binding of the Fc part to one Fc gamma RIIa molecule is required, because a bispecific antibody, 2B1, in which only one 3G8 Fab is present, did not induce neutrophil activation. Moreover, engagement of one Fc gamma RIIa molecule and two Fc gamma RIIb molecules on the same cell is instrumental to achieve activation of the mAb 3G8. The activation of neutrophils by the 3G8 antibody represents a further example of synergistic activation of neutrophils via Fc gamma receptors.

Loss of thrombin-induced Ca2+ mobilization in a subpopulation of platelets during storage.

Thrombin-induced changes in cytosolic free Ca2+ ([Ca2+]i) were studied in human platelets that had been stored for up to 6 days. Changes in [Ca2+]i were measured with Indo-1-loaded platelets and quantitated with two different methods: (i) measurement of the changes in total fluorescence; (ii) measurement of the [Ca2+]i changes in individual platelets in a flow cytometer, allowing the detection of non-responding platelets. The maximal concentration of [Ca2+]i after stimulation with 0.5 U of thrombin/ml decreased from 544 +/- 58 nM (mean +/- SEM, n = 6) on day 0, to 276 +/- 9 nM on day 3 and to 203 +/- 23 nM on day 6. The percentage of platelets responding to 0.5 U of thrombin/ml declined from 90 +/- 2% on day 0 to 72 +/- 4% on day 3, and to 47 +/- 8% on day 6. Nevertheless, also the responding platelets showed a decreased rise in [Ca2+]i. The study shows that during platelet storage a decrease in the rise in [Ca2+]i upon thrombin stimulation occurs. This decrease is partly due to the formation of a subpopulation of platelets that is completely unresponsive and partly due to a decreased responsiveness in the remainder of the platelets; it is not due to a gradual decline in [Ca2+]i rise in all platelets. This phenomenon provides new insight in the functional defect of stored platelets.

The platelet adhesion capacity to subendothelial matrix and collagen in a flow model during storage of platelet concentrates for 7 days.

The influence of storage of platelet concentrates (PC) on the adhesion capacity of platelets was studied. Twenty-four PC, 12 prepared by the buffy coat (BC) method and 12 by the platelet-rich plasma (PRP) method, were stored for 7 days at room temperature. On days 1, 3 and 7 of storage, the platelet adhesion capacity to subendothelial matrix (SEM) and collagen was studied in a rectangular perfusion system under flow conditions in conjunction with the platelet aggregation capacity after stimulation and the adenine nucleotide content. The platelet adhesion capacity to collagen was constant until day 3 of storage and decreased to about 80% of the starting value on day 7 of storage. The adhesion capacity to SEM, however, had already decreased on day 3 to about 75% of the value of day 1 and was even more decreased on day 7 to about 45% of the starting value. On day 1, platelets prepared by the BC method displayed a higher adhesion capacity to collagen and a higher aggregation capacity after stimulation by collagen alone or in combination with ADP, compared to platelets prepared by the PRP method. No other significant differences in adhesion or aggregation capacity were observed between the PC prepared by the two different methods. Both platelet adhesion and aggregation response decreased during storage, as did the total adenine nucleotide content. This study shows that platelet function, as measured by the aggregation and adhesion capacity, of platelets prepared by the PRP method is more severely impaired during the first 3 days of storage as compared to the function of platelets prepared by the BC method.

Stored platelets release nucleotides as inhibitors of platelet function.

It is well known that the function of platelets decreases progressively during storage of platelet concentrates at room temperature. To investigate this phenomenon in more detail, we have resuspended platelets that had been stored for 24 h or 72 h in fresh plasma, and we have measured the aggregation response and the ATP secretion. Conversely, the effect of plasma in which platelet concentrates (PC) had been stored for 24 h or 72 h, was tested on fresh platelets. Both the aggregation response to collagen and ADP and the collagen-induced ATP secretion of stored platelets partially recovered after incubation with fresh plasma (p < 0.05). The same parameters measured with fresh platelets incubated in stored PC-plasma were found to be significantly reduced in comparison with the response of fresh platelets in fresh plasma (p < 0.05). Finally, platelets were stored in a plasma-free medium, suitable for platelet storage and the supernatant was tested. This supernatant inhibited the function of fresh platelets in a storage time-dependent fashion. Boiling of these supernatants did not change the inhibiting capacities, whereas filtration over active charcoal did. Analysis of this supernatant revealed AMP and diadenosine tetraphosphate, which both inhibit platelet function. These data show that stored platelets release nucleotides that inhibit platelet function in a reversible manner. This phenomenon may contribute to the decrease of platelet function during storage and the recovery of platelet function after transfusion.

Apoptosis of neutrophils.

Neutrophils are the most abundant leukocytes and serve as a first line of defense against infectious microorganisms. For this purpose, neutrophils contain granules filled with proteolytic and other cytotoxic enzymes. Neutrophils have the shortest lifespan of all leukocytes. To prevent senescent neutrophils from releasing their toxic contents into the surrounding tissues, these cells become apoptotic and are then internalized by tissue macrophages. Recent studies have revealed more details about effects of cytokines on neutrophil apoptosis and on the uptake of apoptotic neutrophils by macrophages. In addition, the intracellular events leading to apoptosis are slowly being unraveled.

In vitro evaluation of buffy-coat-derived platelet concentrates stored in a synthetic medium.

Human blood platelets, prepared by the buffy-coat method, were prepared and stored in different synthetic media. In a synthetic medium based on gluconate, acetate and citrate (GAC), the pH was 6.8 on day 6. This medium was chosen for further evaluation. The total platelet count and the leukocyte contamination were significantly lower in the platelet concentrates (PCs) prepared in GAC compared to PCs prepared in plasma. Platelets stored in plasma or in GAC were equally functional when tested for aggregation and adenosine triphosphate (ATP) secretion. Only stimuli that act through the arachidonic-acid pathway induced a lower platelet response in GAC. Platelet morphology was quantified by measuring the difference in light transmission during stirring at different rates in an aggregometer; no significant differences for platelets stored in GAC as compared to plasma were observed. Activation of platelets was measured by binding of monoclonal antibodies (McAb) against the Gp IIb/IIIa complex and against activation-dependent antigens (GMP 140 from the alpha-granules and a 53-kD glycoprotein from the lysosomal granules). There was no difference in binding of these McAb between platelets prepared and stored in plasma or GAC. We conclude that platelets prepared by the buffy-coat method and stored in GAC have the same in vitro qualities as platelets stored in plasma, except for the lower aggregation response by the arachidonic-acid pathway. This is probably due to an acetate-induced decrease in intracellular pH.

Prevalence of Streptococcus sobrinus in relation to dental caries in children from Iceland and The Netherlands.

The aim of this study was to investigate the presence of S. sobrinus and S. mutans in specimens of dental plaque and saliva of children five years of age in Reykjavik, Iceland (study 1) and in samples of dental plaque from children nine years of age in Amsterdam, The Netherlands (study 2). The immuneblotting technique (IBT) was a suitable method to evaluate the presence and numbers of S. mutans and S. sobrinus in human dental plaque and saliva. In study 1, eighty-four children were evaluated bacteriologically; of these, 73 percent harbored mutans streptococci in their plaque or saliva. S. sobrinus similarly was present in 29 percent of the children. In study 2 (seventy-two children), the corresponding percentages were 81 percent for S. mutans, and 35 percent for S. sobrinus. The latter was detected in 6 percent of the plaque samples exclusive of S. mutans.

Differences in cariogenicity between fresh isolates of Streptococcus sobrinus and Streptococcus mutans.

Streptococcus sobrinus is known to possess cariogenic properties in vitro. It can produce acid in large amounts and it has the capacity to adhere to enamel and other surfaces. However, most studies on cariogenicity have been performed with laboratory strains that have been subcultured over long periods of time. Therefore, the cariogenicity and acidogenicity of 9 fresh isolates of both S. sobrinus and Streptococcus mutans from human dental plaque were compared. The bacteria were inoculated into the oral cavity of rats. The rats were fed diet SSP 20/5, containing 20% sucrose and 5% glucose. After the experimental period of 42 days, the amount of caries was assessed and bacterial counts were determined using monoclonal antibodies. Four out of 9 S. sobrinus strains and 3 out of 9 S. mutans strains did not colonize the rats. Colonizing strains constituted 39-78% of the total anaerobic cultivable microflora. The numbers of advanced dentinal lesions in the fissures of the rats colonized with S. mutans were significantly lower than those colonized with S. sobrinus (p less than 0.05). S. sobrinus produced acid more rapidly than S. mutans in a pH-stat system at pH values between 6.5 and 5.0 (p less than 0.01). The results indicate that fresh isolates of S. sobrinus are more cariogenic in rats than fresh isolates of S. mutans. This is possibly due to differences in glycolytic properties of these two species.

Tyrosine phosphorylation-dependent activation of phosphatidylinositide 3-kinase occurs upstream of Ca2+-signalling induced by Fcgamma receptor cross-linking in human neutrophils.

The effect of wortmannin on IgG-receptor (FcgammaR)-mediated stimulation of human neutrophils was investigated. The Ca2+ influx induced by clustering of both Fcgamma receptors was inhibited by wortmannin, as was the release of Ca2+ from intracellular stores. Wortmannin also inhibited, with the same efficacy, the accumulation of Ins(1,4,5)P3 observed after FcgammaR stimulation, but did not affect the increase in Ins(1,4,5)P3 induced by the chemotactic peptide, formyl-methionine-leucine-phenylalanine. Because wortmannin is, in the concentrations used here, an inhibitor of PtdIns 3-kinase, these results suggested a role for PtdIns 3-kinase upstream of Ca2+ signalling, induced by FcgammaR cross-linking. Support for this notion was obtained by investigating the effect of another inhibitor of PtdIns 3-kinase, LY 294002, and by studying the kinetics of PtdIns 3-kinase activation. We found translocation of PtdIns 3-kinase to the plasma membrane and increased PtdIns 3-kinase activity in the membrane as soon as 5 s after FcgammaR cross-linking, even before the onset of the Ca2+ response. Moreover, the translocation of PtdIns 3-kinase to the plasma membrane was inhibited by co-cross-linking of either FcgammaRIIa and FcgammaRIIIb with the tyrosine phosphatase, CD45, indicating a requirement for protein tyrosine phosphorylation in the recruitment of PtdIns 3-kinase to the plasma membrane. Taken together, our results suggest a role for PtdIns 3-kinase in early signal transduction events after FcgammaR cross-linking in human neutrophils.

Regulation and kinetics of platelet-activating factor and leukotriene C4 synthesis by activated human basophils.

BACKGROUND: Allergic disease is the result of an interplay of many different cell types, including basophils and mast cells, in combination with various inflammatory lipid mediators, such as platelet-activating factor (PAF) and leukotrienes (LT). LTC4 synthesis by human basophils has been studied quite extensively. However, not much is known about the synthesis of PAF by human basophils. OBJECTIVE: In this study, we have made a comprehensive comparison between the kinetics of PAF and LTC4 synthesis, in highly purified basophils, activated with different stimuli or with combinations of stimuli. METHODS: Synthesis of PAF and LTC4 by human basophils was determined with commercially available assay kits. The basophils were activated with C5a, fMLP, PMA, allergen or anti-IgE, in the absence and presence of IL-3 and/or in combination with elevation of cytosolic free Ca2+ by the sarcoplasmic reticulum Ca2+-ATPase inhibitor thapsigargin. RESULTS: Most stimuli were found to induce both PAF and LTC4 synthesis. PAF synthesis and LTC4 release were enhanced by preincubation of the basophils with IL-3 or by elevation of cytosolic free Ca2+ by thapsigargin. Incubation of human basophils with IL-3 alone or thapsigargin alone did not result in detectable synthesis of PAF and LTC4, whereas the combination of the two resulted in high amounts of PAF and LTC4 synthesis. Depending on the stimulus used, LTC4 release was 5-100-fold higher than PAF synthesis. In addition, PAF, but not LTC4, was transiently detected, probably due to PAF degradation. LTC4 and PAF synthesis was strongly blocked by inhibitors of cytosolic phospholipase A2, indicating that this enzyme is involved in PAF and LTC4 synthesis by activated human basophils. CONCLUSION: This study provides a first comprehensive comparison of PAF and LTC4 synthesis in highly purified human basophils, stimulated with a variety of stimuli.

Human neutrophils lose their surface Fc gamma RIII and acquire Annexin V binding sites during apoptosis in vitro.

We have previously reported that neutrophilic granulocytes rapidly release part of their Fc gamma RIII from the plasma membrane upon in vitro activation, probably by proteolytic cleavage. In plasma and other body fluids, released or soluble Fc gamma RIII has been found in considerable amounts. In the present study, neutrophils were kept in maintenance culture for 18 to 24 hours. Forty percent of the neutrophils completely lost Fc gamma RIII, and the remainder of the cells showed a 60% decrease in Fc gamma RIII expression on their surface. Released Fc gamma RIII was detected in the culture supernatant. Nevertheless, more than 90% of the cells was viable as judged by hydrolysis of fluorescein diacetate. The presence of interferon gamma, granulocyte colony-stimulating factor, or granulocyte-macrophage colony-stimulating factor, but not interleukin-3 (IL-3), IL-6, or IL-8, in the culture medium increased the number of cells that still expressed Fc gamma RIII. We found that this loss of Fc gamma RIII was not the result of cell activation but correlated strongly with apoptosis. The Fc gamma RIII-negative subpopulation exhibited typical morphologic changes, such as nuclear condensation and DNA fragmentation. Furthermore, this subpopulation appeared to have acquired the property of binding Annexin V, a calcium-dependent, phospholipid-binding protein with high affinity for phosphatidylserine. The external exposure of this phospholipid by cells has been reported to occur during apoptosis. The property of Annexin V binding was not shared by the nonapoptotic, Fc gamma RIII-positive subpopulation. In this respect, we identified binding of Annexin V as an convenient marker for apoptotic cells. Our results indicate that soluble Fc gamma RIII in body fluids might be derived for a large part from neutrophils undergoing apoptosis in the tissues.