Cooperation between Fc gamma receptor II and complement receptor type 3 during activation of platelet-activating factor release by cytokine-primed human eosinophils.

After priming with cytokines, such as granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-3, or IL-5, eosinophils are stimulated potently by opsonized particles like serum-treated zymosan (STZ), resulting in activation of the respiratory burst and production of lipid mediators, such as platelet-activating factor (PAF) and leukotriene C4 (LTC4). In the present study, the role of the opsonin receptors Fc gamma RII and CR3 during both STZ-induced activation of the respiratory burst and PAF release by human eosinophils was investigated. Inhibition studies with blocking mAbs (alpha hFc gamma RII: AT10, IV.3; alpha CR3: B2.12, 44a) showed that both Fc gamma RII and CR3 are important for STZ-induced PAF release by cytokine-primed eosinophils. In contrast, CR3 is involved in activation of the respiratory burst, whereas Fc gamma RII seems not to be important, because blocking anti-Fc gamma RII mAbs had no effect. Subsequently, experiments were performed with zymosan particles coated with IgG, iC3b, or a combination of both. IgG-coated particles poorly activated both responses in GM-CSF primed and unprimed cells. iC3b-Zymosan activated the respiratory burst as well as zymosan expressing both opsonins (IgG/iC3b-zymosan). In contrast, iC3b-zymosan induced significantly less PAF release by GM-CSF-primed eosinophils than did IgG/iC3b-zymosan, suggesting synergism between Fc gamma RII and CR3. This synergistic effect was not observed when IgG-zymosan and iC3b-zymosan were added simultaneously. Therefore, these data indicate that on human eosinophils, Fc gamma RII and CR3 act synergistically to activate PAF release, provided that their ligands are in close proximity.

Granulocyte-macrophage colony-stimulating factor, interleukin-3 (IL-3), and IL-5 greatly enhance the interaction of human eosinophils with opsonized particles by changing the affinity of complement receptor type 3.

Eosinophil functions can be modulated by several cytokines such as granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3), and IL-5. We have investigated the modulatory role of these cytokines on the interaction of human eosinophils with opsonized particles (serum-treated zymosan [STZ]). Addition of STZ to eosinophils isolated from the peripheral blood of normal human donors resulted in an interaction of the STZ particles with only 15% to 25% of the cells. Treatment of the eosinophils with GM-CSF, IL-3, or IL-5 strongly enhanced both the rate of particle binding and the percentage of eosinophils binding STZ. The effect of the cytokines is most likely mediated by a change in affinity of the complement receptor type 3 (CR3) on the eosinophils for the complement fragment iC3b on the STZ particles. This is indicated by the observation that (1) the effect of the cytokines on STZ binding was prevented by a monoclonal antibody against the iC3b-binding site on CR3 and (2) the enhanced binding was already apparent before upregulation of CR3 on the cell surface was observed. In a previous study, similar results were obtained with platelet-activating factor (PAF)-primed eosinophils. Because we found that the cytokines strongly enhanced the STZ-induced PAF synthesis, we investigated the role of both released PAF and cell-associated PAF in the priming phenomenon by the cytokines. Cytokine priming appeared to be largely independent of the synthesis of PAF.

Transient exposure of human eosinophils to the protein kinase C inhibitors CGP39-360, CGP41-251, and CGP44-800 leads to priming of the respiratory burst induced by opsonized particles.

We report that a transient incubation of human eosinophils with the protein kinase C (PKC) inhibitor CGP39-360 (staurosporine) or the more PKC-specific inhibitors CGP41-251 and CGP44-800 prior to activation of the respiratory burst with opsonized particles results in priming of this response. This priming effect was concentration dependent and occurred in the range in which the phorbol myristate acetate-induced respiratory burst was inhibited. CGP39-360 priming was minimally affected in Ca(2+)-depleted cells, indicating that an increase in [Ca2+]i is not important. Also, the binding of serum-treated zymosan (STZ) particles was strongly enhanced by the inhibitors. On the other hand, the release of platelet-activating factor (PAF) induced by opsonized particles was enhanced only by CGP39-360 and not by CGP41-251 and CGP44-800. Therefore, priming of the respiratory burst is not due to an aspecific enhancing effect of the inhibitors. These data indicate that different signal transduction routes are involved in priming of the STZ-induced respiratory burst and PAF release in human eosinophils.

Extracellular and intracellular magnesium concentrations in asthmatic patients.

Magnesium deficiency is associated with increased contractility of smooth muscle cells. Since contractility of bronchial smooth muscle is important in patients with asthma, magnesium deficiency could negatively influence the clinical condition. We wanted to assess whether magnesium deficiency exists in patients with asthma. Extracellular (plasma) and intracellular (erythrocytes and mononuclear leucocytes) concentrations of magnesium were determined in 20 mildly symptomatic patients with asthma and compared to 20 healthy controls. In asthmatic patients, the mean +/- SD magnesium level in plasma was 0.81 +/- 0.05 mmol.l-1, in erythrocytes 0.20 +/- 0.02 fmol.cell-1, and in mononuclear leucocytes 5.10 +/- 2.55 fmol.cell-1; these values did not differ significantly from those of the healthy controls: 0.79 +/- 0.06 mmol.l-1, 0.19 +/- 0.02 fmol.cell-1, and 4.61 +/- 1.75 fmol.cell-1, respectively. No evidence for the existence of a magnesium deficit needing chronic magnesium supplementation was, thus, found in these patients.

Effects of nedocromil sodium on in vitro induced migration, activation, and mediator release from human granulocytes.

Using the allergen-induced late-phase asthmatic reaction as a working model, we studied the activity of certain inflammatory cells and their reaction to nedocromil sodium. The processes that were examined in vitro included the following: the chemotaxis of purified neutrophils and eosinophils, the early steps of neutrophil and eosinophil activation, and the release of mediators from these cells. Nedocromil sodium strongly inhibited neutrophil mobilization caused by four chemotactic factors (zymosan activated serum, N-formyl-methionyl-leucyl-phenylalanine platelet-activating factor [PAF], and leukotriene B4 [LTB4] and eosinophil mobilization caused by two factors (PAF and LTB4). In vitro treatment of eosinophils from normal subjects with picomolar concentrations of interleukin-3, interleukin-5, or granulocyte-macrophage colony stimulating factor increased the chemotactic responsiveness toward PAF and LTB4 and induced a chemotactic responsiveness toward N-formyl-methionyl-leucyl-phenylalanine and neutrophil activating factor/interleukin-8. The zymosan activated serum-induced chemotactic responsiveness remained unaltered. Nedocromil sodium inhibited the cytokine-primed chemotactic responsiveness to the various chemotaxins, not the influence of the cytokines on the cells. Activation of granulocytes, as measured by Ca2+ influx, was not inhibited by nedocromil sodium. Mediator formation in eosinophils was modified only slightly. These results suggest that inhibiting the mobilization of inflammatory cells in the lung tissue may be an important action of nedocromil sodium. Therefore these effects may be relevant to the treatment of asthma given the role of airway inflammation in this disease process.

Cytokine priming of the respiratory burst in human eosinophils is Ca2+ independent and accompanied by induction of tyrosine kinase activity.

We report that pretreatment of human eosinophils with GM-CSF, IL-3, or IL-5 enhanced the respiratory burst induced by opsonized particles. In order to gain more insight into the intracellular mechanism(s) involved in cytokine priming, the role of [Ca2+]i and tyrosine kinases was studied. Optimal priming concentrations of GM-CSF, IL-3, and IL-5 did not induce a rise in [Ca2+]i, and Ca(2+)-depleted eosinophils ([Ca2+]i < 20 nM) were still primed after preincubation with these cytokines. GM-CSF, IL-3, and IL-5 induced phosphorylation of two proteins (102 and 122 kd) on tyrosine residues, as deduced from Western blot analysis with an antiphosphotyrosine monoclonal antibody (4G10). This cytokine-stimulated tyrosine phosphorylation was not inhibited under Ca(2+)-depleted conditions. In conclusion, this study demonstrates that GM-CSF, IL-3, and IL-5 priming of the opsonized particle-induced respiratory burst in human eosinophils is completely Ca2+ independent. Moreover the tyrosine phosphorylation of a 102-kd and a 122-kd protein is Ca2+ independent, suggesting that this event might be involved in cytokine priming.

Release of platelet-activating factor is important for the respiratory burst induced in human eosinophils by opsonized particles.

The respiratory burst induced in human eosinophils by serum-treated zymosan (STZ) was found to be almost completely prevented by preincubation of the cells with WEB 2086, an antagonist of platelet-activating factor (PAF). When eosinophils were primed by the addition of 1 mumol/L PAF, subsequent addition of WEB 2086 had only a minor effect on the STZ-induced respiratory burst. These results suggest a role for PAF synthesis and PAF release in the activation of the respiratory burst by STZ. Indeed, supernatant of STZ-stimulated eosinophils was able to prime fresh eosinophils (as did PAF itself), and this effect was again inhibited by WEB 2086. This indicates that eosinophils synthesize and release PAF during STZ stimulation. Measurements of total PAF and PAF release showed that most of the PAF synthesized by eosinophils was released in the extracellular medium. This study shows that synthesis and release of PAF is important for respiratory burst activity induced in human eosinophils by STZ.

Leukotriene C4 formation by enriched human basophil preparations from normal and asthmatic subjects.

Numbers of circulating basophils are increased in asthmatic subjects, compared to normal subjects. Basophil enriched cell preparations from normal and asthmatic subjects were challenged in vitro with the calcium ionophore A23187, anti-IgE, or opsonized zymosan to study leukotriene C4 formation, histamine release, and prostaglandin D2 formation. No prostaglandin D2 formation by basophils was observed. Furthermore, opsonized zymosan was not capable of inducing any mediator formation or release from basophils. At optimal stimulation conditions no differences were found between basophils from normal and asthmatic subjects concerning A23187 or anti-IgE induced leukotriene C4 formation or histamine release. A23187 and anti-IgE induced leukotriene C4 formation were in the range of 1-20 and 0.6-4.8 pmol/10(6) basophils respectively.

Modulation and induction of eosinophil chemotaxis by granulocyte-macrophage colony-stimulating factor and interleukin-3.

Eosinophilia and eosinophil function are regulated by cytokines such as granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3), and IL-5. We have investigated the modulatory role of GM-CSF and IL-3 on the platelet-activating factor (PAF)-, neutrophil-activating factor (NAF/IL-8)-, leukotriene B4 (LTB4)-, N-formyl-methionyl-leucyl-phenylalanine (FMLP)-, and human complement factor C5a-induced chemotaxis of eosinophils from normal individuals. These eosinophils show a chemotactic response toward PAF, LTB4, and C5a, but not to NAF/IL-8 and FMLP. Preincubation of the eosinophils with picomolar concentrations of GM-CSF caused a significant increase in the response toward LTB4 and induced a significant chemotactic response toward NAF/IL-8 and FMLP. Preincubation of the eosinophils with picomolar concentrations of IL-3 also induced a chemotactic response toward NAF/IL-8 and FMLP, and enhanced the PAF-induced chemotaxis response toward C5a was not influenced by both cytokines. Nanomolar concentrations of GM-CSF or IL-3 caused a significant inhibition of the C5a-induced chemotaxis. The LTB4-induced chemotaxis was also significantly inhibited in case of GM-CSF. At these concentrations both GM-CSF and IL-3 acted as chemotaxins for eosinophils were washed after pretreatment with GM-CSF and IL-3 the potentiation of the chemotactic response remained, whereas the inhibitory mode of action disappeared. Our data indicate that at picomolar concentrations the cytokines GM-CSF and IL-3 can modulate eosinophil chemotaxis and at nanomolar concentrations these cytokines can act as chemotaxins for eosinophils.

Inhibition of neutrophil and eosinophil induced chemotaxis by nedocromil sodium and sodium cromoglycate.

1. Neutrophils and eosinophils infiltrate the airways in association with the allergen-induced late phase asthmatic reaction. Mobilization of these cells takes place via lipid-like and protein-like chemotactic factors. In this study platelet-activating factor (PAF), leukotriene B4 (LTB4), zymosan-activated serum (ZAS) and N-formyl-methionyl-leucyl-phenylalanine (FMLP) were used as illustrative examples of both groups. Chemotaxis was studied in human neutrophils and eosinophils. The inhibitory effects of nedocromil sodium and sodium cromoglycate were evaluated. 2. All chemotactic factors tested attracted neutrophils with the following rank order of activity: ZAS greater than PAF identical to FMLP identical to LTB4. Eosinophils were only mobilized by PAF, LTB4 and ZAS with the following rank order of activity: ZAS greater than PAF greater than LTB4. 3. Nedocromil sodium and sodium cromoglycate were equally active as the PAF antagonist BN 52021 in inhibiting the PAF-induced chemotaxis of neutrophils (IC50 approximately 10(-8) M). Both drugs were also equally active in inhibiting the chemotaxis of neutrophils induced by ZAS (IC50 approximately 10(-7)-10(-6) M), FMLP (IC50 approximately 10(-7) M) and LTB4 (IC50 approximately 10(-6) M). 4. Nedocromil sodium significantly inhibited the chemotaxis of eosinophils induced by PAF (IC50 approximately 10(-6) M) and LTB4 (IC50 approximately 10(-7) M). The inhibitory potency of BN 52021 was similar to that of nedocromil sodium on the PAF-induced chemotaxis of eosinophils. Sodium cromoglycate was incapable of eliciting significant inhibition of these chemotactic responses. However, sodium cromoglycate significantly inhibited the chemotaxis of eosinophils induced by ZAS (IC50 approximately 10(-7) M), whereas nedocromil sodium was ineffective.

Inhibition of platelet-activating factor- and zymosan-activated serum-induced chemotaxis of human neutrophils by nedocromil sodium, BN 52021 and sodium cromoglycate.

1. Inflammatory cells such as eosinophils and neutrophils are thought to contribute actively to the pathogenesis of asthma since they infiltrate into the lung tissue. These cells are mobilized by lipid-like and protein-like chemotactic factors. As illustrative examples of both groups, platelet-activating-factor (Paf) and zymosan-activated-serum (ZAS) were used in this study. The inhibitory effects of nedocromil sodium, the Paf antagonist BN 52021 and sodium cromoglycate on Paf- and ZAS-induced neutrophil chemotaxis were evaluated. 2. All tested drugs inhibited Paf-induced neutrophil chemotaxis with approximately the same potency (IC50 approximately 1 nM). 3. Nedocromil sodium and sodium cromoglycate were equally potent in inhibiting ZAS-induced neutrophil chemotaxis (IC50 = 0.1-1 microM), whereas BN 52021 was considerably less potent (IC30 = 10 microM). 4. To find out whether the drugs tested could inhibit early events in cell activation, their capacity to inhibit Paf- and ZAS-induced cytosolic free Ca2+-mobilization was investigated. BN 52021, at a concentration of 100 microM, completely inhibited Paf-induced Ca2+-mobilization and inhibited ZAS-induced Ca2+-mobilization by about 50%. Nedocromil sodium and sodium cromoglycate were ineffective.

Nedocromil sodium inhibits the A23187- and opsonized zymosan-induced leukotriene formation by human eosinophils but not by human neutrophils.

1. Inflammatory cells such as eosinophils and neutrophils are thought to contribute actively to the pathogenesis of asthma by the release of bronchoconstrictor mediators including leukotrienes. Previous studies have revealed the almost exclusive synthesis of leukotriene C4 (LTC4) by human eosinophils and of leukotriene B4 (LTB4), 20-OH-LTB4 and the non-enzymatically formed LTB4-isomers by neutrophils when stimulated in vitro with the calcium ionophore A23187 or opsonized zymosan (OZ). In this study we have investigated whether nedocromil sodium, a new anti-asthma drug, was capable of inhibiting A23187- and OZ-induced leukotriene formation by these cells. 2. Nedocromil sodium inhibited A23187- and OZ-induced LTC4 formation by eosinophils in a concentration-dependent manner (mean IC30 for A23187: 5.6 X 10(-5) M; mean IC30 for OZ: 6.3 X 10(-5) M), whereas it did not inhibit A23187- and OZ-induced LTB4 formation by neutrophils. 3. Extension of the preincubation time of the cells with the drug did not alter the observed inhibitory capacity. The optimal preincubation time was 5 min. 4. The in vitro inhibition of LTC4 formation by eosinophils by nedocromil sodium may be a valuable property of this drug in the treatment of asthma.

Leukotriene C4 formation by purified human eosinophils can be induced by arachidonic acid in the absence of calcium-ionophore A23187.

Addition of arachidonic acid (50 microM) to purified human eosinophils leads to the formation of considerable amounts of LTC4 [11.3 +/- 1.3) x 10(6) molecules/cell, mean +/- SEM, n = 10), 15-HETE [412 +/- 142) x 10(6) molecules/cell, mean +/- SEM, n = 3) and 15-series leukotrienes [35 +/- 15) x 10(6) molecules/cell, mean +/- SEM, n = 3). The ratio of the amounts of LTC4 and 15-lipoxygenase products was found to be strongly dependent on the arachidonic acid concentration, being relatively large at low arachidonic acid concentrations and very small at high arachidonic acid concentrations. Platelet activating factor (1 microM) was able to enhance significantly the production of LTC4 but not that of 15-lipoxygenase products. As arachidonic acid was found to be capable of inducing a fast, transient rise in the cytosolic free Ca2+ concentration, this explains, at least partly, its ability to induce the Ca2+-dependent formation of LTC4.

Inhibitory effects of nedocromil sodium on the in vitro induced migration and leukotriene formation of human granulocytes.

Inflammatory cells, such as neutrophils and eosinophils, are thought to actively contribute to the pathogenesis of asthma since they infiltrate into the lung tissue and may be activated locally to release bronchoconstrictor mediators. In this study we provide evidence that nedocromil sodium is capable of effectively inhibiting the platelet-activating factor (PAF) and zymosan-activated serum (ZAS)-induced chemotaxis of polymorphonuclear granulocytes (PMN) [IC50 approximately 1 nmol/L and 0.1 mumol/L respectively]. The same inhibitory potency was obtained with sodium cromoglycate. Thus, nedocromil sodium may effectively inhibit the mobilisation of inflammatory cells in the lung. Furthermore, nedocromil sodium is capable of inhibiting the formation of the bronchoconstrictor mediator leukotriene-C4 (LTC4) by eosinophils in a concentration-dependent way [IC30 for A23187: 5.6 10(-5) mol/L; IC30 for opsonised zymosan (OZ): 6.3 10(-5) mol/L], whereas this drug is not capable of inhibiting leukotriene-B4 (LTB4) formation by neutrophils. These findings indicate that nedocromil sodium inhibits the release of bronchoconstrictor mediators not only from mast cells but also from eosinophils.

An improved method for the isolation of eosinophilic granulocytes from peripheral blood of normal individuals.

A simple and improved procedure is described for the isolation of human eosinophils from normal individuals with about 2% eosinophils in their peripheral blood. This method comprises a preincubation of a mixed granulocyte preparation with 10 nM fMLP for 10 min at 37 degrees C followed by a one-step density centrifugation on isotonic Percoll. The recovery of eosinophils is 49 +/- 4% at 89 +/- 4% purity. Because of the relatively high rate of recovery, it is now possible to isolate eosinophils from blood samples as small as 20 ml. Because treatment with fMLP may alter the functional activity of the eosinophils, the following metabolic functions were tested: changes in cytosolic free Ca2+, oxygen consumption, chemiluminescence, chemotaxis, and leukotriene C4 formation. We found that 10 nM fMLP does not activate eosinophils in these assays, whereas 1 microM fMLP does (with the exception of chemotaxis). Furthermore, pretreatment of eosinophils with 10 nM fMLP did not influence the response to other stimuli in these assays. The usefulness of this method was evaluated by comparing it with three other previously described procedures. In our hands, only the method presented here enabled us to isolate eosinophils from normal individuals with about 2% eosinophils in their peripheral blood.

Arachidonic acid can induce leukotriene C4 formation by purified human eosinophils in the absence of other stimuli.

Stimulation of purified human eosinophils with 50 microM arachidonic acid leads to the production of leukotriene C4, 15-hydroxy-eicosatetraenoic acid and 15-series leukotrienes. The ratio of the amounts of leukotriene C4 and 15-lipoxygenase products was found to be strongly dependent on the arachidonic acid concentration, being relatively large at low arachidonic acid concentrations and very small at high arachidonic acid concentrations. In the presence of 1 microM platelet-activating factor a significant elevation of leukotriene C4 formation is observed, whereas the formation of 15-lipoxygenase products remains unaltered. As arachidonic acid was found to be capable of inducing a fast, transient rise in the cytosolic free Ca2+ concentration, this explains at least partly its ability to induce the Ca2+-dependent formation of leukotriene C4.

Platelet-activating factor induces leukotriene C4 synthesis by purified human eosinophils.

Platelet-activating factor, at a concentration of 10 microM, was capable of inducing leukotriene C4 synthesis by eosinophils of healthy donors, i.e. (3.1 +/- 0.3) x 10(6) molecules leukotriene C4/cell (n = 31, mean +/- SEM, cell purity 87 +/- 2%). Reversed-phase high performance liquid chromatography analysis demonstrated the exclusive synthesis of leukotriene C4. At a concentration of 1 microM, platelet-activating factor was capable of significantly enhancing the calcium ionophore A23187, the opsonized zymosan or the arachidonic acid induced leukotriene C4 synthesis by eosinophils. These results show that PAF is capable of inducing and enhancing the leukotriene C4 formation by human eosinophils.

Platelet-activating factor (PAF-acether) induced leukotriene C4 formation and luminol dependent chemiluminescence by human eosinophils.

Human eosinophils are capable of synthesizing almost exclusively the strongly spasmogenic compound LTC4 when stimulated with either the calcium ionophore A 23187 or opsonized zymosan (OZ). Although PAF-acether in concentrations ranging from 10 nM to 1 microM is hardly capable of inducing significant LTC4 synthesis itself, it significantly enhances the OZ-induced LTC4 formation at a concentration of 1 microM. However, at a concentration of 10 microM, PAF-acether itself is capable of inducing LTC4 formation comparable with that induced by OZ. PAF-acether, at a concentration of 10 microM (and not at a concentration of 1 microM) is also capable of inducing a luminol dependent chemiluminescent response by eosinophils. The PAF-acether antagonist BN 52021 at a concentration of 0.1 mM not only partially inhibited the PAF-acether induced LTC4 formation but also the OZ induced LTC4 formation. Since an equal inhibition is found the inhibitory mode of action of BN 52021 is most likely directed towards a common pathway. Taken together, these results suggest that eosinophils may be triggered by high locally reached concentrations of PAF-acether to release inflammatory and bronchoconstrictive mediators. This may be of importance for the pathogenesis of the allergen induced late phase asthmatic reaction.

On the optimal conditions of LTC4 formation by human eosinophils in vitro.

The optimal conditions for the -in vitro- LTC4 formation by the human eosinophil, isolated from peripheral blood, have been investigated in detail. LTC4 formation was found strongly Ca2+ and ionophore dependent and was complete after 20 min. Maximal LTC4 production was observed in the presence of 2 mM Ca2+, 10 microM ionophore A23187 and 5 mM glutathione. Addition of arachidonic acid resulted in a significant inhibition of the LTC4-synthesis by human eosinophils. In contrast, the formation of 15-HETE was strongly stimulated by the addition of arachidonic acid. As the LTC4 synthesis was found to be strongly inhibited by the addition of 15(S)-HETE to the incubation medium, this monohydroxy acid may be responsible for the inhibitory activity of arachidonic acid.

Exclusive leukotriene C4 synthesis by purified human eosinophils induced by opsonized zymosan.

Purified human eosinophils were challenged with N-formyl-methionyl-leucyl-phenylalanine, leukotriene B4, platelet-activating-factor, valyl-glycyl-seryl-glutamic acid, phorbol myristate acetate, zymosan, opsonized zymosan and the calcium ionophore A23187 to induce leukotriene synthesis. Reversed-phase high performance liquid chromatography analysis demonstrated the almost exclusive synthesis of leukotriene C4 by eosinophils of 11 healthy donors after challenge with opsonized zymosan [(22 +/- 4) X 10(6) molecules LTC4/cell, mean +/- SE] or the calcium ionophore A23187 [(54 +/- 7) X 10(6) molecules LTC4/cell, mean +/- SE]. The other agents were not capable of inducing leukotriene formation. When in addition to opsonized zymosan N-formyl-methionyl-leucyl-phenylalanine or platelet-activating factor were added a significant increase of the leukotriene C4 synthesis by eosinophils was observed. These results suggest that eosinophils might be triggered to produce considerable amounts of the spasmogenic leukotriene C4 in vivo by C3b- and/or IgG-mediated mechanisms e.g. phagocytosis.