Stimulation of Intracellular Ca2+ Levels in Human Neutrophils by Soluble Immune Complexes
FUNCTIONAL ACTIVATION OF Fcgamma RIIIb DURING PRIMING*

(Received for publication, November 21, 1996, and in revised form, March 12, 1997)

Fiona Watson , Lakhdar Gasmi and Steven W. Edwards Dagger

From the School of Biological Sciences, Life Sciences Building, University of Liverpool, P. O. Box 147, Liverpool L69 3BX, United Kingdom

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES


ABSTRACT

Soluble immune complexes bind to unprimed neutrophils and generate intracellular Ca2+ transients but fail to activate the NADPH oxidase. Following priming of the neutrophils with either tumor necrosis factor alpha or granulocyte-macrophage colony-stimulating factor, stimulation of the cells with the soluble immune complexes leads to an enhanced Ca2+ signal and significant secretion of reactive oxidants. The enhanced Ca2+ signal observed in primed neutrophils results from the influx of Ca2+ from the external environment and is partly sensitive to tyrosine kinase inhibitors. This is in contrast to the Ca2+ signal observed in unprimed neutrophils, which arises from the mobilization of intracellular stores. When the surface expression of Fcgamma RIIIb on primed neutrophils was decreased either through incubation with Pronase or phosphoinositide-specific phospholipase C, the extra enhanced Ca2+ mobilization seen in primed cells was significantly lowered, while the initial rise in intracellular Ca2+ was unaffected. Depletion of Fcgamma RIIIb had no significant effect on the Ca2+ transients in unprimed neutrophils. Cross-linking Fcgamma RII, but not Fcgamma RIIIb, induced increases in intracellular Ca2+ in unprimed neutrophils, while cross-linking either of these receptors increased Ca2+ levels in primed neutrophils. The Fcgamma RII-dependent intracellular Ca2+ rise in primed cells was unaffected by incubation in Ca2+-free medium, whereas the Fcgamma RIIIb-dependent transient was significantly decreased when Ca2+ influx was prevented in Ca2+-free medium supplemented with EGTA. Cross-linking either Fcgamma RII or Fcgamma RIIIb in primed or unprimed cells failed to stimulate substantial levels of inositol 1,4,5-trisphosphate production. These results indicate that following stimulation of primed neutrophils with soluble immune complexes the enhanced Ca2+ mobilization observed is the result of a functional activation of the glycosylphosphatidylinositol-linked Fcgamma RIIIb.


INTRODUCTION

Neutrophils play a major role in host defense via the phagocytosis and destruction of pathogens during acute inflammation. The binding of opsonized bacteria or immune complexes to neutrophil immunoglobulin receptors (Fcgamma R) can activate a number of processes such as phagocytosis, degranulation and activation of the NADPH oxidase (1, 2). However, in addition to this protective role, inappropriate activation of neutrophils to release oxygen metabolites and granule enzymes within tissues can result in tissue damage in inflammatory conditions such as rheumatoid arthritis (1, 2). Furthermore, immune complex deposition followed by subsequent neutrophil activation is important in the pathogenesis of serum sickness, the Arthus reaction, acute glomerulonephritis, and other idiopathic inflammatory diseases (3, 4). Neutrophils are activated within the synovial joints of rheumatoid patients (5-10), and we have shown that synovial fluid from patients with rheumatoid arthritis contains both soluble and insoluble IgG-containing immune complexes, which are capable of activating neutrophils (11-13). However, the soluble immune complexes only activate neutrophils that have been previously primed in vivo or in vitro by GM-CSF1 or gamma -interferon and this activation leads to the secretion of reactive oxidants and granule enzymes (11-13). In contrast, insoluble immune complexes activate both primed and unprimed neutrophils with nearly equal efficacy but the oxidants generated are largely retained intracellularly, i.e. within phagolysosomes. Thus, soluble immune complexes may be of greater importance than insoluble complexes in the destructive processes that occur during inflammatory diseases because they activate the secretion of large quantities of cytotoxic products. The identification of the molecular processes that occur during neutrophil priming to allow activation of oxidant secretion in response to soluble complexes may therefore be of value in identifying new targets for therapeutic intervention.

IgG-containing immune complexes stimulate neutrophils via Fcgamma receptors, two types of which are expressed on freshly isolated control blood cells (14). Fcgamma RII (CD32) is a 40-kDa transmembrane-spanning molecule with a cytoplasmic tail that allows its interaction with G-proteins, whereas Fcgamma RIIIb is a heavily glycosylated molecule of 50-70 kDa, linked to the membrane via a glycosylphosphatidylinositol anchor (15). Thus, although Fcgamma RIIIb has the potential for rapid lateral mobility within the membrane, it is unable to couple directly to either G-proteins or to the cytoskeleton, and it is unclear how ligation to this receptor might lead to the transduction of intracellular signals. Expression of Fcgamma RIIIb on the plasma membrane (100,000-200,000 molecules/cell) is approximately 10-15-fold greater than the expression of Fcgamma RII (7000-15,000 molecules/cell).

There is much debate as to the independent and co-operative roles of Fcgamma RII and Fcgamma RIIIb in neutrophil activation. Neither receptor binds monomeric IgG, but they bind dimers, trimers, immune complexes, and opsonized particles (16, 17). Although reports in the literature are conflicting, it is currently thought that occupancy of Fcgamma RII leads to activation of reactive oxidant production, degranulation, and phagocytosis (18-22). While there have been a few reports of Fcgamma RIIIb occupancy inducing functional responses, its main role is thought to enhance the binding of immune complexes and to augment the function of Fcgamma RII. Similarly, the intracellular signaling molecules generated via Fcgamma RII and Fcgamma RIIIb ligation are undefined. Although it is not clear how Fcgamma RIIIb, which lacks a transmembrane or cytoplasmic domain can generate intracellular signals, it has recently been reported that occupancy of either Fcgamma RII or Fcgamma RIIIb can activate Src family non-receptor tyrosine kinases (23-27). Occupancy of Fcgamma RII is associated with the activation and translocation of Fgr to the Triton-insoluble cytoskeleton, while Fcgamma RIIIb is associated with Hck activation. Ligation of either Fcgamma RII or Fcgamma RIIIb can mediate a cytosolic Ca2+ increase that has been reported to come primarily from the release of intracellular stores, and it has been suggested that occupancy of Fcgamma RII and Fcgamma RIIIb can lead to the generation of both independent and synergistic Ca2+ fluxes (28-30). However, the mechanisms by which Fcgamma R engagement leads to an increase in intracellular Ca2+ are unclear; reports indicate that that it may be pertussis toxin-insensitive and largely independent of inositol 1,4,5-trisphosphate (IP3) accumulation (31, 32). Most observations have, however, been made following stimulation with large insoluble immune complexes or by cross-linking individual Fcgamma receptors. Few reports have examined changes in Fcgamma R function during priming (33), and the method used to isolate neutrophils may influence the results obtained because some separation procedures may result in inadvertent priming of the neutrophil (34, 35).

In this study, we have investigated Ca2+ mobilization in neutrophils stimulated with soluble immune complexes in both primed and unprimed neutrophils. We present evidence that the initial cytosolic Ca2+ increase observed following stimulation with soluble complexes is the result of ligation of Fcgamma RII and that this interaction is insufficient to activate the respiratory burst. When soluble complexes are added to primed neutrophils oxidant secretion is initiated and an enhanced mobilization of Ca2+ results, mainly via influx from extracellular sources. It would appear that this extra intracellular Ca2+ signal results from functional activation of Fcgamma RIIIb during priming. These studies add new insights into the mechanisms by which cellular priming alters the functional responsiveness of neutrophils during inflammatory activation.


EXPERIMENTAL PROCEDURES

Materials

Neutrophil isolation medium was from Cardinal Associates Inc., RPMI 1640 medium from Flow Laboratories; Fluo-3-AM, erbstatin, and genistein were from Calbiochem. rGM-CSF was a non-glycosylated peptide from Glaxo and had an activity of >= 1.5 milliunits/mg of protein in the AML-193 proliferation assay. TNFalpha was from National Institute for Biological Standards and Controls, Potters Bar, United Kingdom. F(ab')2 fragments of monoclonal antibody 3G8 (recognizing Fcgamma RIIIb) and Fab fragments of IV.3 (recognizing Fcgamma RII) were from Medarex, Inc. PI-PLC was from Boehringer Mannheim. All other specialist reagents were from Sigma.

Neutrophil Isolation

Neutrophils were isolated from the venous blood of healthy volunteers by centrifugation on neutrophil isolation medium for 15 min at 400 × g (36). After removal of contaminating erythrocytes by hypotonic lysis, purified neutrophils were suspended in RPMI 1640 medium and counted. Neutrophil purity (assessed by Wright's staining) and viability (assessed by trypan blue exclusion) were >97% and >95%, respectively.

Neutrophil Priming

Neutrophils were primed using either GM-CSF or TNFalpha . Cells (107/ml) were incubated in the presence and absence of GM-CSF (50 units/ml) for 1 h at 37 °C, whereas TNFalpha (50 ng/ml) was added 10 min prior to stimulation.

Immune Complex Preparation

Synthetic immune complexes were made from human serum albumin and rabbit anti-human serum albumin antibodies as described previously (37). The antigen was titrated against constant antibody concentration and the A450 nm measured to identify equivalence. The soluble complexes were formed at 6 × antigen equivalence and were briefly centrifuged (2 min at 13,000 × g in a microcentrifuge) to remove any contaminating insoluble immune complexes that may have been present. Soluble complexes were formed at 180 µg/ml antigen and 125 µg/ml antibody. A 10% (v/v) solution of complexes was used routinely for neutrophil stimulation.

Reactive Oxidant Production

Chemiluminescence was measured at 37 °C in neutrophil suspensions (5 × 105/ml) in RPMI 1640 medium that was supplemented with 10 µM Luminol using an LKB 1251 luminometer (38, 39). For measurements of superoxide secretion, neutrophils were suspended in RPMI 1640 supplemented with 75 µM cytochrome c (40). Absorption changes were recorded at 550 nm using a Bio-Rad 3550 kinetic plate reader. Reference wells contained 30 µg/ml superoxide dismutase.

Intracellular Free Ca2+

Changes in intracellular Ca2+ concentration, [Ca2+]i, were monitored with the fluorescent probe Fluo-3. Neutrophils in RPMI 1640 (2 × 107/ml) were loaded by incubation at 37 °C for 30 min with 2 µM Fluo-3 AM. The cells were then washed twice and resuspended at 2 × 106/ml in Ca2+-free medium (145 mM NaCl, 1 mM Na2HPO4·2H2O, 0.5 mM MgSO4·7H2O, 5 mM glucose, 20 mM Hepes, pH 7.4) to which 1 mM CaCl2 was added as required. In some experiments, 1 mM EGTA was added to media devoid of Ca2+. Fluorescence was then measured at 505 nm excitation and 530 nm emission. Calibration of changes in intracellular Ca2+ levels was as described in Ref. 41 using a Kd for Fluo-3 of 864 nM at 37 °C.

Receptor Cross-linking and mAb Blocking Studies

For measurements of intracellular Ca2+ following the cross-linking of receptors, Fluo-3-loaded neutrophils (2 × 106/ml) were incubated with 1 µg/ml Fab/F(ab')2 fragments of appropriate monoclonal antibodies at 37 °C prior to cross-linking with 40 µg/ml F(ab')2 fragments of goat anti-mouse IgG. For mAb blocking studies, 3 × 106 neutrophils were incubated with 3 µg of Fab/F(ab')2 fragments in a volume of 250 µl for 10 min at 37 °C prior to dilution (to 5 × 105 cells/ml) and stimulation of cells with soluble immune complexes.

Inositol 1,4,5-Trisphosphate Assays

Neutrophils were incubated for 45 min in the presence (primed) and absence (control) of 50 units/ml GM-CSF. Fcgamma RII and Fcgamma RIIIb were then cross-linked (as described above), and samples were removed at time intervals. IP3 was then extracted by rapidly mixing samples with 0.2 volume of ice-cold 20% (v/v) perchloric acid. After neutralizing extracts, IP3 was quantified using a competitive binding assay (Amersham, UK) using D-myo-inositol 1,4,5-trisphosphate as a calibration standard.


RESULTS

Reactive Oxidant Generation Induced by Soluble Immune Complexes

Addition of synthetic soluble IgG immune complexes to freshly isolated neutrophils failed to activate reactive oxidant generation (Fig. 1), as detected by Luminol chemiluminescence. However, if the cells were primed by prior addition of GM-CSF (50 units/ml for 1 h) or TNFalpha (50 ng/ml for 10 min), then the soluble complexes activated a rapid and transient burst of oxidase activity that peaked 2-3 min after addition (Fig. 1, A and C). Luminol-dependent chemiluminescence detects both intra- and extracellular oxidant production (39), but the use of cytochrome c, which is a large basic molecule that cannot permeate the cell, specifically measures Obardot 2 secretion (38). The addition of soluble immune complexes to unprimed cells similarly did not activate Obardot 2 secretion but did lead to a rapid and significant secretion of Obardot 2 in cells that had been primed either with TNFalpha or with GM-CSF (Fig. 1, B and D).


Fig. 1. Effect of priming on reactive oxidant production. In A and C, neutrophils (5 × 105 cells/ml) were incubated in RPMI 1640 medium supplemented with 10 µM Luminol, whereas in B and D they were incubated at the same concentration in RPMI medium supplemented with 75 µM cytochrome c, as described under "Experimental Procedures." Before stimulation, the suspensions were incubated in the absence (open circle ) or presence (bullet ) of 50 units/ml GM-CSF for 60 min (A and B) or 50 ng/ml TNFalpha for 10 min (C and D) prior to the addition of soluble immune complexes. Similar results have been obtained in 10 other experiments.
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Intracellular Ca2+ Mobilization

Resting levels of intracellular Ca2+ were approximately 140 nM (n = 18) in unstimulated neutrophils. When soluble immune complexes were added to unprimed cells, there was a rapid and transient monophasic increase in the level of intracellular Ca2+, which reached a maximal level of 0.9-1.0 µM Ca2+ approximately 30-45 s following stimulation (Fig. 2). This increase in intracellular Ca2+ was comparable to that induced by addition of 0.1 µM fMet-Leu-Phe (data not shown). When the neutrophils were primed by exposure to TNFalpha prior to stimulation with the soluble complexes, there was again a rapid rise in intracellular Ca2+ levels (Fig. 2A), but the kinetics of the increase were altered. The initial increase in intracellular Ca2+ seen in unprimed neutrophils was unaffected, but this was followed by a second, more sustained increase in intracellular Ca2+. Similarly, when the neutrophils were primed with GM-CSF (Fig. 2B), the intracellular Ca2+ mobilization induced by the soluble immune complexes was enhanced and prolonged in comparison to that observed in unprimed cells. Increasing the concentrations of soluble immune complexes (to 20% and 30%, v/v) did not change either the kinetics or the magnitude of the intracellular Ca2+ transients seen in unprimed cells. Thus, priming does not affect the affinity of binding of soluble immune complexes under these conditions.


Fig. 2. Changes in intracellular Ca2+ in primed and unprimed neutrophils. Fluo-3-loaded neutrophils (for details see "Experimental Procedures") were incubated in the absence (unprimed) or presence (primed) of either 50 ng/ml TNFalpha for 10 min (in A) or 50 units/ml GM-CSF for 60 min (in B) prior to addition of 10% (v/v) soluble immune complexes (arrow). Similar results have been obtained in 15 other experiments.
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Effect of EGTA on Intracellular Ca2+ Mobilization

To determine the source of the increased intracellular Ca2+ levels in both unprimed and primed neutrophils, incubations in Ca2+-free medium in the presence of 1 mM EGTA were performed. Fig. 3A shows that when unprimed cells were stimulated with soluble immune complexes in Ca2+-free buffer, there was no significant difference in the kinetics of intracellular Ca2+ changes compared with those obtained in Ca2+-containing buffer. Thus, in unprimed neutrophils, the observed increases in intracellular Ca2+ arise from the mobilization of intracellular stores. In contrast, when primed cells were stimulated with soluble immune complexes Ca2+-free buffer, the initial intracellular Ca2+ increase was unaffected, but the extended "extra" Ca2+ signal only seen in primed cells, was not observed (Fig. 3, B and C). These experiments thus indicate that the enhanced and prolonged increase in intracellular Ca2+ observed only in primed cells is due to Ca2+ influx from external sources.


Fig. 3. Effects of EGTA on intracellular Ca2+ levels in primed and unprimed neutrophils. Fluo-3-loaded neutrophils were incubated in the absence (A) or presence of 50 ng/ml TNFalpha for 10 min (B) or 50 units/ml GM-CSF for 60 min (C) in either Ca2+-containing medium (No EGTA) or Ca2+-depleted medium supplemented with 1 mM EGTA (+ EGTA). 10% (v/v) soluble immune complexes were added at the arrow. Similar results have been obtained in six other experiments.
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Role of Tyrosine Kinases in Ca2+ Mobilization

When unprimed neutrophils were incubated with the tyrosine kinase inhibitor, erbstatin (1 µg/ml), the intracellular Ca2+ response obtained following stimulation with the soluble complexes was inhibited by 30% (± 8%, n = 6). When neutrophils were primed with either TNFalpha (Fig. 4B) or GM-CSF (Fig. 4C), the enhanced intracellular Ca2+ signal seen only in primed neutrophils was similarly decreased. This concentration of erbstatin decreased reactive oxidant generation stimulated by soluble immune complexes in primed neutrophils by >95% (data not shown). Similar results were obtained following addition of another tyrosine kinase inhibitor genistein at 100 µM (data not shown). Addition of erbstatin at 2 µg/ml inhibited the unprimed Ca2+ signal by over 50%, but did not result in greater inhibition of the primed response (data not shown).


Fig. 4. Effects of erbstatin on intracellular Ca2+ transients in primed neutrophils. Fluo-3-loaded neutrophils were incubated in the absence (A, Unprimed) or presence of 50 ng/ml TNFalpha for 10 min (B) or 50 units/ml GM-CSF for 60 min (C) and further incubated in the absence or presence of erbstatin (1 µg/ml for 5 min), prior to the addition of soluble immune complexes (arrow). Similar results have been obtained in four other experiments.
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To determine whether erbstatin exhibited nonspecific toxic effects at these concentrations, the experiments shown in Fig. 5 were performed. After addition of soluble immune complexes to primed neutrophils incubated in the presence and absence of erbstatin, fMet-Leu-Phe was added. The intracellular Ca2+ transients stimulated by fMet-Leu-Phe were unaffected by the inhibitor; this fMet-Leu-Phe-induced response was identical to that observed following its addition to suspensions that had not previously been stimulated by immune complexes.


Fig. 5. Stimulation of intracellular Ca2+ transients by fMet-Leu-Phe following addition of soluble immune complexes and erbstatin. Fluo-3-loaded neutrophils were incubated in the absence or presence of erbstatin (1 µg/ml for 5 min), primed with TNFalpha (50 ng/ml for 30 min), and then stimulated by the addition of soluble immune complexes. When the intracellular (I.C.) Ca2+ levels had returned to prestimulated levels, fMet-Leu-Phe was added (10-7 M). Similar results were obtained in two other experiments.
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Role of Fcgamma Receptors: Depletion of Surface Receptors

When neutrophils were incubated with PI-PLC (at 0.25 units/ml for 30 min), the surface expression of Fcgamma RIIIb, which is glycosylphosphatidylinositol-linked (14, 15), detected by fluorescent-activated cell sorting was decreased to levels that were indistinguishable from nonspecific antibody binding (Fig. 6A, i). This treatment had no effect on the expression of Fcgamma RII (Fig. 6A, ii) or on the expression of CD11b (data not shown). Similarly, incubation with Pronase (0.05 mg/ml for 30 min) also resulted in the decreased surface expression of Fcgamma RIIIb but had no effect on expression of Fcgamma RII or CD11b (data not shown).


Fig. 6. Effect of PI-PLC on surface expression of Fcgamma RII and Fcgamma RIIIb and intracellular Ca2+. In A, neutrophils were incubated at 2 × 106 cells/ml in the presence (PI-PLC) or absence (control) of 0.25 units/ml PI-PLC for 30 min. After washing twice, surface expression of Fcgamma RIIIb (i) and Fcgamma RII (ii) was determined by flow cytometry. Similar results have been obtained in 10 other experiments, and identical profiles were obtained following incubation with 0.05 mg/ml Pronase for 30 min (data not shown). In B, neutrophils were incubated in the absence or presence of 50 units/ml GM-CSF for 60 min and then in the absence or presence of PI-PLC (0.25 units/ml for 30 min); cells were loaded with Fluo-3 during this latter 30-min incubation. Fluorescence measurements were then made after stimulation with soluble immune complexes. Similar results have been obtained in five other experiments and when Fcgamma RIIIb was removed by Pronase (n = 4). Inset shows the effects of PI-PLC incubation on NADPH oxidase activity. Primed neutrophils were incubated in the absence (bullet ) and presence (open circle ) of PI-PLC and NADPH oxidase activity measured using the cytochrome c reduction assay. Similar results were obtained in five other experiments.
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When Fcgamma RIIIb expression on the surface of primed neutrophils was depleted by treatment with either Pronase or PI-PLC, the soluble immune complexes failed to activate the respiratory burst (Fig. 6, inset). Neutrophils treated in this way can, however, still generate reactive oxidants in response to large, insoluble immune complexes or PMA (13).

When unprimed neutrophils were depleted of surface Fcgamma RIIIb by treatment with PI-PLC, there was very little effect on the intracellular Ca2+ rise stimulated by soluble immune complexes (Fig. 6B, i). This result indicates that expression of Fcgamma RIIIb is not required for the transient, intracellular Ca2+ increase that arises from the mobilization of intracellular stores following the binding of soluble immune complexes to the surface of unprimed cells. However, in primed cells, Pronase treatment had a marked effect on the intracellular Ca2+ signals (Fig. 6B, ii). In primed cells depleted of Fcgamma RIIIb, the initial intracellular Ca2+ transient was unaffected, but the later, sustained intracellular Ca2+ signal that arises from Ca2+ influx was not observed. Similar results were obtained when the neutrophils were primed with TNFalpha and when Fcgamma RIIIb was depleted from the cell surface by incubation with Pronase (data not shown). These data thus indicate distinct roles for individual Fcgamma R in the control of these intracellular Ca2+ signals in primed and unprimed neutrophils. Thus, the initial intracellular Ca2+ transient that is seen in both unprimed and primed neutrophils appears to be due to signals generated via occupancy of Fcgamma RII; this intracellular Ca2+ signal arises from the mobilization of intracellular stores. In contrast, the extra intracellular Ca2+ signal that is only seen in primed cells requires Fcgamma RIIIb and arises from Ca2+ influx.

Role of Fcgamma R Ligation of Individual Receptors

To confirm the above conclusions on the roles of Fcgamma RII and Fcgamma RIIIb in the differential intracellular Ca2+ signals, cross-linking Fab/F(ab')2 fragments of specific anti-Fcgamma R mAbs was employed. Incubation of unprimed neutrophils with Fab fragments of IV3 (anti-Fcgamma RII), followed by cross-linking with goat anti-mouse F(ab')2 fragments, resulted in a monophasic increase in intracellular Ca2+ (Fig. 7A). The kinetics of this intracellular Ca2+ increase were unaltered when the cells were primed with either GM-CSF (Fig. 7A) or TNFalpha (data not shown), but a greater intracellular Ca2+ transient was observed in primed cells. The kinetics of these increases in intracellular Ca2+ resemble those observed during incubation of unprimed neutrophils with soluble immune complexes (Fig. 2). However, when unprimed neutrophils were incubated with F(ab')2 fragments of 3G8 (anti-Fcgamma RIIIb) prior to cross-linking, no increases in intracellular Ca2+ were observed (Fig. 7B). Thus, ligation of Fcgamma RIIIb in unprimed neutrophils fails to generate increases in intracellular Ca2+. However, if the cells were primed with either GM-CSF (Fig. 7B) or TNFalpha (data not shown), then cross-linking of 3G8 resulted in increases in intracellular Ca2+. The kinetics of these increases in intracellular Ca2+ were significantly slower than those observed following ligation of Fcgamma RII and very similar to the kinetics of the extra intracellular Ca2+ signal only seen in primed cells (Fig. 2). These results thus clearly show that ligation of Fcgamma RII can lead to rapid intracellular Ca2+ transients in either primed or unprimed neutrophils and the priming process does not alter the kinetics of these transients. In contrast, ligation of Fcgamma RIIIb results in elevations in intracellular Ca2+, but only in cells that are primed. Thus, priming leads to a functional activation of Fcgamma RIIIb that results in its ability to generate intracellular Ca2+ transients via Ca2+ influx.


Fig. 7. Stimulation of intracellular Ca2+ transients by cross-linking Fcgamma Rs. Fluo-3-loaded neutrophils were incubated in the absence (Unprimed) or presence (Primed) of 50 units/ml GM-CSF for 60 min, prior to addition of Fab fragments of anti-Fcgamma RII (IV3) in A or F(ab')2 fragments of anti-Fcgamma RIIIb (3G8) in B, as described under "Experimental Procedures." At the point indicated (GAM), cross-linking was achieved by addition of F(ab')2 fragments of goat anti-mouse IgG. Similar results have been obtained in four other separate experiments and when cells were primed by TNFalpha .
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When intracellular Ca2+ levels were measured by cross-linking Fcgamma RII in primed neutrophils similar responses were observed in Ca2+-containing and in Ca2+-free medium (containing 1 mM EGTA) (Fig. 8A). This result indicates that the Ca2+ signal generated following ligation of this receptor arises largely from the mobilization of intracellular stores. In complete contrast, the Fcgamma RIIIb-dependent intracellular Ca2+ signal was greatly decreased when the cells were suspended in Ca2+-free medium (Fig. 8B), indicating that Ca2+ influx is largely responsible for the intracellular Ca2+ signal following ligation of this receptor.


Fig. 8. Role of extracellular Ca2+ in Fcgamma R mediated intracellular Ca2+ transients. Fluo-3-loaded neutrophils were primed and then incubated as described in the legend to Fig. 7. Suspensions were incubated either in Ca2+-free medium (supplemented with 1 mM EGTA) or Ca2+-containing medium (1 mM), prior to cross-linking Fcgamma Rs.
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The Fab/F(ab')2 fragments were then used to confirm the roles of these Fcgamma Rs in the generation of intracellular Ca2+ signals in response to soluble immune complexes. Addition of 3G8 F(ab')2 fragments (anti-Fcgamma RIIIb) prior to the addition of soluble immune complexes had no effect on the intracellular Ca2+ transients generated in unprimed cells (Fig. 9A), but significantly decreased the extra Ca2+ signal observed in primed cells (Fig. 9B). However, addition of IV3 Fab fragments (anti-Fcgamma RII) prior to the addition of soluble immune complexes inconsistently affected the intracellular Ca2+ transients. It may be that mAb IV3 is readily displaced from Fcgamma RII by the soluble immune complexes. These experiments, however, confirm a role for Fcgamma RIIIb in the extra intracellular signal seen in primed cells.


Fig. 9. Effect of blocking Fcgamma RIIIb on intracellular Ca2+ transients in primed and unprimed neutrophils. Neutrophils were incubated in the absence (A, Unprimed) or presence (B, Primed) of 50 units/ml GM-CSF for 60 min and loaded with Fluo-3 AM during the final 30 min. They were then incubated with F(ab')2 fragments of 3G8 (anti-Fcgamma RIIIb), as described under "Experimental Procedures." At the point indicated by the arrow, 10% (v/v) soluble immune complexes were added. Similar results have been obtained when neutrophils were primed with TNFalpha . Typical results of five separate experiments.
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IP3 Production following Neutrophil Activation

Stimulation of neutrophils with fMet-Leu-Phe resulted in a rapid and transient elevation in the levels of IP3 (Fig. 10A), which peaked within 10-15 s and then declined. However, ligation of either Fcgamma RII (Fig. 10B) or Fcgamma RIIIb (Fig. 10C) failed to activate the production of significant levels of IP3 in either primed or unprimed cells.


Fig. 10. Changes in IP3 levels during neutrophil activation by fMet-Leu-Phe and Fcgamma Rs. In A, neutrophils were incubated in the presence (bullet ) or absence (open circle ) of 0.1 µM fMet-Leu-Phe, and at time intervals samples were removed for the analysis of IP3, as described under "Experimental Procedures." In B and C, neutrophils were incubated in the presence (open circle ) and absence (bullet ) of 50 units/ml GM-CSF for 45 min prior to cross-linking either Fcgamma RII (B) or Fcgamma RIIIb (C), as described "Experimental Procedures." Values are means (± S.D. of triplicate measurements). Similar results were obtained in three separate experiments using different preparations of neutrophils.
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DISCUSSION

In this study we have investigated the mechanisms by which soluble immune complexes activate neutrophils via Fcgamma receptors. Defining this interaction is important because several immune conditions are associated with neutrophil activation via immune complexes (1-3). These soluble immune complexes fail to activate the respiratory burst when added to suspensions of unprimed cells. However, if the cells are primed (as in our study using the cytokines GM-CSF and TNFalpha ), then soluble immune complex binding results in the secretion of large quantities of reactive oxygen metabolites. Because neutrophil function in vivo is invariably regulated by exposure to priming agents and because these soluble immune complexes activate the secretion of potentially tissue-damaging components, these results are important in understanding the molecular pathology of certain neutrophil-mediated conditions. In our study, we have used a neutrophil isolation procedure that induces minimal priming during cell isolation. Many commonly used isolation methods (33, 34, 42), however, inadvertently prime both receptor number/function and NADPH oxidase activity and hence experiments with cells isolated by such methods cannot clearly distinguish between neutrophil responses that are restricted to either the primed or the unprimed state.

Whereas the soluble immune complexes failed to activate the NADPH oxidase in unprimed cells, the use of fluorescein isothiocyanate-labeled complexes showed that they clearly bind to the cell surface (data not shown). These fluorescein isothiocyanate-labeled complexes activated neutrophils in identical ways to the unlabeled complexes, indicating that their molecular properties were unaltered during the labeling process. We have previously shown that the synthetic soluble immune complexes used in these studies and those isolated from rheumatoid synovial fluid, activate primed and unprimed neutrophils by analogous mechanisms (12, 43). The fact that these complexes activated increases in intracellular Ca2+ in unprimed cells is indicative that they bind to functionally active receptors on the cell surface. Hence, we conclude that the key event that occurs during priming that allows the primed cell to generate reactive oxidants in response to soluble immune complexes is not increased complex binding. Indeed, priming results in only small changes in the level of expression of either Fcgamma RII or Fcgamma RIIIb, whereas surface expression of CR3 is greatly up-regulated (44). We thus searched for events downstream of receptor/ligand binding that may account for the altered functional responses of primed cells.

In unprimed cells, the soluble immune complexes activated a transient increase in intracellular Ca2+ that was due to mobilization of intracellular stores and was partly sensitive to inhibition with tyrosine kinase inhibitors. Several other reports have shown that, while intracellular Ca2+ transients are necessary for NADPH oxidase activation, they are in themselves not sufficient to induce activation (45, 46). When the cells were primed, however, an extra intracellular Ca2+ transient was generated. This arose from influx of Ca2+ from external sources and was also partly sensitive to inhibition by tyrosine kinase inhibitors. These data indicate that this extra intracellular Ca2+ signal may be due to signals generated via receptors that become "activated" during the priming process. We thus set out to identify these putative receptors.

Circulating blood neutrophils possess two types of receptors that recognize IgG-containing immune complexes, namely Fcgamma RII and Fcgamma RIIIb (14). Fcgamma RIIIb is linked to the plasma membrane via a glycosylphosphatidylinositol linkage that can be easily cleaved either in vivo or in vitro with Pronase or PI-PLC (15, 17). Our first experiments thus used Pronase and PI-PLC to deplete the cell surface of Fcgamma RIIIb while not affecting expression of Fcgamma RII. Fcgamma RIIIb-depleted (unprimed) neutrophils could generate intracellular Ca2+ transients in response to soluble immune complexes, and the kinetics of these transients were identical with those observed in untreated (Fcgamma RIIIb-expressing) cells. However, the extra intracellular Ca2+ signal normally observed during priming was not observed after Fcgamma RIIIb depletion. Furthermore, this extra intracellular Ca2+ signal was not observed when primed cells were incubated F(ab')2 fragments of 3G8 (anti-Fcgamma RIIIb) prior to addition of soluble immune complexes. Taken together, our experiments indicate that in unprimed neutrophils, soluble immune complexes bind to the cell surface via binding to Fcgamma RII and this results in an increase in intracellular Ca2+: this interaction fails to activate the NADPH oxidase. Upon priming, the soluble complexes now generate an extra intracellular Ca2+ signal that arises via the functional activation of Fcgamma RIIIb. This extra Ca2+ signal arises from Ca2+ influx and requires protein-tyrosine phosphorylation. Experiments cross-linking individual receptors confirm these conclusions that ligation of Fcgamma RII can elevate intracellular Ca2+ via internal store mobilization in either primed or unprimed cells, whereas ligation of Fcgamma RIIIb can only elevate intracellular Ca2+ via Ca2+ influx in primed cells. If the traces of the intracellular Ca2+ transients generated via ligation of Fcgamma RII and Fcgamma RIIIb are combined, they closely resemble the kinetics of intracellular Ca2+ transients observed when primed and unprimed neutrophils are stimulated with soluble immune complexes. It was noteworthy that ligation of either Fcgamma RII or Fcgamma RIIIb failed to activate the synthesis of substantial levels of IP3, in agreement with previous reports (31, 32).

There are numerous reports in the literature regarding the interaction of immune complexes with neutrophil Fc receptors, but none have addressed the interaction of soluble complexes with primed and unprimed cells. In our experiments, the intracellular Ca2+ transients generated by Fcgamma RII ligation were partly sensitive to inhibition with tyrosine kinase inhibitors, confirming the recently reported association of Fcgamma RII with tyrosine kinase activity (23, 24, 26, 27). Our data also confirm the importance of tyrosine kinase activity and Fcgamma RIIIb function (27), and we propose that tyrosine kinase activity becomes associated with Fcgamma RIIIb during priming and regulates Ca2+ influx. We speculate that during priming a Src-like tyrosine kinase (perhaps Hck; Ref. 27) becomes associated with Fcgamma RIIIb. This could then result in the assembly of a new signaling cassette linking Fcgamma RIIIb ligation to cell activation. Several reports have shown that Fcgamma RIIIb can transduce signals, including intracellular Ca2+ transients independently of Fcgamma RII (28-30). A synergistic response of Fcgamma RIIIb and CR3 has previously been reported, whereby CR3 immobilizes Fcgamma RII to the plasma membrane via a cytoskeletal-dependent mechanism and then ligation of Fcgamma RIIIb induces tyrosine activation in the proximity of Fcgamma RII (25). In our experiments using synthetic soluble immune complexes, anti-CR3 antibodies do not affect the ability of primed neutrophils to activate a respiratory burst. Thus, the dependence of the response on CR3 may be diminished in primed neutrophils.

These studies shed new insights into the mechanisms by which soluble immune complexes activate neutrophils in pathological conditions that may result in tissue damage. There is much interest in the pharmacological down-regulation of neutrophil function in inflammatory conditions, but any therapy designed to interfere with neutrophil-mediated responses must be selective; such therapy must specifically block the harmful consequences of neutrophil activation that lead to tissue damage (e.g. result in secretion of cytotoxic products) but not compromise opsono-phagocytosis and host defense against infections. It is interesting to note that individuals with Fcgamma RIIIb deficiency are often asymptomatic and do not generally suffer recurrent infections (47, 48). Therefore, we predict that neutrophils from these individuals will not be able to secrete reactive oxygen metabolites in response to soluble immune complexes, and thus it will be necessary to test this hypothesis using neutrophils from these individuals. If correct, then our data offer the possibility that selective inhibition of the signaling pathway from Fcgamma RIIIb/soluble immune complex ligation to activation of the NADPH oxidase could provide the basis for a novel therapeutic strategy for the treatment of immune disorders involving soluble immune complexes and primed neutrophils.


FOOTNOTES

*   This work was supported by project grants from the Arthritis and Rheumatism Council, United Kingdom.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Dagger    To whom all correspondence should be addressed. Tel.: 44-151-794-4363; Fax: 44-151-794-4349; E-mail: sbir12{at}liverpool.ac.uk.
1   The abbreviations used are: GM-CSF, granulocyte-macrophage colony-stimulating factor; mAb, monoclonal antibody; PI, phosphoinositide; PLC, phospholipase C; TNFalpha , tumor necrosis factor alpha ; IP3, inositol 1,4,5-trisphosphate.

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