©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Distinct Tyrosine Kinase Activation and Triton X-100 Insolubility upon FcRII or FcRIIIB Ligation in Human Polymorphonuclear Leukocytes
IMPLICATIONS FOR IMMUNE COMPLEX ACTIVATION OF THE RESPIRATORY BURST (*)

Ming-jie Zhou , Douglas M. Lublin (1), Daniel C. Link (2), Eric J. Brown (§)

From the (1) Division of Infectious Diseases, Division of Laboratory Medicine, and (2) Division of Hematology, Washington University School of Medicine, St. Louis, Missouri 63110

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Two tyrosine kinase-dependent pathways exist for activation of the respiratory burst by polymorphonuclear leukocyte (PMN) immunoglobulin G Fc receptors. Direct ligation of FcRII activates the respiratory burst, but ligation of the glycan phosphoinositol-linked FcRIIIB does not. Instead, this receptor and the integrin complement receptor CR3 synergize in activation of the respiratory burst (Zhou, M.-J., and Brown, E. J. (1994) J. Cell Biol. 125, 1407-1416). Here we show that direct ligation of FcRII leads to activation and Triton X-100 insolubility of the Src family kinase Fgr, without effect on the related myeloid Src family member Hck. In contrast, adhesion of PMN via FcRIIIB leads to activation and Triton X-100 insolubility of Hck but not Fgr. The exclusive association of FcRIIIB with Hck activation and Triton insolubility is not solely a result of its glycan phosphoinositol anchor, since decay accelerating factor (CD55), another prominent glycan phosphoinositol-anchored PMN protein, is associated with Fgr insolubility to a greater extent than Hck. Ligation of decay accelerating factor, with or without coligation of CR3, does not activate the PMN respiratory burst. Coligation of FcRIIIB with FcRII overcomes the pertussis toxin inhibition of HO production in response to direct ligation of FcRII. These data support the hypothesis that activation of Hck upon FcRIIIB ligation has a role in generation of the synergistic respiratory burst.


INTRODUCTION

Binding of immune complexes to cell receptors for immunoglobulin is a powerful stimulus to activation of phagocytes. Immune complex binding to these cells leads to activation of effector functions of host defense such as phagocytosis, secretion, cytokine synthesis, and the production of toxic oxygen metabolites, which occurs as a result of NADPH oxidase assembly (1) . The cloning of several members of the immunoglobulin Fc receptor family over the past several years has tremendously enhanced understanding of the molecular mechanisms for the functions of these immunologically important receptors. Identification of receptors within the family which associate with a second transmembrane protein, called the chain, and realization that the chain is itself a member of a family of proteins known to be involved in tyrosine kinase-mediated signal transduction has further enhanced understanding of Fc receptor-mediated cell activation. The two Fc receptors expressed on polymorphonuclear leukocytes (PMN),() FcRII and the glycan phosphoinositol (GPI)-linked form of FcRIII (FcRIIIB) are distinct within this family for their failure to associate with the chain, suggesting alternative mechanisms for cell activation by these receptors. Nonetheless, even FcRII appears to activate tyrosine kinases and to be phosphorylated on tyrosine during immune complex-mediated cell activation (2) . FcRII has been found to associate with specific Src family kinases, including Fgr in PMN, and to activate syk kinase (3, 4, 5) . Ligation of FcRII leads to the tyrosine phosphorylation of multiple cellular proteins, including phospholipase C, Shc, and syk, in addition to FcRII itself (6-8).

In contrast, little is known about the molecular mechanisms of signal transduction from the PMN FcRIIIB. This receptor is unique among Fc receptors, since it is expressed on the plasma membrane by a GPI anchor, rather than as a transmembrane protein. It has no association with the chain, which associates with the transmembrane form of FcRIII expressed in NK cells and macrophages, and has no intracytoplasmic domain for direct association with cytosolic signal transduction cascades. Indeed, whether FcRIIIB mediates signal transduction at all remains controversial. Many effects of immune complexes on PMN appear to be mediated exclusively by FcRII (9, 10, 11) , although this is not universally the case (10, 12) . Cross-linking of FcRIIIB can lead to an increase in intracytoplasmic Ca ([Ca] ), although the mechanism for this effect is unclear (13) . Moreover, GPI-linked proteins in several cell types have been associated with membrane domains rich in Src family tyrosine kinases (14) , and evidence has been presented recently for an association of PMN FcRIIIB with tyrosine kinase activity (15, 16) . Thus, the role for FcRIIIB in PMN activation remains uncertain.

Recently, we have developed an assay system which allows a more detailed investigation of the contribution of individual PMN Fc receptors to signal transduction leading to generation of a respiratory burst (15) . We have used PMN adhesion to surfaces coated with monoclonal antibodies (mAb) to individual receptors to assess their contribution to cell activation. We have found that direct ligation of FcRII leads to a respiratory burst, whereas direct ligation of FcRIIIB does not. Instead, FcRIIIB cooperates with the PMN integrin CR3 (Mac-1, CD11b/CD18) to generate what we have termed a synergistic respiratory burst. In the synergistic respiratory burst, the two membrane receptors have distinct roles. Ligation of CR3 immobilizes FcRII to the adherent plasma membrane by a cytoskeleton-dependent mechanism, and ligation of FcRIIIB induces appropriate tyrosine kinase activation in proximity to the immobilized FcRII. Thus, FcRII is required in addition to CR3 and FcRIIIB for the synergistic respiratory burst. In the current work, we have examined the nature of the Src family kinases activated by ligation of the two Fc receptors. We have found that FcRII and FcRIIIB immobilized on the adherent PMN surface by direct ligation lead to the activation and Triton X-100 insolubility of different Src family kinases. FcRII is associated with activation and translocation of Fgr to the Triton-insoluble cell fraction; and FcRIIIB is associated with Hck activation and translocation. The exclusive association of FcRIIIB with Hck activation is not a property of all GPI-linked proteins in PMN, since immobilization of decay accelerating factor (DAF, CD55) leads primarily to Fgr activation at the adherent membrane. Moreover, DAF cannot substitute for FcRIIIB in synergistic activation of the respiratory burst. From these data we conclude that ligation of FcRII and FcRIIIB activate and translocate distinct Src family members in PMN. We hypothesize that the functional consequence of the activation and translocation of distinct kinases is the existence of two separate signal transduction pathways for activation of the PMN respiratory burst by immune complexes.


MATERIALS AND METHODS

Antibodies

The following mAb were used in these studies: IB4 (anti-CD18) (17) , W6/32 (anti-HLA) (18) , B6H12 (anti-CD47) (19) , 3D9 (anti-CD35) (20) , 3G8 (anti-CD16) (21) , IH4 (anti-CD55) (22) , OKM1 (anti-CD11b) (23) , and IV.3 (anti-CD32) (24) . IB4, 3G8, and IV.3 IgG were purified from ascites using octanoic acid as described (25) . W6/32 and B6H12 IgG were prepared using an Amicon Bioreactor (Amicon Inc., Danvers, MA) according to manufacturer's instructions. SDS-PAGE of all purified IgG preparations showed them to be >90% IgG. PY20 (anti-phosphotyrosine) was purchased from Transduction Laboratories (Lexington, KY). Polyclonal anti-Fgr was as described (26, 27) or was purchased from Santa Cruz Biotechnology (Santa Cruz, CA), as was anti-Hck.

Buffers and Other Reagents

Phosphate-buffered saline was from Biowhittaker, Walkersville, MD. Krebs-Ringer buffer (KRPG) was 145 mM NaCl, 4.86 mM KCl, 1.22 mM MgSO, 5.7 mM NaHPO, 0.54 mM CaCl, and 5.5 mM glucose, pH 7.4. Reaction mixture (RM) consisted of 37.5 µM scopoletin, 1.25 mM NaN, 1.25 units/ml HPO in KRPG. Kinase buffer was 40 mM Hepes, pH 7.5, 10 mM MgCl, 3 mM MnCl, 0.1 mM NaVO, 10% glycerol, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride. MBS buffer was 25 mM Mes, pH 6.5, 0.5% Triton X-100, 150 mM NaCl, 1 mM phenylmethylsulfonyl fluoride, 5 mM NaF, 5 mM diisopropyl fluorophosphate, 1 mM NaVO, 1 mM iodoacetamide, 10 µg/ml each of aprotinin, leupeptin, and pepstatin A. Other reagents were obtained from standard sources as described previously (28) .

PMN Isolation

Human PMN were isolated by dextran sedimentation and two-step Hypaque density centrifugation as described (29). PMN were obtained from a integrin-deficient (leukocyte adhesion-deficient, LAD) patient followed at Baylor College of Medicine. This patient has been characterized as having the complete deficiency phenotype. Expression of FcRII and FcRIII on this patient's cells was normal, whereas CR3 was undetectable (not shown). The patient's blood and a normal control were transported and the PMN prepared as described (30) .

Preparation of Coated Plate and HOAssay

96-Well tissue culture plates (Costar, Cambridge, MA) were coated with protein A and then mAb IgG essentially according to the method of Berton et al.(31) , with modifications as described (28) . The microwell HO assay was adapted from the method of De la Harpe and Nathan (32) , as modified by Berton et al.(31) . Data were collected and analyzed exactly as described previously (28) . In each experiment, data were averaged from triplicate wells, which generally varied from each other by 10%. Unless otherwise stated, HO accumulation was measured after 60 min of PMN incubation in wells.

Pretreatment of PMN

PMN at 2.5 10 cells/ml in KRPG were pretreated with 5 µg/ml of Fab or 2.5 µg/ml of F(ab`) of various mAb at 4 °C for 15 min. Without washing, PMN were added to antibody coated plates containing RM (28) . For treatment with pharmacologic agents, PMN were preincubated with herbimycin at 10 µg/ml and then added to RM containing the same concentration of the indicated agent. To pretreat PMN with pertussis toxin, cells in KRPG were incubated with 2 µg/ml of pertussis toxin at 37 °C for 75 min. In experiments examining the effects of cytochalasin, PMN were pretreated with 5 µg/ml cytochalasin D and added to RM containing no additional drug. In all experiments, control PMN were incubated with an identical concentration of nonaqueous diluent as the PMN receiving any drug.

``In Situ'' Tyrosine Phosphorylation

5 10 PMN in KRPG with 0.1 mM NaVO were allowed to adhere to mAb-coated six-well plates at 4 °C for 10 min and then at 37 °C for 15 min. The nonadherent cells were removed by washing with cold MBS buffer. The adherent cells were extracted with 0.5% Triton X-100 in MBS buffer on ice for 8 min, following which the plate was rinsed with kinase buffer containing 0.1 mM NaVO and 0.2% Triton X-100 once. In situ tyrosine phosphorylation was performed by adding 50 µCi of [-P]ATP and 1 µM cold ATP in 500 µl of kinase buffer at room temperature for 15 min. For immunoprecipitation, following washing with the kinase buffer three times, the cell residuals were solubilized with solubilization buffer (20 mM Tris, pH 8.0, 150 mM NaCl, 1% Triton X-100, 1% deoxycholate, 0.1% SDS, 10 µg/ml each of aprotinin, leupeptin, and pepstatin A, 1 mM phenylmethylsulfonyl fluoride, 2 mM diisopropyl fluorophosphate, 1 mM EDTA, 1 mM NaVO) at 4 °C for 60 min with shaking. The lysates were diluted with solubilization buffer without SDS and then precleared with protein A-Sepharose at 4 °C for 2 h. The precleared lysate was incubated with anti-Fgr, -Hck, or -phosphotyrosine IgG captured by protein A-Sepharose at 4 °C for 2 h. The Sepharose mixture was washed once with solubilization buffer and then six times in the same buffer without deoxycholate, SDS, and protease inhibitors. The Sepharose-bound proteins were analyzed by SDS-PAGE as described (33) .

Immune Complex Kinase Assay

To determine whether Fgr and Hck were activated by adhesion, PMN were allowed to adhere to surfaces coated with various mAb for 15 min at 37 °C. After MBS buffer extraction, supernatant was collected and the Triton X-100-insoluble fraction solubilized in solubilization buffer as above. Both the Triton X-100-soluble and -insoluble fractions were immunoprecipitated with anti-Fgr or anti-Hck. Following immunoprecipitation, the immune complexes were incubated with 5 µCi of [-P]ATP in 30 µl of kinase buffer at room temperature containing 20 µg of poly(Glu,Tyr) (Glu:Tyr ratio 4:1, Sigma) for 15 min. The reaction was stopped by adding 6 µl of 10% phosphoric acid and centrifugation. The supernatants were spotted onto Whatman 3MM cellulose filter paper (1.5 1.5 cm), and the filters were washed with 10% trichloroacetic acid four times, 5 mM phosphoric acid, and 80% acetone once each. After air-drying the filters, the P incorporation was measured by scintillation counting. Alternatively, the supernatants were loaded into the sample bucket of a phosphocellulose unit (Pierce) and washed according to the manufacturer's instructions. In experiments in which the Triton X-100-insoluble cell fraction was immunoprecipitated with PY20, cell residuals were incubated with kinase buffer containing 10 mM unlabeled ATP at room temperature for 15 min prior to solubilization and immunoprecipitation.

Western Blot

Following PMN adhesion on mAbs and bovine serum albumin-coated plates, the adherent cells were extracted with 0.5% Triton X-100 in KRPG buffer with protease inhibitors on ice for 5 min. The cell residuals were lysed by solubilization buffer with 0.2% SDS. The recovered lysates were precipitated by adding 5 volumes of cold acetone and kept at -20 °C for 30 min. After centrifugation at top speed in an Eppendorf centrifuge at 4 °C for 10 min, the pellets were washed with 80% acetone twice and allowed to air-dry. The precipitated proteins were dissolved by SDS-sample buffer with 5 mM -mercaptoethanol and 2 mM EDTA and separated by SDS-PAGE, then transferred to polyvinylidene difluoride (Millipore, Bedford, MA). The membranes were incubated overnight in blocking buffer (PBS Tween and 3% bovine serum albumin) and then with anti-Hck and anti-Fgr polyclonal IgG at 1:1000 at room temperature for 4 h. The membranes finally were incubated with horseradish peroxidase-conjugated protein A and developed using the ECL chemiluminescence kit (Amersham Corp.).


RESULTS

FcRII or FcIII Ligation in PMN Leads to Activation of Distinct Src Family Tyrosine Kinases

Our previous studies have shown distinct roles for the two PMN Fc receptors in activation of the respiratory burst. PMN adhesion to an FcRII ligand leads to a respiratory burst, whereas the GPI-linked FcRIIIB is required for the synergistic respiratory burst, but is incapable of activation of the NADPH oxidase on its own. Nonetheless, our previous data demonstrate that both pathways are inhibited by tyrosine kinase inhibitors and suggest that FcRIIIB ligation is associated with tyrosine kinase activity (15) . To begin to understand tyrosine kinase function in the two pathways, we examined activation of two predominant PMN Src family kinases, Fgr and Hck, upon ligation of PMN Fc receptors. As assessed by phosphorylation of poly(Glu,Tyr), ligation of FcRII led to Fgr activation, whereas ligation of FcRIIIB led to Hck activation (Fig. 1A). Fgr activation has been associated previously with FcRII ligation in PMN (4) . Ligation of either HLA or CR3 did not lead to a detectable increase in the activity of either kinase. Total Fgr activity increased approximately 6.5-fold (average of two determinations) in cells adherent via FcRII compared with ligation of HLA, and total Hck activity increased more than 20-fold upon FcRIII ligation (Fig. 1A). A small amount of Fgr kinase activity could be detected in the Triton X-100-soluble fraction in PMN incubated on the control surfaces. However, neither Fgr nor Hck kinase activity was detected in the Triton X-100-insoluble fraction from PMN adherent via HLA or CR3. After either FcRII or FcRIIIB ligation, kinase activity was increased in both Triton X-100 soluble and insoluble cell fractions. Thus, Fgr kinase activity was stimulated by ligation of FcRII; Hck activity was stimulated by ligation of FcRIIIB.


Figure 1: Hck kinase activity is enhanced by FcRIIIB ligation, whereas Fgr kinase activity is enhanced by FcRII ligation. A, Hck activity (left) and Fgr activity (right) were measured in the Triton X-100-soluble and -insoluble fractions from PMN adherent to anti-FcRIIIB, anti-FcRII, anti-CR3, or anti-HLA. Kinase activity was measured using the substrate poly(Glu,Tyr) as described under ``Materials and Methods.'' Shown is a single experiment, performed in duplicate, representative of three. B, Hck (left) and Fgr (right) protein were measured in the Triton X-100-soluble and -insoluble cell fractions from cells adherent to anti-FcRIIIB and anti-HLA (left) or anti-FcRII and anti-HLA (right). 2 10 cell equivalents were used as the starting material for Hck and Fgr immunoprecipitation from the Triton X-100-soluble fraction; 10 10 cell equivalents were used for the immunoprecipitations from the Triton X-100-insoluble fraction. The data are from one of two similar experiments. C, the relative ``specific activities'' of Triton X-100-soluble and -insoluble Fgr and Hck were compared for cells adherent to anti-FcRIII (Hck) and to anti-FcRII (Fgr). Relative specific activities were estimated by dividing kinase activity by protein concentration estimated from densitometry of Western blots. Specific activity of each kinase in the Triton X-100-soluble fraction of PMN adherent to anti-HLA was set to 1. Fgr specific activities in the Triton X-100-soluble and -insoluble fractions of PMN adherent to anti-FcRII were 5.5 and 32, respectively; Hck specific activities in the Triton X-100-soluble and -insoluble fractions were 33.5 and 230. Specific activities of Fgr and Hck are not comparable with each other, because different antibodies were used for the Western blots. The graph depicts data averaged from two experiments.



FcRII or FcRIII Ligation Leads to Association of Distinct Src Family Kinases with the Triton X-100-insoluble Cell Fraction

Src family kinases often associate with the Triton X-100-insoluble cytoskeleton upon activation, and Src-induced oncogenic transformation may require cytoskeletal association (34) , suggesting that important kinase substrates are encountered through translocation to the cytoskeleton. Since Triton X-100 insolubility may reflect this translocation to the cytoskeleton, we examined the differences in the Triton X-100 solubility of Fgr and Hck upon receptor ligation. PMN were adhered to mAb-coated surfaces, briefly solubilized with Triton X-100, and the cell residue analyzed by SDS-PAGE and Western blotting (Figs. 1B and 2). Hck was present in the Triton X-100- insoluble cell fraction if, and only if, PMN were adherent to anti-FcRIIIB (Fig. 2A). Whether or not anti-CR3 also was present on the adherent surface made no difference for Hck localization, even though respiratory burst activation only occurs when both antibodies are present. Fgr was present in the Triton X-100-insoluble fraction only when FcRII was ligated on the adherent surface (Fig. 2B). Unlike FcRIIIB, ligation of a different GPI-linked protein, DAF, led predominantly to Fgr localization in the Triton X-100-insoluble residue. When cells were adherent to anti-CR3 alone, no Fgr or Hck protein was detectable. Specific activity of Fgr and Hck was estimated in both the Triton X-100-soluble and -insoluble fractions after FcR ligation by comparing kinase activity (Fig. 1A) to protein concentration as estimated from the Western blots (Fig. 1B). Interestingly, although active Fgr or Hck was present in both Triton X-100-soluble and -insoluble compartments, the specific activity of both kinases was 6-7-fold greater in the Triton X-100-insoluble fraction after appropriate receptor ligation (Fig. 1C). Presumably, this reflects the fact that not all Triton X-100-soluble kinase has been activated by receptor ligation.


Figure 2: Hck and Fgr protein localization to the Triton X-100-insoluble fraction upon Fc receptor ligation. The Triton X-100-insoluble residue from PMN adherent to various surfaces was solubilized and analyzed by SDS-PAGE and immunoblotting. The mAb used to coat the surfaces to which the PMN were adherent before Triton X-100 extraction and solubilization are indicated in each lane. Each lane contains 10 10 PMN. A, immunoblot with anti-Hck. B, immunoblot with anti-Fgr. Hck was detected if and only if PMN were adherent to anti-FcRIIIB; Fgr was present when PMNs were adherent to anti-FcRII or anti-DAF.



Tyrosine Kinase Activity in the Triton X-100-insoluble Fraction after Ligation of FcRII or FcRIIIB

To examine the phosphorylation patterns in the Triton X-100-insoluble fractions after FcRII or FcRIIIB ligation, an in situ kinase assay was performed after extraction of adherent PMN with Triton X-100. After addition of [P]ATP to the Triton X-100-insoluble residue adherent to the immune complex-coated surfaces, phosphotyrosine-containing proteins were examined by immunoprecipitation of the products of the in vitro kinase assay with anti-phosphotyrosine mAb, PY20, after solubilization with a more stringent detergent (1% Triton X-100, 1% deoxycholate, 0.1% SDS). As shown in Fig. 3, phosphotyrosine-containing proteins with molecular mass of 55-60 kDa were the major products of the in vitro kinase assay when PMN were adherent to either an anti-FcRII- or an anti-FcRIIIB- coated surface. Under the same conditions, these phosphotyrosine-containing proteins were not detected from PMN adherent to anti-HLA- or anti-CD47-coated surfaces. Importantly, no tyrosine kinase activity was found in PMN adherent to an anti-CR3-coated surface (Fig. 3), even though FcRII is present in the Triton X-100-insoluble material (15) , and CR3 participates in the synergistic respiratory burst. No reproducible kinase activity was found in anti-FcRII or anti-FcRIIIB immunoprecipitates from unstimulated, nonadherent PMN (data not shown).


Figure 3: Kinase activity associates with the Triton X-100-insoluble PMN fraction on engagement of Fc receptors. PMN adherent on mAb-coated surfaces were extracted with 0.5% Triton X-100 in MBS buffer with protease inhibitors for 8 min. Following an in situ kinase assay (see ``Materials and Methods''), phosphotyrosine-containing proteins were immunoprecipitated with anti-phosphotyrosine mAb, PY20, and then analyzed by SDS-PAGE.



The autophosphorylated Triton X-100-insoluble material also was immunoprecipitated with monospecific antibodies against several Src family kinases. No radiolabeled proteins were immunoprecipitated by antibodies to Yes, Fyn, Src, or Lck from PMN adherent to any surface (data not shown). Phosphorylated proteins from lysates of PMN adherent to an anti-FcRIIIB coated surface were specifically immunoprecipitated by anti-Hck, but not by anti-Fgr (Fig. 4). As described previously (26, 35) , the autophosphorylated Hck appeared as a doublet. Conversely, a phosphorylated band was immunoprecipitated by anti-Fgr, but not by anti-Hck from the lysate from an anti-FcRII-coated surface. When autophosphorylated lysates from an anti-HLA coated surface were precipitated by both anti-Hck and anti-Fgr, no phosphorylated bands were identified, consistent with the minimal kinase activity associated with the Triton X-100-insoluble material remaining from PMN adherent to this substrate (see Fig. 1A and 3).


Figure 4: Hck and Fgr associated with the Triton X-100-insoluble cell fraction after FcRIIIB or FcRII ligation have kinase activity. Following an in situ kinase reaction using [P]ATP on the Triton X-100-insoluble fractions from PMN adherent to various surfaces (Coating Ab), immunoprecipitation was performed on the products of the kinase reaction with anti-Fgr, anti-Hck, or anti-phosphotyrosine (PY20) (IP Ab).



Hck Is the Predominant Triton X-100-insoluble Tyrosine Kinase Associated with FcRIIIB Ligation, and Fgr Is the Predominant Triton X-100-insoluble Tyrosine Kinase Associated with FcRII Ligation

To determine whether Hck was the predominant tyrosine kinase associated with the Triton X-100-insoluble material from FcRIIIB ligation and Fgr the predominant kinase associated with FcRII ligation, poly(Glu,Tyr) phosphorylation was assayed. When lysates from anti-FcRIII coated surface were immunoprecipitated with anti-Hck, the anti-Hck immune complexes displayed marked tyrosine kinase activity (Fig. 5). Minimal tyrosine kinase activity could be immunoprecipitated by PY20 after anti-Hck preclearing. In contrast, anti-Fgr precipitated almost no poly(Glu,Tyr) phosphorylating activity from the PMN adherent via FcRIIIB. When lysates from anti-FcRII-coated surface were immunoprecipitated with anti-Fgr and anti-Hck, the anti-Fgr immune complexes contained 80% of the tyrosine kinase activity. There was very little detectable Hck tyrosine kinase activity from lysates from anti-FcRII coated surfaces (Fig. 5). Immunoprecipitates with PY20 after anti-Fgr preclearing contained minimal kinase activity toward poly(Glu,Tyr). Thus, we concluded that Hck accounted for almost all the Triton X-100-insoluble tyrosine kinase activity associated with FcRIIIB ligation, and Fgr accounted for the kinase activity associated with FcRII ligation. These data suggest that Hck specifically associates with the Triton-insoluble cell fraction from cells adherent via FcRIIIB, and Fgr kinase specifically associates with the Triton-insoluble cell fraction following adhesion via FcRII.


Figure 5: Hck and Fgr are the major tyrosine kinases associated with the Triton X-100-insoluble fraction after FcR ligation. PMN were lysed with Triton X-100 after adhesion to various surfaces, as described in the legend to Fig. 4. Immunoprecipitations with anti-Hck, anti-Fgr, or anti-phosphotyrosine were performed. Kinase activity in the immunoprecipitates was determined using poly(Glu,Tyr) as substrate. Activity is plotted as substrate-associated counts/min after a 15-min reaction. Data are mean ± S.E. of three experiments. Hck accounts for the tyrosine kinase activity from PMN adherent via FcRIIIB, and Fgr accounts for the predominant kinase activity from PMN adherent via FcRII or DAF.



To determine whether the apparent exclusive association of Hck activity with FcRIIIB ligation was true for all GPI-linked PMN proteins, PMN were allowed to adhere to anti-DAF-coated surfaces and Triton X-100-insoluble kinase activity determined (Fig. 5). Unlike PMN adherent to anti-FcRIIIB, cells adherent to anti-DAF showed more Fgr than Hck activity.

Requirement for Both FcRIIIB and FcRII for Synergistic Respiratory Burst Activation

The signal transduction involved in activation of the respiratory burst by direct ligation of FcRII or by synergy between FcRIIIB and CR3 differ in several respects, including sensitivity to pertussis toxin (PT) and cytochalasin D (15) . Direct ligation of FcRII activates HO production, which is inhibited by PT (Fig. 6A); activation of the synergistic respiratory burst is unaffected by PT. Because our previous studies suggested that the role for CR3 in the synergistic respiratory burst was to immobilize FcRII in proximity to FcRIIIB, we tested whether direct ligation of FcRII and FcRIIIB together could activate the synergistic respiratory burst, by taking advantage of the PT sensitivity of the respiratory burst generated by direct ligation of FcRII. PT inhibited the respiratory burst generated by ligation of FcRII, but coligation of FcRIIIB with FcRII restored the respiratory burst in the presence of PT (Fig. 6B). Ligation of FcRIIIB specifically is required for generation of the respiratory burst with FcRII in the presence of PT, since neither coligation of DAF, another prevalent GPI-linked protein on PMN, nor of HLA, could stimulate respiratory burst in combination with FcRII in the presence of PT (Fig. 6B). Coligation of FcRIIIB and DAF also failed to stimulate the respiratory burst (not shown). Addition of Fab or F(ab`) fragments of mAbs against either FcRII or FcRIIIB specifically inhibited the respiratory burst stimulated by coligation of FcRIIIB with either FcRII or CR3 (Fig. 7A). mAb against CR3 inhibited the PMN respiratory burst by coligation of FcRIIIB with CR3, but had no effect on the respiratory burst generated by coligation of FcRII and FcRIIIB in the presence of PT (Fig. 7A). Thus, coligation of FcRII and FcRIIIB in the presence of PT is a second form of the synergistic respiratory burst in which the need for CR3 has been eliminated.


Figure 6: Fc receptor activation of a PMN respiratory burst through PT-inhibitable and PT-independent pathways. The generation of HO by normal PMN and LAD PMN adherent to wells coated with various mAbs is compared with (dark bar) and without PT (light bar) treatment of the PMN. In A the respiratory burst generated from normal PMN incubated with various single mAb is plotted. In B is plotted the respiratory burst generated from normal PMN incubated with combinations of mAbs coating the wells. C plots the respiratory burst generated by LAD PMN. Data in all panels is HO production at 60 min. Data from normal PMN are means ± S.E. of three independent experiments, each performed in triplicate. A single experiment (of two independent experiments, each performed in triplicate) with LAD PMN is shown in C. PT inhibits the respiratory burst from direct ligation of FcRII. Coligation of FcRIIIB restores the respiratory burst in the presence of anti-FcRII, even though ligation of FcRIIIB alone does not lead to activation of HO production. Maximal respiratory burst stimulated with 50 ng/ml PMA was 2.46 ± 0.42 nmol at 60 min.




Figure 7: Effects of mAbs and pharmacologic reagents in solution on PMN respiratory burst stimulated by surface-bound mAbs. A, Effect of antibodies. PMN were preteated with 5 µg/ml of Fab fragments (anti-FcRII) or 2.5 µg/ml of F(ab`) fragments (anti-CD11b and anti-FcRIIIB). PMN were incubated in wells coated with anti-FcRII or the combination of anti-FcRIIIB and anti-CR3 in the absence of PT or the combination of anti-FcRIIIB and anti-FcRII in the presence of PT. HO production was measured in the presence and absence of anti-receptor antibodies. Anti-CD47, which blocks the integrin -stimulated HO production (28), did not affect any FcR-dependent respiratory burst (data not shown). B, effect of pharmacologic reagents. PMN were pretreated with 2 µg/ml of pertussis toxin, 5 µg/ml of cytochalasin D, or both. Other PMN were pretreated with 10 µg/ml herbimycin A. These PMN were incubated in wells coated with anti-FcRII, anti-FcRIII, and anti-CR3 as in A. HO production was measured after 60-min PMN incubation with the various surfaces. Control HO production, assessed for PMN incubated with diluents for the various reagents, was identical to the buffer control in A (not shown). A single experiment performed in triplicate, representative of three, is shown.



These data suggested that the only role for CR3 in the synergistic respiratory burst was to immobilize FcRII on the adherent surface of the PMN in proximity to the ligated FcRIIIB. To test this hypothesis, we examined LAD PMN which genetically lack CR3. Coligation of FcRIIIB with FcRII activated PT intoxicated LAD PMN normally (Fig. 6C), even though these PMN lacked the synergistic respiratory burst from adhesion to surfaces coated with anti-FcRIIIB and anti-CR3 (15) . As a second test of the hypothesis, we examined the effect of cytochalasin D on the synergistic respiratory burst. Although the synergistic activation by adhesion to anti-CR3 and anti-FcRIIIB is sensitive to cytochalasin D (15) , the PT-insensitive respiratory burst in response to coligation of FcRII and FcRIIIB was not (Fig. 7B). Since cytochalasin has been shown to inhibit CR3-mediated immobilization of FcRII on the adherent PMN membrane (15), this experiment proves that coimmobilization of FcRII and FcRIIIB by antibodies overcomes the need for cytoskeletal assembly in the synergistic respiratory burst.


DISCUSSION

Immune complexes are potent stimuli for activation of PMN. Immune complex deposition and subsequent PMN activation is an important part of the pathogenesis of serum sickness, the Arthus reaction, acute glomerulonephritis, rheumatoid arthritis, and other idiopathic inflammatory diseases. Although these are host-damaging diseases, immune complex-mediated PMN activation also plays an essential role in host defense against bacterial infection. It is clear that in vivo both host defense and host damaging aspects of the PMN-immune complex interaction involve complement activation and deposition onto the immune complexes (36) . The major PMN receptor for the complement deposited onto immune complexes is the leukocyte integrin CR3. Therefore, to understand immune complex activation at a molecular level requires understanding the roles for both Fc receptors and CR3. Although the role for CR3 in this activation process was originally thought to be passive, merely increasing the interaction between IgG and its cellular receptors (37) , there is now good evidence that in some circumstances CR3 ligation contributes actively to signal transduction (11, 38) .

In the current work, we have studied signal transduction during the synergistic respiratory burst and compared it with the signal transduction cascade activated by direct ligation of FcRII. We have shown that the role for CR3 in the synergistic respiratory burst is to immobilize FcRII in proximity to ligated FcRIIIB. Indeed, activation of Fgr and Hck through Fc receptor ligation occurs normally in LAD PMN (data not shown). Thus, unlike the respiratory burst induced by tumor necrosis factor- stimulation of PMN (39) , there is no evidence that CR3 or any other integrin is involved in Fgr activation through FcRII. In this sense, the initial postulate of Ehlenberger and Nussenzweig (37) that complement simply enhances immune complex presentation to phagocyte Fc receptors, is correct. However, it is clear that signal transduction in the synergistic respiratory burst is more complex, since it requires two different Fc receptors, with distinct roles for each.

By using PT, which suppresses the respiratory burst from direct ligation of FcRII, we were able to reconstitute the synergistic respiratory burst in the complete absence of CR3. This experiment proves our early hypothesis that FcRII plays an essential role in the synergistic respiratory burst, even when it is immobilized only by its association with CR3 and not directly ligated (15) . In this situation, we found no tyrosine kinase activity associated with the immobilized FcRII. In contrast, direct ligation of FcRII led to Fgr tyrosine kinase activation and association of a significant fraction of the active kinase with the Triton-resistant adherent cell membrane. An association between FcRII and Fgr in PMN has been reported previously (4) . In our assay system at least, stable association of Fgr with the Triton X-100-insoluble cytoskeleton requires direct ligation of FcRII. Even unactivated Fgr protein is not associated with the Triton X-100-insoluble cytoskeleton in the absence of direct FcRII ligation. Presumably for this reason, FcRII immobilized by association with ligated CR3 is unable to signal assembly of the NADPH oxidase. Because of the nature of the assay, we do not know if the FcRII-Fgr association we detect is direct or indirect. The presence of Fgr in the Triton X-100-insoluble cell residue after FcRII ligation may depend on other Triton-insoluble PMN proteins. It is also possible that active Fgr associates with phosphorylated tyrosine residues in the FcRII cytoplasmic tail (4, 5, 7) and has no direct association with cytoskeletal proteins.

Although direct evidence that Fgr kinase activity is involved in NADPH oxidase assembly is lacking, we believe it very likely that Fgr has a role in generation of the respiratory burst from direct ligation of FcRII. The anti-FcRII-generated respiratory burst is extremely sensitive to tyrosine kinase inhibitors ( Fig. 6and Ref. 15). Since a major difference between direct ligation of FcRII with antibody (which is associated with HO production) and its indirect immobilization through CR3 ligation (which leads to no respiratory burst) is the presence of Triton X-100-insoluble kinase-active Fgr, it is likely that Fgr is involved in initiation of the respiratory burst after direct FcRII ligation. This pathway for generation of the respiratory burst is sensitive to PT. However, the association of active Fgr with FcRII after direct ligation is unaffected by PT, and the in situ kinase assay reveals no different phosphorylated proteins in pertussis-intoxicated cells. Thus, the PT sensitivity of the signal transduction cascade must result from inhibition at a step beyond Fgr association with receptor and its activation.

Immobilized FcRII is required for a respiratory burst even in pertussis-intoxicated PMN. Although FcRII is phosphorylated on tyrosine during Fc receptor-mediated PMN activation (8, 15) , it is unlikely that this is the only phosphorylation essential for cell activation. FcRII phosphorylation has been separated from signal transduction for phagocytosis in PMN (8) . Furthermore, phosphorylation of FcRII itself in response to its direct ligation is unaffected by PT, even though pertussis intoxication inhibits the respiratory burst.() These data suggest that there is another, unidentified, protein associated with FcRII which is needed to signal assembly of the NADPH oxidase. A possible candidate is syk, a kinase known to be involved in immune complex-mediated PMN activation and known to be phosphorylated by FcRII ligation (6, 40) . However, a direct physical association of syk with FcRII or other Triton-insoluble proteins has not been demonstrated.

Ligation of FcRIIIB is associated with Hck kinase activation and translocation to the Triton X-100-insoluble cell fraction. The existence of Hck in PMN is well described (41) , but its specific role has been unclear. This exclusive association of FcRIIIB ligation with Hck activation is not a property of all GPI-linked PMN proteins, since DAF ligation is associated predominantly with Fgr rather than Hck. This may explain why DAF cannot substitute for FcRIIIB in the synergistic respiratory burst. However, other differences, such as the association of extracellular domains of FcRIIIB with specific membrane proteins (42) , may also contribute to the lack of equivalence between DAF and FcRIIIB in the synergistic respiratory burst. Nonetheless, in PMN, whether a particular Src family kinase is activated and becomes Triton insoluble by ligation of a specific GPI-linked protein depends on more than simply the mechanism of membrane attachment of the receptor.

Thus, we hypothesize that the role for ligation of FcRIIIB in the synergistic respiratory burst is to bring Hck into proximity with FcRII, when the FcRII has no associated kinase or has had its kinase activation pathway blocked by PT (Fig. 8). This appears to be an essential early step in activation of the synergistic respiratory burst. Hck activation of the downstream effector pathway for assembly of the NADPH oxidase is not affected by PT, providing an alternative pathway for cell activation. Since FcRII immobilization is required for the synergistic respiratory burst in the absence of associated Fgr, it is tempting to speculate that the downstream target for both Hck in the synergistic respiratory burst and Fgr in direct ligation of FcRII is the same protein physically associated with FcRII. Activation of this target by either a PT-insensitive Hck-dependent pathway or a PT-sensitive Fgr-dependent pathway could then lead to a final common pathway for NADPH oxidase assembly.


Figure 8: Hypothetical signal transduction pathways for Fc receptor activation of the PMN respiratory burst. Ligation of FcRII alone activates a pathway leading to assembly of the NADPH oxidase which is PT-sensitive. The synergistic pathway can be activated by coligation of FcRIIIB with CR3 and is pertussis toxin-insensitive. The role for CR3 in the synergistic respiratory burst is to immobilize FcRII in proximity to FcRIIIB, and its associated Hck kinase, by a cytoskeleton-dependent mechanism. FcRII immobilized on the adherent surface of PMN by direct ligation is associated with the tyrosine kinase Fgr, whereas FcRII immobilized by CR3 ligation is unassociated with a kinase. This suggests that the two pathways for respiratory burst activation, both of which require immobilized FcRII, may be initiated by different Src family kinases.



In summary, we have shown that in PMN ligated FcRII and FcRIIIB are associated with distinct Src family kinases. FcRII leads to Fgr activation and FcRIIIB to Hck activation. The Hck-dependent pathway initiates what we have termed the synergistic respiratory burst. In the absence of pertussis toxin, this pathway for PMN activation apparently requires three different receptors: CR3, FcRII, and FcRIIIB. It is possible that immune complexes deposited in tissue, by ligating FcRII and FcRIIIB simultaneously, could activate the synergistic pathway in the absence of complement. Indeed, the PMN respiratory burst in response to insoluble immune complexes in vitro is PT-insensitive (43, 44) , consistent with activation of the synergistic pathway. Moreover, pathologic immune complexes activate complement (36) , and complement deposition onto immune complexes actually decreases, rather than increases, the efficiency of IgG interaction with Fc receptors (45) . For this reason and because there are about 10-fold more FcRIIIB than FcRII on PMN, the most physiologically relevant interaction of immune complexes with PMN is likely to be via CR3 and FcRIIIB; direct interaction with the less abundant FcRII will likely be minimized by complement deposition. These are precisely the conditions which lead to the synergistic respiratory burst. We believe that the Hck-dependent synergistic pathway for PMN activation is likely to be extremely relevant to immune complex activation of PMN in vivo.


FOOTNOTES

*
This work was supported by Grants AI35811, AI24674, and GM38330 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Division of Infectious Diseases, Campus Box 8051, Washington University School of Medicine, 660 S. Euclid Ave., St. Louis, MO 63110. Tel.: 314-362-2125; Fax: 314-362-9230.

The abbreviations used are: PMN, polymorphonuclear leukocyte; FcRII, IgG Fc receptor, type 2; FcRIII, IgG Fc receptor, type 3; CR3, complement receptor type 3; DAF, decay accelerating factor, CD55; mAb, monoclonal antibody; PT, pertussis toxin; Mes, morpholinoethanesulfonic acid.

M.-J. Zhou and E. J. Brown, unpublished data.


ACKNOWLEDGEMENTS

We are indebted to Dr. Donald Anderson for provision of blood from a patient with LAD and Dr. Andrey Shaw for antibodies against Fyn, Yes, Lck, and Src. We also thank various members of the Brown laboratory for helpful comments during the execution of these experiments.


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