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Originally published online as doi:10.1189/jlb.0504310 on January 20, 2005

Published online before print January 20, 2005
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(Journal of Leukocyte Biology. 2005;77:787-799.)
© 2005 by Society for Leukocyte Biology

Fc{gamma}RIIIB stimulation promotes ß1 integrin activation in human neutrophils

Alejandro Ortiz-Stern and Carlos Rosales1

Immunology Department, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, Mexico City

1 Correspondence: Department of Immunology, Instituto de Investigaciones Biomédicas–UNAM, Apdo. Postal 70228, Cd. Universitaria, México D.F.–04510, Mexico. E-mail: carosal{at}servidor.unam.mx


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ABSTRACT
 
The molecular stimuli involved in receptor-induced integrin activation are still poorly defined. We have investigated the role of receptors for the Fc portion of immunoglobulin G molecules (Fc{gamma}R) on activation of integrins in human neutrophils. Cross-linking of Fc{gamma}RIIA induced an increase in surface expression of ß2 integrins but had no effect on ß1 integrins. In contrast, cross-linking of Fc{gamma}RIIIB not only increased ß2 integrins on the cell surface but also induced ß1 integrin activation, as indicated by an increase in binding to fibronectin and the appearance of an activation epitope detected by the monoclonal antibody 15/7. The Fc{gamma}RIIIB-induced increase of ß2 integrins required Src-family tyrosine kinases, Syk kinase, and phosphatidylinositol-3 kinase (PI-3K), as the corresponding, specific inhibitors, PP2, Piceatannol, and LY294002, completely blocked it. Contrary to this, Fc{gamma}RIIIB-induced ß1 integrin activation was not blocked by PP2 or LY294002. It was, however, enhanced by Piceatannol. After Fc{gamma}RIIIB cross-linking, colocalization of Fc{gamma}RIIIB and active ß1 integrins was detected on the neutrophil membrane. These data show, for the first time, that cross-linking of Fc{gamma}RIIIB induces an inside-out signaling pathway that leads to ß1 integrin activation. This activation is independent of Src-family kinases, and PI-3K and may be induced in part by the interaction of Fc{gamma}RIIIB with ß1 integrins.

Key Words: Fc receptors • inside-out signaling • PI-3K • ERK • Syk • calcium


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INTRODUCTION
 
Human neutrophils (PMN) represent one of the first lines of defense against invading pathogens [1 ]. PMN are recruited early to sites of infection, and their numbers increase notoriously during systemic inflammatory responses. One powerful stimulus for activation of PMN is immune complexes [2 ], which are recognized by PMN through their receptors for the Fc portion of immunoglobulin G (IgG) molecules, Fc{gamma} receptors (Fc{gamma}R) [3 , 4 ]. Human neutrophils present two types of Fc{gamma}R, Fc{gamma}RIIA and Fc{gamma}RIIIB [5 ]. Both receptors have low affinity for monomeric IgG but recognize immune complexes efficiently. Fc{gamma}RIIA consists of a single {alpha} chain, which transverses the membrane. Fc{gamma}RIIA has, within its cytoplasmic region, an immunoreceptor tyrosine-based activation motif (ITAM) [6 ]. The tyrosines within this ITAM get phosphorylated upon receptor cross-linking and serve as docking sites for Src homology 2 domain-containing proteins, which further propagate the signal inside the cell [2 ]. Fc{gamma}RIIIB, conversely, is a glycophosphatidyl inositol (GPI)-linked protein, which does not cross the membrane [5 ]. The signaling mechanism of Fc{gamma}RIIIB is not clear to date, but it has been proposed that interactions with the {alpha}Mß2 integrin or the Fc{gamma}RIIA may be responsible for its signaling capacities [7 8 9 10 11 12 13 ]. Nonetheless, there are responses elicited by Fc{gamma}RIIIB, which are not replicated by the stimulation of {alpha}Mß2 or Fc{gamma}RIIA [14 15 16 17 ], suggesting that Fc{gamma}RIIIB is capable of signaling on its own or requires another yet-uncharacterized signaling partner.

Other important mediators of PMN responses are integrins [18 ], which are heterodimeric transmembrane proteins capable of altering their ligand-binding capacity in response to signals arising from inside the cell [19 , 20 ]. This process of integrin modulation has been termed inside-out signaling [21 ], which allows the cell to rapidly modulate its adhesive state in response to extracellular input. Several integrins have been shown to be subjects of this inside-out signaling regulation [20 , 22 ]. In PMN, the affinity of ß2 and ß1 integrins is tightly regulated [23 24 25 26 27 28 29 30 ].

One important consequence of Fc{gamma}R stimulation in PMN is the activation and up-regulation of ß2 integrins [24 , 31 32 33 ]. This activation of ß2 integrins is important for adhesion to immune complexes, enhanced Fc{gamma}R-mediated phagocytosis, antibody-dependent cell-mediated cytotoxicity, and the production of certain inflammatory mediators [10 , 24 , 34 ]. It has been demonstrated that Fc{gamma}RIIA and Fc{gamma}RIIIB, when cross-linked independently with monoclonal antibodies (mAb), are capable of inducing activation [32 ] and also up-regulation of ß2 integrins [32 ]. This response is dependent on the activity of phosphatidylinositol-3 kinase (PI-3K), as pharmacological inhibition of this enzyme prevents up-regulation and activation of ß2 integrins in response to Fc{gamma}R stimulation [24 ]. In other cell types, stimulation of FcR for IgE (Fc{gamma}RI) has been shown to affect the affinity of ß1 integrins but not the expression levels of ß1 integrins on the cell surface [35 , 36 ]. Fc{gamma}RI stimulation induced activation of {alpha}4ß1 integrins in human basophils [36 ] and of {alpha}5ß1 in mast cells [35 ]. Both responses required the activity of PI-3K [35 , 36 ]. To date, there are no reports describing the effects of Fc{gamma}R stimulation in PMN on the activation and up-regulation of ß1 integrins. We set to explore whether Fc{gamma}R stimulation of PMN affects ß1 integrin activation by selectively cross-linking each Fc{gamma}R with mAb and analyzing the activation state of integrins.

We found that cross-linking of Fc{gamma}RIIIB in addition to inducing up-regulation of ß2 integrin expression also induced ß1 integrin activation. The Fc{gamma}RIIIB-induced signaling pathway to ß1 integrin activation was independent of PI-3K and Src-family tyrosine kinases but may be caused by interaction of Fc{gamma}RIIIB with ß1 integrins on the PMN membrane.


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MATERIALS AND METHODS
 
Antibodies and reagents
The following antibodies were used: anti-extracellular signal-regulated kinase (anti-ERK) rabbit polyclonal IgG and anti-phospho-ERK (anti-pERK) rabbit polyclonal IgG from Santa Cruz Biotechnology (Santa Cruz, CA). Dr. Eric J. Brown (University of California, San Francisco) donated anti-integrin ß2 (mAb IB4). Anti-Fc{gamma}RII (mAb IV.3), anti-Fc{gamma}RIII (mAb 3G8), and anti-major histocompatibility complex (anti-MHC) class I (mAb W6/32) were from American Type Culture Collection (Manassas, VA). Anti-ß1 integrin (mAb MAR4) coupled to Cy-chrome was from BD PharMingen (Torrey Pines, CA); Dr Martin Hemler (Dana Farber Cancer Research Institute, Boston, MA) provided anti-ß1 integrin (mAb TS2/16). Anti-activated ß1 integrin (mAb 15/7) was a kind gift from Dr. Ted Yednock (Elan Pharmaceuticals, San Francisco, CA). The specific PI-3K inhibitors wortmannin and LY294002, the protein kinase A (PKA) inhibitor H89, the PKC inhibitor Staurosporine, and the specific Syk inhibitor Piceatannol were from Calbiochem (San Diego, CA). The Src-family tyrosine kinase inhibitor PP2 was from Upstate Biotechnology (Charlottesville, VA). The mitogen-activated protein kinase kinase 1 (MEK)-1-specific inhibitor PD98059 was from New England Biolabs (Beverly, MA). The glutathione S-transferase (GST)-Fn9-11 fusion protein cloned in the pGEX plasmid was a kind gift from Dr. Mark Ginsberg (Scripps Research Institute, La Jolla, CA) and has been described previously [37 ]. GST alone was purified from plasmid pGEX-4T-1, a kind gift from Dr. Pavel Isa (Instituto de Biotecnologia, Universidad Nacional Autónoma de México, Mexico). All other chemicals were from Sigma Chemical Co. (St. Louis, MO).

Purification of neutrophils
Neutrophils were obtained from heparinized venous blood from healthy adult donors and purified by standard techniques as described previously [38 , 39 ].

Preparation of Fab and F(ab')2 fragments
Pepsin digestion of mAb IV.3, 3G8, and W6/32 was carried out essentially as described [2 ]. Briefly, antibodies were diluted at 2 mg/ml in 0.1 M sodium citrate, pH = 3.5, and pepsin was added at 25 µg/ml. The mixture was incubated at 37°C for 45 min for mAb IV.3, 4 h for mAb 3G8, and 4 h for mAb W6/32. Incubation was stopped by adding 1/10 vol 3 M Tris, pH = 8.6. Fab and F(ab')2 fragments were dialyzed against phosphate-buffered saline (PBS), and undigested IgG was removed with protein G sepharose (Amersham-Pharmacia, Uppsala, Sweden). Purity of Fab and F(ab')2 fragments was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and silver-staining of the gels.

Fluorescein isothocyanate (FITC) labeling of Fab and F(ab')2 antibody fragments
NaHCO3 (1/10 vol; 1 M, pH=9.0) was added to Fab or F(ab')2 antibody fragments in PBS. Isomer I of FITC (iFITC), diluted at 10 mg/ml in dimethyl sulfoxide (DMSO), was added at a molar ratio of 40:1 (iFITC:protein) and shaken continuously for 1 h at room temperature. Excess iFITC was removed by dialysis against PBS. FITC labeling of antibody fragments was assessed by flow cytometry. FITC-labeled fragments retained the properties of unlabeled antibody fragments.

Fc{gamma}R stimulation
PMN (1x107 cell/ml) in PBS (150 mM NaCl, 5.5 mM Na2HPO4, 1.2 mM KH2PO4), containing 2 mM Ca2+ and 2 mM Mg2+, were incubated with 5 µg/ml Fab fragments of mAb IV.3 or with 5 µg/ml F(ab')2 fragments of mAb 3G8 at 4°C during 1 h. For some experiments, PMN were concomitantly incubated with enzyme inhibitors. PMN were then washed and resuspended in the same volume of PBS containing 40 µg/ml F(ab')2 goat anti-mouse IgG (GAM) (ICN/Cappel, Costa Mesa, CA) and immediately placed at 37°C for various periods of time. Stimulation was terminated by the addition of ice-cold PBS.

Western blot
ERK or activated pERK was detected by immunoblotting with the corresponding antibody at 75 ng/ml, as described [40 ].

Fluorescent calcium measurements
PMN were loaded with Fura-2/AM (Calbiochem), and intracellular calcium concentrations were determined and calculated with an LS-55 spectrofluorimeter (Perkin-Elmer, Wellesly, MA), as described previously [41 , 42 ].

Biotinylation
mAb IB4, mAb 15/7, GST, and the GST-Fn9-11 recombinant protein were biotinylated using N-hydroxysulfosuccinimido biotin (NHS-biotin) (Pierce, Rockford, IL), according to the manufacturer’s instructions. Briefly, protein was diluted at a concentration of 1 mg/ml in PBS. Sulfo-NHS-biotin was added at a molar ratio of 20:1 (sulfo-NHS-biotin:protein). The reaction took place for 1 h at room temperature. Excess sulfo-NHS-biotin was removed by dialysis against PBS.

Flow cytometry [fluorescein-activated cell sorter (FACS)]
Stimulated PMN (1x106) were diluted in 100 µl cold flow buffer (1% sucrose, 0.5% bovine serum albumin, in PBS) and incubated with 5 µg/ml of the corresponding, biotinylated mAb at 4°C during 45 min. PMN were then washed three times with cold flow buffer, resuspended in 20 µl flow buffer containing a 1/500 dilution of Cy-chrome streptavidin (Pierce), and incubated at 4°C for 30 min. PMN were then washed three more times with cold flow buffer and finally, fixed in 300 µl 1% paraformaldehyde in PBS.

Purification of GST and GST-Fn9-11
The central cell-binding domain of fibronectin (Fn9-11), containing the integrin Arg-Gly-Asp-binding site [43 ] coupled to GST, was purified as a recombinant protein (GST-Fn9-11) as described previously [43 ]. Briefly, Escherichia coli, carrying a plasmid containing GST-Fn9-11 under the lac promoter, were grown to absorbance at 600 nm = 0.5, 1 mM isopropylthiogalactoside was added, and bacteria were incubated for 2 h more. Bacteria from a 400-ml culture were collected by centrifugation and lysed by sonication in 20 ml cold PBS. Bacterial lysates were clarified by centrifuging 10 min at 10,000 rpm in a JA-20 rotor in a J2-MC centrifuge (Beckman Coulter, Miami, FL). Supernatant was then passed over a glutathione sepharose column (Amersham-Pharmacia), which was washed with PBS until no protein could be recovered. The GST-Fn9-11 recombinant protein was eluted with 10 mM free reduced glutathione. GST-Fn9-11-containing fractions were pooled and dialyzed against PBS. Purity of the recombinant protein was confirmed by SDS-PAGE. Purified protein was finally stored at –70°C until use. The same procedure was used to purify GST alone, albeit bacteria were transformed with a plasmid coding only for recombinant GST.

Binding of GST-Fn9-11 to neutrophils
Fc{gamma}R-stimulated PMN (1x106) in 100 µl cold flow buffer plus Ca2+ and Mg2+ were incubated with 5 µg/ml biotinylated GST-Fn9-11 recombinant protein at 4°C for 45 min. PMN were then washed three times with cold flow buffer and resuspended in 20 µl flow buffer containing a 1/500 dilution of Cy-chrome streptavidin (Pierce). Cells were washed three more times with cold flow buffer and finally fixed in 300 µl 1% paraformaldehyde in PBS for FACS analysis. For GST versus GST-Fn9-11 fluorescence, glutathione sepharose beads were incubated with the biotinylated protein for 10 min, washed three times with cold flow buffer, stained with Cy-chrome streptavidin, washed three more times, fixed in 1% paraformaldehyde in PBS, and analyzed by flow cytometry.

Confocal microscopy
PMN were stimulated as described under Fc{gamma}R stimulation (see above), but FITC-labeled Fab and F(ab')2 antibody fragments were used instead of unlabeled antibody fragments. Stimulated PMN were stained as described under flow cytometry (see above). Once labeled, PMN were allowed to settle overnight, and excess liquid was discarded. The cell pellet was resuspended in 10 µl mounting buffer [10% 1,4-diazabicyclo(2.2.2)octane, 50% glycerol, in PBS], and cells were mounted on slides for microscopical analysis. All images were acquired with an LSM 5 PASCAL confocal microscope (Carl Zeiss, Jenna, Germany), using a Zeiss LSM image examiner. Excitation at 80.6% of the 488 nm channel and 100% of the 633 nm was used. Filters for FITC emission were set to 505–530 nm, and filters for Cy-chrome emission were set to 650 nm.

Up-regulation/activation indexes
For experiments with enzyme inhibitors, the up-regulation and activation indexes were calculated according to the next formula: Index = (MFSI–MFI)/(MFS–MF), where MFSI is the mean fluorescence of inhibitor-treated, stimulated cells; MFI is the mean fluorescence of inhibitor-treated, unstimulated cells; MFS is the mean fluorescence of DMSO-treated, stimulated cells; and MF is the mean fluorescence of DMSO-treated, unstimulated cells. The same cells were analyzed for IB4 and 15/7 binding in each experiment.

Internalization of Fc{gamma}RIIA and Fc{gamma}RIIIB
Human neutrophils were incubated for 1 h at 4°C with the FITC-labeled Fab or F(ab')2 antibody fragments of mAb IV.3 and mAb 3G8, respectively. After this time, cells were left unstimulated or were stimulated as above by cross-linking with goat anti-mouse antibodies for 10 min at 37°C. At the end of this period, cells were washed three times with cold flow buffer and fixed with 1% paraformaldehyde in PBS. Samples were divided in two, and after 30 min, Trypan blue was added to one of the samples to a final concentration of 0.2%, and cells were analyzed by flow cytometry. Fluorescence was normalized to the MFI of the unquenched sample.

Statistical analysis
For experiments with enzyme inhibitors, data were analyzed with a Student’s t-test, using Simple Interactive Statistical Analysis (http://home.clara.net/sisa/t-test.htm). Differences were considered statistically significant when P ≤ 0.01.


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RESULTS
 
Fc{gamma}R cross-linking activates ERK and generates calcium fluxes
It has been reported previously that Fc{gamma}R engagement in neutrophils leads to activation of ERK [14 ] and stimulation of calcium fluxes [12 , 17 , 41 ]. It seems that Fc{gamma}RII or Fc{gamma}RIII causes this effect [14 ]. We decided to confirm these results by selectively cross-linking each Fc{gamma}R with specific mAb. To avoid binding of these mAb through their Fc portions, we prepared Fab fragments of anti-Fc{gamma}RII mAb IV.3 and F(ab')2 fragments of anti-Fc{gamma}RIII mAb 3G8. The mAb IV.3 is sensitive to pepsin digestion, so only Fab fragments can be obtained [44 ]. Cross-linking of Fc{gamma}RII or Fc{gamma}RIIIB caused a strong activation of ERK, as indicated by the appearance of the phosphorylated form of this enzyme (data not shown). As with ERK phosphorylation, cross-linking of Fc{gamma}RII or Fc{gamma}RIIIB induced a rapid and transient raise in intracellular calcium [39 ] (data not shown). Unlike stimulation of Fc{gamma}RII and Fc{gamma}RIIIB, cross-linking of MHC class I molecules did not have an effect on ERK phosphorylation (data not shown) or on intracellular calcium levels (data not shown). These results indicated that Fc{gamma}RII and Fc{gamma}RIIIB were capable of signaling independently of each other when they were cross-linked with Fab fragments of mAb IV.3 and F(ab')2 fragments of mAb 3G8.

Fc{gamma}R cross-linking promotes ß2 integrin up-regulation
As Fc{gamma}RII and Fc{gamma}RIIIB have been shown to promote up-regulation and activation of ß2 integrins [24 , 32 ], we studied the behavior of ß2 and also ß1 integrins after Fc{gamma}R stimulation by flow cytometry. Neutrophils that had been stimulated previously by Fc{gamma}RII or Fc{gamma}RIIIB cross-linking were stained with a biotinylated anti-ß2 integrin mAb IB4 followed by Cy-chrome streptavidin or with a Cy-chrome-conjugated anti-ß1 integrin mAb MAR4. Membrane expression of ß2 integrins increased on the surface of Fc{gamma}RII-stimulated neutrophils (Fig. 1A ). This up-regulation became evident 5 min after stimulation and did not decline for up to 30 min (data not shown). Surface expression of ß2 integrins on the surface of neutrophils also increased after Fc{gamma}RIIIB stimulation (Fig. 1B) . This up-regulation also became evident 5 min after stimulation and did not decline for up to 30 min (data not shown). Unlike ß2 integrins, membrane expression of ß1 integrins on the surface of neutrophils did not change after cross-linking of Fc{gamma}RII or Fc{gamma}RIIIB (Fig. 1C and 1D) . These data indicated that Fc{gamma}RII and Fc{gamma}RIIIB can up-regulate ß2 integrins but not ß1 integrins on PMN.



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  		Figure 1. Fc{gamma}R cross-linking up-regulates ß2 integrins but not ß1 integrins. Human neutrophils were stimulated for 10 min by cross-linking Fc{gamma}RII (A and C) or Fc{gamma}RIIIB (B and D). Cells (1x106) were stained with anti-ß2 integrin mAb IB4 (A and B) or with anti-ß1 integrin mAb MAR4 (C and D) for flow cytometry analysis of integrin surface expression. Dotted line is the isotype-matched, negative staining. Gray area is the integrin surface expression of unstimulated cells, and the bold line is the integrin surface expression after Fc{gamma}R cross-linking. Data are representative of three separate experiments.

Fc{gamma}RIIIB cross-linking promotes ß1 integrin activation
Although there was no change in membrane expression of ß1 integrins on the surface of Fc{gamma}R-stimulated neutrophils (Fig. 1C and 1D) , it was still possible that integrins could be activated to increase their ligand-binding capacity in response to Fc{gamma}R stimulation, as has been shown to occur after Fc{gamma}R stimulation [35 , 36 ]. This increase in the binding capacities of integrins is thought to occur through a conformational change in the molecule [19 , 30 , 45 ]. Certain anti-integrin antibodies have the capacity to recognize these conformational changes. These antibodies are thus useful in identifying activated integrins. One antibody that recognizes activated ß1 integrins is the mAb 15/7 [30 ]. Cross-linking of Fc{gamma}RII produced no change in the binding of mAb 15/7 (Fig. 2A ), indicating that Fc{gamma}RII stimulation does not induce ß1 integrin activation. In contrast, we found that cross-linking Fc{gamma}RIIIB produced a clear increase in the binding of mAb 15/7 (Fig. 2B) . This result showed for the first time that cross-linking Fc{gamma}RIIIB induces ß1 integrin activation. To confirm that this conformational change is indeed responsible for an increase in ß1 integrin ligand-binding capacity, we looked at direct binding of a natural integrin ligand. We used the recombinant protein GST-Fn9-11, which contains the central-binding domain of fibronectin [37 ]. Cross-linking of Fc{gamma}RII had no effect on GST-Fn9-11 binding to PMN (Fig. 2C) , indicating similarly to mAb 15/7, that ß1 integrins are not activated by Fc{gamma}RII stimulation. In contrast, Fc{gamma}RIIIB cross-linking produced a clear increase in the binding of GST-Fn9-11 (Fig. 2D) . This binding depends on the Fn9-11 portion of the GST-Fn9-11 recombinant protein, as no increase in the binding of GST alone could be detected after Fc{gamma}RIIIB stimulation (Fig. 3A ). It is important to notice that binding of GST and GST-Fn9-11 could be detected with equal intensity bound to glutathione sepharose beads (Fig. 3B) . To further strengthen the fact that fibronectin binding after Fc{gamma}RIIIB is dependent on the activation of ß1 integrins, a blockade of ß2 or ß1 integrins with mAb was performed after PMN stimulation but before GST-Fn9-11 binding. The blockade of ß2 integrins had no effect on GST-Fn9-11 binding (Fig. 3C) ; nonetheless, the blockade of ß1 integrins clearly abolished the fibronectin binding (Fig. 3D) . The increased binding of the GST-Fn9-11 in response to Fc{gamma}RIIIB stimulation required Ca2+ and Mg2+, as performing the same experiment in the absence of these ions did not augment binding of the fibronectin fragment (data not shown). Activation of ß1 integrins secondary to Fc{gamma}RIIIB stimulation is evident 3 min after F{gamma}RIIIB cross-linking and declines after 30 min (data not shown). These data confirmed that Fc{gamma}RIIIB cross-linking on PMN induces ß1 integrin activation and that this activation results in a better binding of fibronectin to ß1 integrins.



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  		Figure 2. Fc{gamma}RIIIB cross-linking activates ß1 integrins. Human neutrophils were stimulated for 10 min by cross-linking Fc{gamma}RII (A and C) or Fc{gamma}RIIIB (B and D). Cells (1x106) were stained with anti-activated ß1 integrin mAb 15/7 (A and B) or with GST-Fn9-11 fragment (C and D) for flow cytometry analysis of ß1 integrin activation. Dotted line is the isotype-matched, negative staining; shaded area is the integrin activation status of unstimulated cells; and bold line is the integrin activation status after Fc{gamma}R cross-linking. Data are representative of three separate experiments.



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  		Figure 3. Fibronectin binding after Fc{gamma}RIIIB depends on ß1 integrins. Human neutrophils were stimulated for 10 min by cross-linking Fc{gamma}RIIIB (A, C, and D). Cells (1x106) were stained with GST alone (A) or with GST-Fn9-11 fragment (C and D) for flow cytometry analysis of ß1 integrin activation. Immediately after stimulation, cells incubated for 10 min on ice with an excess amount (20 µg/ml) of (C) anti-ß2 integrin mAb IB4 or (D) anti-ß1 integrin mAb TS2/16 before GST-Fn9-11 binding were assayed. Dotted line, the staining as a result of the unspecific binding of streptavidin (stv) Cy-chrome was used as the negative control; shaded area, fibronectin-binding status of unstimulated cells; and bold line, fibronectin-binding status after Fc{gamma}R cross-linking. (B) Glutathione sepharose beads were incubated with GST alone or with the GST-Fn-9-11 fusion to show that the amount of label was the same for both proteins. Data are representative of three separate experiments.

Fc{gamma}RIIIB-mediated activation of ß1 integrins is independent of PI-3K
Previous reports of FcR-dependent integrin activation, including the up-regulation of ß2 integrins in human neutrophils, suggested the participation of PI-3K in this activation [24 , 35 , 36 ]. To determine whether PI-3K was also used for ß1 integrin activation secondary to Fc{gamma}RIIIB stimulation, we used the PI-3K-specific inhibitors LY294002 and wortmannin. Inhibiting PI-3K activity greatly diminished the Fc{gamma}RIIIB-mediated ß2 integrin up-regulation (Figs. 4A and 5A ). This result is similar to results in previous reports [24 , 32 ] and confirmed the participation of PI-3K in the up-regulation of ß2 integrins in response to Fc{gamma}RIIIB cross-linking on human neutrophils. In contrast, treatment of PMN with LY294002 did not prevent the augmented binding of mAb 15/7 (Figs. 4B and 5B) or the binding of the GST-Fn9-11 protein (Fig. 4C) . Same results were obtained when PI-3K activity was blocked with wortmannin (data not shown). These data clearly showed that Fc{gamma}RIIIB-mediated ß1 integrin activation is independent of PI-3K activity.



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  		Figure 4. ß1 integrin activation secondary to Fc{gamma}RIIIB cross-linking is independent of PI-3K activity. Human neutrophils were stimulated for 10 min by cross-linking Fc{gamma}RIIIB in the presence of only DMSO (bold line) or in the presence of 50 µM LY294002 (thin line). PMN (1x106) were then stained with anti-ß2 integrin mAb IB4 (A), with anti-activated ß1 integrin mAb 15/7 (B), or with GST-Fn9-11 protein (C) for flow cytometry analysis of ß1 integrin activation. Dotted line is the isotype-matched, negative control; shaded area is the binding of unstimulated cells. Data are representative of three separate experiments.



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  		Figure 5. ß1 integrin activation secondary to Fc{gamma}RIIIB cross-linking is independent of PI-3K, Src tyrosine kinases, PKC, PKA, and ERK and is negatively regulated by Syk and the actin cytoskeleton. Human neutrophils were pretreated with DMSO or with increasing doses of LY294002, PP2, Cytochalasin D, or Piceatannol or maximal doses of PD98059, Staurosporine, or H89 before being stimulated for 10 min by cross-linking of Fc{gamma}RIIIB. Cells were then stained with mAb IB4 or mAb 15/7 as in Figure 4 . Up-regulation and activation indexes were calculated as described in experimental procedures. (A) Changes in ß2 integrin cell-surface expression indicated by anti-ß2 integrin mAb IB4 fluorescence in the presence of increasing doses of LY294002, PP2, Cytochalasin D, or Piceatannol. (B) Changes in the ß2 up-regulation index in the presence of 30 µM PD98059 (PD), 10 nM Staurosporine (Stau), or 10 µM H89. (C) Changes in ß1 integrin activation indicated by anti-active ß1 integrin mAb 15/7 fluorescence in the presence of increasing doses of LY294002, PP2, Cytochalasin D, or Piceatannol. (D) Changes in the ß1 activation index in the presence of 30 µM PD98059, 10 nM Staurosporine, or 10 µM H89. (E) Changes in ß1 integrin activation indicated by anti-active ß1 integrin mAb 15/7 fluorescence in the presence of increasing doses of Piceatannol or Resveratrol. Data are mean ± SEM of three independent experiments.

ß1 integrin activation secondary to Fc{gamma}RIIIB stimulation is independent of Src kinases, PKC, PKA, and ERK and is negatively regulated by the cytoskeleton and Syk
As PI-3K was not involved in the activation of ß1 integrins secondary to Fc{gamma}RIIIB cross-linking, we wanted to identify other possible candidates known to modulate Fc{gamma}R-mediated responses [2 , 4 , 28 , 42 ]. For this purpose, we used pharmacological inhibitors of Src-family tyrosine kinases, PKC, PKA, ERK, the actin cytoskeleton, and of Syk kinase to evaluate the signaling pathway from Fc{gamma}RIIIB to integrins. Blocking Src-family tyrosine kinase activity with the inhibitor PP2 eliminated Fc{gamma}RIIIB-mediated ß2 integrin up-regulation in a dose-dependent manner (Fig. 5A) . This response is similar to the one seen after the pharmacological inhibition of PI-3K (Fig. 5A) . In addition, inhibition of Syk with Piceatannol resulted in impaired ß2 integrin up-regulation in a dose-dependent manner (Fig. 5A) . The MEK inhibitor PD98059 and the PKC inhibitor Staurosporine each abolished ~30% of the Fc{gamma}RIIIB-mediated ß2 integrin up-regulation at their maximum doses, but this effect was not statistically significant (Fig. 5B) . The PKA inhibitor H89 abolished ß2 up-regulation ~50% when used at its maximum dose (Fig. 5B) . Contrary to these results, abolishing actin polymerization with Cytochalasin D did not abolish the Fc{gamma}RIIIB-dependent ß2 integrin up-regulation which was actually enhanced mildly (Fig. 5A) . These results suggest that Fc{gamma}RIIIB initiates a signaling transduction pathway leading to ß2 up-regulation, which requires Src-family tyrosine kinases, Syk, and PI-3K and is partially dependent on the activation of PKA. Although Fc{gamma}RIIIB stimulation is capable of activating ERK, a kinase known to be activated by MEK-1, this activation is not required for ß2 integrin up-regulation.

Contrary to these results, inhibition of Src-family kinases or PI-3K was incapable of preventing ß1 integrin activation secondary to Fc{gamma}RIIIB stimulation, even at doses that completely inhibited ß2 integrin up-regulation (Fig. 5C) . It is surprising that inhibition of Syk enhanced Fc{gamma}RIIIB-mediated ß1 integrin activation in a dose-dependent manner. Low doses of Piceatannol did not affect ß1 integrin activation even when ß2 up-regulation was inhibited by as much as 60% (compare Fig. 5 A and C ), but higher doses of Piceatannol, which completely inhibited ß2 integrin up-regulation, increased ß1 integrin activation as much as fivefold (Fig. 5C) . Disruption of the actin cytoskeleton also promoted a slight (twofold) increase in ß1 integrin activation after Fc{gamma}RIIIB stimulation (Fig. 5C) , but this effect was seen only when high concentrations of Cytochalasin D were used. Inhibition of MEK, PKC, or PKA had no effect on the activation of ß1 integrins secondary to Fc{gamma}RIIIB stimulation (Fig. 5D) . To further support the fact that Syk is responsible for the observed increase in ß1 integrin activation when PMN are treated with Piceatannol, we treated the cells with Resveratrol, an analog of Piceatannol, which has no effect on Syk activity [46 ]. Treating the cells with up to 100 µM Resveratrol previous to Fc{gamma}RIIIB stimulation did not promote an increase in ß1 integrin activation equivalent to that produced when cells were treated with Piceatannol (Fig. 5E) . These data suggested that Fc{gamma}RIIIB induces ß1 integrin activation by a signaling pathway, which is independent of Src-family tyrosine kinases, PI-3K, MEK, PKC, and PKA but that may be negatively regulated by Syk.

Fc{gamma}RIIIB, but not Fc{gamma}RII, colocalizes with ß2 integrins after cross-linking
As Fc{gamma}RIIIB seemed to induce ß1 integrin activation independently of a biochemical cascade, it was possible that another mechanism was used by this FcR to activate integrins. It has been reported that Fc{gamma}RIIIB has the capacity to interact on the cell surface with other receptors, and it is also thought that this interaction may be responsible for its signaling capacities. Thus, an interesting possibility was then that Fc{gamma}RIIIB and ß1 integrins interact on the cell surface and that this interaction is responsible for the activation of ß1 integrins. We explored the interaction of Fc{gamma}RII and Fc{gamma}RIIIB with ß1 and ß2 integrins on PMN by confocal microscopy.

We found that Fc{gamma}RII was uniformly distributed on the cell surface of unstimulated PMN (Fig. 6A ). ß2 integrins were also distributed uniformly on the cell surface of unstimulated cells (Fig. 6B and 6H) . In unstimulated cells, Fc{gamma}RII and ß2 integrins did not colocalize (Fig. 6C) . Upon Fc{gamma}RII cross-linking, patches of the receptor appeared on the cell surface, reflecting receptor clustering (Fig. 6D) . In Fc{gamma}RII-stimulated cells, patches of ß2 integrins also appeared on the cell surface (Fig. 6E) ; however, Fc{gamma}RII and ß2 integrins did not colocalize (Fig. 6F) .



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  		Figure 6. Fc{gamma}RIIIB, but not Fc{gamma}RII, colocalizes with ß2 integrins after cross-linking. Human neutrophils were incubated with FITC-labeled Fab fragments of mAb IV.3 (A and D) or FITC-labeled F(ab')2 fragments of mAb 3G8 (G and J). Cells were stimulated for 10 min by the addition of F(ab')2 fragments of goat anti-mouse IgG (D, E, J, and K). Cells were then stained using biotinylated mAb IB4 and streptavidin Cy-chrome to detect ß2 integrins (B, E, H, and K). Merge images are shown in the last column. (L) Arrowheads show colocalization of Fc{gamma}RIIIB with ß2 integrins after cross-linking. Insets show detail at the single-cell level. Over 100 cells were analyzed for each condition in at least two separate experiments. Original scale bar is 10 µm. Representative images are shown.

Similar to Fc{gamma}RII, Fc{gamma}RIIIB was uniformly distributed across the cell surface in unstimulated cells (Fig. 6G) . Also, Fc{gamma}RIIIB and ß2 integrins did not colocalize in unstimulated cells (Fig. 6I) . After Fc{gamma}RIIIB cross-linking, patches of the receptor appeared, reflecting receptor clustering (Fig. 6J) , and ß2 integrin up-regulation occurred, as evidenced by the appearance of ß2 integrin patches (Fig. 6K) . Contrary to Fc{gamma}RII, Fc{gamma}RIIIB and ß2 integrins did colocalize in small patches after cross-linking (Fig. 6L) . It is important to notice that even after Fc{gamma}RIIIB stimulation, the bulk of ß2 integrins does not colocalize with Fc{gamma}RIIIB. These results showed that Fc{gamma}RIIIB and a small fraction of ß2 integrins indeed colocalize, but they do so only after Fc{gamma}RIIIB stimulation.

Fc{gamma}RIIIB colocalizes with active ß1 integrins, and this interaction is enhanced after the inhibition of Syk
In the case of ß1 integrins, we first looked at total ß1 integrins on the cell surface before and after Fc{gamma}RIIIB cross-linking using confocal microscopy. No changes in the distribution of total ß1 integrins were evident after Fc{gamma}RIIIB cross-linking (data not shown). As no changes in total ß1 integrins could be detected, we looked at the distribution of active integrins, evidenced by the binding of mAb 15/7, after Fc{gamma}RIIIB cross-linking. As shown before, Fc{gamma}RIIIB was uniformly distributed on the membrane of unstimulated cells (Fig. 7A ), and as it had been shown by flow cytometry, there was no binding of mAb 15/7 to unstimulated cells (Fig. 7B) , indicating that ß1 integrins were not activated on these cells. Upon Fc{gamma}RIIIB cross-linking, the receptor was aggregated in patches (Fig. 7D) , and small patches of mAb 15/7 staining also appeared on the cell surface (Fig. 7E) . All of these patches of active ß1 integrins colocalized with Fc{gamma}RIIIB (Fig. 7F) . This result suggested that ß1 integrin activation, secondary to Fc{gamma}RIIIB stimulation, might be the result of interactions between Fc{gamma}RIIIB and ß1 integrins on the PMN membrane. As Piceatannol treatment augmented the binding of mAb 15/7 to Fc{gamma}RIIIB-stimulated cells (Fig. 5B) , we looked at Piceatannol-treated cells to detect the increased binding of mAb 15/7. Piceatannol treatment of unstimulated PMN did not elicit any mAb 15/7 binding (Fig. 7H) . However, as shown before, mAb 15/7 binding to PMN after Fc{gamma}RIIIB stimulation was clearly enhanced by Piceatannol treatment (Fig. 7K) . In these conditions, the colocalization of active ß1 integrins and Fc{gamma}RIIIB was more evident (Fig. 7L) . All these data suggested that Fc{gamma}RIIIB might be able to interact with ß1 integrins and in this way, activate them.



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  		Figure 7. Fc{gamma}RIIIB colocalizes with active ß1 integrins. Human neutrophils were stimulated using FITC-labeled F(ab')2 fragments of mAb 3G8 (A, D, G, and J) and cross-linked with F(ab')2 fragments of goat anti-mouse IgG (D, E, J, and K). Stimulated cells were stained with biotinylated mAb 15/7 and streptavidin Cy-chrome (B, E, H, and K). Merge images are shown on the right. Active ß1 integrins and Fc{gamma}RIIIB colocalization are seen as yellow (F). Detail is shown on the inset. Fc{gamma}RIIIB-labeled, unstimulated cells (G) or stimulated cells (J) were also treated with 40 µM Piceatannol. Staining with mAb 15/7 (H and K) shows that Piceatannol only enhances mAb 15/7 binding of stimulated cells (K). This mAb 15/7 binding also colocalizes with Fc{gamma}RIIIB, as shown by the yellow areas of the merged image (L). Over 100 cells were analyzed for each condition in at least two separate experiments. Original scale bar is 10 µm. Representative images are shown.

Fc{gamma}RII is internalized after cross-linking, but Fc{gamma}RIIIB is not, and clusters of Fc{gamma}RIIIB after stimulation only appear on the cell surface
To better understand the nature of the possible interaction between of Fc{gamma}RIIIB and ß1 integrins, we hypothesized that if Fc{gamma}RIIIB were internalized following cross-linking, then a portion of the active ß1 integrins could also be internalized if the interaction between both molecules were strong enough. To study the internalization of FcRs, PMN were stained with FITC-labeled Fab fragments of mAb IV.3 or FITC-labeled F(ab')2 fragments of mAb 3G8. Receptor internalization was evaluated using Trypan blue to quench the extracellular FITC signal in unstimulated and stimulated cells. Our results show that in the steady-state, the amount of Fc{gamma}RII and Fc{gamma}RIIIB that is inaccessible to the quencher remains the same in unstimulated cells after 10 min at 37°C (Fig. 8A ). When receptors are stimulated by the addition of F(ab')2 fragments of goat anti-mouse IgG, receptor internalization is evident for Fc{gamma}RII as a clear increase in the amount of fluorescence that is protected from Trypan blue quenching. In contrast, Fc{gamma}RIIIB does not seem to become internalized after stimulation, as the percentage of fluorescence that is protected from Trypan blue quenching remains the same as before stimulation (Fig. 8A) . As no net change is seen in the amount of Fc{gamma}RIIIB that is internalized after stimulation, it is safe to assume that ß1 integrins, which may associate with this receptor after stimulation, will not undergo internalization either. To further support this notion, we undertook the analysis of Fc{gamma}RIIIB cluster formation after stimulation, using a different cross-linking approach. Neutrophils were incubated with biotinylated F(ab')2 fragments of mAb 3G8 and then cross-linked with Cy-chrome streptavidin (Fig. 8B) ; when cells are stimulated this way, clusters appear on the cell surface and do not seem to become internalized, as in all confocal planes, they colocalized with lipid rafts on the cell membrane, as evidenced by the colocalization with cholera toxin B (Fig. 8B) . As Fc{gamma}RIIIB does not seem to be internalized after stimulation, these data support the hypothesis that interaction between Fc{gamma}RIIIB and ß1 integrins may only occur at the cell surface.



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  		Figure 8. Fc{gamma}RII becomes internalized after cross-linking, but Fc{gamma}RIIIB does not and clusters on the cell surface. (A) Human neutrophils were incubated with FITC-labeled Fab fragments of mAb IV.3 or FITC-labeled F(ab')2 fragments of mAb 3G8. Antibody-labeled cells (IV.3, 3G8) and activated cells (IV.3+GAM, 3G8+GAM) were analyzed for FITC fluorescence after Trypan blue quenching before (open bars) or after (shaded bars) they had been placed at 37°C for 10 min. Data are mean ± SEM of two independent experiments. (B) PMN were incubated with biotinylated F(ab')2 fragments of mAb 3G8 and cross-linked for 10 min with Streptavidin (Stv) Cy-chrome, and cells were washed, labeled with cholera toxin B (CTB)-FITC, and fixed. Over 100 cells were analyzed for each condition in at least two separate experiments. Original scale bar is 10 µm. Representative images are shown.

ß1 integrin activation after Fc{gamma}RIIIB stimulation seems to be independent of lipid rafts
As Fc{gamma}RIIIB is a GPI-linked protein, its primary localization seems to be the cholesterol-enriched lipid rafts of the outer leaflet of the cell membrane (Fig. 8B) . We also detected 15/7 mAb staining in lipid rafts after Fc{gamma}RIIIB stimulation, as evidenced by colocalization with cholera toxin B (data not shown). We set to examine whether lipid rafts and cholesterol play any part on the activation of ß1 integrins secondary to Fc{gamma}RIIIB stimulation. For this, we evaluated the effect of cholesterol-depleting agent methyl-ß-cyclodextrin [47 , 48 ]. It has been reported previously that methyl-ß-cyclodextrin produced shedding of GPI-linked proteins [47 ]. In our case, Fc{gamma}RIIIB was reduced by 30% on the membrane surface after treatment with methyl-ß-cyclodextrin, also suggesting shedding of this receptor (Fig. 9A ). It is surprising that ß1 integrin activation after Fc{gamma}RIIIB stimulation was not affected by the treatment with methyl-ß-cyclodextrin (Fig. 9B) . We then decided to study whether methyl-ß-cyclodextrin affected the colocalization of active ß1 integrins and Fc{gamma}RIIIB after stimulation. When PMN were treated with methyl-ß-cyclodextrin, they became extremely round, and Fc{gamma}RIIIB distribution was altered. Fc{gamma}RIIIB clusters were not seen as such but as a more diffuse membrane distribution (Fig. 9C) . Still, the colocalization between Fc{gamma}RIIIB and active integrins was not disturbed by the treatment with methyl-ß-cyclodextrin (Fig. 9C) . These results suggest that the mechanism responsible for the activation and the colocalization of ß1 integrins with Fc{gamma}RIIIB secondary to Fc{gamma}RIIIB cross-linking is independent of lipid raft integrity.



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  		Figure 9. ß1 integrin activation secondary to Fc{gamma}RIIIB stimulation seems independent of lipid raft integrity. (A) Human neutrophils were incubated with FITC-labeled F(ab')2 fragments of mAb 3G8, and Fc{gamma}RIIIB expression on the cell surface was analyzed by flow cytometry in the absence or presence of 10 mM methyl-ß-cyclodextrin. (B) Activation of ß1 integrins was assessed as in Figure 5 in the absence or presence of 10 mM methyl-ß-cyclodextrin. Data are mean ± SEM of three independent experiments. (C) Human neutrophils were treated and stained as in Figure 7 , but the experiment was performed in the presence of 10 mM methyl-ß-cyclodextrin. Over 100 cells were analyzed for each condition in at least two separate experiments. Original scale bar is 10 µm. Representative images are shown.


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DISCUSSION
 
In this report, we have found that Fc{gamma}RIIIB cross-linking in the human neutrophil activates ß1 integrins. This activation does not depend on an increase in expression of ß1 integrins on the cell membrane but involves an increase in ligand-binding affinity. The Fc{gamma}RIIIB-induced activation of ß1 integrins is independent of a signaling pathway involving PI-3K and Src-family tyrosine kinases but might be negatively regulated by Syk. We also show that Fc{gamma}RIIIB and activated ß1 integrins colocalize on the cell surface, suggesting that the interaction between these molecules may be responsible for activation of ß1 integrins.

The role of integrins in the hematopoietic system is an area of active research [26 , 49 50 51 52 53 54 55 ]. Integrins have emerged as important regulators of leukocyte adhesion, homing, and recruitment of leukocytes to lymphoid organs [49 ], platelet functions [56 ], phagocytocis, and degranulation [34 ]. In addition, in humans, various diseases are caused by integrin deficiencies or malfunctions. For example, ß2 integrin deficiency is a deadly disease characterized by leukocyte malfunction and defective migration [57 ]. Also, the absence of {alpha}IIbß3, the platelet integrin, promotes a bleeding disorder known as Glanzmann’s thrombasthenia [56 ]. Thus, understanding the biology of integrins has relevance in the understanding of the normal functions of the hematopoietic system as well as in human disease.

Integrins exist in two different conformations, inactive and active. The inactive integrin is not capable of interacting with its ligand, and the active integrin can bind to it. Integrins have the capacity to alter their activation state in response to cellular stimulation, thus modulating their adhesive phenotype [21 ]. This integrin activation is essential for the proper function of integrins, as only the active integrin binds its ligand. This essential function of integrins is further underscored by the fact that deficiencies in the ability to activate ß1, ß2, and ß3 integrins result in a disease that mimics ß2 and ß3 absence [55 ]. In addition to integrin activation, it has been appreciated that integrins can up-regulate their numbers on the cell surface and cluster upon stimulation, further adding to their functional capacities [32 ].

The mechanisms of integrin activation and their regulation are not yet clearly defined. Integrin activation seems to involve a conformational change [20 , 45 , 58 ]. This conformational change can be elicited from the inside of the cell or from the outside of the cell with mAb [59 ]. The intracellular events that lead to this conformational change are not clear yet, but several stimuli have been shown to promote integrin activation in different cellular systems. Integrins are known to become activated in response to T cell receptor signaling, chemokines, selectins, phorbol esters, and Fc{gamma}R stimulation [22 ].

FcR stimulation has been identified as an important activator of integrins in neutrophils and other cell types. [24 , 33 34 35 36 ]. In neutrophils, ß2 integrin up-regulation and activation have been shown to occur after Fc{gamma}RII and Fc{gamma}RIIIB stimulation [24 , 32 ]. Fc{gamma}RII- and Fc{gamma}RIIIB-mediated ß2 integrin up-regulation occurred through a biochemical cascade that involves PI-3K [32 ]. Our results described here show that in the case of Fc{gamma}RIIIB stimulation, Src-family tyrosine kinases and Syk kinase, in addition to PI-3K, are involved in the up-regulation of ß2 integrins. This biochemical pathway, emerging from Fc{gamma}RIIIB, is similar to the traditional ITAM-based signaling of Fc{gamma}RII [2 ]. This signaling mechanism for Fc{gamma}RIIIB-mediated ß2 integrin up-regulation makes it attractive to speculate that indeed, Fc{gamma}RIIIB recruits Fc{gamma}RII to mediate intracellular signaling, as has been suggested by other authors [12 ]. However, the fact that Fc{gamma}RIIIB but not Fc{gamma}RII promoted ß1 integrin activation points to the idea that other signaling mechanisms for Fc{gamma}RIIIB must exist.

ß1 integrin responses, after Fc{gamma}R stimulation, were different from those of ß2 integrins. When PMN were stimulated by Fc{gamma}RII or Fc{gamma}RIIIB cross-linking, ß1 integrin expression on the cell surface remained intact. It had been reported previously that ß2 integrins exist preformed in the secretory granules of human neutrophils [60 ]. The degranulation that occurs secondary to Fc{gamma}R stimulation seems to be responsible for the observed increase in the number of ß2 integrins on the cell surface. As ß1 integrin numbers on the cell surface did not increase after Fc{gamma}RII or Fc{gamma}RIIIB stimulation, it can be argued that these preformed vesicles do not contain ß1 integrins.

Although no ß1 integrin up-regulation was observed after Fc{gamma}R stimulation, Fc{gamma}RIIIB promoted ß1 integrin activation that led to increased fibronectin binding. Contrary to Fc{gamma}RIIIB-mediated ß2 integrin up-regulation, Fc{gamma}RIIIB-mediated ß1 activation was independent of the enzymatic activity of PI-3K and Src-family kinases. This suggests that Fc{gamma}RIIIB is capable of activating at least two different signaling pathways in human neutrophils: one that could use a signaling pathway similar to the ITAM signaling machinery of Fc{gamma}RII to promote up-regulation of ß2 integrins and another independent pathway to activate ß1 integrins.

As Fc{gamma}RIIIB activates ß1 integrins independently of a classical, biochemical pathway, we thought that another possibility for this activation was the interaction of the receptors on the cell surface. This idea is supported by the fact that Fc{gamma}RIIIB has been shown to interact with at least Fc{gamma}RII and ß2 integrins [7 , 11 , 33 ]. It is generally perceived that Fc{gamma}RIIIB and ß2 integrins interact almost permanently on the cell surface of the human neutrophil, as has been interpreted by some authors [61 ]. This perception aroused from studies using fluorescence resonance energy transfer (FRET), which showed that Fc{gamma}RIIIB and ß2 integrins were capable of interacting on the cell surface after transfection on fibroblasts [9 , 62 ]. However, these studies did not show that this interaction is permanent. Our colocalization studies show that contrary to this perception, Fc{gamma}RIIIB and ß2 integrins are not together on the cell surface all the time but only colocalize after Fc{gamma}RIIIB cross-linking. A finding that is in accordance with FRET studies but restricts the interactions of Fc{gamma}RIIIB and ß2 integrins to the Fc{gamma}RIIIB-activated neutrophil.

Based on the existence of interactions between Fc{gamma}RIIIB and ß2 integrins after Fc{gamma}RIIIB stimulation, we examined the location of active ß1 integrins on the cell membrane of PMN. We found that Fc{gamma}RIIIB and active ß1 integrins colocalize on the PMN membrane. This finding suggests that interactions between the two receptors may be responsible for the activation of ß1 integrins. Additionally, it was recently found that a urokinase-type plasminogen activator receptor, a GPI-linked receptor, is capable of associating laterally with ß1 integrins on the cell surface [63 ]. As Fc{gamma}RIIIB is also a GPI-linked receptor, this finding further supports the idea that ß1 integrins have the capacity of engaging Fc{gamma}RIIIB in lateral associations on the cell surface.

Although our findings support the notion that Fc{gamma}RIIIB activates ß1 integrins by interactions on the cell surface, other possibilities exist. Small GTPases of several families have been implicated in ß1 integrin activation in several cell types [64 65 66 67 68 69 ]. So, it is possible that some of them may be responsible for the regulation of the signaling to ß1 integrins after Fc{gamma}RIIIB cross-linking.

We found that when cross-linking of Fc{gamma}RIIIB was performed in the presence of Piceatannol, the colocalization between Fc{gamma}RIIIB and the active ß1 integrins was enhanced, suggesting that Syk might negatively regulate this phenomenon. Syk is considered a key mediator of Fc{gamma}R signaling [70 ] and has also been shown recently to be critical for several integrin functions in the human neutrophil [71 ]. Also, Syk has been shown to interact directly with the cytoplasmic tails of ß3 and ß1A integrins [72 73 74 ]. Syk has also been found to regulate the binding cycle of ß2 integrins [75 ]. So, precedents exist where Syk regulates integrin events by interacting directly with the integrin cytoplasmic tails. As with all pharmacological inhibitors, Piceatannol is not completely specific for Syk, and high doses have been shown to interfere with other enzymes [76 ]. Nonetheless, the use of Resveratrol, a Piceatannol analog without activity toward Syk, supports the notion that Syk is actively involved in the regulation of ß1 integrin activation secondary to Fc{gamma}RIIIB stimulation. Although Resveratrol did not affect ß1 integrin activation as much as Piceatannol, high doses also stimulated an increase in ß1 integrin activation, which suggests that other proteins may also regulate ß1 integrin activation. Unfortunately, it is not known if Resveratrol has any specific targets on PMN. Whether blocking Syk affects ß1 integrins directly or interferes indirectly with other elements that modulate integrin activation, such as the cytoskeleton, is an interesting question. Our results suggest that a direct effect seems more possible, as disrupting the actin cytoskeleton did not produce an increase in ß1 activation of the same magnitude as that of the treatment with Piceatannol, albeit there was a minor increase in ß1 integrin activation after treatment with high doses of cytochalasin. Although at these doses, the effects of cytochalasin may be somewhat unspecific, this finding is in agreement with previous reports showing that disruption of the cytoskeleton frees the integrin intracytoplasmic tails and thus facilitates activation.

Fc{gamma}RIIIB is a GPI-linked receptor and is presumed to float on the outer leaflet of the cell membrane in cholesterol-enriched lipid rafts. As a result of this fact, the use of cholesterol-depleting agents could tell us whether lipid rafts play any significant role in the activation of ß1 integrins secondary to Fc{gamma}RIIIB. Our results suggest that ß1 integrin activation and colocalization with Fc{gamma}RIIIB, in this case, are independent of lipid raft function. This result, however, should be examined carefully in light of the fact that cholesterol depletion of PMN has been shown to promote degranulation [77 ], shedding of GPI-linked proteins [47 ], activation of small GTPases [78 ], and polymerization of actin [78 ]; so establishing a clear role for lipids rafts may not be possible using this approach. Still, our results are in agreement with those of Pierini and collaborators [79 ]. This group showed that CD44 (a lipid raft marker) was localized to the uropod of formyl-Met-Leu-Phe-stimulated PMN and that this redistribution upon stimulation was lost when cells were treated with methyl-ß-cyxlodextrin. Our results show that although Fc{gamma}RIIIB stimulation in untreated PMN results in the formation of clusters on the cell membrane, these clusters are no longer seen in methyl-ß-cyclodextrin-treated cells, thus reflecting the importance of lipid raft integrity for the adequate distribution of raft-associated proteins.

It is possible to imagine that after Fc{gamma}RIIIB cross-linking, Fc{gamma}RIIIB may undergo a "conformational change", which may expose certain areas of the molecule so that interactions with ß1 integrins become possible. This association will in turn promote ß1 integrin activation, which would be tightly regulated by the interaction between Syk and the ß1 integrin cytoplasmic tail. When Piceatannol blocks the activity of Syk, the interactions between Fc{gamma}RIIIB and ß1 integrins are no longer regulated, which permits a broader activation of ß1 integrins. The activity of Syk required to maintain the ß1 integrins tightly regulated would be minimal, as only large doses of Piceatannol promote ß1 integrin activation deregulation. Although highly speculative, this model provides a working hypothesis for the activation of ß1 integrins secondary to Fc{gamma}RIIIB cross-linking. A clear demonstration of this hypothesis would come from immunoprecipitation studies if Fc{gamma}RIIIB could be isolated and shown to associate with ß1 integrins after stimulation. These immunoprecipitation studies would also show if interactions between Fc{gamma}RIIIB and ß1 integrins exist even before stimulation, a fact that would explain the apparent absence of a signaling intermediate between Fc{gamma}RIIIB stimulation and ß1 integrin activation. It was not possible, however, to perform this experiment, as the recovery of Fc{gamma}RIIIB after cellular lysis was not feasible using the reagents available to us today.

In the human neutrophil, Fc{gamma}RIIIB expression is 10 times higher than that of Fc{gamma}RIIA [80 ]. This suggests that Fc{gamma}RIIIB is the first Fc{gamma}R that the neutrophil uses to recognize immune complexes. Indeed, evidence for a major role of Fc{gamma}RIIIB in immune complex recognition in rolling neutrophils has been reported recently [16 ]. After Fc{gamma}R stimulation, integrins get activated ([24 ] and this report), generating a more adhesive phenotype for the PMN. It is clear that ß2 integrins are important for transendothelial migration, yet the role for activated ß1 integrins is not as clear. ß1 integrins have been shown to be important for the migration of PMN through the extracellular matrix [51 , 81 82 83 ]. Also, in patients and mice with ß2 integrin deficiency, recruitment of PMN to the lung remains intact [57 , 84 ], suggesting that ß1 integrins could be responsible for this migration. Thus, ß1 integrin activation could be an important mechanism to recruit PMN from the circulation to certain tissues in response to immune complexes.

In conclusion, this report shows for the first time that human neutrophils are capable of activating ß1 integrins in response to Fc{gamma}RIIIB stimulation. This Fc{gamma}RIIIB-mediated ß1 integrin activation is independent of the enzymatic activity of PI-3K and Src-family tyrosine kinases and may be mediated through interactions between Fc{gamma}RIIIB and ß1 integrins.


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ACKNOWLEDGEMENTS
 
This work was supported by Grant 36407-M from Consejo Nacional de Ciencia y Tecnología, Mexico, and by Grant IN220703 from DGAPA-UNAM, Mexico. We thank Nancy Mora for excellent technical assistance, Dr. Ted Yednock for mAb 15/7, Dr. Mark Ginsberg for the plasmid encoding the GST-Fn9-11 recombinant protein, Dr. Pavel Isa for the plasmid encoding GST alone, and all the blood donors who made this work possible.

Received May 27, 2004; revised December 15, 2004; accepted December 19, 2004.


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