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Originally published online as doi:10.1189/jlb.0804479 on May 17, 2005

Published online before print May 17, 2005
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(Journal of Leukocyte Biology. 2005;78:524-532.)
© 2005 by Society for Leukocyte Biology

Signaling through CD16b in human neutrophils involves the Tec family of tyrosine kinases

Maria J. G. Fernandes*,1, Geneviève Lachance{dagger}, Guillaume Paré{dagger}, Emmanuelle Rollet-Labelle{dagger} and Paul H. Naccache{dagger}

Centre de Recherche en Rhumatologie et Immunologie, Centre de Recherche du CHUL,
* Departments of Anatomy and Physiology and
{dagger} Medicine, Laval University, Québec, Canada

1 Correspondence: Centre de Recherche du CHUL, Room T1-49, 2705 Boulevard Laurier, Québec, G1V 4G2, Canada. E-mail: maria.fernandes{at}crchul.ulaval.ca


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Tec kinases belong to the second largest family of nonreceptor tyrosine kinases. Although these kinases are expressed in myeloid cells, little is known about their implication in neutrophil function. We recently reported the participation of Tec kinases in the responses of human neutrophils to the bacterial peptide N-formyl-l-methionyl-l-leucyl-l-phenylalanine via G-coupled protein receptors. In this study, we extended our investigations of Tec kinases to the signaling of the glycosylphosphatidylinositol-linked receptor CD16b, which is highly and specifically expressed in neutrophils. The results obtained indicate that Tec is translocated to the plasma membrane, phosphorylated, and activated upon CD16b cross-linking and that the activation of Tec is inhibited by Src-specific inhibitors as well as by the phosphatidylinositol-3 kinase inhibitor, wortmannin. As no specific inhibitor of Tec exists, the role of Tec kinases was further investigated using a-Cyano-b-hydroxy-b-methyl-N-(2,5-dibromophenyl)propenamide (LFM-A13), a compound known to inhibit Bruton’s tyrosine kinase. We show that this compound also inhibits the kinase activity of Tec and provide evidence that the mobilization of intracellular calcium and the tyrosine phosphorylation of phospholipase C{gamma}2 (PLC{gamma}2) induced upon CD16b engagement are inhibited by LFM-A13. We also show that Tec kinases are important for CD16b-dependent degranulation of neutrophils. In summary, we provide direct evidence for the implication of Tec in CD16b signaling and suggest that Tec kinases are involved in the phosphorylation and activation of PLC{gamma}2 and subsequently, in the mobilization of calcium in human neutrophils.

Key Words: phagocytes • signal transduction • Fc receptors


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The phagocytosis of antigen at the inflammatory site by human polymorphonuclear neutrophils (PMN) involves, in part, the participation of Fc receptors (FcRs) [1 ], which recognize and bind the Fc region of immunoglobulins (Ig) that opsonize the antigen. Two different types of FcRs for IgG are constitutively expressed on the surface of PMN, namely, Fc{gamma}RIIA (CD32) and Fc{gamma}RIIIB (CD16b) [2 , 3 ]. A third type of FcR, Fc{gamma}RI (CD64), is strongly expressed following PMN activation [4 ].

FcRs or their accessory chains contain in their cytoplasmic domain immunoreceptor tyrosine-based activation or inhibition motifs, which are indicative of the signaling events that occur upon receptor engagement [5 ]. Signaling events upon the aggregation of FcRs with immunoreceptor tyrosine-based activation motifs include the activation of several cytoplasmic tyrosine kinases such as those of the Src, Syk, and Tec family of kinases [6 ]. These activated kinases phosphorylate a variety of intracellular substrates that lead to the induction of cellular responses including phagocytosis, respiratory burst, degranulation, and production of inflammatory cytokines [1 ].

CD16b is unique in that it is the only FcR that is linked to the plasma membrane by a glycophospholipid (GPI) anchor [7 ]. Despite the lack of a physical link between the extracellular portion of CD16b and the inner leaflet of the plasma membrane, GPI proteins trigger signaling events within cells [8 ]. Multivalent cross-linking of CD16b elicits downstream signals including the release of Ca2+ from intracellular stores [9 , 10 ], actin filament assembly [9 , 11 ], and the phosphorylation of Hck [12 ], extracellular signal-regulated kinase (ERK) [13 , 14 ], p38 [13 , 14 ], and Pyk2 [14 ]. The effector functions observed after CD16b cross-linking include degranulation [15 ], phagocytosis of concanavalin A-opsonized erythrocytes [16 ], activation of respiratory burst [11 , 17 , 18 ], killing of chicken erythrocytes opsonized with anti-CD16b [4 ], and the recruitment of PMN in immune complex-mediated inflammation [19 ]. The molecular pathways leading up to these effector functions in response to CD16b activation have not, however, been fully characterized.

Recently, we reported the expression of a variety of members of the Tec family of tyrosine kinases in human PMN [20 , 21 ]. This previous study focused on the involvement of Tec kinases in the responses of PMN to chemotactic factors. In the present study, we extend our analysis of the implication of Tec kinases in signal transduction events in human PMN by investigating their role in CD16b signaling.

The Tec kinase protein family is the second-largest group of cytoplasmic protein tyrosine kinases consisting of Tec, Bruton’s tyrosine kinase (Btk)/Bpk/Atk/Emb, Itk/Tsk/Emt, Txk/Rlk, and Bmx/Etk in mammalian cells [22 , 23 ]. Together, these kinases form a protein family by virtue of their common overall structure. The defining feature of these kinases is a pleckstrin homology (PH) domain at their N terminus and an adjacent Tec homology (TH) region, which includes a Btk homology region and one or two proline-rich (PR) regions. Variations in the composition of the TH domain exist between different Tec kinases such as one PR region in Rlk/Txk. The remainder of the protein domain structure of Tec kinases resembles that of the Src family of kinases. The TH domain is followed by Src homology (SH)3, SH2, and tyrosine kinase catalytic domains. Unlike Src kinases, however, Tec kinases lack an N-terminal myristyolation site and regulatory C-terminal tyrosine residues.

The expression of most Tec kinases is restricted to hematopoietic cells with the exception of Etk/Bmx and Tec, which are also expressed in endothelial cells and the liver, respectively [24 ]. Tec kinases have been implicated in signal transduction events in response to a variety of stimuli including those transmitted by growth factor receptors, cytokine receptors, G protein-coupled receptors, antigen receptors in lymphocytes, and integrins.

We provide the first evidence that Tec kinases play a role in CD16b signaling. Moreover, we show that there is a functional link between the activation of Tec kinases and CD16b engagement.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Antibodies
The monoclonal antibody (mAb) against CD16b, which was used in all cross-linking experiments, 3G8 F(ab')2, was purchased from Medarex (Annandale, NJ, Catalog Number 028-203) or Ancell (Bayport, MN, Catalog Number 165-520). The affinity-purified F(ab')2 fragments of the goat anti-mouse IgG F(ab')2 antibody (115-006-072) used for cross-linking the 3G8 F(ab')2 antibody were purchased from Jackson ImmunoResearch (West Grove, PA). The anti-Tec (06-561) antibody, obtained from Upstate Biotechnology (Lake Placid, NY), was used to immunoprecipitate Tec. The antiphosphotyrosine (05-321, clone 4G10) antibody was obtained from Upstate Biotechnology, the anti-Tec (M-20) and antiphospholipase C{gamma}2 (anti-PLC{gamma}2) (sc-407) antibodies from Santa Cruz Biotechnology (CA), and the antiflotillin-1 (610820) antibody from BD Bioscience (Mississauga, Ontario, Canada). These four antibodies were used for immunoblotting, with the exception of the anti-PLC{gamma}2 antibody, which was also used for immunoprecipitation purposes. The secondary antibodies, horseradish peroxidase (HRP)-labeled sheep anti-mouse IgG (NXA931), were obtained from Amersham Bioscience (Baie d’Urfé, Quebec, Canada). The donkey anti-rabbit HRP (711-035-152) was purchased from Jackson ImmunoResearch, and the donkey anti-goat IgG HRP (Sc-2056) was purchased from Santa Cruz Biotechnology.

Chemicals
Dextran T-500, Percoll, and protein A-Sepharose beads were purchased from Amersham Bioscience. Ficoll-Paque was obtained from Wisent (St-Bruno, Quebec, Canada). Diisopropyl fluorophosphate (DFP) was purchased from Serva Electrophoresis (Heidelberg, Germany). Gelatin was purchased from Fisher Scientific (Nepean, Ontario, Canada). Enhanced chemiluminescence (ECL) detection kit was obtained from Perkin Elmer (Woodbridge, Ontario, Canada). Wortmanin, cytochalasin B, Triton X-100, sodium orthovanadate, Trypsin inhibitor, and phenylmethylsulfonyl fluoride (PMSF) were obtained from Sigma Aldrich (Oakville, Ontario, Canada). 4-Amino-5-(4-chlorophenyl)-7(t-butyl)pyrazolo(3,4-d)pyrimidine (PP2), 4-amino-7-phenylpyrazol [3,4 pyramidyne] (PP3), a-Cyano-b-hydroxy-b-methyl-N-(3-fluorophenyl)propenamide (LFM-A11), and LFM-A13 ({gamma}-cyano-ß-hydroxy-ß-methyl-N-(2,5-dibromophenyl)propenamide) were purchased from Calbiochem (San Diego, CA). 3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), aprotinin, and leupeptin were obtained from Roche Diagnostics (Indianapolis, IN). Fura-2-acetoxymethyl ester was obtained from Molecular Probes (Eugene, OR).

Isolation of PMN
Blood was obtained from healthy adult volunteers in tubes containing heparin. After sedimentation of red blood cells in 2% dextran, PMN were purified by centrifugation on Ficoll-Paque cushions. Contaminating erythrocytes were removed by hypotonic lysis, and cells were resuspended in Mg2+-free Hanks’ balanced salt solution (HBSS) containing 1.6 mM CaCl2. The entire procedure was carried out sterilely.

Stimulation of cells with an anti-CD16b antibody
PMN, at 20 x 106 cells/ml or at the indicated concentration, were incubated for 10 min at room temperature with 1 mM DFP prior to a 2-min incubation at 37°C with 2.5 µg of the 3G8 F(ab')2 mAb per 10 x 106 cells. Cross-linking was subsequently performed at 37°C with 20 µg/10 x 106 cells of the F(ab')2, goat anti-mouse F(ab')2, for the times indicated. In some experiments, PMN were preincubated with wortmannin, PP2, LFM-A11, or LFM-A13 prior to CD16b engagement. Briefly, PMN resuspended in HBSS were treated with 200 nM wortmannin, 25 or 50 µM LFM-A13 or LFM-A11 for 10 min, or 5 µM PP2 for 5 min at 37°C prior to CD16b receptor engagement. PMN were also incubated in dimethyl sulfoxide (DMSO) alone prior to CD16b receptor engagement as a negative control.

Translocation assay
Freshly isolated PMN were resuspended at a final concentration of 40 x 106 cells/ml in HBSS containing 1.6 mM CaCl2, and CD16b was cross-linked as described above for 30 s. The cross-linking reaction was terminated by placing the cells on ice. The cells were then centrifuged at 5500 g for 10 s, resuspended in modified relaxation buffer [RLB; 100 mM KCl, 3 mM NaCl, 10 mM Hepes (pH 7.4), 10 µg/ml aprotinin, 10 µg/ml leupeptin, 2 mM sodium orthovanadate, 250 µg/ml trypsin inhibitor, 1 mM PMSF, 3 mM DFP], and sonicated on ice for 22 s. The sonication was followed by a centrifugation at 400 g for 2 min. A volume of 900 µl supernatant was applied on top of a two-step Percoll gradient composed of 1.4 ml 1.12 g/ml solution layered beneath 1.4 ml 1.05 g/ml, as described by Kjeldsen et al. [25 ]. The Percoll gradients were centrifuged for 30 min at 37,000 g at 4°C in a fixed-angle rotor. The top of the gradient contained cytosol and the plasma membranes. Fractions were collected and centrifuged at 100,000 g for 45 min at 4°C to remove the Percoll. The plasma membranes that formed a visible disc above the Percoll pellet were collected, resuspended in RLB, and boiled for 3 min in 2x Laemmli sample buffer prior to analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).

Immunoprecipitation
After CD16b engagement, PMN were centrifuged, and the cell pellets were lysed by adding cold lysis buffer (10 mM Tris-HCl, pH 7.3, 137.2 mM NaCl, 1 mM EDTA, 0.6% CHAPS, 2 mM orthovanadate, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 50 µg/ml soybean trypsin inhibitor, 1 mM PMSF, 1.5 mM DFP) for 9 min on ice. The insoluble material was discarded by centrifugation at 600 g at 4°C for 2 min. We checked that the presence of Tec was undetectable in the pellets. The supernatants were precleared with protein A-Sepharose at 4°C for 30 min. After a 2-min centrifugation at 600 g, the supernatants were incubated at 4°C with 4 µg anti-Tec antibodies per 10 x 106 cells for 2 h followed by a 1-h incubation with protein A-Sepharose beads, which were collected and washed three times with cold lysis buffer, and Laemmli sample buffer (2x) was added prior to boiling for 5 min. The same protocol was used for the immunoprecipitation of PLC{gamma}2. The amount of PLC{gamma}2 antibody used was 1.2 µg/10 x 106 cells.

In vitro autophosphorylation assay
PMN were stimulated, and Tec were immunoprecipitated as described above with or without 50 µM LFM-A13 during the last 30 min of the immunoprecipitation procedure as described in Gilbert et al. [21 ]. The Tec immunoprecipitates were then washed three times in lysis buffer, resuspended at 4°C in kinase buffer (50 mM HEPES, pH 7.0, 10 mM MnCl2, 2 mM MgCl2, 25 µM paranitrophenylphosphate, 10 µM sodium orthovanadate, and 50 µM adenosine 5'-triphosphate), with or without 50 µM LFM-A13, and transferred to 30°C for the indicated period of time. The reactions were stopped at each time by addition of the same volume of Laemmli sample buffer (2x).

Immunoblotting
Samples were loaded onto 7.5–20% gradient SDS-polyacrylamide gels. Separated proteins were transferred from the gels to Immobilon polyvinylidene difluoride membranes as described previously [26 ]. Membranes were blocked with 2% gelatin in Tris-buffered saline (TBS)-Tween [25 mM Tris-HCl (pH 7.8), 190 mM NaCl, and 0.15% Tween 20] for 30 min at 37°C. Primary antibodies were diluted in fresh blocking solution and incubated for 1 h at 37°C. The membranes were rinsed three times in TBS-Tween and incubated with a HRP-labeled secondary antibody for 30 min at 37°C. After three washes in TBS-Tween, membranes were developed using the ECL detection system.

The antibody dilutions used were as follows: 1/1000 for the anti-PLC{gamma}2 and anti-Tec antibodies; the 4G10 and antiflotillin-1 were diluted 1/4000. The secondary antibodies, HRP-labeled sheep anti-mouse IgG, donkey anti-rabbit, and donkey anti-goat IgG, were used at a final concentration of 1/20,000.

Measurement of calcium mobilization
PMN at a concentration of 10 x 106 cells/ml were incubated at 37°C with 1 µM fura-2-acetoxymethyl ester for 30 min. Extracellular probe was removed by washing in HBSS, and cells were resuspended at a concentration of 5 x 106 cells/ml. Cells were stimulated with 2 ug 3G8 F(ab')2 mAb/10 x 106 cells followed by cross-linking with the 80 ug F(ab')2, goat anti-mouse F(ab')2/10 x 106 cells. Fluorescence was monitored at 37°C in a fluorescence spectrophotometer (SLM 8000C) at an excitation wavelength of 340 nm and an emission wavelength of 510 nm. The internal Ca2+ concentrations were calculated as described [27 ].

Degranulation
PMN (20x106 cells/ml) were incubated for 10 min at 37°C with 10 µM cytochalasin B before stimulation [15 ]. LFM-A11 or LFM-A13 (50 µM) was added simultaneously for the same time. PMN were then incubated for a 2-min incubation at 37°C with 1.25 µg 3G8 F(ab')2 mAb/10 x 106 cells, and cross-linking was subsequently performed at 37°C with 20 µg F(ab')2, goat anti-mouse F(ab')2/10 x 106 cells for 15 min. The cross-linking reaction was ended by a quickspin, and the supernatant was harvested. The extent of degranulation was assessed by adding 100 µl supernatant to 1 ml 0.25 mg/ml "Micrococus lysodeikticus" solution prepared in a 0.1 M PO4 buffer. The loss of absorbance was then read at 450 nm. To obtain a percentage value for degranulation, the absorbance values were compared, and the maximum absorbance was obtained by lysing cells with Triton X-100 (0.1%).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Tec translocates to the plasma membrane and is phosphorylated and activated upon CD16b receptor engagement
According to the activation model of Tec kinases by different receptors, the activation of these kinases occurs in two steps. The first involves the translocation of Tec to the plasma membrane, which is regulated by the interaction of its PH domain with phosphatidylinositol 3,4,5-triphosphate (PIP3). The second involves the tyrosine phosphorylation of the activation loop in the kinase domain by Src kinases followed by autophosphorylation. To determine whether Tec translocates to the plasma membrane upon CD16b engagement, CD16b receptors on freshly isolated PMN were cross-linked and the expression of Tec kinase was determined in isolated plasma membranes as described in Materials and Methods. Immunoblot analysis of isolated plasma membranes reveals that Tec is present in larger amounts (2.5x) at the plasma membrane upon the engagement of CD16b. The equal loading of plasma membrane material was confirmed by immunoblotting with an antiflotillin-1 antibody (Fig. 1 ).



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  		Figure 1. Tec is recruited to the plasma membrane upon CD16b cross-linking. Plasma membranes of human PMN were isolated after 30 s of cross-linking of CD16b as described in Materials and Methods. Plasma membranes of PMN stimulated by cross-linking CD16b (+) and those of nonstimulated PMN (–) were fractionated by SDS-PAGE. (A) The quantity of Tec at the plasma membrane was determined by immunoblotting (IB) with an anti-Tec antibody ({alpha}-Tec; upper panel). The membranes were reblotted with an antiflotillin-1 antibody ({alpha}-Flotillin-1) to confirm that an equivalent quantity of plasma membrane was loaded in each well (lower panel). (B) These results were obtained in three independent experiments. The significance among the values obtained was determined using an unpaired t-test. A P value of <0.01 is considered significant.

 
Having observed that Tec is recruited to the plasma membrane, we sought to determine whether the translocation event is accompanied by the phosphorylation of Tec. As shown in Figure 2A (upper panel), immunoprecipitated Tec is phosphorylated upon CD16b cross-linking. Evidence for the specificity of this response is provided by the lack of detection of Tec in precipitates using an isotype-matched, irrelevant antibody. The phosphorylation of Tec induced upon CD16b cross-linking was rapid and time-dependent (Fig. 2B) . Significant levels of tyrosine-phosphorylated Tec were detectable within the first 15 s upon the cross-linking of CD16b, and maximal levels were consistently obtained at 30–60 s.



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  		Figure 2. Tec is phosphorylated upon CD16b cross-linking. (A) Nonstimulated PMN (–) and PMN stimulated by cross-linking CD16b for 30 s (+) were lysed under native conditions and Tec-immunoprecipitated (Tec) from the lysates. Immunoprecipitation was also performed with an isotype-matched antibody as a control (Isotype). The immunoprecipitates (IP) were processed for immunoblotting, as described in Materials and Methods. The level of phosphorylation of Tec was determined by immunoblotting with an antiphosphotyrosine antibody ({alpha}pY; upper panel). The membranes were reblotted with an anti-Tec ({alpha}Tec) antibody to confirm that Tec was immunoprecipitated in equivalent quantities (lower panel). These results were obtained in three independent experiments. (B) The same experiment as in A was performed over a time course of 15 s–2 min. These results were obtained in three independent experiments.

 
We next examined the upstream events potentially involved in the recruitment and activation of Tec kinases following engagement of CD16b. To determine whether the increase in tyrosine phosphorylation of Tec is dependent on PI-3 kinase (PI-3K), PMN were preincubated with wortmannin prior to CD16b cross-linking. As shown in Figure 3A (upper panel), the increase in tyrosine phosphorylation of Tec is significantly inhibited by wortmannin (compare lanes 2 and 4), implicating PI-3K in the signaling events leading to the tyrosine phosphorylation of Tec upon the engagement of CD16b.



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  		Figure 3. The phosphorylation of Tec is inhibited by the PI-3K inhibitor wortmannin and the Src-kinase inhibitor PP2. Nonstimulated PMN (–) and PMN stimulated by cross-linking CD16b for 30 s (+) were preincubated with 200 nM wortmannin (A) or 5 µM PP2 (B) for 10 and 5 min, respectively, at 37°C. The cells were then lysed under native conditions and Tec-immunoprecipitated from the lysates. The immunoprecipitates were processed for immunoblotting as described in Materials and Methods. The level of phosphorylation of Tec was determined by immunoblotting with an antiphosphotyrosine antibody (upper panel). The membranes were reblotted with an anti-Tec antibody to confirm that Tec was immunoprecipitated in equivalent quantities (lower panel). These results were obtained in three independent experiments.

 
It has been proposed in other cellular systems that after coming into proximity with the plasma membrane, Tec kinases are phosphorylated by Src kinases on their tyrosine residues [22 ]. To determine whether a similar scheme is operative upon CD16b engagement, PMN were preincubated with the Src-kinase inhibitor PP2 prior to CD16b cross-linking. As shown in Figure 3B , the increase in Tec phosphorylation is significantly inhibited upon treatment of PMN with PP2 prior to CD16b cross-linking, suggestive of an involvement of a Src kinase in its activation. To determine whether this inhibition is specific, PMN were also incubated with PP3, an inactive analog of PP2. PP3 had no effect on the phosphorylation of Tec in response to the stimulation of the CD16b receptor (data not shown).

To determine whether Tec phosphorylation is accompanied by an increase in kinase activity, PMN were stimulated with anti-CD16b antibodies, lysed under native conditions, and the lysates immunoprecipitated with an anti-Tec antibody. The immunoprecipitated Tec protein was then tested for its ability to phosphorylate IgG as well as itself, as described in Materials and Methods. As shown in Figure 4 , Tec immunoprecipitated from unstimulated cells exhibits a basal level of activity as evidenced by its ability to autophosphorylate itself (top row, lanes 1–4) or IgG (middle row, lanes 1–4). Cross-linking of CD16b increased the in vitro kinase activity of immunoprecipitated Tec toward both substrates (compare lanes 5–8 of top and middle rows with lanes 1–4 of the same rows).



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  		Figure 4. Tec is activated after CD16b cross-linking. The activity of Tec was determined by an in vitro kinase assay described in Materials and Methods. The kinase assay was stopped at 10, 20, 30, and 40 min, and each sample was immunoblotted with an antiphosphotyrosine antibody (top and middle panels) to assess the activity of Tec on itself (top panel) and on IgG (middle panel), respectively. The membranes were also immunoblotted with an anti-Tec antibody to confirm that Tec was immunoprecipitated in equivalent quantities (bottom panel). These results were obtained in five independent experiments.

 
Effects of LFM-A13 on CD16b-induced signaling
The results summarized in Figures 1 2 3 4 provide evidence for the activation of Tec subsequent to the ligation of CD16b. The determination of the functional relevance of this event is made difficult by the lack of a described, specific inhibitor of Tec and the difficulty to adapt the transfection of dominant-negative mutants or RNA interference approaches to human PMN. Conversely, a Btk inhibitor (LFM-A13), without effect on Src kinase or Janus tyrosine kinase, has been described [28 ]. We confirmed its inhibitory activity toward Btk (data not shown) and examined whether it affected the kinase activity of Tec. As shown in Figure 5 , LFM-A13 inhibited, in an in vitro kinase assay performed on immunoprecipitated Tec, the autophosphorylation of Tec (top row) as well as the ability of Tec to phosphorylate the tyrosine residues of IgG heavy chains (middle panel). It may be of relevance to the mechanism of action of LFM-A13 that it inhibited the phosphorylation of an exogenous substrate (the IgG heavy chain) more than the autophosphorylation activity of Tec. Reblots with an anti-Tec antibody confirmed that equal amounts of Tec were loaded into each lane. In the remainder of this study, we therefore used LFM-A13 to test for the involvement of Tec (and possibly other Tec family members) in selected signaling events.



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  		Figure 5. The activity of Tec after CD16b cross-linking is inhibited by LFM-A13. The activity of Tec in the absence or presence of 50 µM LFM-A13 was determined by an in vitro kinase assay for 40 min as described in Materials and Methods. The activity of Tec was determined by immunoblotting the samples of the in vitro kinase assay with an antiphosphotyrosine antibody (top and middle panels). Immunoblotting of the membranes with an anti-Tec antibody confirmed that Tec was immunoprecipitated in equivalent quantities (bottom panel). These results were obtained in five independent experiments.

 
The effect of LFM-A13 on the phosphorylation of intracellular substrates upon CD16b cross-linking was first investigated. Pretreatment of PMN with LFM-A13 did not affect, to a significant extent, the overall tyrosine phosphorylation profile upon CD16b cross-linking as shown in Figure 6 . In contrast to the lack of effect of LFM-A13 on the overall pattern of tyrosine phosphorylation, the inhibitor significantly reduced the mobilization of intracellular calcium observed upon CD16b cross-linking (Fig. 7 ). This was reflected in a decreased peak of cytosolic-free calcium as well as a shorter duration of the calcium spike.



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  		Figure 6. LFM-A13 does not affect the overall tyrosine phosphorylation profile of intracellular substrates upon CD16b cross-linking. PMN were stimulated by cross-linking CD16b for the indicated times after incubation in HBSS in the presence or absence of 25 µM LFM-A13 for 10 min at 37°C and lysed directly in boiling sample buffer. The lysates were then processed for immunoblotting as described in Materials and Methods. The level of tyrosine phosphorylation was determined by immunoblotting with an antiphosphotyrosine antibody. These results were obtained in two independent experiments.

 


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  		Figure 7. The mobilization of intracellular calcium upon CD16b engagement is inhibited by LFM-A13. PMN were preincubated with 25 µM LFM-A13 for 10 min at 37°C prior to stimulation by cross-linking CD16b. The release of calcium stores upon CD16b engagement was determined as described in Materials and Methods. The mobilization of calcium of a representative experiment is shown (A), and a bar graph of the results of three different experiments is shown (B).

 
PLC{gamma}2 is activated upon CD16b cross-linking
To further characterize the signaling pathways activated upon CD16b cross-linking, we took advantage of the inhibitory activity of LFM-A13 on the mobilization of intracellular calcium upon CD16b engagement. Tec kinases are known in B cells to be important for the phosphorylation and activation of PLC{gamma} [24 ], an enzyme implicated in the modulation of Ca2+ stores through the generation of PI(3,4,5)P3. The potential implication of PLC{gamma} in the mobilization of calcium after CD16b engagement was, therefore, investigated.

PLC{gamma} exists in two forms, PLC{gamma}1 and PLC{gamma}2. Immunoblot analysis revealed that human PMN predominantly express PLC{gamma}2 (data not shown), confirming the observations made by other groups [29 ]. To address the hypothesis that PLC{gamma}2 may be involved in the mobilization of calcium upon CD16b cross-linking, PMN were stimulated with anti-CD16b antibodies, and PLC{gamma}2 was immunoprecipitated as described in Materials and Methods. Immunoblotting a membrane containing the PLC{gamma}2 immunoprecipitates with an antiphosphotyrosine antibody revealed that PLC{gamma}2 is rapidly phosphorylated upon CD16b cross-linking with detectable levels of tyrosine phosphorylation as early as in 10 s and peak levels of phosphorylation by 15 s (Fig. 8A ).



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  		Figure 8. PLC{gamma}2 is phosphorylated upon the activation of CD16b. (A) PMN were stimulated by cross-linking CD16b for the times indicated, lysed under native conditions, and PLC{gamma}2 immunoprecipitated from the lysates. The immunoprecipitates were processed for immunoblotting as described in Materials and Methods. The level of phosphorylation of PLC{gamma}2 was determined by immunoblotting with an antiphosphotyrosine antibody. The membranes were reblotted with an anti-PLC{gamma}2 antibody to confirm that PLC{gamma}2 was immunoprecipitated in equivalent quantities. (B) The same experiment was performed with PMN preincubated with 25 µM LFM-A13 for 10 min at 37°C. A bar graph of three different experiments is shown. These results were obtained in three independent experiments.

 
The sensitivity of the phosphorylation of PLC{gamma}2 upon CD16b cross-linking to LFM-A13 was next investigated. As shown in Figure 8B , preincubation of PMN with LFM-A13 significantly diminished the phosphorylation of PLC{gamma}2 after CD16b engagement. These data support the hypothesis that Tec kinases are involved in the activation of PLC{gamma}2, which in turn, plays a critical role in CD16b-dependent mobilization of intracellular calcium in human PMN.

Degranulation induced through CD16b cross-linking is inhibited by LFM-A13
To draw a link between our observations on the implication of Tec in CD16b signaling and functional responses of PMN, we sought to determine whether LFM-A13 inhibits CD16b-associated effector functions. One such function is degranulation. PMN release the contents of azurophilic and specific granules upon CD16b engagement [15 ]. The release of lysozyme, a constituent of azurophilic and specific granules, was measured in PMN incubated with LFM-A13 prior to CD16b cross-linking as described in Materials and Methods. As shown in Figure 9 , degranulation by PMN is inhibited by more than 50% in the presence of LFM-A13 but not its inactive analog LFM-A11. These results indicate that Tec kinases are involved in signaling pathways that control certain functional responses of PMN upon CD16b engagement.



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  		Figure 9. Degranulation by PMN upon CD16b engagement is inhibited by LFM-A13. PMN were incubated in HBSS in the presence of LFM-A13 or LFM-A11 prior to CD16b cross-linking as described in Materials and Methods. The results are expressed as percent of total lysozyme released, determined by lysing the cells with Triton X-100. The significance of the values obtained between samples was determined using the one-way ANOVA test. A P value of <0.001 is considered significant.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Tec kinases are implicated in a variety of biological processes including cell adhesion, cell migration, apoptosis, and the regulation of gene expression, and they play an important role in the development and activation of lymphocytes. Their participation, however, in the myeloid system has been less well characterized, with the exception of mast cells and platelets.

Tec kinases are expressed in human PMN and participate in the responses of PMN to chemotactic factors [20 ]. The present study was initiated to investigate the possible implication of these cytoplasmic kinases in the transmission of signals across the PMN plasma membrane into the cytoplasm via the CD16b receptor. This study led to several novel observations. First, we provide direct evidence for the involvement of Tec in CD16b signaling. Our observations reveal that the activation of Tec following CD16b cross-linking is consistent with the two-step model described for Tec kinases in lymphocytes [23 ]. According to this model, two regulatory steps are required for the activation of Tec kinases upon receptor engagement. Initially, Tec is recruited to the plasma membrane via interaction with PI(3,4,5)P3, generated by PI-3K. We show that upon CD16b engagement, Tec is recruited to the plasma membrane and that the phosphorylation of Tec is inhibited by wortmannin. These results are suggestive of an implication of PI-3K in the activation of Tec upon CD16b engagement. Once recruited to the plasma membrane, Tec is phosphorylated by Src kinases in the activation loop of the kinase domain. The diminution of Tec phosphorylation after CD16b cross-linking in PMN pretreated with the Src inhibitor PP2 provides direct evidence for the participation of Src kinases as upstream effectors of Tec activation. The major Src kinase implicated in CD16b signaling is Hck [12 ]. It is, therefore, possible that this kinase is involved in the phosphorylation of Tec in response to CD16b activation, although this remains to be examined directly.

The phosphorylation of the tyrosine residue in the activation domain of Tec leads to its activation. We show that CD16b engagement leads to the activation of Tec, as it is able to phosphorylate one of its substrates, the heavy chain of IgG. Together, these observations implicate Tec directly in CD16b signaling.

Two other FcRs are expressed on the surface of human PMN, namely, CD32 and CD64. The role of Tec kinases in the signaling of CD32 has been reported in platelets. In contrast, the possible implication of Tec kinases in CD64 signaling remains to be determined. In mast cells, Tec kinases were reported to participate in the Fc{varepsilon}RIß signaling. Btk-deficient mast cells develop normally but upon activation, are impaired in function in vivo and in vitro. Of relevance to the present study, Amoras et al. [30 ] reported that FcR-mediated phagocytosis was impaired in monocytes from X-linked agammaglobunemia (XLA) patients in which the expression of Btk was decreased or absent. The latter investigators did not examine PMN function. Our observations, together with those in the literature, suggest that Tec kinases are generically important for FcR signaling and therefore, FcR function.

We also provide evidence that Tec kinases are implicated in the initiation of at least one critical signaling pathway in response to CD16b ligation, namely, the mobilization of intracellular calcium. This conclusion was derived from the examination of the tyrosine phosphorylation status of PLC{gamma}2 and through the use of LFM-A13, a compound originally described as a Btk inhibitor. The second novel observation reported by this study is, therefore, that LFM-A13 inhibits the mobilization of intracellular Ca2+ after CD16b cross-linking. Ca2+ mobilization was first investigated, as Btk is known to be required for Ca2+ mobilization by phosphorylating and activating PLC{gamma}2 in B lymphocytes. Our data also indicate that LFM-A13 not only inhibits Btk but also, with similar potency, Tec. The use of LFM-A13 does not, therefore, allow unambiguous identification of the member of the Tec family of kinases implicated in the events monitored. As pointed out above, LFM-A13 inhibits, at least, Tec and Btk. It is possible that investigations of the functional responsiveness of PMN from patients with XLA, who have a defective Btk [24 ], may shed some light on this question.

The observation that LFM-A13 inhibits the mobilization of intracellular Ca2+ upon CD16b engagement led to the investigation of the phosphorylation status of PLC{gamma}2 in response to CD16b cross-linking. These experiments led to the third novel observation of this study, namely, that PLC{gamma}2 is involved in the mobilization of intracellular calcium upon CD16b engagement and that its phosphorylation is dependent on Tec kinases. Cross-linking of CD16b leads to the phosphorylation of PLC{gamma}2, which is inhibited by LFM-A13. The molecular mechanisms involved in Tec activation upon receptor engagement, therefore, seem conserved in PMN and lymphocytes for Tec and possibly Btk. A role for Tec in PLC{gamma}2 phosphorylation and Ca2+ mobilization has been reported in Btk-deficient platelets [24 ]. A comparative analysis of platelets isolated from mice deficient in Btk or Tec or both revealed that compared with Btk-deficient platelets, double-deficient platelets display a greater reduction in PLC{gamma}2 phosphorylation. In view of the absence of CD16b in murine PMN, these experiments need to be repeated in human cells to evaluate their relevance to the latter cells.

We also provide evidence that Tec kinases are important for some effector functions mediated by the cross-linking of CD16b. The major response of PMN to the activation of CD16b receptors is the release of the contents of azurophilic and specific granules. Indeed, the inhibition of Tec kinases by LFM-A13 results in a diminution of the exocytosis of granule proteins. This observation is coherent with the fact that the exocytosis of azurophilic and specific granules in human PMN is dependent on a rise in cytosolic Ca2+, as we show that Tec kinases are important for the activation of PLC{gamma}2 and the mobilization of cytosolic Ca2+ in PMN. It has become increasingly clear that Tec kinases influence a wide range of biological processes, such as actin reorganization, transcriptional regulation, cell survival, and cellular transformation [31 ]. To our knowledge, this is the first demonstration that Tec kinases participate in signaling pathways that are involved in the exocytosis of granules in human PMN.

To date, the signalosome formed upon CD16b cross-linking and its components remains to be characterized. Several reports reveal that the Src-kinase, Hck, as well as Syk (our unpublished observations) are involved in the early events of CD16b signaling. Downstream effectors include ERK [13 , 14 ], p38 [13 , 14 ], and Pyk2. This report, therefore, contributes to the identification of signaling molecules and pathways, activated on CD16b engagement. Tec kinases may be involved in a variety of biological processes that are related to the effector functions of CD16b, such as the reorganization of the actin cytoskeleton, cell adhesion, and the regulation of apoptosis. Defining the role of Tec kinases in the effector functions of CD16b will further our understanding of the molecular mechanisms involved in the binding and uptake of opsonized antigen and of immune complexes by human PMN.


   ACKNOWLEDGEMENTS
 
This work was supported in part by grants from the Canadian Institutes of Health Research, the Arthritis Society of Canada, and the Canadian Arthritis Network awarded to M.J.G.F. and P.H.N. P. H. N. is the recipient of the Canada Research Chair on the Physiopathology of the Neutrophil.

Received August 30, 2004; revised April 8, 2005; accepted April 11, 2005.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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