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The Journal of Immunology, 2006, 176: 52-60.
Copyright © 2006 by The American Association of Immunologists

Integrin {alpha}IIb{beta}3 Induces the Adhesion and Activation of Mast Cells through Interaction with Fibrinogen1

Toshihiko Oki*, Jiro Kitaura*, Koji Eto{dagger}, Yang Lu*, Mari Maeda-Yamamoto{ddagger}, Naoki Inagaki§, Hiroichi Nagai§, Yoshinori Yamanishi*, Hideaki Nakajina*, Hidetoshi Kumagai* and Toshio Kitamura2,*

* Division of Cellular Therapy and Division of Hematopoietic Factors, Advanced Clinical Research Center, and {dagger} Laboratory of Stem Cell Therapy, Center for Experimental Medicine, Institute of Medical Science, University of Tokyo, Tokyo, Japan; {ddagger} National Institute of Vegetable and Tea Science, National Agriculture Research Organization, Shizuoka, Japan; and § Department of Pharmacology, Gifu Pharmaceutical University, Gifu, Japan


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Integrin {alpha}IIb, a well-known marker of megakaryocyte-platelet lineage, has been recently recognized on hemopoietic progenitors. We now demonstrate that integrin {alpha}IIb{beta}3 is highly expressed on mouse and human mast cells including mouse bone marrow-derived mast cells, peritoneal mast cells, and human cord blood-derived mast cells, and that its binding to extracellular matrix proteins leads to enhancement of biological functions of mast cells in concert with various stimuli. With exposure to various stimuli, including cross-linking of Fc{epsilon}RI and stem cell factor, mast cells adhered to extracellular matrix proteins such as fibrinogen and von Willebrand factor in an integrin {alpha}IIb{beta}3-dependent manner. In addition, the binding of mast cells to fibrinogen enhanced proliferation, cytokine production, and migration and induced uptake of soluble fibrinogen in response to stem cell factor stimulation, implicating integrin {alpha}IIb{beta}3 in a variety of mast cell functions. In conclusion, mouse and human mast cells express functional integrin {alpha}IIb{beta}3.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Mast cells, derived from circulating CD34+ hemopoietic progenitor cells, differentiate and proliferate in vascularized tissues. These steps are critically regulated by stem cell factor (SCF),3 the ligand for c-kit, which is a receptor tyrosine kinase expressed on the surface of mast cells (1, 2) as well as immature hemopoietic cells.

It is widely accepted that mast cells play a critical role in IgE-mediated immune reactions such as immediate hypersensitivity; mast cells are activated by cross-linking of Ag-specific IgE bound to Fc{epsilon}RI with a multivalent specific Ag. Mast cells secrete preformed and newly synthesized proinflammatory mediators such as histamine, lipids, and cytokines (1, 2, 3). In addition, some sets of monomeric IgE molecules, termed high cytokinergenic (HC) IgE, can also activate mast cells and induce a similar response without specific Ags (1, 4, 5).

In contrast, mast cells also participate in a wide variety of pathological processes independent of IgE, including innate immune response (1, 2, 6), tissue repair (1, 2, 7), acute inflammatory response to implanted biomaterials (8), atherosclerosis (9), and certain autoimmune disorders (10).

Under both IgE-dependent and -independent pathological conditions, cell-extracellular matrix (ECM) interactions mediated by integrins play crucial roles in a variety of mast cell functions such as histamine release (11), cytokine production (3, 5), survival (5), growth (12), and migration (13, 14).

Integrins are heterodimeric type I transmembrane receptors composed of two subunits ({alpha} and {beta}). Integrin {alpha}IIb, recently proven to be a marker for early hemopoietic progenitors (15), was considered to be expressed exclusively on megakaryocyte-platelet lineage as a complex with integrin {beta}3 to form integrin {alpha}IIb{beta}3, also called glycoprotein IIb-IIIa or CD41-CD61, whereas integrin {beta}3 is present on many types of cells as a complex with integrin {alpha}V. In platelets, integrin {alpha}IIb{beta}3 works as a receptor for fibrinogen, von Willebrand factor (vWF), vitronectin (VN), fibronectin (FN), CD40L, and others (16). Integrin {alpha}IIb{beta}3 links platelets to the injured sites of vessels through interaction with fibrinogen and vWF, together with an aggregate to form a platelet plug, leading to hemostasis (16, 17, 18, 19, 20, 21). Eventually, fibrinogen is internalized and stored in granules of megakaryocytes and platelets (22, 23, 24, 25).

The activation of bone marrow-derived mast cell (BMMC) with various stimuli, including IgE and Ag (this mode of stimulation termed IgE plus Ag) (1, 2, 11, 18, 26), monomeric IgE (1, 5, 27), SCF (28, 29), and thrombin (30) induces the adhesion to FN predominantly via integrin {alpha}5{beta}1. BMMC stimulated by TGF-{beta} adhere to laminin-1 via integrin {alpha}7 (31), whereas adhesion of BMMC to VN is mediated by integrin {alpha}V{beta}3 (12). IgE plus Ag-stimulated peritoneal mast cells (PMC), but not BMMC, adhere to type I collagen via integrin {alpha}2{beta}1 (32). However, functions of integrin {alpha}IIb{beta}3 have never been addressed in mast cells.

In the present work, we demonstrate that integrin {alpha}IIb{beta}3 is highly expressed on mast cells using mouse BMMC, PMC, and human cord blood- derived mast cells. We also found that mast cells stimulated by various stimuli adhere to ECM protein such as fibrinogen and vWF in an integrin {alpha}IIb{beta}3-dependent manner. Moreover, the interaction of integrin {alpha}IIb{beta}3 to fibrinogen enhances mast cell functions and induces uptake of fibrinogen into mast cells. Considering that several drugs regulate the function of integrin {alpha}IIb{beta}3 (33), these results might provide a feasible way to overcome mast cell-mediated disorders.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Abs and other materials

The source of Abs are as described. Anti-mouse integrin {alpha}IIb{beta}3 mAb (1B5) was a gift from Dr. B. S. Coller (Rockefeller University, New York, NY) (34). Anti-mouse integrin {alpha}V (8B3) and anti-mouse integrin {beta}3 (8B11) mAbs were gifts from Drs. D. J. Gerber and S. Tonegawa (Picower Center, Massachusetts Institute of Technology, Boston, MA) (35). Anti-human integrin {alpha}IIb{beta}3 mAb (2G12) was a gift from Dr. V. L. Woods (University of California, San Diego, CA) (36). Anti-DNP IgE (SPE-7) was from Sigma-Aldrich. Anti-TNP IgE (C38-2), anti-mouse integrin {alpha}IIb (MWReg30), anti-mouse integrin {alpha}V (RMV-7 and H9.2B8), anti-mouse integrin {alpha}4 (9C10), anti-mouse integrin {alpha}5 (5H10-27), anti-mouse LFA1 (M17/4), anti-mouse integrin {beta}1 (Ha2/5), anti-mouse integrin {beta}2 (GAME-46), and anti-mouse integrin {alpha}V{beta}3 (2C9.G2) mAbs, and others were from BD Biosciences. Cytokines such as mouse IL-3 and SCF were obtained from R&D Systems. Thrombin, bovine serum VN, and DNP-BSA were from Sigma-Aldrich, and human plasma fibrinogen and vWF were from Chemicon International. Human fibrinogen labeled with Alexa Fluor 488 was purchased from Molecular Probes.

Cells

To generate BMMC with 90% purity (c-kit+/Fc{epsilon}RI+ by flow cytometry), bone marrow cells from 6-wk-old male BALB/c or C57BL/6 or CBA mice (Charles River Laboratories) were cultured for 4–8 wk in RPMI 1640/10% FCS supplemented with 2-ME and 10 ng/ml IL-3 (BMMC culture medium).

PMC were generated from peritoneal cells, as described previously (37, 38). In brief, mononuclear cells collected from peritoneal lavage were cultured for 10–14 days in the presence of 10 ng/ml IL-3 and 30 ng/ml SCF, and PMC with 90% purity were obtained.

Skin-derived mast cells were generated from day 16 fetal skin of mice as described (39, 40). Briefly, excised trunk skin was treated with 0.25% of trypsin in HBSS for 30 min at 37°C, and the dispersed cells were cultured in the medium containing 10% FCS, 10 ng/ml IL-3, and 30 ng/ml SCF for 14 days.

Lung-derived mast cells were generated from lung tissue samples of adult mice as described (41). Briefly, the samples were cut into fragments and digested with collagenase and hyaluronidase. The single cell suspension were prepared and cultured in the medium containing 10% of FCS, 10 ng/ml IL-3, and 30 ng/ml SCF for 14 days.

Human cord blood-derived mast cells were generated from CD34+ cord blood cells, as described (42). Mast cells with 90% purity were generated by 8 wk of culture in the medium containing 10% FCS, 100 ng/ml human SCF, and 50 ng/ml human IL-6. Megakaryocytes were generated, as described previously (43). Animal and human studies were approved by the animal care committee and the ethical committee of the Institute of Medical Science, University of Tokyo (Tokyo, Japan).

Bone marrow-derived basophils were prepared as described (44). In brief, bone marrow cells were cultured for 10 days in the presence of 10 ng/ml IL-3. Approximately 30% of the cells were Fc{epsilon}RI+/c-kit– population, which were mainly composed of basophils.

FACS analysis

Cells (3 x 105 cells) were suspended in 2% FCS/PBS, blocked with mouse FcBlock (BD Biosciences), washed with 2% FCS/PBS, incubated with 20 µg/ml primary Abs for 20 min at 4°C, washed twice, incubated with 20 µg/ml secondary Abs for 20 min, washed twice again, then resuspended in 2% FCS/PBS. The samples were then analyzed using a FACSCalibur flow cytometer (BD Biosciences).

RT-PCR

Total RNA was prepared from freshly isolated cells with TRIzol (Invitrogen Life Technologies). Total RNA from each sample was reverse transcribed with SuperScript Reverse Transcriptase kit (Qiagen) and oligo(dT) primer. The primer used for integrin {alpha}IIb in this study was as described previously (45)

Immunoprecipitation assay

Ten million cells of BMMC and BW1457 were surface biotinylated, collected, and lysed in a radioimmunoprecipitation assay buffer. The lysates were immunoprecipitated with a control Ab (hamster IgG or rat IgG) or anti-mouse integrin {alpha}IIb Abs (1B5 or MWReg30) or an anti-mouse integrin {alpha}V{beta}3 Ab (2C9.G2). Immunoprecipitates were run on 7.5% SDS-PAGE gels and transferred to Immobilon membranes. The membranes were blotted using HRP-conjugated streptavidin.

Adhesion assay

Adhesion assay was done as described previously (28). In brief, the 96-well plates (no. 3631; Corning) were coated with 20 or 50 µg/ml VN, vWF, or fibrinogen in PBS for 1 h at 37°C, and washed three times with PBS, followed by blocking with 3% BSA/PBS for 1 h at 37°C and washing three times with RPMI 1640 containing 10 mM HEPES and 0.03% BSA (the assay medium). BMMC, washed four times in the assay medium, were resuspended at 5 x 105 cells/ml in the assay medium and transferred into coated wells (100 µl/well) with or without stimulant including IgE (SPE-7), DNP-BSA (for IgE plus Ag), SCF, and thrombin with the indicated concentration for 1 h at 37°C. For stimulation with IgE plus Ag, BMMC were pretreated with 0.5 µg/ml IgE (C38-2) overnight at 37°C. After washing, cell adhesion was quantitated using CellTiter-Glo (Promega) and a Micro Lumat Plus luminometer (EG&G Berthold), according to the manufacturer’s instructions.

In assays using blocking Abs, BMMC were preincubated with 10–50 µg/ml Abs for 1 h before adding the cells to the plate.

Migration assay

Migration assays were conducted, as previously described (13), using 24-well Transwell chambers with 5-µm polycarbonate filters (Corning). Briefly, the underside of each insert was treated with 30 µg/ml FN or 50 µg/ml fibrinogen for 1 h at 37°C, blocked with 3% BSA/PBS for 1 h at 37°C, then placed back in the Transwell lower chamber containing 0.5 ml of the assay medium, with or without stimulants such as SCF. The washed cells resuspended at 7.5 x 107 cells/ml in 0.2 ml of the assay medium were transferred to the insert. After incubating them for 8 h at 37°C, the cells migrated to lower chambers were counted using a hematocytometer.

Cytokine assay (ELISA)

The cells were transferred into FN- or fibrinogen-coated 96-well plates (1 x 104 cells/well) with or without stimulants. After incubating for 12 h at 37°C, the supernatant of each well was collected, and the concentration of IL-6 was quantified by ELISA with OptiEIA for IL-6 (BD Pharmingen).

Growth assay

The cells were resuspended at 3 x 105 cells/ml in a BMMC culture medium with IL-3, and transferred into fibrinogen-coated 24-well plates with or without SCF. After incubation for the indicated time period at 37°C, the cells were collected and counted using a hematocytometer.

Induced uptake of fibrinogen into SCF-stimulated mast cells

BMMC were suspended at 5 x 105 cells/ml in a Tyrode’s buffer with 10 µg/ml of a control Ab or the anti-integrin {alpha}IIb{beta}3 Ab (1B5). Cell were then incubated for 30 min at room temperature, and incubated with 20 µg/ml fibrinogen labeled with Alexa Fluor 488 in the presence of 100 ng/ml SCF or PBS for 30 min at room temperature. After washing, the cells were analyzed using FACS or a confocal microscope (Olympus Tokyo). For confocal microscopy, the cells attached to microscope slides were fixed with 4% paraformaldehyde, stained with 4',6'-diamidino-2-phenylindole, and then visualized.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
BMMC and PMC express integrin {alpha}IIb{beta}3

We first tested the expression of integrins on two different types of mast cells, BMMC and PMC (Fig. 1, A and B), both of which were >90% Fc{epsilon}RI+/c-kit+ using flow cytometric analysis. We confirmed the expression of integrin {alpha}4 (11, 13, 30, 46, 47), {alpha}5 (5, 11, 26, 28, 30), {alpha}V (12), {alpha}L (48, 49), {beta}1 (11, 26), {beta}2 (50), and {beta}3 (12), as reported (Fig. 1A). Interestingly, the expression of integrin {alpha}IIb was evident on both BMMC and PMC in FACS and RT-PCR (Fig. 1, A and B), the expression level being higher in the former. The expression was not due to megakaryocyte progenitors contaminated in our BMMC preparation because we did not detect a megakaryocyte-specific marker, GPIb-a, GPIb-b, or GPV, in our BMMCs (Fig. 1A and data not shown). We also confirmed the expression on other types of mast cells, such as skin- and lung-derived mast cells (Fig. 1C). As integrin {alpha}IIb heterodimerized solely with integrin {beta}3, integrin {alpha}IIb{beta}3 was expected to be expressed on mouse mast cells. To verify integrin {alpha}IIb{beta}3 expression, BMMC and BW5147 expressing only integrin {alpha}V{beta}3 were surface-labeled by biotin and immunoprecipitated with a specific Ab against integrin {alpha}IIb{beta}3 (1B5). Western blots developed with HRP-conjugated streptavidin gave two bands corresponding to integrin {alpha}IIb and {beta}3 only in the precipitates from BMMC but not from BW5147, suggesting that mouse mast cells express integrin {alpha}IIb{beta}3 on the cell surface (Fig. 1D).



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  		FIGURE 1. Surface expression of integrin {alpha}IIb{beta}3 was detected on mouse mast cells. A, BMMC with >90% purity (Fc{epsilon}RI+/c-kit+) expressed integrin {alpha}IIb as well as integrins {alpha}V, {alpha}4, {alpha}5, {alpha}L, {beta}1, {beta}2, {beta}3 on BMMC, but not platelet-specific markers GPIb-{alpha} or GPIb-{beta}. The x-axis indicates fluorescence intensity as a log scale ranging from 100 to 104. The y-axis indicates the number of the cells. B, PMC with 90% purity (Fc{epsilon}RI+/c-kit+) also expressed integrin {alpha}IIb and {beta}3. RT-PCR confirmed the expression of integrin {alpha}IIb in BMMC, PMC, and P815, a mast cell line, but not in EL-4, a T cell line. C, Skin- and lung-derived mast cells expressed integrin {alpha}IIb. D, Both integrin {alpha}IIb{beta}3 and integrin {alpha}V{beta}3 were expressed on the surface of BMMC, whereas only integrin {alpha}V{beta}3 was expressed on BW5147. The cell lysates of surface biotinylated BMMC and BW5147 were immunoprecipitated with control Ab (lanes 1, 3, 5, and 7) or the Abs against integrin {alpha}V{beta}3 (lanes 2 and 6) or {alpha}IIb{beta}3 (lanes 4 and 8), then analyzed with HRP-conjugated streptavidin. E, The expression of integrin {alpha}IIb on bone marrow-derived cells after different times of culture in the presence of IL-3 was also analyzed using flow cytometry. Dot plot shows Fc{epsilon}RI+/c-kit+ and {alpha}IIb+/c-kit+ cells on day 28. F, The expression of integrin {alpha}IIb was observed on bone marrow-derived basophils. G, The expression of integrin {alpha}IIb on B220+ splenocytes, CD3+ splenocytes, Gr-1+ bone marrow cells, and Mac-1+ bone marrow cells was not detected in FACS analysis.

 
Next, the expression levels of integrin {alpha}IIb were examined on BMMC in the course of culture with IL-3, that is, in the varying developmental stages toward BMMC. The percentage of {alpha}IIb+/c-kit+ cells gradually increased during the culture in parallel with increase in the percentage of Fc{epsilon}RI+/c-kit+ cells indicative of mast cells or mast cell progenitors, and reached ~90% after 28 days (Fig. 1E).

Interestingly, Fc{epsilon}RI+/c-kit– cells, which were mainly composed of bone marrow-derived basophils, expressed integrin {alpha}IIb at the levels comparable to mast cells (Fig. 1F). By contrast, no or negligible expression of integrin {alpha}IIb was observed on T cells (CD3+ splenocytes), B cells (B220+ splenocytes), granulocytes (Gr-1+ bone marrow cells), macrophages (Mac-1+ bone marrow cells, bone marrow-derived macrophages, and peritoneal macrophages), and dendritic cells (bone marrow-derived dendritic cells) (Fig. 1G and data not shown). These results suggest that the expression of integrin {alpha}IIb on cells other than megakaryocytes/platelets was limited to mast cells.

Adhesion of mast cells to immobilized fibrinogen- and vWF-mediated by integrin {alpha}IIb{beta}3

As integrin {alpha}IIb{beta}3 works as a receptor for ECM proteins, such as fibrinogen, vWF, VN, and FN in megakaryocytes and platelets, we examined whether mast cells expressing integrin {alpha}IIb{beta}3 could adhere to these ECM proteins.

Although nonstimulated BMMC did not significantly bind to any ECM proteins, BMMC stimulated by monomeric HC IgE (SPE-7) adhered to all of the ECM proteins (Fig. 2, A and B). Integrin {alpha}IIb{beta}3 on platelets could interact with all these ECM proteins, but several studies revealed that the adhesion of mast cells to FN and VN was mainly mediated by integrin {alpha}5{beta}1 (5, 11, 28, 29, 30, 31) and {alpha}V{beta}3 (12), respectively.



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  		FIGURE 2. Integrin {alpha}IIb{beta}3 on stimulated BMMC mediated their adhesion to fibrinogen and vWF. A and B, Adhesiveness of BMMC stimulated with or without 3 µg/ml HC IgE (SPE-7) for 1 h at 37°C was analyzed on plates coated with or without 20–50 µg/ml ECM proteins (fibrinogen, FN, VN, vWF) by calculating the percentage of the adherent cells (A) or by microscope (magnification, x200 or x400) (B). The results of the functional blockage of integrins with specific anti-integrin Abs (50 µg/ml anti-{beta}1 Ab, 50 µg/ml anti-integrin {alpha}V{beta}3 Ab, 10 µg/ml anti-integrin {alpha}IIb{beta}3 Ab) are also shown. C, The interaction between integrin {alpha}IIb{beta}3 and fibrinogen was observed in response to stimuli, such as 3 µg/ml IgE (SPE-7), 10 ng/ml DNP-BSA (IgE plus Ag), 100 ng/ml SCF, 1 mM MnCl2, and 10 U/ml thrombin, and was blocked by 10 µg/ml anti-integrin {alpha}IIb{beta}3 Ab. D, Interaction blocked by 100 µg/ml RGD peptides but not by RGE peptides. The results shown are the average ± SD of three independent experiments. **, p < 1% (vs the control Ab-treated sample determined by Student’s t test).

 
To clarify the involvement of each integrin in mast cell adhesion to ECM proteins, we used specific blocking Abs against integrin {alpha}IIb{beta}3 (anti-integrin {alpha}IIb{beta}3 Ab), 1B5 and {alpha}V{beta}3 (anti-integrin {alpha}V{beta}3 Ab), 2C9.G2. A hamster mAb, 1B5, raised against mouse integrin {alpha}IIb{beta}3, specifically recognizes mouse integrin {alpha}IIb{beta}3 and completely blocks platelet-fibrinogen interaction, thereby inhibiting platelet aggregation (34). On the contrary, 2C9.G2, a hamster mAb against mouse integrin {alpha}V{beta}3, reacts with integrin {beta}3 in FACS analysis and blocks the adhesion via integrin {alpha}V{beta}3 (11, 51).

BMMC were pretreated with specific blocking Abs, anti-integrin {alpha}IIb{beta}3 Ab, anti-integrin {alpha}V{beta}3 Ab, or anti-integrin {beta}1 Ab, Ha2/5, before the adhesion assay. As shown in Fig. 2, A and B, anti-integrin {alpha}IIb{beta}3 Ab inhibited 90% of the adhesion of mast cells to fibrinogen and 70% of adhesion to vWF, whereas it only weakly inhibited of the adhesion to VN. On the contrary, the anti-integrin {alpha}V{beta}3 Ab profoundly inhibited the adhesion of mast cells to VN, whereas it inhibited only 20–30% of the mast cell adhesion to vWF, and did not significantly inhibit that to fibrinogen. The anti-integrin {beta}1 Ab only partially inhibited mast cell adhesion to VN and vWF, and did not significantly affect that to fibrinogen, indicating that the involvement of integrin {beta}1 in mast cell adhesion to these ECM proteins was limited. However, binding to FN was inhibited by anti-integrin {beta}1 Ab but not by anti-integrin {alpha}IIb{beta}3 Ab or anti-integrin {alpha}V{beta}3 Ab, as reported (4, 5, 11, 28, 29, 30, 31). Similar results were obtained in experiments using PMC (data not shown). Collectively, these results indicate that the interaction of mast cell with fibrinogen was specifically mediated by integrin {alpha}IIb{beta}3 and that the interaction of mast cells with VN was mainly mediated by integrin {alpha}V{beta}3, as reported (12), but that the efficient adhesion to vWF required both integrin {alpha}V{beta}3 and {alpha}IIb{beta}3.

Characterization of mast cell adhesion to fibrinogen

Next, we asked whether other stimuli could induce the same adhesion, and found that BMMC bound to fibrinogen-coated plates upon stimulation with IgE plus Ag, SCF, thrombin, or Mn2+ (Fig. 2C)

The interaction of integrin {alpha}IIb{beta}3 on mast cells to fibrinogen was RGD ((Arg-Gly-Asp)-dependent (Fig. 2D) as noted in platelets, and the kinetics of the adhesion induced by HC IgE (SPE-7) and SCF to fibrinogen showed that the adhesion reached a plateau level from 30 to 60 min after the simulation at 37°C (data not shown). As was the case of mast cell adhesion via integrin {alpha}5{beta}1 (5, 11, 29), there was no change in the cell surface levels of integrins {alpha}IIb, {alpha}V, and {beta}3 following treatment with these reagents (data not shown). Collectively, these results indicate that adhesion of mast cells to fibrinogen requires inside-out signaling followed by increase in the affinity/avidity of integrin {alpha}IIb{beta}3.

SCF induced integrin {alpha}IIb{beta}3-directed migration of mast cells

Accumulating evidence revealed that several kinds of integrins were involved in the migration of mast cells (13, 37, 47, 50), and that the interaction between SCF and c-kit regulated the migration of mast cells via ECM proteins, especially FN (13, 14, 37, 47, 50, 52). We examined SCF-induced migration of mast cells via fibrinogen, and then tested whether integrin {alpha}IIb{beta}3-fibrinogen interaction would induce migration.

As shown in Fig. 3A, the number of migrated BMMC were 5-fold higher via the fibrinogen-treated Transwell membrane surface than the BSA-treated surface in the presence of SCF in the lower wells, and this enhancement was significantly blocked by anti-integrin {alpha}IIb{beta}3 Ab, indicating that SCF enhances migration via fibrinogen in an integrin {alpha}IIb{beta}3-dependent manner.



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  		FIGURE 3. Integrin {alpha}IIb{beta}3 enhanced mast cell function. A, BMMC in upper wells were attracted by 100 ng/ml SCF in lower wells, much more efficiently through fibrinogen or FN-treated Transwell than a BSA-treated surface in an 8-h assay. This enhancement was blocked by 10 µg/ml anti-integrin {alpha}IIb{beta}3. B, BMMC stimulated with 100 ng/ml SCF for 12 h produced IL-6 on FN- or fibrinogen-treated plates. The concentration of IL-6 in culture supernatants was quantified by ELISA. IL-6 production was enhanced on fibrinogen-coated plates, and the enhancement was significantly blocked by 10 µg/ml anti-integrin {alpha}IIb{beta}3 Ab. C, The number of BMMC after 7 days of culture in the presence of 10 ng/ml IL-3 and 100 ng/ml SCF was augmented on fibrinogen-coated plates and significantly blocked with 10 µg/ml anti-integrin {alpha}IIb{beta}3 Abs. The results shown are the average ± SD of three independent experiments. *, p < 5% and **, p < 1% (vs the cells incubated in BSA-coated plates or the control Ab-treated cells, respectively, as determined by Student’s t test).

 
Effects of mast cell-fibrinogen interaction on mast cell functions

Adhesion of mast cells to FN was reported to enhance mast cell functions, such as production of IL-6 or TNF-{alpha} (28), proliferation (12), survival (28), and histamine release (5). We tested whether mast cell adhesion to fibrinogen had any effects on mast cell functions: IL-6 production, growth, and survival and histamine release.

First, IL-6 production was measured using ELISA. BMMC attached to fibrinogen-coated plates produced 50% larger concentrations of IL-6 than those on BSA-coated plates in response to SCF or thrombin, and this enhancement of IL-6 production was clearly blocked by anti-integrin {alpha}IIb{beta}3 Ab, indicating the interaction between integrin {alpha}IIb{beta}3 and fibrinogen enhances IL-6 production of mast cells (Fig. 3B). In contrast, histamine release from BMMC was not enhanced by the interaction with fibrinogen via the integrin (data not shown).

Second, growth and survival of mast cells was examined. The number of BMMC on fibrinogen-coated plates was 45% higher than that on BSA-coated plates after 1 wk of culture, and this increment was also dependent on integrin {alpha}IIb{beta}3 (Fig. 3C). However, we could not find the effects of the binding to fibrinogen on the differentiation of BMMC or bone marrow cells cultured with IL-3 plus SCF (data not shown).

SCF-induced survival was estimated by measuring the percentage of apoptotic cells, induced by the withdrawal of IL-3. However, no significant difference was found between BMMC on fibrinogen-coated and BSA-coated plates in the presence of SCF (data not shown). Thus, the adhesion to fibrinogen enhanced the proliferation mast cells in the presence of SCF but did not prevent the apoptosis of mast cells in the absence of cytokines.

Endocytosis of fibrinogen into mast cells via integrin {alpha}IIb{beta}3

We analyzed the interaction of integrin {alpha}IIb{beta}3 to soluble fibrinogen labeled with Alexa Fluor 488 using FACS. BMMC bound soluble fibrinogen in an integrin {alpha}IIb{beta}3-dependent manner (Fig. 4A) when they were activated by HC IgE, IgE plus Ag, SCF, thrombin, or Mn2+. It was previously shown that activated platelets bound to soluble fibrinogen and internalized it (24). To examine whether fibrinogen internalization occurs in mast cells as well, we analyzed the localization of soluble fibrinogen labeled with Alexa Fluor 488 in activated BMMC using a confocal microscope. Fibrinogen labeled with Alexa Fluor 488 was bound and internalized into the mast cells (Fig. 4B). This phenomenon was blocked by anti-integrin {alpha}IIb{beta}3 Ab, indicating that the activated mast cells can internalize fibrinogen via integrin {alpha}IIb{beta}3.



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  		FIGURE 4. Uptake of soluble fibrinogen in SCF-stimulated BMMC in an integrin {alpha}IIb{beta}3-dependent manner. A and B, BMMC were incubated with 20 µg/ml soluble fibrinogen labeled with Alexa Fluor 488 (FB-Alexa) in the presence of 100 ng/ml SCF. A, In flow cytometry, SCF-stimulated BMMC bound soluble fibrinogen labeled with Alexa Fluor 488. B, In the analysis using a confocal laser microscope, the fibrinogen labeled with Alexa Fluor 488 was incorporated in the cells. This phenomenon was blocked by 10 µg/ml anti-integrin {alpha}IIb{beta}3 Ab.

 
Human mast cells expressed integrin {alpha}IIb{beta}3 and mediated adhesion to fibrinogen

To confirm whether integrin {alpha}IIb{beta}3 is also expressed and is functional on human mast cells, human cord blood-derived mast cells (Fig. 5A) were generated as reported (42). Toluidine blue and tryptase staining confirmed that the purity of the mast cells exceeded 90%. FACS analysis showed that these cells expressed integrin {alpha}IIb{beta}3 (Fig. 5B). In addition, SCF- or MnCl2-stimulated human cord blood-derived mast cells adhered to fibrinogen in an integrin {alpha}IIb{beta}3-dependent manner, as observed in the case of mouse BMMC and PMC (Fig. 5C). An experiment using the blocking Ab (2G12) confirmed that SCF-induced migration of the human mast cells was also enhanced via fibrinogen through integrin {alpha}IIb{beta}3 (data not shown)



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  		FIGURE 5. Human cord blood-derived mast cells express integrin {alpha}IIb{beta}3 and adhered to fibrinogen in an integrin {alpha}IIb{beta}3-dependent manner. A, Typical mast cells developed in the presence of SCF and IL-6 were stained with May-Grünwald Giemsa. B, Surface expression of integrin {alpha}IIb{beta}3 on human cord blood-derived mast cells was analyzed using flow cytometry. C, Adhesion of human cord blood-derived mast cells was induced by 100 ng/ml human SCF or 1 mM MnCl2, and blocked by 60 µg/ml anti-integrin {alpha}IIb{beta}3 Ab (2G12).

 

   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Expression of integrin {alpha}IIb{beta}3 on mast cells

Expression of integrin {alpha}IIb was considered to be restricted to platelets/megakaryocytes. As recently reported, it turned out to be a marker for early hemapoietic progenitors as well (12, 13, 14, 15, 16)

In the present study, we demonstrate that integrin {alpha}IIb{beta}3 is expressed on several types of mouse mast cells, BMMC, PMC, skin- and lung-derived mast cells, and human cord blood-derived mast cells. There are two different types of mast cells, consisting of connective tissue and mucosal mast cells (1, 2). PMC and skin-derived mast cells represent connective tissue mast cells. BMMCs have characteristics similar to mucosal and connective tissue mast cells, depending on which factor their culture medium includes (IL-3 and IL-3 plus SCF, respectively). No remarkable change was observed in the expression level of integrin {alpha}IIb{beta}3 on BMMC under both culture conditions (data not shown). Thus, integrin {alpha}IIb{beta}3 is thought to be expressed on both types of matured mast cells. We observed a low level expression of integrin {alpha}IIb only on a small population of bone marrow cells before the culture (data not shown), but the percentage of integrin {alpha}IIb+/c-kit+ cells and the expression level of integrin {alpha}IIb gradually increased in culture along with the increase of Fc{epsilon}RI+/c-kit+ cells, mast cell-lineage cells (44).

Next, we examined the expression of integrin {alpha}IIb on basophils. However, it was difficult to confirm the expression on freshly isolated basophils from peripheral blood, because of the binding of platelet-derived microparticles, which transfer several kinds of platelet Ags like integrin {alpha}IIb CD62 to other circulating blood cells (53). In contrast, BMMC Fc{epsilon}RI+/c-kit–, mainly composed of basophils and basophil precursors (44, 54), express comparable levels of integrin {alpha}IIb. As Fc{epsilon}RI+/c-kit– cells were generated after 10 days culture, the amount of platelet-derived microparticles binding to these cells was supposed to be negligible, indicating that bone marrow-derived basophils express integrin {alpha}IIb. However it remains to be elucidated whether basophils circulating in peripheral blood express functional integrin {alpha}IIb.

By contrast, no or negligible expression of integrin {alpha}IIb was observed on splenic T cells, splenic B cells, granulocytes, macrophages, and dendritic cells. These results show that high expression of integrin {alpha}IIb on mature cells is restricted to mast cells and bone marrow-derived basophils, except for platelets/megakaryocytes.

Although the precise mechanisms of the regulation of integrin {alpha}IIb expression remain unknown, various transcription factors and their cofactors involved in megakaryocyte development, such as GATA-1, GATA-2, FOG-1, SCL, NF-E2, AML-1, Fli-1, Gfi-1b, MafB, Ets-1, and Ets-2 were implicated in the regulation of integrin {alpha}IIb gene (55, 56). The expression of all these molecules was detected by RT-PCR both in megakaryocytes and mast cells, but not in mature cells of other lineages (data not shown). This result may illustrate, in part, the reason why integrin {alpha}IIb is expressed on mast cells, which is in accordance with a previous report that mast cell progenitors and erythroid/megakaryocyte progenitors are closely related (57). Further investigation is underway to understand the regulatory mechanism of integrin expression.

Adhesion of mast cells to ECM proteins via integrin {alpha}IIb{beta}3

Various integrins are implicated in mast cell adhesion. Our evidence shows that integrin {alpha}IIb{beta}3 on mouse and human mast cells was functionally potent and mediated mast cell adhesion to fibrinogen and vWF. This finding was observed only when these mast cells were activated by some stimuli, including IgE plus Ag, HC IgE, and SCF. In contrast, the blockade of integrin {alpha}IIb{beta}3 by a specific Ab did not alter the adhesion of mast cells to VN and FN. This result is consistent with the previous result that the adhesion of mast cells to VN and FN is mainly mediated by integrins {alpha}V{beta}3 and {beta}1, respectively (5, 11, 28, 29, 30, 31, 56).

Suehiro et al. (58) reported that purified integrin {alpha}V{beta}3 molecule had a higher affinity to VN than did integrin {alpha}IIb{beta}3. Kieffer et al. (59) showed that integrin {alpha}IIb{beta}3 expressed on a melanoma cell line and HEL selectively bound to fibrinogen, not to VN, whereas integrin {alpha}V{beta}3 on these cell lines mediated the adhesion to VN. Collectively, the integrin that is used in the interaction to VN, fibrinogen, and vWF could be determined by the different affinity and expression levels of integrins {alpha}IIb{beta}3 and {alpha}V{beta}3.

Interestingly, bone marrow cells of integrin {alpha}IIb knockout mice are reported to show decreased adhesive capacity to FN caused by impaired adhesive function of integrin {alpha}4{beta}1 and {alpha}5{beta}1, indicating cross-modulation of integrin {alpha}IIb (15). This mode of modulation by integrin {alpha}IIb was not detected in our study using specific blocking Abs, implying that the loss of the expression of integrin {alpha}IIb and functional blockage of integrin {alpha}IIb{beta}3 might lead to distinct results.

Effects of attachment via integrin {alpha}IIb{beta}3 on mast cell functions

Attachment of mast cells to ECM proteins has been reported to enhance various mast cell functions. We demonstrate the evidence that the interaction between integrin {alpha}IIb{beta}3 and fibrinogen regulates mast cell functions in vivo. Recent studies have revealed that SCF and adhesion molecules like integrins are involved in mast cell-associated diseases. In addition, the role of fibrinogen and its degradation product, fibrin, in inflammatory reactions has been given focus. Importantly, SCF is known to critically define the accumulation of mast cells at the site of inflammation (1, 2, 37, 47, 60) including atherosclerotic plaques (9). Gurish et al. (37) showed that integrin {alpha}4{beta}7 is responsible for tissue-specific homing of mast cell progenitors to the small intestine during a helminth infection. Extravascular fibrinogen and fibrin, which also interact with integrin {alpha}IIb{beta}3, are abundant at the site of inflammation, such as arthritis (61), transplant rejection (62), bacterial infection (63, 64, 65), and atherosclerosis (66), where immune cells are recruited and activated (66). Collectively, the interaction of integrin {alpha}IIb{beta}3 with fibrinogen may be involved in mast cell-associated pathological conditions, especially where SCF is highly produced.

Another interesting aspect is that mast cells are known to express a profibrinolytic phenotype and contain fibrinolytic enzymes like tissue plasminogen activator and heparin in their granules (67). Moreover, mast cells are a major source of these enzymes (67). In accordance with these observations, mast cell deficiency leads to experimentally induced-thrombus formation and enhances thrombosis-associated mortality (67). It is tempting to assume that the adhesion of mast cell to fibrinogen augments such profibrinolytic phenotype by enhancing the effects of these fibrinolytic enzymes to lyse fibrinogen. The amount of fibrinogen was thought to be regulated by certain members of integrins using different mechanisms; human monocytes internalized and degraded fibrinogen, independently of plasmin activity via interaction with integrin {alpha}M{beta}2 (Mac-1) to clear fibrinogen and fibrin at injured or inflammatory sites (68). Activated platelets internalized fibrinogen via integrin {alpha}IIb{beta}3 to modulate the coagulation process (22, 23, 24, 25). Interestingly, as we have presented in this paper, activated BMMC can also bind and internalize soluble fibrinogen in an integrin {alpha}IIb{beta}3-dependent manner (Fig. 4B). These results suggest that uptake of fibrinogen and fibrin via integrin {alpha}IIb{beta}3 leads to the clearance of fibrinogen and fibrin in inflammatory sites.

In conclusion, our novel findings show that integrin {alpha}IIb{beta}3 is expressed on mouse and human mast cells, and mediated adhesion to fibrinogen and vWF, resulting in the enhancement of mast cell functions in concert with SCF. A drug that regulates the function of integrin {alpha}IIb{beta}3 (33) may control the accumulation and activation of mast cells, leading to new therapeutic approaches forthcoming for mast cell-mediated diseases.

During our research, Berlanga et al. (69) reported the expression of integrin {alpha}IIb{beta}3 on BMMC. Unlike that report, we detected integrin {alpha}IIb{beta}3 both on BMMC and PMC. It might result from the difference in the affinity of the Abs against the integrin or in the mouse strains used. In fact, the expression levels of several integrins including integrin {alpha}IIb on both types of mast cells were different between BALB/c and C57BL/6 mice (T. Oki, J. Kitaura, Y. Yamanishi, and T. Kitamura, unpublished observation). They suggested the in vivo function of integrin {alpha}IIb{beta}3 expressed on BMMC by showing that the adhesion to VN was increased in BMMC derived from the integrin {alpha}IIb knockout mice. However this alteration was probably due to the compensatory enhanced expression of integrin {alpha}V on BMMC of the integrin {alpha}IIb knockout mice, which we observed and later described; thus the function of integrin {alpha}IIb{beta}3 was not directly addressed. In contrast, we characterized functions of integrin {alpha}IIb{beta}3 on BMMC using a neutralizing Ab in multiple assays. In addition, Berlanga et al. (69) studied the mast cell functions in the absence of stimulation, whereas we investigated those in the presence and absence of the stimulation because the inside-out signaling is required for maximal adhesion of mast cells. Finally, using BMMC from integrin {alpha}IIb knockout mice, a gift from Dr. J. Frampton (Institute of Biomedical Research, Birmingham University, Edgbaston, Birmingham, U.K.), we confirmed the following phenomena: the adhesion of these BMMC to VN was increased due to the enhanced expression of integrin {alpha}V{beta}3, whereas their adhesion to fibrinogen was significantly diminished because of the integrin {alpha}IIb deficiency (data not shown). These results are in accordance with our results obtained from the study using blocking Abs, which indicate that the interaction of BMMC to fibrinogen and VN was mainly mediated by integrin {alpha}IIb{beta}3 and {alpha}V{beta}3, respectively.


   Acknowledgments
 
We greatly thank Drs. B. S. Coller, V. L. Woods, D. J. Gerber, and S. Tonegawa for providing Abs, Dr. J. Frampton for providing the knockout mice, and Dr. S. J. Shattil for careful reading of the manuscript and for useful discussion. We also thank M. Ohara for language assistance.


   Disclosures
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This study was supported by the Ministry of Education, Science, Technology, Sports and Culture and the Ministry of Health and Welfare, Japan. The Division of Hematopoietic Factors is supported by the Chugai Pharmaceutical Company, Tokyo, Japan. Back

2 Address correspondence and reprint requests to Dr. Toshio Kitamura, Division of Cellular Therapy, Advanced Clinical Research Center, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8039, Japan. E-mail address: kitamura{at}ims.u-tokyo.ac.jp Back

3 Abbreviations used in this paper: SCF, stem cell factor; HC, high cytokinergenic; ECM, extracellular matrix; BMMC, bone marrow-derived mast cell; PMC, peritoneal mast cell; vWF, von Willebrand factor; VN, vitronectin; FN, fibronectin. Back

Received for publication May 20, 2005. Accepted for publication October 10, 2005.


   References
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 Abstract
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 Materials and Methods
 Results
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