Eosinophils Adhere to Vascular Cell Adhesion Molecule-1 via ...

Eosinophils Adhere to Vascular Cell Adhesion Molecule-1 via Podosomes Mats W. Johansson, Ming H. Lye, Steven R. Barthel, Allison K. Duffy, Douglas S. Annis, and Deane F. Mosher Departments of Medicine and Biomolecular Chemistry, University of Wisconsin, Madison, Wisconsin

Vascular cell adhesion molecule (VCAM)-1 supports specific eosinophil adhesion via ␣4␤1 integrin. We tested the hypothesis that adhesive contacts formed by eosinophils on VCAM-1 are different from focal adhesions formed by adherent fibroblasts. Eosinophils adherent on VCAM-1 formed punctate adhesions that fit the criteria for podosomes, highly dynamic structures found in adherent transformed fibroblasts, osteoclasts, and macrophages. The structures contained ␤1 integrin subunit, phosphotyrosine-containing proteins, punctate filamentous actin, and gelsolin, a podosome marker. In contrast, nontransformed fibroblasts on VCAM-1 formed peripheral focal adhesions that were positive for ␣4, ␤1, phosphotyrosine, vinculin, talin, and paxillin; negative for gelsolin; and associated with microfilaments. Phorbol myristate acetate or tumor necrosis factor-␣ and interleukin-5 stimulated podosome formation in adherent eosinophils. Because podosomes in tumor cells are associated with extracellular matrix degradation, we analyzed the VCAM-1 layer. VCAM-1 was lost under adherent eosinophils but not under adherent fibroblasts. This loss was inhibited by the metalloproteinase inhibitor ortho-phenanthroline and correlated with expression and podosome localization of a membrane-tethered metalloproteinase, a disintegrin and metalloproteinase domain 8. Podosome-mediated VCAM-1 clearance may be a mechanism to regulate eosinophil arrest and extravasation in allergic conditions such as asthma.

Eosinophils are enriched at sites of inflammation associated with asthma and other allergic diseases (1). Much evidence indicates that adhesion receptors of the integrin family are essential for the selective recruitment of eosinophils from the blood to the airway in patients with asthma. Eosinophils express ␣4␤1, ␣6␤1, ␣L␤2, ␣M␤2, ␣X␤2, ␣D␤2, and ␣4␤7 integrins (2, 3), and thus have receptors for, and the potential capacity to bind to, multiple adhesive ligands. Unstimulated eosinophils adhere specifically to vascular cell adhesion molecule (VCAM)-1 via ␣4␤1 integrin (1, 2), even though ␣4␤7 and ␣D␤2 integrins also can be VCAM-1 receptors (4, 5). The specific interaction between eosinophils and VCAM-1 is of particular interest because the VCAM-1 counter-receptor is expressed in preference to other cytokineinduced counter-receptors on the surface of cultured endothelial

(Received in original form March 19, 2004 and in revised form June 16, 2004) Address correspondence to: Mats W. Johansson, Ph.D., Department of Medicine, University of Wisconsin, 4285A, Medical Sciences Center, 1300 University Avenue, Madison, WI 53706-1532. E-mail: [email protected] Abbreviations: a disintegrin and metalloprotease, ADAM; bovine serum albumin, BSA; cluster of differentiation, CD; Chinese hamster ovary, CHO; fluorescenceactivated cell sorting, FACS; filamentous actin, F-actin; fetal bovine serum, FBS; fluorescein isothiocyanate, FITC; formyl-methionylleucylphenylalanine, FMLP; fibronectin, FN; granulocyte-macrophage–colony stimulating factor, GM-CSF; 12(S)-hydroxy-(5Z,8Z,10E,14Z)-eicosatetraenoic acid, 12(S)-HETE; intercellular cell adhesion molecule, ICAM; immunoglobulin, Ig; interleukin, IL; monoclonal antibody, mAb; matrix metalloproteinase, MMP; ortho-phenanthroline, OP; platelet activating factor, PAF; pAcGP67.coco plasmid, pCOCO; phosphate-buffered saline PBS; phorbol myristate acetate, PMA; phenylmethylsulfonyl fluoride, PMSF; transforming protein of Rous sarcoma virus, Src; Tris-buffered saline, TBS; tumor necrosis factor, TNF; vascular cell adhesion molecule, VCAM. Am. J. Respir. Cell Mol. Biol. Vol. 31, pp. 413–422, 2004 Originally Published in Press as DOI: 10.1165/rcmb.2004-0099OC on June 25, 2004 Internet address: www.atsjournals.org

cells in response to mediators of T helper cell type 2 immunity and in blood vessels of biopsies from patients with asthma (6–8). VCAM-1 supports ␣4 integrin–mediated tethering, rolling, and migration of lymphocytes and rolling of eosinophils (9, 10). A full understanding of the consequences of eosinophil adhesion to VCAM-1 may provide insights into how to control extravasation of eosinophils in conditions such as allergic asthma. Cell contacts are points on the cell surface where adhesion receptors link cells to a substrate of extracellular matrix molecules or counter-receptors on the surface of other cells, and thus influence cytoskeletal remodeling and signaling pathways (11, 12). In adherent cell types, the best characterized contact structure is the focal adhesion, a cell–matrix contact that contains integrins bound extracellularly to the matrix, is associated intracellularly to microfilaments via anchor proteins, such as talin, vinculin, and paxillin, and allows cells to tension the matrix (11–13). The nature of adhesive contacts formed by eosinophils adherent to VCAM-1 is not known. We hypothesized that the contacts may have unique features, and therefore investigated purified human blood eosinophils and eosinophil-like leukemic cell lines (14, 15) adherent to VCAM-1. We found that upon adhesion to VCAM-1, eosinophils form punctate adhesive contacts that have many of the features of podosomes: protrusive, highly dynamic structures that turn over more rapidly than focal adhesions and are considered to be important for directed cell motility (11, 12, 16). Furthermore, we found that adherent eosinophils clear VCAM-1 from underlying substrate by a mechanism blocked by the metalloproteinase inhibitor ortho-phenanthroline (OP) and that a disintegrin and metalloprotease domain (ADAM) 8, (a member of the “a disintegrin and metalloprotease” family), also known as cluster of differentiation (CD) antigen 156a, is highly expressed on eosinophils and localizes to the podosomes upon adherence to VCAM-1.

Materials and Methods Antibodies Anti–␤1 integrin monoclonal antibody (mAb) 13 was obtained as a gift from Kenneth Yamada (National Institutes of Health, Bethesda, MD). Anti-␣D mAb 240I was a gift from ICOS (Bothell, WA). Affinity-purified rabbit antibody against a cytoplasmic ␤1 peptide (17) and anti-fibronectin (FN) Lab mAb (18) have been described. The hybridoma cell line producing anti-␤2 TS1/18 was purchased from American Type Culture Collection (Manassas, VA), and the mAb was purified from serum-free tissue culture supernatant (in HyQ-CCMI medium) on a protein G-agarose column. Anti-gelsolin GS-2C4, anti-vinculin hVIN-1, and anti-talin 8D4 mAbs and rabbit immunoglobulin (Ig) G were purchased from Sigma (St. Louis, MO). Anti-paxillin mAb 349, anti-␣4 9F10, anti-␤2 L130, anti-␤7 Fib504, anti-fibrinogen 262-G7, and isotype controls mouse IgG1, ␬ (anti-keyhole limpet hemocyanin, clone A112–2), mouse IgG2b, ␬ (anti-dansyl, clone 27–35), and rat IgG2a, ␬ (anti-keyhole limpet hemocyanin A110–2) were from BD Biosciences (San Diego, CA). Anti-phosphotyrosine P-Tyr-100 was from Cell Signaling Technology (Beverley, MA). Anti-ADAM8 ectodomain mAb (clone 143338), goat anti-ADAM8 ectodomain polyclonal IgG, and anti-ADAM10 ectodomain mAb (clone 163003) were from R&D Systems (Minneapolis, MN). Anti-␣4 A4-PUJ1 was from Upstate (Charlottesville, VA) and anti-␣4 HP2/1 was from Beckman

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Coulter (Fullerton, CA). Anti–VCAM-1 mAbs 1.G11B1 and P3C4; anti-␣4 P4C2; rhodamine-conjugated goat anti-mouse, goat anti-rat, and rabbit anti-goat IgG; and fluorescein isothiocyanate (FITC)–conjugated goat anti-mouse IgG and rabbit anti-goat IgG were from Chemicon (Temecula, CA). Rhodamine- and FITC-conjugated goat anti-rabbit IgG were from Jackson Immunoresearch Laboratories (Avondale, PA).

Adhesive Ligands Soluble human vascular cell adhesion molecule-1 was produced with a recombinant baculovirus. RNA from Chinese hamster ovary cells transfected with cDNA for the full-length seven-domain form of human VCAM-1 (a gift from Roy R. Lobb, Biogen, Cambridge, MA) was reverse-transcribed and amplified by polymerase chain reaction. A fragment of ⵑ 2,100 bp encoding the extracellular part of mature VCAM-1 was amplified with an XmaI site added at the 5⬘ end and an SpeI site at the 3⬘ end. The pAcGP67.coco plasmid (pCOCO) (19) was digested with XmaI and XbaI, and the polymerase chain reaction product was cloned in frame into pCOCO using standard molecular biology techniques, taking advantage of the compatible cohesive ends left by SpeI and XbaI. After confirmation of the sequence of the insert, the construct was transfected into SF9 cells along with BaculoGold linearized Baculovirus DNA transfection module (BD Biosciences). Viral recombinants were cloned in monolayer, expanded, and used to infect suspended High5 insect cells for protein expression. The recombinant protein contained a signal sequence that was cleaved during secretion, resulting in a protein of the following N- and C-terminal sequences (underlined sequences introduced from pCOCO): ADPGFKIET and YFSPELELVPRGSAAGHHHHHH. The secreted protein was purified from medium with yields of 10–20 mg/L by nickel chelate chromatography and migrated with the expected molecular mass of 76 kD in sodium dodecylsulfate–polyacrylamide gel electrophoresis under reducing conditions. Human plasma FN was purified as described (20). Human plasma fibrinogen and recombinant human intercellular cell adhesion molecule (ICAM)–1 were purchased from Sigma and R&D Systems, respectively. FITC was conjugated to VCAM-1 according to instructions from Molecular Probes (Eugene, OR) and to FN as previously described (20). Alexa Fluor 546–conjugated human plasma fibrinogen was from Molecular Probes, FITC-conjugated porcine gelatin was from Elastin Products Co. (Owensville, MO), and FITC-bovine serum albumin (BSA) was from Sigma.

Other Reagents The following were purchased: percoll, Amersham Pharmacia Biotech (Piscataway, NJ); anti–CD16-conjugated magnetic microbeads, Miltenyi Biotec (Auburn, CA); Diff-Quik solution I, Dade Diagnostics (Aguada, PR); Dulbecco’s modified Eagle’s medium with l-glucose and l-glutamine and RPMI 1,640 with l-glutamine, Mediatech Cellgro (Herndon, VA); fetal bovine serum (FBS) and HyQ-CCMI, HyClone (Logan, UT); protein G-agarose, Life Technologies (Grand Island, NY); recombinant human interleukin (IL)-5, tumor necrosis factor (TNF)-␣, and granulocyte-macrophage–colony stimulating factor (GM-CSF), R&D Systems; prostaglandin D2, Cayman Chemical (Ann Arbor, MI); synthetic eotaxin, Cell Sciences (Canton, MA); platelet activating factor C-18 (PAF), Biomol (Plymouth Meeting, PA); electron microscopy–grade methanolfree paraformaldehyde solution, Electron Microscopy Sciences (Fort Washington, PA); electron microscopy–grade methanol-free formaldehyde solution, Polysciences (Warrington, PA); and Vectashield mounting medium, Vector Laboratories (Burlingame, CA). Additional reagents, including phorbol myristate acetate (PMA), formyl-methionylleucylphenylalanine (FMLP), 12(S)-hydroxy-(5Z,8Z,10E,14Z)-eicosatetraenoic acid (12(S)-HETE), OP (1,10-phenanthroline monohydrate), phenylmethylsulfonyl fluoride (PMSF), aprotinin, rhodamine- and FITC-phalloidin were from Sigma.

Isolation of Human Blood Eosinophils Blood eosinophils were isolated from peripheral blood of normal volunteer donors (n ⫽ 7) and from that of individuals with allergic asthma (n ⫽ 21) and allergic rhinitis (n ⫽ 24) using negative selection for CD16 (21). The purity of the eosinophils was between 94 and 99% as determined by Diff-Quik staining. Viability was at least 99% as assessed by staining with propidium iodide and annexin V-FITC (BD Biosciences). Informed, written consent was obtained from the subjects and

studies were approved by the University of Wisconsin–Madison Health Sciences Human Subjects Committee.

Cells AH1F human foreskin fibroblasts were a gift from Lynn Allen-Hoffmann (University of Wisconsin, Madison, WI) and were grown in Dulbecco’s modified Eagle’s medium with 10% FBS, 100 U/ml penicillin, and 100 ␮g/ml streptomycin, in 10 cm tissue culture dishes (BD Labware, Franklin Lakes, NJ) with splitting 1:4–1:10 when confluent. AML14.3D10 eosinophilic cells (15) were from Cassandra Paul (Wright State University, Dayton, OH) and were grown in RPMI 1,640 with 10% FBS, 50 ␮M ␤-mercaptoethanol, 100 U/ml penicillin, and 100 ␮g/ml streptomycin, in suspension in T25 flasks (Costar, Cambridge, MA) with subculture by dilution to 1 ⫻ 105 cells/ml twice a week. EoL-3 eosinophilic cells (14) were from Richard Lynch (University of Iowa, Iowa City, IA) and grown in RPMI 1,640 with 10% FBS, 100 U/ml penicillin, and 100 ␮g/ml streptomycin, with subculture by dilution to 1 ⫻ 105 cells/ml twice a week.

Fluorescent Staining Round glass coverslips (diameter, 12 mm; Fisher Scientific, Pittsburgh, PA) were placed in wells in 24-well culture plates (Corning, Corning, NY) and coated with 300 ␮l per well of VCAM-1 (5 or 10 ␮g/ml in Tris-buffered saline [TBS]), FN, ICAM-1, fibrinogen (10 ␮g/ml), or FITC–VCAM-1, FITC–FN, Alexa–fibrinogen, FITC–gelatin, or FITC– BSA (all 10 ␮g/ml) for 2 h at 37⬚C. The coverslips were washed with TBS, blocked with 0.1% gelatin (for eosinophils, except after FITC– gelatin coating) or 500 ␮l 1% BSA (for cell lines and fibroblasts) in TBS for 30 min at 37⬚C. Fibroblasts were harvested with 0.02% ethylenediaminetetraacetate and washed with medium. Cells (500 ␮l, containing 4 ⫻ 105 eosinophils or eosinophilic cells or 1 ⫻ 105 fibroblasts, per well) were resuspended in medium with 0.2% BSA and were then added to the coverslip. In some experiments, stimulating agents and/ or inhibitors were added to the cells before addition to the coverslip as indicated. Stimuli included PMA (final concentration, 100 nM, diluted from stock solution prepared by dissolving PMA at 10 mM in dimethyl sulfoxide), IL-5 (final concentration, 100 ng/ml), and/or TNF-␣ (100 ng/ml). Inhibitors included OP (500 ␮M, from 200 mM stock made in methanol), PMSF (100 ␮g/ml, from 10 mg/ml stock made in isopropanol), and aprotinin (2 ␮g/ml, from 10 mg/ml stock made in phosphate-buffered saline [PBS]). Cells were incubated on the coverslip for 1 h at 37⬚C, after which the coverslips were washed three times with TBS. The following steps were performed at room temperature: cells were fixed first for 10 min with 1% paraformaldehyde (diluted from 16% solution, EM Sciences) in 75 mM sodium cacodylate buffer, pH 7.4, containing 0.72% sucrose, and then for 10 min with 3.7% formaldehyde (from 16% solution, Polysciences) in PBS, washed thrice with PBS, permeabilized with 0.05% Triton X-100 in PBS for 4 min, and washed thrice with PBS and once with PBS–3% BSA. Cells were incubated with 30 ␮l primary antibody solution or isotype control in PBS–3% BSA for 1 h, washed twice with PBS and once with PBS–3% BSA, and incubated with rhodamine- or FITC-conjugated secondary antibody for 45 min. Purified primary antibodies were used at 10 ␮g/ml. Antigelsolin, anti-vinculin, and anti-talin antibody containing ascites were diluted 1:100, 1:400, and 1:40, respectively. Polymerized actin was visualized in permeabilized cells by incubation with rhodamine– or FITC– phalloidin (0.17 ␮g/ml). For double-labeling experiments, fixed eosinophils were first incubated with primary antibody and then with a mixture of rhodamine–phalloidin and FITC-conjugated secondary antibody. For visualization of the VCAM-1 layer, coverslips with cells were incubated with 10 ␮g/ml mAb 1.G11B1 to VCAM-1 as primary antibody and FITC-conjugated secondary antibody. Coverslips were washed three times with PBS, dipped in PBS, mounted on microscope slides with Vectashield mounting medium, and sealed with nail polish. Samples were viewed in a BX60 microscope (Olympus, Melville, NY). At least 200 cells were observed on each coverslip. Representative cells were photographed using Kodak TMAX 400 film or a digital camera with the IPLab Spectrum 10P or SPOT RT 3.4 Advanced software (Diagnostic Instruments, Sterling Heights, MI). The effects of agents on podosomes formation were screened by a scoring system of adherent cells stained with FITC–phalloidin. Coverslips

Johansson, Lye, Barthel, et al.: Eosinophil–VCAM-1 Interactions

were scanned and the number and quality of podosomes were scored as ⫺, ⫹, ⫹⫹, or ⫹⫹⫹. To assess the effects more rigorously, at least 200 eosinophils were examined on each coverslip in some experiments. A cell was scored as podosome-containing if it had clear, bright filamentous actin (F-actin)–positive condensations. Scoring and quantitation of podosome formation was performed by both involved and naive laboratory workers. When cells were observed, scored, and quantitated independently by more than one person, whether involved or naive, similar results were obtained.

Flow Cytometry

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staining was diffuse in eosinophils (not shown). The distribution of the ␣D (Figure 2J) and ␤2 (not shown) integrin subunits in unstimulated eosinophils was also diffuse (Figure 2J). These results are consistent with antibody blocking studies that indicate that, although ␣4␤7 and ␣D␤2 integrins are potential VCAM-1 receptors, these integrins do not participate in the adhesion of unstimulated eosinophils to VCAM-1 (S. R. Barthel, M. W. Johansson, and D. F. Mosher, unpublished data). In PMA-treated eosinophils, ␣D localized in a stitch-like pattern (Figure 2K). The ␤2 integrin

Cells (1–5 ⫻ 105) suspended in fluorescence-activated cell sorting (FACS) buffer (PBS with 2% BSA and 0.2% NaN3) were incubated with primary antibody or isotype control for 30 min at 4⬚C, washed with FACS buffer, resuspended, and incubated with secondary antibody for 30 min at 4⬚C. For primary antibody, 0.5 ␮g was added to 200 ␮l cell suspension. FITC-conjugated anti-mouse IgG was used at a final concentration of 20 ␮g/ml. Cells were resuspended in 1% paraformaldehyde, 67.5 mM sodium cacodylate, 113 mM NaCl, pH 7.2, stored at 4⬚C, and within 1 week washed with FACS buffer and analyzed. Data were collected using a FACS Calibur (BD Biosciences, available through the Flow Cytometry Facility, Comprehensive Cancer Center, University of Wisconsin, Madison, WI) from 10,000 cells per condition and analyzed, using the CELLQUEST software (BD Biosciences). Cells were gated based on forward and site scatter.

Adhesion Eosinophil adhesion was assayed as previously described (22).

Statistical Analysis The means of populations were compared using two-tailed Student’s t test. A level of P ⬍ 0.05 was considered statistically significant.

Results Eosinophils Adherent to VCAM-1 Form Podosomes

Eosinophils, either unstimulated or treated with PMA, adhered specifically to VCAM-1 (data not shown). By phase contrast microscopy, unstimulated eosinophils adherent to VCAM-1 were round or elongated and had numerous granules (Figure 1A). PMA stimulation caused flattening and spreading of the eosinophils (Figure 1B). To test the hypothesis that adhesive contacts formed by eosinophils upon adherence to VCAM-1 are different from contacts formed by fibroblasts, we studied eosinophils that had adhered to VCAM-1 in the absence or presence of PMA. Cells were stained for components known to be present in focal adhesions of fibroblasts, which served as reference cells (Figures 1 and 2). To classify the adhesions of eosinophils, we performed doublelabeling immunofluorescence experiments to investigate whether key components colocalize in eosinophils (Figure 3). Eosinophils adherent to VCAM-1, particularly after PMA stimulation, formed large punctate structures in the focal plane of the substrate that contain F-actin, phosphotyrosine-containing proteins, and the ␤1 integrin subunit (Figures 1 and 2). F-actin was also present in unstimulated cells in a cortical distribution at the cell periphery (Figure 1D). The large punctate structures positive for F-actin, phosphotyrosine-containing proteins, and ␤1 were more numerous and better developed after PMA stimulation. PMA-treated eosinophils had very little cortical actin (Figure 1E). Despite repeated attempts with different anti–␣4 integrin mAbs (A4-PUJ1, HP2/1, P4C2, and 9F10), we did not obtain any consistent significant ␣4 staining of adherent eosinophils; no punctate structures could be seen in untreated eosinophils (Figure 2G), and only a few small punctate structures could be seen in some cells after PMA treatment (Figure 2H). In contrast, all the ␣4 mAbs clearly stained EoL-3 cells (see below). ␤7 integrin

Figure 1. Fluorescence localization of F-actin, gelsolin (gels), vinculin (vinc), talin, and paxillin (pax) in eosinophils and fibroblasts adhered to VCAM-1. Micrographs of eosinophils adhered in the absence (left panels) or presence (middle panels) of PMA, or of AH1F fibroblasts (right panels), analyzed by immunofluorescent staining with mAbs to gelsolin (G–I), vinculin (J–L), talin (M–O), or paxillin (P–R), and rhodamine-conjugated secondary antibody (G–R); visualization of F-actin with rhodamine-phalloidin (D–F); or phase contrast microscopy (ph) (A–C). Insets: enlargements to show localization of vinculin, talin, and paxillin in single PMA-treated eosinophils adhered to VCAM-1. Scale bar in R ⫽ 10 ␮m and applies to all larger-scale micrographs. Scale bar in inset in Q ⫽ 5 ␮m and applies to all insets. Representative fields from experiments with eosinophils from 11 donors and 5 experiments with fibroblasts are shown.

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Figure 2. Fluorescence localization of phosphotyrosine (pY) and ␤1, ␣4, and ␣D integrin subunits in eosinophils and fibroblasts adhered to VCAM-1. Micrographs of eosinophils adhered in the absence (left panels) or presence (middle panels) of PMA, or of AH1F fibroblasts (right panels), analyzed by immunofluorescent staining with mAbs to phosphotyrosine (A–C), ␤1 (E, F), ␣4 (G–I), or ␣D (J–L), or with affinity-purified anti-cytoplasmic ␤1 peptide antibody (D ) and rhodamine-conjugated secondary antibody (A–L). Insets: enlargements to show localization of ␤1 integrin in single eosinophils in the absence (D ) or presence of PMA (E ) and of ␣D integrin in a single PMA-treated eosinophil (K ), adhered to VCAM-1. Scale bar in L ⫽ 10 ␮m and applies to all larger-scale micrographs. Scale bar in inset in K ⫽ 5 ␮m and applies to all insets. Representative fields from experiments with eosinophils from 13 donors and 3 experiments with fibroblasts are shown.

subunit remained localized diffusely after PMA treatment. The ␤2 presumably present at the stitch-like sites dimerized to ␣D could not be localized against the diffuse background staining. To classify the large punctate structures, we performed immunofluorescence staining for gelsolin, which has been found previously in podosomes of monocytes, macrophages, osteoclasts, and Rous sarcoma virus–transformed cells, but not in focal adhesions of normal nontransformed fibroblasts (16). Gelsolin staining was bright in a pattern similar to that of F-actin, with cortical staining and also large punctate structures (Figure 1G). Gelsolin staining was most marked in the more numerous and better developed punctate structures of PMA-treated eosinophils than of untreated eosinophils (Figure 1H). In double-labeling experiments performed with rhodamine-labeled phalloidin and FITC– labeled secondary antibodies, there was colocalization between F-actin and gelsolin, in the absence (not shown) or presence (Figures 3A–3C) of PMA. There was also colocalization between F-actin and phosphotyrosine (Figures 3D–3F) and between F-actin and ␤1 integrin (Figures 3G–3I). The presence of gelsolin indicates that the large punctate structures in eosinophils fit the criteria of

Figure 3. Double fluorescence localization of gelsolin (gels), phosphotyrosine (pY), and ␤1 integrin compared with localization of F-actin in eosinophils adhered to VCAM-1. Micrographs of eosinophils adhered in the presence of PMA analyzed by double fluorescent staining with mAbs to gelsolin (A, C ), phosphotyrosine (D, F ), or with anti-cytoplasmic ␤1 peptide antibody (G, I ), and FITC-conjugated secondary antibody; and visualization of F-actin with rhodamine-phalloidin (B, C, E, F, H, I ). Scale bar ⫽ 10 ␮m. Representative fields from experiments with three donors are shown.

podosomes (16). Enhancement of these structures by PMA is consistent with the observation that presence of podosomes in leukemic monoblasts, U937 cells, and macrophages adherent to laminin is favored by treatment of cells with phorbol esters (16). The focal adhesion proteins vinculin (Figure 1J), talin (Figure 1M), or paxillin (Figure 1P) were not detected in adhesive structures of unstimulated eosinophils. Vinculin and talin were diffusely distributed. There was a concentrated focus of anti-paxillin staining in the center of the cells. Bright staining of the same area was obtained with mAb against ␥-tubulin (not shown), indicating that eosinophil paxillin is in the microtubule organizing center, as has been shown for lymphocytes and monocytes (23). Addition of PMA caused the appearance of peripheral stitching positive for vinculin (Figure 1K), talin (Figure 1N), and paxillin (Figure 1Q); ␣D was also present in the stitching (Figure 2K). The positivity for vinculin, talin, and paxillin suggests that the stitching, which occurs only with PMA, has some characteristics of focal adhesions. In the absence of PMA, eosinophils had limited adhesion to FN, ICAM-1, or fibrinogen that was not above background adhesion to gelatin or BSA (not shown). In the presence of PMA, eosinophils adhered more to these substrates as well as to the blocking proteins gelatin and BSA (not shown). Podosomes were present in eosinophils adherent to all substrates, although to varying extents, and were most numerous and largest on VCAM-1 with or without gelatin blocking. Fewer podosomes were found in eosinophils adherent to BSA or gelatin, still fewer in eosinophils adherent to ICAM-1 or fibrinogen, and fewest in eosinophils on FN (not shown). Fibroblasts Adherent to VCAM-1 Form Peripheral Focal Adhesions and Not Podosomes

To compare the structures of eosinophils adherent to VCAM-1 to those of a well-studied, adherent cell type adherent to the


same substrate, we also analyzed fibroblasts on VCAM-1. The fibroblasts adherent to VCAM-1 were well spread and round (Figure 1C). The arrangement of F-actin–containing microfilaments (Figure 1F) was distinctly different from the familiar, more spindle-shaped cell appearance and classical pattern of stress fibers on FN (not shown; see Ref. 13). Punctate F-actin– and gelsolin-positive podosomal structures were absent. Instead, gelsolin was distributed diffusely. The fibroblasts on VCAM-1 formed peripheral structures perpendicular to the perimeter of the cell. These structures were positive for vinculin (Figure 1L), talin (Figure 1O), paxillin (Figure 1R), phosphotyrosine-containing proteins (Figure 2C), ␤1- (Figure 2F), and ␣4- (Figure 2I) integrin subunits. Fibroblasts were negative for ␣D (Figure 2L). The peripheral contacts did not contain gelsolin (Figure 1I), and were associated with the microfilaments (Figure 1F), thus fitting the criteria of focal adhesions. The peripheral distribution of focal adhesions in fibroblasts on VCAM-1 was different from the more widespread distribution of focal adhesions detected in parallel studies of fibroblasts adherent to FN (not shown; see Ref. 13). Eosinophilic Cell Lines Adherent to VCAM-1 Form Small Punctate Structures but Not Gelsolin-Positive Podosomes

To ascertain whether eosinophil-like leukemic cell lines could be used as model systems to study eosinophil adhesive structures, we analyzed AML14.3D10 and EoL-3 cells. Similar to unstimulated eosinophils, these cell lines adhered specifically to VCAM-1. Experiments with blocking antibodies demonstrated that adhesion is mediated by ␣4␤1, which is expressed on both types of leukemic cells (S. R. Barthel, M. W. Johansson, and D. F. Mosher, unpublished; 3). AML14.3D10 cells adherent to VCAM-1 had few small granules (Figure 4A), and EoL-3 were devoid of granules and had a variable morphology, from round to somewhat spread (Figure 4B). Because of the paucity of granules and thus no or little background autofluorescence, adhesive structures of leukemic cells were more easily distinguished than in normal eosinophils.

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F-actin in both cell types was mostly present as cortical actin. In contrast to normal eosinophils, no large punctate structures were observed (Figures 4C and 4D). Furthermore, in contrast to eosinophils, the majority of AML14.3D10 cells stained weakly and diffusely for gelsolin (Figure 4G), and the majority of EoL-3 were gelsolin-negative (Figure 4H). There was a large central focus of bright anti-paxillin staining in the cell lines (Figures 4K and 4L), which was similar to but more distinct than that in eosinophils. Stainings for phosphotyrosine-containing proteins (Figures 4E and 4F) and ␤1 integrin subunit (Figures 4I and 4J) consisted of small punctate structures. ␣4 integrin subunit was also found in small structures in the cell lines (Figures 4M and 4N). ␤2 integrin staining was diffuse in the cell lines. ␤7 integrin staining was diffuse in EoL-3 cells and negative in AML14.3D10 cells (not shown). PMA caused the leukemic cells to be more spread and have more filopodia (not shown). However, neither in the absence nor in the presence of PMA did AML14.3D10 or EoL-3 cells have the type of large punctate structures positive for F-actin, gelsolin, phosphotyrosine-containing proteins, and ␤1 integrin, identified as podosomes in normal eosinophils (not shown). Double-labeling experiments demonstrated that in AML14.3D10 or EoL-3 cells, F-actin did not colocalize with phosphotyrosinecontaining proteins or ␤1 integrin in EoL-3 cells as it did in eosinophils (not shown). IL-5 and TNF-␣ Stimulate Podosome Formation in Eosinophils

To search for substances that replicate stimulation of podosome formation by PMA, we screened the effects of several agents on podosome formation by a ⫺/⫹/⫹⫹/⫹⫹⫹ scoring system of eosinophils on coverslips after phalloidin staining. TNF-␣ and IL-5 were the best stimulators (Table 1). GM-CSF, FMLP, prostaglandin D2, and 12(S)-HETE also enhanced podosome formation, but to a lesser extent than IL-5 or TNF-␣ (Table 1). Eotaxin, PAF C-18, and serum were inactive. Mn2⫹, which activates integrins (4), caused

Figure 4. Fluorescence localization of F-actin, gelsolin (gels), paxillin (pax), phosphotyrosine (pY), and ␤1 and ␣4 integrin subunits in eosinophilic cell lines adhered to VCAM-1. Micrographs of AML14.3D10 (A, C, E, G, I, K, M ) and EoL-3 (B, D, F, H, J, L, N ) cells analyzed by immunofluorescent staining with mAbs to gelsolin (G, H ), paxillin (K, L), phosphotyrosine (E, F ), or ␣4 (M, N ), or with affinity-purified rabbit anti-cytoplasmic ␤1 peptide antibody (I, J ), and FITC-conjugated secondary antibody (E–N); or visualization of F-actin with rhodamine-phalloidin (C, D ); or phase contrast microscopy (ph) (A, B ). Scale bar ⫽ 10 ␮m. Representative fields from 9 experiments with AML14.3D10 cells and 14 experiments with EoL-3 cells are shown.

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more eosinophils to attach (not shown) but did not stimulate podosome formation (Table 1). To compare the activities of IL-5 and TNF-␣ to that of PMA, at least 200 eosinophils were scored as podosomes-containing if a cell had clear, bright F-actin–positive condensations. There was variability among donors, particularly in the percentage of unstimulated eosinophils that were positive (Table 2). In the presence of PMA (100 nM), podosome formation was highly stimulated (Table 2). IL-5 and TNF-␣, optimally together, had a significant effect (Table 2, Figure 5). Furthermore, IL-5– or TNF-␣–treated eosinophils were more spread and had less cortical actin (Figures 5A–5C) than unstimulated cells (Figure 5D). IL-5 and/or TNF-␣ combined with eotaxin did not have a greater effect than IL-5 and/or TNF-␣ without eotaxin (not shown). A number of experiments (not shown) were done to explore determinants of the effects of IL-5, TNF-␣, and PMA. IL-5 and TNF-␣ were active in the range of 0.1–100 ng/ml. Half-maximal activity was obtained at a concentration of 1.5 ng/ml (ⵑ 90 pM) TNF-␣ or 13 ng/ml (ⵑ 400 pM) IL-5, respectively (determined with 0.1, 1, 10, and 100 ng/ml cytokine; n ⫽ 3 for each concentration). In time-course experiments, podosome formation in the absence of an enhancing agent peaked at 30 min after addition of eosinophils to VCAM-1 substrate. In these experiments, the means of the proportion of podosome-containing cells were 11 ⫾ 4% (mean ⫾ SD, n ⫽ 4) at 15 min, 30 ⫾ 4% (n ⫽ 5) at 30 min, 20 ⫾ 9% (n ⫽ 5) at 1 h, 14 ⫾ 4% (n ⫽ 4) at 2 h, and 12 ⫾ 4% (n ⫽ 2) at 4 h. In the presence of PMA or IL-5 ⫹ TNF-␣ added at time of adhesion, stimulation of podosome formation peaked at 1 h. Means for PMA were 18 ⫾ 5% (n ⫽ 3) at 15 min, 27 ⫾ 7% (n ⫽ 3) at 30 min, 57 ⫾ 11% (n ⫽ 3) at 1 h, 41 ⫾ 1% (n ⫽ 2) at 2 h, and 19 ⫾ 6% (n ⫽ 2) at 4 h. Means for IL-5 ⫹ TNF-␣ were 15 ⫾ 3% at 15 min, 18 ⫾ 4% at 30 min, 34 ⫾ 1% at 1 h, and 20 ⫾ 6% at 2 h (n ⫽ 2 at all time points). Preincubation with TNF-␣ caused additional stimulation of podosome formation, resulting in 51% podosome-containing cells at 1 h compared with 34% in the presence of TNF-␣ without preincubation in the same experiment. Preincubation with medium alone resulted in 29% compared with 18% without preincubation. Preincubation with PMA did not cause any additional increase in podosome formation at 1 h, resulting in 70% compared with 75% in the presence of PMA without preincubation. Treatment with IL5 and/or TNF-␣ (or PMA, see above) did not induce podosome formation in AML14.3D10 cells (not shown).

TABLE 1. Effect of various agents on podosome formation in eosinophils on VCAM-1 Agent Eotaxin, ng/ml FBS, % Mn2⫹, mM PAF C-18, ng/ml FMLP, ng/ml GM-CSF, ng/ml 12(S)-HETE, ng/ml Prostaglandin D2, ␮M IL-5, ng/ml TNF-␣, ng/ml PMA, nM

Concentration

Enhancement of Podosome Formation

100 10 1 100 100 100 25 1 100 100 100

– – – – ⫹ ⫹ ⫹ ⫹ ⫹⫹ ⫹⫹ ⫹⫹⫹

Data were obtained after 1 h incubation. Definition of abbreviations: FBS, fetal bovine serum; FMLP, formyl-methionylleucylphenylalanine; GM-CSF, granulocyte–macrophage-colony stimulating factor; 12(S)HETE, 12(S)-hydroxy-(5Z,8Z,10E,14Z)-eicosatetraenoic acid; IL, interleukin; Mn2⫹, manganese; PAF, platelet-activating factor; PMA, phorbol myristate acetate; TNF, tumor necrosis factor.

TABLE 2. Stimulation of podosome formation in eosinophils on VCAM-1 by PMA or IL-5 and/or TNF-␣ Stimulus PMA None IL-5 ⫹ TNF-␣ IL-5 TNF-␣ None

No. of Donors Tested 7 7 3 3 3 3

Percentage of Podosome-Containing Cells 64 17 38 29 30 5

⫾ ⫾ ⫾ ⫾ ⫾ ⫾

4 4 2 5 4 1

Data are means ⫾ SEM and were obtained after 1 h incubation as described in the text. PMA was used at 100 nM and IL-5 and TNF-␣ at 100 ng/ml. The differences between the two “None” control groups are due to individual-to-individual variation among donors. Definition of abbreviations: IL-5, interleukin-5; PMA, phorbol myristate acetate; TNF-␣, tumor necrosis factor-␣.

VCAM-1 Is Cleared from the Substrate under Eosinophils but Not under Eosinophilic Cell Lines or Fibroblasts by a Process Sensitive to a Metalloproteinase Inhibitor

Podosomes of tumor cells are associated with loss of the adhesive ligand (e.g., FN or collagen) in the vicinity of the podosome (16). To investigate whether podosomes of eosinophils also are associated with clearing of ligand, we stained for substrate-bound VCAM-1 by immunofluorescence, using anti-VCAM mAb 1.G11B1. Areas lacking VCAM-1 were present in the VCAM-1 coating after adhesion of eosinophils for 1 h (Figures 6A–6F). Similar results were obtained with another anti–VCAM-1 mAb, P3C4 (not shown). In contrast to the discrete clearing formed around individual podosomes by fibroblasts transformed by the transforming protein of Rous sarcoma virus (Src) on a layer of FN (16), VCAM-1 antigen was missing from relatively large regions under and surrounding spread eosinophils, particularly

Figure 5. Effect of IL-5 and TNF-␣ on fluorescence localization of F-actin in eosinophils adhered to VCAM-1. Micrographs of blood eosinophils adhered in the absence (D ) or presence of IL-5 and TNF-␣ together (A ), IL-5 alone (B ), or TNF-␣ alone (C ) (each cytokine at a final concentration of 100 ng/ml). F-actin was visualized with FITC-phalloidin. Scale bar ⫽ 10 ␮m. Representative fields from experiments with 10 donors are shown.


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Figure 6. Visualization of VCAM-1 substrate after cell adhesion for 1 h and effects of the metalloproteinase inhibitor OP. Micrographs of eosinophils adhered in the absence (A–C) or presence (D–I) of PMA and in the absence (A–F) or presence (G–I) of OP, and micrographs of the eosinophilic cell line AML14.3D10 (J–L) and AH1F fibroblasts (M–O). VCAM-1 previously coated on the substrate was visualized with mAb to VCAM-1 (1.G11B1) and FITC-conjugated secondary antibody (left panels) in relation to adherent cells visualized by phase contrast microscopy (middle panel). Images are fused in the right panel. Scale bar ⫽ 10 ␮m. Representative fields from experiments with eosinophlils from 12 donors, 5 experiments with AML14.3D10 cells, and 4 experiments with fibroblasts are shown.

in the presence of PMA (Figures 6A–6F). There were also areas of VCAM-1 loss where no intact eosinophil remained attached after washing, fixation, and staining. Such areas were most evident in cultures that were not stimulated by PMA (Figures 6A–6C). These may be sites where an eosinophil initially attached, removed VCAM-1, and then detached or moved away. Clearance of substrate VCAM-1 (with or without gelatin blocking) was also observed when coverslips were coated with FITC-conjugated VCAM-1 (not shown). There was more limited clearance of FN or fibrinogen substrate, as assayed with fluorescently conjugated proteins or with immunofluorescent staining and minimal clearance of fluorescently labeled blocking proteins, gelatin, or BSA (not shown). Clearance of VCAM-1, fibrinogen, or FN assayed by immunofluorescent staining with mAbs was more extensive than that assayed with labeled proteins. To learn whether degradation by a certain class of proteinases was responsible for VCAM-1 clearance, we analyzed the effect of proteinase inhibitors. Loss of VCAM-1 under adherent eosinophils was inhibited by the presence of 500 ␮M OP (Figures 6G–6I), but not by PMSF or aprotinin. To quantify VCAM-1 loss, at least 200 eosinophils on each coverslip were counted after adhesion in the presence of PMA and staining of the VCAM-1 layer. To validate quantitation consistency, cells with the same treatment from the same donor were on some occasions observed and scored independently by two persons, one of whom was not involved in the project. Without inhibitor, 96 ⫾ 2% (mean ⫾ SD, n ⫽ 7) of the adherent cells were associated with

loss of VCAM-1 substrate. With OP, 37 ⫾ 3% (n ⫽ 4) of the cells were associated with VCAM-1 loss, and the cleared areas were smaller than in the absence of OP (Figures 6G–6I). With 100 ␮g/ml PMSF, 97 ⫾ 2% (n ⫽ 3) of the cells were associated with VCAM-1 loss, and with aprotinin, 95%. Inhibition by OP was observed both when VCAM-1 clearance was assayed by immunofluorescence (Figures 6G–6I) and when assayed using FITC–VCAM-1 (not shown). These results indicate that one or several metalloproteinases are responsible for the clearance of VCAM-1 under adherent eosinophils. In addition to inhibiting VCAM-1 clearance, OP significantly increased adhesion of nonstimulated eosinophils, with 44 ⫾ 4% cells attaching without OP compared with 67 ⫾ 2% with OP (n ⫽ 3) but did not affect adhesion in the presence of PMA (79 ⫾ 5% with only PMA versus 80 ⫾ 4% with PMA ⫹ OP). In contrast to the VCAM-1 under eosinophils, the VCAM-1 layer was intact under the AML14.3D10 cells (Figures 6J–6L), EoL-3 cells (not shown), or dermal fibroblasts (Figures 6M–6O). Treatment with PMA or IL-5 and/or TNF-␣ did not result in any VCAM-1 clearance associated with AML14.3D10 cells (not shown). The Metalloproteinase ADAM8 Localizes to Podosomes

When we examined expression of proteinase-related genes in the data set generated on purified blood eosinophils using oligonucleotide microarrays from Affymetrix (21), mRNAs for two metalloproteinases of the ADAM family, ADAM8 and ADAM10,

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were found to be expressed in all samples. The signal for ADAM10 was comparable to housekeeping proteases, such as caspase 8, proteasome subunit ␤ type 8, and ubiquitin-specific protease 8, whereas the signal for ADAM8 was 3–4 fold higher. We analyzed these two proteinases as candidate mediators of the OP-inhibitable clearance of VCAM-1 by adherent eosinophils. The signal for cell-surface ADAM8 on eosinophils was intermediate between the signals for ␤1 and ␤2 integrin subunits as assessed by flow cytometry (Figure 7A). AML14.3D10 cells, EoL-3 cells, and fibroblasts, which did not remove VCAM-1 from the substrate upon adherence, had no or little cell-surface ADAM8 (Figure 7B and not shown). Treatment of AML14.3D10 cells with PMA or IL-5 and/or TNF-␣ did not induce cell-surface ADAM8 expression by flow (not shown). Treatment of eosinophils with PMA caused a slight increase (1.2⫻) in ADAM8 expression, whereas IL-5 and/or TNF-␣ did not cause any change (not shown). When eosinophils adherent to VCAM-1 were stained for ADAM8, the proteinase was concentrated in the large F-actin–containing podosomes (Figures 7C–7F). Eosinophils adherent (in the presence of PMA) to FN, ICAM-1, fibrinogen, gelatin, or BSA also had a punctate podosome-like distribution of ADAM8 (not shown). Eosinophils from all donors tested (n ⫽ 19) expressed ADAM8 protein as judged by flow cytometry and/ or immunofluorescence staining. AML14.3D10 and EoL-3 cells were negative for ADAM8 in immunofluorescent staining (not shown). Also, after treatment with PMA or IL-5 and/or TNF-␣, AML14.3D10 cells were negative for ADAM8 by immunofluorescence (not shown). In contrast to the restricted expression of ADAM8 by eosinophils, AML14.3D10, EoL-3, fibroblasts, and eosinophils all expressed cell-surface ADAM10, as measured by flow cytometry (not shown). ADAM10 was present in podosomes in eosinophils on VCAM-1 as well as diffusely distributed (not shown). In AML14.3D10 and EoL-3 cells, ADAM10 was present diffusely and in small punctate structures, similar to the staining for phosphotyrosine-containing proteins or ␤1 shown in Figure 4.

Discussion To understand better how the interaction between circulating eosinophils and VCAM-1, which is expressed on activated endothelium, leads to eosinophil extravasation and recruitment to inflamed tissues, we determined the nature of the adhesive struc-

tures formed by normal blood eosinophils adherent to a substrate coated with the extracellular domain of VCAM-1. Instead of focal adhesions, the well-studied adhesive structure of fibroblasts and other adherent cell types, the principal adhesive structure of the eosinophil was the podosome. Podosomes are dynamic adhesive structures also found in some transformed fibroblasts, osteoclasts, and macrophages, and are believed to be important for directed cell motility and degradation of substrate proteins in transformed cells (11, 12, 16). Like focal adhesions, podosomes contain integrins and phosphotyrosine-containing proteins, but in contrast to focal adhesions, F-actin of podosomes is localized in a discrete punctate pattern. In addition, podosomes contain the actin-severing protein, gelsolin. Podosomes of eosinophils contained no detectable vinculin, talin, or paxillin. In contrast, vinculin, talin, and paxillin have been found in a ring around the podosome core in some other cell types (16). Other unique cell-specific features of podosomes have also been noted. ␤1 integrin subunit is present in the punctate core of lymphocytes, whereas ␤2 and ␤3 integrins localize in the ring in monocytes/macrophages and osteoclasts, respectively (12, 16). There are also differences in podosome-associated metalloproteinases (e.g., membrane type 1-MMP and MMP9 in osteoclasts [16], ADAM12 in Src-transformed fibroblasts [24], and ADAM8 in eosinophils [this study]). Our studies, therefore, characterize a subtype of podosome that seems unique to eosinophils. These structures were found in adherent purified eosinophils from all 32 subjects studied, including 3 normal subjects. We have noted a tendency to a higher proportion of podosome-forming eosinophils from atopic donors than from normal donors and, within the atopic group, a tendency to a higher proportion in subjects with allergic asthma than in subjects with allergic rhinitis. However, because of individual-to-individual and/or purificationto-purification variation (Table 2), an expanded study with more donors, in particular, more normal donors, would be needed to learn if such differences do exist. The type of adhesive structure formed during ␣4␤1 integrin– mediated adhesion depends on both the ligand and cell type (9, 25, 26): Studies with Chinese hamster ovary (CHO) and rhabdomyosarcoma cells transfected with chimeric ␣ integrins have shown that certain cytoplasmic domains (e.g., of ␣5) support focal adhesion formation on FN, whereas the ␣4 cytoplasmic domain negatively influences cell spreading and the formation of

Figure 7. Expression of ADAM8 in eosinophils, and single and double fluorescence localization of ADAM8 in eosinophils adhered to VCAM-1. (A, B ) Flow cytometric analysis of expression of ADAM8 (green area) compared with that of ␤1 (dashed line) and ␤2 (dotted line) integrin subunits on eosinophils (solid line, control) (A ) and AML14.3D10 cells (B ). (C–F) Micrographs of eosinophils adhered in the presence of PMA analyzed by single (C ) or double (D, F ) fluorescent staining with mAb to ADAM8 and rhodamineconjugated (C ) or FITC-conjugated (D, F ) secondary antibody and visualization of F-actin with rhodamine-phalloidin (E, F ). Note the enrichment of ADAM8 in podosomes. Staining of ADAM8 in one podosome is indicated by arrow in D and F, which colocalizes with staining of F-actin in the same podosome (arrow in E and F ). Scale bar ⫽ 5 ␮m. Representative flow cytometry data from experiments with eosinophils from 14 donors and 5 experiments with AML14.3D10 cells and representative microscopic fields from experiments with eosinophils from 4 donors are shown.


focal adhesions and stress fibers, and instead promotes chemotactic, haptotactic, and random migration (9, 25). The ␣4 cytoplasmic tail appears to promote these effects by binding to paxillin or to the Hic-5 and leupaxin–paxillin paralogs (9), and protein kinase A–mediated phosphorylation of the ␣4 integrin subunit, as well as certain mutations of ␣4 cytoplasmic tail, impacts the effects of ␣4 on cell spreading (9). VCAM-1 or an ␣4␤1-binding fragment of FN does not support robust focal adhesion formation in CHO cells (25). In contrast to the CHO cells, however, A375-SM melanoma cells (26) and dermal fibroblasts (this study) do form focal adhesions on the same substrates. The finding that paxillin and ␣4␤1 integrin localized to the peripheral focal adhesions in fibroblasts on VCAM-1 (this study) is consistent with the observation that the cytoplasmic domain of ␣4 interacts with paxillin to promote cell migration and alterations in cytoskeletal organization (9). In eosinophils for which the dominant adhesive structure is the podosome, and in leukemic eosinophils, antipaxillin did not localize in the punctate structures that stained for ␤1, but rather in the ␥-tubulin–positive microtubule-organizing center, as in lymphocytes and monocytes (23). After PMA stimulation, however, some staining with anti-paxillin was found in peripheral stitching. Why the interaction between VCAM-1 and ␣4␤1 integrin leads to the formation of different adhesive structures in different cells is not known. Fibroblasts transformed with constitutively activated Src form podosomes, whereas normal fibroblasts do not (16). PMA stimulated podosome formation in eosinophils. This observation is consistent with earlier reports on phorbol esters in macrophages and other cells (16). Thus, activation of protein kinase C (PKC) also triggers podosome formation. Activation of Src and PKC during adhesion, therefore, may induce podosome formation in eosinophils but not in normal fibroblasts. Recently, it was discovered that the cytoplasmic tails of several metalloproteinases of the ADAM family interact with Fish, a scaffolding protein and Src substrate, and that Fish colocalizes with ADAM12 in the podosomes of Src-transformed fibroblasts (24). Thus, eosinophils may have Fish or another adaptor protein that targets ADAM8 to podosomes. TNF-␣ and IL-5 mimicked the stimulatory effect of PMA on podosome formation. In a survey of substances, GM-CSF, FMLP, and 12(S)-HETE also had some effect, whereas eotaxin and PAF were not stimulatory. The lack of effect of eotaxin and PAF may be compatible with the reported ability of agonists, such as eotaxins and regulated upon activation, normal T cell expressed and secreted (RANTES), to cause detachment of eosinophils from VCAM-1 or FN (27–29). Analyses of the signaling from TNF-␣ and IL-5 receptors may give further insight into the determinants of podosome formation in eosinophils. Metalloproteinases and serine proteinases, including matrix metalloproteinase (MMP)-2, membrane type-1 MMP, and seprase, have been implicated in podosome-directed degradation of adhesive protein substrates in transformed cells (16). Extensive clearance of substrate, with areas of loss similar in size to those noted here with eosinophils on VCAM-1, has been observed under and around membrane type-1 MMP–overexpressing melanoma cells adherent to FN (30). VCAM-1 clearance under eosinophils was diminished by a metalloproteinase inhibitor, but not by the serine proteinase inhibitors PMSF or aprotinin, indicating that eosinophils degrade VCAM-1 in a metalloproteinase-dependent manner. It has been reported that 12–30 mM OP can have general effects on the energy metabolism in hepatocytes or isolated mitochondria (31). However, such concentrations are 24–60-fold higher than the concentration in our experiments that inhibited eosinophil-mediated VCAM-1 loss.

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Eosinophils from all donors had high cell-surface expression of ADAM8, which became concentrated in podosomes upon eosinophil adherence to VCAM-1. ADAM8 was originally described as CD156a, expressed in monocytes and neutrophils (32, 33), and is one of 19 members of the ADAM family of zinc proteinases in humans. ADAM10, which was also expressed by eosinophils, has a broad somatic distribution (32). Cell-surface expression and podosome localization of ADAM8 but not ADAM10 correlated with loss of VCAM-1 substrate in the four cell types studied. ADAMs consist of an extracellular part containing a prodomain, a metalloproteinase domain, a disintegrin domain; a transmembrane segment; and a cytoplasmic tail, which has binding sites for various signaling and adaptor proteins (32, 33). ADAMs are activated through removal of the prodomain, which can be catalyzed by proprotein convertases or by autoproteolysis (32). ADAMs have been implicated in shedding of cell-surface proteins, cell migration, and inflammation (32, 33). The observation of significantly higher neutrophil infiltration in transgenic mice expressing the extracellular region of ADAM8 indicates a role for ADAM8 in leukocyte trafficking (33). Experiments utilizing an ADAM8 knockout mouse were recently reported (34), but its general phenotype has not been described. Substrate recognition and specificities of ADAMs are incompletely cataloged (32, 35–37). Known substrates for ADAM8 include CD23 (low-affinity IgE receptor), CD30 ligand, the neural cell adhesion molecule CHL1, and peptides based on pro– TNF-␣, IL-1 receptor, c-kit ligand, and CD27 ligand (34, 36, 37). Substrates for ADAM10 include EGF receptor, pro–TNF-␣ and CD40 ligand peptides (32, 33, 35, 36), and type IV collagen (32). Proteinases that have been reported to contribute to VCAM-1 proteolysis in other systems are unlikely to be responsible for the VCAM-1 loss that we have observed. The neutrophil serine proteinases elastase and cathepsin G are released in the bone marrow and can cleave VCAM-1 (38). However, VCAM-1 loss under eosinophils was not affected by serine proteinase inhibitors, cathepsin G is not expressed in eosinophils, and neutrophil elastase is not expressed in unstimulated eosinophils (21). ADAM17 on endothelial cells can mediate shedding of VCAM-1 from the same cell (39). However, ADAM17 mRNA is not regularly expressed in eosinophils (21), and flow cytometry of our donors has demonstrated that 30% do not have cell-surface ADAM17 protein on eosinophils (not shown). In contrast, eosinophils from each of the 12 donors tested (of whom 3 were normal, 4 had allergic rhinitis, and 5 had asthma) caused loss of VCAM-1 substrate. These results indicate that ADAM8 is a candidate to be the OP-inhibitable proteinase responsible for VCAM-1 substrate degradation by eosinophils. Further studies, however, are needed to explore whether ADAM8 acts on VCAM-1 directly or, alternatively, activates other proteinase or proteinases that then cleave VCAM-1, and possibly other substrates. The potential importance of ADAM8 in eosinophil trafficking is highlighted by the recently reported microarray analysis of whole lung from mice in which ADAM8 was one of 291 mRNAs upregulated after antigen challenge with ovalbumin or Aspergillus fumigatus (40), and the follow-up study demonstrating that upregulation occurred in a STAT6- and IL-4 receptor ␣–dependent manner (41). In situ hybridization showed that both inflammatory cells, especially eosinophils, and epithelial cells were positive for ADAM8 in allergen-challenged lung (41). T helper cell type 2 immunity mediators, which are secreted in asthma, activate endothelium to express VCAM-1, which initiates the recruitment of eosinophils to the airway, where they are believed to be important for asthmatic inflammation (6–8). There are several possible scenarios concerning the functional significance of the formation of ADAM8-containing podosomes

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upon specific adherence of eosinophils to VCAM-1, the stimulation by cytokines of podosome formation, and metalloproteinasedependent degradation of the VCAM-1 substrate by adherent eosinophils. First, podosomes may be necessary for adhesion and lateral migration of eosinophils on VCAM-1 on activated endothelium. Second, degradation of VCAM-1 may downregulate eosinophil adhesion in the absence of continued endothelial cell synthesis of VCAM-1 and thus may be a mechanism to control eosinophil arrest. Third, degradation of substrates other than VCAM-1 may be necessary for eosinophil transmigration through activated endothelium and subendothelial basement membrane. Future studies are needed to investigate these possibilities. Conflict of Interest Statement: M.W.J. has no declared conflicts of interest; M.H.L. has no declared conflicts of interest; S.R.B. has no declared conflicts of interest; A.K.D. has no declared conflicts of interest; D.S.A. has no declared conflicts of interest; and D.F.M. has no declared conflicts of interest. Acknowledgments: The authors thank Lynn Allen-Hoffmann, Roy Lobb, Richard Lynch, Cassandra Paul, Paul Ponath, and Kenneth Yamada for providing reagents and cell lines; Heather Gerbyshak, Kristyn Jansen, Anne Brooks, and Julie Sedgwick for eosinophil isolation; Bianca Tomasini-Johansson for FITC-FN preparation, and JoAnn Meerschaert for TS1/18 purification; Lara Johansson for help with cell culture; Jennifer Tran for help with fluorescent staining; Anna Huttenlocher and Mary Lokuta for advice on fluorescent staining; Kathleen Schell, Joan Batchelder, and Joel Puchalski for help with flow cytometry data collection and analysis; Becky Kelly and Mary Ellen Bates for advice on staining for flow cytometry; Mary Ellen Bates for sharing eosinophil RNA expression data and Becky Kelly and Lin-Ying Liu for ADAM17 flow cytometry data; Douglas Austin for photographic work; Carol Dizack for help with illlustrations; William Busse for support and critical suggestions; Paul Bertics, Becky Kelly, and Bianca TomasiniJohansson for comments on the manuscript; and Marcy Salmon and Susan Forrester for manuscript preparation. This work was supported by Specialized Center of Research (SCOR) grant HL56396 from the National Institutes of Health.

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