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Resting Murine Neutrophils Express Functional ₄ Integrins that Signal Through Src Family Kinases¹

Department of Laboratory Medicine, University of California, San Francisco, CA 94143

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
There is mounting evidence that ₄ (CD49d) integrins are involved in neutrophil recruitment and function during inflammatory responses. We report that all resting murine neutrophils derived from bone marrow or peripheral blood express easily detectable levels of ₄ integrins on their surface. These ₄ integrins were functional, as demonstrated by stimulation of respiratory burst when neutrophils adhered to surfaces coated with the murine vascular cell adhesion molecule-1 (mVCAM-1). Adhesion occurred via ₄ integrins, as preincubation of neutrophils with an anti-₄-specific Ab inhibited attachment to mVCAM-1. Direct cross-linking of the ₄ integrin subunit by surface-bound mAbs also elicited superoxide release and release of the secondary granule marker, lactoferrin. The functional responses that occurred downstream of ₄ integrin cross-linking required signaling by Src family kinases. Neutrophils derived from hck^-/-fgr^-/-lyn^-/- triple-knockout or hck^-/-fgr^-/- double-knockout mice failed to undergo respiratory burst when plated on mVCAM-1. Triple mutant neutrophils were also defective in release of both superoxide and lactoferrin when plated on surfaces coated with mAbs directed against ₄. Correlated with impaired ₄-induced functional responses, triple-mutant neutrophils also failed to spread and tightly adhere to anti-₄ mAb-coated surfaces. This is the first direct evidence that functional ₄ integrins are expressed by murine PMNs, and that these surface molecules can mediate cellular responses such as tight adhesion, spreading, sustained respiratory burst, and specific granule release in vitro. Moreover the ₄ integrins, like all other integrins tested, use the Src family kinases to transduce intracellular signals.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Cell adhesion plays an important role in the migration of polymorphonuclear leukocytes (PMNs)³ to sites of tissue injury and inflammation. This is a multistep process involving migration through the endothelium and extracellular matrix to the desired site, followed by full activation and the release of effector molecules. Several different adhesion molecules are involved in these events; these include members of the selectin, integrin, and Ig superfamilies (1).

Integrins are the main class of cell adhesion molecules, which mediate attachment to the extracellular matrix as well as direct cell-cell adhesion (2, 3, 4, 5). Integrins are transmembrane proteins consisting of and subunits; there are at least 15 known subunits and eight subunits, which combine into 25 different heterodimers. Integrins are widely expressed and are involved in a number of important biological functions, including embryonic development, wound repair, hemostasis, and prevention of programmed cell death (2, 6). Blood neutrophils as well as all other leukocytes express high levels of the CD18 integrins _L2 (LFA-1), _M2 (Mac-1) and _x2 (p150/95). These molecules mediate firm interactions of PMNs with the endothelium as well as facilitate migration of cells into inflammatory sites in the skin, lung, and reperfusion injuries (7, 8, 9, 10). The CD49d integrins ₄₁ (very late Ag-4) and ₄₇ (LPAM-1) are expressed on lymphocytes, monocytes, and eosinophils (11). In lymphocytes, the principle endothelial cell-associated ligand for ₄₁ is VCAM-1. Although resting human PMNs do not express ₄ integrins, expression has been reported to be induced by stimulation with the potent degranulating agent, dihydrocytochalasin B, in conjunction with fMLP or leukotriene B₄ (12) or by treatment with C5a (13). PMNs induced to express ₄ integrins can tether and adhere to VCAM-1-transfected L cells or endothelium stimulated with TNF- (14). However, these studies depended on the use of an anti-human ₄ integrin-specific Ab, HP2/1, which has recently been reported to contain a contaminating Ab that reacts with an unidentified surface marker on human PMNs (15). Therefore, the expression and use of ₄ integrins by human PMNs remain controversial.

In vivo studies with blocking Abs in rodents have demonstrated a clear role for ₄ integrins in the selective recruitment of PMNs to inflammatory sites. In a murine model of endotoxic shock, increased expression of mVCAM-1 was observed in the liver. Pretreatment with anti-VCAM-1 Ab decreased neutrophil transmigration into the liver parenchyma and attenuated liver tissue damage (16). In an adjuvant-induced arthritis model in rats, treatment with anti-₄ Ab not only modified the disease during the preclinical phase, it also reduced the severity of the disease after joint inflammation had developed (17). In allergen-challenged Brown Norway rats, anti-₄ Ab treatment was found to reduce neutrophil migration into airways, as assessed by bronchoalveolar lavage (18). In another in vivo inflammation model, migration of PMNs into glomeruli could be reduced by anti-₄ treatment in rats with Ab-induced nephritis (19).

In the presence of inflammatory mediators such as TNF- and fMLP, cross-linking of PMN integrins by matrix proteins or cell-associated adhesion molecules induces the formation of focal adhesion structures and PMN spreading. Accompanying integrin-induced cell spreading, PMNs release proteolytic enzymes from the various granules and undergo respiratory burst, resulting in the production of reactive oxygen intermediates (ROI). Some of the intracellular signaling pathways activated by integrin-induced adhesion have been defined. One of the major classes of tyrosine kinases that play a role in regulating integrin signaling is the Src family kinases (20, 21). Of the nine members of this family, Src, Fyn, Yrk, and Yes are expressed in most tissues, while Blk, Fgr, Hck, Lyn, and Lck are found primarily in hemopoietic cells (22). Myeloid cells express Hck, Fgr, and Lyn. In adherent neutrophils, there is increased phosphorylation and an enhancement of the kinase activities of Fgr and Lyn accompanied by localization of these kinases to the actin cytoskeleton (23). Double-mutant hck^-/-fgr^-/- neutrophils show a marked defect in adhesion and cell spreading when plated on murine (m) ICAM-1, a ligand that cross-links ₂ integrins (20). Migration of hck^-/-fgr^-/- neutrophils into tissues during endotoxemia is reduced compared with that of wild-type cells (24), indicating that these kinases are involved in signaling through neutrophil integrins.

In this study we have investigated the expression of ₄ integrins by murine PMNs. We demonstrate that resting murine neutrophils express significant levels of ₄. Further, we show that neutrophils can adhere to surfaces coated with ₄ integrin ligands, such as mVCAM-1 or anti-₄ mAbs, and signal the release of effector molecules. Finally, we show that the myeloid Src family kinases, Hck, Fgr, and Lyn, play a role in ₄ integrin signaling, because mutant neutrophils deficient in all three kinases are defective in transducing signals induced by ₄ integin cross-linking. These results provide the first direct evidence that ₄ integrins, like other integrins expressed by neutrophils, transduce signals leading to downstream effector function in vitro and that the Src family kinases play an important role in intracellular signaling downstream of ₄ integin cross-linking.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Reagents

The following Abs were used in these experiments. Biotinylated Abs against ₄ integrin (R1-2, P/S2, and 9C10), ₁ integrin (Ha2/5), ₂ integrin (C71/16 and M18/2), ₇ integrin (M293), rat IgG2b (A95-1), and rat IgG2a (R35-95) were obtained from PharMingen (San Diego, CA). The rat anti-mouse hybridomas secreting PS/2, and Ly2.2 (anti-CD8; rat IgG2b) were obtained from American Type Culture Collection (Manassas, VA) and A. Weiss (University of California, San Francisco, CA) respectively. The mAbs were purified from culture supernatant by passing over protein G columns. Purified Abs against ₄ integrin (R1-2), ₂ integrin (C71/16 and GAME-46), rat IgG2a (R35-95), rat IgG1 (R3-34), and rat IgG2b (A95-1) were obtained from PharMingen (San Diego, CA). Mouse Ab, AN100226M against human ₄, was a gift from D. Sheppard (University of California, San Francisco, CA). J558L and X63 cells secreting mICAM-1 and mVCAM-1, respectively, were gifts from B. Imhof (Basel Institute of Immunology, Basel, Switzerland). Both secreted proteins contain the first two Ig domains fused to the constant region of the mouse -chain. The proteins were purified from culture supernatant that was passed over rat anti-mouse light chain Sepharose 4B (Zymed, South San Francisco, CA) after passage through a Sephadex G-25 column. Bound protein was eluted in 0.1 M glycine and 0.15 M NaCl (pH 3.4) and immediately neutralized with 0.1 vol of 2 M Tris (pH 8.0). The purified proteins were dialyzed into PBS, and the concentration was determined using the Bradford assay (Bio-Rad, Hercules, CA). Recombinant mVCAM-1 was also purchased from R&D Systems (Minneapolis, MN).

Isolation of bone marrow PMNs

The hck^-/-fgr^-/- and hck^-/-fgr^-/-lyn^-/- knockout mice used in these experiments were backcrossed onto the C57BL/6J genetic background for 15 generations. Congenic C57BL/6J mice were used as wild-type controls for all experiments. Bone marrow PMNs were isolated from 6- to 12-wk-old mice as previously described (25). The isolated PMNs were processed in either of two ways. Those used in experiments with Ab-coated surfaces were washed in Ca²⁺/Mg²⁺-free HBSS supplemented with 10 mM HEPES to maintain the cells in a quiescent state. This was found to reduce the background generated by nonspecific responses elicited by the irrelevant mAbs. When mVCAM-1 or mICAM-1 were used as integrin ligands, the cells were washed in HBSS containing 10 mM HEPES, 0.5 mM CaCl₂, and 1 mM MgCl₂. The washed cells were kept on ice until use. Just before use the cells were diluted to the appropriate concentration in HBSS, Ca²⁺/Mg²⁺, and 10 mM HEPES.

Flow cytometry

Bone marrow and peripheral blood cells were obtained from 6- to 12-wk-old mice. Approximately 10⁶ cells were washed twice in PBS, 2% FBS, and 0.1% sodium azide and stained with biotinylated anti-₄ (R1-2, 9C10, PS/2), anti-₁ (Ha2/5), anti-₇ (M293), or anti-₂ (C71/16) Abs for 10 min at 4°C. Subsequently, the cells were washed twice in wash buffer, then stained with streptavidin-PE and Gr-1-FITC. Following two more washes, the cells were suspended in 1 ml of wash buffer and 1 µg/ml propidium iodide. Viable cells were analyzed with FACScan (Becton Dickinson, San Jose, CA). Gating on the PMN population was performed on the basis of forward/side scatter and Gr-1 staining. It was ascertained that the forward/side light scatter and Gr-1 staining criteria of purified mature bone marrow PMNs (>95% pure as determined by morphological examination) were identical with those used for whole BM PMNs, indicating that the majority of gated cells in bone marrow or peripheral blood samples were mature PMNs. For staining of peripheral human leukocytes, the same procedure was followed as described above, except that cells were stained with mouse mAb, AN100226 M, against human ₄, washed, and then stained with a goat anti-mouse-FITC and CD13/33-PE.

Superoxide release assays

Immulon-4 flat-bottom, 96-well microtiter plates (Dynex, Chantilly, VA) were coated with either cellular adhesion molecules or different mAbs. Each well of the microtiter plate was coated with 100 µl of mICAM-1 or mVCAM-1 at the indicated concentrations at 4°C overnight. The wells were blocked with 200 µl of 20% FBS at room temperature for 1–2 h, then washed three times with PBS. When mICAM-1 or mVCAM-1 were used as integrin ligands, PMNs at a concentration of 7.5 x 10⁶/ml were preincubated with 20 ng/ml mTNF- at room temperature for 10–15 min before being mixed with an equal volume of 200 mM ferricytochrome C (Sigma) in HBSS, Ca²⁺/Mg²⁺, and 10 mM HEPES, then added to the coated wells of a 96-well microtiter plate. The use of these conditions, while different from those previously established (20), resulted in increased stimulation of ROI release and hence a greater difference between unstimulated and stimulated cells during the initial time points of the reaction. The plates were read in an automated microtiter plate reader (Spectramax, Molecular Devices, Menlo Park, CA), and the nanomoles of superoxide produced every 10 min over a period of 2 h was determined as previously described (20). The data are presented as a cumulative assay, and all time points were performed in triplicate.

Coating of Immulon-4 plates with mAbs was essentially as previously described (26). Coating of microtiter wells with biotinylated mAbs was performed as described previously (20) with the following modifications. Plates were incubated with 25 µg/ml streptavidin (50 µl/well) at 4°C overnight. The wells were washed with PBS, incubated with 20 µg/ml biotinylated mAb (50 µl/well) at 4°C for 2–3 h, then blocked with 20% FBS containing 25 µg/ml protein G (Sigma, St. Louis, MO), to block FcR binding, and washed with PBS. Resting PMNs were added directly to mAb-coated wells; preincubation of cells with TNF- was not necessary to induce respiratory burst following plating on mAbs. Where indicated, the plates were warmed at 37°C for 5 min before the addition of 100 nM PMA.

Rapid attachment assays

Rapid attachment assays were conducted in 18-well HTC slides (Cel-Line) coated with cellular adhesion molecules (27). Each well of the slide was coated with 20 µl of mICAM-1 or mVCAM-1 (1 µg/ml) at 37°C for 2–3 h followed by one wash with PBS. The wells were then blocked with 20 µl of 10% BSA at 37°C for 10 min. Cells at 10⁵/ml were preincubated with either blocking mAbs (10 µg/ml) or PBS at room temperature for 20–30 min. Twenty microliters of the cellular suspension was then added to the coated wells and incubated at 37°C for 10 min. The slides were washed in PBS, and adherent cells fixed with 1.5% glutaraldehyde. The slides were stained with modified Wright Giemsa (Sigma) and observed using the x10 objective of a light microscope. Digital images of at least four random fields were obtained, and the number of cells adherent was quantified using NIH Image software (version 1.62). All wells were incubated in triplicate, and results were averaged. Data were plotted relative to the number of cells that adhered to either mICAM or mVCAM (which was defined as 100%) in any individual experiment to facilitate comparison of results from different experiments.

Lactoferrin release assays

Lactoferrin release assays were performed essentially as described previously (25). One hundred microliters of cells at 1.5 x 10⁶/ml were incubated in Ab-coated wells of a microtiter plate at 37°C for 60 min. In experiments in which PMA was used as a stimulant, the plates containing cells were warmed at 37°C for 10 min before the addition of PMA, followed by a 60-min incubation at 37°C. After incubation the samples were transferred to a polypropylene 96-well V-bottom plate and centrifuged at 2000 rpm for 10 min. Twenty-five microliters of supernatant per sample was diluted 4-fold in carbonate buffer (pH 9.6) and incubated overnight at 4°C in an Immulon-4 microtiter plate. All subsequent steps were conducted as previously described (25). Assays were performed in triplicate, and the results were averaged.

Tight adhesion assays

One hundred microliters of bone marrow PMNs at 4 x 10⁶ cells/ml were added to mAb-coated wells of Immulon-4 microtiter plates. Following incubation at 37°C for 60 min the plates were washed three times with warm PBS (37°C), overturned onto 3 MM Whatman paper (Clifton, NJ), and spun in a swinging bucket rotor at 60 x g for 5 min. The washes and centrifugation step were repeated, and the number of adherent PMNs that remained in the bottom of the wells was measured by the membrane enzyme acid phosphatase assay as previously described (20). The percentage of adherent cells was calculated relative to the total input of cells used in the assay. Individual determinations were performed in triplicate, and results were averaged.

Photomicroscopy

The wells of an Immulon-4 plate were coated with different Abs as previously described. One hundred microliters of bone marrow neutrophils at about 5 x 10⁵ cells/ml were added to each well, and the plates were incubated at 37°C for 30–60 min before being photographed under phase contrast on a Nikon microscope (Melville, NY) fitted with a Hoffman contrast modulation adapter (Modulation Optics, Greenvale, NY). When PMA was used as a stimulant, the plate was preincubated at 37°C for 10 min before the addition of PMA to a final concentration of 100 nM.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Resting murine PMNs express ₄ integrins

A role for mVCAM-1, an ₄ integrin ligand, has been established in a murine model of neutrophil-induced liver injury in vivo (16). This finding prompted an analysis of the surface expression of the ₄ integrin subunit on murine PMNs. Flow cytometry was performed on whole blood and bone marrow leukocytes from wild-type mice, using three different ₄ (CD49d)-specific Abs. The expression level of ₄ on the surface of human PMNs was also analyzed. Staining for CD49d expression demonstrated that 100% of murine PMNs, including fully mature cells in the peripheral blood or those in varying maturation states in the bone marrow, expressed ₄ (Fig. 1A). Because ₄ can complex with either ₁ (CD29) or ₇ to form a functional heterodimer, the expression level of these integrin subunits was also examined. About 80–90% of mature PMNs from whole blood expressed ₁ (CD29), while 15–20% expressed low levels of ₇. Bone marrow-derived neutrophils expressed virtually no ₇, indicating that the expression of this integrin subunit may be developmentally regulated (Fig. 1A). One hundred percent of PMNs expressed ₂ (CD18) at all stages of development (Fig. 1A). As has been reported (28), resting peripheral human PMNs did not express CD49d, while the control lymphocyte population demonstrated adequate staining with the AN100226M Ab (Fig. 1B). The above data demonstrate that, unlike human PMNs, all resting murine PMNs express easily detectable levels of ₄ (CD49d) on their surface.

View larger version (29K): [in this window] [in a new window]   FIGURE 1. Resting murine PMNs express 4 integrins. Flow cytometric analysis was conducted by gating on PMNs using forward vs side light scatter and Gr-1 staining. Data shown are from the Gr-1-positive cells only. Open peaks denote staining with control Ab, while shaded peaks indicate staining with specific Abs. A, Resting bone marrow and peripheral blood PMNs from wild-type mice were stained with three different 4-specific Abs (PS/2, 9C10, R1-2) as well as with 1 (Ha2/5), 7 (M293), and 2 (C71/16) Abs. B, Expression of 4 on the surface of resting peripheral human PMNs vs lymphocytes was analyzed by staining whole blood leukocytes with the mouse anti-human AN100226M mAb. Forward/side light scatter and staining with CD13/CD33 were used as a criterion of PMN identity.

Because murine PMNs express significant levels of ₄ integrins, the ability of these cells to adhere to mVCAM-1 and to mount a response in the presence of inflammatory mediators in vitro was tested. Bone marrow PMNs isolated from wild type mice were preincubated with or without TNF-, then added to microtiter wells that had been precoated with either FBS or mVCAM-1 (10 µg/ml). Murine ICAM-1, which is known to support superoxide release by murine PMNs in vitro (20), was used as a positive control. Preincubation of PMNs with TNF- magnified the initial release of O₂^- over the first 30 min of the assay, thus maximizing the difference between resting and stimulated cells. As shown in Fig. 2A, robust superoxide release was observed when PMNs stimulated with TNF- were adherent to either mVCAM-1 or mICAM-1. Respiratory burst was significantly lower when the PMNs adhered to FBS. Concentrations of mVCAM-1 and mICAM-1 as low as 0.1 µg/ml supported adhesion-mediated superoxide release by wild-type PMNs (Fig. 2, B and C). In the absence of TNF- stimulation, the cells did not respond to any concentration of mVCAM-1 or mICAM-1 (10, 1.0, and 0.1 µg/ml). The above data demonstrate that mVCAM-1 is a potent activator of adhesion-dependent respiratory burst by murine PMNs in vitro.

View larger version (34K): [in this window] [in a new window]   FIGURE 2. Superoxide released by wild-type PMNs adherent to cellular adhesion molecules. Wild-type murine PMNs were preincubated (15 min) with or without TNF-, then added to plates coated with mICAM-1 or mVCAM-1. Superoxide release over a 120-min period was monitored as described in Materials and Methods. A, Cells added to plates coated with 20% FBS, mICAM-1 (10 µg/ml), or mVCAM-1 (10 µg/ml). B and C, Concentrations of mICAM-1 (B) and mVCAM-1 (C) ranging from 10–0.1 µg/ml were used to coat sets of microtiter wells. All assays were performed in triplicate, and the results were displayed as an average ± SD.

₄ integrins are involved in the attachment of murine PMNs to mVCAM-1

Among the integrins known to bind VCAM-1 are ₄₁ and ₉₁. While ₄ integrins are not detected on resting human PMNs (28), ₉₁ is highly and selectively expressed (29). To determine whether the interaction between murine PMNs and mVCAM-1 occurs via ₄ (CD49d) integrins, PMNs were tested in a rapid attachment assay. Isolated bone marrow PMNs were plated in the wells of a multiwell slide precoated with BSA, mVCAM-1, or mICAM-1. In the absence of ligand very few PMNs attached to the slide (Fig. 3, A and B). Both mICAM-1 and mVCAM-1 stimulated neutrophil attachment about 5-fold (Fig. 3, A and B). Preincubation of cells with a ₂ (CD18)-specific blocking mAb (GAME-46) diminished attachment of PMNs to mICAM-1 to control levels, while incubation with the irrelevant control mAb (rat IgG1) did not influence attachment (Fig. 3A). Interestingly, preincubation of the PMNs with a CD49d-specific blocking mAb (PS/2) decreased attachment to mVCAM-1 to background levels, indicating that the attachment of murine PMNs to mVCAM-1 occurs predominantly via ₄ integrins (Fig. 3B). In contrast, preincubation of PMNs with a CD18-specific blocking mAb (GAME-46) or a non-ICAM-1-blocking anti-CD18 mAb (C71/16) did not significantly reduce attachment to mVCAM-1, indicating that the ₄-dependent attachment to mVCAM-1 is most likely not mediated through CD18. Preincubation of PMNs with an irrelevant isotype control mAb (IgG2b) did not affect attachment. Preincubation of cells with TNF- did not significantly increase attachment of cells to either ICAM-1 or VCAM-1 and did not alter the effects of mAb treatment (data not shown). We conclude that murine PMNs use predominantly the ₄ integrins to attach to mVCAM-1.

View larger version (45K): [in this window] [in a new window]   FIGURE 3. Preincubation of PMNs with anti-2 and anti-4 integrin Abs, blocks adhesion to mICAM-1 and mVCAM-1, respectively. A, Multiwell slides were coated with mICAM-1 or 10% BSA (indicated as no ligand). Cells were preincubated with either PBS, anti-CD18 blocking Ab (GAME-46) or irrelevant Ab control (rat IgG1; R3-34), then added to coated wells and incubated at 37°C for 10 min. After washing to remove nonadherent cells, four random fields were examined, and the number of attached cells was counted and plotted relative to the number bound in the mICAM-1. B, Multiwell slides were coated with mVCAM-1 or 10% BSA (no ligand). Cells were preincubated with PBS, anti-CD49d blocking Ab (PS/2), anti-CD18 blocking Ab (GAME-46), anti-CD18 nonblocking Ab (C71/16), or irrelevant Ab control (rat IgG2b; A95-1), added to coated wells, allowed to attach, then counted and plotted as described above. Results with isotype control rat IgG2a (R35-95) and rat IgG1 (R3-34) were identical with those obtained with IgG2b (data not shown). Data shown are the average ± SD of triplicate determinations and are representative of three independent experiments.

Double-mutant PMNs derived from hck^-/-fgr^-/- mice fail to spread and undergo respiratory burst when stimulated with inflammatory agonists and plated on mICAM-1-coated surfaces (20), supporting a role for the Src family kinases in integrin signaling. To determine the effect of the loss of the myeloid Src family kinases (Hck, Fgr, and Lyn) on the ability of PMNs to respond to ₄ integrin cross-linking, PMNs from wild-type, double-mutant (hck^-/-fgr^-/-), and triple-mutant (hck^-/-fgr^-/-lyn^-/- or TKO) mice were plated in wells precoated with mVCAM-1. Both the double- and triple-mutant cells were defective in their ability to undergo respiratory burst when plated on mVCAM-1 (Fig. 4), while cells derived from single mutant hck^-/- or fgr^-/- mice responded normally (data not shown). Hence, as observed in studies with the ₂ integrin ligand, mICAM-1 (20), both Hck and Fgr are the principle kinases required for signaling events downstream of ₄ integrins that lead to respiratory burst. In the absence of TNF- stimulation, neither wild-type nor mutant PMNs released significant amounts of superoxide (data not shown). In multiple experiments it was observed that the TKO mutant PMNs were slightly more defective than hck^-/-fgr^-/- double-mutant cells in most superoxide release assays (Fig. 4 and data not shown). Therefore, to firmly address the contributions of these kinases as a gene family to the ₄ integrin signaling events, we concentrated our studies on PMNs derived from the TKO (hck^-/-fgr^-/-lyn^-/-) animals.

View larger version (25K): [in this window] [in a new window]   FIGURE 4. Mutant PMNs, lacking the myeloid Src family kinases, are defective in superoxide production elicited by adhesion to mVCAM-1. PMNs from WT, hck-/-fgr-/- or hck-/-fgr-/-lyn-/- mice were preincubated with TNF- then added to wells coated with mVCAM-1 (10 µg/ml). Superoxide (nanomoles) released over time was determined as described (Materials and Methods). In the absence of TNF-, wild-type, double-mutant (hck-/-fgr-/-) and triple mutant (hck-/-fgr-/-lyn-/-) PMNs released negligible amounts of superoxide (data not shown).

Triple-mutant hck^-/-fgr^-/-lyn^-/- PMNs express wild-type levels of ₄ integrins

To ascertain whether the inability of TKO PMNs to respond to mVCAM-1 was due to decreased expression of ₄, the cells were analyzed by flow cytometry. As shown in Fig. 5 the TKO cells express adequate levels of the ₄ integrin subunit; the expression patterns of all integrin subunits tested were similar to those observed for wild-type PMNs (Fig. 1A). Therefore, the impaired respiratory burst of TKO cells when plated on mVCAM-1 is not the result of reduced ₄ integrin expression.

View larger version (33K): [in this window] [in a new window]   FIGURE 5. PMNs from triple-knockout (hck-/-fgr-/-lyn-/-) mice express adequate levels of 4 integrins. Flow cytometric analysis using blood and bone marrow leukocytes was conducted by gating on PMNs using forward vs side light scatter and Gr-1 staining. Data shown are from the Gr-1-positive cells only. Open peaks denote staining with control Ab, while shaded peaks indicate staining with specific Abs. Three different 4-specific Abs (PS/2, 9C10, and R1-2) as well as with 1 (Ha2/5), 7 (M293), and 2 (C71/16) Abs were used for staining.

Triple-mutant hck^-/-fgr^-/-lyn^-/- PMNs are defective in superoxide release triggered by surface-bound mAbs against ₄ integrins

There is an absolute requirement for costimulus with inflammatory agonists, such as TNF- or fMLP, when eliciting responses from PMNs adherent to cellular adhesion molecules or extracellular matrix (ECM) proteins in vitro (Figs. 1 and 4) (20). In contrast, plate-bound mAbs directed against specific integrin chains expressed by PMNs can trigger superoxide production in the absence of any additional stimulus (20, 26). We tested the ability of plate-bound anti-₄ mAbs to directly stimulate PMN respiratory burst in unstimulated wild-type and TKO cells. To avoid stimulation via Fc receptors, cells were maintained in Ca²⁺/Mg²⁺-free HBSS, and the microtiter wells were blocked with FBS containing soluble protein G to block the Fc region of the Ab (see Materials and Methods). The CD49d (R1-2)-specific mAb triggered strong respiratory burst responses by wild-type PMNs, similar to those mediated by CD18 (C71/16) mAb (Fig. 6A). The matching isotype control rat IgG2b (A95-1) or rat IgG2a (R35-95) Abs did not trigger superoxide release, thus excluding the possibility of any Fc-mediated stimulation (Fig. 6A). PMNs derived from TKO mice were defective in respiratory burst when plated on either plate-bound anti-CD49d or anti-CD18 mAbs (Fig. 6A). The respiratory burst assays were also performed with purified mAbs directed at ₄ (CD49d; PS/2), ₂ (CD18; M18/2), rat IgG2b (A95-1), and rat IgG2a (R35-95) bound to protein G-coated wells via their Fc regions (see Materials and Methods). The results obtained were similar to those with the biotinylated Abs (data not shown). These data demonstrate that adhesion of PMNs to surface-bound anti-₄ mAbs directly stimulates respiratory burst in the absence of additional inflammatory mediator stimuli. Moreover, this PMN functional response to anti-₄ mAbs is lost in Src family kinase-deficient cells.

View larger version (25K): [in this window] [in a new window]   FIGURE 6. PMNs from the triple-knockout (hck-/-fgr-/-lyn-/-) mice are defective in superoxide production on adhesion to plates coated with anti-integrin mAbs. A, PMNs derived from wild-type (WT) or triple-mutant hck-/-fgr-/-lyn-/- (TKO) mice plated were plated in wells coated with CD49d (R1-2), CD18 (C71/16), or irrelevant Ab (IgG2b (A95-1)), and superoxide release was monitored over a 2-h period. B, WT and TKO PMNs were plated on tissue culture (TC) plastic, and the nanomoles of superoxide released was estimated. C, Same as in B, except that the cells were treated with 100 nM PMA. Pooled bone marrow PMNs from two or three mice were used in each experiment. Data shown are the averages (±SD) of triplicate determinations and are representative of at least three independent experiments.

As previously argued in assessing responses of hck^-/-fgr^-/- PMNs to plate-bound mICAM-1 (20), the fact that TKO failed to respond to direct ₄ integrin ligation or to tissue culture plastic supports the model that the impaired responses to plate-bound mVCAM-1 were due to defects in integrin signaling and not to a failure of the cells to respond to TNF-.

Adhesion to surface-bound mAbs against ₄ integrins induces release of the secondary granule marker lactoferrin by wild-type, but not hck^-/-fgr^-/-lyn^-/-, PMNs

One of the consequences of adhesion-dependent activation of PMNs, besides superoxide production, is degranulaton. While secondary granule release can be observed in adherent PMNs on activation with TNF- or GM-CSF, the release of primary granules requires additional treatment with cytochalasin B (30). To test whether direct cross-linking of PMN ₄ integrins could induce secondary granule release, we measured the secretion of the granule protein lactoferrin by cells adherent to surface-bound anti-₄ mAbs. As seen in the respiratory burst assay, the anti-₄ integrin mAb was effective in stimulating lactoferrin secretion by adherent wild-type PMNs to levels similar to those obtained by cross-linking ₂ integrins (Fig. 7A). Uncoated tissue culture plastic, likewise, provided a very strong degranulation stimulus. Similar to results seen in the respiratory burst assay, TKO PMNs failed to release lactoferrin when adherent to anti-₄ mAbs. Lactoferrin release was also impaired when cells were plated on CD18-specific mAb-coated surfaces or tissue culture plastic surfaces (Fig. 7A). However, treatment of the TKO PMNs with PMA elicited robust lactoferrin release, equivalent to that seen in wild-type cells, suggesting that adhesion-independent secondary granule release was unaffected by the loss of these kinases (Fig. 7B). The data demonstrate that direct cross-linking of the ₄ integrins mimics activation by other leukocyte integrins, in that it can elicit secondary granule release by wild-type PMNs. As seen in the respiratory burst functional assay, TKO PMNs are also defective in this aspect of adhesion-mediated activation.

View larger version (53K): [in this window] [in a new window]   FIGURE 7. Adhesion to plate-bound anti-4 mAb induces degranulation in wild-type PMNs, but not in hck-/-fgr-/-lyn-/- cells. A, Wild-type (WT) and triple-knockout (hck-/-fgr-/-lyn-/-; TKO) PMNs were plated in wells coated with biotinylated CD49d (R1-2), CD18 (C71/16), or irrelevant Ab (IgG2b (A95-1)) or on uncoated tissue culture (TC) plastic at 37°C for 60 min. The amount of lactoferrin released by the adherent PMNs was measured as previously described (see Materials and Methods). B, As in A, except that the cells were added to FBS-coated wells and treated with 100 nM PMA before incubation. The concentration of lactoferrin secreted by wild-type PMNs on adhesion to the irrelevant Ab was designated 100%, and the amount of lactoferrin released by PMNs adherent to different surfaces was estimated relative to this value. Data shown are the averages (±SD) of triplicate determinations and are representative of at least three independent experiments. Results with isotype control rat IgG2a (R35-95) were identical with those obtained with IgG2b (data not shown).

Triple mutant hck^-/-fgr^-/-lyn^-/- PMNs are defective in tight adhesion to surface-bound mAbs against ₄ integrins

To determine whether the functional responses of PMNs when adherent to plate-bound anti-₄ mAbs correlated with firm adhesion, we tested the ability of WT and TKO PMNs to resist centrifugal force following plating on anti-integrin mAbs. Wild-type or TKO PMNs were added to microtiter wells precoated with mAbs directed against CD49d and CD18 or isotype control mAbs IgG2b or IgG2a, incubated at 37°C for 60 min, then centrifuged upside down to remove all but the most firmly adherent cells. The percentage of cells that remained tightly adherent was quantified by assaying membrane acid phosphatase activity. In this assay 30–40% of the wild-type PMNs were tightly adherent to surfaces coated with an ₄ integrin-directed mAb, with slightly more adhering to the ₂ integrin-directed mAb (Fig. 8A). Maximal adhesion occurred on uncoated plastic surfaces, while both isotype control mAbs did not stimulate tight adhesion. The TKO PMNs did not adhere tightly to any of the tested surfaces and were easily removed under the conditions used (Fig. 8A). When proximal integrin signaling was bypassed by stimulating the cells with PMA, both wild-type and TKO PMNs were very efficient at tight binding (Fig. 8B). These data demonstrate that direct cross-linking of PMN ₄ integrins by surface-bound mAbs stimulates firm cell adhesion, and as seen with other leukocyte integrins, this process is defective in TKO cells.

View larger version (50K): [in this window] [in a new window]   FIGURE 8. Triple-mutant hck-/-fgr-/-lyn-/- PMNs are defective in tight adhesion to mAb-coated surfaces. A, Wild-type (WT) and triple-knockout (hck-/-fgr-/-lyn-/-; TKO) PMNs were added to wells coated with biotinylated anti-CD49d (R1-2), anti-CD18 (C71/16), irrelevant mAb (IgG2b (A95-1)), or uncoated tissue culture (TC) plastic and incubated at 37°C for 60 min. The percentage of cells that remained adherent to each surface after washes with PBS and centrifugation were determined as described (see Materials and Methods). B, Cells were incubated on FBS-treated plastic after treatment with 100 nM PMA. Data shown are the averages (±SD) of triplicate determinations conducted on pools of bone marrow PMNs isolated from two or three mice. This experiment is representative of three independent experiments. Similar results were obtained with the other isotype control rat IgG2a (R35-95; data not shown).

Triple-mutant hck^-/-fgr^-/-lyn^-/- PMNs do not spread on surfaces coated with mAbs against ₄ integrins

Because firm adhesion by PMNs occurs by cell spreading, we used photomicroscopy to compare the ability of wild-type and TKO PMNs to spread over anti-₄ mAb-coated plates. As shown in Fig. 9, wild-type PMNs did not spread on the isotype control Ab-, IgG2b-, or IgG2a-coated surfaces (data not shown), while they could spread efficiently on anti-₄ or anti-₂ integrin mAbs. The TKO PMNs did not spread on any of the mAb-coated surfaces (Fig. 9). However, both cell types spread extensively when treated with PMA. Taken together, we conclude that PMN adhesion to plate-bound ₄ mAb induces cell spreading, tight adhesion, and functional activation in wild-type, but not hck^-/-fgr^-/-lyn^-/-, cells.

View larger version (75K): [in this window] [in a new window]   FIGURE 9. Triple-mutant hck-/-fgr-/-lyn-/- PMNs fail to spread on integrin-specific mAb-coated surfaces. Unstimulated PMNs isolated from wild-type (WT) or triple-knockout (hck-/-fgr-/-lyn-/-; TKO) mice were incubated in wells coated with biotinylated CD49d (R1-2), CD18 (C71/16), irrelevant Ab (IgG2b (A95-1)), or uncoated plastic. PMNs stimulated with PMA were added to wells blocked with 20% FBS (see Materials and Methods). A, Wild-type PMNs plated on CD49d; B, anti-CD18; C, irrelevant mAb; D, tissue culture plastic; E, treated with PMA before plating on FBS; F–J, as in A–E using TKO PMNs.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

VCAM-1 acts as a ligand for the ₄ integrins as well as ₉₁. While resting human neutrophils do not express ₄ integrins, ₉₁ is highly and selectively expressed (29, 31). Although we have established that murine neutrophils express the ₄ integrins, and preincubation of cells with an anti-₄-blocking Ab reduced attachment to mVCAM-1 to baseline levels, it is conceivable that murine neutrophils also express the ₉₁ integrin. The unavailability of an Ab directed against murine ₉ makes this hypothesis difficult to test. The relative contribution of ₄ vs ₉ integrins to recognition of mVCAM-1 by rodent PMNs remains to be determined.

Plating of TNF--treated PMNs on mICAM-1 and mVCAM-1 leads to robust superoxide release within the first 20 min following an initial lag period. A decrease is observed after that, with a plateau being achieved at about 40 min (Figs. 2 and 4). The respiratory burst kinetics of the reaction are different from those reported previously with other ECM proteins (20). We believe that this difference was due to pretreatment of PMNs with TNF- before plating in the respiratory burst assay, the method used in this study, compared with previous reports in which cells with treated with TNF- simultaneously with plating on ECM-coated surfaces. Superoxide release by untreated PMNs adhering to plate-bound ₄ mAbs is more gradual, with maximal release being attained at 100 min (Fig. 6). A key difference between activation of neutrophils on surfaces coated with ECM proteins or cellular adhesion molecules vs plate-bound mAbs is the absolute dependence on costimulation with an inflammatory agonist such as TNF- or fMLP. The inflammatory agonists activate the ability of integrins to bind their ligands by inducing changes in integrin affinity or avidity, a process referred to as inside-out signaling (2, 32). Because mAbs bind their epitopes on integrin receptors with high affinity, they circumvent the need for inside-out signaling events.

We have used two assays to distinguish the ability of ₄ integrins to mediate attachment vs firm adhesion of PMNs. The rapid attachment assay (Fig. 3) quantifies the ability of cells to bind to surface-bound ligands independently of spreading, whereas the firm adhesion assay (Fig. 8) requires that the cells spread such that they are able to resist a centrifugal force and remain on the adhesive surface. Because cell spreading requires outside-in integrin signaling events, the firm adhesion assay is a functional measure of these signaling responses, similar to the adhesion-dependent activation of respiratory burst or degranulation. Using plate-bound anti-₄ mAbs allows examination of outside-in signaling pathways independent of inside-out events. Hence, our work focused on the use of plate-bound mAbs as the best measure of direct stimulation of murine PMNs via ₄ integrins.

The ₄ integrins are comprised of the very late Ag-4 (₄₁ or CD49d/CD29) and the lymphocyte Peyer’s patch adhesion molecule-1 (LPAM-1, ₄₇, or CD49d/₇). LPAM-1 is mainly found on lymphocytes (28), monocytes (33), eosinophils (34), basophils, and mast cells (35). Although peripheral human PMNs do not express LPAM-1 (33), examination of murine PMNs revealed low levels of ₇ expression (Fig. 1A). The ₇ may be associated with _E, a possibility that cannot be tested due to the unavailability of an anti-mouse _E mAb; however, it is also possible that, unlike human PMNs, a percentage of murine PMNs expresses LPAM-1. It is difficult to directly compare the expression levels of ₄ vs ₁ integrins on cells by flow cytometry (because the mAbs used to detect these proteins may be differentially labeled or may bind with different affinities). However, although a small proportion of the ₄ expressed by murine PMNs may associate with ₇, it is likely that most of the ₄ on resting murine PMNs is associated with ₁.

When integrins adhere to ECM or cell-associated adhesion molecules, the resultant clustering of the integrin molecules in the leukocyte membrane activates tyrosine phosphorylation, leading to changes in cytoskeletal structure that induce the formation of specialized sites of contact, termed focal adhesions. These signaling events involve a number of tyrosine kinases, including the Src family kinases, focal adhesion kinase (36), Pyk2 (37, 38), and Zap70/Syk (39). In turn, these early signaling events lead to the activation of downstream molecules, including lipid kinases (phosphoinositol 3-kinase), small GTPases (Rac, Rho, and Cdc42) (40, 41), and actin-associated proteins (paxillin, talin, and vinculin) (42, 43, 44). Together these pathways contribute to cytoskeletal changes leading to focal adhesion formation, cell spreading, migration, and, in the case of PMNs, respiratory burst and degranulation. The relationship among these tyrosine kinases (which is signaling to which) and whether a specific kinase induces a specific downstream pathway are unknown. However, it is clear that the Src family kinases are required for integrin signaling in both fibroblasts (45) and hemopoietic cells (this work and (20). In macrophages, loss of Hck and Fgr results in disordered formation of cytoskeletal structures and decreased phosphorylation of actin-associated proteins (46). In studies with triple hck^-/-fgr^-/-lyn^-/- macrophages, defects in activation/membrane localization of phosphoinositol 3-kinase have been observed (47). Although not yet directly demonstrated, it is likely that similar signaling pathways are involved in PMN integrin signaling. Based on this work, it appears that murine ₄ integrins use this same initial signaling cascade. Future work will be required to determine whether murine ₄ integrin ligation induces different downstream signaling responses compared with other leukocyte integrins.

The expression of ₄ integrins by human neutrophils remains controversial. It has recently been reported that the commonly used anti-human, ₄ integrin-specific Ab, HP2/1, contains a contaminating Ab that reacts with an unidentified surface marker on human PMNs (15). This contaminant may be responsible for what was thought to be an increase in ₄ expression on activated PMNs. Given this as well as the inability to detect ₄ mRNA by PCR in human PMNs, Kirveskari et al. (15) conclude that neither resting nor activated human neutrophils express ₄ integrins. In the case of rat PMNs there is clearly a role for ₄ integrins in recruitment to sites of inflammation. ₄ is expressed constitutively on resting rat PMNs, and it is shown to mediate neutrophil accumulation in the inflamed joints of rats with adjuvant induced arthritis as well as in dermal inflammation sites (48). Murine neutrophils appear to be similar to rat PMNs. All resting PMNs express ₄ integrins, and as this work demonstrates, these integrins can signal appropriately to induce cell spreading, respiratory burst, and degranulation. The ₄-induced signaling responses depend on Src family kinases analogous to other leukocyte integrins. Additionally, a role for ₄ integrins has been established in a murine liver inflammation model in vivo (16). Murine PMNs, therefore, provide an excellent model system in the study of the contribution of various intracellular signaling molecules in the progress of the inflammatory process when ₄ integrins are engaged. Studies with murine PMNs could provide information on the relative merits of various cellular targets as candidates for therapeutic intervention in the prevention of tissue damage during inflammatory reactions.

	Footnotes

 
¹ This work was supported in part by a California Cancer Research Program Fellowship (to S.P.) and by National Institutes of Health Grant HL54476 (to C.A.L.).

² Address correspondence and reprint requests to Dr. Clifford A. Lowell, Department of Laboratory Medicine, Room HSE 590, University of California San Francisco, 505 Parnassus Avenue, San Francisco, CA 94143-0134.

³ Abbreviations used in this paper: PMN, polymorphonuclear leukocyte; BM, bone marrow; ECM, extracellular matrix; mICAM-1, murine ICAM-1; LPAM-1, lymphocyte Peyer’s patch adhesion molecule-1; TKO, triple-knockout mutant (hck^-/- fgr^-/-lyn^-/-); mVCAM-1, murine VCAM-1.

Received for publication October 11, 2000. Accepted for publication January 10, 2001.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Brown, E.. 1997. Neutrophil adhesion and the therapy of inflammation. Semin. Hematol. 34:319.[Medline]
2. Hynes, R. O.. 1992. Integrins: versatility, modulation, and signaling in cell adhesion. Cell 69:11.[Medline]
3. Springer, T. A.. 1994. Traffic signals for lymphocyte recirculation and leukocyte emigration: the multistep paradigm. Cell 76:301.[Medline]
4. Ruoslahti, E.. 1996. RGD and other recognition sequences for integrins. Annu. Rev. Cell Dev. Biol. 12:697.[Medline]
5. Ruoslahti, E.. 1999. Fibronectin and its integrin receptors in cancer. Adv. Cancer Res. 76:1.[Medline]
6. Bates, R. C., L. F. Lincz, G. F. Burns. 1995. Involvement of integrins in cell survival. Cancer Metastasis Rev. 14:191.[Medline]
7. Price, T. H., P. G. Beatty, S. R. Corpuz. 1987. In vivo inhibition of neutrophil function in the rabbit using monoclonal antibody to CD18. J. Immunol. 139:4174.[Abstract]
8. Nourshargh, S., M. Rampart, P. G. Hellewell, P. J. Jose, J. M. Harlan, A. J. Edwards, T. J. Williams. 1989. Accumulation of ¹¹¹In-neutrophils in rabbit skin in allergic and non-allergic inflammatory reactions in vivo: inhibition by neutrophil pretreatment in vitro with a monoclonal antibody recognizing the CD18 antigen. J. Immunol. 142:3193.[Abstract]
9. Doerschuk, C. M., R. K. Winn, H. O. Coxson, J. M. Harlan. 1990. CD18-dependent and -independent mechanisms of neutrophil emigration in the pulmonary and systemic microcirculation of rabbits. J. Immunol. 144:2327.[Abstract]
10. Vedder, N. B., R. K. Winn, C. L. Rice, E. Y. Chi, K. E. Arfors, J. M. Harlan. 1990. Inhibition of leukocyte adherence by anti-CD18 monoclonal antibody attenuates reperfusion injury in the rabbit ear. Proc. Natl. Acad. Sci. USA 87:2643.[Abstract/Free Full Text]
11. Bochner, B. S., F. W. Luscinskas, Jr M. A. Gimbrone, W. Newman, S. A. Sterbinsky, C. P. Derse-Anthony, D. Klunk, R. P. Schleimer. 1991. Adhesion of human basophils, eosinophils, and neutrophils to interleukin 1-activated human vascular endothelial cells: contributions of endothelial cell adhesion molecules. J. Exp. Med. 173:1553.[Abstract/Free Full Text]
12. Kubes, P., X. F. Niu, C. W. Smith, Jr M. E. Kehrli, P. H. Reinhardt, R. C. Woodman. 1995. A novel ₁-dependent adhesion pathway on neutrophils: a mechanism invoked by dihydrocytochalasin B or endothelial transmigration. FASEB J. 9:1103.[Abstract]
13. Gao, J. X., A. C. Issekutz. 1997. The ₁ integrin, very late activation antigen-4 on human neutrophils can contribute to neutrophil migration through connective tissue fibroblast barriers. Immunology 90:448.[Medline]
14. Reinhardt, P. H., J. F. Elliott, P. Kubes. 1997. Neutrophils can adhere via ₄₁-integrin under flow conditions. Blood 89:3837.[Abstract/Free Full Text]
15. Kirveskari, J., P. Bono, K. Granfors, M. Leirisalo-Repo, S. Jalkanen, M. Salmi. 2000. Expression of ₄-integrins on human neutrophils. J. Leukocyte Biol. 68:243.[Abstract/Free Full Text]
16. Essani, N. A., M. L. Bajt, A. Farhood, S. L. Vonderfecht, H. Jaeschke. 1997. Transcriptional activation of vascular cell adhesion molecule-1 gene in vivo and its role in the pathophysiology of neutrophil-induced liver injury in murine endotoxin shock. J. Immunol. 158:5941.[Abstract]
17. Issekutz, A. C., L. Ayer, M. Miyasaka, T. B. Issekutz. 1996. Treatment of established adjuvant arthritis in rats with monoclonal antibody to CD18 and very late activation antigen-4 integrins suppresses neutrophil and T-lymphocyte migration to the joints and improves clinical disease. Immunology 88:569.[Medline]
18. Schneider, T., T. B. Issekutz, A. C. Issekutz. 1999. The role of ₄ (CD49d) and ₂ (CD18) integrins in eosinophil and neutrophil migration to allergic lung inflammation in the Brown Norway rat. Am. J. Respir. Cell Mol. Biol. 20:448.[Abstract/Free Full Text]
19. Wu, X., A. K. Tiwari, T. B. Issekutz, J. B. Lefkowith. 1996. Differing roles of CD18 and VLA-4 in leukocyte migration/activation during anti-GBM nephritis. Kidney Int. 50:462.[Medline]
20. Lowell, C. A., L. Fumagalli, G. Berton. 1996. Deficiency of Src family kinases p59/61^hck and p58^c-fgr results in defective adhesion-dependent neutrophil functions. J. Cell Biol. 133:895.[Abstract/Free Full Text]
21. Lowell, C. A., G. Berton. 1999. Integrin signal transduction in myeloid leukocytes. J. Leukoyte Biol. 65:313.[Abstract]
22. Bolen, J. B., J. S. Brugge. 1997. Leukocyte protein tyrosine kinases: potential targets for drug discovery. Annu. Rev. Immunol. 15:371.[Medline]
23. Yan, S. R., L. Fumagalli, G. Berton. 1995. Activation of p58^c-fgr and p53/56^lyn in adherent human neutrophils: evidence for a role of divalent cations in regulating neutrophil adhesion and protein tyrosine kinase activities. J. Inflamm. 45:297.[Medline]
24. Lowell, C. A., G. Berton. 1998. Resistance to endotoxic shock and reduced neutrophil migration in mice deficient for the Src-family kinases Hck and Fgr. Proc. Natl. Acad. Sci. USA 95:7580.[Abstract/Free Full Text]
25. Mócsai, A., E. Ligeti, C. A. Lowell, G. Berton. 1999. Adhesion-dependent degranulation of neutrophils requires the Src family kinases Fgr and Hck. J. Immunol. 162:1120.[Abstract/Free Full Text]
26. Berton, G., C. Laudanna, C. Sorio, F. Rossi. 1992. Generation of signals activating neutrophil functions by leukocyte integrins: LFA-1 and gp150/95, but not CR3, are able to stimulate the respiratory burst of human neutrophils. J. Cell Biol. 116:1007.[Abstract/Free Full Text]
27. Laudanna, C., J. J. Campbell, E. C. Butcher. 1996. Role of Rho in chemoattractant-activated leukocyte adhesion through integrins. Science 271:981.[Abstract]
28. Erle, D. J., M. J. Briskin, E. C. Butcher, A. Garcia-Pardo, A. I. Lazarovits, M. Tidswell. 1994. Expression and function of the MAdCAM-1 receptor, integrin ₄₇, on human leukocytes. J. Immunol. 153:517.[Abstract]
29. Taooka, Y., J. Chen, T. Yednock, D. Sheppard. 1999. The integrin ₉₁ mediates adhesion to activated endothelial cells and transendothelial neutrophil migration through interaction with vascular cell adhesion molecule-1. J. Cell Biol. 145:413.[Abstract/Free Full Text]
30. Richter, J., T. Andersson, I. Olsson. 1989. Effect of tumor necrosis factor and granulocyte/macrophage colony-stimulating factor on neutrophil degranulation. J. Immunol. 142:3199.[Abstract]
31. Shang, T., T. Yednock, A. C. Issekutz. 1999. ₉₁ integrin is expressed on human neutrophils and contributes to neutrophil migration through human lung and synovial fibroblast barriers. J. Leukocyte Biol. 66:809.[Abstract]
32. Berton, G., C. A. Lowell. 1999. Integrin signalling in neutrophils and macrophages. Cell. Signalling 11:621.[Medline]
33. Yang, Y., J. E. Harrison, C. G. Print, K. Lehnert, M. Sammar, A. Lazarovits, G. W. Krissansen. 1996. Interaction of monocytoid cells with the mucosal addressin MAdCAM-1 via the integrins VLA-4 and LPAM-1. Immunol. Cell. Biol. 74:383.[Medline]
34. Meerschaert, J., R. F. Vrtis, Y. Shikama, J. B. Sedgwick, W. W. Busse, D. F. Mosher. 1999. Engagement of ₄₇ integrins by monoclonal antibodies or ligands enhances survival of human eosinophils in vitro. J. Immunol. 163:6217.[Abstract/Free Full Text]
35. Wimazal, F., M. Ghannadan, M. R. Muller, A. End, M. Willheim, P. Meidlinger, G. H. Schernthaner, J. H. Jordan, W. Hagen, H. Agis, et al 1999. Expression of homing receptors and related molecules on human mast cells and basophils: a comparative analysis using multi-color flow cytometry and toluidine blue/immunofluorescence staining techniques. Tissue Antigens 54:499.[Medline]
36. Harte, M. T., J. D. Hildebrand, M. R. Burnham, A. H. Bouton, J. T. Parsons. 1996. p130^Cas, a substrate associated with v-Src and v-Crk, localizes to focal adhesions and binds to focal adhesion kinase. J. Biol. Chem. 271:13649.[Abstract/Free Full Text]
37. Astier, A., H. Avraham, S. N. Manie, J. Groopman, T. Canty, S. Avraham, A. S. Freedman. 1997. The related adhesion focal tyrosine kinase is tyrosine-phosphorylated after ₁-integrin stimulation in B cells and binds to p130^cas. J. Biol. Chem. 272:228.[Abstract/Free Full Text]
38. Schaller, M. D., T. Sasaki. 1997. Differential signaling by the focal adhesion kinase and cell adhesion kinase . J. Biol. Chem. 272:25319.[Abstract/Free Full Text]
39. Soede, R. D., Y. M. Wijnands, I. Van Kouteren-Cobzaru, E. Roos. 1998. ZAP-70 tyrosine kinase is required for LFA-1-dependent T cell migration. J. Cell Biol. 142:1371.[Abstract/Free Full Text]
40. Dharmawardhane, S., G. M. Bokoch. 1997. Rho GTPases and leukocyte cytoskeletal regulation. Curr. Opin. Hematol. 4:12.[Medline]
41. Hall, A.. 1998. Rho GTPases and the actin cytoskeleton. Science 279:509.[Abstract/Free Full Text]
42. Hildebrand, J. D., M. D. Schaller, J. T. Parsons. 1995. Paxillin, a tyrosine phosphorylated focal adhesion-associated protein binds to the carboxyl terminal domain of focal adhesion kinase. Mol. Biol. Cell. 6:637.[Abstract]
43. Schaller, M. D., J. T. Parsons. 1995. pp125^FAK-dependent tyrosine phosphorylation of paxillin creates a high-affinity binding site for Crk. Mol. Cell. Biol. 15:2635.[Abstract]
44. Lo, S. H., E. Weisberg, L. B. Chen. 1994. Tensin: a potential link between the cytoskeleton and signal transduction. BioEssays 16:817.[Medline]
45. Klinghoffer, R. A., C. Sachsenmaier, J. A. Cooper, P. Soriano. 1999. Src family kinases are required for integrin but not PDGFR signal transduction. EMBO J. 18:2459.[Medline]
46. Suen, P. W., D. Ilic, E. Caveggion, G. Berton, C. H. Damsky, C. A. Lowell. 1999. Impaired integrin-mediated signal transduction, altered cytoskeletal structure and reduced motility in Hck/Fgr deficient macrophages. J. Cell Sci. 112:4067.[Abstract]
47. Meng, F., C. A. Lowell. 1998. A ₁ integrin signaling pathway involving Src-family kinases, Cbl and PI-3 kinase is required for macrophage spreading and migration. EMBO J. 17:4391.[Medline]
48. Issekutz, T. B., M. Miyasaka, A. C. Issekutz. 1996. Rat blood neutrophils express very late antigen 4 and it mediates migration to arthritic joint and dermal inflammation. J. Exp. Med. 183:2175.[Abstract/Free Full Text]

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