Role of phosphatidylinositol 3-kinase-{gamma} in mediating lung neutrophil sequestration and vascular injury induced by E. coli sepsis

Evan Ong,* Xiao-Pei Gao,* Dan Predescu, Michael Broman, and Asrar B. Malik

Department of Pharmacology and The Center for Lung and Vascular Biology, University of Illinois College of Medicine, Chicago, Illinois

Submitted 20 April 2005 ; accepted in final form 6 July 2005


    ABSTRACT
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 ABSTRACT
 METHODS
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 DISCUSSION
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We addressed the in vivo role of phosphatidylinositol 3-kinase-{gamma} (PI3K-{gamma}) in signaling the sequestration of polymorphonuclear leukocytes (PMNs) in lungs and in the mechanism of inflammatory lung vascular injury. We studied mice with deletion of the p110 catalytic subunit of PI3K-{gamma} (PI3K-{gamma}–/– mice). We measured lung tissue PMN sequestration, microvascular permeability, and edema formation after bacteremia induced by intraperitoneal Escherichia coli challenge. PMN infiltration into the lung interstitium in PI3K-{gamma}–/– mice as assessed morphometrically was increased 100% over that in control mice within 1 h after bacterial challenge. PI3K-{gamma}–/– mice also developed a greater increase in lung microvascular permeability after E. coli challenge, resulting in edema formation. The augmented lung tissue PMN sequestration in PI3K-{gamma}–/– mice was associated with increased expression of the PMN adhesive proteins CD47 and {beta}3-integrins. We observed increased association of CD47 and {beta}3-integrins with the extracellular matrix protein vitronectin in lungs of PI3K-{gamma}–/– mice after E. coli challenge. PMNs from these mice also showed increased {beta}3-integrin expression and augmented {beta}3-integrin-dependent PMN adhesion to vitronectin. These results point to a key role of PMN PI3K-{gamma} in negatively regulating CD47 and {beta}3-integrin expression in gram-negative sepsis. PI3K-{gamma} activation in PMNs induced by E. coli may modulate the extent of lung tissue PMN sequestration secondary to CD47 and {beta}3-integrin expression. Therefore, the level of PI3K-{gamma} activation may be an important determinant of PMN-dependent lung vascular injury.

{beta}3-integrins; CD47; polymorphonuclear leukocyte sequestration; sepsis-induced lung vascular injury


THE MIGRATION OF POLYMORPHONUCLEAR leukocytes (PMNs) to sites of inflammation induced by gram-negative sepsis is an essential requirement of the innate immune response. However, under specific conditions, marked accumulation of PMNs in lung tissue, as in the acute respiratory distress syndrome, may lead to loss of alveolar-capillary barriers and impaired gas exchange function (50). PMN sequestration during sepsis has been shown to promote release of reactive oxygen species, proteolytic enzymes, and other inflammatory mediators that result in lung vascular injury (18, 26, 46). However, the signaling mechanisms responsible for lung PMN sequestration and vascular injury remain incompletely understood (6, 8, 20, 23, 40, 47, 53). Studies have identified phosphatidylinositol 3-kinase (PI3K)-{gamma} as an important lipid kinase regulating PMN migration in response to chemotactic signals (7, 19, 22, 34, 36, 38, 41, 48, 52). Chemotactic defects were seen in PMNs treated with the PI3K inhibitor LY-294002 and PMNs from mice lacking the p110 catalytic domain of PI3K-{gamma} (19, 22, 36, 38). This PI3K heterodimer is unique, because it is found downstream of heterotrimeric G protein-coupled chemokine receptors (8, 19, 34, 41). Activation of chemokine receptors leads to release of the G protein {beta}{gamma}-subunit (G{beta}{gamma}), which induces the membrane localization of PI3K-{gamma} through binding via the p101 adaptor subunit (3, 42, 43). PI3K-{gamma} at the plasma membrane converts the phospholipid phosphatidylinositol 4',5'-bisphosphate to phosphatidylinositol 3',4',5'-trisphosphate, which in turn is involved in establishing the PMN polarity required for chemotaxis. Phosphatidylinositol 3',4',5'-trisphosphate induces the activation of protein kinase B (AKT), leading to the formation of a PMN leading edge (34).

PI3K-{gamma} association with the adhesion protein CD47 may be important in regulating PMN migration (31, 32). CD47, also termed integrin-associated protein, is a 50-kDa membrane protein with an NH2-terminal immunoglobulin domain and a COOH-terminal membrane-spanning region. CD47 may function by promoting PMN adhesion to the Arg-Gly-Asp (RGD)-containing proteins thrombospondin, fibronectin, and vitronectin (9). CD47 was shown to facilitate the binding of these proteins to {beta}1- and {beta}3-integrins (4, 13, 37). Studies have also demonstrated that antibodies to CD47 blocked PMN transmigration across epithelial and endothelial monolayers and interfered with {alpha}v{beta}3-mediated cell adhesion (28).

The in vivo significance of PI3K-{gamma} in lung PMN accumulation and in development of inflammatory lung vascular injury is unknown. In the present study, we used mice lacking the p110 catalytic domain of the leukocyte-specific PI3K-{gamma} (PI3K-{gamma}–/– mice) to address the function of PI3K-{gamma}. Our data demonstrate an increase in PMN CD47 surface expression and promotion of {beta}3-integrin-dependent adhesion of PMNs to vitronectin in PI3K-{gamma}–/– mice challenged with intraperitoneally injected Escherichia coli. In lungs of E. coli-challenged PI3K-{gamma}–/– mice, we observed a marked accumulation of PMNs compared with wild-type (WT) mice and a markedly greater increase in lung vascular permeability and tissue edema formation. Together, these results identify the critical role of PI3K-{gamma} in negatively regulating CD47 and {beta}3-integrin expression in PMNs and, thereby, modulating lung tissue PMN sequestration and resultant lung vascular injury induced by E. coli-induced sepsis.


    METHODS
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 ABSTRACT
 METHODS
 RESULTS
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Mice. PI3K-{gamma}–/– mice, which do not express the catalytic p110 {gamma}-subunit (36), were obtained from Dr. Joseph Penninger (Amgen Institute, Toronto, ON, Canada). WT C57BL/6 mice (Jackson Laboratory, Bar Harbor, ME) were used as controls. The mice (22–26 g body wt, 9–12 wk old) were housed in pathogen-free conditions with free access to food and water in the University of Illinois Animal Care Facility. Studies were conducted in accordance with institutional and National Institutes of Health guidelines. Approval for animal use was obtained from University of Illinois Institutional Review Board.

Materials. Rat monoclonal antibody to CD47 (catalog no. MIAP301), rabbit polyclonal antibody to vitronectin (catalog no. H-270), and goat polyclonal antibody to {beta}3-integrin (catalog no. N-20) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Fluorescein isothiocyanate (FITC)-conjugated goat anti-rat antibody (Santa Cruz Biotechnology) and Alexa Red 568 donkey anti-goat antibody (catalog no. A-11057, Molecular Probes, Eugene, OR) were used as secondary antibodies for flow cytometric analysis of adhesion protein surface expression in PMNs. A goat IgG antibody (Santa Cruz Biotechnology) was used as a negative control in the PMN adhesion assay of vitronectin-coated wells. All other reagents were purchased from Sigma (St. Louis, MO).

E. coli challenge. Mice were challenged intraperitoneally with a defined number of colony-forming units of E. coli per milliliter (1 x 108 live E. coli/100 µl; catalog no. 25922, American Type Culture Collection) (53). This E. coli dose was used to create a standardized model of sepsis-induced lung PMN uptake and acute lung vascular injury. Also this dose did not result in death within the 6-h experimental period after E. coli challenge. Control mice were injected intraperitoneally with an equal volume of PBS. Lungs obtained at different times after challenge were used in the assays described below.

Lung tissue PMN sequestration and myeloperoxidase activity. Lung tissue PMN uptake was assessed by determination of tissue myeloperoxidase (MPO) activity in lungs and morphometric quantification of PMN infiltration in lung tissue as described elsewhere (14, 15).

Electron microscopy. For electron microscopy, lungs were fixed by in situ perfusion with a mixture of freshly prepared 4% formaldehyde, 2.5% glutaraldehyde, 1 mM Ca2+, and 2.5% polyvinylpyrrolidone in 0.1 M PIPES (pH 7.2) and intratracheal instillation of the same mixture without polyvinylpyrrolidone. Excised specimens were further fixed by immersion in a triple fixative (39) for an additional 1 h at room temperature and then postfixed with reduced osmium (1% osmium tetroxide and 1.5% potassium ferrocyanide), stained in the dark (1 h at room temperature) with 7.5% magnesium uranyl acetate, dehydrated in increasing concentrations of ethanol and propylene oxide, and embedded in Epon. Tissue blocks were cured (72 h at 90°C), and ~60-nm-thick sections were obtained with a Leica microtome, counterstained with lead citrate and uranyl acetate, examined, and micrographed in a Jeol 1220 transmission electron microscope. The negatives were scanned, and pictures were obtained using Adobe Photoshop 7.0 run in an Apple G4 computer.

Western blotting and immunoprecipitation. We determined CD47 and {beta}3-integrin expression in lung tissue by Western blotting. Lungs were homogenized in lysis buffer consisting of 1.5% Triton X-100, 0.1% SDS, 0.5% deoxycholic acid, 6 µl/ml protease inhibitor cocktail (Sigma), and 100 mM phenylmethylsulfonyl fluoride at 1:10 (wt/vol, g/ml), and the supernatant was collected. Protein concentration was measured in an aliquot of the tissue homogenate. Homogenates containing equal amounts of protein were subjected to electrophoresis on 10% SDS polyacrylamide gels, transferred to Immobilon-P (Millipore), blocked with 5% nonfat milk, and analyzed by Western blotting using the antibodies described above. For immunoprecipitation, lung lysates were precleared with 50 µl of protein A/G-agarose beads and incubated with 30 µg/ml primary antibody for 2 h at 4°C. The samples were collected by incubation with 50 µl of protein A/G-agarose beads overnight at 4°C with constant shaking. After five separate washes with PBS buffer, the immunoprecipitated samples were resolved on a 4–12% NuPage Bis-Tris gel (Invitrogen, Carlsbad, CA). The bands were then transferred to nitrocellulose membranes at 30 V for 2 h. The membranes were blocked with 5% milk in Tris-buffered saline (TBS) for 1 h and incubated with primary antibodies for 1 h at room temperature. The membranes were washed with TBS + 0.3% Tween 20 before incubation with horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature. After they were washed again with TBS + Tween 20 and TBS, the membranes were exposed to film.

Flow cytometry. PMN CD47 and {beta}3-integrin surface expression was measured in whole blood by flow cytometry as described elsewhere (27, 35, 45). Blood from PI3K-{gamma}–/– and WT mice was obtained via puncture of the right ventricle with a sterile 22-gauge needle on a 1.0-ml syringe at different times after E. coli challenge. Approximately 100 µl of heparinized blood were placed into separate polypropylene tubes. Surface adhesion protein was labeled by incubation with anti-CD47 rat monoclonal antibody (5 µg/µl) or anti-{beta}3-integrin goat polyclonal antibody (5 µg/µl) for 1 h at 4°C and then staining by FITC-conjugated rabbit anti-rat secondary antibody (1 µg/µl) or Alexa Red 568-conjugated anti-goat secondary antibody (1 µg/µl) for 30 min at 4°C. Then 2.0 ml of fluorescein-activated cell-sorting (FACS) lysing solution (Becton Dickinson, San Jose, CA) were added to each sample to hemolyze red blood cells. After they were centrifuged and washed, the leukocytes were resuspended in 500 µl of 1% paraformaldehyde in PBS (Sigma-Aldrich) for fixation. The samples were then immediately analyzed by measurement of fluorescence from the gated leukocyte population using an EPICS Elite ESP (Coulter, Miami, FL). The forward and side light-scatter profiles were adjusted to gate for the PMN population. Fluorescence parameters were collected using four-decade logarithmic amplification. As a negative control, the cells were stained with secondary antibody alone.

PMN adhesion to immobilized vitronectin. PMN adhesion to fibronectin was assessed as described elsewhere (2, 20). After isolation of PMNs by the method described above (44), the cells (5 x 105) were applied to 96-well plates precoated with vitronectin (10 µg/ml) overnight at 4°C and blocked with 1% BSA at 37°C for 1 h. PMNs loaded with calcein-AM (Molecular Probes; 2 µg/ml) for 30 min at 37°C were added to the well after treatment of PMNs with 1 µM formyl-methionine-leucine-phenylalanine (fMLP) in the presence or absence of goat polyclonal anti-{beta}3-integrin antibody (10 µg/ml, 15 min before fMLP). A goat IgG antibody was used as a negative control in the assay of PMN adhesion to vitronectin-coated wells. Adhesion was allowed to proceed for 30 min at 37°C, and the wells were washed twice with PBS to remove nonadherent cells. The fluorescence readings were obtained using a spectrofluorometer (Photon Technology International, Monmouth Junction, NJ) with detection at 485 and 535 nm, respectively. The percentage of adherent PMNs was calculated, and all assays were performed in duplicate.

PMN depletion-repletion experiment. An experiment involving depletion of circulating PMNs followed by repletion of a specific population of PMNs was carried out as described elsewhere (10) to evaluate the role of PMN PI3K-{gamma} in mediating lung sequestration in vivo. Neutropenia was induced in WT mice by intraperitoneal administration of 150 µl of rabbit anti-mouse neutrophil serum (Intercell Technologies, Hopewell, NJ). At 16 h after injection, when circulating PMNs could not be detected, PMNs (~1 x 106 cells) isolated from WT or PI3K-{gamma}–/– mice were used to replete the neutropenic WT mice. PMNs were isolated as described elsewhere (44) using PMN isolation medium (NIM.2, Cardinal Associates, Santa Fe, NM). The isolated PMNs from WT or PI3K-{gamma}–/– mice were injected intravenously into the right jugular vein of neutropenic WT mice and permitted to circulate for 1 h. Lungs of these PMN-repleted mice were obtained at time 0 and 1 h after intraperitoneal E. coli challenge, and MPO activity (a measure of sequestration of repleted PMNs in lungs of the neutropenic WT mice) was determined as described above.

Pulmonary microvascular permeability and isogravimetric lung water determinations. The capillary filtration coefficient (Kf,c) and lung wet weight increases were measured to determine pulmonary microvascular permeability to liquid and edema formation in lungs as described elsewhere (14, 15).

Statistical analysis. Values are means ± SE. Statistical analysis was performed using a two-tailed Student's t-test. P < 0.05 was used as the criterion for significance.


    RESULTS
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Augmented lung tissue PMN sequestration after E. coli challenge of PI3K-{gamma}–/– mice. WT and PI3K-{gamma}–/– mice were challenged with intraperitoneal injection of E. coli, and lung tissue MPO activity was measured to assess lung tissue PMN sequestration. MPO activity increased ~80% in PI3K-{gamma}–/– mice compared with WT mice 1 h after E. coli injection (P < 0.05; Fig. 1A). Thereafter, lung tissue PMN uptake was similar in the two groups of mice (data not shown). We also carried out morphometric analysis to determine lung PMN sequestration in PI3K-{gamma}–/– mice. This value was also increased in lungs from the PI3K-{gamma}–/– mice compared with the WT mice 1 h after E. coli challenge (P < 0.05; Fig. 1B). Representative electron micrographs of lungs from the E. coli-challenged WT and PI3K-{gamma}–/– mice are shown in Fig. 2, A and C. We observed greater accumulation of PMNs in the lung extravascular space of PI3K-{gamma}–/– mice than WT mice. Analysis of approximately equivalent surface areas of lung tissue in WT and PI3K-{gamma}–/– mice (Fig. 3A) showed significant increases in lung vascular and interstitial sequestration of PMNs after E. coli challenge (P < 0.05; Fig. 3, B and C) compared with unchallenged WT and PI3K-{gamma}–/– mice. However, the lung interstitial uptake of PMNs was ~100% greater in PI3K-{gamma}–/– than in WT mice (Fig. 3C). The intravascular uptake of PMNs and interstitial uptake of macrophages did not differ significantly between WT and PI3K-{gamma}–/– mice (Fig. 3, B and D).



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Fig. 1. Deletion of phosphatidylinositol-3-kinase-{gamma} (PI3K-{gamma}–/– mice) results in augmentation of polymorphonuclear leukocyte (PMN) sequestration in mouse lungs induced by Escherichia coli challenge. A: tissue myeloperoxidase (MPO) activity in lungs 1 h after E. coli challenge (1 x 108 live E. coli/100 µl ip) in PI3K-{gamma}–/– and wild-type (WT) mice. B: lung tissue PMN sequestration measured morphometrically 1 h after E. coli challenge in PI3K-{gamma}–/– and WT mice. Values are means ± SE of 6 independent experiments. *P < 0.05 vs. basal. *{dagger}P < 0.05 vs. WT.

 


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Fig. 2. PMN uptake in lung interstitium of WT and PI3K-{gamma}–/– mice before and 1 h after E. coli challenge assessed by electron microscopy. A: full capillary profile from a WT mouse. B: capillary from WT mouse treated with E. coli for 1 h; note PMN attached to endothelial surface. C: capillary from an untreated PI3K-{gamma}–/– mouse; note PMN in the lumen. D: small area from lung alveolar-capillary unit; note increased extravasation of PMNs into lung interstitium (arrows).

 


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Fig. 3. A: lung surface area was similar in E. coli-challenged and unchallenged WT and PI3K-{gamma}–/– mice. B: number of intravascular PMNs in lungs of challenged WT and PI3K-{gamma}–/– mice was increased 6- to 7-fold compared with unstimulated mice; number of intravascular PMNs in challenged WT and PI3K-{gamma}–/– mice was similar. Mean value in unchallenged mice (basal) is sum of measurements in unchallenged WT and PI3K-{gamma}–/– mice. C: number of lung interstitial PMNs after E. coli challenge of WT and PI3K-{gamma}–/– mice was increased 10- to 20-fold compared with unchallenged mice; number of PMNs in lung interstitium was 100% greater in E. coli-challenged PI3K-{gamma}–/– than WT mice. D: number of lung interstitial macrophages was 7- to 8-fold greater in E. coli-challenged WT and PI3K-{gamma}–/– than in unchallenged mice, but there was no difference in number of macrophages between challenged WT and PI3K-{gamma}–/– mice. Values are means ± SE of 5 independent experiments. *P < 0.05 vs. basal. *{dagger}P < 0.05 vs. WT + E. coli.

 
Augmented lung microvascular injury and edema formation in PI3K-{gamma}–/– mice. To address the in vivo effects of increased lung tissue PMN sequestration in the E. coli-challenged PI3K-{gamma}–/– mice, we determined Kf,c, the measure of microvascular permeability to liquid. Although there was no difference in lung microvascular permeability under basal conditions between WT and PI3K-{gamma}–/– mice, the increase in lung vascular permeability was greater in PI3K-{gamma}–/– mice challenged with intraperitoneal E. coli for 1 h than in WT mice (P < 0.05; Fig. 4A). This was also evident in lung edema formation, in that the PI3K-{gamma}–/– mice challenged with intraperitoneal E. coli for 1 h showed rapid and markedly greater increases in tissue water accumulation than E. coli-challenged WT mice (P < 0.05; Fig. 4B). Thus the PI3K-{gamma}–/– mice develop severe lung vascular injury along with increased lung tissue PMN infiltration after E. coli challenge.



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Fig. 4. Increases in lung vascular permeability and edema formation in PI3K-{gamma}–/– mice. A: changes in microvessel liquid permeability (measured as capillary filtration coefficient, Kf,c) in lungs of WT and PI3K-{gamma}–/– mice. *P < 0.05 vs. basal. *{dagger}P < 0.05 vs. WT after E. coli. B: changes in isogravimetric lung water content 0 and 1 h after E. coli challenge in WT and PI3K-{gamma}–/– mice. Lung wet weight was continuously monitored from 0 to 90 after challenge. Values are means ± SE of 6 independent experiments. *P < 0.05 vs. basal (0 min). *{dagger}P < 0.05 vs. WT after E. coli.

 
Greater lung tissue PMN accumulation is the result of PI3K-{gamma} deletion in PMNs. To determine whether the increased lung tissue PMN sequestration in PI3K-{gamma}–/– mice is the result of deletion of PI3K-{gamma} in PMNs, we carried out an in vivo assay in which PMNs isolated from WT or PI3K-{gamma}–/– mice were transfused into WT mice in which neutropenia had been induced by an injection of antineutrophil antibody (10). At 1 h after challenge with intraperitoneal E. coli, lung MPO activity was measured to assess the uptake of PMNs in lungs of the PMN-repleted mice. We observed a fourfold increase in lung PMN uptake in WT mice 1 h after intraperitoneal E. coli challenge compared with basal PMN uptake (Fig. 5). In WT mice in which neutropenia had been induced, the basal lung tissue MPO activity was minimal. We observed increased MPO activity on repletion of PMN isolated from WT mice. Challenging these mice with intraperitoneal E. coli resulted in increased lung PMN uptake similar to the response in WT mice challenged with E. coli. Interestingly, PMN uptake after E. coli challenge of neutropenic WT mice repleted with PMNs isolated from PI3K-{gamma}–/– mice was significantly greater than the response after repletion of WT PMNs. This greater increase in lung PMN uptake was similar to that measured after E. coli challenge of PI3K-{gamma}–/– mice. These results demonstrate that the augmented lung tissue PMN uptake after E. coli challenge was intrinsic to the PI3K-{gamma}–/– PMNs.



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Fig. 5. Increased lung tissue PMN uptake in PI3K-{gamma}–/– mice after E. coli challenge is inherent to PI3K-{gamma}–/– PMNs. Lanes 1 and 2, control response of WT mice before and 1 h after E. coli challenge, respectively. Lane 3, lung PMN uptake in neutropenic WT mice. Lanes 4 and 5, lung PMN sequestration in neutropenic WT mice repleted with PMN isolated from WT mice; note 2-fold increase in MPO activity after 1 h of E. coli challenge, which is similar to control responses (lanes 1 and 2). Lane 7, 4-fold increase in lung PMN uptake in neutropenic WT mice repleted with PMNs isolated from PI3K-{gamma}–/– mice after E. coli challenge (compare with 2-fold increase in WT PMN-repleted mice in lanes 4 and 5); lung tissue PMN uptake in PI3K-{gamma}–/– and WT mice in the absence of E. coli challenge is the same in both groups (lanes 4 and 6). In lane 7, 4-fold increase was similar to lung MPO activity in PI3K-{gamma}–/– mice (lane 8). *P < 0.05 vs. no challenge (lane 1). *{dagger}P < 0.05 vs. WT after E. coli. Values are means ± SE of 4 independent experiments.

 
Augmented expression of CD47 in PI3K-{gamma}–/– PMNs and lungs. Because PI3K-{gamma} regulation of CD47 expression in PMNs may be involved in mediating PMN accumulation (31, 32), we addressed the possibility that the increased lung tissue PMN accumulation in the E. coli-challenged PI3K-{gamma}–/– mice was coupled to CD47 expression. Flow cytometric analysis of PMNs showed increased CD47 surface expression in the PI3K-{gamma}–/– PMNs from mice challenged for 1 h with E. coli compared with WT PMNs (P < 0.05; Fig. 6A). Western blot analysis of lung homogenates also showed much greater CD47 expression in lungs of PI3K-{gamma}–/– mice beginning at 30 min after E. coli challenge than in lungs of control mice (Fig. 6B).



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Fig. 6. CD47 expression in WT and PI3K-{gamma}–/– PMNs and lungs after E. coli challenge. A: flow cytometric analysis of CD47 expression on cell surface of PMNs. Isolated PMNs were labeled with anti-CD47 primary antibody, and an FITC-conjugated secondary antibody was used to determine CD47 expression. Relative mean fluorescence represents average fluorescence from CD47-labeled population of isolated PMNs 1 h after E. coli challenge. There was a markedly greater increase in fluorescence in PMNs isolated from E. coli-challenged PI3K-{gamma}–/– than E. coli-challenged WT mice. MFI, mean fluorescence intensity. Values are means ± SE of 4 independent experiments. *{dagger}P < 0.05 vs. WT after E. coli challenge. B: Western blot analysis of CD47 expression in lung homogenates from E. coli-challenged WT and PI3K-{gamma}–/– mice. Equal amounts of total protein (100 µg) were loaded per lane. Results are representative of 3 independent experiments; 1 of 3 comparable experiments is shown.

 
CD47 interacts with vitronectin and {beta}3-integrins in PI3K-{gamma}–/– mice. We assessed the possibility that the expressed CD47 interacts with the extracellular matrix protein vitronectin and {beta}3-integrins. Immunoprecipitation experiments were carried out to determine interactions with CD47 in lungs. Studies demonstrated increased interaction of CD47 with {beta}3-integrin (Fig. 7A) and {beta}3-integrin with vitronectin (Fig. 7B) in lung tissue of E. coli-challenged PI3K-{gamma}–/– mice compared with WT mice. Increased CD47 association with vitronectin in PI3K-{gamma}–/– lungs was blocked by anti-{beta}3-integrin antibody (Fig. 7C).



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Fig. 7. Association of CD47 with {beta}3-integrins and vitronectin in E. coli-challenged lungs of WT and PI3K-{gamma}–/– mice. A: immunoprecipitation (IP) studies show increased association of CD47 with {beta}3-integrins in lungs of PI3K-{gamma}–/– mice compared with WT mice 1 h after E. coli challenge. IB, immunoblot. B: increased association of {beta}3-integrins with vitronectin 1 h after E. coli challenge in PI3K-{gamma}–/– mice compared with WT mice. C: increased association of CD47 with vitronectin in PI3K-{gamma}–/– mice compared with WT mice 1 h after E. coli challenge without or with pretreatment with antibody against {beta}3-integrins. For immunoprecipitation, equal amounts of protein (2 mg of lung lysate) were added to immunoprecipitation reaction. Results are representative of 3 independent experiments; 1 of 3 comparable experiments is shown.

 
Role of {beta}3-integrin in the mechanism of lung PMN sequestration in PI3K-{gamma}–/– mice. We used FACS analysis to determine {beta}3-integrin surface expression in PMNs obtained 1 h after intraperitoneal E. coli challenge. Results from representative independent experiments showed greater {beta}3-integrin surface expression in PI3K-{gamma}–/– than in WT PMNs (P < 0.05; Fig. 8A). Inasmuch as {beta}3-integrin expression was augmented in PI3K-{gamma}–/– PMNs (Fig. 8A) and {beta}3-integrin and vitronectin interactions with CD47 were increased (Fig. 7), we assessed the possibility that PI3K-{gamma}–/– PMNs would demonstrate greater {beta}3-integrin-dependent adhesion to vitronectin. We observed that unchallenged PMNs isolated from PI3K-{gamma}–/– and WT mice did not differ in their basal adhesion to vitronectin; however, the adhesion of fMLP-stimulated PI3K-{gamma}–/– PMNs to vitronectin-coated wells was twofold greater than adhesion of WT PMNs similarly exposed to fMLP (Fig. 8B). The increased PMN adhesion to vitronectin was blocked by pretreatment of PI3K-{gamma}–/– PMNs with anti-{beta}3-integrin antibody (catalog no. N-20, Santa Cruz Biotechnology), and not by a control antibody (Fig. 8B). Anti-{beta}3-integrin antibody also prevented the E. coli-induced increase in MPO activity and lung tissue PMN accumulation in PI3K-{gamma}–/– mice (Fig. 8, C and D).



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Fig. 8. PMN {beta}3-integrin surface expression induced by E. coli challenge of mice and its effects on PMN adhesion to immobilized vitronectin induced by formyl-methionine-leucine-phenylalanine (fMLP) and lung tissue PMN uptake induced by E. coli challenge of mice. A: flow cytometric analysis of {beta}3-integrin surface expression on PMNs. Note greater rightward shift in fluorescence of PMNs from PI3K-{gamma}–/– mice 1 h after E. coli challenge. Data are representative of 3 separate experiments. B: adhesion to vitronectin in PI3K-{gamma}–/– and WT PMNs. PMNs were stimulated for 30 min with 1 µM fMLP without or with antibody against {beta}3-integrin (10 µg/ml, 15 min before 1 µM fMLP). Values are means ± SE of 3 independent experiments. *{dagger}P < 0.05 vs. WT after E. coli. P < 0.05 vs. fMLP and fMLP + IgG. C and D: tissue MPO activity in lungs and lung PMN sequestration as measured by morphometric analysis 1 h after E. coli challenge with or without antibody against {beta}3-integrin (2 mg/kg iv 30 min before E. coli challenge). Values are means ± SE of 5 independent experiments. *{dagger}P < 0.05 vs. WT after E. coli. P < 0.05 vs. P13K{gamma}–/– after E. coli.

 

    DISCUSSION
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Migration of PMNs into lung tissue, PMN sequestration, and the subsequent generation of oxidants and proteases are key elements in the pathogenesis of sepsis-induced acute lung injury (24, 25). However, the signaling mechanisms responsible for lung tissue PMN accumulation are not fully understood. Previous studies have demonstrated a central role for PI3K-{gamma} in mediating PMN chemotaxis (5, 19, 22, 36, 38, 47); thus, in the present study, we addressed the contribution of PI3K-{gamma} to the mechanism of PMN infiltration in lung tissue PMNs and lung vascular injury induced by gram-negative sepsis in vivo. We observed a doubling of lung tissue PMN uptake as quantified morphometrically within 1 h after E. coli challenge in PI3K-{gamma}–/– mice compared with WT mice. We also observed a greater increase in lung microvascular permeability and lung edema formation in PI3K-{gamma}–/– than in WT mice, consistent with the greater PMN uptake. Our findings implicate an in vivo role of PI3K-{gamma} as an important mechanism of lung tissue PMN sequestration. Previous studies showed that pretreatment with the PI3K inhibitor wortmannin increased susceptibility to polymicrobial sepsis in the cecal ligation-and-puncture model in mice and increased serum levels of proinflammatory cytokines during sepsis (51). It is possible therefore that the greater accumulation of PMNs in lung tissue of PI3K-{gamma}–/– mice may be secondary to release of cytokines. Another possible explanation for the greater lung PMN uptake may be the neutrophilia observed in PI3K-{gamma}–/– mice (22). Thus we carried out a PMN depletion-repletion study using WT mice. After repletion of neutropenic WT mice with PMNs isolated from PI3K-{gamma}–/– mice, we observed that the accumulation of PI3K-{gamma}–/– PMNs lung tissue after E. coli challenge was significantly greater than the uptake of the repleted WT PMNs after E. coli challenge. The lung tissue PMN uptake response in this repletion experiment was similar to that in the control group of E. coli-challenged PI3K-{gamma}–/– mice. These studies show that the augmented lung PMN uptake in the PI3K-{gamma}–/– mice was inherent to the PI3K-{gamma}–/– PMNs, suggesting that PI3K-{gamma} normally modulates the lung PMN sequestration response following gram-negative sepsis.

Because there was marked accumulation of PMNs in the lung interstitium of PI3K-{gamma}–/– mice, we addressed the possibility that the PI3K-{gamma}–/– PMNs may have altered their adhesive properties to the extracellular matrix proteins and that this could account for the inappropriate tissue uptake of these PMNs. Studies have reported no alterations in the ability of PI3K-{gamma}–/– PMNs to adhere to fibronectin-coated plates (22, 36) or increased expression of CD11b/CD18 in the fMLP-stimulated PMN after PI3K-{gamma} inhibition (7). In the present study, we also did not observe increases in the expression of CD18 integrin in E. coli-challenged PI3K-{gamma}–/– mice (data not shown). Other studies have demonstrated that treatment of PMNs with PI3K inhibitors, wortmannin or LY-294002, resulted in increased PMN cell surface expression of the integrin-associated protein CD47 (31, 32). Using flow cytometry, we also observed increased PMN cell surface expression of CD47 in PMNs from PI3K-{gamma}–/– mice. CD47 has been implicated in mediating the infiltration of PMNs to sites of tissue infection (4, 17, 29, 31, 32). CD47 was shown to regulate the ability of PMNs to migrate across the polarized intestinal epithelial barrier (33). PMN migration in mice lacking CD47 was defective soon after infectious challenge (29). Thus it is possible that the increased PMN CD47 expression in PI3K-{gamma}–/– mice contributes to PMN accumulation in the lung interstitium. The mechanism of CD47 regulation of PMN infiltration involves other proteins, including signal regulatory protein-{alpha} and {beta}1- and {beta}3-integrins (4, 12, 16, 32, 49); however, little is known about how these proteins interact with CD47 to regulate PMN migration and uptake in tissues.

The present data suggest that PI3K-{gamma} regulation of CD47 expression may be important in signaling PMN migration through the lung extracellular matrix. To assess the nature of interactions of the PI3K-{gamma}–/– PMNs, we carried out immunoprecipitation assays using an antibody to CD47. Although CD47 has been demonstrated to bind to thrombospondin, fibronectin, and vitronectin (4, 13, 37), we found a significant interaction only with vitronectin in lungs from E.coli-challenged PI3K-{gamma}–/– mice. Thus a basis for the augmented sequestration of PI3K-{gamma}–/– PMNs in lung tissues may involve interaction of the expressed CD47 with vitronectin. In this regard, activation PI3K-{gamma} during sepsis would normally downmodulate CD47 expression and, thereby, minimize the interaction of CD47 with vitronectin.

The present study showed the crucial involvement of {beta}3-integrins in the association of CD47 with vitronectin, consistent with results of previous in vitro assays (13, 16, 21, 30). As with CD47, we observed increased surface expression of {beta}3-integrins in the PI3K-{gamma}–/– PMNs compared with WT PMNs. The increased surface expression of CD47 and {beta}3-integrins, along with greater association of CD47 with vitronectin, may contribute to the augmented adhesive properties of the PI3K-{gamma}–/– PMNs. In fact, adhesion to vitronectin-coated wells was significantly greater for fMLP-stimulated PI3K-{gamma}–/– PMNs than for WT PMNs. Taken together, our data suggest that PMN migration through the extracellular matrix may involve a balance between PI3K-{gamma} activation and CD47-associated {beta}3-integrin expression. Thus the loss of PI3K-{gamma} function would promote the upregulation of the CD47-associated {beta}3-integrin complex and lead to increased adhesion of PMNs within the extracellular matrix and accumulation of PMNs in the lung interstitium. The greater tissue accumulation of PMNs is thereby capable of inducing tissue injury, as evident by the significantly greater increase in lung microvascular permeability and tissue edema formation in E. coli-challenged PI3K-{gamma}–/– mice.

The {beta}3-integrins, which associated with CD47, have an important role in lung PMN sequestration in PI3K-{gamma}–/– mice. We used FACS analysis to show greater {beta}3-integrin surface expression in PI3K-{gamma}–/– PMNs obtained after intraperitoneal E. coli challenge than in WT PMNs. Because {beta}3-integrin expression in the PI3K-{gamma}–/– PMNs was augmented and the {beta}3-integrin and vitronectin interaction with CD47 was increased, we assessed the possibility that PI3K-{gamma}–/– PMNs would demonstrate greater {beta}3-integrin-dependent adhesion to vitronectin. We observed that unchallenged PMNs isolated from PI3K-{gamma}–/– and WT mice did not differ in their basal adhesion to vitronectin; however, the adhesion to vitronectin-coated wells was twofold greater for fMLP-stimulated PI3K-{gamma}–/– PMNs than for WT PMNs exposed to fMLP. The increased PMN adhesion to vitronectin was blocked by pretreatment of PI3K-{gamma}–/– PMNs with anti-{beta}3-integrin antibody. Anti-{beta}3-integrin antibody also prevented the E. coli-induced increase in MPO activity and lung tissue PMN accumulation in PI3K-{gamma}–/– mice. These results point to an important role for PI3K-{gamma} modulation of {beta}3-integrin expression in regulating the lung tissue uptake of PMNs induced by gram-negative sepsis.

The basis of PI3K-{gamma} modulation of CD47/{beta}3-integrin expression is uncertain. PI3K-{gamma} and CD47 have been reported to interact with the {beta}{gamma}-subunits of the heterotrimeric GTP-binding protein Gi (3, 11, 42, 43). PI3K-{gamma} membrane localization and activation depend on the release of G{beta}{gamma} from chemoattractant receptors to create a leading edge in PMNs (3). Inhibitors of G{beta}{gamma} released from Gi were shown to interfere with PMN migration, similar to the inhibition of PI3K-{gamma} (3, 42, 43). Also CD47 was shown to associate with and function through G{beta}{gamma} (11). From immunoprecipitation studies, we found a greater association of CD47 with G{beta}{gamma} in PI3K-{gamma}–/– mice soon after E. coli challenge (data not shown); thus a possible mechanism of activation of CD47 in PI3K-{gamma}–/– PMNs may involve interaction of CD47 with G{beta}{gamma}. At the leading edge, there may exist a balance between PMN chemotaxis and adhesion through competitive binding of G{beta}{gamma} to PI3K-{gamma} and the CD47-{beta}3-integrin complex. Other studies have also suggested that PMN migration involves such cross talk between the chemotaxis machinery and adhesion complexes (1, 5, 20, 26). Our study provides in vivo evidence of the involvement of PI3K-{gamma} and the CD47-{beta}3-integrin complex in the mechanism of PMN accumulation in the lung matrix and the subsequent lung injury.


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This study was supported by National Heart, Lung, and Blood Institute Grants HL-64573, HL-45638, and HL-46350 (A. B. Malik).


    ACKNOWLEDGMENTS
 
We acknowledge Dr. Xiangdong Zhu (Section of Pulmonary and Critical Care Medicine, The University of Chicago) for critical reading and the Flow Cytometry Facility at the Research Resources Center of the University of Illinois at Chicago for technical assistance.


    FOOTNOTES
 

Address for reprint requests and other correspondence: A. Malik, Dept. of Pharmacology, College of Medicine, The Univ. of Illinois, 835 South Wolcott Ave., Chicago, IL 60612-7343 (e-mail: abmalik{at}uic.edu)

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

* E. Ong and X.-P. Gao equally contributed to this work. Back


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