Granulocyte colony-stimulating factor promotes adhesion of neutrophils

Arup Chakraborty1,2, Eric R. Hentzen2,3, Scott M. Seo2,3, and C. Wayne Smith2

1 Section of Infectious Diseases, Department of Medicine, and 2 Section of Leukocyte Biology, Department of Pediatrics, Baylor College of Medicine; and 3 Department of Biomedical Engineering, Rice University, Houston, Texas 77030


    ABSTRACT
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ABSTRACT
INTRODUCTION
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Granulocyte colony stimulating factor (G-CSF) is well known for its ability to drive the maturation and mobilization of neutrophils. G-CSF also appears to have the potential to activate functions of mature neutrophils, influencing recruitment at sites of inflammation and tissue injury. We investigated the ability of G-CSF to stimulate adhesion of isolated blood neutrophils. G-CSF induced significant adherence to intercellular adhesion molecule (ICAM)-1 that was both macrophage antigen-1 (Mac-1) and leukocyte function-associated antigen-1 dependent. The kinetics of G-CSF-stimulated adhesion to ICAM-1 peaked at 11 min without detectable surface upregulation of Mac-1. This was in marked contrast to chemokines, in which peak activation of adhesion is seen within 1 min of stimulation. In contrast to chemokine-induced adhesion, G-CSF stimulation was not inhibited by pertussis toxin. G-CSF also augmented the attachment of neutrophils to activated human umbilical vein endothelial cells (HUVEC) through specific effects on neutrophils, because HUVEC appear to lack functional G-CSF receptors.

granulocyte colony-stimulating factor; intracellular adhesion molecule-1; leukocyte function-associated antigen-1; macrophage antigen-1; adhesion; polymorphonuclear neutrophils


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

GRANULOCYTE COLONY-STIMULATING FACTOR (G-CSF) is a glycoprotein with a molecular mass of 19 kDa produced by a variety of cell types, including endothelial cells activated during bacterial infection and inflammatory conditions (36, 46). G-CSF is a critical cytokine for driving proliferation and differentiation of neutrophil precursors (9) and has been used pharmacologically to enhance the production and mobilization of neutrophils from bone marrow in neutropenic states (30, 31). Administration to healthy volunteers produces few side effects (3, 39) and causes mild transient neutropenia (4, 38). However, numerous reports describe that G-CSF administration can create harmful secondary effects such as pulmonary complications in patients receiving G-CSF during chemotherapy (19, 24, 28, 29, 33). Other examples of complications include severe bronchocentric granulomatosis (17), capillary leak along with hepatocellular injury (10), and neutrophilic dermatoses (41). Experimental studies have implicated G-CSF in the pathogenesis of inflammatory tissue injury. In a model of resuscitated hemorrhagic shock in rats, G-CSF mRNA levels were elevated in lung, liver, and bowel and correlated with the duration of shock (21). G-CSF instillation into the lungs of rats has been shown to result in neutrophil infiltration and lung damage similar to that found in the rat model of resuscitated hemorrhagic shock (21). G-CSF administration has been shown to exacerbate arthritic symptoms in a mouse model (7) and has been found in synovial fluid of patients with rheumatoid arthritis (34),

In addition to its role in driving bone marrow production of neutrophils, G-CSF can stimulate various functions in mature neutrophils in vitro including degranulation (51), adhesion (37, 56), phagocytosis (50), oxidative burst (1, 47), and delayed apoptosis (2), all of which may contribute to the complications that sometimes follow its therapeutic use.

The purpose of the present study is to reexamine the effects of G-CSF on neutrophil adhesion. Some apparently contradictory data have been published, and we are interested in defining the extent to which G-CSF can influence the adhesive mechanisms in human neutrophils. Yong et al. (53-55) reported that G-CSF can promote transendothelial migration of neutrophils without affecting adhesion and that this effect is not dependent on a chemotactic gradient. However, these and other investigators (11) have found that G-CSF stimulates mobilization of CD11b/CD18 [macrophage antigen-1 (Mac-1)] from granule stores, thereby increasing the amount on the cell surface, and Okada et al. (37) and Yuo et al. (56) reported that G-CSF increased adhesion of leukocytes to synthetic fibers (e.g., dacron and nylon wool).

To address this apparent conflict in more detail, we examined the effects of G-CSF on Mac-1 and CD11a/CD18 [leukocyte function-associated antigen-1 (LFA-1)] adhesion to intercellular adhesion molecule (ICAM)-1.


    METHODS
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Materials. Recombinant human G-CSF was obtained from Amgen (Thousand Oaks, CA), human IL-6 was purchased from Peprotech (Rocky Hill, NJ), and formyl-methionyl-leucil-phenylalanin (fMLP), keyhole lympet hemocyanin (KLH), and pertussis toxin were obtained from Sigma (St. Louis, MO). Fluorescein isothiocyanate (FITC)-labeled antibodies to CD45 were purchased from Caltag Laboratories (Burlingame, CA). Anti-CD11a (LFA-1) monoclonal antibody R3.1 and anti-CD11b (Mac-1) monoclonal antibody 60.1 were a generous gift from Dr. Kei Kishimoto (Boehringer-Ingelheim, Ridgefield, CT) and Lora Whitehouse (Repligen, Cambridge, MA), respectively. Anti-Mac-1 monoclonal 2LPM19c (mouse IgG1) was obtained from DAKO (Carpinteria, CA), FITC-labeled anti-Mac-1 (Mab clone ICRF44) was obtained from Serotec (Raleigh, NC), monoclonal anti-human CD18 (clone IB4) was obtained from Ancell (Bayport, MN), and the blocking antibody R15.7, which blocks CD18, was provided by Dr. Robert Rothlein (Boehringer-Ingelheim).

Cells. Human neutrophils were obtained from peripheral blood samples as described (48). Briefly, citrated whole blood was sedimented by 1% dextran to remove red blood cells, and the remaining leukocytes were centrifuged over a Ficoll gradient (Histopaque 1077; Sigma). Neutrophils were collected from the pellet, and remaining red blood cells were eliminated by quick hypotonic lysis in sterile water. Cells were >= 95% neutrophils as evaluated by modified Wright-Geimsa staining and >99% viable as evaluated by the trypan blue dye exclusion method. Neutrophils were kept at 4°C in Ca2+-free HEPES buffer (in mmol/l) (110 NaCl, 10 KCl, 10 glucose, 1 MgCl2, and 30 HEPES, pH 7.35) containing 0.1% human serum albumin (Armour Pharmaceuticals, Kankanee, IL). The murine pre-B lymphocyte cell line 300.19 was stably transfected with human ICAM-1 cDNA and cultured in RPMI1640 medium (Invitrogen, Grand Island, NY) supplemented with 10% heat-inactivated fetal bovine serum (Invitrogen), 1% penicillin-streptomycin (Invitrogen), and 100 µM 2-mercaptoethanol (Sigma). The murine fibroblast cell line, L cells stably transfected with human ICAM-1 cDNA, were grown in RPMI 1640 medium (Invitrogen) supplemented with 10% FBS (Invitrogen) and 1% penicillin-streptomycin (Invitrogen). Human umbilical vein endothelial cells (HUVEC) were isolated by collagenase digestion as previously described (22). In our experiments, HUVEC monolayers were used after single passage (P1). HUVEC monolayers were cultured in M199 (Invitrogen) containing 20% fetal bovine serum (Invitrogen), 1% penicillin-streptomycin (Invitrogen), 1% fungizone (Invitrogen), 1% HEPES buffer (Invitrogen), 1 µg/ml heparin (Sigma), and endothelial cell growth supplement (ECGS) purchased from Fisher Scientific (Pittsburgh, PA). After 4-6 days, HUVEC were passaged onto 35-mm tissue culture dishes (Corning Glass Works, Corning, NY), which were coated with glutaraldehyde-crosslinked gelatin, as described previously (5). Two to four days later, HUVEC were stimulated with 0.2 U/ml recombinant human IL-1beta (Peprotech) at 37°C for 4 h before the adhesion assays.

Static adhesion assay. Static adhesion was performed by using a static adhesion chamber, as described previously (44, 45). Briefly, coverslips covered with monolayer of cells or KLH were placed in a Munz-type adhesion chamber (custom ordered from a local vendor), and another plain coverslip was placed on top of that, separated by a rubber O-ring. Freshly isolated human neutrophils were mixed with the appropriate amount of inducer and injected into the chamber, with the coated side kept to the bottom. After 7 min, the chamber was inverted, and after another 7 min, neutrophils bound to the surface of monolayer or KLH were counted.

Heterotypic aggregation assay. Aggregation assays were performed at 37°C in a Haake VT550 cone-plate viscometer (Haake, Paramus, NJ) as described previously (20). In brief, to determine the cellular composition of aggregates, neutrophils and 300.19 cells were labeled with spectrally distinct fluorescent labels. The 300.19 cells (2 × 106 cells/ml) were stained with nuclear dye LDS-751 (Molecular Probes) for detection in the red fluorescence channel (FL3), and neutrophils (1 × 106 cells/ml) were labeled with anti-CD45-FITC for detection in the green fluorescence channel (FL1). Neutrophils and 300.19 cells were mixed and incubated for 2 min at 37°C in buffer containing 1.5 mM Ca2+ and 0.1% human serum albumin. G-CSF (5 nM) or fMLF (1 uM) was added before shear was applied at the rate of 300 s-1. At prescribed time points, 40-µl samples were withdrawn and the level of aggregation was analyzed by flow cytometry on the basis of characteristic side vs. forward scatter. Quantification of heterotypic aggregation between neutrophils (N) and 300.19 cells (B) was performed by analysis of dot plots of green vs. red fluorescence. Homotypic doublets (N2) or larger aggregates (N3+) are composed solely of neutrophils, whereas heterotypic aggregates are composed of one 300.19 (B) cell and either one (BN1), two (BN2), or three or more (BN3+) neutrophils. The fraction of neutrophils in the heterotypic aggregates was calculated using the following formula
%neutrophils in heterotypic aggregates<IT>=</IT>(BN<SUP>1</SUP><IT>+</IT>2BN<SUP>2</SUP><IT>+</IT>3BN<SUP>3+</SUP>)<IT>/</IT>(N<SUP>1</SUP><IT>+</IT>2N<SUP>2</SUP><IT>+</IT>3N<SUP>3+</SUP><IT>+</IT>BN<SUP>1</SUP><IT>+</IT>2BN<SUP>2</SUP><IT>+</IT>3BN<SUP>3+</SUP>)

Flow cytometric detection of ICAM-1 bead adhesion to neutrophils. We used the same technique described previously (43). Briefly, 1 µm-diameter, protein A-coated, yellow-green fluorescent beads (1 × 1010 ml-1) were washed once at a volume ratio of 1:10 in blocking solution (BlockAid; Molecular Probes) and resuspended at 2 × 109 ml-1 with ICAM-1-Fc chimera (final concentration 20 or 50 µg/ml) for 90 min under bath sonication. Ice was added to the bath sonicator periodically to prevent overheating of protein-bead suspension. To separate bound from unbound soluble ICAM-1/IgG1 (hereafter, ICAM-1), the bead solution was centrifuged and beads were resuspended in 1 × PBS to a final concentration of 1 × 1010 ml-1.

Neutrophils (3 × 105) were preincubated to a final volume of 0.3 ml of HEPES buffer (plus 0.1% HSA and 1.5 mM CaCl2) with 0.04 µg/ml LDS-751 (Molecular Probes), which is a red nucleic acid dye used to identify neutrophils, for 2 min at 37°C in a mixing chamber. Subsequent to all preincubations, beads (stock 1 × 1010 ml-1) were added at a volume ratio of 1:100 to the neutrophil suspension, which was sheared briefly and sampled (15 µl) for the time 0 point. Stimulus was added at a volume ratio of 1:100 to bead-cell suspension, which was immediately sheared by stir bar at a rate of rotation of ~300 rpm, corresponding to shear stresses previously estimated to be <1.0 dyn/cm2. At predetermined time points, 15-µl samples of the suspension were pipetted into 35 µl of ice-cold 0.5% formaldehyde in PBS. These fixed samples were immediately placed on a flow cytometer (3-color FACScan; Becton-Dickinson) to measure bead-cell adhesive events, as previously described. Briefly, using CellQuest software (Becton-Dickinson), we quantified the total number of beads bound to a known number of neutrophils (singlets), from which followed the mean number of beads bound per neutrophil, as previously described.

Electrophoretic mobility shift assay. Whole cell extracts were prepared and analyzed by electrophoretic mobility shift assay (EMSA) as described previously (49). Briefly, cells were incubated with or without G-CSF (5 nM) for 15 min. Cells were lysed in ice-cold, high-salt buffer (20 mM HEPES, pH 7.6, 20 mM NaF, 1 mM Na3VO4, 1 mM Na4P2O7, 1 mM EDTA, 1 mM EGTA, 1mM DTT, 1 mM PMSF, 1 µg/ml leupeptin, 1 µg aprotinin, and 420 mM NaCl, 20% glycerol) and passed three times through freeze-thaw cycles in a dry ice/ethanol bath and then centrifuged at 14,000 g to remove cell debris. Protein (20 µg) was mixed with Salmon sperm DNA (3 µg) and 1 pmol 32P-labeled hSIE duplex oligonucleotide in the presence of binding buffer (13 mM HEPES, pH 7.6, 65 mM NaCl, and 1 mM DTT, 8% glycerol) in a total reaction volume of 20 µl and incubated for 20 min at room temperature. Samples were electrophoresed in a 4% nondenaturing polyacrylamide gel. Gels were dried and DNA-protein complexes were visualized by autoradiography.

Statistical analysis. Data are expressed as means ± SE. Differences between groups were evaluated by using Student's t-test.


    RESULTS
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INTRODUCTION
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RESULTS
DISCUSSION
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G-CSF promotes LFA-1- and Mac-1-dependent adhesion of neutrophils. Three adhesion molecules play a major role in orchestrating firm adhesion of neutrophils to the endothelium. ICAM-1 (CD54) expressed on the endothelium binds to ligands Mac-1 (CD11b/CD18) and LFA-1 (CD11a/CD18) present on neutrophils. To test whether G-CSF promotes adhesion of neutrophils to ICAM-1, we measured the adhesion of G-CSF-stimulated neutrophils to ICAM-1-expressing cells. Murine L cells expressing human ICAM-1 were grown on glass coverslips and placed in the lower wall of a Muntz-type static adhesion chamber. These cells were tested for high levels of ICAM-1 expression by flow cytometry, and G-CSF-stimulated or unstimulated neutrophils did not bind to normal L cells (data not shown). Freshly isolated neutrophils treated with G-CSF or with fMLP (as a positive control) were immediately injected into the adhesion chamber. The result showed that G-CSF-mediated adhesion of neutrophils increased in a dose-dependent manner, peaking at 5 nM. Equivalent adhesion was observed when neutrophils were treated with fMLP (500 nM). We previously (23) found that fMLP concentration (500-1,000 nM) was very effective for activation. G-CSF (5 nM) treatment prompted a significant adhesion (90%) compared with the unstimulated control (15%) (Fig. 1). G-CSF-mediated adhesion of neutrophils could be partially blocked by preincubation of neutrophils with anti-LFA-1 or anti-Mac1 antibody. Preincubation of neutrophils with both the antibodies (Mac-1 and LFA-1) was more effective, and the adhesion was almost completely blocked by antibody against CD18, the common subunit for both Mac-1 and LFA-1 (Fig. 1). This result shows that both LFA-1 and Mac-1 are involved in G-CSF-mediated adhesion of neutrophils to ICAM-1-expressing L cells.


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Fig. 1.   Granulocyte colony stimulating factor (G-CSF)-mediated adhesion of human neutrophils to murine L cells expressing human intercellular adhesion molecule (ICAM)-1. Neutrophils were incubated with G-CSF (5 nM) for 7 min (black) or with formyl-methionyl-leucil-phenylalanin (fMLP; 500 nM) for 1 min (gray) or were unstimulated as a negative control (white) and then injected into the adhesion chamber. To determine the contribution of leukocyte function-associated antigen-1 (LFA-1) and macrophage antigen-1 (Mac-1), neutrophils were also preincubated for 15 min with R3.1 Fab (anti-LFA-1, 20 ug/ml), 60.1 F(ab2) (anti-Mac-1, 20 ug/ml), a combination of both, or R15.7 (anti-beta 2 integrin, the common subunit of both the LFA-1 and Mac-1, IgG1, 20 ug/ml) as indicated (see METHODS). The Y axis represents the percentage of neutrophils that remained adhered to the L cell monolayers after chamber inversion. Data shown are representative of 3 experiments. PMN, polymorphonuclear neutrophils.

To confirm the Mac-1-mediated adhesion during G-CSF-stimulation, we also measured adhesion of neutrophils to KLH-coated surface in the static adhesion chamber (Fig. 2). G-CSF increased adhesion in a dose-dependent manner, reaching a peak at the concentration of 5 nM, and Mac-1 antibody reduced adhesion close to the level of unstimulated control.


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Fig. 2.   G-CSF-stimulated adhesion of human neutrophils to the keyhole lympet hemocyanin (KLH). The percentage of adhesion stimulated with G-CSF (5 nM) (black) or fMLP (500 nM) (gray) or unstimulated (white) was determined in a static adhesion chamber (detailed in METHODS). To block Mac-1-mediated adhesion, neutrophils were also preincubated with anti-Mac-1 MAb 2LPM19c (3 ug/ml) for 15 min as indicated in the figure. Data shown are representative of 4 experiments.

Kinetics of G-CSF-mediated adhesion to ICAM-1-expressing cells under shearing conditions. We previously (20) used cone-plate viscometry to study the kinetics of adhesion and to assess the stability of adhesion under conditions of defined shear stress. A pro-B cell line (300.19) transfected with human ICAM-1 that expresses ~1.7 × 106 ICAM-1/cell was used as a source of ICAM-1. Neutrophils (labeled with anti-CD45-FITC) and 300.19 cells (labeled with LDS-751) were mixed briefly and sheared in a cone-plate viscometer as described previously (20). This apparatus applies a uniform and linear shear field to the entire fluid sample in the gap between the cone and plate. Shear rate and shear stress are related through the fluid viscosity as tau  = µ × G, where tau  is the shear stress in dyne/cm2, G is the shear rate in s-1, and µ is the viscosity in poise. We applied a shear rate of 300 s-1 and samples were taken at different intervals and immediately analyzed by a flow cytometer. ICAM-1-transfected 300.19 cells did not form any homotypic aggregation in response to fMLP or G-CSF. In Fig. 3A, fMLP stimulation produced maximum aggregation of neutrophils and 300.19 cells within 2 min, whereas, in response to G-CSF, aggregation peaked at 11 min. This experiment shows that G-CSF-mediated adhesion is stable enough to withstand a moderate shear rate consistent with that found in postcapillary venules but has different kinetics of forming aggregates compared with fMLP. Other chemokines in a manner similar to fMLP also promote maximum adhesion to ICAM-1 within 2 min (8).


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Fig. 3.   A: kinetics of G-CSF-mediated adhesion under shearing condition. Human neutrophils and human ICAM-1-expressing 300.19 cells were labeled with 2 distinct fluorescent dyes and combined in the cone-plate viscometer. Shear (300 s-1) was initiated after addition of G-CSF (5 mM) () or fMLP (1 uM) () or unstimulated (open circle ). Samples were taken at prescribed time points, and heterotypic aggregation is presented as the percentage of total neutrophils bound to 300.19 cells (y axis). A detailed description can be found in METHODS, Heterotypic aggregation assay. Data shown are representative of 3 experiments. B: adhesion of neutrophils to ICAM-1-coated latex beads. Isolated human neutrophils were suspended with ICAM-1-coated latex beads and left unstimulated or stimulated with either CXC chemokine (IL-8) or G-CSF. Samples were withdrawn at the indicated times (20 s or 4, 9, or 13 min) and analyzed by flow cytometry to determine the extent of bead binding. Results are expressed as fold increases over the extent of bead binding without stimulation. Results are averages of determinations using neutrophils from 2 different individuals.

The delay in the kinetics of adhesion after G-CSF stimulation was confirmed using ICAM-1-coated latex beads [a technique previously used to document the rapid rate of adhesion stimulated in neutrophils with CXC chemokines (Fig. 3B) (43)].

Pertussis toxin treatment did not block G-CSF-mediated adhesion of neutrophils. Next, we investigated whether G-CSF-mediated adhesion is indirectly influenced by chemokine production by neutrophils because G-CSF-mediated adhesion kinetics is slower than chemokines. To test this possibility, we used the trimeric G protein inhibitor pertussis toxin to block chemokine-mediated adhesion signaling because all chemokine receptors and the fMLP receptor require heterotrimeric G protein to signal for adhesion. G-CSF signaling has not been reported to involve heterotrimeric G protein, so it is expected that if G-CSF-stimulated adhesion is mediated indirectly through chemokine(s), pretreatment of neutrophils with pertussis toxin should block the adhesion. Our experiment shows that pretreatment of neutrophils with pertussis toxin has no effect on G-CSF-mediated adhesion to KLH, whereas fMLP-mediated adhesion to KLH was completely abrogated (Fig. 4).


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Fig. 4.   Effect of pertussis toxin on G-CSF and fMLP-mediated adhesion to KLH. Freshly isolated neutrophils were incubated with or without 2 ug/ml pertussis toxin (Ptx) for 2 h at 37°C. Neutrophils were then treated with G-CSF (5 nM) for 7 min or with fMLP (500 nM) for 1 min and injected into an adhesion chamber as described in METHODS. The percentage of neutrophils remaining adhered to the KLH-coated surface is indicated along the y axis. Data shown are representative of 3 experiments.

G-CSF-mediated adhesion is not due to surface upregulation of Mac-1. Previous reports have shown that G-CSF stimulation upregulates surface expression of Mac-1 on neutrophils (11, 56). To determine whether G-CSF-mediated adhesion to ICAM-1, which peaks at 11 min, involves surface upregulation of Mac-1, we tested the surface expression of Mac-1 on neutrophils after G-CSF stimulation at various time points (Fig. 5). Although G-CSF treatment clearly increased Mac-1 surface expression at 30 min, no surface upregulation was detected at earlier time points. This indicates that G-CSF-mediated adhesion takes place before surface upregulation of Mac-1.


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Fig. 5.   Effect of G-CSF on Mac-1 expression on neutrophils. Freshly isolated human neutrophils were stimulated with G-CSF (5 nM) at 37°C for different time periods (x axis) and washed 3 times to remove G-CSF and further incubated with FITC-labeled anti-Mac-1 (monoclonal ICRF44, 5 ug/ml) in such a way that every batch was incubated for 15 min with antibody. The y axis represents the mean channel fluorescence. Data shown are representative of 3 experiments.

G-CSF promotes adherence of neutrophils to interleukin-1-stimulated HUVEC. In previous experiments, the interaction of activated neutrophils with ICAM-1-expressing cells enabled us to identify engagement of specific adhesion molecules involved in G-CSF-mediated firm adhesion of neutrophils. Next, we tested whether G-CSF stimulation promotes adhesion to endothelial cells by using a static adhesion chamber as described previously. HUVEC were grown on coverslips and used as the lower surface of the adhesion chamber. G-CSF (5 nM) was mixed with freshly isolated neutrophils from a healthy volunteer and injected into the adhesion chamber. G-CSF was only marginally effective in promoting adhesion to unstimulated HUVEC monolayers (Fig. 6) and exhibited no further enhancement when HUVEC monolayers were maximally stimulated with interleukin (IL)-1beta (1 U/ml for 4 h) because most of the neutrophils adhered to endothelial cells without any stimulation (data not shown). However, a low degree of HUVEC stimulation (0.2 U/ml IL-1beta for 2 h) resulted in a moderate degree of adhesion of untreated neutrophils. Addition of G-CSF significantly augmented adhesion to these monolayers (Fig. 6) that was blocked completely by combined anti-Mac-1 and anti-LFA-1 monoclonal antibodies.


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Fig. 6.   G-CSF-stimulated adhesion of neutrophils to human umbilical vein endothelial cells (HUVEC). Neutrophils were stimulated with G-CSF (5 nM) for 7 min (black) or unstimulated (white) and injected into a static adhesion chamber for adhesion to HUVEC. HUVEC monolayers were treated with or without IL-1beta (0.2 U/ml for 2 h) as indicated. Neutrophils were also preincubated for 15 min with R3.1 Fab (anti-LFA-1, 20 ug/ml) or anti-Mac-1 Mab 2LPM19c (3 ug/ml) as indicated. The percentage of neutrophils remaining adherent to the HUVEC monolayers is indicated along the y axis. Data shown are representative of 4 experiments.

G-CSF specifically modulates adhesion of neutrophils. Because both neutrophils and HUVEC were exposed to G-CSF in the previous experiment and because one earlier study indicated that G-CSF could activate HUVEC (6), we checked for functional G-CSF receptors in both neutrophils and HUVEC. To do so, we performed EMSA assay with whole cell extract from HUVEC and neutrophils treated with G-CSF. Because the IL-6 receptor is known to be expressed by HUVEC and activate Stat3, we used IL-6 as a positive control. Our results show that G-CSF activated Stat3 in neutrophils but failed do so in HUVEC (Fig. 7).


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Fig. 7.   HUVEC lack a functional G-CSF receptor. To test whether HUVEC express any functional G-CSF receptor, neutrophils and HUVEC were treated with G-CSF (5 nM) or interleukin (IL)-6 (0.5 nM) for 15 min as indicated at top. Cell lysates were analyzed for Stat3 activation by electrophoretic mobility shift assay (EMSA) using a radiolabeled oligonucleotide (hSIE). G-CSF receptor activates a transcription factor Stat3 after receptor-ligand interaction. IL-6 was used as a positive control because IL-6 also activates Stat3. Data shown are representative of 5 experiments.


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The results of the present study support the following conclusions regarding G-CSF effects on adhesion of neutrophils: 1) G-CSF significantly increases adhesion to ICAM-1-expressing target cells, and both Mac-1 and LFA-1 are involved. This increase is equivalent to that induced by optimal stimulation with the chemotactic peptide fMLP. The activation of Mac-1 and LFA-1 adhesion was confirmed using assays distinct for each integrin; 2) kinetic studies reveal that the rate of this adhesion differs significantly from that induced by fMLP. Under shear conditions at a shear rate of 300 s-1, adhesion after stimulation with fMLP peaks within 2 min of stimulation, whereas that induced by G-CSF does not peak until 11 min and appears to show a lag of ~3 to 4 min. This time frame was confirmed separately for LFA-1/ICAM-1 adhesion; 3) the G-CSF-induced adhesion is not inhibited by pertussis toxin, indicating that the lag and induced adhesion were not secondary to the endogenous release of chemokines (e.g., IL-8) (32). Chemokine- and chemotactic factor-induced activation of adhesion is inhibited by pertussis toxin; and 4) in contrast to the conclusions in the Yong (53) study, G-CSF does increase adhesion to endothelial cells. However, this effect requires some level of activation of the endothelial cells, apparently to express increased amounts of ICAM-1. This adhesion involves both Mac-1 and LFA-1. G-CSF failed to stimulate endothelial Stat3 but produced marked Stat3 activation of neutrophils in this functional assay.

In addition to the observations cited above, we found that Mac-1 surface levels were increased after exposure to G-CSF. This is consistent with reports from other laboratories (11, 54, 56), but it is significant to point out that this event did not occur until after the time of peak adhesion to ICAM-1-expressing target cells. A number of years ago, we demonstrated (23) that Mac-1-dependent adhesion in response to fMLF stimulation was limited to the constitutively expressed surface Mac-1 and that the newly mobilized Mac-1 from granular stores failed to participate. A similar phenomenon appears to be true for G-CSF stimulation.

A much earlier report (6) described that G-CSF promotes proliferation and migration of HUVEC. In our experiments, we failed to detect evidence for a functional G-CSF receptor by checking the characteristic G-CSF receptor-mediated Stat3 activation. This is also consistent with a previous report by Yong et al. (52). We also did not observe ICAM-1 upregulation after G-CSF exposure (data not shown).

The delayed induction of adhesion by G-CSF is of interest in the context of the current paradigm for neutrophil emigration at sites of inflammation. This model involves a series of steps in a cascade of events progressing from tethering, rolling, stationary adhesion, and transmigration in postcapillary venules, vessels too large to physically trap activated neutrophils. The transition from rolling to firm adhesion requires an activating stimulus to increase the avidity/affinity of CD18 integrins (Mac-1 and LFA-1). Chemokines (i.e., IL-8) at the endothelial surface are capable of inducing rapid activation, detectable within 10-15 s (43). The lag after G-CSF before there is increased adhesion seems to exclude a contribution of G-CSF as a primary stimulus in this sequence of events because a delay of 3-4 min before stimulation of adhesion would allow rolling cells to pass well beyond the site of inflammation. However, system G-CSF (40) may prime the neutrophil from enhanced response to local chemokines and thereby augment the efficiency of the adhesion cascade. Support for such a concept comes from published studies showing that G-CSF is also known for priming neutrophils for oxidative burst (35). So, at a site of inflammation where other inflammatory mediators are present, neutrophils primed by systemic G-CSF could enter the site in a proadhesive state and may be expected to worsen the tissue damage by triggering respiratory burst.

The adhesion cascade is clearly not the only mechanism for localization of neutrophils at sites of inflammation. Sequestration of neutrophils in vascular beds can occur in an adhesion independent manner, e.g., in the lung (12) and liver (27). This was clearly illustrated in the data obtained by Inano et al. (26). They found that in rabbits, intravenous injection of G-CSF resulted in a rapid profound neutropenia that was maximal within 2 min and sustained for between 90 and 180 min. Histology revealed marked increases in neutrophils sequestered primarily in capillaries. In this setting, physical trapping may prolong the residence time sufficiently to allow G-CSF-induced retention due to adhesion and enhanced transendothelial migration as shown by Yong and coworkers (53-55). The diameters of spherical neutrophils (6-8 µm) are larger than the diameters of many capillary segments (2-15 µm), and ~50% of the capillary segments would thereby require neutrophils to change their shape to pass through (14, 18). Given the large number of capillary segments through which a neutrophil must pass (often >50), most neutrophils must change shape during transit from arteriole to venule. During inflammation, much of the sequestration and infiltration occurs through vessels so narrow that physical tapping is sufficient to stop the flowing neutrophil. Binding of mediators such as chemotactic factors (e.g., the complement fragment C5a) to neutrophil receptors induces a transient resistance of the cells to deformation (25). Because neutrophils must deform to pass through the capillary bed, leukocyte activation by inflammatory mediators could affect further concentration of neutrophils at the alveolar walls. This was found by Inano et al. (26) to be likely for G-CSF stimulation, as well, because G-CSF induced increased resistance of neutrophils to deformation. The role of mechanical factors in the initial sequestration of neutrophils in the alveolar capillaries is supported by evidence that neither L-selectin nor beta 2-integrins are required (13, 16). However, the retention times within this capillary bed are influenced by these adhesion molecules, and this adhesion is likely an interaction of leukocyte adhesion molecules and endothelial adhesion molecules. In the absence of intra-alveolar inflammation, there is little emigration from the vasculature, and neutrophils seem to revert to a basal state of deformability and adhesiveness, thereby moving out of the lung via the blood.

Regarding signaling mechanisms, there is no definitive information regarding the upregulation of CD18 integrin adhesion by G-CSF. Some possible mechanistic insight comes from the observation that G-CSF binding to its high-affinity receptor activates both phosphatidylinositol 3-kinase (PI3K) and PKC (11, 15). Kinetic studies reveal that PKC activation peaks at around 15 min (42), and there is substantial evidence that PKC activation (e.g., with phorbol esters) markedly activates neutrophil adhesion. Evidence that PI3K inhibition has no effect on Mac-1-dependent adhesion would support consideration of the PKC pathway as primarily important in G-CSF augmentation of adhesion.


    ACKNOWLEDGEMENTS

We thank Scott M. White for reviewing the manuscript critically, Robert A. Bowden for showing how to do static adhesion assay, and Prasenjit Guchhait for helping in cell culture. We also thank Texas Women's Hospital, Harris County Hospital District, and St. Luke's Episcopal Hospital.


    FOOTNOTES

Address for reprint requests and other correspondence: A. Chakraborty, Children's Nutrition Research Center, 1100 Bates, Rm. 6014, Houston, TX 77030-2600 (E-mail: arupc{at}bcm.tmc.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.

First published September 18, 2002;10.1152/ajpcell.00165.2002

Received 11 April 2002; accepted in final form 16 September 2002.


    REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
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DISCUSSION
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