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
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ABSTRACT |
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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
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INTRODUCTION |
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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.
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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-1
(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 s1. 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
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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 ml1) 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.
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.
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RESULTS |
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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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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 = µ × G, where
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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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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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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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)-1 (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-1
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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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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DISCUSSION |
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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 s1,
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 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.
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ACKNOWLEDGEMENTS |
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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.
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FOOTNOTES |
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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.
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