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*
Cardiovascular Research Group, Division of Clinical Sciences (Northern General Hospital), University of Sheffield, Sheffield, United Kingdom; and
Histopathology Department, Northern General Hospital, Sheffield, United Kingdom
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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1-1 (0.02 mg/mouse), significantly
(p < 0.05) inhibited both LPS (0.3 µg)- and KC
(0.3 µg)-induced neutrophil migration. A second mAb to CD18 (GAME-46)
and both F(ab')2 and Fab of HM1-1 produced similar
results to those above, while coadministration of mAbs did not result
in greater inhibition. Electron microscopy studies showed that CD29 was
involved in the movement of neutrophils from the interstitium into
alveoli. The effect of mAbs to CD49 (
integrin) subunits of CD29 was
also examined. mAbs to CD49e and CD49f inhibited both responses, while
anti-CD49b and CD49d significantly inhibited responses to KC only.
These data suggest that CD29 plays a critical role in neutrophil
migration in pulmonary inflammation and that CD49b and CD49d mediate
CD18-independent neutrophil accumulation. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Neutrophils migrate from vessels in response to a stimulus at the
inflammatory site. This process of migration has many stages and is
regulated at each by a range of inflammatory mediators (for reviews,
see Refs. 5, 6, 7). The onset of inflammation results in
increased expression, or altered avidity of adhesion molecules, and
thus increases the adhesiveness of both the circulating cells and the
endothelium (for reviews, see Refs. 8, 9). Neutrophil
migration occurs in the postcapillary venules in tissues such as skin
(10), mesentery (11), and upper airways
(12, 13). This process has been characterized in detail
using intravital microscopy and involves selectin-mediated capture and
rolling of neutrophils along the vessel wall, followed by CD18
(2) integrin-mediated firm adhesion and
transendothelial migration (for review, see Ref. 14).
Blocking mAbs directed against CD18 are effective inhibitors of
neutrophil migration in various inflammatory situations in vivo
(15, 16, 17). In addition, genetic deficiency of CD18 results
in a marked reduction of neutrophil migration from the systemic
circulation in a number of species despite a frequent blood
neutrophilia (18, 19, 20, 21).
Neutrophil migration in the pulmonary circulation, however, appears to
be different. In the alveolar region, neutrophil migration is thought
to occur not only in the capillaries (22, 23), but several
in vivo studies have shown there is a CD18-independent component in
response to certain stimuli, including C5a (17),
Streptococcus pneumoniae (24), and
ischemia-reperfusion (25). Moreover, postmortem
observations in a child with leukocyte adhesion deficiency type I, a
disease in which neutrophils do not express surface CD18, showed that
neutrophils were present in the lung at sites of acute inflammation,
yet there was no evidence of neutrophil emigration to inflamed sites
elsewhere in the body (26). Similar findings have been
made in CD18-deficient cattle with Pasturella
hemolytica-induced pneumonia (27) and in mice bearing
CD18-deficient neutrophils after intratracheal instillation of S.
pneumoniae (28). In contrast, migration of
neutrophils to the lung in response to LPS, IL-1, or
Pseudomonas aeruginosa is dependent on CD18, as assessed by
effects of blocking mAbs or absence of CD18 on circulating neutrophils
(17, 24, 28, 29, 30).
Following migration across the capillary endothelium, neutrophils cross
the basal lamina and enter the interstitium. It has been suggested that
neutrophils then adhere to fibroblasts, which can direct their movement
toward the alveoli (31). Neutrophil adhesion to
fibroblasts is mediated by the 1 integrin CD29
(32, 33, 34). This forms heterodimers with the integrin,
CD49, and neutrophils have been shown to express CD49d, e, and f at
levels sufficient to mediate adhesion to extracellular matrix proteins
(35, 36, 37). Recent data have shown extravasated neutrophils
to express CD49b and that a blocking mAb inhibited neutrophil
locomotion in extravascular tissue in vivo (49).
In this study, we have investigated the role of CD29/CD49 integrin heterodimers in neutrophil migration in both CD18-dependent and CD18-independent pulmonary inflammation. In the first part of the study, we have shown that intratracheal instillation of LPS induced neutrophil migration that was CD18 dependent, while the response to instillation of the murine CXC chemokine KC was unaffected by CD18 blockade. We then used blocking mAbs against murine CD29 and CD49 subunits and, having demonstrated a role for CD29, determined at what stage of the neutrophil migration process this integrin was involved.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Male BALB/c mice (8–10 wk) were purchased from Harlan (Oxford, U.K.) and were kept in a temperature-controlled environment and allowed access to food and water ad libitum. Each animal was anesthetized with ketamine (100 mg/kg) and acepromazine (2.5 mg/kg) i.p. and allowed to recover in a supine position after surgery in a heated cage under constant supervision.
Materials
LPS from P. aeruginosa serotype 10 and rat IgG were
obtained from Sigma (Poole, U.K.). Mouse recombinant KC was obtained
from PeproTech (London, U.K.). mAbs to mouse Ly-6G (RB6-8C5, labeled
with PE), CD29 (HM1-1), CD49a (HA31/8), CD49b (HM2), CD49e
(5H10-27), and CD49f (GoH3) were obtained from PharMingen (Oxford,
U.K.). Anti-CD49d (PS/2) was a gift from M. Robinson (Celltech, Slough,
U.K.). All mAbs used i.v. were obtained in low azide/no endotoxin form
if possible and those that contained azide were dialyzed before in vivo
use. Hamster IgG was obtained from ICN Pharmaceuticals (Basingstoke,
U.K.). FITC-labeled rat IgG2a, rat IgG2b, and hamster IgG were obtained
from Serotec (Kidlington, U.K.). Sterile bottled PBS was purchased from
BioWhittaker (Wokingham, U.K.). Sterile saline was purchased from
Fresenius Kabi (Warrington, U.K.). The HM1-1 clone was a gift from
Hideo Yagita (Juntendo University, Tokyo, Japan), and
F(ab')2 and Fab fragmentation was conducted by
Cymbus Biotechnology (Chandlers Ford, U.K.).
Intratracheal instillation
The necks of anesthetized mice were first shaved and then opened
by a midline incision of 
1 cm on the ventral aspect. The trachea was
exposed by careful dissection of the surrounding tissues with blunt
forceps. Once exposed, a catheter (24 gauge, Jelco; Johnson & Johnson
Medical, Ascot, U.K.) was placed 0.5 cm into the trachea, and agents
(1.5 µl/g) were instilled into the lungs using a pipette with a
gel-loading tip. The agents were flushed through the catheter with air
(4.5 µl/g). Pinching the surrounding tissue together closed the
incision. Anti-integrin or control mAbs were administered i.v. 5 min
before intratracheal instillations. Animals remained on their backs in
a warmed cage until conscious and were then given food and water. After
4 h, the experiment was terminated by giving animals an overdose
of sodium pentobarbitone.
Bronchoalveolar lavage
Animals were given an overdose of anesthetic, as described. The chest cavity was carefully opened to allow the lungs to fully expand. The trachea was exposed and catheterized at the same point of entry as was previously used to instill test agents. The catheter was tied in place, and heparinized saline (10 U/ml) was instilled in 4x 1-ml aliquots. Lavage fluid was recovered and placed on ice. Ten microliters of each bronchoalveolar lavage (BAL)3 sample was stained with Kimura, and total cell counts were made using a Neubauer chamber. Cytospins were made for each BAL sample (100 µl) and stained with Diff-Quick rapid staining set (Merck, Dorset, U.K.) to allow differential cell counts to be made.
Determination of Ab doses for in vivo studies and integrin expression on peripheral blood neutrophils
Doses of anti-CD18 mAbs 2E6 and GAME-46 were the same as
those used in earlier work that demonstrated attenuation of neutrophil
adhesion and/or migration in vivo (16, 38). Saturating
concentrations of other mAbs were determined by flow cytometry.
Peripheral blood was collected by cardiac puncture and incubated with
primary mAb at various concentrations for 30 min at 4°C. After
washing, the blood was incubated in the dark for another 30 min at
4°C with a secondary, FITC-labeled Ab directed against the primary.
RBC were then lysed with Pharmlyse (PharMingen, San Diego, CA) for 15
min at room temperature in the dark. Cells were washed, placed on ice,
and analyzed on a FACScan flow cytometer (Becton Dickinson, Oxford,
U.K.). Gating was based on forward and side scatter parameters, and
PE-conjugated anti-mouse Ly-6G Ab was used to specifically gate on
neutrophils, as previously described (28). Fluorescent
intensity of 1 x 105 neutrophils was
analyzed and compared with nonspecific background fluorescence. The
concentrations of mAb that caused maximal increase in fluorescence
intensity were chosen for subsequent experiments, both in vitro and in
vivo. For mAbs that have been shown to bind to cells other than
leukocytes, saturating doses were also confirmed by administering the
mAb i.v. 15 min before cardiac puncture. The removed blood was then
incubated with and without further mAb, as described above, to
determine whether the i.v. dose saturated leukocytes. This was repeated
until there was no further shift in fluorescence, and this dose was
used in subsequent experiments.
Effect of Abs on circulating neutrophil numbers
All Abs used in these studies were assessed for their effects on circulating neutrophil numbers. Blood samples were taken before and 0.5, 1, 2, 3, and 4 h after i.v. Ab administration. Total and differential cell counts were performed on whole blood, and circulating neutrophil numbers were calculated. None of the mAbs used in this study reduced circulating neutrophil numbers.
Histologic analysis of neutrophil migration
Animals were anesthetized and lungs were instilled as described with KC (0.3 µg/mouse). Anti-mouse CD29 (20 µg) or hamster IgG (20 µg) was given i.v. via a tail vein. A control group of uninstilled mice with no i.v. treatment was also used. Four hours after KC instillation, animals were killed with an overdose of sodium pentobarbitone, and the lungs exposed and perfusion fixed with 3% glutaraldehyde. Sections (0.5 µm) were stained with toluidene blue and viewed under light microscopy. Inflamed areas were selected, cut into 0.1-µm sections, and analyzed under transmission electron microscopy (Philips 400T; Philips, Eindhoven, Holland). Viewed at a magnification of x2800, it was possible to identify neutrophils in the capillaries, the interstitium, and the alveoli within the alveolar septum. These were determined in sections taken from three sets of lungs for each of the treatments described. Any neutrophils captured in the process of transmigration, and therefore occupying more than one compartment, were excluded from calculations. The total number of neutrophils in each section of lung examined was recorded, and the percentage of neutrophils present in the capillaries, interstitium, and alveoli was calculated. A running mean was recorded and samples examined until the mean was unaltered by increasing the n number.
Statistical analysis
Results were analyzed for statistical significance using a one-way ANOVA, followed by Bonferroni’s test for multiple comparisons or Dunnett’s t test. Results were considered significant if p < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The effect of increasing doses of LPS (0.1–1 µg/mouse) and KC
(0.03–0.3 µg/mouse) on neutrophil numbers in BAL fluid 4 h
after intratracheal administration is shown in Fig. 1
. Intratracheal administration of both
agents resulted in a dose-dependent increase in neutrophil numbers that
was significant at the two higher doses of each compared with PBS
control. A dose of 0.3 µg of each was used in all subsequent
experiments.
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Fig. 2A shows the
differential effect of i.v. administration of anti-CD18 (2E6; 500
µg/mouse) and anti-CD29 (HM1-1; 20 µg/mouse) mAbs on LPS-
and KC-induced neutrophil migration in mouse lungs. Anti-CD18 inhibited
the response to LPS by 82% (p < 0.01), but
had no effect on neutrophil migration induced by KC. To confirm this
observation, experiments were repeated with GAME-46, another
anti-CD18 mAb. LPS-induced neutrophil accumulation
(x105 neutrophils in BAL) in the presence of
control mAb was 7.5 ± 1.4, and this was inhibited 72% by i.v.
treatment with 30 µg GAME-46. In contrast, KC-induced neutrophil
migration was 7.6 ± 1.9 and 7.5 ± 0.5 in control mAb- and
GAME-46-treated mice, respectively, confirming the CD18-independent
nature of response to KC.
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A).
Coadministration of anti-CD18 and anti-CD29 did not result in
more inhibition than that seen with administration of either alone,
suggesting that the two molecules do not act in synergy. As indicated
above, there was no inhibitory effect of mAbs on circulating neutrophil
numbers. Effect of F(ab')2 and Fab fragments of anti-CD29 on LPS- and KC-induced migration
To ensure the inhibition of LPS- and KC-induced neutrophil
migration by anti-CD29 mAb (HM1-1) was not due to an effect of
the whole Ab, we investigated the effects of
F(ab')2. Fig. 2B shows that both
responses were significantly (p < 0.01)
inhibited by 20 µg of the fragmented Ab, confirming a large
CD29-dependent component of LPS- and KC-induced migration. To rule out
the possibility that cross-linking of CD29 by
F(ab')2 could explain the results, additional
experiments were conducted using 20 µg of single Fab fragments. LPS-
and KC-induced neutrophil accumulation (x105
neutrophils in BAL) in the presence of control mAb were 9.3 ± 1
and 9.4 ± 1.7, and these were reduced to 0.3 ± 0.1 and
0.2 ± 0.1 by i.v. treatment with HM1-1 Fab, respectively,
adding further support for a role of CD29.
Site of action of anti-CD29 mAb on KC-induced neutrophil migration
Electron micrographs of lung sections taken from a KC-instilled
mouse are shown in Fig. 3. Using sections
such as these, we were able to determine the effects of i.v.
anti-CD29 mAb on the sequential steps of neutrophil migration in
the lung. All neutrophils were counted in each section, and two
sections from each set of lungs from three animals in each treatment
group were analyzed. The mean total number of neutrophils counted in
each section from each treatment group were: control, 37 ± 9; KC
plus control Ab, 91 ± 21; KC plus anti-CD29 mAb, 93 ±
28 neutrophils. Using these numbers and their distribution in
capillary, interstitial, or alveolar compartments, the percentage of
neutrophils in each area of lung was calculated. These data, shown in
Fig. 4, demonstrate that in control lungs
>94% of neutrophils are in capillaries, with the remainder in the
interstitium. No neutrophils were found in the alveolar space. In
contrast, 4 h after KC instillation, approximately one-half of the
observed neutrophils were in capillaries, with 30% in the interstitium
and 20% in the alveoli. Thus, while KC induces effective migration of
neutrophils to airspace after 4 h, the interstitium appears to be
a major compartment for these migrating cells. In the presence of the
anti-CD29 mAb, neutrophil migration to the alveoli was markedly
(p < 0.01) inhibited, as reflected in the
lavage data presented in Fig. 2, A and B.
Although the percentage of neutrophils present in the capillaries was
slightly, but significantly (p < 0.05),
increased by anti-CD29 mAb, neutrophils were not prevented from
entering the interstitial space. Thus, it would appear that CD29 is
involved primarily in the migration of neutrophils from the
interstitium into the alveoli and has a relatively minor role in the
migration of neutrophils across the capillary wall.
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Flow cytometric analysis was used to determine the expression of
CD49a, b, d, e, and f on unstimulated peripheral blood neutrophils, as
shown in Fig. 5. Neutrophils expressed
all of the subunits, but at varying levels compared with the relevant
isotype controls.
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Having established a role for CD29 in both LPS- and KC-induced
neutrophil migration into alveoli, we investigated the effects of mAbs
to the CD49 () subunits of CD49/CD29 integrin, as shown in Fig. 6. All Abs were either hamster or rat IgG
isotypes; controls were therefore hamster IgG or rat IgG. Since there
was no significant difference between responses to PBS (0.46 ±
0.14, i.v. hamster IgG; 0.47 ± 0.17, i.v. rat IgG), LPS (8.9
± 1.1, i.v. hamster IgG; 7.6 ± 0.8, i.v. rat IgG), or KC
(6.6 ± 0.9, i.v. hamster IgG; 7.2 ± 1.6, i.v. rat IgG) in
these control groups, the responses were pooled. The results show that
responses to both LPS and KC were significantly
(p < 0.01) inhibited by mAbs to CD49e (20
µg) and CD49f (10 µg). However, mAbs to mouse CD49b (20 µg) and
CD49d (40 µg) had a significant (p < 0.01
and p < 0.05, respectively) effect only on the
response to KC instillation. Anti-CD49a (20 µg) had no effect on
either LPS- or KC-induced neutrophil migration. Again, these inhibitory
effects were not due to a reduction in the number of circulating
neutrophils.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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was largely CD18 dependent (17). In this study, we
developed a mouse model, similar to that described by Doerschuk
(28, 39, 40, 41), that used a preformed chemoattractant, in
this case KC, to induce neutrophil emigration that was CD18
independent. We also found that intratracheal instillation of human C5a
in mouse lungs caused CD18-independent neutrophil migration, but the
response was less reliable and required a relatively high dose (1
µg/mouse) to induce a response approximately one-half the magnitude
of that induced by LPS or KC (data not shown). KC, a CXC chemokine, is
equivalent to human Gro and is known to be a potent inducer of
neutrophil migration in vivo. Based on published work on
Escherichia coli and LPS from E. coli (24, 29, 30), we predicted that neutrophil migration induced by LPS
would be CD18 dependent. Indeed, we found that the same dose of
anti-CD18 mAb that had no effect on KC-induced neutrophil migration
inhibited the response to LPS by >80%.
The anti-CD18 mAb chosen for these studies was 2E6 since this has
been used at similar doses as a CD18 function blocker in vivo. For
example, Borgstrom et al. (15) showed that 2E6
significantly inhibited leukotriene B4-induced
leukocyte adhesion and transmigration in TNF--stimulated mouse
venules. The same mAb has also been shown to significantly reduce the
number of neutrophils recovered in peritoneal lavage fluid after i.p.
injection of Chlamydia trachomatis (16).
However, to confirm that the results with 2E6 were indeed due to
functional inhibition of CD18, we used another anti-mouse CD18 mAb,
GAME-46 (42). This was used in a recent study
(38) to attenuate leukocyte adhesion in TNF--stimulated
venules of wild-type mice and confirms observations made in vessels of
CD18-/- mice. Thus, inhibition of LPS- but not
KC-induced neutrophil migration in mouse lung by 2E6 or GAME-46
provides strong evidence of a CD18-independent mechanism of
migration.
Why there should be a CD18-independent pathway for neutrophil migration in pulmonary inflammation remains uncertain. One explanation may be a differential up-regulation of ICAM-1 expression on capillary endothelium. A study by Burns et al. (39) showed that mouse pulmonary capillary endothelial cells show little constitutive ICAM-1 expression that was not altered after instillation of S. pneumoniae, a stimulus previously shown to induce CD18-independent migration (24). In contrast, LPS increases ICAM-1 on pulmonary capillary endothelial cells and on alveolar epithelial cells (43, 44), presumably resulting in CD18/ICAM-1-dependent migration. Indeed, in vivo studies have shown that Abs to ICAM-1 inhibit neutrophil migration to the lung 4 h after LPS instillation (45, 46). Since instilled KC would be expected to up-regulate neutrophil CD18 expression and function (47), the absence of endothelial ICAM-1 is the likely explanation for the emigration process showing complete independence on CD18.
Having established a model of CD18-independent neutrophil migration in
mouse lungs, we investigated the role of the 1
integrin CD29. Neutrophils express CD29 integrins, and these are known
to be involved in adhesion and migration of neutrophils across
extracellular matrix proteins (34, 35, 48, 49), and may
therefore facilitate migration of neutrophils through the alveolar
interstitium. Neutrophil migration to both LPS and KC was markedly
reduced by the anti-CD29 mAb HM1-1. This effect could not be
attributed to an FcR-mediated pathway or cross-linking since both
F(ab')2 and Fab fragments of the same mAb were
effective. We also showed that the effect of CD29 inhibition was not
increased when CD18 was blocked in combination, suggesting that these
integrins do not mediate the same component of the migration
pathway.
To determine at what stage of the migration process CD29 was involved,
we used electron microscopy to examine the effect of HM1-1 on
KC-induced neutrophil migration. The data indicate that although CD29
inhibition has a small effect on the migration of neutrophils across
capillary endothelium in response to KC, the greatest effect is on cell
movement from the interstitium to the airspace. That there was no
significant accompanying increase in neutrophils in the interstitial
compartment when CD29 was blocked was an unexpected finding. One
explanation is that there is space in the interstitium for a defined
number of neutrophils only so that when CD29 is blocked, no more
neutrophils can enter and are held up in the capillary. Behzad et al.
(31), studying CD18-independent (induced by S.
pneumoniae) neutrophil migration in rabbit lung, presented
compelling evidence that interstitial fibroblasts play a crucial role
in providing directional information to migrating neutrophils. They
found that 70% of the surface of migrating neutrophils was in close
proximity to fibroblasts or matrix proteins; since neutrophil adhesion
to these substrates is CD29 dependent (35), this is a
likely explanation for the effect of the CD29 mAb in our studies.
Moreover, Werr et al. (34) showed that neutrophil
migration in extravascular tissue of rat mesentery in response to
topical application of chemoattractant (platelet-activating factor
(PAF)) was markedly reduced by HM1-1. Migration in response to LPS
was not investigated by these authors but, because the response to LPS
in our study was attenuated by anti-CD29, we assume that the
mechanism by which neutrophils negotiate the interstitium to enter the
airspace is the same, regardless of the stimulus. Therefore, we
speculate that the difference between LPS- and KC-induced neutrophil
migration is up-regulation of ICAM-1 and dependency on CD18 for passage
across the capillary endothelium.
The CD29 integrin forms a noncovalent dimer with CD49 chains, of which
a number have been shown to be involved in inflammation. In a recent
study by de Fougerolles et al. (50), Abs to CD49a and
CD49b, both of which inhibit lymphocyte binding to collagen, inhibited
inflammation in murine models of delayed-type hypersensitivity, contact
hypersensitivity, and arthritis. In the present study, CD49a (which
preferentially binds type IV collagen) does not appear to have a
significant role in LPS- or KC-induced neutrophil migration. In
contrast, anti-CD49b mAb reduced KC- but not LPS-induced neutrophil
migration in the lung, thus revealing an adhesion pathway for
CD18-independent neutrophil migration in the pulmonary circulation. We
presume this pathway involves neutrophil adhesion to type I collagen.
Although not a direct comparison, our data are consistent with the data
of Werr et al. (49), who extended their earlier study and
showed that CD49b was the major integrin involved in PAF-induced
CD29 (or 1 integrin)-dependent extravascular
neutrophil motility in rat mesentery. They also found that local
administration of CD49b mAb with PAF in a mouse air pouch model was an
effective inhibitor of neutrophil recruitment, although they did not
investigate the involvement of this integrin in responses to LPS. We
found evidence of CD49b on circulating neutrophils, and others have
shown this molecule is up-regulated on migrated neutrophils
(49).
We also found that CD49d was involved in KC- but not LPS-induced responses, suggesting that CD18-independent neutrophil migration utilizes CD49d in addition to CD49b. Why CD49d and CD49b should be involved in KC- but not LPS-induced neutrophil migration is not clear. It can be speculated that as KC is a preformed chemokine, it induces a response much more rapidly than LPS. Moreover, the intensity of the stimulus may be far greater for instilled chemoattractant compared with KC released by lung cells in response to LPS stimulation (51, 52). Therefore, instilled KC may induce up-regulation of CD49b and CD49d function and/or expression on migrated neutrophils. VCAM-1 is a major ligand for CD49d and is present on lung fibroblasts (53, 54), but its role in this study was not investigated. CD49d also binds to the alternatively spliced variant of fibronectin, connecting segment-1 (CS-1), and although there is evidence for increased expression of CS-1 in inflamed kidneys and rheumatoid joints (55, 56), we do not know about its presence in the alveolar interstitium. In vitro studies have shown that CD49d facilitates C5a-induced neutrophil migration across lung fibroblast monolayers (48), although a role for VCAM-1 or CS-1 was not investigated. In rat lung, Li et al. (57) have shown that anti-CD18 and anti-CD49d Ab given individually had no effect on LPS-induced neutrophil accumulation in airspaces after 18 h. However, when given in combination, there was a significant reduction in neutrophil numbers in BAL fluid. Although our data are consistent with these findings with respect to anti-CD49d mAb, the results are in direct contrast with the present study and others showing that blockade of CD18 inhibits LPS-induced neutrophil migration in pulmonary inflammation (24, 29, 30). The apparent differences in results may reflect the time at which BAL fluid was taken (i.e., 4 h vs 18 h).
The relative roles of CD49e and CD49f in neutrophil migration in the lung were also investigated. CD49e, like CD49d, binds to fibronectin (35, 58), whereas CD49f has been shown to mediate binding to laminin (35). We found that either integrin could mediate migration of neutrophils in mouse lung, regardless of the stimulus. Both CD49e and CD49f contribute to C5a-induced migration of neutrophils across cultured lung fibroblasts in vitro (48), but this effect is seen only when CD18 is also blocked. In contrast to CD49d expression on mouse neutrophils, expression of CD49e and CD49f is high and not further increased by PMA (35). Thus, exposure of interstitial neutrophils to instilled KC or chemoattractant generated in the lung in response to LPS may not result in up-regulation of these integrins as we speculate might occur with CD49b and CD49d.
We conclude from the data presented that CD29 is required for neutrophil migration to alveolar airspaces whether or not CD18-ICAM-1 interactions have been employed. CD29 appears to be important in facilitating neutrophil migration across the interstitium and into the alveoli. There are, however, differences in the CD49 integrins required for CD18-dependent migration compared with those that are needed for CD18-independent migration to occur, and we have identified that CD49b and CD49d are involved in the latter.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Victoria Ridger, Cardiovascular Research Group, Clinical Sciences Center (NGH), University of Sheffield, Herries Road, Sheffield, S5 7AU, U.K.
3 Abbreviations used in this paper: BAL, bronchoalveolar lavage; CS-1, connecting segment-1; PAF, platelet-activating factor.
Received for publication August 3, 2000. Accepted for publication December 20, 2000.
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J. E. Ip, Y. Wu, J. Huang, L. Zhang, R. E. Pratt, and V. J. Dzau Mesenchymal Stem Cells Use Integrin beta1 Not CXC Chemokine Receptor 4 for Myocardial Migration and Engraftment Mol. Biol. Cell, August 1, 2007; 18(8): 2873 - 2882. [Abstract] [Full Text] [PDF]
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 J. G. Moreland and G. Bailey Neutrophil transendothelial migration in vitro to Streptococcus pneumoniae is pneumolysin dependent Am J Physiol Lung Cell Mol Physiol, May 1, 2006; 290(5): L833 - L840. [Abstract] [Full Text] [PDF]
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S. Ghosh, M. R. Wilson, S. Choudhury, H. Yamamoto, M. E. Goddard, B. Falusi, N. Marczin, and M. Takata Effects of inhaled carbon monoxide on acute lung injury in mice Am J Physiol Lung Cell Mol Physiol, June 1, 2005; 288(6): L1003 - L1009. [Abstract] [Full Text] [PDF]
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L. Allen, D. H. Dockrell, T. Pattery, D. G. Lee, P. Cornelis, P. G. Hellewell, and M. K. B. Whyte Pyocyanin Production by Pseudomonas aeruginosa Induces Neutrophil Apoptosis and Impairs Neutrophil-Mediated Host Defenses In Vivo J. Immunol., March 15, 2005; 174(6): 3643 - 3649. [Abstract] [Full Text] [PDF]
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C.-H. Woo, J.-H. Lim, and J.-H. Kim VCAM-1 upregulation via PKC{delta}-p38 kinase-linked cascade mediates the TNF-{alpha}-induced leukocyte adhesion and emigration in the lung airway epithelium Am J Physiol Lung Cell Mol Physiol, February 1, 2005; 288(2): L307 - L316. [Abstract] [Full Text] [PDF]
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S. Choudhury, M. R. Wilson, M. E. Goddard, K. P. O'Dea, and M. Takata Mechanisms of early pulmonary neutrophil sequestration in ventilator-induced lung injury in mice Am J Physiol Lung Cell Mol Physiol, November 1, 2004; 287(5): L902 - L910. [Abstract] [Full Text] [PDF]
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S. J. Skerrett, H. D. Liggitt, A. M. Hajjar, R. K. Ernst, S. I. Miller, and C. B. Wilson Respiratory epithelial cells regulate lung inflammation in response to inhaled endotoxin Am J Physiol Lung Cell Mol Physiol, July 1, 2004; 287(1): L143 - L152. [Abstract] [Full Text] [PDF]
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B. T. Edelson, Z. Li, L. K. Pappan, and M. M. Zutter Mast cell-mediated inflammatory responses require the {alpha}2{beta}1 integrin Blood, March 15, 2004; 103(6): 2214 - 2220. [Abstract] [Full Text] [PDF]
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D. H. Dockrell, H. M. Marriott, L. R. Prince, V. C. Ridger, P. G. Ince, P. G. Hellewell, and M. K. B. Whyte Alveolar Macrophage Apoptosis Contributes to Pneumococcal Clearance in a Resolving Model of Pulmonary Infection J. Immunol., November 15, 2003; 171(10): 5380 - 5388. [Abstract] [Full Text] [PDF]
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 K. Sasaki, T. Tsuji, T. Jinushi, J. Matsuzaki, T. Sato, K. Chamoto, Y. Togashi, T. Koda, and T. Nishimura Differential regulation of VLA-2 expression on Th1 and Th2 cells: a novel marker for the classification of Th subsets Int. Immunol., June 1, 2003; 15(6): 701 - 710. [Abstract] [Full Text] [PDF]
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 A. R. Burns, C. W. Smith, and D. C. Walker Unique Structural Features That Influence Neutrophil Emigration Into the Lung Physiol Rev, April 1, 2003; 83(2): 309 - 336. [Abstract] [Full Text] [PDF]
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S. J. Rowe, L. Allen, V. C. Ridger, P. G. Hellewell, and M. K. B. Whyte Caspase-1-Deficient Mice Have Delayed Neutrophil Apoptosis and a Prolonged Inflammatory Response to Lipopolysaccharide-Induced Acute Lung Injury J. Immunol., December 1, 2002; 169(11): 6401 - 6407. [Abstract] [Full Text] [PDF]
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G. Andonegui, S. M. Goyert, and P. Kubes Lipopolysaccharide-Induced Leukocyte-Endothelial Cell Interactions: A Role for CD14 Versus Toll-Like Receptor 4 Within Microvessels J. Immunol., August 15, 2002; 169(4): 2111 - 2119. [Abstract] [Full Text] [PDF]
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 S. Tasaka, S. E. Richer, J. P. Mizgerd, and C. M. Doerschuk Very Late Antigen-4 in CD18-Independent Neutrophil Emigration during Acute Bacterial Pneumonia in Mice Am. J. Respir. Crit. Care Med., July 1, 2002; 166(1): 53 - 60. [Abstract] [Full Text] [PDF]
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R.-F. Guo, N. C. Riedemann, I. J. Laudes, V. J. Sarma, R. G. Kunkel, K. A. Dilley, J. D. Paulauskis, and P. A. Ward Altered Neutrophil Trafficking During Sepsis J. Immunol., July 1, 2002; 169(1): 307 - 314. [Abstract] [Full Text] [PDF]
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U. MAUS, J. HUWE, L. ERMERT, M. ERMERT, W. SEEGER, and J. LOHMEYER Molecular Pathways of Monocyte Emigration into the Alveolar Air Space of Intact Mice Am. J. Respir. Crit. Care Med., January 1, 2002; 165(1): 95 - 100. [Abstract] [Full Text] [PDF]
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X.-p. Gao, N. Xu, M. Sekosan, D. Mehta, S. Y. Ma, A. Rahman, and A. B. Malik Differential Role of CD18 Integrins in Mediating Lung Neutrophil Sequestration and Increased Microvascular Permeability Induced by Escherichia coli in Mice J. Immunol., September 1, 2001; 167(5): 2895 - 2901. [Abstract] [Full Text] [PDF]
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, B). Resident lung macrophages
(mØ; B) were seen in the alveolar spaces of all lungs
examined. Neutrophils from each lung section were counted according to
which area of the lung they occupied. It was then possible to determine
the total number and proportion of cells in each compartment, as shown
in Fig. 4
