| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
*
Speros P. Martel Laboratory of Leukocyte Biology, Department of Pediatrics,
Department of Molecular and Human Genetics, and
Department of Medicine, Baylor College of Medicine, Houston, TX 77030;
§
Institute of Biosciences and Bioengineering, Rice University, Houston, TX;
¶
Department of Comparative Medicine, University of Alabama, Birmingham, AL 35294; and
||
Department of Pathology, Washington University School of Medicine, St. Louis, MO 63110
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
was reduced
by 67% and 59% in LFA-1- and CD18-deficient mice but increased by
198% in Mac-1-deficient mice. Genetic deficiencies demonstrate that
both LFA-1 and Mac-1 contribute to adhesion of neutrophils to
endothelial cells and ICAM-1, but adhesion through LFA-1 overshadows
the contribution from Mac-1. Neutrophil extravasation in response to
TNF- in LFA-1-deficient mice dramatically decreased, whereas
neutrophil extravasation in Mac-1-deficient mice markedly
increased. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
L2), CD11b/CD18 (Mac-1, CR3,
M2), CD11c/CD18 (p150,95,
x2), and CD11d/CD18
(d2) (2, 3, 4). Although leukocyte
adhesion deficiency I has given insight into the functional importance
of the CD11/CD18 integrins, the relative contributions of each of the
CD11 integrins remain unclear. LFA-1 and Mac-1 are the two most abundant CD11/CD18 integrins on neutrophils. A large number of studies have been performed both in vitro and in vivo to understand the functions of Mac-1 (5), with less evidence for the functional role of LFA-1 in neutrophils (6). Both these molecules have been shown to play roles in the firm adhesion of leukocytes to the endothelium, but LFA-1 appears dominant in transendothelial migration. Although CD11a and CD11b share structural similarities, are both located in a gene cluster on human chromosome 16, and function as ligands for ICAM-1 (CD54), these molecules clearly have differences in expression and unique functions that are not overlapping. LFA-1 cooperates with the TCR in Ag-stimulated T cell priming, and Mac-1 is an important receptor on phagocytic cells that recognizes ligands such as complement fragment iC3b. Surface levels of Mac-1 and LFA-1 on human neutrophils are approximately equal. However, levels of Mac-1 but not of LFA-1 can be rapidly increased after chemotactic stimulation because of the transport of Mac-1 to the cell surface from secretory granules. Although early in vivo studies using Abs against Mac-1 suggested that Mac-1 plays a dominant role in neutrophil emigration in vivo (7, 8), subsequent studies using Abs have demonstrated the importance of LFA-1 in neutrophil emigration (6, 9, 10).
Recently, mice deficient in Mac-1 have been developed by our group and others and found to have normal migration of neutrophils in the peritoneal cavity after chemical stimulus (6, 11). To define better the functions of LFA-1 that are unique and those that are overlapping with Mac-1, we have now generated mice deficient in LFA-1 by targeted homologous recombination in embryonic stem cells and compared neutrophil function both in vitro and in vivo with mice deficient in Mac-1, mice deficient in CD18, which therefore lack both Mac-1 and LFA-1, and wild-type (WT)3 mice.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
All animal studies were approved by the Institutional Review Board of Baylor College of Medicine.
Targeting construct and generation of LFA-1-deficient mutant mice
Murine CD11a cDNA was used as a probe to isolate two partially
overlapping genomic phage clones from a 129/Sv mouse lambda library
(Stratagene, La Jolla, CA). The genomic clones were characterized (Fig. 1
A) by restriction mapping,
Southern blot analysis, and DNA sequencing. A 7.0-kb genomic
restriction fragment containing exons 3–6 and a 3.1-kb genomic
restriction fragment upstream of exon 1 were ligated into the
polylinker of pBluescript SK+ (Stratagene). A neomycin cassette driven
by the mouse RNA polymerase II promoter was inserted between the two
fragments (Fig. 1B). The construct was linearized by
digestion with NotI, which cleaves in the polylinker
downstream of the CD11a sequence. The construct was designed so that
targeted homologous recombination with the murine CD11a gene
will result in a 2.1-kb genomic fragment containing exons 1 and 2 being
replaced with the 1.8-kb neomycin cassette (Fig. 1C).
|
C. Cells confirmed by Southern blotting to carry the
replacement mutation were injected into day 3.5 C57BL/6 blastocysts and
transferred into foster mothers. Chimeric males were mated with C57BL/6
females, and germline transmission was confirmed by Southern blotting
of tail DNA after digestion with EcoRI as indicated in Fig. 1D. Mac-1- and CD18-deficient mutant mice
Mac-1-deficient mice were generated as described by Lu et al. (6). CD18-null mice, which do not have CD11a or CD11b on the cell surface (14), were generated as described by Scharffetter-Kochanek et al. (15) and were back-crossed for at least 7 generations with C57BL/6J mice. The animals used for these experiments were 8- to 16-wk-old males. WT littermates were used as controls for mixed-background homozygous mutant mice (Mac-1 (-/-) and LFA-1 (-/-)), and WT C57BL/6J mice (Harlan, Indianapolis, IN) were used as controls for CD18-null mice.
Flow cytometric determination of neutrophil receptor expression
Isolated neutrophils were suspended at a concentration of 3
x 105 cells/ml in Hanks HEPES buffer (NaCl, 7.15
g/L; KCl, 0.75 g/L; MgCl2, 2.03 g/L; glucose, 1.8
g/L; HEPES, 7.15 g/L; pH, 7.35) containing 0.1% (w/v) human serum
albumin and 1.5 mM CaCl2. The cells were treated
with and without zymosan-activated serum (ZAS; 10% (v/v)) at 37°C
for 10 min. Thereafter, cells were maintained at 4°C and labeled with
fluorescence-conjugated mAbs specific for the cell adhesion molecules
(final concentration 4 µg/ml, 1 h) and analyzed by FACScan flow
cytometry (Becton Dickinson Immunocytometry, San Jose, CA). The
following rat mAbs to mouse cell adhesion molecules were used:
LFA-1-FITC (KBA (IgG2a), kindly provided by Hideo Yagita, Juntendo
University School of Medicine, or M17/4 (IgG2a), PharMingen, San Diego,
CA), Mac-1-FITC (M1/70.15 (IgG2b), CalTag Laboratories, Burlingame,
CA), CD18-PE (C71/16 (IgG2a), CalTag Laboratories), CD62L-FITC (MEL-14
(IgG2a), CalTag Laboratories), CD23-FITC (IgG2a, PharMingen), and
CD-45-Cy-Chrome (-CY3) (IgG2a, CalTag Laboratories). For acquisition,
the collection conditions were 3000 gated cells, the same settings
(forward scatter voltage E00, amplification gain 1.00; side scatter
voltage 290, amplification gain 3.17; fluorescence channel 1 (FL1) and
2 (FL2) voltage 650, amplification gain 8; and fluorescence channel 3
(FL3) voltage 600, amplification gain 1.00), and linear mode. The
neutrophil population was gated on in the forward-side scatter plot and
controlled by characteristic emission of CD45-CY3. The mean
fluorescence intensity (MFI) for the labeled receptors was quantified
on the green fluorescence channel (FL1) for FITC-labeled Abs, on the
red fluorescence channel (FL2) for the PE-labeled Ab, and on the orange
fluorescence channel (FL3) for the CY3-labeled Ab, using linear mode.
Analysis of cellular events was performed using FACScan analysis
software (Becton Dickinson). Quantitation of receptor number on
neutrophils under resting and ZAS-stimulated conditions was found by
comparing the binding of fluorescently conjugated anti-CD11a and
anti-CD11b to the cells with the binding to Quantum Simply Cellular
(7) anti-rat microbeads (Flow Cytometry Standards, San
Juan, Puerto Rico). These beads were derivatized with a range of rat
IgG-binding capacities (bead 1: 3674; bead 2: 11,878; bead 3: 40,487;
bead 4: 123,050). Bead suspensions were labeled with the Abs under
identical conditions as for the cells. Beads were analyzed by FACScan
using the same settings as for cells in the linear mode such that
resolution was 
10,000 binding sites. A linear relation of MFI to the
number of sites was obtained for each Ab. The relative receptor number
under resting and stimulated conditions was computed by subtracting the
fluorescence due to nonspecific Ab binding (CD23) from that for
anti-CD11a and anti-CD11b and applying the MFI vs site number
relation. All data are presented as mean ± SEM, n
= 2 separate neutrophil preparations.
Peripheral white blood cell counts
Blood was drawn from the tail vein. Ten µl of blood was added to Isoton II diluent (Coulter, Miami, FL), and erythrocytes were lysed with Manual Lyse (Stephens Scientific, Riverdale, NJ). The total number of leukocytes was determined by Coulter counter ZM (Coulter Electronics, Hiateach, FL). Blood smears were prepared with Neat stain (Midlantic Biomedical, Paulsboro, NJ) and analyzed under a Nikon phase contrast microscope.
Static adhesion assay
An adhesion assay was performed as described previously
(16) in a static adhesion chamber. One of the coverslips
of the chamber was either covered with an endothelial monolayer or
coated with either purified ICAM-1, fibrinogen (plasminogen free,
human; CalBiochem, La Jolla, CA), or keyhole limpet hemocyanin (KLH;
Sigma, St. Louis, MO). Mouse endothelial cell M1END1 was kindly
provided by Dr. R. Hallmann at Friedrich-Alexander University,
Erlangen, Germany. M1END1 cells were plated on 0.2% gelatin-coated
coverslips and incubated in DMEM with 10% FBS at 37°C until
confluent. Unilamellar liposomes containing purified mouse ICAM-1 were
prepared as previously described (17). Mouse ICAM-1 was
purified from the C3F6 mouse B lymphoma cell line and incorporated into
egg lecithin bilayers as previously described (17). To
prepare ligand-coated coverslips, 5 µl of ICAM-1-liposome suspension
(ICAM-1 density 600 molecules/µm2), fibrinogen
(1 mg/ml), or KLH (0.5 mg/ml) was spread onto a coverslip. The
coverslip was incubated in tissue culture incubator at 37°C for 30
min. The M1END1-covered or ICAM-1-liposome-coated coverslip was rinsed
twice with Dulbecco’s PBS (D-PBS) and assembled in the chamber for
adhesion assay. Mouse bone marrow neutrophils were prepared and
isolated as previously described (6). One milliliter of
neutrophil suspension (1 x 106 cells/ml)
was injected into the chamber. In certain experiments, neutrophils were
activated by adding ZAS to a final concentration of 1%. The cells were
allowed to settle on the coated coverslips for 500 s. The total
number of cells was recorded by counting the cells in 3 fields (x40
objective) under a Nikon phase contrast microscope. The chamber was
inverted for an additional 500 s. The number of cells remaining
adherent in the same field was recorded. The percentage of adherent
neutrophils was calculated as described previously (16).
In Ab-blocking experiments, Abs against CD11a (KBA), CD11b (M1/70), and
4 (PS2), and IgG2 control Ab SFDR5 (anti-HLA) were added to the
neutrophils at a concentration of 20 µg/ml. The suspensions were
mixed and incubated at 25°C for 20 min. ZAS was added immediately
before the cell suspension was injected into the chamber.
Detachment assay
The detachment assay was performed in a parallel flow chamber
connected to a hydrodynamic flow system and a Nikon phase contrast
microscope equipped with a video camera. Mouse endothelial cells were
cultured in 35-mm tissue culture dishes and stimulated with mouse
recombinant TNF- (5 ng/ml; R&D Systems, Minneapolis, MN) for 4
h. The endothelial monolayer was assembled onto the flow chamber.
Isolated bone marrow neutrophils (3 x 106)
were injected into the chamber and allowed to settle on the monolayer
for 5 min. Afterward, D-PBS was perfused into the system for 1 min at a
flow rate that resulted in a shear stress of 2.0
dynes/cm2. In the following 4 min, the shear
stress was doubled every minute until it reached 32
dynes/cm2. The entire experiment was conducted at
37°C. The number of firmly adherent cells was monitored under the
microscope and videotaped for analysis. The videotaped images were
analyzed with Optimas image analysis software (Bioscan, Edmonds, WA).
The percentage of firmly adherent cells that detached was calculated.
Firmly adherent cells were defined as cells that moved less than half
the cell diameter in a 10-s interval.
Leukocyte extravasation in subcutaneous air pouch
The air pouch was prepared by dorsal subcutaneous injection of
3.0 ml air on day 0 and day 3 (18). On day 6, 40 ng of
mouse recombinant TNF- (R&D Systems) in 1 ml of D-PBS solution was
injected into the matured air pouch. To enhance leukocyte response to
TNF-, we injected the dose of TNF- with 0.5%
carboxymethylcellulose (CMC), an inert carrier (19). At 2,
4, and 6 h after administration of TNF-, the mice were
sacrificed by injections of overdose of combined anesthetics (ketamine,
xylazine, and acepromazine). The air pouches were flushed with 8 ml of
D-PBS. Lavage fluids were centrifuged at 500 x g for
10 min at 4°C. The cells were resuspended in 0.5 ml D-PBS. The
total number of leukocytes was counted by Coulter counter ZM (Coulter
Electronics). Differential cell analysis was performed under a Nikon
phase contrast microscope after cytospinning and Neat staining
(Midlantic Biomedical). The air pouch wall was carefully dissected and
spread onto a clean slide. The tissue was fixed with a solution
containing ethanol, chloroform, and acetic acid (6:3:1, v/v/v) and
stained with 1% Alcian blue and 0.1% eosin (20).
Leukocyte DNA laddering
DNA laddering was performed as described by Lee et al. previously (21) with minor modification. In brief, leukocyte suspension from air pouch lavage was centrifuged at 500 x g for 10 min at 4°C. Leukocyte pellets were resuspended in 0.5 ml of STE buffer (100 mM NaCl, 10 mM Tris, pH 8.0, 1 mM EDTA, pH 8.0) containing 1% SDS and 0.2 mg/ml proteinase K. After incubation at 55°C overnight, samples were exacted twice with phenol, chloroform, and isoamyl alcohol mixture (25:24:1 v/v/v). DNA was precipitated with 2 vol of cold alcohol and washed once with 500 µl 70% cold alcohol. DNA pellets were air dried and resuspended in TE buffer (10 mM Tris, pH 8.0; 1 mM EDTA, pH 8.0). DNA was digested with 100 µg/ml DNase-free RNase at 37°C for 15 min and analyzed by 2% agarose gel electrophoresis using 1x TAE buffer (40 mM Tris-acetate, pH 8.0; 1 mM EDTA).
Statistical analysis
Results are presented as mean ± SD. Statistical analysis was performed using one-way ANOVA with Bonferroni correction for multiple comparisons, two-way ANOVA, and t test. A value of p < 0.05 was considered significant.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Embryonic stem cells with a targeted event had an 8.5-kb
EcoRI fragment identified by the 5'-flanking probe on
Southern blot compared with the 12.0-kb fragment in WT 129/Sv mice
(Fig. 1D). Targeted embryonic stem cell clones were injected
into C57BL/6J blastocysts, and male chimeric offspring that were >90%
agouti coat color were bred to C57BL/6J mice. Germline transmission was
confirmed by Southern blotting studies. The expected targeted allele of
8.5 kb was identified by the 5'-flanking probe on Southern blots of
mice heterozygous for the mutation (Fig. 1E). In addition, a
probe derived from exon 2 was also used in Southern blots of DNA from
mice homozygous for the mutation, which confirmed that exon 2 was
deleted in the mutant mice but present in the WT mice (data not
shown).
Homozygous and heterozygous mutant mice were born in expected ratio and
were fertile. Mutant mice did not demonstrate any gross abnormalities
in growth and development and did not develop an increased rate of
spontaneous infections when maintained in microisolator cages. Mice
that were homozygous for the targeted mutation in CD11a (CD11a (-/-))
had a significantly increased peripheral white blood cell count
compared with WT siblings that was primarily due to increased
neutrophils (Table I).
|
Flow cytometric analysis of isolated mouse neutrophils confirmed
that mAb recognition of LFA-1 in LFA-1-deficient mutants was identical
to the background level bead staining (Fig. 2A) and the nonimmune control.
To examine whether targeted mutations in either LFA-1 or Mac-1 would
lead to a compensatory increased expression of the other integrin
subunit, we quantitated the number of binding sites per neutrophil by
using flow cytometric analysis and beads calibrated with known numbers
of binding sites. Unstimulated neutrophils from mice deficient in LFA-1
had similar numbers of binding sites for Mac-1 compared with WT
littermates, and mice deficient in Mac-1 had similar numbers of binding
sites for LFA-1 compared with WT littermates (Fig. 2B).
After stimulation with 10% ZAS, the number of binding sites for Mac-1
increased by greater than 3-fold on WT and LFA-1-deficient neutrophils,
whereas there was no change in the number of sites for LFA-1 on either
WT or Mac-1-deficient neutrophils. LFA-1 was not detected above
nonspecific background on neutrophils from LFA-1-deficient mice, and
Mac-1 was not detected on neutrophils from Mac-1-deficient mice. CD62L
was expressed at similar levels on LFA-1-deficient and Mac-1-deficient
neutrophils and was equivalent to WT controls. Activation with ZAS was
accompanied by a loss of CD62L surface expression from all the mice,
reflecting a similar level of cell stimulation elicited across all the
strains (data not shown).
|
To specifically address the interaction of LFA-1 and Mac-1 with
ICAM-1, we studied the static adhesion of neutrophils from mice
deficient in LFA-1, Mac-1, and CD18 to purified mouse ICAM-1.
Neutrophils were isolated from the bone marrow of LFA-1 (-/-), Mac-1
(-/-), CD18 (-/-), and WT mice. The purity of neutrophils was
>75% as determined by Neat (Midlantic Biomedical) stained cytospin
preps before use in an adhesion assay. With ZAS stimulation, the
adhesion of WT neutrophils to ICAM-1 was increased by 166%. The
adhesion of CD18-deficient neutrophils was not increased with ZAS
stimulation (Fig. 3A), as
would have been predicted because these neutrophils lack both Mac-1 and
LFA-1. Although LFA-1-deficient neutrophils demonstrated activation by
ZAS as evidenced by up-regulation of Mac-1 on the cell surface,
adhesion to purified ICAM-1 was not increased after ZAS stimulation. In
contrast, adhesion of Mac-1-deficient neutrophils to ICAM-1 was
significantly increased after ZAS stimulation by 60% from basal
level, but the number of adherent cells was still significantly reduced
compared with that of WT neutrophils (Fig. 3A).
|
B). However, the adhesion
of Mac-1-deficient neutrophils was significantly increased with ZAS
stimulation, although the adhesion after activation was significantly
lower than that of WT neutrophils (see Fig. 3B). Thus,
stimulated neutrophils lacking LFA-1 did not show increased adhesion to
either purified ICAM-1 or endothelial cells despite evidence of ZAS
activation with increased surface Mac-1.
To determine the contribution of very late activation Ag 4 (VLA-4)
(CD49d/CD29, 4/ß1) in
neutrophil adhesion and to examine whether blocking mAbs gave results
similar to the genetic absence of the molecule, we used Abs against
LFA-1 (KBA), Mac-1 (M1/70), and CD49d (PS2) to block the adhesion of
ZAS-stimulated neutrophils to purified ICAM-1 and endothelial cells.
IgG2 control Ab SFDR5 did not affect neutrophil adhesion. Treatment of
WT neutrophils with Ab against LFA-1 blocked 80% of neutrophil
adhesion to purified ICAM-1 and 40% of adhesion to endothelial cells,
whereas anti-Mac-1 Ab blocked only about 30% of adhesion to
purified ICAM-1 and endothelial cells (Fig. 3C). Anti-CD49d
Ab PS2 had no significant inhibition on the adhesion of ZAS-stimulated
neutrophils (Fig. 3C). In addition, anti-CD49d did not
decrease the basal level of adhesion of unstimulated neutrophils to
ICAM-1 or endothelial cells (data not shown). Adhesion studies of
CD18-deficient human neutrophils (23) to stimulated
endothelium have previously demonstrated a CD18-independent mechanism
for neutrophil adhesion similar to that observed in the mouse (Figs. 2 and 3).
To examine the possibility that LFA-1 is required for Mac-1 to function
as an adhesion molecule, we examined the adhesion of neutrophils to two
Mac-1-dependent ligands, fibrinogen (24, 25) and KLH
(6). Adhesion to fibrinogen and KLH was the same for
neutrophils from LFA-1 (-/-) mice as for WT controls (Fig. 4), whereas adhesion of neutrophils from
Mac-1 (-/-) mice was completely abolished. Thus, in the absence of
LFA-1, Mac-1 on the neutrophil surface remained functional as an
adhesive molecule.
|
The strength of adhesion through LFA-1, Mac-1, and CD18 was
evaluated by the ability of neutrophils to maintain firm attachment
with increasing shear stress. When neutrophils and endothelial cells
were activated by ZAS and TNF- respectively, the adhesion strength
was resistant to incrementally increased shear stress in WT
neutrophils. Even at highest shear stress of 32
dynes/cm2, 70% of neutrophils remained
adherent. CD18-deficient neutrophils showed the greatest decrease in
adhesion; with an initial increase in shear stress to 2
dynes/cm2, 44% of neutrophils detached. At 32
dynes/cm2, 73% of neutrophils detached, more
than twice as many as WT controls (Fig. 5c). Neutrophils from
LFA-1-deficient mice also exhibited a substantial decrease in adherent
neutrophils. At 2 dynes/cm2, 33% of neutrophils
detached, with 62% detachment at 32 dynes/cm2
(Fig. 5a). Neutrophils deficient in Mac-1 showed a
statistically significant decrease in adhesion
(p 
0.05), but the increase in detachment was
much more modest and required higher shear forces than that observed
for either CD18- or LFA-1-deficient neutrophils (Fig. 5b).
|
-induced leukocyte extravasation in subcutaneous pouch
Leukocyte extravasation in a subcutaneous pouch is a
well-characterized model in regard to structure (26) and
function (18) used to examine leukocyte emigration in
response to a specific stimulus. Injection of TNF- into the pouch
induced a reproducible and dose-dependent leukocyte influx (data not
shown). The number of accumulated leukocytes in the pouch reached its
peak at 4 h after injection of TNF- and started to decrease at
6 h (data not shown). The predominant component of infiltrated
leukocytes at 4 h after injection of TNF- was neutrophils,
which comprised more than 80% of total leukocytes. Monocytes accounted
for about 15%, and eosinophils and fibroblast-like cells made up <5%
of the total cells.
To compare directly the contribution of LFA-1, Mac-1, and CD18 to
leukocyte extravasation, we injected 40 ng of TNF- into the air
pouches of LFA-1-, Mac-1-, and CD18-deficient mice and analyzed
leukocyte influx at 4 h after injection. In LFA-1- and
CD18-deficient mice, leukocyte influx was decreased by 67% and 59%,
respectively, compared with WT mice. Leukocyte influx in
Mac-1-deficient mice was increased by 198% (Fig. 6). The percentage of neutrophils,
monocytes, and other cells in the pouch was similar for all groups
(data not shown). There was no difference in the number of mast cells
attached to or in the wall of the air pouch as assessed by Alcian blue
staining.
|
)
(27). The results in Table II, which compare the relative change of
the emigration ratio, show that, in CD18-deficient mice, emigration
ratios of total leukocytes and neutrophils were decreased by 89% and
93%, respectively, whereas in LFA-1-deficient mice, emigration ratios
were decreased by 85% and 96%, respectively. Taken together, the data
suggest that LFA-1 is responsible for almost all the leukocyte influx
contributed by CD11/CD18 integrins in response to TNF-. In contrast
to LFA-1- and CD18-deficient mice, in Mac-1-deficient mice the
emigration ratios of total leukocytes and neutrophils were increased by
238% and 270%, respectively. To examine whether the increase in
leukocytes noted in Mac-1-deficient mice was due to reduced
apoptosis, we examined DNA laddering. As shown in Fig. 7, there was no evidence of apoptosis as
assessed by DNA laddering in leukocytes obtained 4 h after TNF-
stimulation in the air pouch, whereas DNA laddering was clearly evident
in leukocytes 48 h after TNF- stimulation.
|
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The number of binding sites for Abs to LFA-1 and Mac-1 was
approximately equal in unstimulated murine neutrophils, which is
similar to human neutrophils. A deficiency of either LFA-1 or Mac-1 did
not cause a compensatory increase in the number of binding sites for
the other ligand, and neutrophils deficient in LFA-1 exhibited a
similar increase in Mac-1 on the cell surface after ZAS stimulation
compared with WT neutrophils. The CD11/CD18 integrins have previously
been shown to play a critical role in neutrophil adhesion and
emigration, and neutrophils from CD18-deficient mice, which lack both
LFA-1 and Mac-1, did not show any increased adhesion to either murine
endothelial cells or ICAM-1 with ZAS stimulation. ZAS stimulation of
neutrophils from LFA-1-deficient mice also did not show increased
adhesion to either murine endothelial cells or ICAM-1, even though the
surface expression of Mac-1 increased severalfold. In summary, we found
that, after ZAS stimulation of neutrophils, increased expression of
Mac-1 in the absence of LFA-1 is not sufficient to increase adhesion to
either endothelial cells or ICAM-1, whereas the absence of Mac-1
modestly but significantly reduced adhesion when LFA-1 was present.
These results strongly suggest that both LFA-1 and Mac-1 are involved
in the adhesion of neutrophils to endothelial cells, but that adhesion
through LFA-1 overshadows the contribution from Mac-1. Our observation
is consistent with a recently published study by Andrew et al.
(29) that also showed that LFA-1 was important for
neutrophils to adhere to either resting or TNF--activated murine
endothelial cells. Cytokine activation of endothelial cells induces a
number of other cell adhesion molecules that may potentially influence
neutrophil-endothelial adhesion. Utilization of adhesion assay with
neutrophils and purified ICAM-1 allowed us to examine specifically
whether the absence of either LFA-1 or Mac-1 influences adhesion to
purified ICAM-1.
We considered several potential mechanisms that may explain why LFA-1 had a more dominant role than Mac-1. First, we ruled out the possibility that LFA-1 was more abundant than Mac-1 on the surface of murine neutrophils by quantitation of cell surface receptors by Ab-labeled beads. Although immunobead quantitation studies revealed that there are far more binding sites for Mac-1 than for LFA-1 on ZAS-stimulated neutrophils, these studies do not rule out the possibility that more molecules of LFA-1 have undergone a conformational change to a "high-avidity" conformation. However, this would not explain why the absence of LFA-1 would completely abolish the increase in adhesion, unless initial binding through LFA-1 were required to observe the separate contribution of Mac-1 adhesion.
We also examined whether LFA-1 is required for Mac-1 to function as an adhesion ligand. Neutrophils deficient in LFA-1 exhibited the same adhesion to fibrinogen and KLH as did WT neutrophils, but adhesion to these substrates was absent in neutrophils deficient in Mac-1. Thus, LFA-1 is not absolutely required for Mac-1 on activated neutrophils to function as an adhesion molecule to its ligands other than ICAM-1.
Finally, we examined whether LFA-1 may provide stronger adhesion to ICAM-1 than Mac-1 under shear. ICAM-1 is a common ligand for LFA-1 and Mac-1. It belongs to the Ig superfamily and consists of five Ig-like domains. In humans, LFA-1 binds to the first Ig domain of ICAM-1 (30), whereas Mac-1 binds to the third Ig domain (31). In the mouse, ICAM-1 has been identified as a ligand of LFA-1, resembling the human system (32, 33). Nevertheless, few studies have been conducted to characterize systematically the interaction of murine Mac-1 and ICAM-1. Lub et al. (34) examined the adhesion of murine macrophage cell lines that express varying amounts of LFA-1 and Mac-1 and found that both subunits can function as a ligand for ICAM-1 in the mouse as in the human. However, Mac-1-dependent adhesion to ICAM-1 was observed only when LFA-1 was absent. When LFA-1 and Mac-1 are coexpressed on the macrophage surface at similar levels, the adhesion is predominantly mediated by LFA-1. Therefore, Lub et al. concluded that there is competition between LFA-1 and Mac-1 for ICAM-1 binding. Although our studies also show that LFA-1 can itself mediate the majority of murine neutrophil adhesion to ICAM-1, neutrophils deficient in Mac-1 did have reduced adhesion in both static and shear flow conditions compared with WT neutrophils. Our observations are not consistent with the hypothesis that LFA-1 competes with Mac-1 for binding, because activated neutrophils that lacked LFA-1 and had high levels of Mac-1 did not show increased binding to purified ICAM-1 after ZAS stimulation.
We believe that these observations are consistent with the hypothesis that LFA-1 has greater avidity for ICAM-1 than does Mac-1. Once adhesion is initiated by the interaction of LFA-1 to ICAM-1, the more abundant but lower-avidity binding of Mac-1 to ICAM-1 may strengthen the adhesion. In support of this hypothesis, the absence of Mac-1 led to a significant increase in detachment of neutrophils under shear stress, but the increase in detachment was much more modest and required higher shear forces than that observed for LFA-1-deficient neutrophils. Another possibility is that the expression of LFA-1 and Mac-1 is influenced by receptor topography, though studies of LFA-1 and Mac-1 topography on mouse neutrophils are lacking. Previous studies that examined the topography of Mac-1 on human neutrophils (35) and LFA-1 on human lymphocytes (36) have shown Mac-1 and LFA-1 primarily on the membrane of the cell body and not clustered on microvilli; the topography of LFA-1 on neutrophils is unknown.
Chemical peritonitis induced by thioglycollate has been used in
evaluating leukocyte extravasation in LFA-1- (37), Mac-1-
(6), and CD18-deficient mice (14). With this
model, total extravasated leukocytes in LFA-1-deficient mice were
reduced by more than 50% at 4 h (37), whereas
leukocyte extravasation in Mac-1-deficient mice was not changed
significantly at 4 h (6) but increased substantially
at 12 h after administration of thioglycollate, mainly because of
an increase in neutrophils (11). However, in
CD18-deficient mice, leukocyte accumulation was reported to be
increased at 24 h after injection of thioglycollate
(14). These results are difficult to compare because each
knockout was studied in different experimental protocols and there was
no attempt to correct for baseline leukocytosis in the knockout.
Furthermore, the molecular signals that lead to leukocyte influx into
the peritoneum after thioglycollate injection have not been
characterized. Therefore, in this study we examined the effects of a
specific cytokine, TNF-, using a subcutaneous air pouch model, to
compare leukocyte extravasation directly in LFA-1-, Mac-1-, and
CD18-deficient mice. A pouch created by repeated injection of air
results in a connective tissue-lined space resembling the synovial
cavity (24). Injection of TNF- into the pouch triggers
the release of a number of chemokines including macrophage inflammatory
protein-2 (MIP-2), MIP-1, and JE. These chemokines, especially
MIP-2, induce acute leukocyte influx, which is dominated by neutrophils
(18). In LFA-1-deficient mice, leukocyte accumulation was
decreased by about 67%, whereas in Mac-1-deficient mice, leukocyte
accumulation increased by 198%, compared with WT controls.
Furthermore, the reduction in leukocyte accumulation in LFA-1-deficient
mice was very similar to that in CD18-deficient mice. The effect of
leukocyte migration in vivo that was observed in mice deficient in
LFA-1 was consistent with in vitro studies that showed that adhesion of
activated neutrophils deficient in LFA-1 to either ICAM-1 or
endothelial cells was similar to that of activated neutrophils
deficient in CD18. Although the in vitro studies suggested that the
absence of Mac-1 would not lead to a marked reduction in leukocyte
extravasation, the marked increase in leukocytes in the pouch at an
early time (4 h) after TNF- stimulation was surprising. Coxon et al.
(11) observed that, in thioglycollate-induced peritonitis,
neutrophil accumulation was increased by 3-fold at 10.5 h.
Extravasated neutrophils exhibited a significant decrease in apoptosis
at 10.5 h in Mac-1-deficient mice, suggesting that the absence of
Mac-1 could facilitate the accumulation of extravasated neutrophils
because of a reduction in programmed neutrophil death. Although reduced
apoptosis may play a role in the accumulation of neutrophils at later
time points, they found no evidence of apoptosis in
thioglycollate-induced peritonitis at 4 h. We also found no
evidence of apoptosis after 4 h in the TNF--stimulated air
pouch model by assessing DNA laddering, which was clearly evident at
48 h (see Fig. 7). In addition, mice deficient in Mac-1 have been
described to have significantly reduced numbers of mast cells resident
in the peritoneal cavity, peritoneal wall, and dorsal skin
(38). We found no difference in the number of mast cells
attached to or in the wall of the air pouch as assessed by Alcian blue
staining. Thus, the increased number of neutrophils in the pouch is
more likely due to an increased rate of influx. One potential
explanation for this observation is that Mac-1 may serve as a
"brake" after the neutrophil migrates below the endothelium as it
moves through the interstitial space and subsequently crosses the
mesothelium to enter the pouch. LFA-1 has been shown to support
migration of lymphocytes on purified ICAM-1 at a speed of 10 µm/min
(17), and WT murine neutrophils also exhibit a similarly
rapid migration on purified ICAM-1 (M. L. Dustin, unpublished
data). Therefore, it is possible that not only adhesion strength but
also the migration rate supported by LFA-1 vs Mac-1 on ICAM-1 or
cellular substrata would have to be considered to account for the in
vivo results. In contrast to neutrophil-endothelium interactions, the
molecular mechanisms that regulate neutrophil migration through the
interstitium remain poorly characterized.
In summary, these data both confirm and extend our knowledge of the
role that LFA-1 and Mac-1 play in neutrophil adhesion and
extravasation. Although both LFA-1 and Mac-1 have structural
similarities, function as ligands to ICAM-1, and play a role in the
adhesion of neutrophils to endothelial cells, genetic deficiencies in
LFA-1 and Mac-1 clearly demonstrate the unique contribution of each
molecule to neutrophil adhesion and extravasation. Direct comparison of
neutrophil adhesion in vitro demonstrated that both LFA-1 and Mac-1 are
involved in the adhesion of neutrophils to endothelial cells and
ICAM-1, but that adhesion through LFA-1 overshadows the contribution
from Mac-1. Neutrophil extravasation in response to TNF- in
LFA-1-deficient mice was dramatically reduced, whereas extravasation of
neutrophils in Mac-1-deficient mice was markedly increased.
Mac-1 has been previously shown to play an important role in the
adhesion of neutrophils to parenchymal cells (39, 40),
which is unique from LFA-1. The marked difference in neutrophil
extravasation observed in mice deficient in LFA-1 and Mac-1 in response
to TNF- further demonstrates the unique role that these molecules
play in neutrophil extravasation. LFA-1 appears to be more important in
neutrophil adhesion, which is an obligate step preceding extravasation,
whereas Mac-1 may be more important in events occurring after
transendothelial migration, including regulating adhesive interactions
of the neutrophil as it moves through the interstitial space.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Christie M. Ballantyne, Baylor College of Medicine, 6565 Fannin Street, M.S. A-601, Houston, TX, 77030. E-mail address:
3 Abbreviations used in this paper: WT, wild-type; KLH, keyhole limpet hemocyanin; ZAS, zymosan-activated serum; MFI, mean fluorescence intensity; CMC, carboxymethylcellulose; MIP, macrophage inflammatory protein; D-PBS, Dulbecco’s PBS.
Received for publication May 20, 1999. Accepted for publication August 13, 1999.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
. J. Immunol. 159:3595.[Abstract]
receptor III on human neutrophils: possible role of lectin-like interactions. J. Immunol. 150:3030.[Abstract]
4 integrins mediate lymphocyte attachment and rolling under physiologic flow. Cell 80:413.[Medline]
This article has been cited by other articles:
|
|
 |
|
  G. B. Menezes, W.-Y. Lee, H. Zhou, C. C. M. Waterhouse, D. C. Cara, and P. Kubes Selective Down-Regulation of Neutrophil Mac-1 in Endotoxemic Hepatic Microcirculation via IL-10 J. Immunol., December 1, 2009; 183(11): 7557 - 7568. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. T. Lefort, Y.-M. Hyun, J. B. Schultz, F.-Y. Law, R. E. Waugh, P. A. Knauf, and M. Kim Outside-In Signal Transmission by Conformational Changes in Integrin Mac-1 J. Immunol., November 15, 2009; 183(10): 6460 - 6468. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Szczur, Y. Zheng, and M.-D. Filippi The small Rho GTPase Cdc42 regulates neutrophil polarity via CD11b integrin signaling Blood, November 12, 2009; 114(20): 4527 - 4537. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. E. Byeseda, A. R. Burns, S. Dieffenbaugher, R. E. Rumbaut, C. W. Smith, and Z. Li ICAM-1 Is Necessary for Epithelial Recruitment of {gamma}{delta} T Cells and Efficient Corneal Wound Healing Am. J. Pathol., August 1, 2009; 175(2): 571 - 579. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. Li, J. J. Molldrem, and Q. Ma LFA-1 Regulates CD8+ T Cell Activation via T Cell Receptor-mediated and LFA-1-mediated Erk1/2 Signal Pathways J. Biol. Chem., July 31, 2009; 284(31): 21001 - 21010. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Wang, D. Li, R. Nurieva, J. Yang, M. Sen, R. Carreno, S. Lu, B. W. McIntyre, J. J. Molldrem, G. B. Legge, et al. LFA-1 Affinity Regulation Is Necessary for the Activation and Proliferation of Naive T Cells J. Biol. Chem., May 8, 2009; 284(19): 12645 - 12653. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Asai, F. Okajima, K. Nakagawa, D. Ibusuki, K. Tanimura, Y. Nakajima, M. Nagao, M. Sudo, T. Harada, T. Miyazawa, et al. Phosphatidylcholine hydroperoxide-induced THP-1 cell adhesion to intracellular adhesion molecule-1 J. Lipid Res., May 1, 2009; 50(5): 957 - 965. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. Y. Choi, E. Chavakis, M. A. Czabanka, H. F. Langer, L. Fraemohs, M. Economopoulou, R. K. Kundu, A. Orlandi, Y. Y. Zheng, D. A. Prieto, et al. Del-1, an Endogenous Leukocyte-Endothelial Adhesion Inhibitor, Limits Inflammatory Cell Recruitment Science, November 14, 2008; 322(5904): 1101 - 1104. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. von Vietinghoff and K. Ley Homeostatic Regulation of Blood Neutrophil Counts J. Immunol., October 15, 2008; 181(8): 5183 - 5188. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S.-H. Lee, J. E. Prince, M. Rais, F. Kheradmand, C. M. Ballantyne, G. Weitz-Schmidt, C. W. Smith, and D. B. Corry Developmental Control of Integrin Expression Regulates Th2 Effector Homing J. Immunol., April 1, 2008; 180(7): 4656 - 4667. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. Y. Choi, V. V. Orlova, S. C. Fagerholm, S. M. Nurmi, L. Zhang, C. M. Ballantyne, C. G. Gahmberg, and T. Chavakis Regulation of LFA-1-dependent inflammatory cell recruitment by Cbl-b and 14-3-3 proteins Blood, April 1, 2008; 111(7): 3607 - 3614. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Zarbock and K. Ley Mechanisms and Consequences of Neutrophil Interaction with the Endothelium Am. J. Pathol., January 1, 2008; 172(1): 1 - 7. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Z. Li, A. R. Burns, R. E. Rumbaut, and C. W. Smith {gamma}{delta} T Cells Are Necessary for Platelet and Neutrophil Accumulation in Limbal Vessels and Efficient Epithelial Repair after Corneal Abrasion Am. J. Pathol., September 1, 2007; 171(3): 838 - 845. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 X. Tai, F. Van Laethem, A. H. Sharpe, and A. Singer Induction of autoimmune disease in CTLA-4 / mice depends on a specific CD28 motif that is required for in vivo costimulation PNAS, August 21, 2007; 104(34): 13756 - 13761. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. Segura, C. Guerin, N. Hogg, S. Amigorena, and C. Thery CD8+ Dendritic Cells Use LFA-1 to Capture MHC-Peptide Complexes from Exosomes In Vivo J. Immunol., August 1, 2007; 179(3): 1489 - 1496. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. Graf, T. Bushnell, and J. Miller LFA-1-Mediated T Cell Costimulation through Increased Localization of TCR/Class II Complexes to the Central Supramolecular Activation Cluster and Exclusion of CD45 from the Immunological Synapse J. Immunol., August 1, 2007; 179(3): 1616 - 1624. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. A. Clemens, L. E. Lenox, T. Kambayashi, N. Bezman, J. S. Maltzman, K. E. Nichols, and G. A. Koretzky Loss of SLP-76 Expression within Myeloid Cells Confers Resistance to Neutrophil-Mediated Tissue Damage while Maintaining Effective Bacterial Killing J. Immunol., April 1, 2007; 178(7): 4606 - 4614. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
X.-P. Gao, X. Zhu, J. Fu, Q. Liu, R. S. Frey, and A. B. Malik Blockade of Class IA Phosphoinositide 3-Kinase in Neutrophils Prevents NADPH Oxidase Activation- and Adhesion-dependent Inflammation J. Biol. Chem., March 2, 2007; 282(9): 6116 - 6125. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. Varga, S. Balkow, M. K. Wild, A. Stadtbaeumer, M. Krummen, T. Rothoeft, T. Higuchi, S. Beissert, K. Wethmar, K. Scharffetter-Kochanek, et al. Active MAC-1 (CD11b/CD18) on DCs inhibits full T-cell activation Blood, January 15, 2007; 109(2): 661 - 669. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Szczur, H. Xu, S. Atkinson, Y. Zheng, and M.-D. Filippi Rho GTPase CDC42 regulates directionality and random movement via distinct MAPK pathways in neutrophils Blood, December 15, 2006; 108(13): 4205 - 4213. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. F. Smith, T. L. Deem, A. C. Bruce, J. Reutershan, D. Wu, and K. Ley Leukocyte phosphoinositide-3 kinase {gamma} is required for chemokine-induced, sustained adhesion under flow in vivo J. Leukoc. Biol., December 1, 2006; 80(6): 1491 - 1499. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Phillipson, B. Heit, P. Colarusso, L. Liu, C. M. Ballantyne, and P. Kubes Intraluminal crawling of neutrophils to emigration sites: a molecularly distinct process from adhesion in the recruitment cascade J. Exp. Med., November 27, 2006; 203(12): 2569 - 2575. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Z. Li, A. R. Burns, and C. W. Smith Lymphocyte Function-Associated Antigen-1-Dependent Inhibition of Corneal Wound Healing Am. J. Pathol., November 1, 2006; 169(5): 1590 - 1600. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Basit, J. Reutershan, M. A. Morris, M. Solga, C. E. Rose Jr., and K. Ley ICAM-1 and LFA-1 play critical roles in LPS-induced neutrophil recruitment into the alveolar space Am J Physiol Lung Cell Mol Physiol, August 1, 2006; 291(2): L200 - L207. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Ghosh, A. A. Chackerian, C. M. Parker, C. M. Ballantyne, and S. M. Behar The LFA-1 adhesion molecule is required for protective immunity during pulmonary Mycobacterium tuberculosis infection. J. Immunol., April 15, 2006; 176(8): 4914 - 4922. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. E. Green, U. Y. Schaff, M. R. Sarantos, A. F. H. Lum, D. E. Staunton, and S. I. Simon Dynamic shifts in LFA-1 affinity regulate neutrophil rolling, arrest, and transmigration on inflamed endothelium Blood, March 1, 2006; 107(5): 2101 - 2111. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. A. Francis, X. Shen, J. B. Young, P. Kaul, and D. J. Lerner Rho GEF Lsc is required for normal polarization, migration, and adhesion of formyl-peptide-stimulated neutrophils Blood, February 15, 2006; 107(4): 1627 - 1635. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. P. Pavlick, D. V. Ostanin, K. L. Furr, F. S. Laroux, C. M. Brown, L. Gray, C. G. Kevil, and M. B. Grisham Role of T-cell-associated lymphocyte function-associated antigen-1 in the pathogenesis of experimental colitis Int. Immunol., February 1, 2006; 18(2): 389 - 398. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Z. Liu, M. Zhao, N. Li, L. A. Diaz, and T. N. Mayadas Differential roles for beta2 integrins in experimental autoimmune bullous pemphigoid Blood, February 1, 2006; 107(3): 1063 - 1069. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
W. Jia, H. Li, and Y.-W. He The extracellular matrix protein mindin serves as an integrin ligand and is critical for inflammatory cell recruitment Blood, December 1, 2005; 106(12): 3854 - 3859. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. Heit, P. Colarusso, and P. Kubes Fundamentally different roles for LFA-1, Mac-1 and {alpha}4-integrin in neutrophil chemotaxis J. Cell Sci., November 15, 2005; 118(22): 5205 - 5220. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Guerau-de-Arellano, J. Alroy, D. Bullard, and B. T. Huber Aggravated Lyme Carditis in CD11a-/- and CD11c-/- Mice Infect. Immun., November 1, 2005; 73(11): 7637 - 7643. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Yang, R. M. Froio, T. E. Sciuto, A. M. Dvorak, R. Alon, and F. W. Luscinskas ICAM-1 regulates neutrophil adhesion and transcellular migration of TNF-{alpha}-activated vascular endothelium under flow Blood, July 15, 2005; 106(2): 584 - 592. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Soda, Y. Kano, T. Nakamura, K. Kasono, M. Kawakami, and F. Konishi Spermine, a Natural Polyamine, Suppresses LFA-1 Expression on Human Lymphocyte J. Immunol., July 1, 2005; 175(1): 237 - 245. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 X.-P. Gao, Q. Liu, M. Broman, D. Predescu, R. S. Frey, and A. B. Malik Inactivation of CD11b in a mouse transgenic model protects against sepsis-induced lung PMN infiltration and vascular injury Physiol Genomics, April 14, 2005; 21(2): 230 - 242. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. D. Cowden Dahl, S. E. Robertson, V. M. Weaver, and M. C. Simon Hypoxia-inducible Factor Regulates {alpha}v{beta}3 Integrin Cell Surface Expression Mol. Biol. Cell, April 1, 2005; 16(4): 1901 - 1912. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. M. Watts, F. J. M. Beurskens, I. Martin-Padura, C. M. Ballantyne, L. B. Klickstein, M. B. Brenner, and D. M. Lee Manifestations of Inflammatory Arthritis Are Critically Dependent on LFA-1 J. Immunol., March 15, 2005; 174(6): 3668 - 3675. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Liu, S. A. Caldwell, and S. I. Abrams Cooperative Disengagement of Fas and Intercellular Adhesion Molecule-1 Function in Neoplastic Cells Confers Enhanced Colonization Efficiency Cancer Res., February 1, 2005; 65(3): 1045 - 1054. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. A. Solovjov, E. Pluskota, and E. F. Plow Distinct Roles for the {alpha} and {beta} Subunits in the Functions of Integrin {alpha}M{beta}2 J. Biol. Chem., January 14, 2005; 280(2): 1336 - 1345. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. W. Calderwood, J. M. Williams, M. D. Morgan, G. B. Nash, and C. O. S. Savage ANCA induces {beta}2 integrin and CXC chemokine-dependent neutrophil-endothelial cell interactions that mimic those of highly cytokine-activated endothelium J. Leukoc. Biol., January 1, 2005; 77(1): 33 - 43. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. K. Shaw, S. Ma, M. B. Kim, R. M. Rao, C. U. Hartman, R. M. Froio, L. Yang, T. Jones, Y. Liu, A. Nusrat, et al. Coordinated Redistribution of Leukocyte LFA-1 and Endothelial Cell ICAM-1 Accompany Neutrophil Transmigration J. Exp. Med., December 20, 2004; 200(12): 1571 - 1580. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. Ren, M. A. McCrory, C. Pass, D. C. Bullard, C. M. Ballantyne, Y. Xu, D. E. Briles, and A. J. Szalai The Virulence Function of Streptococcus pneumoniae Surface Protein A Involves Inhibition of Complement Activation and Impairment of Complement Receptor-Mediated Protection J. Immunol., December 15, 2004; 173(12): 7506 - 7512. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. V. Arumugam, J. W. Salter, J. H. Chidlow, C. M. Ballantyne, C. G. Kevil, and D. N. Granger Contributions of LFA-1 and Mac-1 to brain injury and microvascular dysfunction induced by transient middle cerebral artery occlusion Am J Physiol Heart Circ Physiol, December 1, 2004; 287(6): H2555 - H2560. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Collin, A. Rossi, S. Cuzzocrea, N. S. A. Patel, R. Di Paola, J. Hadley, M. Collino, L. Sautebin, and C. Thiemermann Reduction of the multiple organ injury and dysfunction caused by endotoxemia in 5-lipoxygenase knockout mice and by the 5-lipoxygenase inhibitor zileuton J. Leukoc. Biol., November 1, 2004; 76(5): 961 - 970. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. G. Kevil, M. J. Hicks, X. He, J. Zhang, C. M. Ballantyne, C. Raman, T. R. Schoeb, and D. C. Bullard Loss of LFA-1, but not Mac-1, Protects MRL/MpJ-Faslpr Mice from Autoimmune Disease Am. J. Pathol., August 1, 2004; 165(2): 609 - 616. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Wu, J. R. Rodgers, X.-Y. D. Perrard, J. L. Perrard, J. E. Prince, Y. Abe, B. K. Davis, G. Dietsch, C. W. Smith, and C. M. Ballantyne Deficiency of CD11b or CD11d Results in Reduced Staphylococcal Enterotoxin-Induced T Cell Response and T Cell Phenotypic Changes J. Immunol., July 1, 2004; 173(1): 297 - 306. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. L. Dunne, R. G. Collins, A. L. Beaudet, C. M. Ballantyne, and K. Ley Mac-1, but Not LFA-1, Uses Intercellular Adhesion Molecule-1 to Mediate Slow Leukocyte Rolling in TNF-{alpha}-Induced Inflammation J. Immunol., December 1, 2003; 171(11): 6105 - 6111. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. S. Orange, K. E. Harris, M. M. Andzelm, M. M. Valter, R. S. Geha, and J. L. Strominger The mature activating natural killer cell immunologic synapse is formed in distinct stages PNAS, November 25, 2003; 100(24): 14151 - 14156. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Wu, J. E. Prince, C. F. Brayton, C. Shah, D. Zeve, S. H. Gregory, C. W. Smith, and C. M. Ballantyne Host Resistance of CD18 Knockout Mice against Systemic Infection with Listeria monocytogenes Infect. Immun., October 1, 2003; 71(10): 5986 - 5993. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Brickson, L. L. Ji, K. Schell, R. Olabisi, B. St. Pierre Schneider, and T. M. Best M1/70 attenuates blood-borne neutrophil oxidants, activation, and myofiber damage following stretch injury J Appl Physiol, September 1, 2003; 95(3): 969 - 976. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Gabrijelcic, A. Acuna, M. Profita, A. Paterno, K.F. Chung, A.M. Vignola, and R. Rodriguez-Roisin Neutrophil airway influx by platelet-activating factor in asthma: role of adhesion molecules and LTB4 expression Eur. Respir. J., August 1, 2003; 22(2): 290 - 297. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Emoto, Y. Emoto, V. Brinkmann, M. Miyamoto, I. Yoshizawa, M. Staber, N. van Rooijen, A. Hamann, and S. H. E. Kaufmann Increased Resistance of LFA-1-Deficient Mice to Lipopolysaccharide-Induced Shock/Liver Injury in the Presence of TNF-{alpha} and IL-12 Is Mediated by IL-10: A Novel Role for LFA-1 in the Regulation of the Proinflammatory and Anti-Inflammatory Cytokine Balance J. Immunol., July 15, 2003; 171(2): 584 - 593. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Miyamoto, M. Emoto, Y. Emoto, V. Brinkmann, I. Yoshizawa, P. Seiler, P. Aichele, E. Kita, and S. H. E. Kaufmann Neutrophilia in LFA-1-Deficient Mice Confers Resistance to Listeriosis: Possible Contribution of Granulocyte-Colony-Stimulating Factor and IL-17 J. Immunol., May 15, 2003; 170(10): 5228 - 5234. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. L. Borjesson, S. I. Simon, E. Hodzic, H. E. V. DeCock, C. M. Ballantyne, and S. W. Barthold Roles of neutrophil beta 2 integrins in kinetics of bacteremia, extravasation, and tick acquisition of Anaplasma phagocytophila in mice Blood, April 15, 2003; 101(8): 3257 - 3264. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Schramm, T. Schaefer, M. D. Menger, and H. Thorlacius Acute mast cell-dependent neutrophil recruitment in the skin is mediated by KC and LFA-1: inhibitory mechanisms of dexamethasone J. Leukoc. Biol., December 1, 2002; 72(6): 1122 - 1132. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Kadono, G. M. Venturi, D. A. Steeber, and T. F. Tedder Leukocyte Rolling Velocities and Migration Are Optimized by Cooperative L-Selectin and Intercellular Adhesion Molecule-1 Functions J. Immunol., October 15, 2002; 169(8): 4542 - 4550. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. Heit, S. Tavener, E. Raharjo, and P. Kubes An intracellular signaling hierarchy determines direction of migration in opposing chemotactic gradients J. Cell Biol., October 14, 2002; 159(1): 91 - 102. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Kupatt, R. Wichels, J. Horstkotte, F. Krombach, H. Habazettl, and P. Boekstegers Molecular mechanisms of platelet-mediated leukocyte recruitment during myocardial reperfusion J. Leukoc. Biol., September 1, 2002; 72(3): 455 - 461. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Wang, H. Hayashi, R. Harrison, B. Chiu, J. R. Chan, H. L. Ostergaard, R. D. Inman, J. Jongstra, M. I. Cybulsky, and J. Jongstra-Bilen Modulation of Mac-1 (CD11b/CD18)-Mediated Adhesion by the Leukocyte-Specific Protein 1 Is Key to Its Role in Neutrophil Polarization and Chemotaxis J. Immunol., July 1, 2002; 169(1): 415 - 423. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. F. H. Lum, C. E. Green, G. R. Lee, D. E. Staunton, and S. I. Simon Dynamic Regulation of LFA-1 Activation and Neutrophil Arrest on Intercellular Adhesion Molecule 1 (ICAM-1) in Shear Flow J. Biol. Chem., May 31, 2002; 277(23): 20660 - 20670. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A W Stadnyk, C Dollard, T B Issekutz, and A C Issekutz Neutrophil migration into indomethacin induced rat small intestinal injury is CD11a/CD18 and CD11b/CD18 co-dependent Gut, May 1, 2002; 50(5): 629 - 635. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. A. Bowden, Z.-M. Ding, E. M. Donnachie, T. K. Petersen, L. H. Michael, C. M. Ballantyne, and A. R. Burns Role of {alpha}4 Integrin and VCAM-1 in CD18-Independent Neutrophil Migration Across Mouse Cardiac Endothelium Circ. Res., March 22, 2002; 90(5): 562 - 569. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. L. Dunne, C. M. Ballantyne, A. L. Beaudet, and K. Ley Control of leukocyte rolling velocity in TNF-alpha -induced inflammation by LFA-1 and Mac-1 Blood, January 1, 2002; 99(1): 336 - 341. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. B. Forlow, J. R. Schurr, J. K. Kolls, G. J. Bagby, P. O. Schwarzenberger, and K. Ley Increased granulopoiesis through interleukin-17 and granulocyte colony-stimulating factor in leukocyte adhesion molecule-deficient mice Blood, December 1, 2001; 98(12): 3309 - 3314. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. M. Seo, L. V. McIntire, and C. W. Smith Effects of IL-8, Gro-alpha , and LTB4 on the adhesive kinetics of LFA-1 and Mac-1 on human neutrophils Am J Physiol Cell Physiol, November 1, 2001; 281(5): C1568 - C1578. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Kanwar, C. W. Smith, F. R. Shardonofsky, and A. R. Burns The Role of Mac-1 (CD11b/CD18) in Antigen-Induced Airway Eosinophilia in Mice Am. J. Respir. Cell Mol. Biol., August 1, 2001; 25(2): 170 - 177. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Bouvard, C. Brakebusch, E. Gustafsson, A. Aszodi, T. Bengtsson, A. Berna, and R. Fassler Functional Consequences of Integrin Gene Mutations in Mice Circ. Res., July 30, 2001; 89(3): 211 - 223. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. B. Henderson, L. H.K. Lim, P. A. Tessier, F. N.E. Gavins, M. Mathies, M. Perretti, and N. Hogg The Use of Lymphocyte Function-Associated Antigen (Lfa)-1-Deficient Mice to Determine the Role of Lfa-1, Mac-1, and {alpha}4 Integrin in the Inflammatory Response of Neutrophils J. Exp. Med., July 16, 2001; 194(2): 219 - 226. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. E. Prince, C. F. Brayton, M. C. Fossett, J. A. Durand, S. L. Kaplan, C. W. Smith, and C. M. Ballantyne The Differential Roles of LFA-1 and Mac-1 in Host Defense Against Systemic Infection with Streptococcus pneumoniae J. Immunol., June 15, 2001; 166(12): 7362 - 7369. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Tyagi, L. B. Klickstein, and A. Nicholson-Weller C5a-stimulated human neutrophils use a subset of {beta}2 integrins to support the adhesion-dependent phase of superoxide production J. Leukoc. Biol., November 1, 2000; 68(5): 679 - 686. [Abstract] [Full Text]
|
|
|
|
|
|
F. Leite, J. F. Brown, M. J. Sylte, R. E. Briggs, and C. J. Czuprynski Recombinant Bovine Interleukin-1beta Amplifies the Effects of Partially Purified Pasteurella haemolytica Leukotoxin on Bovine Neutrophils in a beta 2-Integrin-Dependent Manner Infect. Immun., October 1, 2000; 68(10): 5581 - 5586. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. W. Smith Introduction: functional polarity of motile neutrophils Blood, April 15, 2000; 95(8): 2459 - 2461. [Full Text] [PDF]
|
|
|
|
|
|
R. A. Bowden, Z.-M. Ding, E. M. Donnachie, T. K. Petersen, L. H. Michael, C. M. Ballantyne, and A. R. Burns Role of {alpha}4 Integrin and VCAM-1 in CD18-Independent Neutrophil Migration Across Mouse Cardiac Endothelium Circ. Res., March 22, 2002; 90(5): 562 - 569. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
