* Department of Microbiology and Immunology;
Speros P. Martel Laboratory of Leukocyte Biology, Department of Pediatrics; and
Section of Cardiovascular Science, The Methodist Hospital, Department of Medicine, Baylor College of Medicine, Houston, Texas
Correspondence: C. Wayne Smith, M.D., Section of Leukocyte Biology, Children’s Nutrition Research Center, Room 6014, 1100 Bates, Houston, TX 77030. E-mail: cwsmith{at}bcm.tmc.edu
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: adhesion molecules • canine • ICAM-1 • endothelial cells • CD11b/CD18 • Mac-1
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Berton et al. [7 ] have raised the possibility that LFA-1 may also function as a signaling molecule for the production of H2O2 by neutrophils. They found that when neutrophils were allowed to settle onto plastic surfaces coated with anti-CD11a mAb, substantial H2O2 release could be detected. This observation has been confirmed by Menegazzi et al. [8 ]. The anti-CD11a mAb was apparently sufficient to induce the respiratory burst, because the experiments did not include added stimulants such as chemotactic factors. Signaling through LFA-1 may be biologically relevant, because it is clearly established that neutrophils utilize LFA-1 to adhere to [5 ] and migrate through endothelial monolayers [5 , 6 ]. However, studies of Berton et al. did not provide evidence that LFA-1 signals H2O2 production by neutrophils following binding to a natural ligand.
In the current study, we have evaluated H2O2 production by neutrophils following LFA-1 binding to ICAM-13. To reduce possible signaling through Mac-1 adhering to ICAM-1, we developed a chimeric ICAM-1 construct of canine domains 1 and 2 and of human domains 3–5. We found in preliminary experiments that canine neutrophils failed to recognize human ICAM-1 (hICAM-1) but bound efficiently to canine ICAM-1 (cICAM-1). Previous studies have shown that the human LFA-1 binding site on hICAM-1 involves domains 1 and 2 [9 ], and the human Mac-1 binding site involves hICAM-1 domain 3 [9 ]. We observed that canine neutrophils release H2O2 after adhesion to the chimeric ICAM-1 that was blocked by anti-CD11a mAb but not by anti-CD11b mAb. H2O2 production occurred without added chemotactic stimulation.
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mAbs
R15.7 [immunoglobulin G1 (IgG1)], anti-CD18
[10
], R7.1 (IgG1), anti-CD11a [11
,
12
], R6.1, and CA7 (IgG1) specific for human ICAM-1
domains 2 and 5 [13
] were kindly provided by Dr. R.
Rothlein (Boehringer Ingelheim Pharmaceutical, Ridgefield, CT). MY904
[IgG1 and F(ab')2], anti-CD11b, was obtained from Lilly
(Indianapolis, IN). CL18/6 and CL18/1 [IgG1 and
F(ab')2], anti-canine ICAM-1 [14
], and
F(ab')2 of R7.1 and R15.7 were made using the
ImmunoaffinityPAK kit (Pierce).
Culture of canine endothelial cells
Canine jugular vein endothelial cells (CJVEC) were isolated and
cultured as described [10
], and confluent monolayers on
0.1% gelatin-coated, 25 mm glass coverslips were prepared from the
first through the fourth passages.
Isolation of canine polymorphonuclear neutrophil (PMN)
Neutrophils were isolated from healthy mongrel dogs as described
previously [14
]. Blood samples were anticoagulated with
citrate phosphate dextrose (Abbot, Chicago, IL) (0.14 ml/ml blood) and
sedimented in 1% (wt/vol in 0.87% NaCl) Dextran (Spectrum Chemical,
New Brunswick, NJ) for 45 min at room temperature.
Leukocyte-rich plasma was layered on Ficoll-Hypaque gradients, and
neutrophils were recovered, washed, and resuspended in DPBS, pH 7.4,
containing 0.2% dextrose. Final leukocyte suspensions contained >95%
neutrophils and were used immediately for H2O2
assays or maintained at 4°C before use in adhesion assays.
Adhesion assay
Transfected L cells or CJVEC were plated onto 0.1%
gelatin-pretreated, 25 mm coverslips and allowed to become visually
confluent. A visual static adhesion assay was described in detail
previously [15
]. In studies designed to evaluate the
involvement of ß2-integrins or ICAM-1 in neutrophil
adhesion, cells were preincubated as follows: Coverslip with
transfected L cells or CJVEC monolayer was treated with anti-ICAM-1
mAbs at 20 µg/ml concentration in 1 ml of PBS for 30 min at room
temperature and was mounted in the adhesion chamber directly.
Neutrophils were incubated with antibodies specific for integrin
subunits at 2–4 times the saturating concentration at room temperature
for 30 min. Chemotactic stimuli of 1% zymosan-activated-serum (ZAS; as
previously described) [10
] for canine neutrophils was
added immediately before injecting the cell mixture into the adhesion
chamber.
Measurement of hydrogen peroxide production
Hydrogen peroxide production was quantitated by a modification
of the method described by Nathan [1
]. Briefly, L-cell
plates (Linbro, Flow, McLean, VA) were treated with 200 µl of 0.1%
gelatin for 1 h at room temperature before adding trypsinized
L-cell transfectants. Cells reached confluency in 2–3 days, as judged
by phase contrast microscopy. Plates were washed three times with
prewarmed DPBS or Kreb-Ringer Phosphate (KRP) to 37°C by gentle
blotting onto a paper towel. For Mac-1-dependent adhesion, plates were
coated with 150 µl (slightly greater than final volume of
experimental incubation) of a 0.5 mg/ml KLH (Sigma) solution in DPBS
for at least 60 min at 37°C [3
]. Plates were washed
three times with DPBS or KRP by flicking out contents. The assay
reaction mixture (100 µl/well), prepared from 10x stock solutions,
was 24 µM scopoletin, 5 µg/ml horseradish peroxidase, 1 mM sodium
azide, and 5 mM glucose in low-phosphate KRP. F(ab')2
fragments of mAbs were added to wells (20 µl/well) from 7x solutions
to achieve final saturating concentrations. Unless otherwise indicated,
10–20 µg/ml was used. For canine experiments, 1.4 µl ZAS was added
to 1% of the final volume. Neutrophils were added in a volume of 20
µl to achieve a final concentration of 2 x
104/well. Wells not receiving antibody or chemotactic
factor received equal volumes of appropriate vehicles so that final
incubation volumes were 
140 µl. Experiments were performed in
replicates of four or eight each, and fluorescence of scopoletin was
determined immediately after addition of neutrophils and at 30-min
intervals thereafter in a Titertek Fluroskan II fluorometer (Flow) with
excitation and emission wavelengths of 355 nm and 460 nm, respectively.
Plates were maintained at 37°C in the absence of CO2.
Values of hydrogen peroxide production (decrease in scopoletin
fluorescence compared with baseline at time zero) were plotted vs.
time, and the areas under the curve for each experimental condition
were determined using a digitizer pad and Sigma Scan software (Jandel
Scientific, Sausalito, CA). Statistical comparisons were made by
analysis of variance.
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subunits of LFA-1 and Mac-1. Antibody
R7.1 (CD11a) inhibited adhesion as effectively as R15.7 (anti-CD18),
while MY904 (anti-CD11b) was without effect. Confirmation that the
adhesion was dependent on interaction with ICAM-1 was obtained using
antibodies against canine ICAM-1. We recently analyzed the binding
characteristics of mAbs CL18/1 and CL18/6 Hydrogen peroxide induces
LFA-1-dependent neutrophil adherence to cardiac myocytes (H. Lu,
K. Youker, C. Ballantyne, M. Entman, C. W. Smith, unpublished
results). These mAbs bound to L cells expressing full-length
canine ICAM-1. CL18/1 and CL18/6 did not bind to L cells expressing
wild-type human ICAM-1. The binding specificities of these two
antibodies were characterized by using two chimeric ICAM-1 constructs,
C1,2:H3–5 and H1,2:C3–5. CL18/1 bound to L cells expressing
H1,2:C3–5 but not to C1,2:H3–5, and CL18/6 bound to C1,2:H3–5 but
not to H1,2:C3–5. As shown in Figure 1
, CL18/6 used in the current
experiment inhibited neutrophil adhesion to chimeric ICAM-1
(C1,2:H3–5) and anti-CD11a, while CD18/1 was without effect,
indicating that the LFA-1-dependent adhesion involved the chimeric
ICAM-1 (C1,2:H3–5) on the L cells. Unstimulated neutrophils exhibited
minimal adhesion to mock-transfected L cells.
 View larger version (30K): [in a new window] |
Figure 1. Adhesion of unstimulated canine neutrophils to C1,2:H3–5 chimeric
ICAM-1-transfected L cells. Coverslips (25 mm) with confluent L-cell
transfectants were mounted to adhesion chambers. Freshly isolated
canine neutrophils were injected into the adhesion chamber and allowed
to adhere at 37°C for 15 min. Effects of adhesion molecules were
evaluated in the presence of the following mAbs: 10 µg/ml R7.1
(anti-CD11a), 20 µg/ml MY904 (anti-CD11b), 20 µg/ml R15.7
(antiCD18), 20 µg/ml CL18/1 (anti-ICAM-1, nonblocking), and 20
µg/ml CL18/6 (anti-ICAM-1). Values represent mean ±
SEM, n = 3, *P < 0.05.
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Figure 2. Adhesion of unstimulated canine neutrophils to LPS-stimulated CJVEC
monolayers. Confluent canine endothelial cell monolayers were treated
with 30 ng/ml of LPS for 18–24 h, washed by dipping in PBS, and
incubated with PBS (control), or 20 µg/ml CL18/1 (anti-ICAM-1,
nonblocking) or CL18/6 (anti-ICAM-1). These monolayers were then placed
in adhesion chambers. Isolated canine neutrophils were incubated at
room temperature in PBS, 10 µg/ml R7.1 (anti-CD11a), 20 µg/ml MY904
(anti-CD11b), 20 µg/ml R15.7 (antiCD18), or R7.1 and MY904 combined,
and the cell suspension containing antibody was injected into the
adhesion chamber. Adherence was determined using a visual assay at room
temperature. Values represent mean ± SEM,
n = 5, *P < 0.05.
|
800 pmoles per 2 x
104 neutrophils were seen (Fig. 3)
. To evaluate the
possible adhesion molecules involved, F(ab')2 fragments
of mAbs specific for ß2 integrins and ICAM-1 were added
(Fig. 4
). R7.1 (anti-CD11a) significantly inhibited the hydrogen peroxide
secretion, and CL18/6 was as effective as R7.1. MY904 (anti-CD11b) was
without effect as was the anticanine ICAM-1 (CL18/1) that fails to bind
to this chimeric ICAM-1. The blocking efficacy of MY904
F(ab')2 was tested on Mac-1- dependent neutrophil
adhesion and H2O2 production. MY904
F(ab')2 at 10 µg/ml significantly inhibited
ZAS-stimulated canine neutrophil adherence (Fig. 5A
) to KLH and H2O2 production (Fig. 5B)
. These
results indicate that LFA-1-dependent adherence is an important
contributor to the hydrogen peroxide production when unstimulated
canine neutrophils were interacting with canine ICAM-1 domains 1 and 2
expressed on L cells. When parent L cells, mock-transfected L cells, or
those expressing chimeric ICAM-1 were evaluated for
H2O2 production in the absence of added
neutrophils, none was found. In addition, crosslinking the expressed
C1,2:H3–5 chimeric ICAM-1 on L-cell monolayers by first binding CL18/6
and then adding goat antimouse antibody also failed to result in
H2O2 production (unpublished results).
 View larger version (19K): [in a new window] |
Figure 3. Kinetics of hydrogen peroxide production when unstimulated canine
neutrophils were exposed to C1,2:H3–5 chimeric ICAM-1-transfected L
cells and mock-transfected L cells. Ninety-six-well plates with
confluent monolayers of C1,2:H3–5 chimeric ICAM-1-transfected L cells
and mock-transfected L cells were rinsed with warm PBS three times
before neutrophils were added. Neutrophils at 2 x
104/well were incubated with the L cells up to 3 h.
Hydrogen peroxide production was measured by the scopoletin assay.
Results were represented as values above the background. Values are
mean ± SEM, n = 7, and the curves are
significantly different, P < 0.01.
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Figure 4. Effect of anti-CD11a, anti-CD11b, and anti-ICAM-1 on hydrogen peroxide
production from unstimulated canine neutrophils exposed to C1,2:H3–5
chimeric ICAM-1-transfected L cells. Ninety-six-well plates with
confluent monolayers of C1,2:H3–5 chimeric ICAM-1-transfected L cells
were rinsed with warm PBS three times before neutrophils were added.
Neutrophils were incubated with chimeric ICAM-1-transfected L cells in
quadruplicate for 3 h in the presence of PBS (control) or
F(ab')2 fragments of 10 µg/ml R7.1 (anti-CD11a), 20
µg/ml MY904 (anti-CD11b), 20 µg/ml R15.7 (anti-CD18), 20 µg/ml
CL18/1 (anti-ICAM-1, nonblocking), and 20 µg/ml CL18/6 (anti-ICAM-1).
Hydrogen peroxide production was measured by the scopoletin assay.
Results were represented as values above L-cell basal level. Values are
mean ± SEM, n = 7, *P < 0.01.
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 View larger version (31K): [in a new window] |
Figure 5. Blocking effect of mAb MY904 (anti-CD11b). (A) KLH-coated coverslips
were mounted into adhesion chambers. Canine neutrophils stimulated with
1% ZAS were injected into adhesion chamber in the presence of PBS or
F(ab')2 fragments of MY904. Adhesion of neutrophils was
measured. (B) Canine neutrophils were added to KLH-coated 96-well
plates in the presence of 1% ZAS and F(ab')2 fragments
of MY904. Hydrogen peroxide production was measured by scopoletin
assay. Values represent mean ± SEM, n = 3. Values for MY904 concentrations above 2.5 µg/ml were
significantly different from controls, P < 0.01.
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 View larger version (28K): [in a new window] |
Figure 6. Effect of NIF on the hydrogen peroxide production from neutrophils
exposed to C1,2:H3–5 chimeric ICAM-1 or KLH. KLH or L cell transfected
with C1,2:H3–5 chimeric ICAM-1 was prepared in 96-well plates.
Unstimulated neutrophils were added to chimeric ICAM-1 in the presence
of PBS or NIF. Neutrophils added on KLH were stimulated with ZAS (1%
vol) and incubated in the presence of NIF. Hydrogen peroxide production
was measured for 3 h by scopoletin assay. Values represent
mean ± SEM, n = 3, *P < 0.05.
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 View larger version (18K): [in a new window] |
Figure 8. Kinetics of hydrogen peroxide production from neutrophils exposed to
C1,2:H3–5 chimeric ICAM-1 and KLH. KLH or L cell transfected with
C1,2:H3–5 chimeric ICAM-1 was prepared in 96-well plates. Unstimulated
neutrophils were added to chimeric ICAM-1. Neutrophils added on KLH
were stimulated with ZAS (1% vol). Hydrogen peroxide production was
measured for 3 h by scopoletin assay. (A) Expanded view of the
first 60 min of the experiment shown in (B). Values represent mean ± SEM, n = 7; *, P < 0.01.
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 View larger version (18K): [in a new window] |
Figure 9. Effect of cytochalasin B or H7 on hydrogen peroxide production from
neutrophils exposed to C1,2:H3–5 chimeric ICAM-1 or KLH. KLH or L cell
transfected with C1,2:H3–5 chimeric ICAM-1 was prepared in 96-well
plates. (A) Unstimulated neutrophils were added to chimeric ICAM-1 in
the presence of PBS (control) or cytochalasin B at 2 µg/ml, 5
µg/ml, or 10 µg/ml. Neutrophils added on KLH were stimulated with
ZAS (1% vol) and incubated in the presence of PBS (control) or
cytochalasin B. Hydrogen peroxide production was measured after 3 h by scopoletin assay. Values represent mean ± SEM,
n = 3, *P < 0.05. (B)
Unstimulated neutrophils were added to chimeric ICAM-1 in the presence
of PBS (control) or H7 and HA1004 at 150 µM (HA1004 as a chemical
control). Neutrophils added on KLH were stimulated with ZAS (1% vol)
and incubated in the presence of PBS (control) or H7 and HA1004.
Hydrogen peroxide production was measured for 3 h by scopoletin
assay. Values represent mean ± SEM, n = 3, *P < 0.05.
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 View larger version (22K): [in a new window] |
Figure 7. Effect of R7.1 F(ab')2 (anti-CD11a) on the hydrogen
peroxide production from neutrophils exposed to L cells expressing
C1,2:H3–5 chimeric ICAM-1 or KLH-coated plastic. KLH and L cells were
prepared in 96-well plates. Unstimulated neutrophils were added to
wells containing L cells in the presence of PBS or 10 µg/ml of R7.1
F(ab')2. Neutrophils added on KLH were stimulated with
ZAS (1% vol). Hydrogen peroxide production was measured at 15 min, 30
min, and 60 min by scopoletin assay. Values are mean ±
SEM, n = 3, *P < 0.01.
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These findings are of potential interest in light of recent data
showing that neutrophil LFA-1 and Mac-1 have several functional
distinctions. Isolated neutrophils exhibit negligible Mac-1-dependent
adhesion unless exposed to concentrations of chemotactic factors in the
chemokinetic range [5
]. In contrast, LFA-1-dependent
adhesion to ICAM-1 can be seen without added chemotactic factors or
with markedly low stimulus levels [5
, 10
,
28
]. Emigration, in vitro and in
vivo, appears to require LFA-1-dependent adhesion much more than
Mac-1-dependent adhesion. Transmigration, in vitro through
endothelial monolayers stimulated with IL-1ß, TNF-, or endotoxin
[29
30
31
] or in response to chemotactic gradients, is
more efficiently blocked by anti-LFA-1 mAbs than anti-Mac-1 mAbs
[5
, 32
]. Studies of mice with targeted
deletions of CD11a or CD11b reveal significantly reduced emigration of
neutrophils in thioglycollate-induced peritonitis in CD11a-deficient
mice [33
] but no impairment of emigration in mice with
CD11b deficiency [34
]. Rutter et al.
[35
] and Graf et al. [36
] have
found in rabbit models of peritonitis that mAbs blocking Mac-1 were
ineffective in preventing neutrophil emigration, and mAbs blocking
LFA-1 markedly reduced neutrophil emigration. In a rat model of dermal
inflammation, Issekutz and Issekutz [37
] found that
anti-CD11a mAb was more effective than anti-CD11b mAb in reducing
neutrophil localization, and Argenbright et al.
[11
] found that anti-CD11a mAb was as effective as
anti-CD18 in reducing firm adhesion of leukocytes in an intravital
model of inflammation in the rabbit mesentery. Similar observations in
a rat model of uveitis were made by Rosenbaum and Boney
[38
]. Thus, it appears that Mac-1 is relatively less
important than LFA-1 for neutrophil emigration in many experimental
models with canine, rat, mouse, rabbit, and human cells, although
optimal transmigration requires cooperation of LFA-1 and Mac-1
[4
, 5
, 32
, 37
].
In contrast to the dominant roles of LFA-1, the cytotoxic activity of neutrophils for parenchymal cells appears to be heavily dependent on Mac-1 in vivo and in vitro [4 , 10 , 39 40 41 ], a process probably linked to secretory activity markedly augmented by Mac-1-dependent adhesion.
The functional significance of LFA-1-dependent triggering of the
oxidative burst in neutrophils is obscure. Because the kinetics are
more rapid than with Mac-1-dependent adhesion, oxygen radical
production seems likely to immediately follow the process of
transendothelial migration. For example, neutrophils contacting
endothelial monolayers stimulated in vitro for 3 h with
IL-1ß will undergo LFA-1-dependent transmigration in high numbers
within <15 min [29
]. Individual cells attaching to the
endothelial monolayer under conditions of shear, transmigrate within
1 min after forming a stable adhesion [42
]. There is
evidence from studies in vivo that oxidant stress can occur
in cells of the vascular wall at sites of leukocyte adhesion. Using
carboxydichlorofluorescein (CDCF) as a probe for intracellular oxidant
stress, Suematsu et al. [43
] observed the
mesenteric microcirculation in rats following administration of an
inhibitor of nitric oxide synthesis, NG-nitro-L-arginine
methy ester (L-NAME). Significant increases occurred in leukocyte
adhesion, a phenomenon first reported by Kubes et al.
[44
], and in CDCF fluorescence in endothelium.
Anti-ICAM-1 and anti-CD18 mAb significantly attenuated leukocyte
adhesion and oxidant stress. Similar results were obtained after
superfusion of normal mesenteric vessels with formyl-Met-Leu-Phe
(fMLP). Although these authors did not assess the relative
contributions of LFA-1 and Mac-1, our results and those of others
showing that LFA-1 is heavily involved in the emigration of neutrophils
raise the possibility that LFA-1-dependent oxidative burst may be
occurring near endothelial cells at the time of transmigration.
Received July 21, 1999; revised January 19, 2000; accepted January 20, 2000.
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