ICAM-1-CD18 interaction mediates neutrophil cytotoxicity
through protease release
Carlton C.
Barnett Jr.1,
Ernest E.
Moore1,
Gary W.
Mierau2,
David A.
Partrick1,
Walter L.
Biffl1,
David J.
Elzi3, and
Christopher C.
Silliman3
1 Department of Surgery, Denver
Health Medical Center and University of Colorado Health Sciences
Center, Denver 80204; 2 Department
of Pathology, Children's Hospital, Denver 80218; and
3 Bonfils Blood Center and
Department of Pediatrics, University of Colorado Health Sciences
Center, Denver, Colorado 80220
 |
ABSTRACT |
Interaction of
the
2-integrin complex on the
polymorphonuclear neutrophil (PMN) with intercellular adhesion
molecule-1 (ICAM-1) has been implicated in PMN-mediated cytotoxicity.
This study examined interaction of the CD11a, CD11b, and CD18 subunits
of the
2-integrin with ICAM-1,
transfected into Chinese hamster ovarian (CHO) cells to avoid effects
of other adhesion molecules. Incubation of quiescent PMNs with
wild-type and ICAM-1-transfected CHO cells produced nominal cell lysis.
Similarly, when phorbol myristate acetate (PMA)-activated PMNs were
incubated with wild-type CHO cells, minimal cytotoxicity was produced.
However, when ICAM-1-transfected CHO cells were incubated with
PMA-activated PMNs, 40% cell lysis occurred. Blockade with a
monoclonal antibody (MAb) to ICAM-1 or MAbs to CD11a, CD11b, or CD18
reduced PMN-mediated cytotoxicity to baseline. To examine the role of
adhesion in cytotoxicity, we studied
2-integrin-mediated PMN
adhesion to ICAM-1-transfected CHO cells and found that MAbs for CD11a,
CD11b, and CD18 all abrogated PMN cytotoxicity despite disparate
effects on adhesion. To assess the role of CD18,
2-integrin subunits were
cross-linked, and CD18 alone mediated protease release. Moreover,
ICAM-1 was immunoprecipitated from transfected CHO cells and incubated
with PMNs. This soluble ICAM-1 provoked elastase release, similar to
PMA, which could be inhibited by MAbs to CD18 but not MAbs to other
2-integrin subunits. In
addition, coincubation with protease inhibitors eglin C and AAPVCK
reduced PMN-mediated cytotoxicity to control levels. Finally,
ICAM-1-transfected CHO cells were exposed to activated PMNs from a
patient with chronic granulomatous disease that caused significant cell
lysis, equivalent to that of PMNs from normal donors. Collectively,
these data suggest that ICAM-1 provokes PMN-mediated cytotoxicity via
CD18-mediated protease release.
transfection; adhesion molecules; integrins; neutrophil
degranulation
 |
INTRODUCTION |
THE
2-integrin
(CD11-CD18) complex on the polymorphonuclear neutrophil (PMN) consists
of a constant
-region (CD18) that is noncovalently linked to
variable
-units: CD11a, CD11b, and CD11c (42). The pairing of these
-units with the constant
-unit leads to PMN cell surface
expression of the CD11a-CD18 (LFA-1), CD11b-CD18 (MAC-1), or CD11c-CD18
(p150,95) receptor complex. The neutrophil is unique in expressing all
three of these
2-integrin complexes. These complexes are known to play an important role in
normal microbicidal function, as aberrant expression of the
2-integrin complex known as
leukocyte adhesion deficiency syndrome is associated with overwhelming
infections (2).
In addition to its microbicidal role, the
2-integrin complex is known to
interact specifically with the intercellular adhesion molecule-1
(ICAM-1), which is expressed on endothelial cells and various other parenchymal cells (12). This interaction has been identified as a pivotal event in PMN-mediated tissue injury (11, 13,
15, 23, 34). Despite intensive study, the relative contribution of the
specific
- and
-subunits of the
2-integrin complex in
PMN-mediated cytotoxicity is not clear. It is known that CD11a-CD18 and
CD11b-CD18 bind to different domains on ICAM-1, with the LFA-1 complex
binding to the D1 amino terminal and MAC-1 binding the D3 amino
terminal (43). The significance of these
2-integrin complexes binding to
different ICAM-1 domains has not been fully elucidated. Whereas the
surface expression of CD11a is not increased numerically by
inflammatory stimuli (11), the avidity of the LFA-1 complex for ICAM-1
is increased by various cytokines via conformational changes (5, 20).
Moreover, CD11a has been shown to be involved in PMN cytotoxicity (14).
The binding of CD11b to ICAM-1 has been shown to promote PMN adhesion,
migration, and respiratory burst (22). In addition, CD11b is known to
be upregulated following PMN exposure to diverse inflammatory mediators
[interferon-
(IFN-
), lipopolysaccharides (LPS), and tumor
necrosis factor (TNF)] (11). In addition, CD11b binds to other
cell surface ligand(s) (28) that may play a significant role in
cytotoxicity. Recently, it has been shown that CD11a and CD11b have
compartmentalized functions (35). Intravenous monoclonal antibody (MAb)
to CD11a attenuates lung injury, whereas intratracheal administration
has no effect. Conversely, MAb to CD11b is able to abrogate lung injury
when given intratracheally but not if delivered intravenously (35).
Thus it appears that both CD11a and CD11b play important roles in
PMN-mediated tissue injury.
The specific function of the constant
-region (CD18) in PMN-mediated
cytotoxicity has not been studied as extensively. CD18 has been shown
to mediate release of azurophilic granules and augment superoxide
generation (36, 48). CD18 has been implicated as a triggering protein
in tyrosine phosphorylation (6) and may also mediate intracellular
signaling through the intimate association of its intracytoplasmic
portion with
-actinin (36).
Recently, we have shown that the coincubation of activated neutrophils
with ICAM-1-transfected Chinese hamster ovary (CHO) cells is sufficient
to provoke PMN-mediated cytotoxicity (4). PMN-mediated injury is
generally attributed to the coordinated effects of the respiratory
burst and degranulation, but the relative contribution of these
processes has been debated (50). Moreover, the regulation of reactive
oxygen metabolite generation and protease release appears to be
compartmentalized, with both playing important and potentially
complementary roles in PMN-mediated tissue injury (5, 25, 41, 45, 51).
It has been shown in certain patient populations, e.g., thermal injury
(38) and sepsis (30), that PMNs have deficient NADPH oxidase function
and therefore decreased ability to generate reactive oxygen
metabolites. However, these patients may still develop acute
respiratory distress syndrome (ARDS) and multiple organ failure
syndrome, in which neutrophils are thought to play a central role (33).
These observations lead us to postulate that factors other than
reactive oxygen metabolites play a prominent role in PMN-mediated
cytotoxicity.
The purpose of this study was to characterize specific interactions
between human ICAM-1 and the pertinent subunits of the
2-integrin complex (CD11a,
CD11b, and CD18) on human PMNs and, more importantly, to examine the
roles of reactive oxygen metabolites and proteases in PMN cytotoxicity.
An in vitro model was developed in which human ICAM-1 became the
specific adhesion molecule for the neutrophil to engage without
interference from other ligands or adhesion molecules involved in the
PMN-endothelial interaction. Our hypothesis was that ICAM-1 provokes
PMN cytotoxicity via CD18-mediated protease release.
 |
METHODS |
Reagents, hardware, and MAbs.
CHO cells were purchased from American Type Culture Collection
(Rockville, MD). The Rc/CMV plasmid was obtained from Invitrogen (San
Diego, CA). Dulbecco's PBS solution, RPMI 1640, L-glutamine, penicillin,
streptomycin, and trypsin were purchased from Mediatech (Herndon, VA);
heat-inactivated FCS was purchased from Gemini Bio-Products (Calabasas,
CA); methoxy-succinyl-alanyl-alanyl-prolyl-valyl-chloromethyl ketone
(AAPVCK),
methoxy-succinyl-alanyl-alanyl-prolyl-valyl-p-nitroanilide (AAPVPNA), catalase, chloroquine, cytochrome
c, DMSO, eglin C, HEPES,
paraformaldehyde, Percoll, phorbol myristate acetate (PMA), sodium
citrate, SDS, superoxide dismutase (SOD), Triton X-100, aprotinin,
iodoacetamide, Lowry protein assays, phenylmethylsulfonyl fluoride, and
Tris base were obtained from Sigma Chemical (St. Louis, MO). Dextran
T-500 was purchased from Pharmacia (Uppsala, Sweden), mannitol was
obtained from Abbott Laboratories (North Chicago, IL), chromium-51
(51Cr) was purchased from Amersham
(Arlington Heights, IL), and scintillation cocktail was obtained from
National Diagnostics (Atlanta, GA). Unless otherwise noted, reagents
were purchased from Sigma. T-25 flasks, 96-well plates, 48-well plates,
and microplates were obtained from Falcon BD Labware (Lincoln Park,
NJ). FITC-labeled 84H10, unlabeled 84H10, and 25.3.1 were purchased
from Immunotech (Westbrook, ME). Unconjugated and FITC-labeled
IgG1,
IgG2a, and
IgG2b nonspecific controls were
from Sigma; Cris-3, BF10, LM2/1, and goat anti-mouse F(ab')2 (GAM) were from
Biosource (Camarillo, CA). TS1/18 and 31H8 were obtained from Endogen
(Boston, MA). RR1/1 was obtained from BioDesign (Kennebunk, ME), and 44 was from Pharmingen (San Diego, CA). Amicon Spin-X filters were
purchased from Life Science Products (Denver, CO), and anti-ICAM-1
ELISA kits were obtained from Endogen (Boston, MA). Protein A beads and
affinity columns were purchased from Pierce (Rockford, IL). Sodium
cacodylate, osmium tetroxide, glutaraldehyde, EMbed 812, uranyl
acetate, and lead citrate were purchased from Electron Microscopy
Sciences (Fort Washington, PA). A model EM-10CA transmission electron
microscope was obtained from Carl Zeiss (Oberkochen, Germany).
Immunoaffinity and specificity of MAbs.
Specific data for MAbs to CD11a, CD11b, and CD18, which were employed
in this study for epitope specificity, and their inhibition of
aggregation and ability to immunoprecipitate and neutralize ligands
were obtained from the manufacturer as well as from published data from
the Organizing Committee of the Third and Fourth International Conferences on Human Leucocyte Differentiation Antigens and other sources (24, 32). Cris-3 is specific for CD11a and inhibited leukocyte
aggregation by 49.5% (24); however, its ability to immunoprecipitate
and neutralize CD11a has not been evaluated. The 25.3.1 was reported to
be specific for and able to immunoprecipitate CD11a (24, 32).
Immunotech reports positive neutralization of CD11a by 25.3.1;
inhibition of aggregation was not tested. LM2/1 was reported to be
specific for CD11b, and, although it did not inhibit aggregation, it
was able to immunoprecipitate CD11b (32). Biosource International
reports neutralization of CD11b by LM2/1; 44 was reported to be
specific for and able to immunoprecipitate CD11b (24, 32); inhibition
of aggregation and neutralization were not tested. TS1/18 was reported
to be specific for CD18 and caused 94% inhibition of aggregation (32); immunoprecipitation and neutralization were not tested. 31H8 was reported to be specific for CD18 by Endogen; PMN aggregation, immunoprecipitation, and neutralization were not tested.
ICAM-1 gene transfection.
CHO cells were grown to confluence with RPMI 1640 supplemented with 2 mM L-glutamine, 100 U/ml
penicillin, 1,000 U/ml streptomycin, and 10% FCS at 37°C, 5%
CO2. The human ICAM-1 expression
vector CD1.8 as well as the Rc/CMV vector for resistance to G418 were transfected into CHO cells by a calcium phosphate method (4). The
vector Rc/CMV was also transfected alone into CHO cells for wild-type
control. ICAM-1 transfection efficiency was confirmed by flow cytometry
using 10 µg/ml of anti-CD54 FITC-labeled antibody (84H10) or
FITC-labeled isotypic antibody as a negative control. Mean fluorescent
intensity (MFI) was determined as experimental MFI divided by negative
control MFI. ICAM-1-transfected CHO cells were then trypsinized and
serially diluted in a 1:10,000 volume of complete medium with G418 (250 µg/ml) until suitable transfectant colonies were isolated. These were
maintained in G418-containing medium at 37°C, 5%
CO2 (39).
Neutrophil isolation.
Neutrophil isolation was performed as previously described (18). The
cell population was >98% PMNs by differential staining (Stat Stain,
San Francisco, CA) and >99% viable by trypan blue exclusion.
Neutrophil isolation from a patient with p67-phox chronic granulomatous
disease was performed using a modified Ficoll/Hypaque technique (1).
PMN-mediated cytotoxicity via ICAM-1.
CHO cells transfected with Rc/CMV alone as well as with ICAM-1 were
labeled with 2 µCi/ml 51Cr and
grown to confluence in 96-well plates at 37°C, 5%
CO2. Cells were washed twice in
PBS, after which they were placed in 200 µl of PBS + 1% dextrose
(PBS+D) per well. PMNs were added to all experimental wells immediately
after MAbs or inhibitors. Because the
2-integrin is not
constituitively avid for ICAM-1 and requires activation to enable this
high-affinity interaction (29, 37), PMA was chosen as an activating
agent and was added to wells at a final concentration of 1 µM (37).
CHO cells plus MAbs or inhibitors were incubated with activated PMNs
for 4 h at 37°C, 5%
CO2. Postincubation culture plates
were centrifuged at 200 g for 10 min
to pellet nonadherent cells. To determine total
51Cr counts, 50 µl of 0.5% SDS
were added to lyse-selected, labeled CHO cells not exposed to PMNs, and
the spontaneous 51Cr counts were
assessed from one-half of the volume of supernatant from selected
wells. Experimental counts were determined from one-half of the volume
of supernatant in all other wells.
51Cr counts were measured in a
scintillation counter (Beckman, Irvine, CA). Percent cell lysis, equal
to percent 51Cr release, was
determined as follows: (experimental
51Cr counts)
(spontaneous
51Cr counts/total
51Cr counts) × 100%.
To determine the role of the
2-integrin subunits in
promoting PMN-mediated cytotoxicity, subsets of ICAM-1-transfected CHO cells were coincubated with individual and combinations of MAbs specific for
2-subunits CD11a,
CD11b, or CD18 15 s before addition of activated PMNs. In addition, in
selected experiments, transfected CHO cells were incubated with an MAb
to ICAM-1 (84H10) 15 s before the addition of activated PMNs.
To study the role of proteases, ICAM-1-transfected CHO cells were
coincubated with protease inhibitors AAPVCK (50 µM) or eglin C (20 µM). Two protease inhibitors were chosen to reduce the possibility that results were caused by an isolated effect of the inhibitor on the
neutrophil or CHO cell. The initial studies with these inhibitors used
concentrations from 5-100 µM of both AAPVCK and eglin C,
resulting in inhibition of the cleavage of the AAPVPNA beginning at 5 µM and maximal at 20 µM for eglin C and 50 µM for AAPVCK with no
differences beyond these concentrations (results not shown). Therefore,
doses of 50 µM AAPVCK and 20 µM eglin C were utilized. Chloromethyl
ketones have been shown to cause a significant degree of their
inhibition via the hemiketal formed from interaction of the active site
serine with the carbonyl group on the inhibitor, making it more
specific to the active site of human leukocyte protease (44). Eglin C
differs from other inhibitors in its intramolecular structure
surrounding the primary binding segment, forming unique secondary
contact structures that allow electrostatic and hydrogen bonds to form
at the active site of the enzyme (7). To determine whether these
protease inhibitors had nonspecific effects that altered neutrophil
generation of superoxide, PMNs were incubated with AAPVCK (50 µM) or
eglin C (20 µM) for 5 min before being activated with either PMA (1 µM) or platelet-activating factor (PAF; 200 nM) followed by
formyl-methionine-leucine-phenylalanine (fMLP; 1 µM). Superoxide
generation was determined in 96-well plates by SOD-inhibitive reduction
of cytochrome c as described (40).
To study the role of reactive oxygen metabolites, ICAM-1-transfected
CHO cells were preincubated with catalase for a dose response curve
from 100 to 10,000 units with and without 10 µg/ml of SOD (results
for 10,000 units are discussed). An additional subset of
ICAM-1-transfected CHO cells was incubated with heat-inactivated PMNs
(17). To further investigate the role of reactive oxygen metabolites,
ICAM-1-transfected CHO cells were coincubated with PMA-activated PMNs
obtained from a well-described patient with an autosomal recessive
p67-phox deficiency. PMA-activated neutrophils from this patient do not
reduce cytochrome c (40) and
demonstrate no shift in mean channel fluorescence of intracellular
dihydrorhodamine by flow cytometry (47).
Neutrophil adhesion.
PMN adhesion was determined by a modified McClay adhesion assay (31).
Wild-type and ICAM-1-transfected CHO cells were grown to confluence in
48-well plates and exposed to 2 × 105 PMNs labeled with
51Cr for 1 h (10 µCi/1 × 107 cells) in complete medium.
PMNs were then added to each well. PMA was added to a final
concentration of 1 µM, and cells were incubated for 30 min at
37°C, 5% CO2. Experimental
plates were coincubated with saturating doses of MAbs to the
2-integrin subunits, as
determined by flow cytometric analysis. Plates were sealed with an
adhesive covering and centrifuged in an inverted position at 250 g for 5 min, and the covering was
removed with plates in the inverted position. Two hundred microliters
of 0.5% SDS were then added to each well, and 100 µl of lysate were
subsequently removed from each well for scintillation counting. PMN
adhesion was determined by 51Cr
counts in the lysate of experimental wells, which was compared with the
counts in the lysate of control wells.
Qualitative analysis of the adhesion of PMNs to ICAM-1-transfected
CHO cells by electron microscopy.
The adhesive interactions between PMNs and ICAM-1-transfected CHO cells
were qualitatively assessed, employing conventional transmission
electron microscopy techniques (19). Briefly, both vector-transfected
and ICAM-1-transfected CHO cells were grown to confluence in 12-well
plates. The cells were washed and overlayed with warm Krebs Ringer
phosphate dextrose buffer. A saturating dose of the TS1/18 MAb, which
binds to a nonadhesive locus of the CD18 adhesion molecule, or vehicle
control was added to the reaction mixture 15 s before the addition of
freshly isolated PMNs (1 × 106). The PMNs were stimulated
by the addition of 1 µM PMA for 30 min. After trypsinization, the CHO
cells were fixed overnight with 0.1 M sodium cacodylate-buffered 2.5%
glutaraldehyde, pH 7.2, and then for 1 h in 2% sodium
cacodylate-buffered osmium tetroxide. The cells were dehydrated
in a graded series of ethanols, embedded in EMbed 812, and
sectioned at a thickness of 80 nm. Sections were stained with uranyl
acetate and lead citrate and examined with a Zeiss EM-10CA transmission
electron microscope at 60 kV accelerating voltage.
Immunoprecipitation of soluble ICAM-1 from transfected CHO cells.
Soluble ICAM-1 (sICAM-1) was purified from transfected CHO cells using
a standard immunoprecipitation technique (3). Briefly, ICAM-1-transfected CHO cells were lysed with Triton X-100, and the
resulting lysates were incubated with protein A beads covalently coupled to an MAb to ICAM-1 (RR1/1). The beads were then placed in a
column, and ICAM-1 was eluted from the beads at pH 2.8. The sICAM-1 was
concentrated, and a modified Lowry protein assay was completed to
determine the amount of protein. To assess the presence of sICAM-1 in
the immunoprecipitate, the pooled fractions were separated by 10%
SDS-PAGE, transferred to nitrocellulose, and immunoblotted with an MAb
to ICAM-1 (84H10). The bands were visualized using enhanced
chemiluminescence followed by autoradiography.
Superoxide generation and protease release via cross-linking
2-integrins.
Cross-linking of
2-integrin
subunits CD11a, CD11b, or CD18 was accomplished by incubating PMNs in
suspension with saturating doses of MAb specific for the
2-subunits or nonspecific and
isotypic controls. PMNs were incubated with MAbs to individual
2-subunits on ice for 30 min
and washed twice in PBS+D. The
2-integrin subunits were
cross-linked by incubating cells with 20 µg/ml of GAM. Additional experiments investigated the effects of PMN incubation with sICAM-1 isolated from transfected CHO cells at a final concentration of 75 ng/ml as determined by a modified Lowry protein assay.
Superoxide generation was determined in 96-well plates by
SOD-inhibitive reduction of cytochrome
c. Experimental wells contained 20 µg/ml of GAM to cross-link
2-integrin-binding MAbs. PMNs
were incubated without GAM as negative control. Blank wells contained 1 µg/ml of SOD. PMA (1 µM)-stimulated release of superoxide was determined from PMNs not exposed to GAM for positive control. Superoxide generation was determined kinetically as described over 60 min at 37°C.
Protease release was determined by incubating 2 × 105 PMNs in suspension with 20 µg/ml of GAM at 37°C for 60 min after saturation with MAbs
specific for
2-subunits or with
nonspecific and isotypic immunoglobulin controls. Cells were pelleted,
and supernatant was assayed colorimetrically for protease activity.
Supernatant (25 µl) was incubated in a 96-well plate at room
temperature for 60 min with 1 mM AAPVPNA, 0.1 M HEPES, and 0.5 M NaCl
(pH 7.5) in a total volume of 150 µl. Basal protease release was
determined from PMNs not exposed to GAM. Absorbance was measured at 405 nm (Molecular Devices, Palo Alto, CA). An extinction coefficient of 8.8 × 103 was used to determine
nanomoles substrate cleaved per hour. PMNs from each donor were lysed
with Triton X-100 to obtain total PMN protease content. Protease
release is expressed as percentage of total PMN protease.
Statistical analysis.
Data are presented as means ± SE, with each study group repeated
five times unless otherwise noted. One-way ANOVA testing was performed
to determine the significance of observed differences. Scheffé's
F procedure was used for post hoc
comparisons. Significance at the 95% confidence interval is
represented by P < 0.05.
 |
RESULTS |
ICAM-1 transfection into CHO cells.
Flow cytometric analysis demonstrated 62.5 ± 2.5 to 87.1 ± 3.1% of CHO cotransfected with CD1.8 and Rc/CMV expressed ICAM-1 on
their cell surface compared with 0.3 ± 0.3% in the wild type (Rc/CMV only). MFI was 33.3 in the transfected cells vs. 1.03 in the
wild-type cells. These results compare favorably to previously observed
transfection efficiency for ICAM-1 (49).
PMN cytotoxicity for CHO cells.
Incubation of ICAM-1-transfected CHO cells with quiescent PMNs produced
3.2 ± 1.8% cell lysis, compared with 16.0 ± 1.5% for PMA-activated PMNs. Furthermore, incubation of wild-type CHO cells with
PMA-activated PMNs produced minimal cytotoxicity (4), despite the fact that 1 µM PMA stimulation of PMNs generated
significant elastase release (30 ± 4.2% of total cellular
elastase). PMA alone was not cytotoxic to CHO cells. As additional
controls, 10 µg of purified human neutrophil elastase were added to
51Cr-labeled wild-type CHO cells
to determine if these wild-type CHO cells were susceptible to free
elastase. This dose has been reported to cause endothelial cell injury
in vitro (10). Incubation of wild-type CHO cells with purified elastase
resulted in 2.71 ± 0.37% cell lysis, which was not statistically
different from the cytotoxicity observed for wild-type CHO cells
incubated with PMA-activated neutrophils, 2.3 ± 0.4%.
Preincubation of transfected CHO cells with combinations of MAbs for
CD11a, CD11b, and CD18 (MAbs 25.3.1, 44, and TS1/18, respectively)
decreased cell lysis via PMA-activated PMNs to baseline: 2.7 ± 1.0%, 2.5 ± 1.0%, and 3.8 ± 1.5%, respectively. Because
combinations of MAbs to the
2-integrin subunits were
effective in preventing PMN-mediated cytotoxicity, individual MAbs for
CD11a (25.3.1 or Cris-3), CD11b (44 or LM2/1), or CD18(TS1/18 or 31H8)
were also tested for their ability to block PMN cytotoxicity.
Nonspecific MAb for the PMN receptor CD13 as well as isotypic MAbs for
IgG1,
IgG2a, and
IgG2b were used as controls.
Preincubation of transfected CHO cells with individual MAbs to CD11a,
CD11b, or CD18 resulted in 2.1 ± 2.0% and 1.57 ± 1.56% cell
lysis, respectively, for CD11a pretreatment, 3.4 ± 2.2% and 5.0 ± 2.2%, respectively, for CD11b, and 0.4 ± 0.4% and 0.01 ± 0.01%, respectively, for CD18. In addition, preincubation of
transfected CHO cells with an MAb to ICAM-1 (84H10) also decreased PMN
cytotoxicity of transfected CHO cells to 0.44 ± 0.17% specific 51Cr release. Because firm
adhesion via
2-integrins and
their ligands on target cells is required for PMN-mediated
cytotoxicity, these results, e.g., the blockade of PMN cytotoxicity by
MAbs to the
2-integrin subunits
or their ligand on the transfected CHO cells, confirm the work of
others (14, 15, 21, 35). Thus preincubation with MAbs to CD11a, CD11b,
CD18, or ICAM-1 was sufficient to reduce PMN cytotoxicity in this
model. Conversely, coincubation with an MAb to the nonspecific PMN
receptor CD13 as well as isotypic controls had no effect on
PMN-mediated cytotoxicity (Fig.
1).

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Fig. 1.
Blockade of each 2-integrin
subunit abrogates polymorphonuclear neutrophil (PMN)-mediated
cytotoxicity. Intercellular adhesion molecule-1 (ICAM-1)-transfected
Chinese hamster ovary (CHO) cells, labeled with
51Cr, were incubated with
quiescent or phorbol myristate acetate (PMA; 1 µM)-activated PMNs.
Subsets of CHO cells were coincubated with monoclonal antibodies (MAbs)
to ICAM-1 (84H10), CD11a (25.3.1 or Cris-3), CD11b (44 or LM2/1), or
CD18 (TS1/18 or 3H18) and exposed to PMA-activated PMNs. CHO cells were
also coincubated with MAb to the nonspecific PMN receptor CD13 as well
as isotypic control MAbs for IgG1,
IgG2a, and
IgG2b. Neutrophil cytotoxicity
(%cell lysis) is expressed as percent
51Cr released from CHO cells
(means ± SE). For all groups, n = 5. * Significant difference from ICAM-1-transfected CHO cells
treated with PMA-activated PMNs, P < 0.05.
|
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Neutrophil adhesion.
Adhesion of PMA-activated PMNs to wild-type CHO was 12,935 ± 1,017 dpm (Table 1) compared with
28,013 ± 3,477 dpm for PMA-activated PMNs to ICAM-1-transfected CHO
cells. Blockade of the CD11a or CD11b
2-integrin subunits on
PMA-activated PMNs resulted in adhesion that was not different from
activated PMNs to wild-type CHO cells. Blockade of CD11a (13,058 ± 1,065 dpm with 25.3.1, 9,666 ± 823 dpm with Cris-3) and CD11b
(14,526 ± 2,641 dpm with 44, 10,392 ± 821 dpm with LM2/1) on
PMA-activated PMNs yielded analogous results. In contrast, blockade of
CD18 on PMA-activated PMNs by two different MAbs elicited disparate
results. The MAb TS1/18 was unable to reduce adhesion of PMA-activated
PMNs to ICAM-1-transfected CHO cells (22,852 ± 2,767 dpm with
TS1/18 vs. 28,013 ± 3,477 dpm for control PMNs), whereas the 31H8
MAb to CD18 reduced the adhesion of activated PMNs to transfected CHO
cells to 10,423 ± 1,068 dpm. Average
51Cr (in dpm) in an equal volume
of radiolabeled PMNs (2 × 105) was 33,397 ± 2,368. Lastly, it is important to note that blockade of ICAM-1 with an MAb
(84H10) also abrogated PMN adherence in the transfected CHO cells:
12,658 ± 1,047 dpm vs. 12,9305 ± 1,017 dpm PMN
adherence to wild-type CHO cells.
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Table 1.
PMA-activated neutrophil adherence to CHO cells and ICAM-1-transfected
CHO cells with 2-integrin MAb blockade
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Qualitative analysis of adhesion of PMNs to vector and
ICAM-1-transfected CHO cells by electron microscopy.
To determine if the adhesive events between ICAM-1-transfected CHO
cells and freshly isolated PMNs were affected by incubation with the
TS1/18 MAb to a nonadhesive locus of CD18, PMNs were incubated with
both vector and ICAM-1-transfected CHO cells in the presence or absence
of saturating doses of the TS1/18 MAb and stimulated for 30 min with 1 µM PMA. Electron microscopy of vector-transfected CHO cells incubated
with activated PMNs did not demonstrate any intimate cell surface
interactions (data not shown). In contrast, ICAM-1-transfected CHO
cells incubated with activated PMNs resulted in numerous PMNs becoming
tightly adherent to the CHO cells (Fig. 2).
Preincubation with the TS1/18 MAb to CD18 did not block the adhesion of
activated PMNs to ICAM-1-transfected CHO cells (Fig. 2).

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Fig. 2.
Electron microscopy of the adhesive interactions of activated PMNs with
ICAM-1-transfected CHO cells. PMNs and ICAM-1-transfected CHO cells
were preincubated with buffer (A) or
saturating doses of TS1/18 MAb to CD18
(B) 15 s before activation of the
PMNs with 1 µM PMA for 30 min. Specialized modifications of the cell
surface at sites of adhesion between the PMNs and transfected CHO cells
were observed in both the buffer and MAb treatment groups
(×7,500). A higher-power view (×16,000,
bottom) better demonstrates the
complex interdigitation of cytoplasmic processes in this zone of
interaction. These results confirmed that PMNs and ICAM-1-transfected
CHO cells firmly adhere despite pretreatment with the TS1/18 MAb to
CD18. Observations were confirmed with replicate experiments.
|
|
Cross-linking
2-integrins
for superoxide production.
Superoxide generation results over 60 min (expressed in
nmol · 2.0 × 105
PMN
1 · min
1)
caused by saturating PMNs in suspension with MAb and cross-linking with
GAM are displayed in Table 2. Neither
saturation with MAb nor cross-linking with GAM caused an increase in
superoxide generation beyond that of buffer-treated controls (release
of 0.5 ± 0.3 nmol · 2 × 105
PMN
1 · min
1).
Activation with PMA demonstrated an intact neutrophil capacity for
superoxide generation (7.6 ± 1.6 nmol · 2.0 × 105
PMN
1 · min
1).
Cross-linking
2-integrins
for protease release.
Protease release caused by MAb saturation and cross-linking with GAM is
shown in Fig. 3. Cross-linking CD18
receptors with MAbs specific for the CD18 moiety alone (TS1/18 and
31H8) provoked significant increases in protease release (8.9 ± 1.4 and 8.8 ± 1.4%, respectively) compared with basal release (2.8 ± 0.3%, P < 0.05). These
results are similar to elastase release in response to fMLP (Fig. 3).
Cross-linking CD11a and CD11b receptors, however, caused no increase in
protease release beyond buffer-treated control PMNs. These results
imply that the CD18 subunit may play a role mediating neutrophil
protease release.

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Fig. 3.
2-Integrin subunit
cross-linking via MAb. PMNs (2.0 × 105) were incubated with
saturating doses of mouse anti-human MAb to CD11a, CD11b, and CD18 as
well as nonspecific and isotypic controls for 30 min on ice and then
cross-linked with goat anti-mouse
F(ab')2 (GAM). Protease
release was determined colorimetrically via cleavage of AAPVPNA.
Hatched and crosshatched bars represent basal PMN protease release;
open bars represent PMNs saturated with MAb alone; solid bars represent
PMNs treated with GAM. For all groups,
n = 5. * Difference from basal,
P < 0.05.
|
|
sICAM-1-provoked protease release.
ICAM-1 was immunoprecipitated from the transfected CHO cells, and
immunoblotting of the separated proteins of the relevant fractions
demonstrated a single band of protein at 73.5 kDa (Fig. 4). Incubation of PMNs with a final
concentration of 75 ng/ml of the immunoprecipitated sICAM-1 produced
significant amounts of elastase release from isolated PMNs in
comparison to buffer-treated controls or an irrelevant fraction from
the sICAM-1 elution (19.2 ± 2.8% vs. 2.4 ± 0.5%,
P < 0.05, n = 5) (3). PMA-induced protease release elicited similar elastase release, 25.6 ± 6.9%, to that of
sICAM-1. MAbs to CD11a, CD11b, and CD18 did not cause elastase release
compared with buffer-treated controls (1.8 ± 0.2 to 3.1 ± 0.5%
vs. 2.4 ± 0.5%, n = 5). However,
preincubation with MAbs to CD18 (TS1/18 or 31H8) abrogated sICAM-1
elastase release from isolated PMNs (19.2 ± 2.8% for sICAM-1 vs.
4.3 ± 1.0% for TS1/18-sICAM-1 and 5.5 ± 1.4% for 31H8-ICAM-1;
P < 0.05, n = 5), whereas MAbs to CD11a or CD11b
did not affect protease release (19.2 ± 2.8% for sICAM-1 vs. 16.5 ± 6.2 to 25.6 ± 6.9% for CD11a or CD11b MAbs with sICAM-1;
n = 5).

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Fig. 4.
Immunoprecipitation of soluble ICAM-1 from transfected CHO cells. A
Western blot shows a single band of immunoreactivity (73.5 kDa) to a
mouse MAb to human ICAM-1 (84H10) from immunoprecipitates of whole cell
lysates of ICAM-1-transfected CHO cells utilizing the mouse MAb R/R-1
to ICAM-1. Protein (6, 12, and 24 ng) was loaded from left to right,
respectively. Arrows and numbers correspond to the exact positions of
the 97- and 66-kDa molecular mass standards.
|
|
PMN cytotoxicity for CHO cells: investigation of proteases.
To evaluate the role of proteases in PMN cytotoxicity of
ICAM-1-transfected CHO cells, the protease inhibitors AAPVCK and eglin
C were employed. In control experiments, neither AAPVCK nor eglin C
caused lysis of wild-type or ICAM-1-transfected CHO cells as determined
by trypan blue exclusion. Furthermore, no effects on PMN viability were
seen with the concentration of these inhibitors used in the following
experiments. In addition, both a time course of inhibition (immediate,
10 min) and a range of concentrations were tested in similar
experiments, as well as in experiments quantifying PMN elastase release
using the reduction of AAPVPNA. Maximal inhibition of elastase was
documented for 50 µM AAPVCK and 20 µM eglin C with a 5-min
incubation time (results not shown). Preincubation of
ICAM-1-transfected CHO cells with the protease inhibitors AAPVCK and
eglin C reduced cell lysis to 4.85 ± 2.03% and 2.7 ± 1.2% in
response to PMA-activated PMNs, respectively, analogous to control
levels and equivalent to cell lysis mediated by activated PMNs on
wild-type CHO cells (Fig. 5). These results
differ significantly from ICAM-1-transfected CHO cells treated with
PMA-activated PMNs, P < 0.05 (Fig.
5). In addition, incubation of ICAM-1-transfected CHO cells with
buffer-treated PMNs resulted in 1.4 ± 0.9% cell lysis, whereas
PMA-activated PMNs caused 22.2 ± 3.2% cell lysis (Fig. 5).

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Fig. 5.
Inhibition of protease abrogates PMN-mediated cytotoxicity.
ICAM-1-transfected CHO cells, labeled with
51Cr, were incubated with
quiescent or PMA (1 µM)-activated PMNs for 4 h at 37°C, 5%
CO2. Subsets of CHO cells were
coincubated with either AAPVCK (50 µM) or eglin C (20 µM) to
inhibit PMN protease activity. Additional subsets of CHO cells were
coincubated with mannitol, catalase (10,000 units), and catalase + superoxide dismutase (SOD; 10 µg/ml) and were also incubated with
heat-inactivated PMNs. Neutrophil cytotoxicity (%cell lysis) is
expressed as percent 51Cr released
from CHO cells (means ± SE). For all groups,
n = 5. * Difference from
ICAM-1-transfected CHO exposed to PMA-activated PMNs,
P < 0.05.
|
|
Superoxide generation in the presence of protease inhibitors.
To rule out the possibility that the protease inhibitors employed
abrogated PMN cytotoxicity by inhibition of the oxidase, superoxide
anion production was evaluated in the presence of these inhibitors to
both receptor-linked and non-receptor-linked soluble stimuli.
Pretreatment of PMNs with the protease inhibitors AAPVCK and eglin C
had no effect on superoxide generation mediated by the receptor-linked
signaling of PAF followed by fMLP or via PMA. PMA activation caused the
generation of 4.8 ± 0.6 nmol · 2.0 × 105
PMN
1 · min
1
superoxide, whereas pretreatment with AAPVCK or eglin C before PMA
activation caused 6.1 ± 1.3 and 4.6 ± 1.0 nmol · 2.0 × 105
PMN
1 · min
1,
respectively. Treatment of PMNs with fMLP alone resulted in superoxide
generation of 2.1 ± 0.3 nmol · 2.0 × 105
PMN
1 · min
1.
Priming of PMNs with PAF followed by fMLP produced 8.0 ± 1.5 nmol · 2.0 × 105
PMN
1 · min
1
of superoxide, whereas pretreatment with AAPVCK or eglin C before priming with PAF and activation by fMLP led to superoxide generation of
7.0 ± 1.5 and 7.5 ± 1.5 nmol · 2.0 × 105
PMN
1 · min
1,
respectively; n = 4 for all groups.
Thus AAPVCK and eglin C do not adversely reduce or increase the ability
of the PMN to generate superoxide via receptor-dependent or
-independent mechanisms.
Furthermore, neutralization of reactive oxygen metabolites was
performed using mannitol, catalase, catalase with SOD, and heat
inactivation of PMNs. Mannitol treatment yielded 20.4 ± 7.0% cell
lysis; catalase at 10,000 units produced 24.9 ± 7.0% cell lysis;
SOD (10 µg/ml) + catalase (10,000 units) yielded 21.5 ± 3.7%
cell lysis; heat-inactivated PMNs produced 17.1 ± 3.3% cell lysis.
These results did not differ from ICAM-1-transfected CHO treated with
PMA-activated PMNs. These data suggested that reactive oxygen
metabolites did not play a conspicuous role mediating cytotoxicity in
this in vitro model.
To explore the role of proteases in the absence of reactive oxygen
metabolites, PMNs were isolated from a patient with p67-phox autosomal
recessive, chronic granulomatous disease and compared with PMNs from
healthy donors. Incubation of transfected CHO cells with PMA-activated
PMNs from normal donors and from this patient with chronic
granulomatous disease resulted in 38 ± 12.2% lysis and 40.9 ± 8.1% cell lysis, respectively, compared with buffer-treated PMNs from
both the healthy donors (0.6 ± 0.3% cell lysis) and the chronic
granulomatous disease patient (2.1 ± 0.95% cell lysis). Moreover,
pretreatment of chronic granulomatous disease PMNs with the most
effective protease inhibitor eglin C significantly reduced PMN
cytotoxicity (Fig. 6).

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Fig. 6.
ICAM-1 provokes PMN-mediated cytotoxicity from p67-phox-deficient,
chronic granulomatous disease (CGD) neutrophils. ICAM-1-transfected CHO
cells, labeled with 51Cr, were
incubated with quiescent or PMA (1 µM)-activated PMNs from a patient
with CGD or PMNs from normal donors for 4 h at 37°C, 5%
CO2. Subsets of CHO cells were
coincubated with eglin C (20 µM) to inhibit protease activity from
CGD PMNs. Neutrophil cytotoxicity (%cell lysis) is expressed as the
percent 51Cr released from CHO
cells (means ± SE). CGD cell incubations were repeated 3 times with
a different normal control each time. * Difference from
ICAM-1-transfected CHO exposed to PMA-activated CGD PMNs,
P < 0.05.
|
|
 |
DISCUSSION |
Neutrophil priming, adhesion, emigration, chemotaxis, and activation
are considered important regulatory processes in PMN function (9, 46).
Induction and control of these events involve the expression and
binding of adhesion molecules on the PMN to cellular ligands (9). There
appear to be three distinguishable phases of PMN-ligand interaction
that involve different families of adhesion molecules (9, 16, 46). The
first is a low-affinity process thought to be mediated by expression of
selectins that produce rolling of PMNs along the endothelium (16). The
intermediate step is initiated by circulating agents or, alternatively,
membrane-expressed factors that, on binding PMN receptors, transduce a
signal that can result in PMN priming and increased surface expression
of the
2-integrin (9). The
third stage is a high-affinity, strong adhesion between
2-integrins on PMNs and their
counterreceptors, believed to be predominantly ICAM-1 (9). The
neutrophil has been implicated during this high-affinity interaction in
the pathogenesis of hyperinflammatory states leading to the ARDS and
multiple organ failure (33).
The importance of ICAM-1 in PMN-mediated tissue injury has been shown
by blocking the interaction between the CD11-CD18 complex and ICAM-1 or
by creating ICAM-1 deficiency, which reduced tissue injury
and organ dysfunction following ischemia/reperfusion,
hemorrhagic shock, and endotoxin challenge in animal models (2, 11, 21, 53). Although the interaction between
2-integrin complex and ICAM-1
has been recognized as playing a role in PMN-mediated cytotoxicity, the
mechanism of this cytotoxicity has not been fully established. Moreover, the precise function of the individual subunits in the
2-integrin complex has not been
elucidated, as there is apparently conflicting data regarding the
significance of the CD11a and CD11b subunits in neutrophil-mediated
cytotoxicity (14, 22). In general, CD18 has been considered only as the
constant
-unit in the LFA-1 and MAC-1 complexes. However, we and
others have shown, by cross-linking CD18 receptors, that this region is
physiologically active (36, 48). Consequently, the purpose of this
study was to examine the role of the CD11a, CD11b, and CD18 subunits of the
2-integrin using a model in
which ICAM-1 is the single specific adhesion molecule for these
subunits to engage.
In the present study, ICAM-1 was transfected successfully into CHO
cells. It has been previously shown in this model that cell lysis
requires both the presence of ICAM-1 and activated PMNs (4). Moreover,
ICAM-1 is sufficient to induce cytotoxicity without the involvement of
alternative PMN counterreceptors (4). In an attempt to further
investigate the physiological role of the CD11a, CD11b, and CD18
subunits of the
2-integrin
complex, alternate combinations of MAbs for these subunits were
coincubated with PMA-activated PMNs and ICAM-1-transfected CHO cells.
However, all combinations of MAbs were equally effective in reducing
PMN-mediated cytotoxicity. Consequently, multiple MAbs to CD11a, CD11b,
and CD18 were tested individually, and all appeared important in
blocking cell lysis (Fig. 1). Although all three
2-integrin subunits were apparently involved in PMN-mediated cytotoxicity, the specific role of
each subunit in causing target cell lysis was unclear. To further
examine this question, PMN adhesion to ICAM-1-transfected CHO cells was
tested. In these experiments, an MAb (84H10) to ICAM-1 was able to
effectively block PMN adhesion to transfected cells. In addition, the
MAbs to the
2-integrin subunits
were all effective in reducing cytotoxicity. Finding that one MAb for CD18 (TS1/18) was apparently unable to block PMN adhesion to these ICAM-1-transfected CHO cells, as confirmed by electron microscopy, despite effectively reducing cytotoxicity, led us to postulate an
expanded role for CD18 in the cytotoxic process. To investigate the
functional capacities of CD11a, CD11b, and CD18, we employed cross-linking of these
2
subunits and incubations with sICAM-1 immunoprecipitated from the
transfected CHO cells as a surrogates for binding to cell-associated
ICAM-1 and measured PMN generation of superoxide and protease release.
Cross-linking CD11a, CD11b, and CD18 produced no increase in superoxide
generation beyond buffer-treated controls. It is known that
cross-linking PMN Fc receptors via specific MAb causes superoxide
generation, increases in intracellular
Ca2+, and tyrosine phosphorylation
(27). However, because cross-linking of CD11a, CD11b, and CD18 did not
produce an increase in superoxide production compared with
buffer-treated controls, it is unlikely that nonspecific PMN-IgG
interaction occurred. Additionally, cross-linking CD11a and CD11b
caused no increase in protease release above basal levels; however,
cross-linking CD18 produced a significant increase in protease release
(Fig. 3). Moreover, sICAM-1 provoked protease release similar to PMA,
which could be abrogated by preincubation with MAbs that blocked CD18
but not by antibodies to either CD11a or CD11b. Taken collectively,
these data have suggested that CD18 plays a pivotal role in PMN
cytotoxicity via protease release.
To determine whether neutrophil protease release contributes to
PMN-mediated cytotoxicity, we returned to our original model. Coincubating ICAM-1-transfected CHO cells with multiple inhibitors of
reactive oxygen metabolites while they were exposed to PMA-activated PMNs did not attenuate PMN-mediated lysis of CHO cells. Conversely, two
chemically different protease inhibitors were able to reduce PMN-mediated cytotoxicity (Fig. 5), suggesting the importance of
proteases in PMN-mediated cytotoxicity. Finally, to examine the role of
neutrophil proteases in ICAM-1 provoked PMN cytotoxicity without the
confounding of incomplete inhibition of reactive oxygen metabolites,
neutrophils were obtained from a patient with an autosomal recessive
deficiency for p67-phox who has been shown to produce no superoxide
anion. With the use of the same cytotoxicity model, these neutrophils
from the chronic granulomatous disease patient were able to effectively
kill ICAM-1-transfected CHO cells, equivalent to that of PMNs from
normal controls. Furthermore, this cytotoxicity from chronic
granulomatous disease neutrophils is also reduced by protease
inhibitors (Fig. 6). This model can be extrapolated to represent the
isolated environment which is created when the neutrophil is tightly
adherent to a target cell, implicating proteases as important mediators
of neutrophil cytotoxicity (24, 40, 49).
The present study suggests the pathological upregulation of ICAM-1
could place tissues expressing this receptor in double jeopardy. By
providing a site for neutrophil adhesion, presumably via CD11a or
CD11b, a favorable environment for CD18 cross-linking with subsequent
neutrophil degranulation could be created (52). This protease release
may further upregulate the CD18 receptor and, consequently, incite
greater tissue injury (52). Clinical implications of the present study
may extend to both primary inflammatory sites and secondary distal
tissue beds. ICAM-1 upregulation occurs in response to a variety of
inflammatory agents that also stimulate the PMN, including LPS, TNF,
interleukin-1, interleukin-6, and IFN-
(12). In addition to its
presence on the endothelium, ICAM-1 has been identified as a surface
receptor on cardiac myocytes, hepatocytes, and type I pneumocytes (8,
12, 26). Thus, during either localized inflammatory processes
(reperfused organ) or conditions of the systemic inflammatory response
syndrome, blocking ICAM-1 or the PMN processes that it provokes may
attenuate secondary tissue injury.
 |
ACKNOWLEDGEMENTS |
We gratefully acknowledge the gift of CD1.8 from Dr. Timothy A. Springer. We thank Harry L. Malech, MD, for experimental design and
data analysis, Daniel R. Ambruso, MD, for neutrophils from the p67-phox
chronic granulomatous disease patient and data analysis, Ron Harbeck,
PhD, for data analysis, and Andrew Hiester for dihydrorhodamine assays
for superoxide anion generation.
 |
FOOTNOTES |
This research was supported in part by National Institute of General
Medical Sciences Grants P50-GM-49222-0 and T32-GM-08315, a
Clinical Associate Physician Award (no. 5 M01-RR00069) from the General
Clinical Research Centers Program, National Center for Research
Resources, National Institutes of Health, and a grant from the National
Blood Foundation.
Address for reprint requests: E. E. Moore, Dept. of Surgery, Denver
General Hospital, 777 Bannock St., Denver, CO 80204.
Received 24 June 1997; accepted in final form 18 March 1998.
 |
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