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
Top
Abstract
Introduction
Methods
Results
Discussion
References

Interaction of the beta 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 beta 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 beta 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, beta 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 beta 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
Top
Abstract
Introduction
Methods
Results
Discussion
References

THE beta 2-integrin (CD11-CD18) complex on the polymorphonuclear neutrophil (PMN) consists of a constant beta -region (CD18) that is noncovalently linked to variable alpha -units: CD11a, CD11b, and CD11c (42). The pairing of these alpha -units with the constant beta -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 beta 2-integrin complexes. These complexes are known to play an important role in normal microbicidal function, as aberrant expression of the beta 2-integrin complex known as leukocyte adhesion deficiency syndrome is associated with overwhelming infections (2).

In addition to its microbicidal role, the beta 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 alpha - and beta -subunits of the beta 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 beta 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-gamma (IFN-gamma ), 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 beta -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 alpha -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 beta 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
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Abstract
Introduction
Methods
Results
Discussion
References

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 beta 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 beta 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 beta 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 beta 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 beta 2-integrins. Cross-linking of beta 2-integrin subunits CD11a, CD11b, or CD18 was accomplished by incubating PMNs in suspension with saturating doses of MAb specific for the beta 2-subunits or nonspecific and isotypic controls. PMNs were incubated with MAbs to individual beta 2-subunits on ice for 30 min and washed twice in PBS+D. The beta 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 beta 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 beta 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
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Abstract
Introduction
Methods
Results
Discussion
References

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 beta 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 beta 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 beta 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 beta 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.

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 beta 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 beta 2-integrin MAb blockade

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 beta 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).

                              
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Table 2.   PMN generation of superoxide via cross-linking beta 2-subunits

Cross-linking beta 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.   beta 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
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Abstract
Introduction
Methods
Results
Discussion
References

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 beta 2-integrin (9). The third stage is a high-affinity, strong adhesion between beta 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 beta 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 beta 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 beta -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 beta 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 beta 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 beta 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 beta 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 beta 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-gamma (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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Abstract
Introduction
Methods
Results
Discussion
References

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Am J Physiol Cell Physiol 274(6):C1634-C1644
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