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(Journal of Leukocyte Biology. 2000;68:679-686.)
© 2000 by Society for Leukocyte Biology

C5a-stimulated human neutrophils use a subset of ß2 integrins to support the adhesion-dependent phase of superoxide production

ShivRaj Tyagi*,{dagger}, Lloyd B. Klickstein{dagger},{ddagger} and Anne Nicholson-Weller*,{dagger}

* Divisions of Infectious Diseases and Allergy and Inflammation, Department of Medicine, and Charles A. Dana Research Institute and Harvard-Thorndike Laboratory, Beth Israel Deaconess Medical Center; and
{ddagger} Division of Rheumatology, Immunology and Allergy, Brigham and Women’s Hospital,
{dagger} Harvard Medical School, Boston, Massachusetts

Correspondence: Dr. Anne Nicholson-Weller, M.D., Dana 617, Beth Israel Deaconess Medical Center, 330 Brookline Avenue, Boston, MA 02215. E-mail: anichols{at}caregroup.harvard.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Isolated human polymorphonuclear neutrophils (PMN) responded to human C5a with an immediate, transient release of superoxide lasting from 0.5 to 5 min. This was followed by a second release of superoxide, which began at 10 min after addition of C5a, was sustained for more than 30 min, and required ICAM-1 immobilized in the wells. F(ab')2 monoclonal antibody (mAb) preparations were used to dissect the role of individual ß2 integrins and to avoid the confounding effects of ligating Fc receptors. Anti-CD18 mAb treatment of the PMN had no effect on the immediate first phase but completely inhibited the second, adhesion-dependent phase of superoxide production. Anti-CR3 mAb only inhibited the adhesion phase of superoxide production partially, implying that other ß2 integrins were involved. A mixture of anti-CD11a, anti-CD11b, and anti-CD11c was not able to block superoxide production completely, suggesting a role for {alpha}d/ß2. Surprisingly, blocking anti-LFA-1 mAb had no effect on superoxide production. Consistent with this observation, immobilized, purified ICAM-2, a specific counter-receptor for LFA-1, did not support the adhesion-dependent phase ofsuperoxide production. Thus, PMN treated with C5a used signals via CR3, P150/95, and {alpha}d/ß2, but not LFA-1, to support superoxide production. LFA-1 has been shown by others to mediate most of the adhesion necessary for transendothelial migration in vivo. The inability of LFA-1 ligation to stimulate superoxide production may be an important means of preventing blood-vessel damage when PMN migrate across the endothelium.

Key Words: human • complement • free radicals • inflammatory mediators


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Superoxide (O2-)1 production by polymorphonuclear neutrophils (PMN) is an essential element of the antimicrobial repertoire of the cell. Children born with chronic granulomatous disease, a deficiency in an element of the reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase responsible for O2- generation, suffer recurrent and serious infections [1 ]. Multiple complement activators of PMN have been shown to trigger O2- production, including the soluble agonists C3a [2 ] and C5a [3 , 4 ], which bind to G protein-coupled receptors, and insoluble C1q [5 ], where the PMN receptor is unknown. The cumulative O2- production over 40 min was found to correlate with doses of C5a that induced adherence of PMN to plastic [6 ]. Subsequently, monoclonal antibodies (mAbs) to leukocyte ß2 integrins have been shown to block O2- production by PMN treated with most receptor-mediated agonists; however, the integrins do not serve as receptors for the agonists. Instead, in the case of C1q– and tumor necrosis factor {alpha} (TNF-{alpha})-triggered O2- production, the ß2 integrin provides an essential second signal for superoxide production [7 , 40 ]. Similarly, ß2 integrins are required for PMN-mediated phagocytosis also [8 , 9 ].

To determine whether ß2 integrins play a role in PMN O2- production triggered by a soluble complement agonist, we studied the effects of blocking mAbs to ß2 integrins on C5a-triggered O2- production. In our assay, the C5a was added to PMN in suspension, and the cells were allowed to settle by gravity onto the surface of microtiter wells that had been coated with specific proteins. Kinetic assays revealed two phases of O2- production by these C5a-stimulated PMN. The first was characterized by a high rate of O2- production of short duration that was independent of ß2 integrins. The second phase, which began at about 10 min as the cells settled on the surface of the microtiter well, featured a lower rate of O2- production, but production was sustained over 30 min. This phase was dependent entirely on selective ß2 integrin signaling, namely CR3, P150,95, and probably {alpha}d/ß2, but not lymphocyte function-associated antigen-1 (LFA-1). The second phase was independent of further G protein-coupled signal transduction, because the cells were refractory to additional C5a. The ß2 integrin-dependent phase accounted for >50% of the total O2- produced by the cells. We conclude that ß2 integrin-mediated signaling is essential for sustained C5a-triggered O2- production by adherent PMN. Finally, we propose a model where the selective inability of LFA-1 to mediate this signal when the PMN are stimulated with a chemotactic agent, in contrast to a stimulus such as C1q where LFA-1 is able to support adhesion-dependent O2- production, provides a mechanism by which PMN can adhere to and cross the endothelium without damaging it by sustained release of O2-.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents
The following reagents were purchased as noted: cytochrome c (horse heart), formyl-Met-Leu-Phe (fMLP), phorbol 12-myristate 13-acetate (PMA), superoxide dismutase, gelatin (type A from porcine skin), and hen ovalbumin from Sigma Chemical (St. Louis, MO); bicinchoninic acid protein assays reagent, Pierce Chemical (Rockford, IL); and Hanks’ balanced saline solution (HBSS)= (without Ca++ and Mg++) and HBSS++ (0.5 mM Ca++ and 0.5 mM Mg++) buffer, Gibco-BRL, Life Technologies (Grand Island, NY).

Antibodies and preparation of F(ab')2 fragments
Sources of mAbs are noted in Table I [10 11 12 13 14 15 16 ].


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  		Table 1. Sources of Monoclonal Antibodies

 
All mAbs were purified from hybridoma-conditioned media by 50% ammonium sulfate precipitation followed by protein A or protein G affinity chromatography. Each mAb preparation was digested to F(ab')2 using the ImmunoPure F(ab')2preparation kit (pepsin digest) or the F(ab')2preparation kit (ficin digest) for immunoglobulin G1 (IgG1), following the manufacturer’s (Pierce Chemical) protocol. The completeness of the digestion was followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using a 10% gel and Coomassie staining. The exclusive use of F(ab')2fragments in these experiments avoided considerations of Fc{gamma}R engagement, which is known to modify O2- production [17 ].

Human proteins
C1q was isolated from fresh human serum by fractional euglobulin precipitation and gel filtration chromatography, as described [18 ]. C1q purity was analyzed by SDS-PAGE, and the functional activity of the C1q was assessed by hemolytic assay [19 ]. Recombinant human C5a was purchased from Sigma. Recombinant soluble intercellular adhesion molecule-1 (ICAM-1) was kindly provided by Dr. Robert Rothlein, Boehringer Ingelheim, Pearl River, NY. ICAM-2 was purified from human platelets and was homogeneous as judged by SDS-PAGE with silver staining and by functional assays (unpublished results).

Isolation of human PMN
Venous blood (20–25 ml) was collected from normal volunteers in a syringe containing 2.0 ml acidified citrate (pH 5.2) and 8.0 ml dextran (6% Dextran 70 in 0.9% NaCl; Kendall McGaw Laboratories, Irvine, CA). After the erythrocytes had sedimented for 1 h at room temperature, the leukocyte-rich supernatant was removed, added to a new tube, underlaid with 15 ml Ficoll-Paque (Pharmacia Biotech, Piscataway, NJ), and centrifuged for 25 min at 1000 g at 4°C. The cells were maintained at 4°C during all subsequent steps. PMN (bottom pellet) were resuspended in 1 ml HBSS=, and contaminating erythrocytes were lysed with hypotonic saline. Finally, 95–98% pure PMN were resuspended in HBSS= supplemented with 0.1% ovalbumin. Cells were used within 2–3 h of venipuncture. Just before the start of the assay, the cells were resuspended in HBSS++ and warmed to 37°C.

Microplate assay of O2- production
C1q, ICAM-1, or ICAM-2 was added to 96-well plates (Immulon-2HB, Dynex Technologies, Chantilly, VA), was allowed to bind at 37°C for 1 h or at room temperature for 2–3 h, and then the wells were washed twice with HBSS=. All wells, whether or not specific ligands had been added previously, were blocked with gelatin HBSS= (0.5% gelatin in HBSS=) for 1 h. Finally, the wells were washed twice in HBSS= before starting the assay. All reagents were equilibrated to 37°C. A typical reaction assay was 150 µl HBSS++ containing 80 µM cytochrome c, 4 x 105 PMN, and the reaction was started by the addition of C5a (33 nM). Immediately after the assay was started, the microplate was placed in the reading chamber, and absorbance was read at 550 nm in the kinetic mode with intermittent agitation at 37°C. O2- generation was calculated from the superoxide dismutase-inhibitable reduction of ferricytochrome c in a microplate assay [20 ] using a Thermomax kinetic microplate reader (Molecular Devices, Sunnyvale, CA). In all experiments, fMLP (1.0 µM) and PMA (100 nM) were included as positive controls (triplicates or more). Calculations were based on an extinction coefficient of 20 AU/µmol • cm [20 ]. In more than 10 experiments, we used parallel assays in the presence of superoxide dismutase, and the color change was inhibited greater than 95%, an indication that nearly all of the cytochrome c reduction was a result of O2- production.

Adhesion assay
Tip-plate adhesion assays for PMN adherence to purified ICAMs were performed as described [21 ] on plastic petri dishes (Nunc Lab Tek, Fisher Scientific, Medford, MA).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
There are two phases of O2- generation in C5a-treated PMN
The rate of O2- generation was determined over 45 min after stimulation of purified PMN at time = 0 min with optimal doses of C1q (100 µg/ml for coating wells) or C5a (33 nM). C1q triggered production of O2- after a 5-min lag period, the maximal rate of production was 0.53 nmol/min at 25 min, and O2- production was measurable for the duration of the assay (Fig. 1a ). In contrast, C5a triggered an immediate O2- response with a maximal rate of 1.3 nmol/min; however, the O2- production returned to baseline at 4 min (Fig. 1b) . This rapid phase of NADPH oxidase activity has been noted previously with C5a stimulation [22 , 23 ]. The presence of recombinant, soluble ICAM-1, adsorbed to the plastic well before triggering the PMN with C5a, did not change the initial production of O2-, termed the first phase. However, C5a also induced a delayed and sustained O2- production when ICAM-1, the specific counter receptor for ß2 integrins, was present (Fig. 2 ). This second, delayed phase of O2- production, which was temporally correlated with the cells settling on the bottom of the well, has been described as an adhesion-dependent phase of O2- production [6 , 24 ]. The requirement for ICAM-1 permits the more specific designation of the second O2- production phase as the ß2 integrin-dependent phase.



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  		Figure 1. a) Immobilized C1q stimulated O2- production by PMN. Microtiter wells were precoated with C1q (100 µg/ml) in HBSS, washed, and blocked, and PMN in HBSS++ (4x105/well) were added to start the reaction. The rate of cytochrome c reduction (mOD/min after each 5 min interval) was recorded, and O2- production was calculated as described in Materials and Methods. C1q stimulated O2- production (•—•); unstimulated PMN were added to wells that were blocked with gelatin HBSS= ({square}—{square}). The mean ± SE, n = 4–6, was plotted. Results are representative of 10 experiments. b) Soluble C5a stimulated O2- production by PMN. Microtiter wells were precoated with 0.5% gelatin and washed with HBSS. PMN and C5a (33 nM) were then added simultaneously to start the reaction and O2- assay as described above. The "Buffer" negative control was unstimulated cells in gelatin-blocked wells ({square}—{square}). The mean ± SE, n = 5–6, was plotted. Results are representative of 10 experiments.

 


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  		Figure 2. Modulation of the C5a response by immobilized ICAM-1: Microtiter wells were precoated with recombinant ICAM-1 (3 µg/ml) for 2 h at 37°C and blocked with 0.5% gelatin. The assay was performed otherwise as in Fig. 1 . The mean ± SE, n = 4–6, was plotted. ICAM-1 alone in any concentration used did not stimulate O2- production; whereas the combination of C5a plus ICAM-1 stimulated a sustained adhesion-dependent phase of O2- production. The "Buffer" negative control was unstimulated cells in gelatin-blocked wells ({square}—{square}). This experiment was repeated twice with similar results.

 
Selective ß2 integrin-dependent production of O2- in C5a-treated PMN
C5a is well-recognized to activate leukocyte integrins, and PMN express all four ß2 integrins, namely LFA-1 (CD11a/CD18), CR3 (CD11b/CD18), P150/95 (CD11c/CD18), and {alpha}d/ß2 (CD11d/CD18). LFA-1, CR3, and P150/95 are the recognized counter-receptors for ICAM-1 [13 , 21 , 25 ]. Preincubation of PMN with F(ab')2 fragments of the anti-CD18-blocking mAb, TS1/18, inhibited almost completely the second phase of O2- production but had no effect on the first phase (Fig. 3 ). Similar results were obtained with another blocking F(ab')2, anti-CD18 mAb, IB4 (unpublished results). There was no inhibition seen with a nonblocking, anti-CD18 mAb F(ab')2 preparation of CBR-LFA 1/7 (Fig. 3) .



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  		Figure 3. Pretreatment of PMN with blocking anti-ß2 integrin-CD18 F(ab')2 fragments inhibited the adhesion-dependent phase of O2- production that was stimulated by C5a plus ICAM-1. Microtiter wells were coated with recombinant ICAM-1 (3.0 µg/ml) or gelatin HBSS=. PMN were pretreated with 10 µg/ml F(ab')2 fragments of blocking (TS1/18) or nonblocking (CBR LFA-1/7) mAbs, or buffer for 15 min at room temperature. Subsequently, the cells were challenged with C5a (33 nM) and O2- production assayed. Unstimulated, buffer-treated PMN were added to wells that were blocked with gelatin HBSS= ({square}—{square}). The mean values ± SE, n = 4–6, were plotted from a representative experiment. In a repeat experiment, IB4, another blocking anti-CD18 mAb, inhibited 95%, and TS1/18 inhibited 91%; CBR LFA-1/7 did not inhibit O2- production.

 
Additional studies were performed to identify which ß2 integrins were involved in C5a-stimulated O2- production. CR3 was recruited by C5a, indicated by the ability of pretreating PMN with F(ab')2 fragments of blocking mAb LM2/1 or CBR-M1/29 to inhibit O2- production by about 50% (Fig. 4 ). There was no inhibition afforded by pretreatment of PMN with F(ab')2 fragments of the nonblocking anti-CR3 mAb, CBR M1/20, which indicated a critical role for ligand binding by CR3. In contrast to the involvement of CR3 in the second phase of C5a-stimulated O2- production, F(ab')2 fragments of the blocking mAb to LFA-1 (TS1/22) had no effect on O2- production when compared with the nonblocking, control mAb, TS2/4 (Fig. 5a ). To confirm that PMN stimulated with C5a could not use LFA-1 to support the adhesion-dependent production of O2-, an experiment was performed using immobilized ICAM-2, which is a specific counter-receptor for LFA-1 [26; and unpublished results]. C5a-stimulated PMN in wells coated with ICAM-2 did not demonstrate the adhesion phase of superoxide production (Fig. 5b) , whereas the same preparation of PMN was able to use ICAM-1 to stimulate superoxide production. Controls were performed to determine that there was an equivalent number of ICAM-1 and ICAM-2 molecules immobilized/µm2 of plastic surface (unpublished results).



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  		Figure 4. The effect of anti-CD11b F(ab')2 fragments on C5a/ICAM-1-stimulated, second-phase O2- production in PMN. Microtiter wells were coated with recombinant ICAM-1 (3.0 µg/ml) or gelatin HBSS=. PMN were pretreated with 10 µg/ml F(ab')2 fragments of blocking anti-CD11b CBR M1/29 ({blacklozenge}—{blacklozenge}), or LM2/1 ({blacktriangleup}—{blacktriangleup}), or nonblocking anti-CD11b (CBR M1/20) mAbs. Subsequently, the cells were challenged with C5a (33 nM), and O2- production was assayed. Unstimulated, buffer-treated PMN were added to wells containing immobilized ICAM-1 ({circ}—{circ}) or were blocked with gelatin HBSS= ({square}—{square}). Both of the blocking mAbs inhibited O2- production by 50%, and the nonblocking mAb yielded results that were similar to the non-mAb-treated group (unpublished results). The mean values ± SE, n = 5–6, were plotted from a single experiment. This experiment was repeated with similar results.

 


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  		Figure 5. a) Effect of anti-CD11a F(ab')2 fragments on C5a/ICAM-1-stimulated O2-. Microtiter wells were precoated with recombinant ICAM-1 (3 µg/ml) for 2 h at 37°C and blocked with 0.5% gelatin. PMN were pretreated with 10 µg/ml F(ab')2 fragments of blocking (TS1/22), or nonblocking anti-CD11a (TS2/4), mAbs or buffer. Subsequently, the cells were challenged with C5a (33 nM) and O2- production assayed. Unstimulated, buffer-treated PMN were added to wells containing immobilized ICAM-1 ({circ}—{circ}) or blocked with gelatin HBSS= ({square}—{square}). The blocking and nonblocking mAbs did not inhibit the rate of O2- production significantly. The mean values ± SE, n = 5–6, were plotted from a single experiment. This experiment was repeated twice with similar results. b) ICAM-1, but not ICAM-2, supported the adhesion-dependent phase of O2- production in C5a-treated PMN. Microtiter wells were precoated with recombinant ICAM-1 (3 µg/ml), or a dilution of purified platelet ICAM-2 that yielded an equivalent number of active binding, for 2 h at 37°C. Subsequently, the wells were blocked by 0.5% gelatin. These conditions yielded 450–500 ICAM molecules/mm2 plastic surface (see Materials and Methods). The cells were stimulated with 33 nM C5a, and the O2- production was measured. The mean ± SE, n = 4–6, was plotted. PMN stimulated with C5a + ICAM-1 (•—•) but not C5a + ICAM-2 ({blacktriangleup}—{blacktriangleup}) supported O2-. When the PMN were unstimulated, ICAM-1 ({blacksquare}—{blacksquare}) or ICAM-2 ({blacklozenge}—{blacklozenge}) in any concentration used did not stimulate O2- production. This experiment was repeated with similar results.

 
C5a-treated PMN adhere to ICAM-2, hence the absence of an adhesion-dependent phase of O2- production is because of a lack of LFA-1-mediated signaling
Adhesion experiments were performed to determine whether C5a preferentially activated CR3 over LFA-1 to mediate adhesion or whether C5a-stimulated PMN selectively responded to CR3-mediated signals but not those generated by LFA-1. PMN were found to bind avidly to ICAM-2, and essentially all binding to ICAM-2 was inhibited by pretreating the PMN with the anti-LFA-1-blocking mAb, TS1/22 (Fig. 6 ). Thus, the inability of C5a-stimulated PMN to generate the adhesion-dependent phase of O2- production when adherent via LFA-1 is a consequence of a selective inability of the PMN to generate or use the LFA-1-mediated signal.



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  		Figure 6. Purified PMN adhere to ICAM-2 in an LFA-1-dependent manner. ICAM-1 and ICAM-2 were coated on demarcated spots on 35 mm plastic petri dishes for 2 h at room temperature. The plates were blocked and washed with HBSS++ containing 10 mM HEPES, pH 7.3, and 0.5% heat-inactivated human serum albumin (HSA). C5a-treated PMN were added (1.5x106 total in 1.5 ml), and the plates were left undisturbed for 4 min at room temperature. Nonadherent cells were removed, and the plates were washed with a transfer pipet. Adherent cells were counted in three locations for each spot using a calibrated ocular grid. Results are shown as mean ± SD from a representative experiment.

 
Other ß2 integrins bind ICAM
Two lines of evidence supported the functional participation of P150,95 and/or {alpha}d/ß2 on PMN binding to ICAM-1. First, the presence of blocking anti-CD18 mAb fully inhibited O2- production by PMN, although anti-CR3 mAb inhibited it by only 50%, and anti-LFA-1 didn’t inhibit at all. Second, there was specific binding of PMN to immobilized ICAM-1 even when PMN were treated with the combination of blocking mAbs to LFA-1 and CR3 (Fig. 6) . Consequently, we tested whether a blocking anti-CD11c mAb was able to inhibit the adhesion-dependent phase of superoxide production and found partial inhibition when the C5a-treated PMN were assayed in wells coated with ICAM-1. Although we lacked the reagents to test directly for the participation of {alpha}d/ß2, a mixture of blocking anti-CD11a, anti-CD11b, and anti-CD11c was unable to inhibit as completely as anti-CD18. Because there was variability between donors, results from PMN isolated from two different donors are shown (Fig. 7a and b ). Thus, these data suggest that all four ß2 integrins may bind ICAM-1, and all but LFA-1 may mediate the adhesion-dependent signal for O2- production by C5a-treated PMN.



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  		Figure 7. The effect of anti-CD11c alone and in combination with anti-CD11a and anti-CD11b on C5a/ICAM-1-stimulated, second-phase O2- production in PMN, representing experiments performed with cells from two different donors. Microtiter wells were coated with recombinant ICAM-1 (3.0 µg/ml) or gelatin HBSS=. PMN were pretreated with 10 µg/ml F(ab')2 fragments of blocking anti-CD11c (3.9) or a combination of 10 µg/ml each of anti-CD11c (3.9) + anti-CD11a (TS1/22) + anti-CD11b mAb (CBR M1/29); or blocking anti-CD18 (TS1/18) mAb ({blacktriangleup}—{blacktriangleup}) or buffer ({blacksquare}—{blacksquare}). Subsequently, each group of cells was stimulated with C5a (33 nM). Unstimulated, buffer-treated PMN were added to wells blocked with gelatin HBSS= ({square}—{square}). The mean values ± SE, n = 5–6, were plotted from a single experiment. This experiment was repeated with similar results. In both donors, anti-CD11c inhibited a fraction of the C5a-stimulated second phase of superoxide. Of note, the combination of blocking anti-CD11a, anti-CD11b, and anti-CD11c still did not block as efficiently as anti-CD18.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
This study demonstrated that C5a-treated PMN require a specific, adhesion-dependent signal mediated by a subset of ß2 integrins for sustained production of O2-. In particular, LFA-1 did not mediate this signal, although this integrin was able to support cell adhesion. LFA-1 is important for transendothelial migration in vivo; hence, we propose that the selective inability of chemotaxin-treated PMN to use signals from LFA-1 helps prevent O2- generation while the cells exit the vasculature.

Two phases of oxygen radical formation have been noted previously in PMN stimulated with C5a and analyzed in a chemiluminescence assay [22 ]. In this assay, the oxidation of luminol, which can be taken up in the cell, is followed in a scintillation counter [27 ]. In an experiment where the exogenous luminol was washed away from the luminol-loaded cells, the subsequent addition of C5a stimulated only the second peak of chemiluminescence. This was interpreted to mean that the first peak of chemiluminescence was detecting oxygen radicals released outside the cell, whereas the second, prolonged phase of oxygen-radical formation was being generated within the cell [22 ]. In our studies, the delay in the onset of the C5a-induced, second adhesion-dependent phase of O2- production may be a result of the time necessary for the cells to settle onto the ICAM-1. This feature of our experimental assay allowed the separate analysis of the two distinct phases of O2- production. The delayed and prolonged C5a-stimulated, adhesion-dependent O2- production has not been well-studied. However, when PMN are stimulated with fMLP, which, like C5a, uses a G protein-coupled receptor, ß2 integrins are activated and subsequently recruit members of the Src family tyrosine kinases, hck and c-fgr (reviewed in [28 ]).

The specificity of the blocking antibodies used in our experiments was rigorously controlled. We used exclusively F(ab')2 fragments of predominantly isotype-matched antibodies (Table 1 ) and found that only adhesion-blocking mAb inhibited O2- production. mAbs that bound to CD18 at a site away from the ligand-binding site and do not block ß2 integrin-mediated adhesion ("nonblocking" mAb) did not inhibit O2- production (Fig. 3 4 5a) . This indicates that the anti-ß2 mAbs that inhibited PMN O2- generation in vitro must have blocked a ligand-receptor interaction, as opposed to mediating a dominant negative signal for O2- generation because of cross-linking of the integrin by the bivalent F(ab')2 fragments.

The observation that blocking anti-CD18 mAb completely inhibited the adhesion phase of C5a-stimulated O2- production, although anti-CR3 mAb afforded only partial inhibition (Fig. 4) , and anti-LFA-1 gave no inhibition, strongly suggested that additional ß2 integrins must be involved. Consistent with this finding, there was significant PMN adhesion to ICAM-1 despite treatment of the cells with the combination of anti-CR3 + anti-LFA-1 (Fig. 6) . PMN express two other ß2 integrins, namely P150,95 and {alpha}d/ß2, and P150,95 has been shown to bind ICAM-1 [13 ] and thus was the leading candidate. Blocking anti-CD11c directly demonstrated a role for P150,95, but the incomplete inhibition provided by a mixture of anti-CD11a, anti-CD11b, and anti-CD11c—compared with the complete inhibition seen with anti-CD18—inferred a role for {alpha}d/ß2 (Fig. 7a and 7b) . The integrin {alpha}d/ß2, when assessed as a recombinant protein expressed on Chinese hamster ovary cells (CHO), bound to wells coated with ICAM-1-Fc reportedly twice as well as to plastic coated with bovine serum albumin (BSA) and better than the control CHO transfectants [29 ]. Hence, although the binding of {alpha}d/ß2 to ICAM-1 is much less robust than to ICAM-3, it is possible that all four ß2 integrins may recognize ICAM-1. Furthermore, it is possible that immobilized ICAM-1 triggers a ligand-induced binding site on {alpha}d/ß2 expressed on PMN, as it does on lymphocyte LFA-1 [30 ], and the ligand-activated {alpha}d/ß2 may then bind well to ICAM-1. This has not been tested to our knowledge. Alternatively, although only ICAM-1 was immobilized in the wells, it is possible that the PMN bind to adjacent cells via ICAM-3 and {alpha}d/ß2. The PMN are observed to be touching each other in the periphery of the microtiter wells, and PMN express high levels of ICAM-3. This seems less likely because ICAM-3 binding to {alpha}d/ß2 should occur in the absence of immobilized ICAM-1; however, adhesion-dependent production of O2- does not occur under these conditions. An additional possibility is that LFA-1 binding to ICAM-1 may direct the upregulation of CR3 and P150,95 from intracellular stores, as suggested previously [14 ], and these ("unblocked") integrins may mediate adherence and signaling.

The ability of C5a-treated PMN to use LFA-1 for adherence but not for the production of superoxide was unexpected. This was shown first by the inability of blocking LFA-1 mAb to affect O2- production (Fig. 5a) . Confirmation that LFA-1 was not involved came from the inability of C5a-stimulated PMN to generate an adhesion-dependent phase of O2- production when ICAM-2 was immobilized on the plastic instead of ICAM-1(Fig. 5b) . Although some controversy exists [31 ], most studies indicate that ICAM-2 binds only LFA-1 among the ß2 integrins [21, 26, 32; and unpublished results]. Our results are also consistent with ICAM-2 serving as the specific counter-receptor for LFA-1 (Fig. 6) .

During the isolation procedure, PMN became sufficiently activated for CR3 and LFA-1 to mediate adhesion (unpublished data). However, this activation alone was not sufficient to induce O2- production (Fig. 1b) ; an additional signal from C5a in our experiments was necessary. Data from other laboratories indicate that C5a stimulation should enhance LFA-1 avidity [32 ]. In contrast to the results using C5a as the agonist, when CR3 and LFA-1 are activated by immobilized C1q, both integrins participate in signaling for O2- production, confirming that the LFA-1 downstream signaling pathway for O2- production does exist in PMN [40 ]. Thus, the most likely explanation is that, although LFA-1 is activated by C5a, there is a selective inability of LFA-1 to signal the C5a-treated PMN for the second phase of O2- production. This may be the first example of PMN selectively using a downstream signaling pathway from activated CR3, P150/95, and {alpha}d/ß2 but not LFA-1. This observation parallels the activation-induced upregulation from PMN internal stores of CR3, P150/95, and {alpha}d/{alpha}2 but not LFA-1. There is precedent for the selective activation of ß2 integrins for adhesion in monocytes [33 ] and eosinophils [34 ], and in PMN, there are data that indicate CR3 can be activated by a phosphotidyinositol (PI) 3-kinase-dependent or PI 3-kinase-independent pathway [35 ]. These findings in aggregate suggest that the cellular interactions of CR3, P150/95, and {alpha}d/ß2 on PMN for "inside-out" and "outside-in" signaling are fundamentally different from those of LFA-1.

There are important functional implications for the differential activation of integrins by C5a. In responding to a tissue infection, PMN must contact and move through the endothelium without damaging the endothelial cells with released O2-. The C5a-induced surface expression of P-selectin [36 ] on the endothelial cell has the potential to block CR3-ICAM-1 binding-induced O2- production [37 ]. Although C5a-stimulated PMN would activate LFA-1 and CR3 for transendothelial migration, the higher density of ICAM-2 compared with ICAM-1 on the initially resting endothelial cells [38 ] might selectively engage LFA-1, which would not support O2- production. This hypothesis is supported by recent in vivo studies, showing that in response to a local TNF-{alpha} stimulus, the LFA-1 knockout mouse and the CD18 knockout mouse show comparable abnormally low levels of PMN in the tissue, whereas the CR3 knockout mouse had higher than normal numbers of migrating PMN [39 ]. Thus, LFA-1 is the dominant ß2 integrin involved in the adhesion necessary for transendothelial migration. In summary, in addition to the previously described inhibitory role for P-selectin, our description of the ability of PMN to use LFA-1 for transendothelial migration, but not for O2- production, is a second mechanism that may prevent PMN-mediated endothelial damage when these cells migrate across the vessel wall.


   ACKNOWLEDGEMENTS
 
These studies were supported by NIH grants R01 AI42987 and R01 HL33768 to A. N. W., and NIH grant R03 AR45025 and an Arthritis Foundation/ACR Investigator Award to L. B. K.; S. T. was supported by NIH training grant AI 07061.The authors are grateful to Drs. Nancy Hogg and Timothy A. Springer for providing mAbs as listed in Table 1 and Dr. Robert Rothlein for providing recombinant soluble ICAM-1.

Received December 9, 1999; revised May 30, 2000; accepted May 31, 2000.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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