Journal of Leukocyte Biology Myeloid cells, immune suppression, tumor immunology
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(Journal of Leukocyte Biology. 2002;72:718-726.)
© 2002 by Society for Leukocyte Biology

Evidence that TNF-induced respiratory burst of adherent PMN is mediated by integrin {alpha}Lß2

Eva Decleva, Pietro Dri, Renzo Menegazzi, Sara Busetto and Rita Cramer

Department of Physiology and Pathology, University of Trieste, Italy

Correspondence: Eva Decleva, Department of Physiology and Pathology, University of Trieste, via A. Fleming, 22, 34127 Trieste, Italy. E-mail: declevae{at}univ.trieste.it


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Polymorphonuclear leukocytes (PMN) respond to tumor necrosis factor (TNF) with a respiratory burst (RB) only after adherence to surfaces coated with extracellular matrix proteins such as fibronectin and fibrinogen (permissive substrates) but not with others such as laminin or collagen (nonpermissive substrates). As PMN adherence to both types of surfaces is dependent on ß2 integrins, we investigated the molecular basis of the different metabolic response to TNF. In particular, we evaluated the relative role of each ß2 integrin ({alpha}Lß2, {alpha}Mß2, and {alpha}Xß2) in adherence and O2- production of PMN residing on fibronectin- and laminin-coated surfaces, which were considered as models of permissive and nonpermissive surfaces, respectively. By using {alpha} chain-specific monoclonal antibodies (mAb), we show that {alpha}Mß2 and {alpha}Xß2 mediate adherence to fibronectin and laminin; {alpha}Lß2 is not involved in adherence to laminin and has only a minimal contribution in adherence to fibronectin. Furthermore, production of O2- in response to TNF was induced by immobilized anti-{alpha}Lß2 but not anti-{alpha}Mß2 or anti-{alpha}Xß2 mAb. A strong correlation was also found between expression of {alpha}Lß2 and TNF-induced RB on fibronectin. Lastly, PMN responded to TNF on laminin with a RB after the inclusion of {alpha}L-specific mAb in the laminin coat. Thus, we conclude that TNF-induced RB by PMN residing on fibronectin is mediated by {alpha}Lß2 and that {alpha}Mß2 and {alpha}Xß2 are likely to play an ancillary role to the signaling activity of {alpha}Lß2 by facilitating its recruitment to sites of adherence. The nonpermissiveness of laminin appears to be a consequence of its inability to act as a ligand for {alpha}Lß2.

Key Words: adherence • ß2 integrins • neutrophils • superoxide anion


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The extracellular matrix (ECM) plays a central role in the regulation of cell behavior and function during tissue formation, organization, and homeostasis. The functional responses of polymorphonuclear leukocytes (PMN), the predominant cells of the acute inflammatory reaction, are also profoundly modulated by the ECM. This is well established, for example, in the respiratory burst (RB), which can be elicited in cells in suspension by several agonists such as N-formylpeptides and phorbol 12-myristate 13-acetate. However, the RB requires adherence to the ECM when tumor necrosis factor (TNF) and other molecules of relevant biological interest [granulocyte macrophage-colony stimulating factor (GM-CSF) and G-CSF] are used [1 2 3 4 5 6 ].

We have shown that at least two costimulatory events are needed for the activation of the RB by TNF, namely commitment of the 55-kDa TNF receptor [7 , 8 ] and interaction of the integrins with components of the ECM [5 ]. More recently, we have demonstrated that a decrease in intracellular concentration of chloride anions is an early and essential signaling step for PMN activation under these conditions [9 , 10 ]. Questions still remain, however, about the mechanisms accounting for the differential responses of PMN exposed to TNF on substrates coated with various ECM proteins and about the role played by cytoskeletal remodeling and cell spreading, known to be necessary for the activation of the RB of PMN treated with TNF on ECM-coated surfaces [3 4 5 6 ]. In fact, it is well established that ECM proteins such as fibronectin (FN) and fibrinogen support TNF-induced triggering of PMN spreading and RB, whereas others such as laminin (LM), type IV and type I collagens, do not [3 , 5 , 11 , 12 ].

In the present study, we have attempted to define the molecular basis of the differential oxidative response to TNF by PMN residing on FN- and LM-coated substrates through the use of two complementary approaches. First, we have used soluble and immobilized anti-integrin monoclonal antibodies (mAb) to examine the relative role of each ß2 integrin ({alpha}Lß2, {alpha}Mß2, {alpha}Xß2) in the adherence and in the O2- production of PMN stimulated with TNF. Second, we have used models in which cytoskeletal reorganization is augmented or inhibited to investigate the role of actin polymerization and cell spreading in TNF-induced activation of the RB. Our results demonstrate that adherence of TNF-treated PMN to FN- and LM-coated surfaces is mediated by integrins {alpha}Mß2 and {alpha}Xß2 and that the different metabolic response observed on the two kind of surfaces, i.e., responsiveness on FN and unresponsiveness on LM, depends on the engagement of {alpha}Lß2 on the former surface but not on the latter. Finally, our observations convey the idea that PMN spreading on ECM is functional to TNF-mediated RB only if a sufficient recruitment of {alpha}Lß2 is ensured.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents
Cytochrome c (type VI, from horse heart), bovine serum albumin (BSA; fraction V), glutaraldehyde (grade II), recombinant protein G, genistein (4',5,7-trihydroxyisoflavone, synthetic), CB (from Helminthosporium dematioideum), and ficin (from fig tree latex) attached to beaded agarose were obtained from Sigma Chemical Co. (St. Louis, MO). FN was purified from human plasma by affinity chromatography on gelatin as described by Ruoslahti et al. [13 ]. LM, type 1, isolated from the Engelbreth-Holm-Swarm mouse tumor (>90% purity), was provided by Becton Dickinson (Bedford, MA). Casein, formaldehyde, and ethanol were from Merck (Darmstadt, Germany). Human recombinant TNF-{alpha} was purchased from Bissendorf Biochemicals (Hannover, Germany). Percoll and D-[1-14C] glucose (57.0 mCi/mmol) were obtained from Amersham Pharmacia Biotech (Little Chalfont, Buckinghamshire, England). 3,3', 5,5'-tetramethylbenzidine (TMB) was provided by Serva Feinbiochemica (Heidelberg, Germany). Liquid scintillation cocktail (ReadySafeTM liquid) was from Beckman Instruments Inc. (Fullerton, CA). All other reagents and chemicals were of the highest purity grade available. All solutions were made in endotoxin-free water for clinical use.

Antibodies
Purified mAb 13 [rat immunoglobulin G (IgG)2a], specific for the CD29 subunit (common ß1 chain) of the very late antigen complex [14 ], was kindly donated by Dr. K. Yamada (National Institutes of Health, Bethesda, MD). mAb 60.3 (mouse IgG2a) against the CD18 subunit (common ß2 chain) of the CD11/CD18 complex [15 ] was a gift of Dr. J. M. Harlan (University of Washington, Seattle). The anti-CD18 mAb TS1/18 (mouse IgG1) [16 ], mAb TS1/22 (mouse IgG1) specific for CD11a ({alpha}L) [17 ], and mAb BB7.5 (mouse IgG1) specific for a common determinant on the human leukocyte antigen (HLA)-A, -B, -C (class I) molecules [18 ] were affinity-purified from ascites fluids recovered from mice injected with the corresponding cell lines purchased from American Type Culture Collection (ATCC; Manassas, VA). The anti-CD11b mAb M1/70 (rat IgG2b) [19 ] was affinity-purified from culture supernatants of the corresponding cell line provided by ATCC. mAb 3.9 (mouse IgG1), specific for CD11c ({alpha}X) [20 ], was a generous gift of Dr. N. Hogg (Imperial Cancer Research Fund, London, UK). F(ab')2 fragments of mAb TS1/18, TS1/22, 3.9, and BB7.5 were prepared by ficin-digestion of the corresponding antibody as previously described [21 ]. F(ab')2 fragments of mAb M1/70 were obtained by pepsin-digestion using the ImmunoPure® F(ab')2 Preparation Kit (Pierce, Rockford, IL).

PMN isolation
PMN were isolated from the peripheral blood of healthy donors by density gradient centrifugation over Percoll as described by Metcalf et al. [22 ] with slight modifications. Briefly, fresh blood collected in 4 mM EDTA was layered over a two-step Percoll gradient [62% and 75% isotonic Percoll in phosphate-buffered saline (PBS), 300–310 mOsM, density 1.078 g/ml, and 1.103 g/ml, respectively] and was centrifuged at 200 g for 10 min and at 400 g for an additional 15 min at 20°C. PMN, collected at the interface between 62% and 75% Percoll, were washed once in HEPES-buffered saline solution containing BSA (140 mM NaCl, 5 mM KCl, 5 mM glucose, 5 mM HEPES, pH 7.4, and 0.2% BSA), and contaminating erythrocytes were lysed by a brief hypotonic treatment (10 s with 3 vol 1 mM sodium phosphate buffer solution, pH 7.4, followed by addition of 7 vol 1.3% NaCl solution in 1 mM sodium phosphate buffer, pH 7.4, to restore isotonicity). The resulting cell population was resuspended in HEPES-buffered saline solution and contained 94–97% neutrophils, 2–5% eosinophils, and 0.5–1% mononuclear cells.

Preparation of FN- and LM-coated surfaces
Flat-bottom 96-well plates (F16 MaxiSorp Loose Nunc-Immuno Modules, Nunc, Roskilde, Denmark) or 24-well plates, tissue-culture treated (Corning Costar Corporation, Cambridge, MA), were coated with FN or LM as described elsewhere [23 ]. Briefly, 50 or 200 µl FN or LM solution (20 µg/ml in PBS) was put in each well, and the plate was left at 37°C for 1–2 h in a humidified incubator. Just before use, the wells were rinsed three times with PBS.

Preparation of surfaces coated with immobilized and oriented mAb
mAb were immobilized in an oriented manner by affinity binding of the Fc portion to protein G-coated surfaces as previously described [10 ] with some modifications. Briefly, flat-bottomed microwells (Carbohydrate Binding, 8-well Strip Plates, Corning Costar) were first coated with protein G by treating each well with 50 µl of a 2.5% glutaraldehyde solution for 15 min at room temperature (RT). After washing three times with PBS and incubating with 50 µl of a 5 µg/ml solution of protein G in PBS for 60 min at RT, the wells were rinsed three times with PBS. Unreacted aldehyde groups were quenched by overnight incubation at 4°C with 50 µl 2 mg/ml casein in PBS. After extensive washing with PBS, 50 µl of each mAb at 10 µg/ml in PBS or 50 µl of a mixture of two mAb, each at 5 µg/ml, was added to the wells and incubated for 60 min at RT. Unreacted sites were finally saturated with FN (20 µg/ml) or LM (20 µg/ml) as described above. For scanning electron microscopy experiments, plastic dishes (13 mm {phi} Thermanox, Nunc) were coated with protein G (20 µg/ml in PBS) by an overnight incubation at 4°C, followed by incubation with casein for 1 h at 37°C. The subsequent steps were identical to those described above. Binding of mAb to the wells was measured by enzyme-linked immunosorbent assay (ELISA) with horseradish peroxidase (HRP)-conjugated sheep anti-mouse IgG F(ab')2 (Sigma Chemical Co.) or HRP-conjugated goat anti-rat IgG F(ab')2 (Sigma Chemical Co.), using 2 mM TMB as a substrate. Readings were taken at 405 nm with a microplate reader (Multiskan MCC/340, Labsystem Oy, Helsinki, Finland) after blocking the reaction with 2 N H2SO4.

Assay of O2- production
O2- production was measured by the superoxide dismutase (SOD)-inhibitable cytochrome c reduction assay as previously described [7 ]. Briefly, PMN were suspended at 1.5 x 106 cells/ml in HEPES-buffered saline solution supplemented with 1 mM CaCl2 and 1 mM MgCl2 (Ca2+/Mg2+ HEPES) or with 1 mM EDTA (EDTA-HEPES) and were incubated for 10 min at 37°C in a shaking water bath. Aliquots (50 µl) were then added to FN-, LM-, or mAb-coated wells containing 0.1 ml of the same medium supplemented with 0.18 mM cytochrome c (final concentration, 0.12 mM) and when required, TNF, mAb, or F(ab')2 fragments at a concentration 1.5 times higher than the desired final one. In the experiments with genistein or CB, PMN were pretreated in suspension with 50 µM genistein or 5 µg/ml CB for 10 min at 37°C and were then transferred into the wells containing the corresponding inhibitor at the same concentration used in suspension in addition to cytochrome c and TNF. Cells were then incubated at 37°C in a humidified incubator, and the plate was read at 550 nm and 540 nm at the desired times. The amount of reduced cytochrome c was calculated from the absorbance difference between 550 nm and 540 nm, using an absorbance of 0.037 optical density (OD) units for 1 nmol reduced cytochrome c as a standard.

Assay of adherence
The number of PMN adherent to FN, LM, or immobilized mAb was assessed in the same wells in which O2- release had been measured after the last absorbance reading as described elsewhere [9 ]. Briefly, wells were filled with PBS, sealed with 8-cap strips, and centrifuged upside-down for 5 min at 200 g to remove nonadherent cells. The strips were then removed, the wells were flicked empty, and the number of PMN remaining in the wells was quantified by an enzymatic assay based on the measurement of myeloperoxidase activity, as previously described [24 ].

Hexose monophosphate shunt activity
The oxidation of glucose via the hexose monophosphate shunt was measured as described [22 ]. Briefly, 3 x 106 PMN/ml was prewarmed for 10 min at 37°C in glucose-free Ca2+/Mg2+-HEPES and then added (100 µl/well) to FN- or LM-coated wells (24-well clusters, Corning Costar) containing 0.5 µCi D-[1-14C] glucose in 0.5 ml of the same medium and when required, TNF (10 ng/ml final concentration) and MnCl2 (1 mM final concentration). To avoid 14CO2 leakage, the wells were immediately sealed with disposable rubber stoppers with a plastic center well (Kontes Glass, Vineland, NJ) containing 200 µl 10% KOH as a CO2 trapping agent. The plate was incubated for 1 h at 37°C, and the reaction was stopped by injecting 0.5 ml 1 N HCl/well through the stoppers. After an overnight equilibration at 4°C, 150 µl KOH contained in each center well was transferred into scintillation vials (ReadySafeTM, Beckman Instruments). Vials were shaken vigorously and counted in a ß-scintillation counter (LS6000TA, Beckman Instruments). The nmol glucose oxidized in each experimental condition (run in duplicate) was calculated by measuring the radioactivity (counts per minute) of a D-[1-14C] glucose standard solution.

Immunofluorescence flow cytometry
PMN (0.25x106 in 0.5 ml Ca2+/Mg2+-HEPES) were cooled at 4°C and incubated for 45 min with 4 µg/ml mAb TS1/22. After two washes with ice-cold PBS, the cells were incubated for an additional 30 min with 5 µg/ml AlexaFluorTM546 goat anti-mouse IgG, F(ab')2 fragment conjugate (Molecular Probes, Eugene, OR). After two additional washings, PMN were suspended in PBS containing 1% formaldehyde and analyzed by a FACScan flow cytometer (FACSCaliburTM, Becton Dickinson, Mountain View, CA).

Scanning electron microscopy
PMN (3x106 cells/ml in Ca2+/Mg2+-HEPES, 0.1 ml/well) were plated on FN-, LM-, or mAb-coated 13 mm {phi} plastic dishes placed at the bottom of the wells of 24-well plates and were incubated for 30 min at 37°C in Ca2+/Mg2+-HEPES or EDTA-HEPES in a final volume of 0.6 ml. Nonadherent PMN were then removed by two gentle washes with PBS, and the remaining cells were fixed for 15 min at RT with 2% (v/v) glutaraldehyde in PBS. The samples were rinsed with PBS, serially dehydrated in graded ethanol (30-50-70-90-100%), and finally processed using conventional scanning electron microscopy techniques.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Adherence of TNF-treated PMN to FN- and LM-coated surfaces is ß2 integrin-dependent
When exposed to TNF, PMN adhere to FN- and LM-coated surfaces (Fig. 1C and 1D ). This adherence is accompanied by a massive production of O2- on FN but not on LM (Fig. 1A and 1B) , in accordance with previous results obtained by us [5 , 25 ] and others [26 ]. Figure 1 also shows that TNF-induced adherence to both surfaces is completely inhibited by the anti-ß2 mAb 60.3, and it is unaffected by the anti-ß1 mAb 13, an antibody that has been shown to inhibit adherence in other cellular systems [14 ]. As expected, TNF-induced O2- production on FN is inhibited by mAb 60.3 but not by mAb 13 (Fig. 1A) . Thus, TNF-treated PMN adhere to FN- and LM-coated surfaces in a ß2 integrin-dependent manner but respond with a RB only on FN.



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  		Figure 1. Effects of anti-integrin ß chains mAb on O2- production (A, B) and adherence (C, D) of TNF-treated PMN. PMN suspensions (1.5x106 cells/ml in Ca2+/Mg2+-HEPES) were prewarmed at 37°C and added in 0.05 ml aliquots to microtiter plate wells coated with FN or LM and containing 0.18 mM cytochrome c, 15 ng/ml TNF, and 15 µg/ml mAb 13 (anti-ß1) or mAb 60.3 (anti-ß2) in 0.1 ml Ca2+/Mg2+-HEPES. Following incubation for 45 min at 37°C, O2- production was determined after spectrophotometrically measuring the amount of reduced cytochrome c, as detailed in Materials and Methods. For determination of adherence, the 96-well plates were centrifuged upside-down to remove nonadherent cells after the cytochrome c readings were taken. Adherence was quantified by measuring myeloperoxidase activity as detailed in Materials and Methods. Data are mean ± SEM of six duplicate experiments. Statistical analysis (one-tailed Student’s t-test on unpaired data): TNF-treated PMN versus TNF-treated PMN in the presence of mAb. ***, P < 0.001; NS, not significant.

 
Modulation of spreading: effects on TNF-induced RB
Previous reports have indicated that the RB of TNF-treated PMN residing on biological surfaces is dependent on spreading [3 4 5 6 ], i.e., on a particularly broad and tight contact of the cells with the substrate (see, for example, Fig. 2A ). Table 1 shows that CB and the tyrosine kinase inhibitor genistein, which inhibit cytoskeletal polymerization and cell spreading [1 , 27 ], virtually abolish O2- generation while leaving TNF-induced adherence to FN quantitatively unaltered.



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  		Figure 2. Effect of Mn2+ on the morphology of PMN exposed to TNF on FN- and LM-coated surfaces. PMN suspensions (2.0x106 cells/ml in Ca2+/Mg2+-HEPES) were prewarmed at 37°C and added in 0.15 ml aliquots to 13 mm {phi} plastic dishes coated with FN (A, C) or LM (B, D). After incubation for 30 min at 37°C with 10 ng/ml TNF in the presence (C, D) or absence (A, B) of 1 mM MnCl2, the dishes (set on the bottom of 24-well plates) were washed twice with PBS, and adherent PMN were fixed with 2% glutaraldehyde. Samples were then processed for scanning electron microscopy as described in Materials and Methods. (A, B) TNF-treated PMN residing on FN (A) and LM (B; original bars=10 µm). Insets, Resting PMN (same magnification). (C, D) TNF-treated PMN residing on FN (C) and LM (D) in the presence of 1 mM MnCl2 (original bars=1 µm). Insets, Low magnification view of the same samples (original bars=10 µm).

 

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  		Table 1. Effect of Cytoskeletal Polymerization Inhibitors on O2- Production and Adherence of PMN Stimulated with TNF on FN-Coated Surfaces

 
PMN adherent to LM exhibit a less-pronounced spread phenotype than PMN adherent to FN (Fig. 2A and 2B ). As this could explain the lack of RB on this substrate, we hypothesized that by strengthening the interaction of PMN with LM, a RB with TNF might be observed even with PMN on this surface. We tested this possibility by using Mn2+, a potent activator of ß2 integrin functions [28 29 30 ], to enhance spreading of TNF-treated PMN to LM. Figure 2 shows that spreading on FN, already high in control cells (Fig. 2A) , remained nearly unchanged in the presence of Mn2+ (Fig. 2C) , whereas on LM, TNF-treated PMN exhibited a morphology (Fig. 2D) comparable with that of PMN on FN. On both substrates, the cation enhanced flattening of resting PMN (data not shown).

As expected, the effects of Mn2+ on PMN morphology were paralleled by those on cell adherence. As shown in Figure 3 , in fact, this cation increased the adherence to LM of resting and TNF-treated PMN. Nonetheless, the activation of ß2 integrins induced by Mn2+, although enhancing the interaction of TNF-treated PMN with LM-coated surfaces, did not result in a RB, as assessed by measuring glucose oxidation via the hexose monophosphate shunt (Fig. 3) . In these experiments, the SOD-inhibitable cytochrome c reduction test could not be used, as MnCl2 heavily interfered with the assay (data not shown), presumably as a result of a quenching effect on the superoxide anion. The addition of MnCl2 to the medium did not interfere, however, with the measurement of glucose oxidation, as demonstrated by the comparable amounts of glucose oxidized by TNF-stimulated PMN in the absence or presence of MnCl2 on FN-coated substrates (2.13±0.6 nmol/106 PMN/60 min and 1.83±0.4 nmol/106 PMN/60 min in the absence and in the presence of 1 mM MnCl2, respectively; mean±SEM of five experiments).



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  		Figure 3. Effect of Mn2+ on adherence (left) and hexose monophosphate shunt activity (right) of resting and TNF-treated PMN residing on LM-coated surfaces. For adherence, PMN (7.5x104/well) were incubated in LM-coated wells in a final volume of 0.15 ml. After 45 min at 37°C, cell adherence was assessed as described in Figure 1 . TNF, 10 ng/ml; MnCl2, 1 mM. Data are mean ± SEM of five duplicate experiments. Statistical analysis (two-tailed Student’s t-test on unpaired data): **, P < 0.01. For glucose oxidation (ox. gluc.), PMN (3.0x105/well) in 0.6 ml glucose-free Ca2+/Mg2+-HEPES containing 0.5 µCi D-[1-14C] glucose were dispensed in LM-coated wells and were incubated for 60 min at 37°C with or without TNF, 10 ng/ml, and MnCl2, 1 mM. The wells were sealed with rubber stoppers provided with a center well containing 0.2 ml 10% KOH. The reaction was stopped by adding 0.5 ml 1 N HCl to the reaction mixture, and the radioactivity (14CO2) absorbed in the KOH of the center well was measured by liquid scintillation. The nmol of oxidized glucose/106 PMN was calculated on the basis of a D-[1-14C] glucose standard solution. Data are means ± SEM of five duplicate experiments performed in parallel with the adherence experiments.

 
Selective blockade of {alpha}Lß2, {alpha}Mß2, and {alpha}Xß2 differentially affects O2- production and adherence of TNF-treated PMN
As the previous results indicated that the differential metabolic responses to TNF of PMN residing on LM or FN could not be accounted for by the involvement of different integrin subfamilies (ß1 or ß2; Fig. 1 ) or to an inadequate ß2 integrin-dependent adherence and/or spreading on LM, we examined the possible role of each member of the ß2 integrins, i.e., {alpha}Lß2, {alpha}Mß2, and {alpha}Xß2, in TNF-stimulated adherence and O2- generation. Figure 4 shows that the anti-{alpha}M mAb M1/70 strongly inhibited PMN adherence to LM (Fig. 4A) and to FN (Fig. 4B) , whereas the anti-{alpha}L mAb TS1/22 was ineffective on LM and only slightly inhibited adherence to FN. The anti-{alpha}X mAb 3.9 inhibited adhesion to FN by about 25% and to a lesser extent adherence to LM. As expected, the anti-ß2 mAb TS1/18 was the most effective adherence inhibitor. Under these conditions, all mAb inhibited TNF-induced O2- production by PMN residing on FN, although to a different degree (Fig. 4C) .



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  		Figure 4. Effect of anti-ß2 integrin chain mAb on adherence to FN and LM and on O2- production on FN by TNF-treated PMN. For experimental conditions, see Figure 1 . F(ab')2 fragments of the indicated mAb were used at 20 µg/ml. After incubation for 45 min at 37°C, O2- production and adherence were determined as described in the legend to Figure 1 . Data are mean ± SEM of five duplicate experiments. (A, B) Data are reported as percent inhibition of TNF-induced adherence in the absence of mAb, which was 29.3 ± 4.4% on LM and 89.8 ± 4.5% on FN. (C) Data are reported as percent inhibition of TNF-induced O2- production in the absence of mAb, which was 26.7 ± 1.8 nmol O2-/45 min/106 PMN. Two-tailed, one-sample Student’s t-test was used to analyze significance of mAb effects. *, P < 0.05; **, P < 0.01; ***, P < 0.001; NS, not significant.

 
Role of individual ß2 integrins in the TNF-induced RB and adherence of PMN residing on FN
To further assess the ability of each ß2 integrin to mediate adherence and RB, PMN were allowed to interact with FN by means of a single integrin, using mixtures of anti-{alpha} chain mAb. Figure 5A shows that adherence to FN was observed only when {alpha}Mß2 or {alpha}Xß2 was allowed to freely interact with the substrate, i.e., with a combination of mAb TS1/22 + mAb 3.9 or mAb TS1/22 + mAb M1/70 included in the incubation medium. Integrin {alpha}Lß2 (experiments with mAb M1/70 and 3.9 added in combination) was unable to sustain any increase in adherence over the values observed in the presence of anti-ß2 mAb TS1/18 (no ß in Fig. 5 ) or mAb TS1/22, M1/70, and 3.9 added in combination (no {alpha} in Fig. 5 ). Figure 5B shows that a RB could not be observed when each ß2 integrin was independently allowed to interact with the substrate, suggesting that cooperation among ß2 integrins is needed for this response.



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  		Figure 5. Role of {alpha}Lß2, {alpha}Mß2, and {alpha}Xß2 in adherence and in O2- production of PMN exposed to TNF on FN. For experimental conditions, see Figure 1 . Each mAb was used as F(ab')2 fragment at 20 µg/ml. (only {alpha}L) M1/70 + 3.9; (only {alpha}M) TS1/22 + 3.9; (only {alpha}X) TS1/22 + M1/70; (no {alpha}) TS1-22 + M1/70 + 3.9; (no ß) TS1/18. Measurement of adherence (A) and of O2- production (B) were performed as described in Materials and Methods. Data are mean ± SEM of three duplicate experiments. Two-tailed Student’s t-test on paired data was used to analyze the significance of differences versus no ß (anti-ß2 mAb TS 1/18). *, P < 0.05; **, P < 0.01; NS, not significant.

 
Effect of ß2 integrins cross-linking on O2- release by TNF-treated PMN
In an attempt to gain more insight into the nature of possible cooperation between ß2 integrins, we devised a model in which TNF-treated PMN were exposed to solid-phase, immobilized {alpha}-chain-specific mAb to establish the individual signaling capability of ß2 integrins. These experiments were carried out in the presence of EDTA to prevent the divalent cation-dependent binding of ß2 integrins [28 29 30 31 ] to the FN used to saturate the wells where mAb were immobilized. Indeed, the presence of 1 mM EDTA completely blocked TNF-induced adherence to FN (2.5±1.2% adherence in the presence of EDTA vs. 77.4±12.3% adherence of control cells) and the associated RB (1.0±0.8 nmol O2-/45 min/106 PMN in the presence of EDTA vs. 38.7±8.5 nmol O2-/45 min/106 of control PMN; mean±SEM, n=3). Figure 6 shows that under these experimental conditions, a substantial production of O2- was induced only by the anti-{alpha}L mAb TS1/22, alone or in combination with the anti-{alpha}M or the anti-{alpha}X mAb. The anti-{alpha}M mAb M1/70 and the anti-{alpha}X mAb 3.9 were ineffective in this respect, individually or in combination.



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  		Figure 6. Effect of cross-linking of ß2 integrins on O2- production by TNF-treated PMN. PMN (7.5x104) in EDTA-HEPES were incubated with 10 ng/ml TNF in wells coated with the indicated mAb (see Materials and Methods for experimental details) and containing 0.12 mM cytochrome c in a final volume of 0.15 ml. After 45 min, O2- production was measured as described in Materials and Methods. Adherence was also measured, and the amount of O2- produced/106 cells was calculated on the basis of the number of adherent cells. Data are mean ± SEM of six duplicate experiments. Antibody binding to protein G-coated wells (ELISA, OD405 nm) was as follows: mAb BB7.5, 0.147 ± 0.033; mAb TS1/22, 0.155 ± 0.032; mAb M1/70, 0.123 ± 0.005; mAb 3.9, 0.134 ± 0.050.

 
Role of the cytoskeleton in the RB induced by {alpha}Lß2 cross-linking in TNF-treated PMN
Table 2 shows the effect of genistein on O2- production, cell attachment, and morphology of TNF-treated PMN exposed to surface-bound {alpha}L-chain-specific mAb TS1/22. In the presence of this compound, which does not affect PMN attachment to the immobilized mAb but reverses the slightly altered morphology induced by TNF, stimulated PMN still generate more than 50% of the O2- produced in the absence of the inhibitor. This indicates that triggering of the RB by {alpha}Lß2 cross-linking is only partially dependent on cytoskeleton polymerization, in striking contrast with what is observed when PMN are stimulated with TNF on FN-coated surfaces (Table 1) .


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  		Table 2. Effects of Genistein on the Respiratory Burst and on the Morphology of TNF-Treated PMN on Surfaces Coated with Anti-{alpha}L mAb TS1/22

 
The RB of PMN stimulated with TNF on FN-coated surfaces correlates with {alpha}Lß2 expression levels
Having established that after cross-linking {alpha}Lß2 signals for TNF-induced O2- production, we addressed the question as to whether the metabolic response to TNF observed in various PMN populations correlates with the level of expression of this integrin. Figure 7 shows that when TNF-induced O2- production by PMN on FN is plotted as a function of the corresponding {alpha}Lß2 expression, a sigmoidal curve is obtained, suggesting a cooperative interaction among {alpha}Lß2 integrins in signaling for the RB.



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  		Figure 7. Correlation between TNF-induced O2- production by PMN residing on FN and expression of {alpha}Lß2. PMN suspensions (1.5x106 cells/ml) from different donors were prewarmed at 37°C and added in 0.05 ml aliquots to the wells of microtiter plates coated with FN and containing 0.12 mM cytochrome c and 10 ng/ml TNF in a final volume of 0.15 ml Ca2+/Mg2+-HEPES. After 45 min of incubation, O2- production was measured as described in Materials and Methods. For expression of integrin {alpha}Lß2, 2.5 x 105 cells were incubated with mAb TS1/22 for 45 min on ice and after washing, with a rhodamine-labeled goat anti-mouse IgG F(ab')2 as described in Materials and Methods. Goodness of fit (R2) was determined by nonlinear regression analysis using GraphPad Prism.

 
A RB by TNF-treated PMN can be induced on LM-coated surfaces by artificially engaging {alpha}Lß2
On the basis of the previous results, it is possible that the absence of a RB in TNF-treated PMN on LM (Fig. 1) might be attributed to a low or absent involvement of {alpha}Lß2 integrins. If correct, it thus follows that a RB should also be elicited on this surface by artificially engaging {alpha}Lß2. This possibility was evaluated by exposing TNF-treated PMN to wells coated with LM and mAb TS1/22. Figure 8 shows that under these conditions PMN generate substantial amounts of O2-, even on LM. As predicted, no O2- was produced by TNF-treated PMN when the anti-{alpha}M mAb M1/70 or the anti-{alpha}X mAb 3.9 was included in the LM-coat.



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  		Figure 8. PMN residing on LM respond to TNF with a RB after integrin {alpha}Lß2 cross-linking. PMN suspensions (1.5x106 cells/ml in Ca2+/Mg2+-HEPES) were prewarmed at 37°C and added in 0.05 ml aliquots to the wells of microtiter plates coated with the indicated mAb and LM (see Materials and Methods). O2- production was measured as described in Materials and Methods. Data are mean ± SEM of four duplicate experiments and are expressed as the difference between the O2- produced in wells coated with LM plus the various mAb and the O2- produced in wells coated with LM only (8.5±1.2 nmol O2-/45 min/106 PMN). Two-tailed Student’s t-test on paired data was used to analyze significance of the differences versus O2- release on the control anti-HLA mAb BB7.5. *, P < 0.05. Antibody binding to protein G-coated wells was similar to that described in the legend to Figure 6 .

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The role of the ECM in the metabolic response of PMN to physiologic soluble stimuli such as G-CSF, complement fragment 5a, or TNF has almost exclusively been dealt with in terms of "permissiveness" or "nonpermissiveness" [1 2 3 4 5 6 ]. According to this view, surfaces coated with ECM proteins (such as FN or fibrinogen) onto which stimulated PMN respond with a RB have been defined as "permissive". Surfaces coated with ECM proteins such as LM, type IV or type I collagens, where PMN do not respond with a RB to these stimuli, have been termed "nonpermissive". However, the molecular basis of the differential modulation of this PMN response by ECM proteins remains largely unknown.

In the present work, insights into this problem have been obtained by studying the effect of integrin-specific mAb and inhibitors of cytoskeleton polymerization on the RB, adherence, and spreading of PMN exposed to TNF on FN and LM, which have been considered to be models of permissive and nonpermissive surfaces, respectively. Our main conclusion is that the key determinant of permissiveness of FN versus nonpermissiveness of LM is the ability of the former ECM protein, but not the latter, to interact with PMN {alpha}Lß2 integrin. Three sets of data support this conclusion: Although to a different degree, adherence to FN is inhibited by mAb directed against the {alpha} chains of all three ß2 integrins, whereas adherence to LM is inhibited by anti-{alpha}M and anti-{alpha}X mAb but not by anti-{alpha}L mAb (Fig. 4) ; PMN can be rendered responsive to TNF even on LM-coated surfaces after the insertion of immobilized {alpha}L-specific mAb on these surfaces (Fig. 8) ; and a direct relationship is demonstrable between the expression of {alpha}Lß2 and production of O2- by TNF-treated PMN on FN (Fig. 7) .

Furthermore, results of experiments aimed at defining the relative role of the three ß2 integrins in TNF-induced RB give additional support for this conclusion. Two experimental models have been used in these studies. In the first, mAb recognizing the I domain of the {alpha} chain (i.e., the integrin region involved in binding to ECM ligands, refs. [32 33 34 ]) of each of the three ß2 integrins were tested for their effect on TNF-induced RB and adherence of PMN residing on FN. The results of these experiments (Figs. 4 and 5) provide evidence for a comparable relevance of the three integrins in the metabolic response to TNF and for a predominant role of {alpha}Mß2 and {alpha}Xß2 over {alpha}Lß2 in adherence. In the second one, the direct ability of each integrin to signal for a RB by TNF-treated PMN was evaluated using I domain-specific anti-{alpha} chain mAb immobilized on protein G-coated surfaces. From the results of these experiments, it clearly emerges that only {alpha}Lß2 is able to mediate activation of the RB (Fig. 6) . This finding diverges from the seemingly equivalent role of {alpha}Lß2, {alpha}Mß2, and {alpha}Xß2 in the TNF-induced RB of PMN residing on FN, which can be inferred from the results shown in Figures 4 and 5 . This apparent inconsistency can be explained considering the striking proadhesive properties of {alpha}Mß2, and {alpha}Xß2. It can be envisaged, in fact, that the promotion of adherence and spreading by these two integrins facilitates the interaction of {alpha}Lß2 integrin with the substrate, making it competent for signaling for the RB. In fact, it is well known that cell spreading is an essential requirement for the occurrence of a RB on biologic surfaces [3 4 5 6 ]. Our data, showing that the cytoskeleton polymerization inhibitors genistein and CB prevent the TNF-induced RB on FN (Table 1) , further support this notion. At the same time, we extend this concept to show that cytoskeleton polymerization and spreading, although necessary, are not always sufficient for activation of a RB in adherent PMN. This is exemplified in the experiments reported in Figures 2 and 3 , showing that PMN laying on LM do not respond to TNF with a RB even after the massive spreading induced by Mn2+ addition. These latter results also indicate that the nonpermissiveness of LM is not only linked to its inability to adequately support cell spreading.

The results of our experiments with immobilized mAb differ from those reported by other authors [35 ] who showed that in the absence of costimulators (e.g., TNF used in the present study), mAb to {alpha}Lß2 and {alpha}Xß2 integrins stimulated activation of the RB. These authors found an extensive spreading with the immobilized antibodies against either ß2 integrin {alpha} chain, leaving open the possibility that other integrins might interact with the substrate in addition to those cross-linked by the immobilized mAb. We believe, therefore, that the RB observed following {alpha}Xß2 cross-linking [35 ], rather than representing a direct signaling ability of this integrin, might be the result of an unexpected involvement of {alpha}Lß2. In the present studies, the problem of a possible nonspecific involvement of integrins, other than those ligated by the immobilized mAb, has been overcome by the inclusion of EDTA in the incubation medium. Under these conditions, we show the following: PMN exposed to TNF generate O2- following {alpha}Lß2 cross-linking, even in the absence of spreading (Table 2) ; the TNF-induced activation of RB after {alpha}Lß2 cross-linking is only partially inhibited by genistein (Table 2) , which suggests an ability of this integrin to signal for metabolic activation, even in the absence of cytoskeleton polymerization; and a small but significant production of O2- is observed on immobilized anti-{alpha}L mAb TS1/22, also in resting cells (7.1±2.3 nmol O2-/45 min/106 PMN on mAb TS1/22 vs. 0.5±0.4 nmol O2-/45 min/106 PMN on the control mAb BB7.5; n=3; P<0.05), thus confirming the intrinsic transducing ability of {alpha}Lß2 [35 , 36 ], which is strongly increased by addition of TNF (Table 2) .

As far as the mechanism by which TNF enhances {alpha}Lß2 signaling activity is concerned, it can be excluded that this depends on an increase in {alpha}Lß2 levels, as expression of this integrin does not change following cell stimulation (refs. [37 , 38 ]; data not shown). The sigmoidal curve describing the relationship between TNF-induced O2- production and {alpha}Lß2 expression (Fig. 7) suggests that TNF favors cooperation among {alpha}Lß2 integrin members in the PMN response. Whether this cooperation derives from an enhanced clustering of {alpha}Lß2 at sites of cell contact with the substrate, a phenomenon known to increase the avidity of this integrin in other cellular systems [39 40 41 ] remains to be elucidated.


   ACKNOWLEDGEMENTS
 
This work was supported by grants from Ministry of University and Scientific and Technological Research (Cofinanziamento ex 40%) from University of Trieste (ex 60%) and from Fondo Commissario del Governo. We are grateful to Dr. F. Rapagna and C. Cigana for their excellent technical assistance and to Mr. T. Ubaldini for performing scanning electron microscopy. We also thank Dr. K. Yamada (National Institutes of Health, Bethesda, MD), Dr. J. M. Harlan (University of Washington, Seattle), and Dr. N. Hogg (Imperial Cancer Research Fund, London, UK) for the generous gift of anti-integrin mAb.

Received May 9, 2002; revised June 19, 2002; accepted June 21, 2002.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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