Hydrogen peroxide augments eosinophil adhesion via β₂ integrin

Abstract

During eosinophil (EOS) accumulation at sites of allergic inflammation, an initial step is the binding of EOS to adhesion molecules expressed on vascular endothelial cells (EC). We have previously observed that adhesion of peripheral blood EOS to recombinant human vascular cell adhesion molecule-1 (rh-VCAM-1) stimulates the respiratory burst of EOS. Although the biological consequence of this activation remains to be elucidated, reactive oxygen species such as hydrogen peroxide (H₂O₂) may modify the adhesive property of EOS. In the present study, we examined whether H₂O₂ modifies the adhesive property of EOS. EOS were isolated from the peripheral blood of healthy subjects. Adhesion of the EOS to paraformaldehyde-fixed human umbilical vein EC (HUVEC), stimulated or not stimulated with tumour necrosis factor-α (TNF-α; 100 pm for 24 hr), was examined in the presence or absence of H₂O₂. H₂O₂ significantly enhanced adhesion of EOS to both resting and TNF-α-stimulated fixed HUVEC (P < 0·01, respectively). Such enhancing effects were inhibited by anti-β₂ integrin antibody or anti-CD11b antibody, but not by anti-CD11a or anti-α₄ integrin antibody. H₂O₂ also enhanced EOS adhesion to rh-intracellular cell adhesion molecule-1 (ICAM-1) but not to rh-VCAM-1. Finally, H₂O₂ enhanced the expression of both CD11b and CD18 on EOS. These results indicate that H₂O₂ directly augments the adhesive property of EOS through β₂ integrin.

Introduction

Eosinophils (EOS) preferentially accumulate at sites of allergic inflammation and because of this probably play crucial roles in the pathophysiology of diseases, such as asthma, through the release of a variety of inflammatory mediators.^1,2 For EOS to participate in allergic inflammation, it is necessary that they migrate from the peripheral circulation into the tissues. An initial step of this process is the interaction of circulating EOS with adhesion proteins expressed on the surface of vascular endothelial cells (EC). Accumulating evidences have established that EOS integrin adhesion molecules such as α₄ integrins, including α₄β₁ (very late activation antigen-4 [VLA-4], CD49d/CD29), and β₂ integrins, including α_Lβ₂ (lymphocyte function-associated antigen-1 [LFA-1], CD11a/CD18) and α_Mβ₂ (Mac-1, CD11b/CD18), play important roles in the adhesion to, and the transmigration through, EC.^3,4 Vascular cell adhesion molecule (VCAM)-1, a counter ligand for α₄β₁, is a potent inducer of spontaneous adhesion of blood EOS in vitro.^5,6 Administration of anti-α₄β₁ antibodies (Ab) in vivo inhibits the accumulation of EOS induced by soluble inflammatory mediators and the antigen-induced EOS infiltration into guinea-pig skin or mouse trachea.^7,8 Monoclonal Ab (mAb) against intracellular cell adhesion molecule-1 (ICAM-1), a ligand for β₂ integrins, also inhibited EOS airway infiltration in a primate model of asthma.⁹ Although recombinant human (rh)-ICAM-1 requires second signals, such as granulocyte–macrophage colony-stimulating factor (GM-CSF) or Mn²⁺, for inducing adhesion of blood EOS,^10–12 it has been shown that the β₂ integrin/ICAM-1 pathway is essentially involved in EOS transmigration across EC monolayers in vitro.^13,14

We have previously reported that adhesion of EOS to rh-VCAM-1 activates a respiratory burst of EOS.⁶ This suggests that the interaction between EOS α₄ integrin and endothelial VCAM-1 is an important step, not only for selective EOS recruitment into the tissue but also as an initial action in the activation of EOS effector functions. The biological consequences of the activation of such a specific effector function remains unknown; however, it is possible that the released oxygen species modify either the morphological or functional status of EC, such as the permeability, adhesion molecule expression and adhesiveness to leucocytes.^15–17 Oxygen species also alter the functional status of leucocytes. For example, hydrogen peroxide (H₂O₂) promotes adhesion of neutrophils¹⁸ and monoblastoid U-937 cells¹⁹ via CD11b/CD18 molecules. Here we report that H₂O₂ enhances EOS adhesion to both resting and tumour necrosis factor-α (TNF-α)-stimulated EC through the enhanced expression and activation of β₂ integrin of EOS.

Materials and methods

Reagents

Percoll was purchased from Pharmacia (Uppsala, Sweden). Hanks' balanced salt solution (HBSS), newborn calf serum (NCS), fetal calf serum (FCS), trypsin–EDTA, l-glutamine and penicillin–streptomycin were obtained from Life Technologies (Grand Island, NY). Anti-CD16 mAb-coated magnetic microbeads were obtained from Miltenyl Biotec (Auburn, CA). H₂O₂ was obtained from Wako Pure Medical Co. (Osaka, Japan). Human umbilical vein endothelial cells (HUVEC) and endothelial cell basal medium (EBM) medium were obtained from Clonetics (Palo Alto, CA). rh-TNF-α, rh-VCAM-1 and rh-ICAM-1 were obtained from R & D Systems (Minneapolis, MN). Other reagents were purchased from Sigma (St. Louis, MO) unless stated otherwise.

Separation of EOS

EOS were isolated from peripheral blood specimens, of normal volunteers, that contained less than 5% EOS in the total leucocytes. Subjects ranged in age from 26 to 39 years, and gender distribution was equal. Isolation of EOS was performed by negative immunomagnetic bead selection, as previously described.¹⁴ Briefly, heparinized blood was diluted with HBSS without Ca²⁺ and centrifuged for 20 min at 700 g over 1·090 g/ml Percoll. Plasma, the mononuclear cell band and Percoll were removed, and the red blood cells in the pellet were lysed by hypotonic shock. The resulting granulocytes were washed with HBSS (chilled to 4°) supplemented with 2% NCS (HBSS/NCS), then incubated with anti-CD16 Ab-coated magnetic beads for 40 min at 4°. The cells were filtered through a steel wool column in a magnetic field (Miltenyl Biotec) to remove neutrophils bound to magnetic beads. CD16-negative EOS (> 98% pure and > 99% viable) were collected, washed and resuspended in HBSS supplemented with 5% FCS (HBSS/FCS).

Preparation of HUVEC

HUVEC were cultured on type-IV collagen-coated tissue-culture flasks. When confluent, HUVEC were passaged into collagen-coated 96-well-tissue culture plates, and either medium alone or TNF-α (at 100 pm) was added and culture continued for 24 hr in 5% CO₂ at 37°. Following culture, the incubated mixture was decanted and the HUVEC were washed three times with HBSS. HUVEC were fixed with 100 µl of 1% paraformaldehyde in phosphate-buffered saline (PBS) at ambient temperature for 15 min to block the generation of mediators. After washing these cells three times with HBSS, 200 µl of 1% glycine in HBSS was added and incubated at ambient temperature for 1 hr to quench the residual paraformaldehyde. Plates were then decanted and washed three times with HBSS before use.

EOS adhesion assay

EOS adhesion was assessed as residual EOS peroxidase (EPO) activity of adherent EOS, as previously described.^6,10,14 Briefly, EOS (100 µl of 1 × 10⁵ cells/ml in HBSS/FCS) were placed onto HUVEC monolayers or rh-adhesion molecule-coated plates, in the presence or absence of an activator, and incubated for 30 min at 37°. After five washes with HBSS (preincubated to 37°), 100 µl of HBSS/FCS was added to the reaction wells. As standards, 100 µl of serially diluted cell suspension (1 × 10³, 3 × 10³, 1 × 10⁴, 3 × 10⁴ and 1 × 10⁵ cells/ml) was added to empty wells. EPO substrate (Tris buffer, pH 8·0, containing 1 mm o-phenylenediamine, 1 mm H₂O₂ and 0·1% Triton-X-100) was then added to all of the wells. After a 30-min incubation at room temperature, 50 µl of 4-m H₂SO₄ was added to stop the reaction, and the absorbance at 490 nm was measured. Percent EOS adhesion was calculated from the log dose–response curve. EOS viability after the incubation exceeded 98%, as assessed by using Trypan Blue dye exclusion.

Expression of CD11b and CD18 on EOS

The expression of CD11b and CD18 on EOS was examined by flow cytometric analysis. EOS were exposed to either 1 µm H₂O₂ or buffer (HBSS/5% FCS) alone for 30 min at 37°. The cells were washed three times and resuspended in PBS (chilled to 4°) supplemented with 2% bovine serum albumin (BSA) and 0·2% sodium azide (fluorescence-activated cell sorter [FACS] buffer). Cells (1 × 10⁵/100 µl) were incubated with fluorescein isothiocyanate (FITC)-conjugated mouse anti-human CD18 (clone MHM23, immunoglobulin G1 [IgG1]; Dako A/S, Glostup, Denmark) and/or phycoerythrin (PE)-conjugated mouse anti-human C3bi (CD11b) (clone 2LPM19c, IgG1; Dako A/S) on ice for 30 min. PE- and FITC-conjugated mouse IgG1 (Dako A/S) were used as isotype-matched controls. Cells were then washed and resuspended in FACS buffer. Mean fluorescence was measured on at least 10 000 events using a fluorescence-activated cell sorter (FACScan; Becton-Dickinson, San Jose, CA, USA). Relative mean fluorescence was determined by subtraction of values for the IgG1 isotype-matched control.

Statistics

Data are presented as mean ± SEM. For statistical analysis, the Student's t-test was used for paired comparisons and analysis of variance (anova) for repeated measures; Scheffe constants were used for comparison of more than two variables to determine a significance. A P-value of less than 0·05 was considered significant.

Results

Effect of H₂O₂ on EOS adhesion

Initial experiments were conducted to determine whether H₂O₂ modifies adhesion of EOS to paraformaldehyde-fixed HUVEC. After 30 min of incubation, 0·01–1 µm H₂O₂ significantly enhanced adhesion of EOS to TNF-α-stimulated (100 pm, 24 hr) HUVEC (0·01 µm, P < 0·05; 0·1 µm, P < 0·05; 1 µm, P < 0·01; n = 8, respectively; Fig. 1a). Similarly, H₂O₂ promoted adhesion of EOS to resting HUVEC (0·01 µm, P < 0·05; 0·1 µm, P < 0·05; 1 µm, P < 0·01; n = 8, respectively; Fig. 1b). In both experiments the maximum effect of H₂O₂ was obtained at 1 µm (% adhesion: TNF-α-stimulated HUVEC, 27·4 ± 2·6%; resting HUVEC 10·8 ± 2·5%) and was comparable to the maximum effect of N-formyl-methionyl-leucyl-phenylalanine (FMLP, 1 µm: TNF-α-stimulated HUVEC, 27·0 ± 2·2%; resting HUVEC, 10·9 ± 3·3%, n = 5). Although 10 µm H₂O₂ did not augment EOS adhesion, the cell viability exceeded 98% under this experimental condition. Moreover, 1–10 µm H₂O₂ did not modify the pH of the assay buffer (data not shown). To confirm the specificity of the H₂O₂ effect, EOS were exposed to 1 µm H₂O₂ in the presence or absence of catalase (from Aspergillus nigar; Sigma), which rapidly degrades H₂O₂. The adhesion to both TNF-α-stimulated and resting HUVEC was then determined: EOS adhesion augmented by 1 µm H₂O₂ was blocked by 10 U/ml catalase (% inhibition of adhesion to TNF-α-stimulated HUVEC, 90·7 ± 3·4%, Fig. 2a; % inhibition of adhesion to resting HUVEC, 97·2 ± 2·6%, n = 5, respectively, Fig. 2b). H₂O₂ at 1 µm also augmented eosinophil adhesion to unfixed TNF-α-stimulated and resting HUVEC, and adhesion to both was blocked by 10 U/ml catalase (data not shown).

Figure 1.

Effect of H₂O₂ on eosinophil (EOS) adhesion to tumour necrosis factor-α (TNF-α)-stimulated (a) and resting (b) fixed, human umbilical vein endothelial cells (HUVEC). EOS were incubated in the presence or absence of H₂O₂ for 30 min at 37°. *P < 0·05, †P < 0·01 versus control (no H₂O₂), n = 8, respectively.

Figure 2.

Effect of 10 U/ml of catalase on eosinophil (EOS) adhesion to tumour necrosis factor-α (TNF-α)-stimulated (a) and resting (b) fixed, human umbilical vein endothelial cells (HUVEC). Open bars represent adhesion of EOS in the presence of 1 µm H₂O₂. Closed bars represent adhesion of EOS in the absence of H₂O₂. *P < 0·01, n = 5, respectively.

To examine whether H₂O₂ directly affects the adhesive property of EOS, EOS adhesion to 1% BSA-coated microassay plates was examined: 1 µm H₂O₂ augmented the adhesion (buffer control, 16·2 ± 2·5% versus H₂O₂, 25·1 ± 2·3%; P = 0·001, n = 6).

The reaction kinetics of the H₂O₂ effect on adhesion of EOS was examined using TNF-α-stimulated HUVEC. The H₂O₂ effect was observed at 5 min after addition of H₂O₂ and lasted for at least 60 min (P < 0·05 at each time-point examined, n = 4; Fig. 3a). The H₂O₂ effect on adhesion of EOS to the resting HUVEC was observed at 10 min after addition of H₂O₂ and lasted for 60 min (P < 0·05 at each time-point examined except 5 min, n = 4; Fig. 3b).

Figure 3.

Kinetics of the H₂O₂ effect on adhesion of eosinophils (EOS) to tumour necrosis factor-α (TNF-α)-stimulated-(a) (n = 4) and resting (b) (n = 4) human umbilical vein endothelial cells (HUVEC). Open bars represent adhesion of EOS in the presence of 1 µm H₂O₂. Closed bars represent adhesion of EOS in the absence of H₂O₂. *P < 0·05.

Effect of anti-integrin mAbs on the H₂O₂-induced adhesion of EOS

To identify the EOS integrin(s) involved in the H₂O₂ effect, EOS were pretreated with either anti-α₄ integrin mAb (clone HP1/2, mouse IgG1, 3 µg/ml; Cosmo Bio., Tokyo, Japan), anti-β₂ integrin mAb (clone L130, mouse IgG1, 3 µg/ml; Becton-Dickinson) or an isotype-matched control mouse IgG1 (3 µg/ml; Cappel, Aurora, Ohio, USA) at ambient temperature for 15 min, and then EOS adhesion was examined. H₂O₂ at 1 µm significantly enhanced the adhesion of EOS to TNF-α-stimulated HUVEC in the presence of mouse IgG1 or anti-α₄ Ab (P < 0·01, n = 5, respectively, Fig. 4a). In contrast, the H₂O₂ effect was blocked by anti-β₂ mAb (Fig. 4a). Similarly, H₂O₂ enhanced the EOS adhesion to resting HUVEC in the presence of mouse IgG1 or anti-α₄ Ab (P < 0·01, n = 3, respectively), but the effect was blocked by anti-β₂ Ab (Fig. 4b). To further confirm whether H₂O₂ enhanced the adhesion via β₂ integrin, adhesion of EOS to immobilized rh-ICAM-1, a ligand for β₂ integrin, or to rh-VCAM-1, a ligand for α₄ integrin, was examined: 1 µm H₂O₂ enhanced adhesion of EOS only to rh-ICAM-1 (rh-ICAM-1, 3·5 ± 0·7% in medium alone versus 6·8 ± 1·3% in medium + H₂O₂, P = 0·01, Fig. 5a; rh-VCAM-1, 41·8 ± 3·6% in medium alone versus 41·6 ± 4·0% in medium + H₂O₂, P = NS, n = 5, Fig. 5b).

Figure 4.

Effects of anti-β₂ integrin monoclonal antibody (mAb) (L130) and anti-α₄ integrin mAb (HP1/2) on adhesion of eosinophils (EOS) to tumour necrosis factor-α (TNF-α)-stimulated (a) and resting (b) human umbilical vein endothelial cells (HUVEC). Mouse immunoglobulin G1 (IgG1) is an isotype-matched control. Open bars represent adhesion of EOS in the presence of 1 µm H₂O₂. Closed bars represent adhesion of EOS in the absence of H₂O₂. *P < 0·01, †P < 0·05, n = 5, respectively.

Figure 5.

Effect of 1 µm H₂O₂ on adhesion of eosinophils (EOS) to recombinant human intracellular cell adhesion molecule-1 (rh-ICAM-1) (a) and rh-vascular cell adhesion molecule-1 (VCAM-1) (b). Open bars represent adhesion in the presence of 1 µm H₂O₂. Closed bars represent adhesion in the absence of H₂O₂. n = 5, respectively.

To further identify EOS β₂ integrin(s) involved in the H₂O₂ effect, EOS were pretreated with either anti-CD11a mAb (clone G25.2, mouse IgG2a, 5 µg/ml; Becton-Dickinson), anti-CD11b mAb (clone D12, mouse IgG2a, 5 µg/ml; Becton-Dickinson), or isotype-control mouse IgG2a (5 µg/ml; Cappel), and then EOS adhesion was examined in the presence or absence of 1 µm H₂O₂. The EOS adhesion augmented by H₂O₂ was partially inhibited by anti-CD11b antibody (% inhibition: adhesion to TNF-α-stimulated HUVEC, 60·3 ± 18·1%; adhesion to resting HUVEC, 68·6 ± 16·0%; P < 0·01, n = 5, respectively; results not shown). On the other hand, neither anti-CD11a antibody nor mouse IgG2a modified the augmented EOS adhesion to TNF-α-stimulated or resting HUVEC (data not shown). The effect of the combination of anti-CD11a and anti-CD11b did not differ from anti-CD11b antibody alone (data not shown).

Effect of H₂O₂ on EOS expression of CD11b and CD18

To examine whether H₂O₂ modifies the number of CD11b or CD18 molecules on EOS, the expression of those molecules was determined by flow cytometric analysis. Following stimulation with 1 µm H₂O₂, the expression of both CD11b and CD18 was enhanced (mean fluorescence index [MFI]: CD11b, 204·0 ± 26·3, control, versus 236·0 ± 24·0, H₂O₂, P < 0·05; CD18, 48·0 ± 8·3, control, versus 56·0 ± 6·3, H₂O₂, P < 0·01, n = 3, respectively; results not shown).

Role of H₂O₂ as an autocrine mediator for EOS adhesion

Because EOS are capable of generating radical oxygen species in response to a variety of inflammatory mediators, endogenous H₂O₂ may play roles in the development of stimulus-dependent adhesion of EOS in an autocrine mechanism. To examine this possibility, adhesion of EOS to paraformaldehyde-fixed resting HUVEC in response to GM-CSF, phorbol myristate acetate (PMA), FMLP, or platelet-activating factor (PAF), was examined in the presence or absense of 10 U/ml of catalase. Catalase did not modify spontaneous-, 100 pm GM-CSF-induced, or 1 ng/ml phorbol 12-myristate 13-acetate (PMA)-induced adhesion (control versus catalase: spontaneous, 5·2 ± 0·9% versus 5·1 ± 0·8%; GM-CSF, 12·2 ± 1·4% versus 12·5 ± 2·8%; PMA, 21·6 ± 3·7% versus 22·2 ± 4·2%; n = 5, P = NS, respectively; results not shown). In contrast, EOS adhesion induced by both FMLP and PAF (both at 1 µm) was partially inhibited by 10 U/ml of catalase (control versus catalase: FMLP, 14·8 ± 1·4% versus 8·0 ± 1·3%, P < 0·01, Fig. 6a; PAF, 15·4 ± 1·6% versus 9·0 ± 1·4%, P < 0·05, n = 5, Fig. 6b, respectively).

Figure 6.

Effect of 10 U/ml catalase on adhesion of eosinophils (EOS) induced by N-formyl-methionyl-leucyl-phenylalanine (FMLP) (a) and platelet-activating factor (PAF) (b) (both at 1 µm). Open bars represent adhesion of EOS in the presence of 1 µm H₂O₂. Closed bars represent adhesion of EOS in the absence of H₂O₂. n = 5, respectively.

Discussion

Our findings extend earlier observations concerning interaction between EOS and endothelial adhesion molecules,^3,4,6,10 and suggest the potentially important biological consequence of the EOS respiratory burst during this process. First, nanomolar concentrations of H₂O₂ induced EOS adhesion to resting EC. Second, H₂O₂ also promoted adhesion of EOS to EC activated with a proinflammatory cytokine, TNF-α. The H₂O₂ effects appear to involve EOS β₂ integrins, particularly CD11b/CD18. The counter ligands on HUVEC for the enhanced adhesion are postulated to be ICAMs, because CD11b/CD18 binds to ICAM-1, ICAM-2, and ICAM-3.^3,4 Indeed, H₂O₂ augmented adhesion of EOS to rh-ICAM-1. Although EOS α₄ integrin(s) could also interact with endothelial adhesion proteins such as VCAM-1, the anti-α₄ mAb did not affect the H₂O₂ -induced EOS adhesion. Furthermore, H₂O₂ did not enhance adhesion of EOS to rh-VCAM-1, indicating that α₄ integrin is not involved in the H₂O₂-induced adhesion. Finally, we have provided evidence that H₂O₂ directly enhances eosinophil expression of CD11b and CD18. Although both the number and functional state of integrins can be regulated by a variety of mediators^3,4,20,21 our data suggest that the increased expressions of these molecules are, at least in part, involved in the enhancing effects for EOS adhesion. Intracellular signalling mechanisms associated with the H₂O₂ effect were not evaluated in this study, but should be clarified in future work.

At the sites of allergic inflammation, multiple inflammatory cells, including EOS and macrophages, may generate oxygen species.^22,23 It has been reported that bronchoalveolar lavage (BAL) cells obtained following an antigen challenge generated 8–10 µm superoxide anion.²³ EOS obtained from BAL following a segmental antigen challenge possess a greater ability to generate superoxide anion, as well as to induce adhesion and expression of CD11b, than that shown by EOS in peripheral circulation.²⁴ H₂O₂ can be detected at micromolar concentrations in normal human sera.²⁵ Although the precise concentration of oxygen species at the inflammatory sites remains unknown, these findings suggest that it is possible to achieve an H₂O₂ concentration similar to that used in this study at an allergic inflammation site.

As EOS are capable of generating radical oxygen species in response to a wide range of inflammatory mediators, EOS-derived H₂O₂ may play roles in the development of adhesive property of the cells. In this context, we observed that both FMLP- and PAF-induced EOS adhesion was partially inhibited by catalase while such an inhibitory effect was not observed when GM-CSF or PMA was the stimulant. These results suggest that EOS-derived H₂O₂ may be involved in the development of EOS adhesion as an autocrine/paracrine mediator in vivo when certain inflammatory mediators, such as FMLP or PAF, are involved.

Our observations agree with earlier work on other types of cells. Skogkund et al. reported that 30–300 µm H₂O₂ induced the adhesion of monoblastoid U-937 cells to plastic, and that this adhesion was inhibited by mAb against CD11b and CD18, but not by mAb against CD11a or CD11c.¹⁹ Fraticelli et al. reported that H₂O₂ caused the up-regulation of CD11b and CD18 on neutrophils concurrently with a shedding of l-selectin and increased the adherence of neutrophils to EC.¹⁸ These reports and our present observations suggest that H₂O₂ can induce a direct proadhesive effect on certain types of leucocytes, including EOS, via enhanced expression of CD11b/CD18.

Our observations raise possibilities as to the biological significance of the expression and function of EOS β₂ integrin during interaction with H₂O_2. When activated, a variety of inflammatory cells, including EOS, are capable of generating radical oxygen species at sites of allergic inflammation, and therefore EOS will probably be exposed to H₂O₂. Meanwhile, H₂O₂ enhances the expression of ICAM-1 on EC and increases the ability of EC to potentiate leucocyte adhesion.^15,16 Thus, H₂O₂ alters the adhesive property of both EOS and EC, and thereby augments the interaction between these two kinds of cells in a β₂ integrin-dependent manner. The effect of H₂O₂ on EOS β₂ integrin may also modify the subsequent transmigration of EOS across EC, because it has been well documented that the β₂ integrin/ICAM-1 pathway is crucial in this process.^13,14 Thus, H₂O₂ may modify the adhesion and transendothelial migration of EOS, and contribute to the phenotypic change of EOS in the airway. These effects would contribute to the eventual manifestations of inflammation in allergic diseases such as asthma.

Glossary

Abbreviations

EC

endothelial cells

EOS

eosinophils

EPO

eosinophil peroxidase

FMLP

N-formyl-methionyl-leucyl-phenylalanine

H₂O₂

hydrogen peroxidase

HUVEC

human umbilical vein endothelial cells

ICAM-1

intracellular cell adhesion molecule-1

PAF

platelet-activating factor

VCAM-1

vascular cell adhesion molecule-1