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Am. J. Respir. Cell Mol. Biol., Volume 19, Number 1, July 1998 158-166

Granulocyte Macrophage Colony-stimulating Factor Augments ICAM-1 and VCAM-1 Activation of Eosinophil Function

Makoto Nagata, Julie B. Sedgwick, Hirohito Kita, and William W. Busse

Section of Allergy/Clinical Immunology, Department of Medicine, University of Wisconsin, Madison, Wisconsin; Pulmonary Division, Second Department of Internal Medicine, Saitama Medical School, Saitama, Japan; and Department of Immunology, The Allergy Disease Research Laboratory, Mayo Clinic and Foundation, Rochester, Minnesota


    Abstract
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) are members of the immunoglobulin superfamily adhesion molecules on vascular endothelium and important in the development of eosinophil (EOS) accumulation in allergic inflammation. To define the role of these adhesion proteins in EOS inflammation, peripheral blood EOS from allergic donors were incubated in either buffer (control)-, recombinant human (rh)-VCAM-1-, or rh-ICAM-1-coated plates, and the effects of these adhesion proteins on EOS effector functions were determined. VCAM-1 induced spontaneous EOS adhesion whereas EOS adhesion to ICAM-1 required a second signal, such as granulocyte macrophage colony-stimulating factor (GM-CSF). Although only VCAM-1 stimulated EOS superoxide anion (O2-) generation, the addition of GM-CSF (100 pM) to the reactions resulted in a greater and equivalent production of O2- with VCAM-1 and ICAM-1. In the presence of GM-CSF, ICAM-1 and VCAM-1 caused significant release of EOS-derived neurotoxin (EDN). Moreover, only ICAM-1 (no GM-CSF) promoted calcium ionophore A23187 (0.2 µM)-induced EOS leukotriene C4 (LTC4). Enhanced O2- generation, EDN release, and LTC4 generation observed with ICAM-1 and VCAM-1 were significantly inhibited by anti-beta 2-integrin antibody. These results suggest that ICAM-1 and VCAM-1 are important in determining the eventual function of airway EOS.


    Introduction
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Eosinophils (EOS) contribute to allergic inflammation through a variety of mechanisms (1). For example, EOS generate oxygen metabolites and release granule-derived proteins which are toxic to airway tissue (6, 7). In addition, EOS generate and release leukotriene C4 (LTC4) to contract bronchial smooth muscle (8). Finally, EOS have the capacity to express a variety of proinflammatory cytokines, including granulocyte macrophage colony-stimulating factor (GM-CSF), which can promote cell survival and enhance selective effector cell functions (9, 10).

EOS participation in allergic inflammation involves several steps, including cell adhesion to and transmigration through vascular endothelium. Constitutively expressed intercellular adhesion molecule-1 (ICAM-1) on epithelium and endothelium is increased following interleukin (IL)-1beta and/or tumor necrosis factor alpha  (TNF-alpha ) exposure and may be a primary adhesion molecule in cell transmigration (11, 12). Interest in the role of ICAM-1 in the pathogenesis of eosinophilic inflammation in asthma was heightened by the finding that anti-ICAM antibody inhibited increased bronchial responsiveness and pulmonary eosinophilia following antigen challenge of sensitized monkeys (13). Among other adhesion proteins expressed on cytokine-activated airway endothelium, vascular cell adhesion molecule-1 (VCAM-1) may be crucial not only for the selective adhesion and directed migration of EOS to the airways, but also as a contributor to the eventual function of adherent and recruited cells (14, 15). However, in the in vivo environment of allergic inflammation, EOS are also exposed to cytokines, including GM-CSF, which may promote this cell's phlogistic properties, including superoxide anion (O2-) generation (16). Therefore, the enhanced inflammatory functions observed in airway EOS (14) are likely determined by cell interactions with multiple factors including adhesion molecules and cytokines, such as GM-CSF.

We have reported that EOS adhesion to VCAM-1 stimulates O2- generation and enhances activation of the respiratory burst by the chemotactic peptide formylmethionylleucylphenylalanine (FMLP), but does not cause granular protein release (15). Moreover, GM-CSF primes EOS function in response to FMLP but by itself cannot activate EOS (21). Based upon these observations, we hypothesized that the eventual function of EOS in allergic inflammatory reactions is determined by its interactions with cytokines and adhesion molecules, and that the effect these various factors have on cell function will be distinct and unique to each adhesion molecule. In the following report, we evaluated the effects of GM-CSF, a cytokine generated in allergic inflammation and likely to be present in the milieu associated with cell adhesion and migration, on EOS function when these cells are exposed to either VCAM-1- or ICAM-1-coated surfaces.

    Materials and Methods
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Human Subjects

EOS were isolated from the peripheral blood of subjects with allergic rhinitis or mild atopic asthma. Subjects ranged in age from 20 to 50 yr, and gender distribution was equal. Immediate hypersensitivity was demonstrated by at least one positive skin reaction (> 3 mm) by the prick-puncture technique to extracts of common allergens including ragweed, house dust mite, grass pollen, cat dander, and dog dander. Except for as-needed inhaled beta -agonist use, the subjects took no medications at the time of study. Informed consent was obtained before participation in the study.

Reagents

Percoll was purchased from Pharmacia (Piscataway, NJ). Hanks' balanced salt solution (HBSS), phosphate-buffered saline (PBS), newborn calf serum (NCS), and fetal calf serum (FCS) were obtained from Life Technologies/ BRL (Grand Island, NY). Soluble recombinant human (rh) VCAM-1 (22) and mouse antihuman alpha 4-integrin monoclonal antibody (mAb) (HP1/2; IgG1) (23) were generous gifts from Dr. Roy Lobb (Biogen, Inc., Cambridge, MA). Soluble rh-ICAM-1 was obtained from Dr. Ted Widek (Boehringer Ingelheim, Ridgefield, CT). Mouse antihuman beta 2-integrin mAb (clone L130; IgG1) and mouse IgG1 were obtained from Becton-Dickinson (San Jose, CA). Ficoll, dextran, superoxide dismutase (SOD), phorbol myristate acetate (PMA), gelatin, and horse-heart ferricytochrome C (type VI) were obtained from Sigma Chemical Co. (St. Louis, MO). Hypaque was purchased from Sanofi-Winthrop Pharmaceuticals, New York, NY. The rh-GM-CSF was obtained from R&D Systems (Minneapolis, MN).

Cell Separation

To isolate peripheral blood EOS, multiple discontinuous density Percoll gradients were used as previously reported (24). Briefly, following dextran sedimentation and ficoll-hypaque centrifugation, granulocytes (40 × 106 cells/gradient in HBSS with 5% NCS) were layered onto Percoll gradients consisting of four density solutions: 1.085, 1.090, 1.095, and 1.100 g/ml. After centrifugation (700 × g) for 20 min, EOS (>=  90% purity on a Wright-stained cytocentrifuge preparation) from the 1.095/1.100 g/ml interface were collected. The cells were washed and resuspended in HBSS supplemented with 0.1% gelatin (HBSS/gel). EOS were > 95% viable by trypan blue exclusion; the only contaminating cells were neutrophils.

Preparation of VCAM-1- and ICAM-1-coated Microassay Plates

Soluble rh-VCAM-1 or rh-ICAM-1 was dissolved in 0.05 M NaHCO3 coating buffer (15 mM NaHCO3, 35 mM Na2CO3, pH 9.2) (22). VCAM-1, ICAM-1, or coating buffer alone (50 µl/well) was added to flat-bottomed 96-well immunoassay plates (Immunlon II; Dynatech, Chantilly, VA) and incubated at 4°C overnight (23). Residual coating fluid was decanted and 200 µl/well of HBSS/gel was added to the treated and control wells to reduce nonspecific EOS adhesion and activation. After a 2-h incubation at ambient temperature, the wells were decanted and ready for the addition of EOS. A colorimetric assay based on bovine serum albumin (BSA) standards (Micro BCA; Pierce, Rockford, IL) was used to determine the amount of VCAM-1 or ICAM-1 protein bound to the wells before and after HBSS/gel blocking.

EOS Adherence Assay

EOS adherence was assessed as eosinophil peroxidase (EPO) activity of adherent cells as previously described (14, 15). Briefly, 100 µl of EOS (1 × 105 cells/ml in HBSS/ gel) and 100 pM GM-CSF (where noted) were placed in VCAM-1-, ICAM-1- or control (buffer)-treated, HBSS/gel-blocked wells and incubated for 30 min (VCAM-1) or 60 min (ICAM-1) at 37°C. Following three rigorous washes with HBSS (37°C) to remove nonadherent cells, 100 µl of HBSS/gel was added to the reaction wells, while 100 µl of the original cell suspension was added to empty wells as a measure of total EPO activity. EPO substrate (1 mM H2O2, 1 mM o-phenylenediamine HCl, and 0.1% Triton in 55 mM Tris buffer, pH 8.0) was then added to all wells. After a 30-min incubation at room temperature, 50 µl of 4 M H2SO4 was added to stop the reaction. Absorbance was measured at 490 nm in a microplate spectrophotometer (EL309; Biotec, Winooski, VT). In selected experiments, EOS were preincubated with 1 µg/ml HP1/2 (anti-alpha 4 mAb), 3 µg/ml L130 (anti-beta 2 mAb), or an equivalent concentration of the corresponding isotype mouse IgG1 at 4°C for 30 min before addition of cells into wells. Adherence was calculated:
% adherence=<FR><NU>(activated Abs 490 nm)−(spontaneous Abs 490 nm)</NU><DE>(total Abs 490 nm)</DE></FR>×100

The detection of EPO by this assay was linear between the concentrations of 102 and 104 EOS/well as determined by a standard curve. This assay does not cross-react with neutrophil myeloperoxidase and correlated significantly with cell adhesion as measured by 51Cr radioactivity (14).

O2- Generation

O2- generation was measured as previously described (25). Briefly, 1 × 105 cells/well in HBSS/gel and 0.1 µM cytochrome C were added to VCAM-1-, ICAM-1-, or control buffer-coated 96-well plates in the presence or absence of 100 pM GM-CSF. The reaction mixture was incubated at 37°C and absorbance was measured at 550 nm over 3 h. Each reaction condition was performed in duplicate and against an identical control reaction containing 20 µg/ml of SOD. Due to the 1:1 stoichiometry of cytochrome C reduction and O2- generation, the data were calculated using an extinction coefficient of 21.1 M-3 cm-1 for cytochrome C reduction and expressed as nmol of O2-/5 × 105 cells minus SOD control and spontaneous O2- generation. EOS viability after the 3-h activation exceeded 95% by trypan blue exclusion.

Eosinophil-derived Neurotoxin (EDN) Release

Following the 3-h incubation to measure O2- generation, the 96-well plates were immediately centrifuged (700 × g) at 4°C for 15 min and cell-free supernates recovered for EDN analysis. EDN levels were quantitated by a double antibody competitive radioimmunoassay (RIA) using radiolabeled EDN, rabbit anti-EDN antibody, and burro antirabbit IgG as reported (26, 27).

LTC4 Release

Eosinophils were suspended, 2 × 106 cells/ml, in PBS containing 20 mM serine and 5 mM glutathione. Using ICAM-1-, VCAM-1-, or buffer-coated, HBSS/gel-blocked 96-well immunoassay plates, EOS (2.5 × 105/well) were incubated at 37°C for 60 min in the presence or absence of 100 nM GM-CSF. EOS activation was terminated by the addition of 100 pM of 4°C assay buffer (LTC4 RIA kit; Dupont/NEN Co., Boston, MA). The 96-well plates were centrifuged (700 × g) at 4°C for 15 min and supernates recovered and stored at -70°C for later LTC4 analysis. In selected experiments, EOS were incubated on ICAM-1, VCAM-1, or control wells for 60 min at 37°C and then 0.2 µM calcium ionophore A23187 was added as activator. After a 20-min incubation at 37°C, the reaction was stopped and processed as above. LTC4 was quantitated by RIA per manufacturer's instructions.

Statistical Analysis

Data are presented as mean ± SEM. The Student's t test was used for paired comparisons. Maximum values of O2- generation for the 3-h reaction were used to assess differences. Analysis of variance (ANOVA) with repeat measures and Scheffe's post-hoc test (AbStat; Anderson-Bell, Corp., Parker, CO) were used for comparison of more than two variables to determine significance. P < 0.05 was considered significant.

    Results
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

EOS Adherence

Based on BSA standards, coating of the reaction wells with 5 µg/ml VCAM-1 or 10 µg/ml ICAM-1 resulted in equivalent amounts of protein binding. High background levels of nonspecific (presumably by beta 2-integrins) EOS adhesion (> 20%) and activation of O2- generation were observed when EOS were exposed to unblocked, uncoated, plastic tissue-culture reaction wells (data not shown). To minimize both nonspecific EOS adhesion and activation, HBSS/gel was used as a blocking agent and assay buffer (15). Gelatin-blocking resulted in low and equivalent levels of baseline EOS adhesion as observed with blocking with FCS or human serum albumin, but did not yield the high baseline values of O2- generation as found with these other commonly used blocking substrates (data not shown). HBSS/gel blocking also resulted in equivalent levels of protein retained in control, VCAM-1-coated, and ICAM-1-coated wells (data not shown). Several concentrations of GM-CSF (1 to 1,000 pM) were assessed for their effect on VCAM-1 adhesion, with maximal enhancement observed at 100 pM. Increasing GM-CSF to 1 nM did not further enhance VCAM-1 or ICAM-1 activation of EOS (data not shown).

Kinetic experiments were performed to determine the incubation time required for optimal EOS adhesion to VCAM-1 or ICAM-1 in the absence or presence of GM-CSF. By 15 min, there was significant adherence of EOS to VCAM-1-coated wells compared with buffer-coated control wells (18.7 ± 3.6% versus 3.3 ± 1.2% adhesion, respectively; n = 10, P = 0.0001 by ANOVA and Scheffe constants, Figure 1A). The addition of GM-CSF (100 pM) moderately, but significantly, enhanced EOS adhesion to VCAM-1 (25.2 ± 4.5% adhesion, P < 0.02 versus VCAM-1 alone). Compared with VCAM-1-coated wells, EOS adhered poorly to wells coated with ICAM-1 (Figure 1B). To achieve significant EOS adhesion to ICAM-1-coated wells, 100 pM GM-CSF was required; even then, the level of adherence associated with GM-CSF was modest and reached significance (versus buffer-control) only after 60 min of incubation. The failure of EOS to adhere to ICAM-1 was observed even with larger concentrations of ICAM-1 (up to 40 µg/ml; data not shown). Consequently, a 30-min incubation period was used to determine EOS adhesion to VCAM-1-coated wells and a 60-min incubation to ICAM-1-coated wells. No release of EPO or EDN was observed during incubations up to 1 h, suggesting that EOS degranulation did not contribute to the changes in EOS adhesion.


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  		Figure 1.   Comparison of EOS adhesion with VCAM-1 (A) and ICAM-1 (B) in the presence and absence of 100 pM GM-CSF. EOS and GM-CSF (or buffer in control) were added to VCAM-1- or ICAM-1-coated wells. Adhesion was assessed by quantitation of EPO in adherent cells. n = 4. *P < 0.0001 versus buffer control ± GM-CSF by ANOVA and Scheffe constants; +P < 0.02 versus VCAM-1; #P < 0.05 versus buffer control.

O2- Generation

EOS generated small, but significant and immediate (30 min), amounts of O2- when incubated in VCAM-1-coated wells (15). In the absence of VCAM-1, 100 pM GM-CSF did not activate O2- generation (1.0 ± 0.3 nmol O2-/5 × 105 cells, P = NS versus control). However, when 100 pM GM-CSF was added to EOS in VCAM-1-coated wells, a bimodal increase in O2- generation was observed (Figure 2A). The O2- reaction kinetics revealed that rapid but low levels of activation occurred by 30 min, consistent with the VCAM-1-alone response. With GM-CSF present, this initial activation was followed by a second, and much greater, activation which reached a maximal response by 2.5 h (10.2 ± 1.3 nmol O2-/5 × 105 cells, P < 0.0001 by ANOVA versus other three conditions); therefore, the development of GM-CSF-enhanced EOS adhesion to VCAM-1 (optimal at 15 min) preceded the "second wave" of O2- generation. In contrast, EOS incubated in ICAM-1-coated wells did not generate O2- (Figure 2B). Only when 100 pM GM-CSF was added to EOS in ICAM-1-coated wells were significant amounts of O2- generated, equivalent to VCAM-1 + GM-CSF (Figure 2B: 1.0 ± 0.4 nmol/ 5 × 105 cells for uncoated [CTL], 12.6 ± 1.5 nmol/5 × 106 cells for ICAM-1 + GM-CSF, P < 0.0001 versus CTL, n = 7).


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  		Figure 2.   Generation of O2- anion by EOS incubated for 3 h in VCAM-1- (A) or ICAM-1- (B) coated wells (or buffer control) in the absence or presence of 100 pM GM-CSF. n = 7. +P = 0.023 versus buffer control; *P < 0.0001 versus buffer control.

EDN Release

Cell-free O2- reaction supernates were evaluated for EOS degranulation of EDN following a 3-h incubation in VCAM-1- or ICAM-1-coated wells in the absence or presence of GM-CSF. Neither ICAM-1 nor VCAM-1 alone stimulated EOS degranulation of EDN (Figure 3A). In the presence of 100 pM GM-CSF, both ICAM-1 and VCAM-1 stimulated significant release of EDN when compared with controls; however, EDN release by ICAM-1 + GM-CSF was significantly greater than VCAM-1 + GM-CSF (Figure 3A: 40.9 + 11.0 ng/105 EOS for ICAM-1 + GM-CSF versus 28.1 ± 8.6 ng/105 cells for VCAM-1 + GM-CSF; P = 0.028, n = 5). Degranulation of EDN occurred only when both GM-CSF and adhesion molecules were present and when increased EOS adhesion occurred.


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  		Figure 3.   Effect of VCAM-1 or ICAM-1 on EOS function. (A) EDN degranulation was assessed after EOS (1 × 105) were incubated 3 h at 37°C in uncoated (CTL), VCAM-1-coated or ICAM-1-coated wells, in the absence or presence of 100 pM GM-CSF. n = 5. +P < 0.05 versus CTL and ICAM-1-coated wells without GM-CSF; *P < 0.03 versus all other conditions. (B) EOS were incubated in uncoated (CTL), ICAM-1-coated, or VCAM-1-coated reaction wells for 60 min at 37°C and then activated with 0.2 µM A23187. After a 20-min incubation at 37°C, cell-free supernates were assessed by RIA for LTC4 generation. n = 5. *P < 0.02 versus CTL and VCAM-1.

LTC4 Release

Neither ICAM-1 nor VCAM-1, in the presence or absence of GM-CSF, stimulated EOS production of detectable amounts of LTC4 after a 3-h incubation (< 0.25 ng/106 cells, n = 3). When EOS were incubated in control-, ICAM-1-, or VCAM-1-coated wells at 37°C (× 1 h) and then exposed to 0.2 µM A23187 (× 20 min), only EOS incubated with ICAM-1 released significant amounts of LTC4 (Figure 3B; CTL: 3.4 ± 1.9 ng/106 cells; ICAM-1: 7.9 ± 3.1 ng/106 cells, P < 0.02 versus CTL or VCAM-1; VCAM-1: 4.5 ± 2.6 ng/ 106 cells, n = 5).

Effect of Anti-integrin Antibodies on ICAM-1-modulated EOS Adhesion

To determine which EOS adhesion molecules were involved in the VCAM-1 and ICAM-1 interactions, EOS were preincubated (4°C × 30 min) with either anti-alpha 4 integrin mAb (HP1/2, 1 µg/ml), anti-beta 2 mAb (L130, 3 µg/ml), or an isotype control mouse IgG1 (3 µg/ml) at 4°C for 30 min. The cells were then exposed to control-, ICAM-1-, or VCAM-1-coated wells in the presence or absence of 100 pM GM-CSF (for adhesion, O2- generation, and EDN release) or 0.2 µM A23187 (for LTC4 production).

Spontaneous adhesion to VCAM-1-coated wells was decreased to baseline levels by anti-alpha 4 mAb (13.4 ± 2.0% adhesion for VCAM-1 without mAb versus 5.3 ± 0.7% with anti-alpha 4; n = 6, P = 0.009, Figure 4A). Anti-beta 2 mAb had no effect either alone or when added with anti-alpha 4. In contrast, EOS adhesion to VCAM-1-coated wells in the presence of GM-CSF was only partially inhibited by either anti-alpha 4 or anti-beta 2 mAb; both mAbs were required to inhibit EOS adhesion completely (24.7 ± 3.0% adhesion for VCAM-1 + GM-CSF with no Ab versus 15.7 ± 2.0% by anti-alpha 4, P = 0.019, 12.5 ± 1.6% by anti-beta 2, P = 0.002, and 7.8 ± 1.6% by the combination of mAbs, P < 0.05 versus no Ab). Interestingly, the GM-CSF component of enhanced EOS adhesion to VCAM-1-coated wells appeared to be totally dependent on the beta 2 integrin, since anti-beta 2 mAb decreased the adhesion to the level of binding to VCAM-1 alone; higher concentrations (10 µg/ml) of anti-alpha 4 and anti-beta 2 mAbs did not further affect cell adhesion (data not shown).


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  		Figure 4.   Effect of anti-alpha 4 and anti-beta 2 integrin mAbs on EOS adhesion to VCAM-1- (A) or ICAM-1- (B) coated wells in the absence or presence of 100 pM GM-CSF. n = 3. *P < 0.05 versus VCAM-1 with no Ab; +P < 0.05 versus VCAM-1 + GM-CSF with no Ab. An IgG1 isotype control had no effect on EOS adhesion (data not shown).

EOS adhesion to ICAM-1 in the presence of GM-CSF was completely inhibited by anti-beta 2 mAb; anti-alpha 4 mAb had no effect (Figure 4B; ICAM-1 + GM-CSF + no Ab = 21.5 ± 8.8% adhesion; + anti-alpha 4 = 22.0 ± 6.6%, P = NS; + anti-beta 2 = 6.8 ± 2.4%, P < 0.02 versus other conditions, n = 3). Addition of a mouse IgG1 isotype control to the reaction wells did not affect EOS adhesion to VCAM-1-, ICAM-1-, or control-coated wells in the presence or absence of GM-CSF (data not shown).

Effect of Anti-integrin Antibody Inhibition on EOS O2- Generation

Anti-alpha 4 mAb inhibited only the immediate (30 min) generation of EOS O2- generation to VCAM-1 plus 100 pM GM-CSF (Figure 5A). A higher dose of anti-alpha 4 mAb (10 µg/ml) did not further affect O2- generation (data not shown). In contrast, anti-beta 2 mAb (L130) significantly inhibited the immediate and late (> 60 min) EOS O2- generation to VCAM-1 plus GM-CSF (2.9 ± 1.2 nmol cytochrome C/5 × 105 cells at 180 min, P < 0.0001 versus 11.8 ± 0.9 without mAb). The combination of anti-alpha 4 and anti-beta 2 mAbs did not further modify these results, and residual levels (4 nmol) of O2- remained unaffected by either mAb. Only anti-beta 2 mAb abrogated EOS O2- generation stimulated by ICAM-1 when GM-CSF was present (Figure 5B) Mouse IgG1 isotype control had no effect on O2- generation to VCAM-1 or ICAM-1 alone or in combination with GM-CSF (data not shown).


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  		Figure 5.   Effect of anti-alpha 4 and anti-beta 2 integrin mAbs on EOS O2- generation stimulated by VCAM-1 (A) or ICAM-1 (B) plus 100 pM GM-CSF. n = 3. *P < 0.002 versus ICAM-1 or VCAM-1 + no Ab. An IgG1 isotype control had no effect on EOS O2- generation (data not shown).

Effect of Anti-integrin Antibodies on EDN and LTC4 Release

Only anti-beta 2 mAb significantly inhibited degranulation of EDN stimulated by VCAM-1 or ICAM-1 in the presence of 100 pM GM-CSF (Figure 6); a mouse IgG1 isotype control did not affect EOS degranulation (data not shown). Finally, anti-beta 2, but not anti-alpha 4 or IgG1, significantly decreased EOS LTC4 generation to ICAM + 0.2 µM A23187 (Figure 7).


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  		Figure 6.   Effect of anti-alpha 4 and anti-beta 2 integrin mAbs on EOS EDN release stimulated by 100 pM GM-CSF + VCAM-1 (A) or + ICAM-1 (B). n = 3. *P < 0.05 versus VCAM-1 + GM-CSF and no Ab; +P < 0.02 versus ICAM-1 + GM-CSF and no Ab. An IgG1 isotype control had no effect on EOS EDN release (data not shown).


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  		Figure 7.   Effect of anti-integrin antibodies on EOS LTC4 production stimulated by 0.2 µM A23187 and ICAM-1. n = 3. *P < 0.05 versus ICAM-1 and no Ab. An IgG1 isotype control had no effect on EOS LTC4 generation (data not shown).

    Discussion
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Our observations have identified the combined effects of adhesion to rh-VCAM-1 or rh-ICAM-1 and interaction with GM-CSF on peripheral blood EOS function (Table 1). While contact with VCAM-1 induced spontaneous EOS adhesion and O2- generation, these functions required the presence of GM-CSF to achieve a significant response with ICAM-1. Therefore, our data support the observations that granulocytes do not spontaneously adhere to ICAM-1 but require a secondary signal such as Mn2+, FMLP, or platelet-activating factor (PAF) (28). Although 1 µM PAF induced higher levels of EOS adhesion to wells coated with 10 µg/ml lCAM-1 (42% adhesion), 100 pM GM-CSF was equivalent to the effects of 0.5 mM MnCl2 or 0.1 µM FMLP (data not shown).

                              
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TABLE 1
Comparison of VCAM-1 and ICAM-1 effects on EOS function*

The magnitude of GM-CSF-enhanced EOS adhesion to ICAM-1 was modest compared with VCAM-1 enhancement, and the kinetics of this interaction were slower. Despite these differences in EOS adhesion, ICAM-1 and VCAM-1 were equally effective in stimulating EOS O2- generation when GM-CSF was present. In addition, the combination of ICAM-1 and GM-CSF stimulated EOS EDN degranulation that was significantly greater than that observed with activation by VCAM-1 and GM-CSF. Finally, EOS adhesion to ICAM-1 promoted LTC4 generation when A23187 was added as a stimulus, whereas GM-CSF and VCAM-1 had no effect on this function.

The differences noted between the effects of ICAM-1 and VCAM-1 on EOS adhesion and function may be related to the expression of these substrates on vascular endothelium. ICAM-1 is constitutively expressed at high levels on endothelium, whereas VCAM-1 expression is low and requires cytokine stimulation to reach appreciable levels (12, 31). Under noninflammatory conditions, circulating EOS are exposed to constitutively expressed ICAM-1 but do not adhere, and inflammatory events are not initiated. During inflammation, EOS integrins are activated and cell adhesion to ICAM-1 would potentiate EOS migration to the airways. In contrast, when endothelial VCAM-1 is expressed, EOS, but not neutrophils (32), adhere and become activated, even in the absence of additional stimuli. These different processes help explain both the cell specificity and process of inflammation during allergic reactions.

If GM-CSF levels increased in the peripheral circulation during an allergic or asthmatic exacerbation, our findings would suggest that EOS are functionally activated as soon as they adhere to the cytokine-stimulated endothelium. However, it is generally accepted that EOS are not activated until they have reached the interstitial matrix and/or epithelium. This supposition is not actually inconsistent with our results. Reports on the changes in serum/ plasma GM-CSF during inflammatory responses are unclear. Increases in both GM-CSF levels in bronchoalveolar lavage fluid (BALF) and BAL cells expressing GM-CSF mRNA have been reported (33, 34). Robinson and associates (35) observed increased proportions of BAL cells positive for GM-CSF mRNA in subjects with atopic asthma; these values also were associated with increases in airflow restriction and bronchial hyperresponsiveness. Virchow and colleagues (36) found increased levels of GM-CSF protein in the BALF of atopic asthma patients 18 h after segmental allergen provocation, whereas Shaver and associates (37) observed increased GM-CSF in BALF 2 to 16 d after challenge. Although GM-CSF-positive cells and protein were increased in the BALF and correlated with BAL EOS in mild, stable asthmatic patients compared with nonasthmatic control subjects (38), Woolley and coworkers (39) found no differences in serum or BALF levels. Only patients with severe asthma (40) or rheumatoid arthritis (41), exacerbation of chronic bronchitis (42), or a patient with tryptophan-independent episodic eosinophilia-myalgia (43) have been reported to have higher serum GM-CSF than healthy control subjects. Further study is required to determine the parameters for changes in serum GM-CSF.

Based on the findings that elevation in serum GM-CSF is not observed in patients with infections (44), it has been proposed that this cytokine plays a role in the immediate vicinity of the cells in which it is secreted. This is consistent with our findings that alternating the sequence of EOS exposure of GM-CSF and adhesion protein did not affect the degree of cell activation. Exposure of EOS to VCAM-1 before (or after) 100 pM GM-CSF resulted in the same enhanced functional responses once both agonists were present (data not shown). Therefore, adhesion to VCAM-1 and later exposure to sufficiently high levels of GM-CSF in the inflamed matrix could result in increased EOS activation at this site.

Although the levels of GM-CSF-enhanced adhesion and EOS function may appear low, they were comparable with cell activation previously reported. We have reported that normal EOS generated 8 to 12 nmol O2- when activated by 1 µM A23187, 0.1 µM FMLP, 0.5 mg/ml serum-opsonized zymosan (25, 47), or 1 µM PAF (48); levels similar to those stimulated by GM-CSF and adhesion. Likewise, ICAM-1/GM-CSF-stimulated degranulation reached levels comparable with 1 µM PAF and only slightly less than sIgA-coated beads (49). Finally, EOS generation of LTC4 by a suboptimal concentration of A23187 (1 µM) was enhanced by ICAM-1 adhesion to exceed a level usually observed only with a higher concentration of ionophore (10 µM) (50) or 1 µM FMLP (51). Therefore, EOS activation by the combination of GM-CSF and adhesion to VCAM-1 or ICAM-1 could be a relevant pathologic mechanism.

VCAM-1 binds to the integrin complex alpha 4beta 1 (VLA-4) on EOS surface membranes (52, 53). Although anti-alpha 4 mAb completely blocked EOS adhesion to VCAM-1 (15), its effect on the synergistic activation of these cells with VCAM-1 and GM-CSF was minimal; anti-alpha 4 mAb inhibited only the immediate (30 min) generation of O2- and did not significantly affect later (3-h) O2- generation and EDN release. In contrast, the adhesion molecule/GM-CSF enhancement of EOS O2- generation, degranulation, and LTC4 release were all inhibited by anti-beta 2-integrin antibody, suggesting that these events were dependent on a specific interaction with EOS beta 2-integrins. This is consistent with reports by others that beta 2-integrin is required for degranulation of adherent EOS by GM-CSF, PAF (54), or PMA (55), and IgG-stimulation of the respiratory burst (56). These observations suggest that the modulation of beta 2-integrin via GM-CSF was the mechanism of adhesion-dependent EOS functional upregulation. Like VCAM-1, EOS adhesion to fibronectin is not dependent on beta 2-integrin expression, but such adhesion can enhance beta 2-dependent GM-CSF- or TNF-alpha -stimulated EOS respiratory burst (57, 58). Moreover, Sung and coworkers (59) recently reported that GM-CSF enhances the adhesion strength of EOS and VCAM-1. The mechanism for beta 2-integrin participation in these responses was not determined but may include changes in receptor number (60, 61), activation (62), ligation (57), and/or signal transduction events such as tyrosine phosphorylation (63).

Interaction of EOS integrins with ICAM-1 or VCAM-1 in the presence of GM-CSF was sufficient to change the cell's phenotype to that which more closely resembles an airway EOS (14). Therefore, cell-surface-membrane adhesion molecules are important not only for selective EOS migration but also as modulators of cell function. Our observations suggest that ICAM-1 and VCAM-1, in conjunction with GM-CSF, promote EOS effector functions that affect not only this cell's accumulation in the inflamed tissue but also the inflammatory phenotype of the migrated EOS, and are thus important to the eventual manifestation of bronchial disease in asthma.

    Footnotes

Abbreviations: bronchoalveolar lavage, BAL; eosinophil-derived neurotoxin, EDN; eosinophil(s), EOS; eosinophil peroxidase, EPO; formylmethionylleucylphenylalanine, FMLP; granulocyte macrophage colony-stimulating factor, GM-CSF; intercellular adhesion molecule-1, ICAM-1; leukotriene C4, LTC4; monoclonal antibody(ies), mAb(s); superoxide anion, O2-; platelet-activating factor, PAF; recombinant human, rh; radioimmunoassay, RIA; vascular cell adhesion molecule-1, VCAM-1.

(Received in original form April 21, 1997 and in revised form August 12, 1997).

Acknowledgments: This work was supported by NIH grant AI23181.
    References
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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