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Published ahead of print on February 2, 2006, doi:10.1165/rcmb.2005-0303OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 34, pp. 760-765, 2006
© 2006 American Thoracic Society
DOI: 10.1165/rcmb.2005-0303OC

Eosinophil Trans-Basement Membrane Migration Induced by Interleukin-8 and Neutrophils

Izumi Kikuchi, Shinya Kikuchi, Takehito Kobayashi, Koichi Hagiwara, Yoshio Sakamoto, Minoru Kanazawa and Makoto Nagata

Department of Respiratory Medicine, Saitama Medical School, Saitama, Japan; and Department of Allergy and Respiratory Medicine, Kanto Central Hospital of the Mutual Aid Association of Public School Teachers, Tokyo, Japan

Correspondence and requests for reprints should be addressed to Makoto Nagata M.D., Ph.D., Department of Respiratory Medicine, Saitama Medical School, Saitama, Japan. E-mail: favre4mn{at}saitama-med.ac.jp


   Abstract
Top
 Abstract
Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
Neutrophilic inflammation observed with severe asthma is often associated with interleukin-8 (IL-8). Neutrophils can secrete a variety of mediators that may augment the migration of eosinophils. We have reported a positive correlation between the concentrations of neutrophils and eosinophils in sputum from subjects with severe asthma, suggesting a possible role of neutrophils in regulating eosinophilic inflammation. The aim of this study was to investigate whether neutrophils stimulated with IL-8 modify the trans-basement membrane migration (TBM) of eosinophils. Eosinophils and neutrophils were isolated from peripheral blood drawn from healthy donors or subjects with mild asthma. The TBM of eosinophils in response to IL-8 was evaluated in the presence or absence of neutrophils using the chambers with a Matrigel-coated transwell insert. Neither IL-8 alone nor the presence of neutrophils alone induced the TBM of eosinophils. However, when eosinophils were coincubated with neutrophils and stimulated with IL-8, the TBM of eosinophils was significantly augmented. This augmented TBM of eosinophils was inhibited by a matrix metalloproteinase-9 inhibitor, a leukotriene B4 receptor antagonist, platelet-activating factor antagonists, or an anti–TNF-{alpha} monoclonal antibodies. These results suggest that neutrophils migrated in response to IL-8 may lead eosinophils to accumulate in the airways of asthma and possibly aggravate this disease.

Key Words: eosinophils • growth-related oncogene-{alpha} • interleukin-8 • neutrophils • trans-basement membrane migration


   Introduction
Top
 Abstract
 Introduction
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
Eosinophils, inflammatory cells predominantly found in the airways of patients with asthma, likely contribute to airway remodeling or airflow limitation observed with asthma (1–4). The mechanism by which eosinophils accumulate in the airways is a complex process that is mainly regulated by cytokines, chemokines, and adhesion molecules. This process is likely to be inhibited by corticosteroid treatment via the suppression of cytokines/chemokines productions from corticosteroid-sensitive cells such as Th2 cells. In a subgroup of patients, accumulation of neutrophils is found in their airways even in the absence of apparent infection. Asthma in such patients is often severe and chronic and is refractory to corticosteroid therapy (5–8). Based on a recent report from the European network study for understanding mechanisms of severe asthma (ENFUMOSA), patients with severe asthma have greater sputum neutrophilia and evidence of ongoing eosinophil-derived mediator release, compared with patients with mild to moderate asthma, suggesting that both neutrophilic and eosinophilic inflammation persists in the airways of severe asthma (9). In this context, we have recently reported a positive correlation between the concentrations of neutrophils and eosinophils in induced sputum from patients with severe persistent asthma who are treated with medicines including systemic corticosteroid (10).

Functions of neutrophils are not effectively suppressed by corticosteroids (11, 12), suggesting that neutrophils may play a role in the pathophysiology of the disease in such patients. There are many reports which suggest that neutrophils are exposed to a variety of inflammatory mediators in the airways of patients with asthma. For example, interleukin-8 (IL-8), which acts as a chemoattractant for neutrophils, is found in bronchoalveolar lavage fluid and serum from patients with asthma (13–16). Concentration of IL-8 has been shown to be correlated with accumulation of neutrophils in the airways of asthma (6), and therefore this chemokine may be an essential molecule responsible for the development of neutrophilic inflammation in asthma. Activated neutrophils can secrete a variety of mediators (e.g., matrix metalloproteinases [MMPs], leukotriene B4 [LTB4], platelet-activating factor [PAF], and TNF-{alpha}) that can induce digestion of basement membrane, or migration or activation of eosinophils (17, 18), and may thus contribute to the pathophysiology of asthma.

We framed a hypothesis that neutrophils stimulated with and migrated to IL-8 play roles in the regulation of eosinophilic inflammation in asthma. Trans-basement membrane migration (TBM) is one of the key processes by which circulating eosinophils accumulate in the airways of asthma. Here we report that a combination of IL-8 and neutrophils augment the TBM of eosinophils.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 References
 
Reagents
Anti-CD16 antibody–coated magnetic beads were purchased from Miltenyi Biotec (Auburn, CA). Percoll was purchased from Pharmacia (Uppsala, Sweden). HBSS was purchased from GIBCO BRL (Grand island, NY). BIIL260, an LTB4 receptor antagonist, and WEB2086 and WEB2170, PAF receptor antagonists, were provided by Boehringer Ingelheim (Ridgefield, CT). MMP-9 inhibitor and GM1489, an MMP inhibitor, was purchased from Calbiochem (San Diego, CA). AA861, a 5-lipoxygenase (LO) inhibitor, and PBS were obtained from Wako (Osaka, Japan). IL-8, Growth-related oncogene-{alpha} (GRO-{alpha}), TNF-{alpha}, eotaxin, regulated upon activation, normal T cell expressed and secreted (RANTES), and anti–CXC chemokine receptor 2 (CXCR2) antibody were purchased from R&D Systems (Minneapolis, MN). LTB4 was purchased from Cayman Chemical (Ann Arbor, MI). PAF, Phorbol 12-myristate 13-acetate(PMA), o-phenylenediamine (OPD), and BSA were obtained from Sigma (St. Louis, MO). Anti–TNF-{alpha} monoclonal antibody (mAb) (clone Mab11, mouse IgG1) was purchased from Becton Dickinson (Franklin Lakes, NJ). Mouse IgG1, an isotype control for anti–TNF-{alpha} mAb, and newborn calf serum (NCS) were purchased from ICN Biomedicals, Inc. (Aurora, OH). The acetoxy methyl ester of 2'-7'-bis (2-carboxy-ethyl)-5(6)-carboxyfluorescein (BCECF-AM) was purchased from Dojin Laboratory (Kumamoto, Japan).

Preparation of Neutrophils and Eosinophils
Neutrophils and eosinophils were isolated from peripheral blood collected from nonatopic healthy donors whose eosinophil content was < 5% of their peripheral leukocytes. In some experiments, cells isolated from individuals with mild intermittent asthma were also used. The numbers of males and females were comparable among donors, with similar age distributions ranging from 20–38 yr. Informed consent was obtained before collection of each blood sample. Neutrophils and eosinophils were separated by the combination of Percoll density gradient centrifugation and negative immunomagnetic bead selection as previously described (19, 20). Briefly, 40 ml of dextran were added to 160 ml of heparinized blood, and erythrocytes were removed as sediment. The remaining suspension of leukocytes was layered onto Percoll gradients of 1.080, 1.085, and 1.090 g/ml in density. After centrifugation at 700 x g for 20 min, neutrophils (purity exceeded 95%) were collected from 1.085/1.090 g/ml interface, and suspended in HBSS containing 0.2% BSA (HBSS/BSA buffer). After the removal of Percoll, the red blood cells in the pellet were lysed by hypotonic shock and removed by washing with cold PBS. The remaining cells were washed with 4°C HBSS supplemented with 2% NCS (HBSS/NCS), then incubated with anti-CD16 antibody–coated magnetic beads for 30 min at 4°C, and were then filtered with a column containing steel wool placed in a magnetic field (Miltenyi Biotec). Eosinophils (> 98% purity and > 99% viability), which passed through the column, were collected and washed, and the number of cells was adjusted to 2.5 x 105 cells/ml by using HBSS/BSA buffer.

TBM
The TBM of neutrophils and eosinophils was examined using a modified Boyden's chamber method (21). The study was conducted in duplicate. Briefly, neutrophils were suspended in loading buffer with BCECF-AM at a final concentration of 1 µM and incubated for 30 min at 37°C while shading the light (22, 23). The cells retain the label at least for 90 min (23) and superoxide anion generation in response to PMA (0.5ng/ml) was not modified by BCECF-AM (our unpublished observation). Labeled neutrophils (0.5 x 105 cells), eosinophils (0.5 x 105 cells), or a combination thereof (0.5 x 105 cells plus 0.5 x 105 cells) in a 200-µl medium were added to the upper compartment of a chamber with a Matrigel-coated transwell insert (pore size 3 µm; Becton Dickinson Labware). Either the control medium (500 µl) or a medium that contained one of activators (IL-8, GRO-{alpha}, eotaxin, RANTES, and LTB4) was added to the lower compartment of the chamber. After a 2-h incubation in 5% CO2 at 37°C, the medium in the upper compartment of the chamber and the inserts between the chambers were gently removed. The peroxidase activity of eosinophils in the medium in the lower compartment of the chamber was determined, and the number of migrated eosinophils was calculated from the activity of the standard media which contained known numbers (5 x 103, 1.5 x 104, 5 x 104, 1.5 x 105, and 5 x 105 cells) of eosinophils. To determine the peroxidase activity of eosinophils, the medium was incubated with a substrate (1 mM OPD, 1 mM H2O2, and 0.1% Triton X-100 in Tris-HCl, pH 8.0) for 30 min at room temperature (21). The reaction was stopped by adding 100 µl of 4N H2SO4, and absorbance at 490 nm was determined (21). The effect of neutrophils on the outer density value in this assay was negligible: 0.046 ± 0.002 for 0% control and 0.064 ± 0.006 for 100% control of neutrophils, respectively (n = 4). Similarly, addition of neutrophils to 100% control of eosinophils did not modify the outer density value (1.566 ± 0.023 by addition of 0% control versus 1.569 ± 0.012 by addition of 100% control of neutrophils (n = 4, P = n.s.). The numbers of migrated neutrophils were determined by the measurement of fluorescence in the medium using the Fluoromark (Bio-Rad Laboratories, Hercules, CA) microplate fluorometer (23). The number of migrated neutrophils determined by this method was highly correlated with those counted by hemocytometer (n = 13, P < 0.001, r = 0.9, Pearson's correlation coefficient). The viability of both eosinophils and neutrophils after migration exceeded 98% by trypan blue exclusion.

Blocking Study
Both eosinophils and neutrophils were incubated in a medium containing BIIL260, WEB2086, WEB2170 or AA861 for 15 min at 37°C, in a medium containing anti–TNF-{alpha} mAb (clone Mab11) or an isotype matched control mouse IgG1 for 15 min at ambient temperature, or in a medium containing MMP-9 inhibitor for 30 min at 37°C. The media containing the cells were then transferred to the upper compartment of the chamber, and the assay was performed as described above.

Statistical Analysis
Values are expressed as means ± SEM. Student's t test was conducted to compare two groups, and repeated-measures ANOVA with Scheffé's constants were used to compare more than two groups. A value of P < 0.05 was considered statistically significant.


   RESULTS
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 References
 
Effects of Neutrophils on the TBM of Eosinophils
To investigate whether stimulated neutrophils affect the TBM of eosinophils, we coincubated a mixture of eosinophils and neutrophils in the presence or absence of IL-8, a CXC chemokine that selectively stimulates chemotactic response of neutrophils. Preliminary experiments confirmed that 10 nM of IL-8 is sufficient to induce the TBM of neutrophils (data not shown). Neither a coincubation with neutrophils nor IL-8 (10 nM) alone induced the TBM of eosinophils (migrated eosinophils: 0.9 ± 0.4% by medium control, 2.2 ± 0.8% by coincubation with neutrophils, P = n.s.; 1.9 ± 0.5% by IL-8 alone, P = n.s.; n = 10) (Figure 1). However, when eosinophils were coincubated with neutrophils and stimulated with IL-8, a significant TBM of eosinophils was observed (migrated eosinophils: 12.9 ± 3.1%, P < 0.01 versus the other three conditions; n = 10) (Figure 1). Checkerboard analysis confirmed that the effect of IL-8 on the TBM of eosinophils coincubated with neutrophils is chemotactic (n = 3, data not shown). When the transmigrations of neutrophils by IL-8 and eosinophils by a combination of IL-8 and neutrophils were simultaneously examined, the capacity of eosinophils to migrate was significantly correlated with the number of neutrophils that migrate (P = 0.002, r = 0.54, n = 6). The time-course profile of TBM of eosinophils traced that of neutrophils: the TBM of neutrophils reached a plateau within the first 15 min, whereas eosinophil TBM mainly occurs 15–60 min after the initiation of reaction (n = 6, data not shown). In selected experiments, where the effect of IL-8 and neutrophils on the TBM of eosinophils was examined using both eosinophils and neutrophils from donors with mild asthma provided similar results: only a combination of IL-8 and neutrophils induced the TBM of eosinophils (n = 3, data not shown). Furthermore, when eosinophils were co-incubated with neutrophils from different donors, a similar phenomenon was observed: only a combination of IL-8 and neutrophils, but not IL-8 alone or neutrophils alone, induced the TBM of eosinophils (n = 3, data not shown). GRO-{alpha} (10 nM), another CXC chemokine, showed similar results: the TBM of eosinophils was significantly induced only when eosinophils were coincubated with neutrophils and stimulated with GRO-{alpha} (migrated eosinophils: 0.6 ± 0.3 by medium control, 1.2 ± 0.4 by a coincubation with neutrophils, P = n.s.; 0.8 ± 0.7 by GRO-{alpha} alone, P = n.s.; 7.1 ± 2.3 by a combination of GRO-{alpha} and coincubation with neutrophils, P < 0.05 versus the other three conditions, n = 4; Figure 2A). LTB4 (100nM), which is chemotactic for both eosinophils and neutrophils, directly induced the TBM of eosinophils; however, the TBM of eosinophils was enhanced when both eosinophils and neutrophils were coincubated (migrated eosinophils: 1.0 ± 0.8 by medium control, 1.2 ± 0.9 by a coincubation with neutrophils, P = n.s.; 12.9 ± 4.6 by LTB4 alone, P < 0.05 versus control; 23.6 ± 6.0 by a combination of LTB4 and coincubation with neutrophils, P < 0.05 versus the other three conditions, n = 5; Figure 2B). Finally, eotaxin (3 nM), a CC chemokine that selectively stimulates chemotactic response of eosinophils, induced the TBM of eosinophils (migrated eosinophils: 2.9 ± 0.7 by medium control versus 59.5 ± 3.8 by eotaxin alone, P < 0.01). Coincubation with neutrophils did not modify the TBM of eosinophils in response to eotaxin (migrated eosinophils: 62.3 ± 4.5, P = n.s. versus eotaxin alone, n = 5; Figure 2C). Similar results were obtained when RANTES, another CC chemokine, was an activator (n = 3, data not shown).


Figure 1
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  		Figure 1. Effects of neutrophils and IL-8 (10 nM) on the TBM of eosinophils. The means ± SEM of 10 experiments using cells from different donors are shown. N.S., not significant.

 

Figure 2
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  		Figure 2. Effects of neutrophils and a variety of agonists on the TBM of eosinophils. Molecules used were GRO-{alpha} (10 nM, A), LTB4 (100 nM, B), and eotaxin (10 nM, C). The means ± SEM of four to five experiments using cells from different donors are shown. N.S., not significant.

 
MMP-9, LTB4, PAF, and TNF-{alpha} Are Involved in the Augmentation of Eosinophil TBM by IL-8–Stimulated Neutrophils
The results shown the above sections suggest that neutrophils act as regulators to affect TBM of eosinophils. Suppression of neutrophil-derived protease may lead to the suppression of TBM of eosinophils. To test this hypothesis, we inhibited MMPs, which work when cells digest the basement membrane during intrusion. An MMP-9 inhibitor (10 µM) could inhibit the augmented TBM of eosinophils in the presence of IL-8–activated neutrophils (Figure 3A). Similar results were observed when GM1489, an inhibitor of MMPs, was tested (n = 5, data not shown).


Figure 3
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  		Figure 3. Effects of MMP-9 inhibitor (10 µM, A); BIIL260, an LTB4-antagonist (1 µM, B); WEB2086, a PAF-antagonist (10 µM, C); and an anti–TNF-{alpha} antibody (clone Mab11, isotype mouse IgG1, 3 µg/ml; D) on the TBM of eosinophils in the presence of neutrophils stimulated by IL-8. The means ± SEM of five to seven experiments using cells from different donors are shown.

 
Activated neutrophils may secrete stimulatory molecules for eosinophils and thus augment their TBM as observed above. LTB4, PAF, and TNF-{alpha} are the representative molecules secreted by neutrophils and are capable of activating eosinophils. To investigate whether they are involved, we inhibited the activity of these molecules and observed whether the TBM of eosinophils was suppressed. Reagents used were BIIL260, an LTB4 receptor antagonist; WEB2086, a PAF receptor antagonist; and an anti–TNF-{alpha} monoclonal antibody (anti–TNF-{alpha} mAb, clone Mab11, mouse IgG1, 3 µg/ml). All of these reagents partially suppressed the TBM of eosinophils in the presence of IL-8–activated neutrophils but showed no effect in the absence (Figures 3B–3D). Similar results were obtained in the studies using AA861, a 5-LO inhibitor that suppresses the production of LTB4, and WEB2070, another PAF receptor antagonist (data not shown). These results indicate that the augmentation of eosinophil TBM due to IL-8–stimulated neutrophils is partly mediated by LTB4, PAF, and TNF-{alpha}. The effects of these inhibitors on TBM of neutrophils alone or eosinophils alone were also examined: IL-8 (10 nM)–induced TBM of neutrophils was not modified by inhibitors for MMP9, LTB4, PAF, or TNF-{alpha} (n = 6, P = n.s., data not shown). Similarly, eotaxin (3 nM)-induced TBM of eosinophils was not modified by inhibitors for MMP9, LTB4, or PAF (n = 6, P = n.s., data not shown). Anti–TNF-{alpha} mAb slightly but significantly reduced eotaxin-induced TBM of eosinophils (migrated eosinophils: 48.1 ± 3.5 by isotype mouse IgG1 versus 39.3 ± 2.4 by eotaxin alone, n = 6, P = 0.02), suggesting that TNF-{alpha} may act as an autocrine activator and partly contribute to the TBM of eosinophils in this system. Finally, pretreatment of only neutrophils, but not eosinophils, with the various inhibitors did not modify the subsequent TBM of eosinophils in the presence of IL-8–activated neutrophils (n = 3, P = n.s., data not shown).

The Conditioned Medium from IL-8-Stimulated Neutrophils Induces the TBM of Eosinophils
To further examine whether the neutrophils transmigrated to IL-8 produce chemoattractants for eosinophils, neutrophils were added to the upper compartment of a chamber with a Matrigel-coated transwell insert and either the control medium or IL-8 (10 nM) was added to the bottom compartment. After a 2-h incubation in 5% CO2 at 37°C, the bottom compartments were centrifuged at 4°C for 20 min at 700 x g. The supernatants were gently recovered and then examined for their ability to induce the TBM of eosinophils. The conditioned medium from a combination of IL-8 and neutrophils significantly induced the TBM of eosinophils as compared with the control medium or the condition medium from neutrophils in the absence of IL-8 (migrated eosinophils: 7.0 ± 0.6%, P < 0.05 versus the other two conditions, n = 5).

Anti-CXCR2 Antibody Does Not Modify the Augmentation of Eosinophil TBM by IL-8–Stimulated Neutrophils
The augmented TBM of eosinophils observed with IL-8–stimulated neutrophils may be a consequence of modification of the CXCR2, an IL-8 ligand, which is expressed on activated eosinophils (24). Although addition of the anti-CXCR2 antibody partly attenuated the TBM of neutrophils in response to IL-8 (% inhibition: 53.3 ± 8.6%, n = 5, P < 0.01 versus isotype control), the augmented TBM of eosinophils in the presence of IL-8 and neutrophils was not significantly modified by this antibody (n = 5, P = n.s., data not shown). An anti-CXCR1 antibody provided similar results (n = 2, data not shown).


   DISCUSSION
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
References
 
We showed that neutrophils stimulated with chemoattractant such as IL-8 augment the TBM of eosinophils. The reaction kinetics of the augmented TBM of eosinophils traced that of neutrophils. The augmented TBM of eosinophils by a combination of neutrophils and IL-8 was inhibited by an MMP-9 inhibitor, an LTB4 receptor antagonist, PAF-antagonists, or an anti–TNF-{alpha} mAb. LTB4, a chemotactic factor for both neutrophils and eosinophils (18, 25), by itself induced the TBM of eosinophils, and this TBM is augmented by the presence of neutrophils. There results provide a mechanism for our previous observation that a positive correlation between the concentrations of neutrophils and eosinophils in sputum from subjects with severe asthma (10), and suggest that neutrophils can regulate the accumulation of eosinophils through these mechanisms in the airways of asthma.

The mechanism by which neutrophils enhance the TBM of eosinophils is important. We showed that the inhibition of MMP-9 effectively suppresses the augmented TBM of eosinophils. There is evidence that IL-8 stimulates the release of MMP-9 from neutrophils (26). Moreover, the cellular source of MMP-9 may include eosinophils (27). The MMP-9 inhibitor, which is a selective inhibitor of MMP-9 (IC50 = 5 nM) (28), did not modify the TBM of neutrophils to IL-8 or eosinophils to eotaxin, suggesting that MMP-9 is not required for TBM induced by these ordinary and potent chemoattractants in these experimental conditions. Nonetheless, our results suggest that digestion of membrane by MMP-9 is involved in the mechanisms of augmented TBM of eosinophils induced by IL-8–stimulated neutrophils. The process of TBM of eosinophils would also require the presence of an activator that acts as either chemotactic or chemokinetic for eosinophils. Zuurbier and colleagues (29) reported that eosinophil migration across monolayers of lung epithelial cells in response to complement fragment 5a (C5a), but not to RANTES, PAF, or IL-8, was increased in the presence of neutrophils. They explained that neutrophils, but not eosinophils, rapidly inactivated C5a and decreased the activity of C5a that had diffused into the upper compartment, and thereby maintain a proper C5a chemotactic gradient in their trans-epithelial migration model. In contrast, our study suggested that neutrophils enhanced the TBM of eosinophils, at least in part, via the generation of activators for eosinophils: the inhibition of LTB4, PAF, or TNF-{alpha} actions partially suppressed TBM of eosinophils. In addition to LTB4, PAF is a chemotactic factor for both neutrophils and eosinophils (18, 24). The pharmacologic inhibitors may reduce migration of neutrophils and subsequently reduce migration of eosinophils, instead of acting to disrupt the effects of neutrophil-derived mediators on eosinophils. From this point of view, we observed that IL-8–induced TBM of neutrophils in this system was not modified by inhibitors for LTB4 or PAF. Furthermore, pretreatment of neutrophils with the various inhibitors did not modify the subsequent TBM of eosinophils in the presence of IL-8–activated neutrophils. TNF-{alpha} is not a chemotactic agent for neutrophils or eosinophils itself, but has been shown to be an activator for functions of both neutrophils and eosinophils such as adhesion or respiratory burst (25, 30). Our results that TNF-{alpha} is involved in the augmented TBM of eosinophils suggest that the activation of effector function(s) of either neutrophils or eosinophils may be sufficient to augment the TBM of eosinophils. We observed that anti–TNF-{alpha} mAb slightly but significantly reduced eotaxin-induced TBM of eosinophils, but not IL-8–induced TBM of neutrophils, suggesting some role of this cytokine as an autocrine activator for eosinophil migration in this system.

Increased concentrations of LTB4 (31, 32), PAF (33), and TNF-{alpha} (34) have been reported in the airways of asthma. Our results suggest that neutrophils are activated via a combination of IL-8 and basement membrane and generate these mediators. In this context, we found that the conditioned medium from a combination of IL-8 and neutrophils, but not neutrophils alone or medium alone, induced the TBM of eosinophils, indicating that the neutrophils transmigrated to IL-8 produce chemoattractant(s) that result in subsequent migration of eosinophils. Taken together, the augmented TBM of eosinophils is likely a complex of the effects of both MMP-9, on the basement membrane, and eosinophil activators and chemoattractants such as LTB4, PAF, and TNF-{alpha}.

Our assay simulates the initial phase of inflammation involving neutrophils and eosinophils, in which cells are stimulated and migrate out of blood vessels to accumulate at the inflammation site. In the later phase of inflammation, the expression of surface receptors of cells may be modified to become responsive to molecules that they were initially unresponsive. A good example is the expression of CXCR2, an IL-8 ligand, in activated eosinophils (24). The augmented TBM of eosinophils in the presence of IL-8 and neutrophils was not significantly modified by anti-CXCR2 antibody, ensuring in this assay that eosinophils are activated not directly by IL-8 but via the activation of neutrophils. However, this antibody partly attenuates the TBM of neutrophils to IL-8. Similar results were observed with an anti-CXCR1 antibody. These results suggest that migration of neutrophils is not a sole contributing factor for the augmented eosinophil migration. Not only MMP-9 or chemoattractants released from transmigrated neutrophils, but also the eosinophil activators released from neutrophils activated by IL-8 and integrin-mediated signaling before transmigration, may be important in the subsequent migration of eosinophils. We speculated that a complex consisting of this priming process, and release of MMP-9 and chemoattractants from migrated neutrophils, results in the eventual manifestation of the enhanced transmigration of eosinophils.

Neutrophils may actively participate in the development of airway disease of asthma. Our results suggest that neutrophils migrated to IL-8 may lead eosinophils to accumulate in the airways of asthma and possibly aggravate this disease. Therefore, therapies that suppress functions or accumulation of neutrophils may be effective for severe asthma. Further study will be warranted to elucidate the detailed roles of neutrophils in the pathophysiology of the disease.


   Acknowledgments
 
The authors are thankful to Professor Masumi Akita, Ms. Noriko Murai, Assistant professor Yasushi Sakamoto, and Dr. Koji Tsuchiya for their cooperation in the studies. The authors also thank Ms. Akemi Yokote and Ms. Nozomi Nozaki for their excellent technical assistance.


   Footnotes
 
Originally Published in Press as DOI: 10.1165/rcmb.2005-0303OC on February 2, 2006

Conflict of Interest Statement: None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

Received in original form August 4, 2005

Accepted in final form January 18, 2006


   References
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 

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