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Migration of Eosinophils Across Endothelial Cell Monolayers: Interactions Among IL-5, Endothelial-Activating Cytokines, and C-C Chemokines¹

Syed Shahabuddin^*, Paul Ponath and Robert P. Schleimer^2,*

^* Department of Medicine, Division of Clinical Immunology, Johns Hopkins Asthma and Allergy Center, Johns Hopkins University School of Medicine, Baltimore, MD 21224; and LeukoSite, Inc., Cambridge, MA 02142

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Eosinophils are the predominant cell type recruited in inflammatory reactions in response to allergen challenge. The mechanisms of selective eosinophil recruitment in allergic reactions are not fully elucidated. In this study, the ability of several C-C chemokines to induce transendothelial migration (TEM) of eosinophils in vitro was assessed. Eotaxin, eotaxin-2, monocyte chemotactic protein (MCP)-4, and RANTES induced eosinophil TEM across unstimulated human umbilical vein endothelial cells (HUVEC) in a concentration-dependent manner with the following rank order of potency: eotaxin eotaxin-2 > MCP-4 RANTES. The maximal response induced by eotaxin or eotaxin-2 exceeded that of RANTES or MCP-4. Preincubation of eosinophils with anti-CCR3 Ab (7B11) completely blocked eosinophil TEM induced by eotaxin, MCP-4, and RANTES. Activation of endothelial cells with IL-1ß or TNF- induced concentration-dependent migration of eosinophils, which was enhanced synergistically in the presence of eotaxin and RANTES. Anti-CCR3 also inhibited eotaxin-induced eosinophil TEM across TNF--stimulated HUVEC. The ability of eosinophil-active cytokines to potentiate eosinophil TEM was assessed by investigating eotaxin or RANTES-induced eosinophil TEM across resting and IL-1ß-stimulated HUVEC in the presence or absence of IL-5. The results showed synergy between IL-5 and the chemokines but not between IL-5 and the endothelial activator IL-1ß. Our data suggest that eotaxin, eotaxin-2, MCP-4, and RANTES induce eosinophil TEM via CCR3 with varied potency and efficacy. Activation of HUVEC by IL-1ß or TNF- or priming of eosinophils by IL-5 both promote CCR3-dependent migration of eosinophils from the vasculature in conjunction with CCR3-active chemokines.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Accumulation of eosinophils is observed in a variety of human diseases, including asthma and allergic rhinitis, and is considered to play a key role in pathogenesis of these conditions (1, 2). The mechanism of eosinophil migration into tissues from the vasculature has been studied by several laboratories (3, 4, 5). Eosinophil recruitment to sites of allergic inflammation is believed to be dependent on the local production of eosinophil-priming cytokines (e.g., IL-3, GM-CSF, and IL-5) (6, 7), endothelial-activating cytokines (e.g., IL-1ß, TNF-, IL-4, and IL-13) (8, 9, 10, 11), and C-C chemokines (e.g., eotaxin, eotaxin-2, RANTES, monocyte chemotactic protein-3 (MCP-3),³ and MCP-4) (12, 13, 14, 15, 16, 17). To study eosinophil recruitment, in vitro models of chemotaxis, adhesion, and transendothelial migration (TEM) have been developed (8, 9, 18, 19, 20, 21, 22). Studies using an in vitro model of TEM have established that eosinophil migration across endothelial cell monolayers is induced after activation of endothelial cells with IL-1ß, TNF-, or IL-4 (8, 9, 20, 21, 22). This process is disrupted by single Abs directed against ß2 integrins on the surface of the eosinophils or by a cocktail of Abs against several endothelial adhesion molecules (VCAM-1, ICAM-1, and E-selectin) (8). Eosinophil-activating cytokines, (e.g., IL-3, IL-5, and GM-CSF) also potentiate eosinophil TEM; eosinophils purified from the lungs of allergic volunteer subjects who underwent experimental Ag challenge display a similarly potentiated TEM response (9).

Numerous chemokines have been identified that have the ability to cause selective eosinophil migration, including RANTES, MCP-3, MCP-4, macrophage-inflammatory protein-1, eotaxin-1, eotaxin-2, and macrophage-derived chemokine (14, 15, 16, 17, 23, 24, 25, 26, 27, 28). Studies in humans and animals indicate that MCP-3, RANTES, and eotaxin are all present at significantly increased levels in either airways or skin of allergic subjects after allergen challenge (29, 30, 31). In vivo challenge models using RANTES or eotaxin have resulted in an eosinophil-rich inflammatory infiltrate in both animals (32) and humans (33, 34). Furthermore, the expression of these chemokines is associated with the accumulation of eosinophils at the sites of allergic inflammation (12, 35, 36, 37). The importance of several chemokines in eosinophil recruitment has been demonstrated by in vivo studies in which neutralizing Abs to chemokines or their receptors blocked eosinophil trafficking to sites of inflammation (38, 39, 40).

The integrated roles of cytokines and chemokines during the process of eosinophil TEM remain unclear. Several studies with neutrophils indicate that there is often a synergism between a leukocyte-activating cytokine, e.g., GM-CSF, and a chemoattractant such as f-Met-Leu-Phe (41, 42). Recent reports have suggested a cooperation between IL-5 and eotaxin during eosinophil accumulation in animal models, which is reminiscent of what we observed in vitro in the TEM assay (18, 43). Although endothelial-activating cytokines, eosinophil-activating cytokines, and C-C chemokines each have the ability to induce eosinophil migration across endothelium, the magnitude of the response is likely to depend on the combination of these stimuli. In the present study, we have investigated the roles played by the priming cytokine IL-5, the endothelial activators IL-1ß or TNF-, and several C-C chemokines, independently and in concert. Using the in vitro TEM model, we demonstrate that eotaxin, eotaxin-2, MCP-4, and RANTES induce eosinophil TEM. Activation of endothelial cells by cytokines such as IL-1ß or TNF- or priming of eosinophils by IL-5 both promote CCR3-dependent migration of eosinophils across an endothelial monolayer in conjunction with these chemokines.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Monoclonal Abs

Anti-human CCR3 mAb (7B11; IgG2a), which selectively blocks CCR3 (44), and anti-human CCR1 mAb (2D4; IgG1) were produced by LeukoSite (Cambridge, MA). F(ab')₂ preparation of mAb recognizing very late Ag (VLA)-4 (HP2/1; IgG1; Immunotech, Minneapolis, MN), Dß2 (240I; IgG1; ICOS, Bothell, WA), E-selectin (ENA 2; IgG1; Caltag Laboratories, Burlingame, CA), and platelet endothelial cell adhesion molecule (PECAM)-1 (CD31) (IP-66; IgG1; Centocor, Malvern, PA) were used as blocking Abs. A mAb against HLA class I (W6/32; IgG2a; Becton Dickinson, Mountain View, CA; and 12D10; IgG1; Centocor, Malvern, PA) were used as a negative control.

Endothelial cell cultures

Human umbilical vein endothelial cells (HUVEC) were isolated and cultured according to the methods described by Jaffe et al. (45) with some modifications (8). HUVEC (2 x 10⁴ cells) from the first or second passage were cultured on 2% gelatin-coated Transwell culture inserts (6.5-mm diameter polycarbonate membranes with 5-mm pores; Costar, Cambridge, MA) according to the method of Morzycki et al. (8, 46). Endothelial cell culture medium (0.2 ml M199; Life Technologies, Grand Island, NY) supplemented with 20% FCS, 2 mM L-glutamine, penicillin (100 U/ml), streptomycin (100 U/ml) (Life Technologies), endothelial cell growth supplement (20 µg/ml; Collaborative Research, Bedford, MA), and heparin sodium (90 µg/ml; Sigma, St. Louis, MO) was added to the Transwell inserts only. To confirm the confluency of the HUVEC on the membrane, sample monolayers were routinely stained with Diff-Quik (American Scientific Products, McGraw Park, IL) before use in experiments.

Purification of eosinophils

Human eosinophils were isolated from EDTA-anticoagulated venous blood of normal donors or patients with asymptomatic allergic rhinitis or asthma by percoll (1.090 g/ml) gradient centrifugation at room temperature. After centrifugation, all procedures were conducted at 4°C to prevent cell activation. RBCs were removed by hypotonic lysis before removal of CD16-positive cells (neutrophils) using immunomagnetic beads (Dynal, Oslo, Norway) (8, 47). Eosinophil purity (based on examination of Diff Quik-stained cytocentrifugation preparations) was 98 ± 1%, and viability (erythrosin B dye exclusion) was 99 ± 1% (n = 30). Eosinophils were labeled with ⁵¹Cr, washed extensively, and resuspended (2 x 10⁶/ml) in enriched medium (M199 medium supplemented with 20% FCS, antibiotics, and L-glutamine).

Transendothelial migration assay

The TEM assays were performed as previously reported with some modification (8). HUVEC monolayers grown in Transwell inserts were pretreated for 4 h with indicated concentrations of human rIL-1ß (R&D Systems, Minneapolis, MN), human rTNF- (R&D Systems), or culture medium (control) and then were rinsed twice with PAGCM buffer (PIPES buffer (25 mM PIPES, 110 mM NaCl, 5 mM KCl, pH 7.4) containing 0.003% human serum albumin, 0.1% D-glucose, and 1 mM each of MgCl₂ and CaCl₂ (Sigma). These treatments did not change the state of confluence, as determined by microscopic examination of Diff-Quik-stained filters. Enriched medium (0.6 ml) with or without eotaxin, eotaxin-2, MCP-4, or RANTES (R&D Systems) was added to the wells of a 24-well plate, and 0.1 ml of eosinophil suspension was added to each chamber before immersion of the Transwell chamber. In experiments to examine the effects of IL-5 on spontaneous or chemokine-induced eosinophil TEM across resting, IL-1ß, or TNF--stimulated HUVEC, ⁵¹Cr-labeled eosinophils were incubated with 5 ng/ml of IL-5 for 30 min at 37°C before being added along with IL-5 to the upper chamber. The effect of blocking Abs on TEM was examined by preincubating ⁵¹Cr-labeled eosinophils with 10 µg/ml anti-CCR3, 2 µg/ml anti-VLA-4, 220 µg/ml anti-Dß₂, or 10 µg/ml anti-HLA in enriched medium at 4°C for 30 min and then adding cells to the upper chamber along with the medium containing the Ab. To evaluate the effect of anti-PECAM-1 Ab on TEM, HUVEC were treated with anti-PECAM Ab (10 µg/ml), or 12D10 (10 µg/ml) at 37°C for 30 min before ⁵¹Cr-labeled eosinophils were placed in the upper chamber. In all experiments, TEM was allowed to proceed for 2 h, after which nonadherent cells in the upper chamber were removed. The undersurface of the filter was rinsed with 0.6 ml ice-cold PAG (PIPES buffer with 0.003% human serum albumin and 0.1% D-glucose) containing 5 mM EDTA to dislodge adherent transmigrated cells. The cells that had migrated and fallen into the lower chamber were combined with those detached from the filter and ⁵¹Cr radioactivity was determined. All determinations were conducted in triplicate. Migration was calculated as follows: [(cpm migrated)/(total cpm added)] x 100. Background eosinophil TEM was less than 5% in all experiments; this was subtracted from the total and the values are expressed as net TEM unless indicated otherwise.

Statistical analysis

The results are presented as mean ± SEM. The statistical significance of differences between groups was determined using Student’s (two-tailed) unpaired t test.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
The ability of several C-C chemokines to induce TEM of eosinophils was assessed. As shown in Fig. 1, the C-C chemokines eotaxin, eotaxin-2, MCP-4, and RANTES induced eosinophil TEM across resting HUVEC. The results showed a concentration-dependent increase in eosinophil migration with the following rank order of potency: eotaxin eotaxin-2 > MCP-4 RANTES (maximun TEM: 41.5 ± 0.9% for eotaxin at 100 ng/ml; 38 ± 3.6% for eotaxin-2 at 100 ng/ml; 21 ± 3.5% for MCP-4 at 500 ng/ml; and 20 ± 2.3% for RANTES at 500 ng/ml). In addition to the fact that RANTES and MCP-4 were less potent, they also appeared to produce a lower maximal response than the CCR3-specific chemokines eotaxin and eotaxin-2 did. The CXC chemokines IL-8 (100 ng/ml) and growth-related oncogene- (100 ng/ml) did not induce eosinophil TEM (data not shown). Similar experiments were performed in Transwell chambers without the presence of an endothelial monolayer. In these experiments, the potencies were similar to those in Fig. 1, but the maximal responses for the four chemokines were similar to each other (i.e., 20.0 ± 1.0 and 19.5 ± 0.5 for eotaxin and eotaxin-2 at 100 ng/ml, and 18.5 ± 3.5 and 16.5 ± 0.5 for RANTES and MCP-4 at 500 ng/ml; n = 2). These results indicate that the differences in maxima seen in Fig. 1 are dependent upon the presence of an endothelial monolayer.

View larger version (20K): [in this window] [in a new window]   FIGURE 1. Effect of C-C chemokines on eosinophil TEM. HUVEC were grown as confluent monolayers on polycarbonate Transwell inserts. Indicated C-C chemokines were placed in the lower chamber. 51Cr-labeled eosinophils were placed in the upper chamber and transendothelial migration of eosinophils was determined after a 2-h incubation at 37°C in a 5% CO2 incubator as described in Materials and Methods. Data shown are net TEM after subtracting baseline (<5%) and represent mean ± SEM from at least three experiments.

View larger version (19K): [in this window] [in a new window]   FIGURE 2. Transendothelial migration of eosinophils across IL-1ß- or TNF--activated HUVEC. Monolayers of HUVEC grown on Transwell inserts were stimulated for 4 h with either medium or indicated concentrations of IL-1ß or TNF- and then rinsed. 51Cr-labeled eosinophils were placed in the upper chamber and enriched medium 199 was added to the lower chamber. Eosinophil TEM was determined over 2 h and expressed as in Fig. 1 from at least five experiments. *, p < 0.05 vs control.

View larger version (13K): [in this window] [in a new window]   FIGURE 3. Induction of eosinophil TEM across TNF--stimulated HUVEC by chemokines. Endothelial cells were grown as a monolayer in Transwell inserts, stimulated for 4 h with medium or indicated concentrations of TNF-, and rinsed. 51Cr-labeled eosinophils were placed in the upper chamber, and enriched medium 199 (control), eotaxin (10 ng/ml), or RANTES (100 ng/ml) was added to the lower chamber. Eosinophil TEM was determined and expressed as in Fig. 1 from at least three experiments. *, p < 0.05 vs expected value (combined net values).

View larger version (25K): [in this window] [in a new window]   FIGURE 5. Effect of the anti-CCR3 Ab 7B11 on C-C chemokine-induced eosinophil transendothelial migration. Indicated C-C chemokines (100 ng/ml) were placed in the lower chamber. 51Cr-labeled eosinophils were preincubated with enriched medium, anti-CCR3 Ab (10 µg/ml), or anti-HLA Ab (negative control) at 4°C for 30 min and then were placed in the upper chamber of the TEM assay. Eosinophil TEM was determined as in Fig. 1. Data shown represent mean ± SEM from three experiments in which control migration was 4.5 ± 0.4%. *, p < 0.05 vs control.

View larger version (16K): [in this window] [in a new window]   FIGURE 6. Effect of the anti-CCR3 Ab 7B11 on eotaxin-induced eosinophil TEM across TNF--activated HUVEC. Endothelial cells were grown as a monolayer in Transwell inserts, stimulated for 4 h with either enriched medium (control) or 25 ng/ml of TNF-, and rinsed. 51Cr-labeled eosinophils were preincubated with medium, anti-CCR3 Ab (10 µg/ml), or anti-HLA Ab (negative control) at 4°C for 30 min and then were placed in the upper chamber. Medium control or eotaxin (10 ng/ml) was added to the lower chamber. Eosinophil TEM was determined as in Fig. 1. Data shown represent mean ± SEM from three experiments in which control migration was 3.7 ± 0.3%. *, p < 0.05 vs control.

View larger version (18K): [in this window] [in a new window]   FIGURE 7. Effect of the blocking mAb against VLA-4, Dß2, and PECAM-1 on eotaxin-induced eosinophil TEM across TNF--activated HUVEC. Endothelial cells were grown as a monolayer in Transwell inserts, stimulated for 4 h with either enriched medium (control) or 25 ng/ml of TNF-, and rinsed. A, 51Cr-labeled eosinophils were preincubated with medium, anti-VLA-4 Ab (2 µg/ml), anti-Dß2 (220 µg/ml), or anti-HLA Ab (negative control) at 4°C for 30 min and then were placed in the upper chamber. B, Medium or TNF--stimulated HUVEC was treated with medium, anti-PECAM Ab (10 µg/ml), or 12D10 (negative control) at 37°C for 30 min, and then 51Cr-labeled eosinophils were placed in the upper chamber. Medium control or eotaxin (10 ng/ml) was added to the lower chamber. Eosinophil TEM was determined as in Fig. 1. Data shown represent mean ± SEM from three experiments in which control migration was 4.0 ± 0.6%. *, p < 0.05 vs control.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

We observed that IL-1ß was significantly (100- to 1000-fold) more potent than TNF- in inducing TEM of eosinophils. Neither TNF- nor IL-1ß has been found to be directly chemotactic for eosinophils across endothelial barrier (56). However, previous studies have demonstrated increased eosinophil TEM across HUVEC activated with IL-1ß; this response is inhibited by Abs against ß1 and ß2 integrins or with a combination of anti-ICAM-1, anti-E-selectin, and anti-VCAM-1 (8, 19, 20). In addition to inducing adhesion molecules, these cytokines also induce the release of other cytokines, including GM-CSF and RANTES (57, 58, 59). GM-CSF is known to be chemokinetic for eosinophils, and previous studies have shown that 50% of the eosinophil TEM response to IL-1ß is inhibited by an Ab to GM-CSF, indicating that IL-1ß induction of GM-CSF production by HUVEC is involved in the response (9). The difference between IL-1ß and TNF- in inducing eosinophil TEM could be due to their potency in generating GM-CSF from HUVEC. IL-1ß is more potent than TNF- in this regard (60). However, TEM induced by TNF- or IL-5 was probably independent of endothelial-derived chemokines because the presence of anti-CCR3 or anti-CCR1 (data not shown) did not inhibit eosinophil TEM by any of these cytokines in the absence of added chemokine.

Synergism between IL-1ß or TNF- with RANTES or eotaxin in inducing eosinophil TEM is a distinctive finding of this study. Activation of endothelial cells by these proinflammatory cytokines results in induction of ICAM-1, VCAM-1, and E-selectin together with GM-CSF and RANTES, which could enhance eosinophil adhesion and transmigration in the presence of chemokine on the basal side of the monolayer. Abs directed to Dß₂ or VLA-4 had no effect on TEM induced by eotaxin alone or in the presence of TNF-, suggesting that their ligands such as VCAM-1 and ICAM-3 are not involved in the response. PECAM-1 has previously been shown to participate in TEM of monocytes (61). Interestingly, a specific Ab directed against PECAM-1 failed to inhibit eotaxin-induced TEM but completely inhibited the increase of TEM induced by TNF- when combined with eotaxin. This suggests that eotaxin-induced TEM is independent of PECAM-1 but that the synergistic response induced by TNF- with eotaxin requires participation of PECAM-1.

Preincubation of eosinophils with IL-5 significantly enhanced RANTES- and eotaxin-induced TEM across resting HUVEC, confirming and extending our previous findings (18). In addition, the results also indicate a clear synergy between IL-5 and eotaxin (Table I) and among IL-1ß, IL-5, and eotaxin. A similar synergy between IL-5 and eotaxin has been recently reported in tissue eosinophilia in animal models (39, 62). We observed a less than additive response using IL-1ß and IL-5. The lack of synergy between IL-1ß and IL-5 is not surprising in light of the fact that IL-1-activated HUVEC produce GM-CSF, which has an activity similar to that of IL-5 on eosinophils (see above). Taken together, these results suggest that maximal eosinophilia will occur in a tissue in which endothelial activators, eosinophil priming cytokines, and eosinophil-active chemokines are expressed simultaneously.

The findings in the present study have numerous implications for the mechanism of eosinophil recruitment as well as therapeutic strategies for disrupting this process. The observation of greater eosinophil TEM with selective CCR3 agonists (eotaxin and eotaxin-2) than nonselective agonists (RANTES and MCP-4) suggests that receptors other than CCR3 may dampen the response to RANTES or MCP-4 (e.g., CCR1 or CCR2). It is possible that receptors on endothelium can modify the response. In a separate study we found that HUVEC express CCR3, CCR4, CCR5, and CXCR4 (63, 64). Thus, differential responses to chemokines on the part of the endothelial cells may influence the outcome of the response. The potential role of these chemokine receptors either in endothelial responses or posssibly as chemokine presentation molecules is a subject currently under investigation. Our results may also be explained by difference in intrinsic activity of the chemokines involved. Recent studies by Farzan et al. (65) and Oppermann et al. (66) have shown a dissociation between activation of various signaling pathways (e.g., receptor binding affinity, calcium signals vs protein tyrosine kinase activation) among chemokines binding to the same receptor. Chemokine responses are now known to be influenced by glycosylation of the agonist as well as by the state of chemokine receptor tyrosine sulfation (65). The differential ability of chemokines to induce receptor activation, phosphorylation, desensitization, and internalization may provide yet another mechanism for the agonist-induced attenuation of chemokine receptor signaling (66). Further studies are needed to better understand the potency and efficacy differences between eotaxin/eotaxin-2 and RANTES/MCP-4 in inducing eosinophil TEM.

Huber et al. (67) found that IL-1ß-induced TEM of neutrophils is dependent on endothelial-derived IL-8. The failure of anti-CCR3 to inhibit eosinophil TEM induced by TNF- or IL-5 indicates that TNF-activated endothelial cells are not producing CCR3 agonists that are involved in the TEM response in vitro. Because there was a trend of anti-CCR3-dependent inhibition of eosinophil TEM across IL-1ß-treated HUVEC, the role of chemokines in the IL-1 response remains uncertain. These findings also imply that the clinical efficacy of CCR3 antagonists in the therapy of allergic inflammation may be limited in cases in which the inflammatory response is heavily driven by the production of IL-5 or endothelial activators, particularly TNF-. In such a case, our findings indicate that the TEM response is mediated independently of CCR3. However, on a more encouraging note is the finding that anti-CCR3 profoundly inhibited the response induced by the combination of a chemokine with an endothelial activator, IL-5, or both. Because this is likely to be the cytokine mixture found in vivo, clinical studies with CCR3 antagonists are clearly warranted.

View larger version (13K): [in this window] [in a new window]   FIGURE 4. Induction of eosinophil TEM across IL-1ß-stimulated HUVEC by chemokines. Endothelial cells were grown as a monolayer in Transwell inserts, stimulated for 4 h with medium or indicated concentrations of IL-1ß, and rinsed. 51Cr-labeled eosinophils were placed in the upper chamber, and enriched medium 199 (control), eotaxin (10 ng/ml), or RANTES (100 ng/ml) was added to the lower compartment. Eosinophil TEM was determined and expressed as in Fig. 1 from at least three experiments. *, p < 0.05 vs expected value (combined net values).

	Acknowledgments

	Footnotes

 
¹ This work was supported by Grants RO1 AR31891 and AI44885 from the National Institutes of Health.

² Address correspondence and reprint requests to Dr. Robert P. Schleimer, Johns Hopkins Asthma and Allergy Center, 5501 Hopkins Bayview Circle, Baltimore, MD 21224. E-mail address:

³ Abbreviations used in this paper: MCP, monocyte chemotactic protein; TEM, transendothelial migration; VLA, very late Ag; PECAM, platelet endothelial cell adhesion molecule; HUVEC, human umbilical vein endothelial cells; CXCR, CXC chemokine receptor.

Received for publication August 24, 1999. Accepted for publication January 31, 2000.

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