Selective eosinophil transendothelial migration triggered by eotaxin via modulation of Mac-1/ICAM-1 and VLA-4/VCAM-1 interactions

Gui-Quan Jia1,5, Jose-Angel Gonzalo1,5, Andres Hidalgo3, Denisa Wagner2, Myron Cybulsky4 and Jose C. Gutierrez-Ramos1,5

1 The Center for Blood Research, Inc. and
2 The Department of Genetics and Pathology, Harvard Medical School. Boston, MA 02115, USA
3 Centro de Investigaciones Biologicas, Madrid 28006, Spain
4 Toronto Hospital, 200 Elizabeth Street, CCRW 1–855, Toronto, Ontario, M5G 2C4, Canada
5 Millennium Pharmaceuticals, Inc. Cambridge, MA 02139, USA

Correspondence to: G.-Q. Jia, Millennium Pharmaceutical Inc., 640 Memorial Drive, Cambridge, MA 02139, USA


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
We have recently cloned eotaxin, a highly efficacious eosinophilic chemokine involved in the development of lung eosinophilia during allergic inflammatory reactions. To understand more precisely how eotaxin facilitates the specific migration of eosinophils, we have studied which adhesion receptors are essential for eotaxin action both in vivo and in vitro. Experiments using mice genetically deficient in adhesion receptors demonstrated that molecules previously reported to be involved in both leukocyte tethering/rolling (P-selectin and E-selectin) and in sticking/transmigration (ICAM-1 and VCAM-1) are required for eotaxin action in vivo. To further elucidate the mechanism(s) involved in this process, we have used an in vitro transendothelial chemotaxis model. mAb neutralization studies performed in this system suggest that the integrins Mac-1 (CD11b/18), VLA-4 ({alpha}4ß1) and LFA-1 (CD11a/18) are involved in the transendothelial chemotaxis of eosinophils to eotaxin. Accordingly, the expression of these integrins on eosinophils is elevated by direct action of this chemokine in a concentration-dependent manner. Taken together, our results suggest that eotaxin-induced eosinophil transendothelial migration in vivo and in vitro relies on Mac-1/ICAM-1 and VLA-4/VCAM-1 interactions, the latter ones becoming more relevant at later time points of the eotaxin-induced recruitment process.

Keywords: chemokine, eotaxin, eosinophil, integrin, transendothelial migration


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
Eosinophil accumulation is a prominent feature of allergic inflammatory disorders. However, eosinophils represent only a small percentage of circulating leukocytes, suggesting the existence of mechanisms for selective eosinophil recruitment in vivo. The mechanism(s) leading to the selective accumulation of eosinophils in allergic inflamed tissue are at present unclear. The recruitment of subpopulations of circulating leukocytes to the sites of inflammation has been proposed to be a complex process involving the coordinated action of various primary adhesion receptors that mediate tethering/rolling of leukocytes along the endothelial wall and of activation-dependent adhesion receptors that mediate sticking/transmigration across the vascular endothelium.

However, it is unlikely that the differential expression of adhesion receptors is sufficient to fully explain the selective recruitment of eosinophil into tissue during allergic inflammation. Eosinophils express L-selectin (1,2), ligands for P-selectin and E-selectin (2) as well as the integrins LFA-1 (CD11a/18) and Mac-1 (CD11b/18) which interact with ICAM-1. However, these adhesion molecules are also expressed on a wide variety of other leukocyte subset(s). It is likely that during the development of the inflammatory response, additional factors are required to regulate the selective accumulation of eosinophils.

Recently, chemokines have been suggested to play an important role in the selective transmigration of leukocyte subsets (3). Eotaxin is a chemokine which is able to mediate chemotaxis of peripheral blood eosinophils. This chemokine differs from most other chemokines because it is highly specific for eosinophils (4,5). Injection of eotaxin into guinea pig skin or into mouse peritoneum induces a profound selective eosinophilic infiltrate (4,5). The expression of eotaxin also parallels the accumulation of eosinophils in the lung during the course of a mouse model of lung allergic inflammation (5). The importance of eotaxin in the development of lung eosinophilia has recently been demonstrated using selective anti-eotaxin mAb or eotaxin gene knockout mice (6,7). In view of the critical role of this chemokine, the precise adhesive mechanism(s) by which eotaxin induces a selective eosinophil migration become an important issue.

In the present study, we demonstrate, using both adhesion molecule-deficient mice and an in vitro transendothelial chemotactic migration model, that eotaxin modulates the surface expression/function of adhesion molecules to achieve selective eosinophil transendothelial migration. Mac-1/ICAM-1 interactions are dominant at early time points during eotaxin-induced eosinophil recruitment, whereas VLA-4/VCAM-1 become predominant at later time points.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
Reagents and mice
The mAb used for detection of surface adhesion molecules were purchased from PharMingen (San Diego, CA). For the blocking experiments, the following mAb were used: rat anti-LFA-1 mAb M17/4 (IgG2a) (8,9), rat anti-Mac-1 mAb M1/70 (IgG2b) (10) and anti-VLA-4 mAb PS/2 (IgG2b) (11). Adhesion molecule-deficient mice used in this study were: P-selectin mutant mice purchased from the Jackson laboratory (Bar Harbor, ME), L-selectin mutant mice provided by Dr M. H. Siegelman (12), and E-selectin mutant mice, ICAM-1 mutant mice (13), VCAM-1-hypomorphic mutant mice (14) and mice deficient for both P-selectin and ICAM-1 or P-selectin and E-selectin (15). Since VCAM-1-null mutant mice are embryonic lethal (16), VCAM-1-hypomorphic mutant mice were used for these studies (14). These mutant mice were generated by a targeted deletion in domain 4 of the VCAM-1 molecule (which eliminates one {alpha}4 integrin binding site). Based on previous work, a very low affinity of this truncated protein to {alpha}4 can be predicted (17). The mutation resulted in 20-fold decrease of expression (hypomorphic ) of the domain 4-deficient (D4D) VCAM-1 gene, as found by Northern blot analysis and increased survival rates of one-third (not shown and (14)). Mice designated as wild-type in the results are littermates of these mutants that have a mixed genetic background 129svxC57BL/6J.

Endothelial cell culture
The mouse endothelial cell line b-End-2 cells (18) was cultured in IMDM with 10% FCS. Cells (2x 104) from well grown passage were seeded on transwell inserts (6.5 mm diameter polycarbonate membrane with 5 µm pores; Costar, Cambridge, MA) and cultured for a further 2 days. The media were added into the transwell inserts to prevent the formation of an endothelial bilayer (i.e. a monolayer both above and below the filter). After 2 days, the endothelial cell-coated wells were used for transendothelial migration assays described below.

Purification of eosinophils
For the in vitro studies, eosinophils were purified from the peritoneum of IL-5 transgenic mice as previously described (5). Eosinophil purity was 92 ± 2% based on Giemsa staining and viability was >95% based on Trypan blue exclusion for the experiments in this article except some experiments for Fig. 3Go, where unpurified population cells were used as input for the chemotaxis assay.



View larger version (56K):
[in this window]
[in a new window]
  		Fig. 3. Eotaxin-induced eosinophil transendothelial migration. Purified eosinophils were placed in the upper chamber of the assay with (I) or without (II) endothelial cells and recombinant mouse eotaxin was placed in the lower chamber at the concentration indicated. After 2 h incubation, the migration of eosinophils was analyzed as described in Methods. Data represent one of four experiments performed.

 
Adherence assay
The ability of eotaxin to induce adhesiveness on the integrin ligands was determined as follows. Isolated eosinophils from IL-5 transgenic mouse were labeled with BCECF-AM at room temperature for 30 min and then preincubated with or without neutralized antibodies to VLA-4, Mac-1 or LFA-1 (10 µg/ml) for 20 min at 4°C. After that, the cells were incubated with or without eotaxin (500 ng/ml) for varying times at 37°C and subsequently subjected to adherence assay in 96-well plates pre-coated with a recombinant fragment of fibronectin (5 µg/ml), containing the CS1 binding site specific for VLA-4 (positive control), VCAM-1 (1.25 µg/ml), ICAM-1 (2.5 µg/ml) or BSA (5 µg/ml) (negative control) for 30 min at 4°C to reduce background binding. After incubation, the cells were gently washed with PBS and read by fluorescence intensity.

In vitro transendothelial migration assay
The migration of eosinophils to recombinant eotaxin through the endothelial cell layer was evaluated in duplicate as previously described (5) with the membrane coated with endothelial cells described as above. The endothelial cells in transwell inserts were washed once with sera-free medium and 2x105 eosinophils in 0.1 ml sera-free medium were added. After 2 h incubation, the transwells were removed and the number of cells per well counted in the FACScan by passing each sample for a constant predetermined time period. Contaminating endothelial cells were gated out and a constant gate was assigned for the eosinophil population in the side/forward scatter window and used for every sample. In the blocking experiments, eosinophils were preincubated with anti-integrin antibodies or with control antibody at 37°C for 15 min before their addition to the transwell inserts.

Immunofluorescence and flow cytometry
Flow cytometry was performed to determine the expression of cell surface adhesion molecules in selected experiments. The migratory cells from the assay were incubated with anti-Fc-receptor mAb (2.492; PharMingen, San Diego, CA) and then stained with each one of the following antibodies individually: ICAM-1, Mac-1 (CD11b), LFA-1 (CD11a), VLA-4 (CD49d), VLA-5 or CD44, conjugated with FITC or phycoerythrin (PharMingen, San Diego, CA). Leukocyte cell populations were identified by forward scatter and side scatter gating. Dead cells were excluded by propidium iodide (Sigma, St Louis, MO) incorporation. Flow cytometry data were acquired with a FACScan cytometer (Becton Dickinson) and analyzed with CellQuest software. Fluorescence data were collected on a log scale and the level of expression was determined by the relative fluorescence intensity as the mean fluorescence channel for each individual surface adhesion molecule.

Intraperitoneal injection of eotaxin
The migration of eosinophils in vivo in response to eotaxin was determined by injection of recombinant murine eotaxin protein (1 µg/200 µl PBS per mouse; Peprotech, Rocky Hill, NJ) into the peritoneum of the different adhesion receptor-deficient mice. Mice injected with the same volume of PBS were used as controls. Peritoneal exudates were collected at different time points as indicated or for 2 h (10 mice per group) or 6 h (five mice per group) after eotaxin administration. The number of eosinophils recovered was then determined as described below.

Quantitation of eosinophils in peritoneal exudates.
Peritoneal cells (5x105/slide) were applied to glass slides by cytocentrifugation, stained with Wright–Giemsa (Fisher Diagnostics, Pittsburgh, PA), rinsed with distilled water, air dried and mounted. The percentage of eosinophils was determined by counting their number in eight high-power fields (x40 magnification; total area 0.5 mm2) per area randomly selected and dividing this number by the total number of cells per high power field. To obtain the absolute number of infiltrating peritoneal eosinophils, these percentages were multiplied by the total number of cells recovered from the exudate of this organ.


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
The accumulation of eosinophils in response to eotaxin is affected in mutant mice lacking adhesion molecules
To characterize the peritoneal accumulation of eosinophils in response to eotaxin, eosinophils were quantified in the peritoneal exudates of mice 0, 1, 2, 3 or 4 h after injection with different doses of eotaxin (Fig. 1Go). Eotaxin-induced eosinophil accumulation happened in both a dose- and time-dependent manner with maximum peritoneal eosinophilia occurring at 2 h for all doses (Fig. 1Go). At this time point 3, 1.5 and 0.5 µg/mouse eotaxin induced a 22-, 13- and 4-fold increase respectively in eosinophils compared to PBS-treated controls (Fig. 1Go).



View larger version (20K):
[in this window]
[in a new window]
  		Fig. 1. Eotaxin-induced peritoneal eosinophilia. Peritoneal exudate was collected at the time points indicated following injection of eotaxin (closed symbols) or PBS (empty symbols). Three different doses of eotaxin were administered: 3 µg/mouse (squares), 1.5 µg/mouse (circles) or 0.5 µg/mouse (triangles). The number of eosinophils in the peritoneal exudate was determined as described in Methods. The error bars represent differences between the values obtained from five mice per time point.

 
To study which adhesion molecules are essential during the selective accumulation of eosinophils mediated by eotaxin in vivo, we used a panel of mice that had been made genetically deficient in adhesion molecules. The deficient mice were injected i.p. with 1 µg/mouse of recombinant murine eotaxin. The eosinophil accumulation detected in the peritoneal cavity 2 h after eotaxin injection of wild-type mice was ~1.5x105 eosinophils and was considered 100%. The percentage of eosinophil accumulation in response to eotaxin in the different mutants are always referred to this 100% in the wild-type strain. At the same time point, similar numbers of eosinophils to the wild-type strain were found in the peritoneum of L-selectin-deficient and E-selectin-deficient mice in response to eotaxin (Fig. 2Go). In contrast, eosinophil accumulation was markedly reduced (29.7 ± 4.5% of wild-type) in P-selection-deficient mice 2 h after eotaxin administration when compared with wild-type mice (Fig. 2Go). Injection of eotaxin in mice that were double mutants for P-selectin + ICAM-1 and P-selectin + E-selectin resulted in 14.0 ± 2.1 and 26.9 ± 7.8% of the peritoneal eosinophil accumulation observed in wild-type mice respectively (Fig. 2Go). To evaluate whether leak of the expression of these adhesion receptors results in a delay in the eosinophil migration process to the peritoneum rather than impairment, the numbers of eosinophils were also determined in the peritoneum of these mutant mice 6 h after eotaxin administration. No differences were observed in peritoneal eosinophil recruitment between L-selectin-deficient mice and wild-type littermates 6 h after injection with eotaxin (Fig. 2Go). Interestingly, P-selectin-deficient mice that showed a significant reduction in peritoneal eosinophilia 2 h after eotaxin administration were comparable to wild-type mice 6 h after eotaxin injection (Fig. 2Go). This indicates that eotaxin-induced eosinophil accumulation in the peritoneum is only initially delayed in the P-selectin-deficient mice and that alternative mechanisms exist for eosinophil recruitment in the absence of P-selectin. Our data favors an important role for both ICAM-1 and P-selectin as the P-selectin + ICAM-1 double-deficient mice had a 21.1 ± 3.3% of the peritoneal accumulation of eosinophils observed in the wild-type mice at any time point analyzed (Fig. 2Go). A similar phenomenon occurs in the P-selectin + E-selectin double-deficient mice. Eotaxin-injected P-selectin + E-selectin double-deficient mice exhibited only 10.2 ± 2.4% of the eosinophil migration observed in wild-type mice 6 h after injection (Fig. 2Go). This correlates with the low range of eosinophil migration (25.7 ± 2.6%) detected in the peritoneum of E-selectin-deficient mice at the same time point after eotaxin injection (Fig. 2Go).



View larger version (46K):
[in this window]
[in a new window]
  		Fig. 2. Eotaxin-induced peritoneal recruitment of eosinophils in different adhesion receptor-deficient mice. The total number of peritoneal eosinophils (circles) and the percentage of eosinophil migration to this organ (gray bars) were evaluated 2 and 6 h after the administration of eotaxin (1 µg/mouse, i.p.). Each circle represents a single mouse. Eotaxin-treated wild-type or test mice are represented by closed circles. PBS-treated wild-type and deficient mice are represented by open circles. Black short lines indicate the mean of each group. Errors bars indicate the SD of the average of the percentage of eosinophil migration in the different groups of mice. Percentages of migration shown here are calculated as a percentage of total eosinophils detected in the peritoneum of wild-type mice 2 h after eotaxin injection which was considered to be 100%. The genotype of wild-type, single or double adhesion receptor-deficient mice is represented by black squares in the matrix. Differences between groups were tested for significance by the Mann–Whitney Wilcoxon test using Xlstat in Microsoft Excel 5.0.

 
ICAM-1-deficient mice and VCAM-1-deficient mice exhibited 62.7 ± 6.1 and 75 ± 2.7% respectively of the peritoneal eosinophil accumulation observed in wild-type littermates 2 h after eotaxin injection (Fig. 2Go). More interestingly, similar numbers of eosinophils were found in the peritoneum of both ICAM-1-deficient mice and wild-type littermates 6 h after eotaxin administration (Fig. 2Go). In contrast, peritoneal eosinophilia was reduced by half in the VCAM-1-mutant littermates when compared with that observed in wild-type controls 6 h after eotaxin injection (Fig. 2Go). This suggests that these two adhesion molecules could exert a differential contribution to this process; VCAM-1 being more critical at late stages (6 h) in the response to eotaxin.

Eotaxin induces selective eosinophil transendothelial migration in vitro
Transendothelial chemotaxis in vitro has been established as an in vitro model of leukocyte migration across postcapillary venules (19,20). This assay allows quantitative evaluation of the mechanisms by which eotaxin induces selective eosinophil migration, as well as to study its effects on adhesion molecules both on eosinophils and endothelial cells (19,20). Recombinant mouse eotaxin was able to induce significant eosinophil transendothelial migration in vitro, although the actual percentage of eosinophils that migrated to eotaxin was lower in the assay with an endothelial cell layer (Fig. 3, IGo) than in that performed without endothelial cells (Fig. 3, IIGo). The transendothelial migration of eosinophils responding to eotaxin occurred in a dose-dependent manner. Almost no eosinophil transendothelial migration was found in the absence of eotaxin (Fig. 3Go). Eotaxin-induced transmigration was selective for eosinophils since in the migrated cell population almost 100% were eosinophils [which increased from 70% eosinophils in the input of an unpurified cell population; data not shown and (5)]. These data together with our previously reported results showing that in vivo eotaxin induced the recruitment of eosinophils to the peritoneum (Fig. 1Go) (5) demonstrate that eotaxin alone can selectively induce migration of eosinophils across endothelial beds in vivo and in vitro.

Integrins are required for the transmigration of eosinophils in vitro
Based on our results in vivo, we studied if the integrin ligands for ICAM-1 and VCAM-1, Mac-1, LFA-1 and VLA-4 participated in the transendothelial migration of eosinophils induced by eotaxin. Firstly, we determined if there is any effect of eotaxin on the functional activity of the integrins. We used the adherence assay as a tool to examine eotaxin action because adherence of eosinophils is an initial step for its transmigration. In these experiments, we found that eotaxin was able to increase the eosinophil adherence to ICAM-1 and VCAM-1 (Fig. 4Go). This specific binding was abrogated by specific antibodies to CD11b or CD49d, but not by control antibodies. The eotaxin-induced eosinophil adherence to ICAM-1 is mostly attributable to CD11b (Mac-1) (Fig. 4Go).



View larger version (33K):
[in this window]
[in a new window]
  		Fig. 4. Eotaxin transiently increases VLA-4- and Mac-1-dependent binding of eosinophils to FN-H89, VCAM-1 and ICAM-1. Isolated eosinophils (purity > 90%) were preincubated with eotaxin (500 ng/ml) (bars) or without (circles) for varying times at 37°C and subsequently subjected to adhesion assays in 96-well plates pre-coated with a recombinant fragment of fibronectin (5 µg/ml), containing the CS1 binding site specific for VLA-4,, VCAM-1 (1.25 µg/ml), ICAM-1 (2.5 µg/ml) or BSA (5 µg/ml) for 30 min at 4°C to reduce background binding. Previously, cells were labeled with BCECF-AM and preincubated without or with antibodies to VLA-4, Mac-1 or LFA-1 (10 µg/ml) for 20 min at 4°C. Data are mean ± SEM of a representative experiment out of three, performed in triplicate.

 
Secondly, the contribution of the integrins during the transmigration was examined with the chemotaxis. Chemotaxis assays were performed in the presence of neutralizing antibodies against these three molecules. As shown in Fig. 5Go, mAb to CD11a (LFA-1), to CD11b (Mac-1) or to {alpha}4 (VLA-4) alone can significantly inhibit eosinophil transendothelial migration induced by eotaxin to different extents. Blockade of CD11b (Mac-1) had a more marked effect than the other two [CD11a (LFA-1) and {alpha}4 (VLA-4)] in inhibiting eosinophil transendothelial migration (up to 60–70% reduction). Blocking of LFA-1 in these experiments resulted in a much lower reduction than that after blocking of Mac-1 during eosinophil transendothelial migration (only 15% reduction; Fig. 5Go). These results demonstrated that eotaxin-induced eosinophil transendothelial migration relied more heavily on Mac-1 than on LFA-1 and VLA-4. ß1 and ß2 integrins belong to different integrin subfamilies and bind different ligands; however, the combination of neutralizing antibodies to these three molecules did not further significantly inhibit eosinophil transmigration (Fig. 5Go). It is still possible that both ß1 and ß2 integrins, as well as other molecules such as CD31, are needed for optimal migration of eosinophils in vivo.



View larger version (15K):
[in this window]
[in a new window]
  		Fig. 5. Effects of anti-Mac-1, anti-LFA-1 and anti-VLA-4 on eotaxin-induced eosinophil transendothelial migration. Assays were performed as described in the legend to Fig. 3Go with endothelial cells except eosinophils were preincubated for 15 min at 37°C with the indicated anti-integrin mAb at a concentration of 5 µg/ml. Values represent mean ± SEM from three independent experiments. The migration of eosinophils in response to eotaxin with control mAb was considered 100% and then used to normalize the experimental data. *P > 0.05 versus control antibody; **P > 0.01 versus control antibody.

 
The expression of integrins is increased on eosinophils that transmigrate to eotaxin across endothelial cells
The effect of eotaxin on the expression of adhesion molecules both on eosinophil and endothelial cells during the process of transendothelial migration was evaluated using immunofluorescence and flow cytometry (21). On eosinophils, among the adhesion molecules possibly involved in the transmigration process (Mac-1, LFA-1, VLA-4, VLA-5, CD44, CD31 and ICAM-1), only Mac-1, LFA-1 and VLA-4 were found to undergo significant changes in their surface expression levels on eotaxin-induced migratory eosinophils compared to the input cell population (Fig. 6Go and data not shown). All adhesion molecules mentioned above were expressed on eosinophils (data not shown). The expression of ICAM-1 had minor changes that were not statistically significant (Fig. 6Go). These results suggested that eotaxin could induce the up-regulation of selected adhesion molecules on eosinophils during the transmigration process. Since we did not find two populations of eosinophil with different expression levels of integrins in the input population used for chemotaxis, it ruled out the possibility that only the eosinophils with higher expression levels of these integrins were selected to migrate to eotaxin through the endothelial cell layer during the transmigration process (data not shown). Thus, eosinophil transendothelial migration induced by eotaxin correlates with a selective modulation of Mac-1, LFA-1 and VLA-4 expression. The elevated levels of expression on the migratory eosinophils followed the order Mac-1 > LFA-1 > VLA-4 as indicated by the mean fluorescence channel values. On endothelial cells, using the b-End2 brain endothelial cell line as a model system, ICAM-1 and ICAM-2 were found to be expressed at a high level, and VCAM-1 was expressed at a low level, but no significant changes in their expression occurred after stimulation with eotaxin (data not shown).



View larger version (22K):
[in this window]
[in a new window]
  		Fig. 6. Expression of adhesion molecules is increased on the migratory eosinophils. In each of the above transendothelial migration assays, input cells and migrated cells in the lower chamber were stained with antibodies against different adhesion molecules and analyzed by flow cytometry. The data was collected as described in Methods. The results of one experiment are shown from four experiments. The number on each peak indicates the relative mean channel number for comparison with the data in Fig. 7Go.

 
Different concentrations of eotaxin are required for the up-regulation of integrins on eosinophils
To determine whether the changes observed in the expression of the adhesion molecules mentioned above on eosinophils were the result of direct eotaxin action rather than events secondary to cell–cell interaction with endothelial cells (22), eotaxin was tested for its capacity to directly induce changes in the surface expression of these molecules. Stimulation of eosinophils directly with eotaxin in the absence of endothelial cells resulted in statistically significant up-regulation of the expression of Mac-1, LFA-1 and VLA-4, but not ICAM-1 on eosinophils (Fig. 7AGo). This expression up-regulation induced by eotaxin was dose dependent in the range of 2–500 ng/ml (after 1 h of incubation; Fig. 7AGo). Since the eotaxin-mediated modulation of adhesion molecule expression on eosinophils was similar regardless of the presence or absence of endothelial cells, we can exclude the possibility that eotaxin-mediated up-regulation of adhesion molecules is a consequence resulting from their migration across endothelial cells (Fig. 3Go) (5,23). Eotaxin-induced Mac-1 expression occurred already at a low concentration of eotaxin, whereas the increase of LFA-1 expression required higher concentrations (Fig. 7AGo). In addition, the increase (~47% total increase of mean fluorescence channel MFC) of Mac-1 expression was higher than that (27% in total increase) of LFA-1 expression at any dose of eotaxin stimulation (Fig. 7AGo). Although eotaxin was shown to have an effect on expression of these molecules already at 2 ng/ml, significant chemotactic activity of eotaxin was only achieved from 10 to 500 ng/ml, suggesting that a certain level of adhesion molecule expression or engagement of some other system (e.g. cytoskeletal rearrangement) is essential for chemotactic migration.



View larger version (23K):
[in this window]
[in a new window]
  		Fig. 7. Dose-dependent enhancement of eosinophil surface expression of adhesion molecules (A) and the time course of induction of adhesion molecules (B) by eotaxin. (A) Eosinophils were incubated with eotaxin at different concentrations as indicated for 1 h and stained as described in Methods. Results are expressed as the mean of duplicates from one of three experiments and the bars indicate the range for the duplicates. (B) Eosinophils were incubated with (squares) or without (triangles) eotaxin at a concentration of 200 ng/ml for various time periods as indicated. Then, the experiments followed the same procedure as described in (A). Data is representative of three similar experiments with the range of duplicates indicated by bars.

 
Eotaxin induces a rapid up-regulation of ß2 integrin expression and a delayed up-regulation of VLA-4
Studies on the kinetics of expression up-regulation for the three adhesion molecules studied above (Mac-1, LFA-1 and VLA-4) on eosinophils were conducted using a fixed concentration of eotaxin (200 ng/ml; Fig. 7BGo). Small changes, if any, in the expression of all three molecules were observed after incubation with eotaxin at 37°C for 10 min. However, the expression of the two ß2 integrins was increased after 30 min of stimulation (Fig. 7BGo), reached a peak after 1 h of stimulation, declining 2 h later. In contrast, VLA-4 expression showed a steady increase up to 2 h after stimulation with eotaxin (Fig. 7BGo).


   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
We have been interested in the molecular and cellular mechanisms that result in the highly selective accumulation of eosinophils during allergic inflammatory reactions. The discovery, cloning and study of eotaxin has been a key step in the understanding of this process due to the specificity and efficacy of this CC chemokine on eosinophil migration (6,23–25). In addition, eotaxin has patterns of mRNA and protein expression during allergic inflammatory reactions that are well correlated with the accumulation of eosinophils in the target organ (6,26). Recent studies, using genetic-deficient mice or treatment with neutralizing mAb suppresses lung eosinophilc inflammation (6,7), suggest a pivotal role of this chemokine in this event.

The rationale for the work presented here is based on the assumption that eotaxin exerts its specific task of recruiting eosinophils to the inflammatory site by inducing the activation/up-regulation of certain combination(s) of adhesion receptor that allow(s) eosinophils to go across an endothelial barrier allowing them to migrate preferentially at inflammation sites. This rationale has its origin in a hypothesis proposed by others based mainly on elegant in vitro studies (27,28). Moreover, clinical studies have further supported this hypothesis in that eosinophils at inflammation sites express considerably elevated levels of CD11b, ICAM-1 and HLA, whereas non-recruited blood eosinophils do not express those molecules (22,29).

It has been our intention to answer as many questions as possible using in vivo models because we are well aware of the complexity of the leukocyte migration process and the difficulty of reproducing it entirely in vitro (28,30). The use of adhesion receptor-deficient mice has allowed us to examine molecules and cellular processes as they occur in vivo during acute inflammatory events. On the other hand, we have also taken advantage of informative in vitro transendothelial migration assays that allowed us to perform mechanistic experiments in a more controlled assay system (19,20).

Eosinophils express L-selectin as well as ligands for P-selectin and E-selectin (1,2). We have found that the absence of L-selectin does not affect eotaxin-induced eosinophil accumulation in the peritoneum, whereas in the absence of P-selectin eotaxin-induced eosinophils recruitment is delayed and suboptimal when compared to that observed in wild-type mice (Fig. 2Go). Although 2 h after eotaxin administration eosinophil recruitment was basically absent in P-selectin-deficient mice, 6 h after the administration of this chemokine there was a significant degree of eosinophil recruitment (Fig. 2Go), suggesting that other molecules compensated partially for the loss of P-selectin at later time points. Among the possible surface molecules, E-selectin and ICAM-1 seem to contribute to the delayed recruitment of eosinophils as both P-selectin/E-selectin or P-selectin/ICAM-1 double mutants examined 6 h after eotaxin administration exhibited a marked reduction in eotaxin-induced accumulation of eosinophil (Fig. 2Go). These molecules are likely to participate at different stages of the eosinophil extravasation process: P-selectin is dominant at early stages in the initial rolling process (tethering), whereas E-selectin participates later in eosinophil tethering/rolling. This is consistent with the observation that E-selectin is only expressed several hours after induction (31). In this regard, others have shown an anti-E-selectin mAb only effectively inhibited the accumulation of neutrophils in both the peritoneum and lung at time points later than 4 h after the induction of an inflammation (32). This correlates with the fact that accumulation of eosinophils in the peritoneum of E-selectin-deficient mice in response to eotaxin is more impaired at late time points (6 h) than at early time points (2 h) after eotaxin administration (Fig. 2Go). ICAM-1-mediated interactions also took part in reinforcing eosinophil interactions to the endothelium, since in the double-mutant P-selectin/ICAM-1 eosinophil accumulation was completely abrogated at any time point (Fig. 2Go). These observations support our previous data that ICAM-1 and VCAM-1 were required in vivo for the accumulation of eosinophils in the lung in a murine model of lung allergic inflammation (6).

However, the kinetics of eosinophil accumulation is different in these two mutants in that whereas in the ICAM-1-deficient mice injection of eotaxin results in impaired eosinophil accumulation in the peritoneum which is slightly more pronounced at early time points (non-significant difference), in the VCAM-1 mutant it is at later time points when the genetic deficiency results in a more pronounced impairment (reduction in eosinophil accumulation was ~25% of wild-type levels at 2 h and ~50% of wild-type levels at 6 h after eotaxin injection, statistical significance P < 0.05). Interestingly this observation correlates with the kinetics of eotaxin-induced up-regulation of expression for the ligands for these molecules. Thus, eotaxin-induced up-regulation of CD11 on eosinophils was maximal at 1 h, whereas that of VLA-4 reached a peak after 2 h (Fig. 7BGo). Since eosinophils used in this study were purified from IL-5 transgenic mice (33), it is possible that the expression of some adhesion molecules on eosinophils has been elevated to some degree as a consequence of the exposure to IL-5. We found that eotaxin can further elevate the expression level of adhesion molecules on IL-5 transgenic eosinophils, strongly suggesting that a high level of adhesion molecule expression on eosinophils might be required for the eotaxin-induced transmigration through endothelial cells, leading to selective recruitment.

The interpretation of these phenotypes does not allow us to determine if the impairment in eotaxin-induced peritoneal eosinophilia in mutant lacking these adhesion molecules reflects the participation of eotaxin in the rolling as well as in the sticking of eosinophils to the endothelial walls (34). Alternatively, activating events mediated by eotaxin could be involved solely in the sticking process and the phenotype observed in some mutant mice reflects the need of previous interactions (tethering, initial rolling) that are not eotaxin dependent for the proper action of eotaxin in the sticking step per se. Experiments are underway to determine further the specific participation of each one of the adhesion receptors studied here in eotaxin-mediated rolling and sticking of eosinophils.

These adhesion molecules are central for the action of chemokines other than eotaxin. Results from a parallel study have shown that the lack of, at least, L-selectin, P-selectin and/or ICAM-1 has a similar impact on the recruitment of eosinophils to the peritoneum in response to MIP-1{alpha} injection (data not shown)

We also examined the adhesion receptors that are essential during the eotaxin-induced eosinophil transendothelial migration in vitro. The blockade of specific adhesion receptors in the in vitro assay revealed which adhesion receptors are essential for this process. We found that (i) eotaxin alone is able to support transendothelial migration in vitro (Fig. 3Go), (ii) eotaxin up-regulates the expression and functional activity of the adhesion receptors indicated below on eosinophils directly rather than acting indirectly through endothelial cells (Figs 4 and 7AGoGo), and (iii) transendothelial migration of eosinophils to eotaxin is mainly CD11b (Mac-1) dependent, and to a lesser extent CD11a (LFA-1) (Figs 4 and 5GoGo) and VLA-4 (Fig. 5Go) dependent. The effect of eotaxin on eosinophils during the transmigration process started at the early step of increasing adherence (Fig. 4Go). The increase in the expression of integrins on eosinophils induced by eotaxin is still observed in the presence of an inhibitor of protein synthesis, cycloheximide (data not shown), but to a lesser extent, indicating that a certain amount of integrins increased on the eosinophil surface transported from cell granules. The eotaxin-induced eosinophil transendothelial migration correlated with an increase in the expression of CD11a and CD11b, and to a less extent of VLA-4, after eotaxin pretreatment in vitro (Fig. 7AGo) (21,35). This observation could be related to previous reports that showed that eosinophils infiltrating the bronchial submucosa of asthmatics with air flow limitation have strongly up-regulated expression of CD11a, CD11b and VLA-4 (36). We have previously reported that lung epithelial cells express high levels of eotaxin protein during the development of an allergic response (6,37). Thus, it is most likely that in vivo eosinophils are exposed to eotaxin and this could account for their up-regulation of adhesion molecule expression. In this study, the elevated levels of expression of these three adhesion receptors on eosinophils were dependent on the dose of eotaxin, notably that of CD11b (Fig. 7BGo). This is in contrast to RANTES, another CC chemokine that supports chemotaxis of eosinophils in vitro (38), but does not induce the up-regulation of CD11/CD18 (20), suggesting that the mechanism of RANTES-induced eosinophil migration is different from that of eotaxin-induced eosinophil migration.

In summary, we have dissected the adhesion receptors that are involved in the recruitment of eosinophils mediated by eotaxin in vivo and in vitro. Our work reveals that (i) eotaxin alone is able to induce eosinophil transendothelial migration in vivo and in vitro; and (ii) eotaxin function in vivo and in vitro relies mainly on Mac-1/ICAM-1 interactions and on VLA-4/VCAM-1 interactions at later time points of the recruitment process. These results imply that Mac-1 and LFA-1 might play an important role in eotaxin-induced eosinophil transmigration, whereas VLA-4 could play a more important role at later stages of this response.


   Acknowledgments
 
This work has been funded by NIH grants HL 148675-02, DK1543, DK33506 and HL94-10-B, and by CiCyT PB93-0317 and the Aplastic Anemia Foundation of America grants. G.-Q. J. was from Shanghai Medical University. J.-A. G. is a recipient of a postdoctoral fellowship from the Spanish Ministry for Science. J.-C. G.-R. is the Amy C. Potter fellow.


   Notes
 
Transmitting editor: C. Martinez-A

Received 29 May 1998, accepted 17 September 1998.


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

  1. Lewinsohn, D. M., Bargatze, R. F. and Butcher, E. C. 1987. Leukocyte–endothelial cell recognition: evidence of a common molecular mechanism shared by neutrophils, lymphocytes and other leukocytes. J. Immunol. 138:4313.[Abstract/Free Full Text]
  2. Resnick, M. B. and Weller, P. F. 1993. Mechanisms of eosinophil recruitment. Am. J. Resp. Cell Mol. Biol. 8:349.[ISI][Medline]
  3. Schall, T. 1991. Biology of the RANTES/SIS cytokine family. Cytokine 3:165.[ISI][Medline]
  4. Jose, P. J., Griffiths-Johnson, D. A., Collins, P. D., Walsh, D. T., Moqbel, R., Totty, N. F., Truong, O., Hsuan, J. J. and Williams, T. J. 1994. Eotaxin: a potent eosinophil chemoattractant cytokine detected in a guinea pig model of allergic airways inflammation. J. Exp. Med. 179:881.[Abstract]
  5. Gonzalo, J. A., Jia, G.-Q., Aguirre, V., Friend, D., Coyle, A. J., Jenkins, N. A., Lin, G. S., Katz, H., Litchman, A., Copeland, N., Kopf, M. and Gutierrez-Ramos, J. C. 1996. Mouse eotaxin expression parallels eosinophil accumulation during lung allergic inflammation but it is not restricted to a Th2-type response. Immunity 4:1.[ISI][Medline]
  6. Gonzalo, J. A., Lloyd, C. L., Kremer, L., Finger, E., Martinez-A, C., Siegelman, M. H., Cybulski, M. and Gutierrez-Ramos, J. C. 1996. Eosinophil recruitment to the lung in a murine model of allergic inflammation: the role of T cells, chemokines and endothelial adhesion receptors. J. Clin. Invest. 98(10):2332.[Abstract/Free Full Text]
  7. Rothenberg, M. E., MacLean, J. A., Pearlman, E., Luster, A. D. and Leder, P. 1997. Targeted disruption of the chemokine eotaxin partially reduces antigen-induced tissue eosinophilia. J. Exp. Med. 185, no. 4:785.[Abstract/Free Full Text]
  8. Springer, T. A., Davignon, D., Ho, M., Kurzinger, K., Martz, E. and Sanchez-madrid, F. 1982. LFA-1 and Lyt-2,3, molecules associated with T lymphocyte-mediated killing; and Mac-1, an LFA-1 homologue associated with complement receptor function. Immunol. Rev. 68:171.[ISI][Medline]
  9. Wuthrich, R. P. 1992. Monoclonal antibodies targeting murine LFA-1 induce LFA-1/ICAM-1-independent homotypic lymphocyte aggregation. Cell. Immunol. 144:22.[ISI][Medline]
  10. Beller, D. I., Springer, T. A. and Schreiber, R. D. 1982. Anti-Mac-1 selectively inhibits the mouse and human type three complement receptor. J. Exp. Med. 156:1000.[Abstract]
  11. Miyake, K., Weissman, I. L., Greenberger, J. S. and Kincade, P. W. 1991. Evidence for a role of the integrin VLA-4 in lympho-hemopoiesis. J. Exp. Med. 173:599.[Abstract]
  12. Catalina, M. D., Carroll, M. C., Arizpe, H., Takashima, A., Estess, P. and Siegelman, M. H. 1996. The route of antigen entry determines the requirement for L-selectin during immune responses. J. Exp. Med. 184:2341.[Abstract/Free Full Text]
  13. Xu, H., Gonzalo, J. A., St. Pierre, Y., Williams, I. R., Kupper, T. S., Cotran, R. S., Springer, T. A. and Gutierrez-Ramos, J.-C. 1994. Leukocytosis and resistance to septic shock in intercellular adhesion molecule 1-deficient mice. J. Exp. Med. 180:95.[Abstract]
  14. Friedried, C., Cybulsky, M. I. and Gutierrez-Ramos, J. C. 1996. Vascular cell adhesion molecule-1 expression by hematopoiesis-supporting stromal cells is not essential for lymphoid or myeloid differentiation in vivo or in vitro. Eur. J. Immunol. 26:2773.[ISI][Medline]
  15. Frenette, P. S., Mayadas, T. N., Rayburn, H., Hynes, R. O. and Wagner, D. D. 1996. Susceptibility to infection and altered hematopoiesis in mice deficient in both P- and E-selectins. Cell 84:563.[ISI][Medline]
  16. Gurtner, G. C., Davis, V., Li, H., McCoy, M. J., Sharpe, A. and Cybulsky, M. I. 1995. Targeted disruption of the murine VCAM1 gene: essential role of VCAM-1 in chorioallantoic fusion and placentation. Genes Dev. 9:1.[Abstract]
  17. Chuluyan, H. E., Osborn, L., Lobb, R. and C., I. A. 1995. Domains 1 and 4 of vascular cell adhesion molecule-1 (CD106) both support very late activation antigen-4 (CD49d/CD29)-dependent monocyte transendothelial migration. J. Immunol. 155:3135.[Abstract]
  18. Hahne, M., Jäger, U., Isenmann, S., Hallmann, R. and Vestweber, D. 1993. Five tumor necrosis factor-inducible cell adhesion mechanisms on the surface of mouse endothelioma cells mediate the binding of leukocytes. J. Cell Biol. 121:655.[Abstract]
  19. Moser, R., Schleiffenbaum, B., Groscurth, P. and Fehr, J. 1989. Interleukin 1 and tumor necrosis factor stimulate human vascular endothelial cells to promote transendothelial neutrophil passage. J. Clin. Invest. 83:444.[ISI][Medline]
  20. Ebisawa, M., Yamada, T., Bickel, C., Klunk, D. and Schleimer, R. P. 1994. Eosinophil transendothelial migration induced by cytokines. Effect of the chemokine RANTES1. J. Immunol. 153:2153.[Abstract/Free Full Text]
  21. Palecek, S. P., Loftus, J. C., Ginsberg, M. H., Lauffenburger, D. A. and Horwitz, A. F. 1997. Integrin-ligand binding properties govern cell migration speed through cell-substratum adhesiveness. Nature 385:537.[ISI][Medline]
  22. Walker, C., Rihs, S., Braun, R. K., Betz, S. and Bruijnzeel, P. L. B. 1993. Increased expression of CD11b and functional changes in eosinophils after migration across endothelial cell monolayers. J. Immunol. 150:4061.[Abstract/Free Full Text]
  23. Rothenberg, M. E., Luster, A. D. and Leder, P. 1995. Murine eotaxin: an eosinophil chemoattractant inducible in endothelial cells and in interleukin 4-induced tumor suppression. Proc. Natl Acad. Sci. USA 92:8960.[Abstract]
  24. Griffith-Johnson, D. A., Collins, P. D., Rossi, A. G., Jose, P. J. and Williams, T. J. 1993. The chemokine, eotaxin, activates guinea-pig eosinophils in vitro and causes their accumulation into the lung in vivo. Biochem. Biophys. Res. Commun. 197:1167.[ISI][Medline]
  25. Ponath, P. D., Qin, S., Post, T. W., Wang, J., Wu, L., Gerard, N. P., Newman, W., Gerard, C. and Mackay, C. R. 1996. Molecular cloning and characterization of a human eotaxin receptor expressed selectively on eosinophils. J. Exp. Med. 183:2437.[Abstract]
  26. Rothenberg, M. E., Ownbey, R., Mehlhop, P. D., Loiselle, P. M., van de Rijn, M., Bonventre, J. V., Oettgen, H. C., Leder, P. and Luster, A. D. 1996. Eotaxin triggers eosinophil-selective chemotaxis and calcium flux via a distinct receptor and induces pulmonary eosinophilia in the presence of interleukin 5 in mice. Mol. Med. 2:334.[ISI][Medline]
  27. Springer, T. A. 1994. Traffic signals for lymphocyte recirculation and leukocyte emigration: The multistep paradigm. Cell 76:301.[ISI][Medline]
  28. Butcher, E. C. and Picker, L. J. 1996. Lymphocyte homing and homeostasis. Science 272:60.[Abstract]
  29. Hansel, T. T., Braunstein, J. B., Walker, C., Blaser, K., Bruijnzeel, J. C., Virchow, J., J. C. and Virchow, S., C. 1991. Sputum eosinophils from asthmatics express ICAM-1 and HLA-DR. Clin. Exp. Immunol. 86:271.[ISI][Medline]
  30. Girard, J.-P. and Springer, T. A. 1995. High endothelial venules (HEVs): specialized endothelium for lymphocyte migration. Immunol. Today 16:449.[ISI][Medline]
  31. Lawrence, M. B. and Springer, T. A. 1993. Neutrophils roll on E-selectin. J. Immunol. 151:6338.[Abstract/Free Full Text]
  32. Mulligan, M. S., Varani, J., Dame, M. K., Lane, C. L., Smith, C. W., Anderson, D. C. and Ward, P. A. 1991. Role of endothelial–leukocyte adhesion molecule 1 (ELAM-1) in neutrophil-mediated lung injury in rats. J. Clin. Invest. 88:1396.[ISI][Medline]
  33. Tominaga, A., Takaki, S., Koyama, N., Katoh, S., Matsumoto, R., Migita, M., Hitoshi, Y., Hosoya, Y., Yamauchi, S., Kanai, y., Miyazaki, J., Usuku, G., Yamamura, K. and Takatsu, K. 1991. Transgenic mice expressing a B cell growth and differentiation factor gene (interleukin 5) develop eosinophilia and autoantibody production. J. Exp. Med. 173:429.[Abstract]
  34. Das, A. M., Flower, R. J. and Perretti, M. 1997. Eotaxin-Induced eosinophil migration in the peritoneal cavity of ovalbumin-sensitized mice. J. Immunol. 159:1466.[Abstract]
  35. Weber, C., Kitayama, J. and Springer, T. A. 1996. Differential regulation of ß1 and ß2 integrin avidity by chemoattractants in eosinophil. Proc. Natl Acad. Sci. USA 93:10939.[Abstract/Free Full Text]
  36. Ohkawara, Y., Yamauchi, K., Maruyama, N., Hoshi, H., Ohno, I., Shirato, K. and Ohtani, H. 1995. In situ expression of the cell adhesion molecules in bronchial tissues from asthmatics with air flow limitation: in vivo evidence of VCAM-1/VLA-4 interaction in selective eosinophil infiltration. Am. J. Respir. Cell Mol. Biol. 12:4.[Abstract]
  37. Ponath, P. D., Qin, S., Ringler, D. J., Clark-Lewis, I., Wang, J., Kassam, N., Smith, H., Shi, X., Gonzalo, J.-A., Newman, W., Gutierrez-Ramos, J.-C. and Mackay, C. R. 1996. Cloning of the human eosinophil chemoattractant, eotaxin. J. Clin. Invest. 97:604.[Abstract/Free Full Text]
  38. Rot, A., Krieger, M., Brunner, T., Bischoff, S. C., Schall, T. J. and Dahinden, C. A. 1992. RANTES and macrophage inflammatory protein 1 alpha induce the migration and activation of normal human eosinophil granulocytes. J. Exp. Med. 176:1489.[Abstract]