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(Journal of Leukocyte Biology. 2003;73:65-73.)
© 2003 by Society for Leukocyte Biology

P-selectin mediates IL-13-induced eosinophil transmigration but not eotaxin generation in vivo: a comparative study with IL-4-elicited responses

Karen Y. Larbi*, John P. Dangerfield*, Fiona J. Culley{dagger}, Diane Marshall*, Dorian O. Haskard*, Peter J. Jose{dagger}, Timothy J. Williams{dagger} and Sussan Nourshargh*

* BHF Cardiovascular Medicine Unit, National Heart & Lung Institute, Faculty of Medicine, Imperial College of Science, Technology and Medicine, Hammersmith Hospital, London, United Kingdom; and
{dagger} Leukocyte Biology Section, Division of Biomedical Sciences, Faculty of Medicine, Imperial College of Science, Technology and Medicine, London, United Kingdom

Correspondence: Dr. Karen Larbi, BHF Cardiovascular Medicine Unit, National Heart and Lung Institute, Faculty of Medicine, Imperial College, Hammersmith Hospital, DuCane Road, London W12 0NN, U.K. E-mail: k.larbi{at}ic.ac.uk


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The study investigated the role of P-selectin in the responses of eosinophil transmigration and eotaxin generation in vivo elicited by interleukin (IL)-13, as compared with IL-4. Two murine models of leukocyte transmigration were used, migration into cytokine-stimulated peritoneal cavities and through stimulated cremasteric venules, as observed by intravital microscopy. In mice lacking P-selectin, eosinophil infiltration elicited by the cytokines in the peritonitis model was totally inhibited. In the cremaster muscle, however, although spontaneous leukocyte-rolling flux and stimulated leukocyte firm adhesion were inhibited by ~97% and ~48%, respectively, stimulated transmigration was unaffected. However, IL-13-induced leukocyte transmigration was totally blocked in P-selectin-deficient mice treated with an anti-{alpha}4 integrin monoclonal antibody (mAb; PS/2). In comparison, treatment of wild-type mice with the anti-{alpha}4 integrin mAb resulted in only partial suppression of IL-13-induced leukocyte transmigration. Significant levels of eotaxin were detected in response to IL-13/IL-4 in both tissues in P-selectin-deficient animals. In conclusion, the regulatory role of P-selectin in leukocyte transmigration elicited by IL-13 appears to be tissue-specific, a phenomenon that is independent of the ability of the cytokine to stimulate eotaxin generation.

Key Words: cytokines • chemokines • adhesion molecules • eosinophils • in vivo animal models


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Interleukin (IL)-13 and IL-4 are products of activated CD4+ T lymphocytes of the T helper cell type 2 (Th2) subset [1 ] and are also released by a number of other inflammatory cell types. The local expression profile of the mRNA and/or protein of these cytokines in allergic conditions in patients [2 3 4 5 ], in conjunction with the profile of their biological properties, has strongly implicated IL-13 and IL-4 as key molecules in the development of allergic inflammatory disorders. In this context, the cytokines exhibit numerous shared, immunomodulatory properties such as regulation of immunoglobulin (Ig)E isotype-switching in B cells, up-regulation of major histocompatibility complex class II, and the low-affinity IgE receptor (Fc RII/CD23) expression on monocytes/macrophages as well as on B cells [6 , 7 ]. The overlap in biological functions of IL-13 and IL-4 is at least partly a result of the fact that the IL-4 receptor (R) {alpha}-chain forms an important functional, signaling component of the IL-4R and IL-13R complexes. Indeed, a truncated murine (m)IL-4 mutant protein that binds as an antagonist to the IL-4R {alpha}-chain can inhibit IL-4- and IL-13-elicited signal transduction (i.e., phosphorylation of signal transducer and activator of transcription 6) and IgE production [8 ], and neither cytokine can induce airway eosinophilia or airway hyper-reactivity in IL-4R {alpha}-chain-deficient mice [9 ].

In addition to their immunoregulatory roles, endogenously generated IL-13 and IL-4 have been implicated in leukocyte infiltration, in particular, accumulation of eosinophils into sites of allergic inflammation in humans and in animal models. For example, in nasal polyps, intranasal administration of glucocorticoids reduced the number of cells expressing mRNA for IL-13 and IL-4 and the number of infiltrating eosinophils [10 ]. In experimental animals, lung eosinophilia observed upon allergen challenge post-sensitization was significantly suppressed in IL-4-deficient mice [11 ], IL-4R {alpha}-chain-deficient mice [9 ], or wild-type mice treated with blockers of IL-4 and/or IL-13 [9, 12 13 14 ]. Furthermore, in a pulmonary granuloma model, induced with Schistosoma mansoni eggs, as lung eosinophil infiltration was suppressed in IL-13- or IL-4-deficient mice, the response was only totally abolished in mice deficient in both cytokines [15 ].

Delineating the mechanism by which IL-13 and IL-4 elicit a relatively selective eosinophil accumulation response has attracted much interest and is believed to be associated with the ability of these cytokines to stimulate the generation of eosinophil-specific chemoattractants and to induce the activation/expression of adhesion pathways that favor eosinophil adhesion to endothelial cells. For example, in vitro, IL-13 and IL-4 stimulate eotaxin (CCL11) mRNA and/or protein in cultured dermal fibroblasts [16 , 17 ] and cultured keratinocytes [18 ]. In vivo, intranasal administration of IL-13 or IL-4 stimulates eotaxin production in mouse lungs [19 ], and transgenic mice selectively expressing IL-13 or IL-4 in the lungs express high levels of locally generated eotaxin or eotaxin-2 mRNA—eotaxin-2 (CCL24) being a novel, eosinophil-specific chemokine [20 , 21 ]. Finally, cultured human endothelial cells stimulated with IL-13 or IL-4 exhibited the mRNA for a third member of the eotaxin family, a new eosinophil-specific CC chemokine termed eotaxin-3 (CCL26), as identified by differential display analysis [22 ].

IL-13 and IL-4 can also induce eosinophil migration through their ability to directly activate endothelial cells to express adhesion molecules such as vascular cell-adhesion molecule 1 (VCAM-1) [23 24 25 ] and P-selectin [26 , 27 ], molecules that have been associated with eosinophil/endothelial cell interactions in vitro [24 , 28 29 30 31 32 33 ] and in vivo [34 35 36 37 38 39 40 ].

The existence of limited reports on the profile of eosinophil migration in response to exogenously administered IL-13 led us to perform the present study in which the initial aim was to characterize the profiles of IL-13-induced eosinophil infiltration and eotaxin generation, in comparison with responses elicited by IL-4 in two distinct but commonly used murine in vivo models, namely inflamed peritoneal cavity and cremaster muscle. Furthermore, as there have been no in vivo investigations into the role of P-selectin and {alpha}4 integrins in eosinophil migration induced by IL-13, the study aimed to extend the observations of a previous investigation into the role of P-selectin in leukocyte migration in response to IL-4 [41 ] by comparing the responses elicited by IL-13 and IL-4 in P-selectin-deficient mice. Our findings demonstrate that although IL-13-induced eosinophil migration in the peritoneal cavity is entirely P-selectin-dependent, leukocyte transmigration through IL-13-stimulated mouse cremasteric venules is dependent on P-selectin and {alpha}4 integrins. In addition, our findings indicate that the genetic deletion of P-selectin does not have an inhibitory effect on IL-13- or IL-4-induced generation of the eosinophil-specific chemoattractant eotaxin.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals
Control C57BL/6 mice were purchased from Harlan-Olac (Bicester, Oxfordshire, UK), and P-selectin-deficient C57BL/6J-selp (tm1 bay) mice were obtained from Jackson Laboratories (Bar Harbor, ME).

Reagents
Recombinant (r)mIL-13 and rmIL-4 were purchased from R&D Systems Europe Ltd. (Abingdon, Oxfordshire, UK) and Serotec Ltd. (Kidlington, Oxfordshire, UK), respectively. Anti-{alpha}4 integrin monoclonal antibody (mAb) PS/2 (IgG2b) was obtained from Serotec Ltd., Ketamine (Ketalar) was from Parke Davis (Eastleigh, Hants, UK), and Xylazine (Rompun) was from Bayer PLC (St. Edmunds, Suffolk, UK). Sterile, pyrogen-free heparin sodium (1000 IU/ml) was purchased from CP Pharmaceuticals Ltd. (Wrexham, UK). Saponin, toluidine blue, and light green SF yellowish May-Grünwald/Giemsa were all purchased from Sigma Chemical Co. (Dorset, UK).

Quantification of leukocyte migration into the peritoneal cavity
Age-matched mice of >20 g were injected intraperitoneally (i.p.) with saline (1 ml, control group), IL-13 (0.3–1 µg/ml/cavity), or IL-4 (1 µg/ml/cavity). Post-injection (4 or 24 h), the animals were killed by asphyxiation with CO2, and the peritoneal cavity was opened via midline incision and lavaged with 3 ml modified saline solution [0.9% saline containing 0.25% bovine serum albumin (BSA) and 2 mM EDTA]. Total cell counts were determined following staining exudate samples with Kimura stain [42 ]. Differential cell analysis was determined in exudate smears prepared in a cytocentrifuge (Cytospin-3, Shandon, Cheshire, UK) and stained with May-Grünwald/Giemsa stains. For quantification, 500 cells per slide were counted, and the results were expressed as the number of total leukocytes, mononuclear cells, neutrophils, and eosinophils recovered from each cavity.

Intravital microscopy
Intravital microscopy on the mouse cremaster muscle was performed as described previously [43 ]. Briefly, male mice (>20 g) were injected intravenously (i.v.) with saline, anti-{alpha}4 integrin mAb (PS/2), or an isotype-matched control Ab (all at 3 mg/kg) 15 min before intrascrotal (i.s) administration of 400 µl saline (control mice), IL-13 (30–300 ng/animal), or IL-4 (300 ng/animal). The mice were anaesthetized by i.p. administration of ketamine (100 mg/kg) and xylazine (10 mg/kg; 4 or 24 h later) and were placed on a custom-built, heated (37°C) microscope stage where the surgical procedure was performed. Following incision of the scrotum, one testis was gently withdrawn to allow the cremaster muscle to be incised and was pinned out flat over the window in the microscope stage. The cremaster muscle was kept warm and moist by continuous application of warmed Tyrodes balanced salt solution.

Post-capillary cremasteric venules (20–40 µM in diameter) were viewed on an upright fixed-stage microscope (Axioskop FS, Carl Zeiss Ltd., Welwyn Garden City, Herts, UK), fitted with water-immersion objectives. Images were then captured using a color video camera (JVC, Model KY-F55BE) and stored by videocassette recorder (Model MD830E, Panasonic UK Ltd., Bracknell, Berks).

Rolling cells were defined as those cells moving slower than the flowing erythrocytes, and rolling flux was then quantified as the number of rolling cells moving past a fixed point on the venular wall per minute for 5 min. Firmly adherent leukocytes were defined as those cells that remained stationary for at least 30 s within a 100-µm segment of a venule. Transmigrated leukocytes were defined as those cells in the perivenular tissue adjacent to but remaining within a distance of 50 µm of a 100-µm vessel segment under study. In each animal, three to five vessel segments and three to four vessels were quantified, and averages were taken. At the end of the quantification period, the cremasteric tissues were excised and stored at -80°C for analysis of eotaxin activity at a later date or were fixed (4% paraformaldehyde) and stained with May-Grünwald/Giemsa for determining the subtype of emigrated leukocytes. With respect to the latter, differential leukocyte counts were performed by counting 100–200 cells per tissue, and the results were expressed as % of mononuclear cells, neutrophils, and eosinophils.

Quantification of eotaxin in peritoneal exudates and cremaster muscle
Eotaxin levels in peritoneal exudates or homogenized cremaster muscle tissues were quantified using an enzyme-linked immunosorbent assay (ELISA) system incorporating a commercially available antimurine eotaxin-matched antibody pair (R&D Systems Europe). Briefly, samples were prepared as follows: Peritoneal exudates were centrifuged at 2000 rpm for 10 min at 4°C, supernatants were collected and stored at -80°C for subsequent quantification of eotaxin, and dissected cremaster muscles were weighed and homogenized at 0.5% w/v in cold phosphate-buffered saline (PBS), using an ultratirrax homogenizer. The homogenates were then centrifuged in a microfuge at 13,000 rpm for 5 min, and supernatants were collected and stored at -80°C for subsequent quantification of eotaxin. For the ELISA, 96-well plates were coated with primary antibody overnight, washed, and blocked for 1 h with PBS containing 1% BSA. Samples and standards were diluted in PBS with 0.1% BSA and added to plates in duplicates and incubated overnight at 4°C. The plates were then washed, and the biotinylated detector antibody was added for 2 h. This was then followed by the addition of neutravidin-horseradish peroxidase conjugate for a further hour. At each stage, the plates were washed thoroughly and were finally developed for 30 min using K-Blue peroxidase substrate (Neogen Corp., Lexington, KY). The optical density was read at 450 nm, and sample concentrations were determined by reference to the standard curve. Levels of eotaxin in peritoneal exudates and cremaster tissues were expressed as fmol/ml exudate and fmol/mg cremaster tissue, respectively.

Statistical analysis
Results are expressed as the mean ± SEM for n animals. Data were analyzed by Student’s t-test and one-way ANOVA, followed by Neuman Keuls for comparison test. With all statistical tests, P < 0.05 was considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Characterization of leukocyte migration induced by IL-13 and IL-4 in the mouse peritoneal cavity
In this first series of experiments, the comparative effects of IL-13 and IL-4 in inducing leukocyte migration into the mouse peritoneal cavity were investigated. Using a 24-h in vivo test period, both cytokines when administered via the i.p. route (IL-13 at 0.3–1 µg/cavity; IL-4 at 1 µg/cavity) induced an increase in the % of infiltrating eosinophils as compared with the small level of responses detected in animals injected with i.p. saline (Fig. 1A ). Although differential cell counts demonstrated that the resident leukocytes in the peritoneal cavity were predominantly mononuclear leukocytes (>90%), cytokine-stimulated infiltrating leukocytes were all eosinophils. Hence, in contrast to the increased eosinophil infiltration detected in IL-13- or IL-4-stimulated cavities, no significant increase in mononuclear leukocyte (saline-treated animals, 95%, 1 µg/cavity; IL-13-treated animals, 94%, 1 µg/cavity; IL-4-treated animals, 93%) or neutrophil accumulation (saline-treated animals, 0%; IL-13/IL-4-treated animals, 1%) was noted following administration of either cytokine.



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  		Figure 1. IL-13- and IL-4-induced eosinophil accumulation into the mouse peritoneal cavity. (A) Saline (1 ml/cavity; open bars; n=6), IL-13 (0.3 or 1 µg/cavity in 1 ml; solid bars; n=5), or IL-4 (1 µg/cavity in 1 ml; cross-hatched bar; n=8) was injected into the mouse peritoneal cavity, and eosinophil migration was quantified 24 h later. Eosinophil accumulation is expressed as % eosinophils per cavity, and the results are presented as mean ± SEM. A significant difference from saline-treated levels is indicated by *P< 0.05 or **P< 0.01. (B) Mice were injected i.p. with saline (1 ml/cavity; open bars; at 4 h, n=6, and at 24 h, n=8), IL-13 (1 µg/cavity in 1 ml; solid bars; at 4 h, n=6, and at 24 h, n=5), or IL-4 (1 µg/cavity in 1 ml; cross-hatched bars; at 4 h, n=4, and at 24 h, n=8) and eosinophil migration into the peritoneal cavity quantified at different times post-injection. Results are expressed as % eosinophils per cavity and presented as mean ± SEM. A significant difference from saline-treated levels is indicated by **P < 0.01 or ***P < 0.001.

 
We next examined the time-course profiles of IL-13 and IL-4 in eliciting eosinophil migration into the mouse peritoneal cavity. For these experiments, both cytokines were injected via the i.p. route at a dose of 1 µg/cavity, and responses were quantified 4 h and 24 h later. As shown in Figure 1B , no significant eosinophil infiltration was noted into the peritoneal cavity 4 h post-administration of IL-13 or IL-4, but significant responses were observed 24 h post-injection of either cytokine.

Characterization of leukocyte responses in IL-13- and IL-4-stimulated mouse cremasteric venules
As described for the peritonitis model, dose-response profiles of IL-13 and IL-4 in inducing leukocyte responses in the mouse cremaster muscle, as observed by intravital microscopy, were investigated. Figure 2 shows that using a 24-h in vivo test period, i.s. administration of IL-13 (30–300 ng/mouse) induced a dose-dependent increase in leukocyte firm adhesion (Fig. 2A) and transmigration (Fig. 2B) as compared with animals injected with saline. Local injection of IL-4 (300 ng/mouse) induced comparable adhesion and transmigration responses to those observed with IL-13. In contrast, however, neither cytokine produced a significant effect on leukocyte-rolling flux (data not shown). Time-course studies demonstrated that IL-13 and IL-4 stimulated significant levels of leukocyte adhesion and transmigration at 4 and 24 h post-injection of the cytokines (data not shown).



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  		Figure 2. Leukocyte responses induced by IL-13 and IL-4 in mouse cremasteric venules. Mice were treated with i.s. saline (400 µl/cavity; open bars; n=4), IL-13 (30–300 ng/mouse; solid bars; n=4), or IL-4 (300 ng/mouse; hatched bars; n=5). Leukocyte responses were quantified at 24 h post-injection. The data represent mean ± SEM. A significant difference from saline-treated levels is indicated by **P< 0.01 and ***P < 0.001.

 
Role of P-selectin in IL-13-induced leukocyte migration in the mouse peritoneum and cremaster muscle
The profile of IL-13-elicited leukocyte migration in the peritoneal cavity and cremaster muscle of wild-type and P-selectin-deficient mice was investigated 24 h post-administration of the cytokine. Figure 3 shows that in contrast to responses obtained in wild-type mice, no significant increase in % eosinophil infiltration into the peritoneum of P-selectin-deficient animals was observed following i.p. administration of IL-13 (1 µg/cavity). Similar results were observed following i.p. administration of IL-4 (data not shown).



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  		Figure 3. IL-13-induced eosinophil migration into the peritoneum of wild-type (WT) and P-selectin-deficient (P-sel ko) mice. Mice were injected i.p. with saline (1 ml/cavity; open bars; n=5) or IL-13 (1 µg/cavity; solid bars; n=6), and 24 h later, eosinophil migration into the cavities was quantified. Results are expressed as % eosinophils, and the data are presented as mean ± SEM. A significant difference from saline-treated levels is indicated by **P < 0.01. In addition, significant differences between the two strains of mice are indicated by lines.

 
In the mouse cremaster muscle, leukocyte-rolling flux was almost totally inhibited (95%), IL-13-stimulated firm adhesion was partially suppressed (41%), and transmigration was unaltered in P-selectin-deficient mice as compared with wild-type controls (Fig. 4 ). Similar findings were obtained with IL-4 in P-selectin-deficient animals in that leukocyte-rolling flux was inhibited by 98%, IL-4-induced firm adhesion was suppressed by 55%, and IL-4-induced leukocyte transmigration was unaffected (data not shown). Of interest, contrary to the eosinophil-specific effects of IL-13 and IL-4 in the peritonitis model, differential staining of IL-13-stimulated cremaster muscles demonstrated that in wild-type animals, the transmigrated leukocytes were composed of eosinophils (19±4.95%), mononuclear leukocytes (80±4.85%), and neutrophils (0.99±0.21%). Of importance, no significant change in this profile of leukocyte transmigration was noted in the P-selectin-deficient mice (22.6±2.5% eosinophils, 75.1±3.5% mononuclear leukocytes, and 2.3±1.3% neutrophils).



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  		Figure 4. IL-13-stimulated leukocyte responses in cremasteric venules of wild-type (WT) and P-selectin-deficient (P-sel ko) animals. Mice were treated with i.s. saline (400 µl/cavity; open bars; wild-type/P-selectin-deficient, n=4) or IL-13 (300 ng/mouse; solid bars; wild type, n=4; P-selectin-deficient, n=3), and 24 h later, leukocyte responses were quantified. The data represent mean ± SEM. A significant difference from saline-treated levels is indicated by *P < 0.05, **P< 0.01, and ***P< 0.001. In addition, significant differences between the two strains of mice are indicated by lines.

 
Role of {alpha}4 integrins in IL-13-induced leukocyte migration through mouse cremasteric venules
As no defect in IL-13-induced leukocyte transmigration was observed in cremasteric venules of P-selectin-deficient mice, the following series of experiments was performed to investigate the potential role of {alpha}4 integrins in this response. For this purpose, the effect of the anti-mouse {alpha}4 integrin mAb PS/2 (3 mg/kg) on IL-13-induced leukocyte migration through cremasteric venules of wild-type and P-selectin-deficient mice was investigated. In agreement with the results presented in Figure 4 , in P-selectin-deficient mice, IL-13-induced leukocyte firm adhesion was partially suppressed, and transmigration was unaltered, as compared with the responses observed in wild-type animals (Fig. 5 ). Furthermore, although in wild-type mice, {alpha}4 integrins blockade partially suppressed IL-13-stimulated leukocyte firm adhesion and transmigration, pretreatment of P-selectin-deficient mice with PS/2 (but not a control antibody) resulted in total inhibition of these responses. It is interesting that in wild-type mice, basal leukocyte-rolling flux was partially suppressed in animals pretreated with PS/2. In P-selectin-deficient mice, leukocyte-rolling flux was inhibited by 98%, going down to 100% inhibition following pretreatment of mice with the anti-{alpha}4 integrin mAb.



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  		Figure 5. Effect of an anti-{alpha}4 mAb (PS/2) on leukocyte transmigration through IL-13-stimulated mouse cremasteric venules of wild-type (WT) and P-selectin-deficient (Psel KO) mice. Mice were treated with i.s. saline (open bars; n=3) or IL-13 (300 ng/animal; solid bars; n=3) 24 h before surgical preparation. In additional groups of mice, animals were pretreated with i.v. mAb PS/2 (anti-{alpha}4 integrin; hatched bars; n=4) or isotype-matched control mAb (horizontal-lined bars; n=3) at the dose of 3 mg/kg, 15 min before i.s. injection of IL-13. A significant difference from responses obtained from saline-treated animals is shown by *P < 0.05 and **P< 0.01. Additional statistical comparisons are indicated by lines.

 
Role of P-selectin in eotaxin generation
One explanation for the lack of eosinophil infiltration into IL-13-stimulated peritoneal cavities of P-selectin-deficient mice may be that eotaxin generation was reduced in these animals. To enable us to directly compare the profiles of cytokine-induced leukocyte migration with eotaxin generation, a similar time-course profile to that established for leukocyte migration was established for IL-13- and IL-4-stimulated eotaxin generation in models in wild-type and P-selectin-deficient animals (Fig. 6 ). In wild-type mice, significant levels of eotaxin were detected in peritoneal cavities (Fig. 6A) and cremaster muscles (Fig. 6B) 4 h post-administration of either cytokine, with responses declining to basal levels by 24 h. Similar profiles of responses were also detected in the P-selectin-deficient mice. Surprisingly, however, at the 4-h time-points, there was a trend toward a greater level of eotaxin generation in the P-selectin-deficient mice as compared with the wild-type mice in the peritonitis model, although this was significant only in IL-4-stimulated peritoneal exudate samples (P<0.001).



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  		Figure 6. IL-13- and IL-4-induced eotaxin generation in peritoneal exudates and cremaster muscles of wild-type (WT) and P-selectin-deficient (P-sel ko) mice. (A) Wild-type (solid bars) and P-selectin-deficient (open bars) mice were injected i.p. with saline (1 ml/cavity, at 4 or 24 h, n=8), IL-13 (1 µg/cavity, at 4 h, n=4, and at 24 h, n=6), or IL-4 (1 µg/cavity, at 4 or 24 h, n=6). At 4 or 24 h post-injection, the peritoneal cavity was lavaged, and levels of eotaxin in exudates were quantified using an ELISA. Results, presented as fmol/ml exudate, are mean ± SEM. A significant difference from saline-treated levels is indicated by **P< 0.01. (B) Wild-type (solid bars) and P-selectin-deficient (open bars) mice were injected i.s. with saline (400 µl/mouse, at 4 or 24 h, n=8), IL-13 (300 ng/mouse, at 4 h, WT, n=8, KO, n=3; and at 24 h, n=8), or IL-4 (300 ng/mouse, at 4 h, n=5; and at 24 h, n=6). At these time points following injection, the cremaster muscles were excised and homogenized, and eotaxin levels were quantified by ELISA. Results, presented as fmol/mg cremaster muscle tissue, are mean ± SEM. A significant difference from saline-treated levels is indicated by *P < 0.05 and **P < 0.01.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, we have compared and characterized the ability of the Th2 cytokines IL-13 and IL-4 in eliciting eotaxin generation and eosinophil accumulation in two murine models of leukocyte migration. In addition, in light of the growing interest in the role of P-selectin in eosinophil/endothelial cell interactions, the present study provides the first direct investigation into the role of P-selectin in responses induced by IL-13, in comparison with an investigation into the P-selectin dependency of IL-4-induced eotaxin generation and leukocyte migration. The results indicate that IL-13 and IL-4 are equipotent and exhibit very similar time-course profiles in inducing eotaxin generation and leukocyte migration in the mouse peritoneal cavity and cremaster muscle. Furthermore, experiments performed in P-selectin-deficient mice indicated a role for P-selectin in IL-13- and IL-4-elicited eosinophil migration into the peritoneal cavity but not the cremaster muscle; IL-13 induced leukocyte migration into the latter tissue involves P-selectin and {alpha}4 integrins. Collectively, these observations suggest the existence of tissue-specific requirements for P-selectin in regulation of eosinophil migration in vivo, as we have previously demonstrated for VCAM-1 [44 ]. Finally, despite the inhibition of eosinophil migration detected in the peritoneum of P-selectin-deficient mice, no reduction in eotaxin generation was observed, demonstrating that P-selectin does not have a critical role in stimulating the generation of the chemokine eotaxin.

This study provides the first direct comparison of the effects of IL-13 and IL-4 in inducing leukocyte migration into the mouse peritoneal cavity as compared with the cremaster muscle, two commonly investigated models of leukocyte infiltration. Intraperitoneal administration of IL-13 or IL-4 induced selective accumulations of eosinophils into the mouse peritoneal cavity. The cytokines appeared equipotent and exhibited a similar time-course profile. In the cremaster muscle, IL-13 and IL-4 induced increased levels of leukocyte firm adhesion and transmigration in a dose-dependent manner, and significant levels of leukocyte responses were detected at 4 and 24 h post local administration of the cytokines. Ex vivo analysis of IL-13-stimulated cremaster muscles by standard staining protocols demonstrated that ~20% of the infiltrating leukocytes were eosinophils, and the remainder were mononuclear leukocytes.

IL-13 and IL-4 can stimulate endothelial cells for increased expression of adhesion molecules VCAM-1 [23 24 25 ] and P-selectin [26 , 27 ], molecules that support eosinophil/endothelial cell interactions. In this context, although VCAM-1 has received much attention [24 , 28 , 29 , 34 35 36 37 , 45 ], increasing evidence has implicated P-selectin as a key adhesion molecule in eosinophil infiltration into sites of inflammation. Hence, in vitro eosinophils have been shown to adhere to purified and cell-associated P-selectin under static conditions [30, 33 ], and P-selectin has been shown to support eosinophil rolling under conditions of flow [27 , 31 , 46 , 47 ]. In vivo, lipopolysaccharide-induced eosinophil migration into the pleural cavity of mice was inhibited by an anti-P-selectin mAb by ~50% [38 ], and eosinophil recruitment into lungs of ovalbumin-challenged, P-selectin-deficient mice was inhibited by ~70% post-allergen challenge [39 ]. Furthermore, eosinophil rolling, firm adhesion, and transmigration were suppressed in an eosinophilic peritonitis model using ragweed allergen in P-selectin-deficient mice [40 ]. To extend the above observations and to specifically investigate the role of P-selectin in eosinophil migration induced by IL-13, in comparison to responses elicited by IL-4, responses induced by these cytokines in the peritonitis model and the cremaster muscle of P-selectin-deficient mice were quantified. In agreement with the findings of the above studies, eosinophil accumulation into the mouse peritoneal cavity was severely impaired in P-selectin-deficient mice following local administration of IL-13 or IL-4. This observation was most likely a result of an inhibition of eosinophil rolling in IL-13- or IL-4-stimulated venules of the peritoneal microcirculation. In direct contrast, in cytokine-stimulated mouse cremaster muscles, although leukocyte-rolling flux was almost totally inhibited, leukocyte firm adhesion was only partially suppressed, and transmigration was unaffected (confirmed for eosinophils by ex vivo staining of cremaster muscles). However, pretreatment of the P-selectin-deficient mice with an anti-{alpha}4 integrin mAb resulted in an almost total inhibition of IL-13-induced leukocyte firm adhesion and transmigration. These results demonstrate that in the cremaster muscle, IL-13/IL-4-induced leukocyte migration is mediated by P-selectin and {alpha}4 integrins, the latter presumably interacting with induced VCAM-1. The exact mechanism by which P-selectin glycoprotein ligand (PSGL)-1/P-selectin and {alpha}4 integrins/VCAM-1 adhesion pathways may interact to mediate IL-13/IL-4-elicited responses is at present unclear but may be associated with the combined expression profiles of P-selectin and VCAM-1 on IL-4/IL-13-stimulated endothelial cells and the potential requirement for PSGL-1 and {alpha}4 integrins in the establishment of stable leukocyte-adhesive interactions, prerequisites to leukocyte transmigration.

The results of the present study further indicate and support the existence of organ-specific, adhesive mechanisms regulating leukocyte infiltration into sites of inflammation; e.g., local antigen challenge has been found to induce expression of P-selectin but not E-selectin in mouse cremasteric venules, whereas the reverse was detected in venules of mouse skin [48 ]. In addition, we have previously reported that although endothelial cell VCAM-1 expression is elevated in the rat pleural membrane and skin following stimulation with IL-4, VCAM-1 played a functional role in eosinophil migration into rat skin, and its expression was dissociated from eosinophil recruitment into the pleural cavity [44 ]. Although in the present study, P-selectin was not found to be critical for IL-13-induced leukocyte transmigration in the cremaster muscle, it appeared to act in a cooperative manner with {alpha}4 integrins. Furthermore, there is evidence for a functional role for P-selectin in this tissue following induction of inflammation with other stimuli such as antigen [48 ], collectively suggesting that the relative contribution of P-selectin to eosinophil infiltration is stimulus- and tissue-specific. Other factors that can contribute to tissue-specific mechanisms of leukocyte infiltration may include the responsiveness, characteristics, and/or density of local inflammatory cells such as mast cells or the contribution of other barrier cells such as mesothelial cells, a cell type that could clearly act as a distinct barrier to transmigrating leukocytes in the peritonitis model [49 , 50 ].

As well as directly supporting cell migration, there is currently much interest in the potential role of adhesion molecules as regulators of chemokine generation [51 ], a mechanism that may have contributed to the observed inhibition of eosinophil migration in the peritoneal cavity of P-selectin-deficient mice. To address the role of P-selectin in eotaxin generation in vivo, eotaxin levels were quantified in peritoneal exudates and for comparison, in cremaster muscle tissues of wild-type and P-selectin-deficient mice following local administration of IL-13 or IL-4. In all samples analyzed from the genetically modified mice, no significant inhibition of eotaxin generation was detected as compared with wild-type mice. Indeed, surprisingly, overall, there appeared to be a trend toward a general increase in the levels of eotaxin in samples obtained from the P-selectin-deficient mice in the peritonitis model, and this achieved significance only in peritoneal samples obtained from IL-4-stimulated cavities at 4 h post-administration of cytokine. The reason for this trend toward increased chemokine generation in samples from mice lacking P-selectin is unclear but may be associated with a potential regulatory role for P-selectin in the generation of anti-inflammatory mediators involved in terminating the induction and/or synthesis of chemokines such as eotaxin. Of interest, platelet P-selectin has been shown to play a critical role in the generation of lipoxin A4 [52 ], an endogenous bioactive eicosanoid with anti-inflammatory activities [53 ], including an ability to inhibit the generation of eotaxin [54 ]. Furthermore, in P-selectin-deficient mice, the severity of a number of murine models of inflammation is significantly enhanced—observations that may also be related to reduced levels of anti-inflammatory mediators in mice lacking P-selectin [55 ].

In summary, using two distinct murine models of inflammation, we have demonstrated differing dependencies on P-selectin in IL-13-induced eosinophil infiltration, highlighting the increasing evidence for tissue-specific mechanisms that govern eosinophil recruitment. Furthermore, our findings demonstrate that genetic deletion of P-selectin has no inhibitory effects on IL-13/IL-4-induced generation of the eosinophil-specific chemoattractant eotaxin and may in fact result in enhanced generation of the chemokine.


   ACKNOWLEDGEMENTS
 
This work was supported by The Wellcome Trust, UK, and the British Heart Foundation, UK.

Received March 13, 2002; revised September 11, 2002; accepted October 13, 2002.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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