The CC chemokine eotaxin, originally purified from bronchoalveolar lavage fluid of sensitized guinea pigs following allergen challenge, is a potent eosinophil-selective chemoattractant. In the present study, we have used 111In-eosinophils and human eotaxin to characterize the profile of chemokine-induced eosinophil accumulation in vivo in rat skin. Intradermally injected eotaxin caused a dose-dependent accumulation of 111In-eosinophils. Time course studies indicated that the response was rapid, since all the accumulation occurred within the first 1 to 2 h of eotaxin injection. The i.v. administration of anti-intercellular adhesion molecule-1, anti-vascular cell adhesion molecule-1, or anti-α4 integrin mAbs significantly inhibited the eosinophil accumulation induced by 100 pmol of human eotaxin by 73, 43, and 67%, respectively. Further, when 111In-eosinophils were pretreated in vitro with anti-α4 integrin or anti-β2 integrin mAbs, or with a combination of both mAbs, eotaxin-induced responses in vivo were reduced by 52, 49, and 68%, respectively. Eosinophil accumulation induced by intradermal IL-4, but not that induced by TNF-α or leukotriene B4, appeared to be mediated in part by endogenously generated eotaxin. Anti-eotaxin Abs significantly inhibited (54%) the later phases (24–28 h) but not the early phase (0–4 h) of the response to IL-4. This was consistent with eotaxin mRNA expression peaking at 18 h after IL-4 injection. Our findings show that human eotaxin is a potent inducer of eosinophil accumulation in vivo, this response being dependent on α4 integrin/vascular cell adhesion molecule-1 and β2 integrin/intercellular adhesion molecule-1 adhesion pathways. Further, the eosinophil accumulation in response to IL-4 is partly mediated by endogenously generated eotaxin.

Selective tissue infiltration of eosinophils at sites of inflammation is one of the striking features of allergic disease states such as asthma, rhinitis, or atopic dermatitis, but the mechanisms involved in specific eosinophil recruitment are not yet fully understood. In this context, leukocyte extravasation appears to follow a series of sequential leukocyte-endothelial cell interactions involving the selectins, the integrins, and the chemokines as the major molecular regulators (1, 2).

Eotaxin, a CC chemokine originally purified from the bronchoalveolar lavage fluid of allergen-challenged guinea pigs, is a potent and selective eosinophil chemoattractant that is active both on guinea pig and human cells (3, 4). Guinea pig (5, 6), mouse (7, 8), human (9, 10), and rat (11) eotaxin have now been cloned. Human eotaxin exhibits a similar high potency and selectivity for human eosinophils, inducing the elevation of intracellular free calcium concentrations, chemotaxis, actin polymerization, production of reactive oxygen species, and αMβ2 (CD11b/CD18) up-regulation in vitro (9, 10, 12, 13). Although eotaxin is constitutively expressed at low levels in most human organs, marked up-regulation in protein or message has been detected in the epithelium and submucosa of nasal polyp tissues (9) and in the intestine of patients with inflammatory bowel diseases (10), where there is a clear eosinophil infiltration. The major cellular sources of eotaxin are thought to be the epithelium, the endothelium, and activated infiltrating leukocytes such as monocytes and eosinophils (9, 10, 14). Human eotaxin has been demonstrated by histologic examination to induce eosinophil accumulation in monkey skin (9). We found that human eotaxin was inactive on guinea pig eosinophils, both in vitro and in vivo (data not shown) despite the observation that guinea pig eotaxin is highly active on human eosinophils (4). We then found that 125I-human eotaxin bound to rat eosinophils (data not shown). Therefore, we have used a previously described rat skin model (15, 16) to examine inflammatory responses induced by human eotaxin in vivo.

The initial interaction between leukocytes and the vascular endothelium, namely rolling, is a transient and reversible adhesion mainly mediated by the selectins and their carbohydrate counterligands (17, 18), although recently the α4 integrins, α4β1 (very late activation Ag-4) and α4β7, have also been implicated in this process (19, 20). Rolling is thought to result in the exposure of leukocytes to chemoattractants, such as chemokines, presented on the surface of endothelial cells (21). The chemokines then trigger a firm adhesion of leukocytes that is thought to be mediated through the interaction of integrins with their endothelial counterreceptors: vascular cell adhesion molecule (VCAM4)-1 for α4 integrins (for review, see 22 and intercellular adhesion molecule (ICAM)-1 and ICAM-2 for β2 integrins (23, 24). These adhesive interactions serve to facilitate the subsequent transendothelial migration of leukocytes along chemoattractant gradients (25, 26).

Interactions of α4 integrins with VCAM-1 and fibronectin can mediate eosinophil accumulation at sites of inflammation because eosinophils, but not normally neutrophils, express α4 integrins on their surface (22), and mAbs against the α4 integrin subunit or VCAM-1 inhibit eosinophil accumulation in several in vivo models of allergic and nonallergic inflammation (16, 27, 28, 29). Further, the recent cloning of the human eotaxin receptor, CCR3, and its high expression on eosinophils (30, 31) provide a molecular basis for the eosinophil selectivity of eotaxin. Hence, all these findings have led us to hypothesize that the combination of the chemoattractant activity of eotaxin together with its possible role in integrin activation might determine the selective eosinophil traffic observed at sites of eosinophilic inflammation. Our findings show that human eotaxin is a potent inducer of eosinophil accumulation in vivo, and that this response is dependent on α4 and β2 integrins and their endothelial cell counterligands. Further, we provide evidence that endogenously generated eotaxin is involved in eosinophil accumulation induced by IL-4.

Male Sprague Dawley cell donor rats (400–500 g) and male Sprague Dawley in vivo assay rats (200–300 g) were purchased from Harlan-Olac, Bicester, Oxfordshire, U.K.

Pentobarbitone sodium (Sagatal, 60 mg/ml) was purchased from May and Baker, Dagenham, Essex, U.K. Hypnorm (0.315 mg/ml fentanyl citrate and 10 mg/ml fluanisone) was from Janssen Pharmaceutical, Grove, Oxford, U.K. Hypnovel (5 mg/ml midazolam hydrochloride) was from Roche Products, Welwyn Garden City, U.K. 111InCl3 (10 mCi/ml in pyrogen-free 0.04 N hydrochloric acid), 125I-human serum albumin (HSA; 20 mg of albumin per ml of sterile isotonic saline, 50 μCi/ml), [α-32P]dCTP, Multiprime DNA labeling system, and Hybond N hybridization transfer membranes were from Amersham International, Amersham, U.K. BSA (<0.1 ng of endotoxin per mg), 2-mercaptopyridine N-oxide, control mAb MOPC-21 (mouse myeloma IgG1), platelet-activating factor (PAF), glycogen, and Freund’s complete and Freund’s incomplete adjuvants were from Sigma Chemical, Poole, Dorset, U.K. Sterile HBSS, HEPES, Tyrode’s salt solution, and TRIzol were from Life Technologies, Paisley, U.K. Percoll and protein A-Sepharose were from Pharmacia Fine Chemicals, Uppsala, Sweden. Pyrogen- and preservative-free heparin sodium (5000 U/ml) was from Pabyrn Laboratories, Greenford, U.K. Restriction enzymes and RNA m.w. markers were from Promega, Southampton, U.K. Fura-2 acetoxymethyl ester was from Cambridge Bioscience, Cambridge, U.K. Leukotriene B4 (LTB4) was from Cascade Biochemicals, Reading, U.K. Recombinant human TNF-α was from Biogen, Cambridge, MA. Recombinant rat IL-4 was from Serotec, Oxford, U.K. Synthetic human eotaxin (9) was from Leukosite, Cambridge, MA.

Anti-rat β2 mAb WT.3 (mouse IgG1) was from AMS Biotechnology, Oxford, U.K. and was generated as described elsewhere (32). Anti-rat ICAM-1 mAb 1A29 (mouse IgG1) was generated by immunization of mice with endothelial cells obtained from high venules of rat lymph nodes as described in detail in a previous study (33). The anti-human α4 integrin mAb HP2/1 (mouse IgG1), recognizing rat α4 (34), and the anti-rat VCAM-1 mAb 5F10 (mouse IgG2a) (16) were from Biogen.

Rat leukocytes were elicited and purified as previously described (15, 16). Eosinophils were used only when their purity, as determined by Kimura staining, was >94%. The predominant contaminating cell type was mononuclear and a major exclusion criterion was the presence of neutrophils. When these cells were compared by FACS analysis to eosinophils immunostained in whole blood, there was no significant difference in the expression of α4 integrins and L-selectin (16). Mononuclear cells (>97% pure at the upper interface) were prepared from the same donors and gradients as neutrophils (>98% pure at the lower interface).

Rat eosinophils, neutrophils, and mononuclear leukocytes (107 cells/ml in Ca2+/Mg2+-free PBS containing 0.1% BSA) were loaded with fura-2 acetoxymethyl ester (2.5 μM, 30 min at 37°C). After two washes, cells were resuspended at 106 cells/ml in Ca2+/Mg2+-free PBS containing 10 mM HEPES/0.25% BSA/10 mM glucose, pH 7.4. Aliquots were dispensed into quartz cuvettes and the external Ca2+ concentration adjusted to 1 mM with CaCl2. Changes in fluorescence were monitored at 37°C using an LS50 fluorescence spectrophotometer (Perkin-Elmer, Beaconsfield, U.K.) at excitation wavelengths 340 nm and 380 nm, and emission wavelength 510 nm. [Ca2+]i levels were calculated using the ratio of the readings at the two excitation wavelengths and a Kd for Ca2+ binding at 37°C of 224 nM (35). Responses were monitored for 3 min, and data are expressed as maximal increase in [Ca2+]i over the basal levels.

Rat eosinophils or neutrophils were radiolabeled with 111In as previously described (15, 16). Briefly, the cells (1–2 × 107) were incubated with 111InCl3 (approximately 100 μCi chelated with 40 μg 2-mercaptopyridine-N-oxide in 0.1 ml of 50 mM sodium phosphate, pH 7.4) for 15 min at 20°C. The labeled leukocytes were washed three times and resuspended (1 × 107 cells/ml) in HBSS, pH 7.4, containing cell-free citrated rat plasma to a final concentration of 10%. The final cell suspension normally carried approximately 60% of the total radioactivity used, and 5 × 106 111In-cells were injected into each recipient rat.

Leukocyte infiltration and edema formation were simultaneously measured using the local accumulation of i.v.-injected 111In-labeled cells and 125I-HSA, as previously described (15). Briefly, rats were anesthetized with a mixture of Hypnorm (0.1 ml/rat) and Hypnovel (0.1 ml/rat), injected i.p., and their dorsal skin shaved. 111In-eosinophils or 111In-neutrophils (5 × 106 cells mixed with 2.5 μCi 125I-HSA) were then injected i.v. via a tail vein. Five minutes later, the agents under investigation were injected (100 μl/site, in Tyrode’s salt solution containing 0.1% BSA) into the back skin. At the end of a 4-h test period, or 1 h for the time course experiment, the animals were reanesthetized and a cardiac blood sample was collected for determination of cell and plasma radioactivity. The animals were then killed by an overdose of sodium pentobarbitone, the back skin removed, and the injection sites punched out with a 17-mm-diameter punch. Skin, blood cell pellet, and plasma samples were counted in a Cobra Auto Gamma counter (Canberra Packard, Pangbourne, U.K.) and counts were cross-channel corrected for the two isotopes. The 111In count per cell was determined and used to express eosinophil or neutrophil accumulation in each skin site in terms of the number of labeled leukocytes. Edema formation at each site was expressed as microliters of plasma by dividing skin sample 125I counts by 125I counts in 1 μl of plasma.

After their last wash, 111In-eosinophils were divided into four groups for pretreatment with mAbs: 1) MOPC-21 (10 μg/5 × 106 cells/0.5 ml), 2) HP2/1 (5 μg/5 × 106 cells), 3) WT.3 (5 μg/5 × 106 cells), and 4) WT.3 + HP2/1 (each at 5 μg/5 × 106 cells). These mAb concentrations were determined by indirect immunostaining and FACS analysis to be saturating. After incubation for 20 min at 20°C, the cells were injected i.v. into recipient animals.

Rabbits were immunized with human synthetic eotaxin (0.2 mg/animal) emulsified in Freund’s complete adjuvant. Booster injections in incomplete adjuvant were given at 3 and 6 wk and the animals bled out 12 days after the last boost. The IgG fraction of the antiserum was purified by binding to protein A-Sepharose; elution with 0.1 M glycine-HCl, pH 3.0; collection into sodium phosphate, pH 7.4, to limit exposure to acid; and dialysis against saline. The IgG was adjusted to the original volume of serum, filtered, and stored at −20°C in aliquots.

A full-length rat eotaxin cDNA of 980 bp, isolated from an inflamed lung cDNA library (11), was excised from the vector pBKCMV (Stratagene, Cambridge, U.K.) by restriction digestion with BamHI and XhoI. To control for RNA loading, a full-length rat glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA of 1272 bp was used.

IL-4 (5000 U/100 μl) and Tyrode’s/0.1% BSA vehicle were injected into the skin of three rats 24, 18, 8, and 4 h before killing with an anesthetic overdose. The skin sites were quickly punched out and frozen at −80°C. Total RNA was extracted using TRIzol according to the manufacturer’s instructions. RNA samples (10 μg) were separated on 1% agarose gels, containing 2.2% formaldehyde, transblotted onto nylon membranes, and fixed by UV irradiation. Membranes were hybridized with the rat eotaxin cDNA probe (labeled using [α-32P]dCTP and Multiprime kits). Membranes were washed at a final stringency of 0.1× SSC, 0.1% SDS, 55°C (2 × 20 min) and autoradiographed at −70°C. The membranes were stripped, rehybridized using the 32P-labeled rat GAPDH cDNA probe, and washed at the same high stringency. Autoradiographs were assessed by laser densitometry. Data are expressed as the ratio of eotaxin to GAPDH mRNA: the sample in each rat with the highest ratio being given an arbitrary value of one and other samples expressed relative to this.

Results are expressed as the mean ± SEM for n animals or cell preparations. For the in vivo experiments, each datum unit is the average of responses in duplicate skin sites. Data were analyzed by two-way ANOVA of log-transformed data and statistical significance determined with the Newman-Keuls procedure for repeated comparisons. A p value <0.05 was considered statistically significant.

Human eotaxin induced a rapid and dose-dependent increase in [Ca2+]i in rat eosinophils with a significant effect at 1, 10, and 30 nM, the maximal effect being at 10 nM (Fig. 1). Responses peaked within 30 s and returned to basal levels (76.1 ± 2.2 nM Ca2+i) by approximately 2 min. The nonselective chemoattractant, PAF, used as a positive control, caused a similar increase in [Ca2+]i when assayed at 10 nM (Fig. 1).

FIGURE 1.

Dose response of human eotaxin-induced elevation of [Ca2+]i in rat eosinophils. The response to 10 nM PAF is shown for comparison. Results are expressed as the mean ± SEM increase in [Ca2+]i above basal levels (76.1 ± 2.2 nM) in eosinophils from three cell donors.

FIGURE 1.

Dose response of human eotaxin-induced elevation of [Ca2+]i in rat eosinophils. The response to 10 nM PAF is shown for comparison. Results are expressed as the mean ± SEM increase in [Ca2+]i above basal levels (76.1 ± 2.2 nM) in eosinophils from three cell donors.

Close modal

In contrast to the results with eosinophils, human eotaxin had no significant effect on the [Ca2+]i of rat neutrophils and mononuclear cells. The positive control, 10 nM PAF, induced large and significant responses in both cell types. The [Ca2+]i for neutrophils were: basal (88.4 ± 4.3 nM, n = 4 cell preparations), 10 nM eotaxin (6.9 ± 4.1 nM increase, n = 4), and 10 nM PAF (425.2 ± 65.8 nM increase, n = 4, p < 0.01). The results for mononuclear cells were: basal (203.1 ± 20.9 nM, n = 4), 10 nM eotaxin (5.3 ± 1.4 nM increase, n = 4), and 10 nM PAF (500.7 ± 168.6 nM increase, n = 4, p < 0.01).

Using a 4-h in vivo time period, intradermal (i.d.) administration of human eotaxin caused a dose-dependent accumulation of 111In-labeled eosinophils in rat skin with significant responses being achieved at 10 and 100 pmol/skin site (Fig. 2,A). Intradermal PAF at 100 pmol/skin site also induced a significant eosinophil accumulation (Fig. 2,A). In contrast, human eotaxin did not induce any edema formation (Fig. 2 B) or 111In-neutrophil accumulation (data not shown) while PAF (100 pmol/site, used as a positive control) induced significant responses in each case. Histologic examination of the skin injected with human eotaxin (100 pmol/site) demonstrated the presence of an eosinophil infiltrate, with no neutrophil infiltrate, after a 4-h in vivo test period in animals not injected i.v. with labeled cells (data not shown).

FIGURE 2.

Dose response of human eotaxin-induced 111In-eosinophil accumulation (A) and edema formation (B) in rat skin. 111In-eosinophils and 125I-HSA were injected i.v. and human eotaxin, PAF, or Tyrode’s/0.1% BSA vehicle was injected i.d. into the back skin. Responses were measured over a 4-h accumulation period. Cell accumulation is expressed as number of 111In-eosinophils per site per 5 × 106 cells injected and edema formation as microliters of plasma exudation per site. Results are the mean ± SEM for six rats. A significant difference from measurements in vehicle-injected sites (dashed line) is indicated by ** p < 0.01.

FIGURE 2.

Dose response of human eotaxin-induced 111In-eosinophil accumulation (A) and edema formation (B) in rat skin. 111In-eosinophils and 125I-HSA were injected i.v. and human eotaxin, PAF, or Tyrode’s/0.1% BSA vehicle was injected i.d. into the back skin. Responses were measured over a 4-h accumulation period. Cell accumulation is expressed as number of 111In-eosinophils per site per 5 × 106 cells injected and edema formation as microliters of plasma exudation per site. Results are the mean ± SEM for six rats. A significant difference from measurements in vehicle-injected sites (dashed line) is indicated by ** p < 0.01.

Close modal

Human eotaxin at a dose of 10 pmol/skin site or Tyrode’s/BSA vehicle was injected i.d. into different skin sites 240, 120, 60, 30, and 0 min before the i.v. injection of 111In-eosinophils. After a 1-h in vivo test period, the animals were killed and the responses quantified. Significant cell accumulation in response to the chemokine was detected within the first 2 h (i.e., at 0–60, 30–90, and 60–120 min) but not at later time points (Fig. 3).

FIGURE 3.

Time course of human eotaxin-induced 111In-eosinophil accumulation in rat skin. Intradermal injections of human eotaxin (10 pmol/skin site, •) or Tyrode’s/BSA vehicle (○) were given at different time points before i.v. injection of the 111In-eosinophils. Cell accumulation during a 1-h period was measured. Results are expressed as number of 111In-eosinophils per site per 5 × 106 cells injected and presented as mean ± SEM for four rats. A significant difference from vehicle-injected sites is indicated by * p < 0.05, ** p < 0.01.

FIGURE 3.

Time course of human eotaxin-induced 111In-eosinophil accumulation in rat skin. Intradermal injections of human eotaxin (10 pmol/skin site, •) or Tyrode’s/BSA vehicle (○) were given at different time points before i.v. injection of the 111In-eosinophils. Cell accumulation during a 1-h period was measured. Results are expressed as number of 111In-eosinophils per site per 5 × 106 cells injected and presented as mean ± SEM for four rats. A significant difference from vehicle-injected sites is indicated by * p < 0.05, ** p < 0.01.

Close modal

To study the adhesion pathways involved in eotaxin-induced eosinophil accumulation in vivo, the effects of mAbs directed against different adhesion molecules were determined. Abs were i.v. administered, 15 min before injecting the labeled cells, at 3.5 mg/kg for anti-α4 integrin mAb (HP2/1) and 5 mg/kg for anti-ICAM-1 mAb (1A29), anti-VCAM-1 mAb (5F10), and the control mAb (MOPC-21). In a previous study, these i.v. doses of mAbs were found to be sufficient to give the maximum attainable inhibitory effects on TNF-α-induced eosinophil accumulation (16). Once the cells were injected, human eotaxin at 10 and 100 pmol/skin site was i.d. administered and responses allowed to develop for 4 h. Anti-ICAM-1 mAb significantly reduced the eosinophil accumulation elicited by eotaxin (Fig. 4,A), the response to the top dose being reduced by 73%. Similarly, the anti-VCAM-1 and anti-α4 integrin mAbs also significantly inhibited the eosinophil accumulation induced by human eotaxin; at 100 pmol eotaxin/skin site, responses were reduced by 43 and 67%, respectively (Fig. 4, B and C). These inhibitory effects could not be explained by a reduction in the circulating 111In-eosinophils numbers, as the anti-α4 integrin mAb had no significant effect on the circulating numbers, while the anti-ICAM-1 and the anti-VCAM-1 mAbs increased circulating 111In-eosinophils (by 46 and 61%, respectively).

FIGURE 4.

Effect of anti-rat ICAM-1 mAb 1A29, anti-rat VCAM-1 mAb 5F10, and anti-human α4 integrin mAb HP2/1 on human eotaxin-induced 111In-eosinophil accumulation in rat skin. Animals were pretreated i.v. with mAb 15 min before the i.v. injection of the 111In-eosinophils. Eotaxin at 10 and 100 pmol/skin site or the Tyrode’s/BSA vehicle were injected into the back skin 5 min after the i.v. injection of 111In-eosinophils and responses were allowed to develop for 4 h. PanelA shows the effect of 1A29 (5 mg/kg, open bars) vs control mAb MOPC-21 (5 mg/kg, closed bars). PanelB shows the effect of 5F10 (5 mg/kg, open bars) vs control mAb MOPC-21 (5 mg/kg, closed bars). PanelC shows the effect of HP2/1 (3.5 mg/kg, open bars) vs control mAb MOPC-21 (3.5 mg/kg, closed bars). Results are expressed as number of 111In-eosinophils per skin site per 5 × 106 injected cells, corrected by subtraction of the values detected in vehicle-injected sites. Results are the mean ± SEM for n = 4 to 6 pairs of animals. ** p < 0.01, compared with responses in the paired control MOPC-21-treated rats.

FIGURE 4.

Effect of anti-rat ICAM-1 mAb 1A29, anti-rat VCAM-1 mAb 5F10, and anti-human α4 integrin mAb HP2/1 on human eotaxin-induced 111In-eosinophil accumulation in rat skin. Animals were pretreated i.v. with mAb 15 min before the i.v. injection of the 111In-eosinophils. Eotaxin at 10 and 100 pmol/skin site or the Tyrode’s/BSA vehicle were injected into the back skin 5 min after the i.v. injection of 111In-eosinophils and responses were allowed to develop for 4 h. PanelA shows the effect of 1A29 (5 mg/kg, open bars) vs control mAb MOPC-21 (5 mg/kg, closed bars). PanelB shows the effect of 5F10 (5 mg/kg, open bars) vs control mAb MOPC-21 (5 mg/kg, closed bars). PanelC shows the effect of HP2/1 (3.5 mg/kg, open bars) vs control mAb MOPC-21 (3.5 mg/kg, closed bars). Results are expressed as number of 111In-eosinophils per skin site per 5 × 106 injected cells, corrected by subtraction of the values detected in vehicle-injected sites. Results are the mean ± SEM for n = 4 to 6 pairs of animals. ** p < 0.01, compared with responses in the paired control MOPC-21-treated rats.

Close modal

Anti-α4 integrin mAb HP2/1 (16) and anti-β2 integrin mAb WT.3 (data not shown) bind to rat eosinophils. The effect of in vitro pretreatment of 111In-eosinophils with these mAbs, before i.v. injection of the cells, on their accumulation in eotaxin-injected skin sites is shown in Figure 5. Anti-α4 integrin and anti-β2 integrin mAbs significantly inhibited 111In-eosinophil accumulation in response to eotaxin by 52 and 49%, respectively. Although the combination of both Abs appeared to cause a larger reduction in the chemokine-elicited responses (68%), this effect was not significantly different from the effects of either of the Abs alone.

FIGURE 5.

Effect of anti-α4 integrin mAb HP2/1 and anti-β2 integrin mAb WT.3 on human eotaxin-induced 111In-eosinophil accumulation in rat skin. Each 111In-labeled cell preparation was divided into four aliquots of 5 × 106 cells/0.5 ml and pretreated in vitro with the following mAbs before i.v. injection (one treatment/recipient animal): MOPC-21 (control, 10 μg), HP2/1 (anti-α4, 5 μg), WT.3 (anti-β2, 5 μg), or HP2/1 and WT.3 (5 μg each). Five minutes later, human eotaxin at 10 or 100 pmol/skin site, or Tyrode’s/BSA vehicle, were i.d. injected into the back skin. Responses were measured over a 4-h accumulation period. Results are expressed as number of 111In-labeled eosinophils per site per 5 × 106 cells injected and have been corrected for the values detected in vehicle-injected sites. Results are presented as mean ± SEM for n = 4 recipients. A significant difference from responses in the rats receiving control mAb MOPC-21-treated cells is shown by * p < 0.05, ** p < 0.01.

FIGURE 5.

Effect of anti-α4 integrin mAb HP2/1 and anti-β2 integrin mAb WT.3 on human eotaxin-induced 111In-eosinophil accumulation in rat skin. Each 111In-labeled cell preparation was divided into four aliquots of 5 × 106 cells/0.5 ml and pretreated in vitro with the following mAbs before i.v. injection (one treatment/recipient animal): MOPC-21 (control, 10 μg), HP2/1 (anti-α4, 5 μg), WT.3 (anti-β2, 5 μg), or HP2/1 and WT.3 (5 μg each). Five minutes later, human eotaxin at 10 or 100 pmol/skin site, or Tyrode’s/BSA vehicle, were i.d. injected into the back skin. Responses were measured over a 4-h accumulation period. Results are expressed as number of 111In-labeled eosinophils per site per 5 × 106 cells injected and have been corrected for the values detected in vehicle-injected sites. Results are presented as mean ± SEM for n = 4 recipients. A significant difference from responses in the rats receiving control mAb MOPC-21-treated cells is shown by * p < 0.05, ** p < 0.01.

Close modal

Northern blot analysis showed a time-dependent eotaxin mRNA expression at sites of IL-4 injection (5000 U/skin site). Eotaxin message was virtually undetectable in vehicle-injected sites but was increased, beginning at 4 h and reaching a peak at approximately 18 h, after IL-4 injection (Fig. 6).

FIGURE 6.

Northern blot analysis of eotaxin mRNA expression in rat skin. RNA samples from skin sites injected with IL-4 (5000 U, •) or Tyrode’s/BSA vehicle (○), at 24, 18, 8, and 4 h before killing, were hybridized with a rat eotaxin cDNA probe. After stripping, the membranes were probed for the constitutive GAPDH message and laser densitometric analysis of the autoradiographs expressed as the ratio of eotaxin/GAPDH (n = 3 rats).

FIGURE 6.

Northern blot analysis of eotaxin mRNA expression in rat skin. RNA samples from skin sites injected with IL-4 (5000 U, •) or Tyrode’s/BSA vehicle (○), at 24, 18, 8, and 4 h before killing, were hybridized with a rat eotaxin cDNA probe. After stripping, the membranes were probed for the constitutive GAPDH message and laser densitometric analysis of the autoradiographs expressed as the ratio of eotaxin/GAPDH (n = 3 rats).

Close modal

To determine whether eosinophil accumulation induced by IL-4 in rat skin is mediated by the generation of endogenous eotaxin, we used a polyclonal antiserum raised against human eotaxin. The IgG fraction of this rabbit antiserum, coinjected with i.d. agonist, inhibited eosinophil accumulation induced by human eotaxin but not that induced by LTB4 or TNF-α (Fig. 7). IL-4 (5000 U/skin site) was coinjected with control rabbit IgG or anti-human eotaxin IgG at two different time points: −24 h and 0 h, each in the same animal so that each rat acted as its own internal control. Labeled cells were then injected i.v. and responses allowed to develop for 4 h. Anti-human eotaxin IgG reduced the 111In-eosinophil accumulation induced by IL-4 injected at −24 h (i.e., the 24- to 28-h response) in all rats tested (n = 8, p < 0.05) but had no effect on the 0- to 4-h eosinophil accumulation response to IL-4 (Fig. 8).

FIGURE 7.

Effect of anti-human eotaxin IgG on eotaxin-, LTB4-, and TNF-α-induced 111In-eosinophil accumulation in rat skin. 111In-eosinophils were injected i.v. into recipient animals and 5 min later agonists (human eotaxin, LTB4, or human TNF-α, each at 10 pmol/skin site) or the Tyrode’s/BSA vehicle were coinjected together with anti-human eotaxin IgG or control rabbit IgG into the skin. Responses were measured over a 4-h accumulation period. Results are expressed as number of 111In-eosinophils per site per 5 × 106 injected cells and presented as mean ± SEM for four animals. A significant difference between the responses to agonist in control IgG and anti-eotaxin-treated sites is shown by * p < 0.05, ** p < 0.01.

FIGURE 7.

Effect of anti-human eotaxin IgG on eotaxin-, LTB4-, and TNF-α-induced 111In-eosinophil accumulation in rat skin. 111In-eosinophils were injected i.v. into recipient animals and 5 min later agonists (human eotaxin, LTB4, or human TNF-α, each at 10 pmol/skin site) or the Tyrode’s/BSA vehicle were coinjected together with anti-human eotaxin IgG or control rabbit IgG into the skin. Responses were measured over a 4-h accumulation period. Results are expressed as number of 111In-eosinophils per site per 5 × 106 injected cells and presented as mean ± SEM for four animals. A significant difference between the responses to agonist in control IgG and anti-eotaxin-treated sites is shown by * p < 0.05, ** p < 0.01.

Close modal
FIGURE 8.

Effect of an anti-human eotaxin IgG on IL-4-induced 111In-eosinophil accumulation in rat skin. IL-4 (5000 U/site) mixed with 100 μl of anti-eotaxin or control IgG was injected i.d. in different sites of the same animal 24 h before or 5 min after the i.v. injection of 111In-eosinophils. 111In-eosinophil accumulation was measured over a 4-h test period; thus, responses were at 24 to 28 h (n = 8 rats) or 0 to 4 h (n = 4 rats) after IL-4 injection. Human eotaxin (10 pmol/site, 0–4 h response, n = 8 rats) was used as a positive control for the Ab. Results are expressed as the response in the presence of anti-eotaxin as a percentage of the the control response in the same animal (mean ± SEM). A significant difference between the responses in control IgG- and anti-human eotaxin IgG-treated sites (calculated from the original data of 111In-eosinophils/skin site) is shown by * p < 0.05, ** p < 0.01.

FIGURE 8.

Effect of an anti-human eotaxin IgG on IL-4-induced 111In-eosinophil accumulation in rat skin. IL-4 (5000 U/site) mixed with 100 μl of anti-eotaxin or control IgG was injected i.d. in different sites of the same animal 24 h before or 5 min after the i.v. injection of 111In-eosinophils. 111In-eosinophil accumulation was measured over a 4-h test period; thus, responses were at 24 to 28 h (n = 8 rats) or 0 to 4 h (n = 4 rats) after IL-4 injection. Human eotaxin (10 pmol/site, 0–4 h response, n = 8 rats) was used as a positive control for the Ab. Results are expressed as the response in the presence of anti-eotaxin as a percentage of the the control response in the same animal (mean ± SEM). A significant difference between the responses in control IgG- and anti-human eotaxin IgG-treated sites (calculated from the original data of 111In-eosinophils/skin site) is shown by * p < 0.05, ** p < 0.01.

Close modal

Chemokines comprise a family of small m.w proteins (8–10 kDa) that have been implicated in the selective recruitment of leukocyte subsets observed in different inflammatory disease states. Among all the CC chemokines described to date, eotaxin is the only member that acts on a single receptor, CCR3. This receptor is present in high numbers on eosinophils, and the high affinity interaction can explain the high specificity of eotaxin for eosinophils (3, 4, 8, 9, 10, 36). Although human eotaxin has been tested extensively in vitro, little is known of its activity in vivo.

To characterize the eosinophil accumulation elicited by human eotaxin, we have used an in vivo assay system to measure 111In-eosinophil accumulation and edema formation in rat skin (15, 16). Preliminary experiments showed that 125I-human eotaxin bound to rat eosinophils in vitro (data not shown). This binding caused a rapid and dose-dependent increase in intracellular calcium levels (Fig. 1). In contrast, human eotaxin failed to elicit changes in intracellular calcium concentrations in either rat neutrophils or rat mononuclear cells. This demonstrates the eosinophil selectivity of eotaxin in the rat, in agreement with previous in vitro studies in other species (8, 9, 10, 36). Next, we demonstrated that human eotaxin elicits a potent and selective (Fig. 2) eosinophil accumulation in rat skin; no neutrophil accumulation or edema formation was seen. The eosinophil accumulation was a rapid event, significant responses occurring within the first hour (Fig. 3). Taken together with the in vitro data on calcium mobilization in isolated cells, this rapid eosinophil accumulation suggests a direct effect of eotaxin on eosinophils in vivo. Chemokines such as eotaxin are thought to be presented on the microvascular endothelium and stimulate adhesion (21).

The adhesive mechanisms that regulate chemokine-induced accumulation of leukocytes in vivo remain largely uncharacterized. As part of the present study, we have investigated the roles of the β2 integrins/ICAM-1 and α4 integrins/VCAM-1 adhesion pathways in the process of eosinophil accumulation elicited by eotaxin (Figs. 4 and 5). Pretreatment of 111In-eosinophils in vitro with a saturating concentration of a neutralizing anti-β2 integrin mAb, before their administration into the recipient animals, significantly suppressed the eotaxin-induced 111In-eosinophil accumulation. Further, the i.v. administration of a neutralizing anti-ICAM-1 mAb also caused an inhibition of this response. These results suggest that eotaxin-induced leukocyte accumulation is at least partly mediated via the direct effects of the chemokine on the leukocytes causing up-regulation or activation of β2 integrins in vivo. β2 integrins can then mediate the firm attachment of the eosinophils to venular endothelial cells via basally expressed ICAM-1. Previous studies have demonstrated the importance of the β2 integrins or ICAM-1 in eosinophil accumulation in other models of inflammation (37, 38, 39, 40). We have found that eotaxin up-regulates the expression of β2 integrins on rat peripheral blood eosinophils in vitro (K. Nagai et al., unpublished observations). If this up-regulation also occurs in vivo, the newly expressed β2 integrins will not be blocked by the in vitro pretreatment of the eosinophils with mAb. This would explain why we found that blocking the existing β2 integrins in vitro (Fig. 5) was less effective than blocking the ICAM-1 counterligand in vivo (Fig. 4).

Neutralizing mAbs directed against α4 integrins or VCAM-1 also suppressed the eosinophil accumulation induced by i.d. eotaxin. The anti-α4 integrin mAb was effective at suppressing this response whether it was used to pretreat the 111In-eosinophils in vitro or given i.v. These results suggest that, in addition to activating β2 integrins, the eotaxin-induced eosinophil accumulation may be mediated via α4 integrins although the mechanism by which eotaxin may activate α4 integrins is still unknown. VCAM-1, one of the principal endothelial cell ligands for α4 integrins, is generally considered to be expressed only on cytokine-activated endothelial cells (22). However, as an anti-VCAM-1 mAb was effective at attenuating the rapid eosinophil accumulation induced by eotaxin, our results suggest the presence of low levels (that may be undetectable by immunostaining) of basally expressed VCAM-1 on venular endothelial cells that are sufficient to mediate the accumulation of eosinophils. These results are in agreement with our previous findings in which we reported that an anti-α4 integrin mAb could inhibit the eosinophil accumulation induced by rapidly acting chemoattractants such as LTB4 and C5adesArg (27).

The pretreatment of 111In-eosinophils with a combination of anti-α4 integrin and anti-β2 integrin mAbs did not cause a significantly larger level of inhibition than that seen with either mAb alone. This differs from the additive effect seen with two similar Abs, which completely inhibited the eosinophil accumulation induced in guinea pig airways in response to i.v. Sephadex particles (29). It is possible that α4 and β2 integrins may be involved in different but sequential stages of the eotaxin-induced eosinophil migration, namely rolling and firm adhesion, respectively. In this respect, there is in vivo evidence for the involvement of α4 integrins in eosinophil rolling (19), as well as in the subsequent phase of firm adhesion, whereas β2 integrins are not thought to contribute to eosinophil rolling.

Recent in vitro studies have addressed the mechanisms involved in chemokine-mediated leukocyte adhesion and transendothelial cell migration. Eosinophil transmigration across unstimulated endothelial cell monolayers induced by RANTES can be virtually abolished by mAbs directed against β2 integrins (41). Although this effect was reported to occur without an apparent increase in β2 integrin expression on the leukocyte surface, recent reports have demonstrated that RANTES, monocyte chemoattractant protein (MCP)-3, and eotaxin can stimulate the up-regulation of αMβ2 integrin (CD11b/CD18) on human eosinophils (13, 42). In addition, RANTES and MCP-3 can trigger a strong and prolonged adhesion of human eosinophils to ICAM-1, this effect being primarily mediated by direct activation of β2 integrins (42). Eosinophils can also adhere to either VCAM-1 or fibronectin upon stimulation with chemokines (25, 26, 42, 43). This event was shown to be rapid and transient, and occurred without apparent changes in α4β1 integrin expression on the leukocyte surface (26, 42), although it was correlated with f-actin polymerization (26, 42), uropod-like formation, and asymmetrical distribution of very late activation Ag-4 (26). Hence, the above in vitro studies largely support the present in vivo findings and suggest that the eotaxin-induced eosinophil migration may be mediated by changes in the expression, affinity states, or signaling pathways of α4 and β2 integrins.

IL-4 is a Th2 cytokine that selectively induces VCAM-1 expression on endothelial cells with little or no effect on ICAM-1 or E-selectin expression (44, 45, 46). This feature of IL-4 has been associated with the selective adhesion and transmigration of eosinophils, as opposed to neutrophils (47, 48, 49, 50), across the vascular endothelium mediated by α4 integrins on the eosinophil surface (22). In vivo, i.p. or i.d. injection of IL-4 induces selective eosinophil accumulation in nude mice and rats (49, 51). In agreement with in vitro studies, the IL-4-induced eosinophil accumulation in vivo appears to be α4 integrin/VCAM-1 dependent (51). Besides inducing VCAM-1 expression, IL-4 can exert eosinophil accumulation through the endogenous generation of secondary inflammatory mediators. In other systems, IL-4 has been implicated in the release of stem cell factor from alveolar macrophages (52), in the increased expression of C10 in murine resident peritoneal macrophages (53), and in the synthesis and secretion of MCP-1 by cultured HUVECs (54).

Eotaxin message has been detected after transplantation of IL-4-secreting tumor cells in mice, an effect that occurs within 6 h and persists for 7 days (7). We conducted a series of experiments to detect the possible involvement of eotaxin generation in eosinophil accumulation induced by i.d.-injected IL-4 in the rat. Firstly, Northern blot analysis showed that eotaxin message began to increase at 4 h in IL-4-injected sites and was still present at 24 h (Fig. 6). Secondly, we used a neutralizing anti-human eotaxin Ab to block part of the response to IL-4. 111In-eosinophil accumulation observed 24 to 28 h after, but not 0 to 4 h after, IL-4 injection was significantly suppressed when the cytokine was coinjected with the Ab (Fig. 8). These effects of anti-eotaxin are consistent with the mRNA data, which suggest the likely presence of eotaxin protein during the 24- to 28-h 111In-eosinophil accumulation period but little or no eotaxin release during the 0- to 4-h period. Hence, we conclude that endogenous eotaxin generation partly mediates the later phases of eosinophil accumulation induced by IL-4. This response to eotaxin may, in turn, be dependent on the up-regulation of VCAM-1 on the endothelial cell surface in response to IL-4, as discussed above.

Th2 cytokines appear to play a key role in regulating eotaxin-induced eosinophil accumulation, since IL-5 has been shown to cooperate with eotaxin to induce eosinophil infiltration in the skin and in the lungs by mobilizing eosinophils from the bone marrow (10, 14, 55), and eosinophil infiltration induced by IL-4 can be partly mediated through eotaxin generation, as discussed above. Indeed, in two mouse models of Ag-induced pulmonary eosinophilia, it has recently been shown that elimination of T cell accumulation results in a reduction of eosinophil recruitment associated with eotaxin down-regulation (56, 57).

In conclusion, we have shown that human eotaxin is a potent and selective inducer of eosinophil accumulation in rat skin and provide the first in vivo evidence that eosinophil accumulation induced by chemokines can be abrogated by mAbs directed against both leukocyte and endothelial cell surface adhesion molecules. We have also demonstrated that the secondary generation of endogenous eotaxin is involved in the later phases of eosinophil accumulation in response to IL-4.

1

This work was supported by The Wellcome Trust, U.K., and The National Asthma Campaign, U.K.

4

Abbreviations used in this paper: VCAM, vascular cell adhesion molecule; PAF, platelet-activating factor; LTB4, leukotriene B4: ICAM, intercellular adhesion molecule; i.d., intradermal; HSA, human serum albumin; MCP, monocyte chemoattractant protein; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

1
Springer, T. A..
1994
. Traffic signals for lymphocyte recirculation and leukocyte emigration: the multistep paradigm.
Cell
76
:
301
2
Butcher, E..
1991
. Leukocyte-endothelial cell recognition: three (or more) steps to specificity and diversity.
Cell
67
:
1033
3
Griffiths-Johnson, D. A., P. D. Collins, A. G. Rossi, P. J. Jose, T. J. Williams.
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
4
Jose, P. J., D. A. Griffiths-Johnson, P. D. Collins, D. T. Walsh, R. Moqbel, N. F. Totty, O. Truong, J. J. Hsuan, T. J. Williams.
1994
. Eotaxin: a potent eosinophil chemoattractant cytokine detected in a guinea-pig model of allergic airways inflammation.
J. Exp. Med.
179
:
881
5
Jose, P. J., I. M. Adcock, D. A. Griffiths-Johnson, N. Berkman, T. N. C. Wells, T. J. Williams, C. A. Power.
1994
. Eotaxin: cloning of an eosinophil chemoattractant cytokine and increased mRNA expression in allergen-challenged guinea-pig lungs.
Biochem. Biophys. Res. Commun.
205
:
788
6
Rothenberg, M. E., A. D. Luster, C. M. Lilly, J. M. Drazen, P. Leder.
1995
. Constitutive and allergen-induced expression of eotaxin mRNA in the guinea pig lung.
J. Exp. Med.
181
:
1211
7
Rothenberg, M. E., A. D. Luster, P. Leder.
1995
. Murine eotaxin: an eosinophil chemoattractant inducible in endothelial cells and in interleukin 4-induced tumor suppression.
Proc. Natl. Acad. Sci. USA
92
:
8960
8
Gonzalo, J., G. Jia, V. Aguirre, D. Friend, A. J. Coyle, N. A. Jenkins, G. Lin, H. Katz, A. Lichtman, N. Copeland, M. Kopf, J. Gutierrez-Ramos.
1996
. Mouse eotaxin expression parallels eosinophil accumulation during lung allergic inflammation but it is not restricted to a Th2-type response.
Immunity
4
:
1
9
Ponath, P. D., S. Qin, D. J. Ringler, I. Clark-Lewis, J. Wang, N. Kassam, H. Smith, X. Shi, J. Gonzalo, W. Newman, J. Gutierrez-Ramos, C. R. Mackay.
1996
. Cloning of the human eosinophil chemoattractant, eotaxin: expression, receptor binding and functional properties suggest a mechanism for the selective recruitment of eosinophils.
J. Clin. Invest.
97
:
604
10
Garcia-Zepeda, E. A., M. E. Rothenberg, R. T. Ownbey, J. Celestin, P. Leder, A. D. Luster.
1996
. Human eotaxin is a specific chemoattractant for eosinophil cells and provides a new mechanism to explain tissue eosinophilia.
Nat. Med.
2
:
449
11
Williams, C. M. M., D. J. Newton, S. A. Wilson, T. J. Williams, J. W. Coleman, B. F. Flanagan.
1998
. Conserved structure and tissue expression of rat eotaxin.
Immunogenetics
47
:
178
12
Elsner, J., R. Hochstetter, D. Kimmig, A. Kapp.
1996
. Human eotaxin represents a potent activator of the respiratory burst of human eosinophils.
Eur. J. Immunol.
26
:
1919
13
Tenscher, K., B. Metzner, E. Schöpf, J. Norgauer, W. Czech.
1996
. Recombinant human eotaxin induces oxygen radical production, Ca2+-mobilization, actin reorganization, and CD11b upregulation in human eosinophils via a pertussis toxin-sensitive heterotrimeric guanine nucleotide-binding protein.
Blood
88
:
3195
14
Humbles, A. A., D. M. Conroy, S. Marleau, S. M. Rankin, R. T. Palframan, A. E. I. Proudfoot, T. N. C. Wells, L. Dechun, P. K. Jeffery, D. A. Griffiths-Johnson, T. J. Williams, P. J. Jose.
1997
. Kinetics of eotaxin generation and its relationship to eosinophil accumulation in allergic airways disease: analysis in a guinea pig model in vivo.
J. Exp. Med.
186
:
601
15
Sanz, M., V. B. Weg, M. A. Bolanowski, S. Nourshargh.
1995
. IL-1 is a potent inducer of eosinophil accumulation in rat skin: inhibition of response by a PAF antagonist and an anti-human IL-8 antibody.
J. Immunol.
154
:
1364
16
Sanz, M., A. Hartnell, P. Chisholm, C. Williams, D. Davies, V. B. Weg, M. Feldmann, M. A. Bolanowski, R. R. Lobb, S. Nourshargh.
1997
. TNFα-induced eosinophil accumulation in rat skin is dependent on α4 integrin/vascular cell adhesion molecule-1 adhesion pathways.
Blood
90
:
4144
17
Lawrence, M. B., T. A. Springer.
1991
. Leukocytes roll on a selectin at physiologic flow rates: distinction from and prerequisite for adhesion through integrins.
Cell
65
:
859
18
Bevilacqua, M. P., R. M. Nelson.
1993
. Selectins.
J. Clin. Invest.
91
:
379
19
Sriramarao, P., U. H. von Andrian, E. C. Butcher, M. A. Bourdon, D. H. Broide.
1994
. L-selectin and very late antigen-4 integrin promote eosinophil rolling at physiological shear rates in vivo.
J. Immunol.
153
:
4238
20
Berlin, C., R. F. Bargatze, J. J. Campbell, U. H. von Andrian, M. C. Szabo, S. R. Hasslen, R. D. Nelson, E. L. Berg, S. L. Erlandsen, E. C. Butcher.
1995
. α4 integrins mediate lymphocyte attachment and rolling under physiologic flow.
Cell
80
:
413
21
Rot, A..
1992
. Endothelial cell binding of NAP-1/IL-8: role in neutrophil emigration.
Immunol. Today
13
:
291
22
Lobb, R. R., M. E. Hemler.
1994
. The pathophysiologic role of α4 integrins in vivo.
J. Clin. Invest.
94
:
1722
23
Carlos, T. M., J. M. Harlan.
1994
. Leukocyte-endothelial adhesion molecules.
Blood
84
:
2068
24
Bochner, B. S., R. P. Schleimer.
1994
. The role of adhesion molecules in human eosinophil and basophil recruitment.
J. Allergy Clin. Immunol.
94
:
427
25
Campbell, J. J., S. Qin, K. B. Bacon, C. R. Mackay, E. C. Butcher.
1996
. Biology of chemokine and classical chemoattractant receptors: differential requirements for adhesion-triggering versus chemotactic responses in lymphoid cells.
J. Cell Biol.
134
:
255
26
Weber, C., R. Alon, B. Moser, T. A. Springer.
1996
. Sequential regulation of α4β1 and α5β1 integrin avidity by CC chemokines in monocytes: implications for transendothelial chemotaxis.
J. Cell Biol.
134
:
1063
27
Weg, V. B., T. J. Williams, R. R. Lobb, S. Nourshargh.
1993
. A monoclonal antibody recognising very late activation antigen-4 (VLA-4) inhibits eosinophil accumulation in vivo.
J. Exp. Med.
177
:
561
28
Pretolani, M., C. Ruffie, J. R. Lapa-e-Silva, D. Joseph, R. R. Lobb, B. B. Vargaftig.
1994
. Antibody to very late antigen 4 prevents antigen-induced bronchial hyperreactivity and cellular infiltration in the guinea pig airways.
J. Exp. Med.
180
:
795
29
Das, A. M., T. J. Williams, R. R. Lobb, S. Nourshargh.
1995
. Lung eosinophilia is dependent on IL-5, and the adhesion molecules CD18 and VLA-4 in a guinea-pig model.
Immunology
84
:
41
30
Ponath, P. D., S. Qin, T. W. Post, J. Wang, L. Wu, N. P. Gerard, W. Newman, C. Gerard, C. R. Mackay.
1996
. Molecular cloning and characterization of a human eotaxin receptor expressed selectively on eosinophils.
J. Exp. Med.
183
:
2437
31
Daugherty, B. L., S. J. Siciliano, J. DeMartino, L. Malkowitz, A. Sirontino, M. S. Springer.
1996
. Cloning, expression and characterization of the human eosinophil eotaxin receptor.
J. Exp. Med.
183
:
2349
32
Tamatani, T., M. Kotani, M. Miyasaka.
1991
. Characterization of the rat leukocyte integrin, CD11/CD18, by the use of LFA-1 subunit-specific monoclonal antibodies.
Eur. J. Immunol.
21
:
627
33
Tamatani, T., M. Miyasaka.
1990
. Identification of monoclonal antibodies reactive with the rat homolog of ICAM-1, and evidence for a differential involvement of ICAM-1 in the adherence of resting versus activated lymphocytes to high endothelial cells.
Int. Immunol.
2
:
165
34
Yednock, T. A., C. Cannon, C. Fritz, F. Sanchez-Madrid, L. Steinman, L. Karin.
1992
. Prevention of experimental autoimmune encephalomyelitis by antibodies against α4β1 integrin.
Nature
356
:
63
35
Grynkiewicz, G., M. Poenie, R. Y. Tsien.
1985
. A new generation of Ca2+ indicators with greatly improved fluorescence properties.
J. Biol. Chem.
260
:
3440
36
Rothenberg, M., R. Ownbey, P. Mehlhop, P. Loiselle, M. Van de Rijn, J. Bonventre, H. Oettgen, P. Leder, A. Luster.
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
37
Wegner, C. D., R. H. Gundel, P. Reilly, N. Haynes, L. G. Letts, R. Rothlein.
1990
. Intercellular adhesion molecule-1 (ICAM-1) in the pathogenesis of asthma.
Science
247
:
456
38
Sun, J., W. Elwood, A. Haczku, P. J. Barnes, P. G. Hellewell, K. F. Chung.
1994
. Contribution of intercellular adhesion molecule-1 (ICAM-1) to allergen-induced airways hyperresponsiveness and inflammation in sensitised Brown-Norway rats.
Int. Arch. Allergy Immunol.
104
:
291
39
Teixeira, M. M., S. Reynia, M. Robinson, A. Shock, T. J. Williams, F. M. Williams, A. G. Rossi, P. G. Hellewell.
1994
. Role of CD18 in the accumulation of eosinophils and neutrophils and local oedema formation in inflammatory reactions in guinea pig skin.
Br. J. Pharmacol.
111
:
811
40
Walsh, D. T., V. B. Weg, T. J. Williams, S. Nourshargh.
1995
. Substance P-induced inflammatory responses in guinea-pig skin: the effect of specific NK1 receptor antagonists and the role of endogenous mediators.
Br. J. Pharmacol.
114
:
1343
41
Ebisawa, M., T. Yamada, C. Bickel, D. Klunk, R. P. Schleimer.
1994
. Eosinophil transendothelial migration induced by cytokines. III: Effect of the chemokine RANTES.
J. Immunol.
153
:
2153
42
Weber, C., J. Kitayama, T. A. Springer.
1996
. Differential regulation of β1 and β2 integrin avidity by chemoattractants in eosinophils.
Proc. Natl. Acad. Sci. USA
93
:
10939
43
Lloyd, A. R., J. J. Oppenheim, D. J. Kelvin, D. D. Taub.
1996
. Chemokines regulate T cell adherence to recombinant adhesion molecules and extracellular matrix proteins.
J. Immunol.
156
:
932
44
Thornhill, M. H., S. M. Wellicome, D. L. Mahiouz, J. S. S. Lanchbury, U. Kyan-Aung, D. O. Haskard.
1991
. Tumor necrosis factor combines with IL-4 or IFN-γ to selectively enhance endothelial cell adhesiveness for T cells: the contribution of vascular cell adhesion molecule-1-dependent and -independent binding mechanisms.
J. Immunol.
146
:
592
45
Thornhill, M. H., D. O. Haskard.
1990
. IL-4 regulates endothelial cell activation by IL-1, tumor necrosis factor, or IFN-γ.
J. Immunol.
145
:
865
46
Gundel, R., D. Lindell, P. Harris, M. Fournel, G. Jesmok, M. E. Gerritsen.
1996
. IL-4-induced leucocyte trafficking in cynomolgus monkeys: correlation with the expression of adhesion molecules and chemokine generation.
Clin. Exp. Allergy
26
:
719
47
Sano, H., N. Nakagawa, H. Nakajima, S. Yoshida, I. Iwamoto.
1995
. Role of vascular cell adhesion molecule and platelet activating factor in selective eosinophil migration across vascular endothelial cells.
Int. Arch. Allergy Immunol.
107
:
533
48
Schleimer, R. P., S. A. Sterbinsky, J. Kaiser, C. A. Bickel, D. A. Klunk, K. Tomioka, W. Newman, F. W. Luscinskas, M. A. Gimbrone, B. W. McIntyre, B. S. Bochner.
1992
. IL-4 induces adherence of human eosinophils and basophils but not neutrophils to endothelium: association with expression of VCAM-1.
J. Immunol.
148
:
1086
49
Moser, R., P. Groscurth, J. M. Carballido, P. L. B. Bruijnzeel, K. Blaser, C. H. Heusser, J. Fehr.
1993
. Interleukin-4 induces tissue eosinophilia in mice: correlation with its in vitro capacity to stimulate the endothelial cell-dependent selective transmigration of human eosinophils.
J. Lab. Clin. Med.
122
:
567
50
Moser, R., J. Fehr, P. L. B. Bruijnzeel.
1992
. IL-4 controls the selective endothelium-driven transmigration of eosinophils from allergic individuals.
J. Immunol.
149
:
1432
51
Sanz, M., R. R. Lobb, S. Nourshargh.
1995
. IL-4-induced eosinophil accumulation in rat skin is VCAM-1-dependent.
Inflamm. Res.
44
:
223
(Abstr.).
52
Lukacs, N. W., R. M. Strieter, P. Lincoln, E. Brownell, D. M. Pullen, H. J. Schock, S. W. Chensue, D. D. Taub, S. L. Kunkel.
1996
. Stem cell factor (c-kit ligand) influences eosinophil recruitment and histamine levels in allergic airway inflammation.
J. Immunol.
156
:
3945
53
Orlofsky, A., E. Y. Lin, M. B. Prystowsky.
1994
. Selective induction of the β chemokine C10 by IL-4 in mouse macrophages.
J. Immunol.
152
:
5084
54
Rollins, B. J., J. S. Pober.
1991
. Interleukin-4 induces the synthesis and secretion of MCP-1/JE by human endothelial cells.
Am. J. Pathol.
138
:
1315
55
Collins, P. D., S. Marleau, D. A. Griffiths-Johnson, P. J. Jose, T. J. Williams.
1995
. Co-operation between interleukin-5 and the chemokine, eotaxin, to induce eosinophil accumulation in vivo.
J. Exp. Med.
182
:
1169
56
MacLean, J. A., R. Ownbey, A. D. Luster.
1996
. T cell-dependent regulation of eotaxin in antigen-induced pulmonary eosinophilia.
J. Exp. Med.
184
:
1461
57
Gonzalo, J., C. M. Lloyd, L. Kremer, E. Finger, C. Martinez-A., M. H. Siegelman, M. I. Cybulsky, J. Gutierrez-Ramos.
1996
. Eosinophil recruitment to the lung in a murine model of allergic inflammation: the role of T cells, chemokines and adhesion receptors.
J. Clin. Invest.
98
:
2332