Leukotriene B4 is essential for selective eosinophil recruitment following allergen challenge of CD4+ cells in a model of chronic eosinophilic inflammation

Life Sciences 83 (2008) 214–222

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Life Sciences j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / l i f e s c i e

Leukotriene B4 is essential for selective eosinophil recruitment following allergen challenge of CD4+ cells in a model of chronic eosinophilic inflammation Alessandra Bonacini Cheraim a, Pedro Xavier-Elsas a,⁎, Sandra Helena Penha de Oliveira e, Tiago Batistella b, Momtchilo Russo c, Maria Ignez Gaspar-Elsas d, Fernando Queiroz Cunha b a

Depto. de Imunologia, Instituto de Microbiologia Prof. Paulo de Góes, UFRJ, Rio de Janeiro, Brazil Laboratório de Farmacologia da Inflamação e da Dor, Depto. de Farmacologia, FMRP-USP, Ribeirão Preto, SP, Brazil Depto. de Imunologia, Instituto de Ciências Biomédicas, USP, São Paulo, Brazil d Laboratório de Fisiopatologia Humana, Instituto Fernandes Figueira/FIOCRUZ, Rio de Janeiro, Brazil e Laboratório de Farmacologia, Depto. de Ciências Básicas, Faculdade de Odontologia de Araçatuba, UNESP, Araçatuba, SP, Brazil b c

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Article history: Received 21 February 2008 Accepted 6 June 2008 Keywords: Eosinophils Leukotrienes Chemotaxis Inflammation Chemokines

A B S T R A C T Subcutaneous heat-coagulated egg white implants (EWI) induce chronic, intense local eosinophilia in mice, followed by asthma-like responses to airway ovalbumin challenge. Our goal was to define the mechanisms of selective eosinophil accumulation in the EWI model. EWI carriers were challenged i.p. with ovalbumin and the contributions of cellular immunity and inflammatory mediators to the resulting leukocyte accumulation were defined through cell transfer and pharmacological inhibition protocols. Eosinophil recruitment required Major Histocompatibility Complex Class II expression, and was abolished by the leukotriene B4 (LTB4) receptor antagonist CP 105.696, the 5-lipoxygenase inhibitor BWA4C and the 5-lipoxygenase activating protein inhibitor MK886. Eosinophil recruitment in EWI carriers followed transfer of: a) CD4+ (but not CD4−) cells, harvested from EWI donors and restimulated ex vivo; b) their cell-free supernatants, containing LTB4. Restimulation in the presence of MK886 was ineffective. CC chemokine receptor ligand (CCL)5 and CCL2 were induced by ovalbumin challenge in vivo. mRNA for CCL17 and CCL11 was induced in ovalbumin-restimulated CD4+ cells ex vivo. MK886 blocked induction of CCL17. Pretreatment of EWI carriers with MK886 eliminated the effectiveness of exogenously administered CCL11, CCL2 and CCL5. In conclusion, chemokine-producing, ovalbumin-restimulated CD4+ cells initiate eosinophil recruitment which is strictly dependent on LTB4 production. © 2008 Elsevier Inc. All rights reserved.

Introduction Eosinophil recruitment into sites of allergic inflammation depends various mechanisms to ensure selectivity (Bochner, 2000; Bochner and Schleimer, 2001; Broide and Srimarao, 2001). Eosinophils (along with basophils, mast cells and CD4+ T cells), selectively express CC chemokine receptor 3 (CCR3), and migrate in response to the CCR3 ligands, CC chemokine receptor ligand 11 (CCL11, previously termed eotaxin), CCL5 (previously termed RANTES), CCL8, CCL7 and CCL13 (previously referred to as macrophage chemoattractant proteins -2, -3 and -4) (Giembycz and Lindsay, 1999; Ochi et al., 1999). However, many cytokines and chemokines, including Interleukin (IL)-3, IL-4, IL-5, IL-6, IL-9, IL-13, granulocyte-macrophage colony stimulating factor (GMCSF), and CCL17 (previously termed TARC), were shown to induce eosinophil migration (Giembycz and Lindsay, 1999). Only a few of these ⁎ Corresponding author. Depto. de Imunologia, Instituto de Microbiologia Prof. Paulo de Góes, UFRJ, Cidade Universitária, CCS Bloco I sala 066, Rio de Janeiro, CEP 21941-590, Brazil. Tel./fax: +55 21 25541731. E-mail addresses: elsas@iff.fiocruz.br, [email protected] (P. Xavier-Elsas). 0024-3205/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2008.06.004

ligands truly display eosinophil-selective chemoattractant activity, or bind to receptors expressed predominantly in eosinophils. Selectivity may arise from an interaction of eosinophil-selective and -nonselective ligands: for instance, IL-5, the eosinophil-selective hemopoietic growth factor, is a weak chemoattractor by itself, but is required for the pulmonary eosinophilia induced by IL-13, a cytokine with a broad spectrum of targets (Pope et al., 2001). Eosinophils may also selectively accumulate in vivo, in response to the lipid mediators, Leukotriene (LT) B4, Platelet Activating Factor (PAF), and LTD4 (Tager et al., 2000; Giembycz and Lindsay, 1999). Strong evidence implicates the Leukotriene B(4) receptor (BLTR) in eosinophil recruitment to the peritoneal cavity induced by thioglycollate in mice (Tager et al., 2000). For PAF, the mechanisms underlying apparent eosinophil selectivity in vivo have yet to be defined. In the case of LTD4, secondary induction of the eosinophil-selective cytokine, IL-5, may play an important role. IL-5 also enhances the chemotactic response to LTB4 in vitro, and eosinophilia induced by LTB4 has been frequently documented in allergic subjects (Giembycz and Lindsay, 1999). In this study, we addressed the mechanism of the chronic eosinophilic inflammation induced in mice by subcutaneous egg

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white implants (EWI). This model involves surgical implantation of a piece of heat-coagulated hen egg white, which is followed by chronic, intense eosinophilic inflammation at the implant site, accompanied by a large increase in bone-marrow eosinophilopoiesis lasting several weeks (Xavier Elsas et al., 2004). EWI carriers, when challenged with ovalbumin in the airways, develop asthma-like manifestations, associated with intense eosinophil infiltration (Carvalho et al., 1999; Siqueira et al., 1997; Russo et al., 1997). Although much remains to be established concerning the mechanisms of this peculiar type of inflammatory reaction, it is clearly dependent on a protein antigen (ovalbumin), which must be administered in large amounts, although in insoluble form, which ensures a long half-life at the implant site. Because the development of delayed type hypersensitivity to proteins is similarly favoured by their inoculation as a mixture with Freund's adjuvant, which forms a persisting deposit, one would expect CD4+ cells, which are essential in delayed hypersensitivity (Teixeira et al., 2001), to play in important role in the EWI model as well. We assessed the contribution of CD4+ cells to eosinophil recruitment following ovalbumin challenge, by quantifying eosinophil migration into the peritoneal cavity of EWI carriers, and obtained evidence for an interaction between CD4+ cells and LTB4, in promoting selective eosinophil accumulation. Methods Animals and animal handling Male Balb/c mice, weighing 20–25 g, were used throughout the study. Where indicated, we used mice of the B6.129S2-C2tatm1Ccum/J strain (referred to as MH CII −/− mice hereafter), that are profoundly deficient in mature CD4+ T lymphocytes in the peripheral tissues, due to a mutation in the Class II transactivator gene that prevents expression of Class II molecules in dendritic cells and B cells (Fikrig et al., 1997). MH CII −/− mice were obtained from Jackson Laboratory (Bar Harbor, Maine, USA). Mice were handled following the standard procedures for the use of laboratory animals approved by the Department of Pharmacology, School of Medicine of Ribeirão Preto, University of São Paulo, Brazil. Egg white implants Dehydrated hen egg white was from Ito Avicultura (São Paulo, SP, Brazil). Pellets were prepared as previously described (Xavier Elsas et al., 2004), by dissolving hen egg white in distilled water, to a 100 g/l final concentration, and dispensing 45 µl per well in a 96-well plate (4.5 mg/well). Heat coagulation was carried out on the plate in a microwave oven (90 s at 900 Hz). Pellets were recovered and dehydrated in absolute ethanol for 48 h. The dehydrated pellets were allowed to dry at room temperature for 24 h and stored at 4 °C until use. Mice were anesthesized with 1,3,4-tribromoethanol (250 mg/kg, i.p.) and one pellet was implanted through a 0.5 cm surgical opening under the skin of the dorsum, before suture. Control (sham-implanted, SHAM) mice were submitted to the surgery, but received no implant. At days 15, 21, and 30 after surgery, different groups of animals were challenged with ovalbumin (3.3, 10 or 30 µg in a 0.2 volume), KLH (10 µg) or PBS (0.2 ml) (see Results for dose– response relationships). Animals were sacrificed by cervical dislocation at the indicated times after challenge i.p. (see Results for kinetics of the response). Drugs and drug treatments To evaluate the effect of different drugs on eosinophil migration, mice were injected with the indicated doses of each drug (see below) 1 h before challenge and 24 h after challenge with ovalbumin, and cell migration was evaluated 48 h after challenge (hence 24 h after the

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second drug injection). All drugs except MK 886 (see below) were injected s.c. in a 0.2 ml volume. Dexamethasone (Decadronal™, MSD) was dissolved in PBS and administered at 1 mg/kg; indomethacin (Sigma) was dissolved in Tris buffer and administered at 2 mg/kg; MK886 (Merck) was dissolved in 0.1% methylcellulose and administered orally at 1 mg/kg, in a 0.5 ml volume; CP 105.696, a potent and selective antagonist of the BLT1 receptor in humans (Murray et al., 2003) and mice (Kim et al., 2006), generously donated by Prof. M. M. Teixeira (Universidade Federal de Minas Gerais, Brazil), was dissolved in PBS containing 5% dimethylsulfoxide (DMSO), and administered at 3 mg/kg; BWA4C (Merck) was dissolved in PBS containing 10% DMSO, and administered at 20 mg/kg; BN 52021 (Institut Henri Beaufour), was dissolved in PBS and administered at 20 mg/kg; Thalidomide was dissolved in PBS containing 1% DMSO, and administered at 45 mg/kg; Aminoguanidine (RBI) was dissolved in PBS, and administered at 50 mg/kg; N-nitro-L-arginine (Sigma) was dissolved in PBS and administered at 50 mg/kg; Hoe 140 (Hoechst) was dissolved in PBS and administered at 2 mg/kg. Migration assays Leukocyte migration was evaluated at 6, 12, 24 and 48 h after challenge with OVA, KLH or PBS. Mice were sacrificed and their peritoneal cavities were washed with 3 ml of PBS/EDTA. The volumes recovered were comparable in all experimental groups and were approximately 95% of the injected volume. Total counts were performed in an automated cell counter (COULTER® Ac T 8; Beckman Coulter Corporation; Miami, Florida, USA). Differential cell counts were done on May–Grunwald-Giemsa stained cytocentrifuge smears under oil immersion (1000× magnification). Data are presented as percent eosinophils, neutrophils or mononuclear cells (which include both macrophages and lymphocytes). Cell separation, culture, transfer and analysis by flow cytometry For transfer experiments, peritoneal exudate cells were harvested from EWI carriers or from naive control mice, 15 days after surgery. Cells from individual donor mice were washed in RPMI 1640 medium (Gibco), pH 7.2, adjusted to 2.5 × 106 cells/ml, and stimulated with 10 µg/ml ovalbumin for 1 h, at 37 °C, in 5% CO2, 95% air. They were then washed 2×, resuspended in medium and injected i.p. in individual syngeneic recipient mice, which were either EWI carriers at day 15 or naive controls. In selected experiments, cells cultured as above were washed and further incubated for in vitro release of soluble mediators, for 6 h, at 37 °C, in a 15 ml tube, with constant stirring. Cell-free supernatants were collected and injected i.p. into recipient mice, which were either EWI carriers at day 15 or naive controls, keeping the ratio of one donor to one recipient. Where indicated, peritoneal cells that had been stimulated with ovalbumin as detailed above were submitted to further separation with the help of anti-CD4 (L3T4) antibody-coated magnetic beads using the MACS system (Magnetic Cell Sorting, Miltenyi Biotech, Germany). Cells were incubated with beads for 20 min at 4 °C, before passage through the column. Positively and negatively selected cells were washed, resuspended in medium and used either for immediate transfer or for further incubation to provide culture supernatants for transfer (as described above), keeping as above the one donor to one recipient ratio. In all cases, cell migration to the peritoneum was evaluated 48 h after transfer. The effectiveness of the separation procedure was evaluated by flow cytometry: positively and negatively selected cell populations were incubated (0.5 × 106 106 cells) in 10 µl of normal mouse serum for 20 min, washed and further incubated with fluoresceinated anti-CD4 antibody conjugated with FITC (10 µl at a 1:5 dilution) for 30 min at 4 °C. The cells were washed twice, resuspended in 1% paraformaldehyde-PBS), and analysed in a FACScan (Becton Dickinson), using an argon laser, in the 530–670 nm range.

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Mediator detection

Results

The concentrations of JE/CCL2 and RANTES/CCL5 in the supernatant of OVA-stimulated peritoneal cells obtained from EWI mice were detected by an in-house ELISA using reagents and standards from R&D Systems, USA. Briefly, microtiter plates were coated overnight at 4 °C with 1 µg/ml of an immunoaffinity-purified polyclonal sheep antibody against JE/CCL2 or against RANTES/CCL5. After blocking the plates, various dilutions of recombinant murine JE/ CCL2 or RANTES/CCL5 or duplicated samples were added and incubated overnight at 4 °C. Rabbit biotinylated immunoaffinitypurified pAb (0.5 µg/ml), anti-JE or anti-RANTES were added, followed by incubation at room temperature for 1 h. Finally, 50 µl of avidin-HRP conjugate (1:5000 dilution; DAKO A/S, Denmark) was added to each well; after 30 min the plates were washed and the chromogen OPD (200 µg/well; Sigma) was added. After 15 min, the reaction was stopped with 1 M H2SO4 and the optical density (O.D.) measured at 490 nm. The results were expressed as pg/ml of MCP-1 and RANTES, based on standard curves. The concentration of LTB4 in supernatants of OVA-stimulated peritoneal cells from EWI mice was measured by a specific EIA kit (Caymann Chemical, Ann Arbor, MI, USA), according to the manufacturer's instructions. Briefly, recombinant murine LTB4 standards at various dilutions and the samples were added to a microtiter plate pre-coated with mouse monoclonal antibody. Then, LTB4 acetylcholinesterase tracer and antiserum to LTB4 were added to each well. The plate was incubated at room temperature overnight. The plate was washed, 200 µl of substrate to acetylcholinesterase (Ellman's Reagent) added to each well, and the plate developed for 60–90 min. O.D. was determined at 405 nm, and the results were expressed as pg of LTB4/ml, based on a standard curve.

Selective eosinophil accumulation induced by ovalbumin challenge in EWI carriers

Reverse transcription (RT)-PCR analysis Total RNA was extracted from 2.5× 106 unseparated cells or from 106 CD4+ cells, stimulated or not with ovalbumin, using Trizol reagent (Life Technologies). Synthesis of first-strand cDNA was performed in 20 µl of reaction mixture containing 2 µg RNA, 1 µl dNTP 100 mM, 5 µl oligo (dT) 12–18 primer 0.5 mg/ml, 5 µl RNase inhibitor 40 U/ml, 5 µl AMV reverse transcriptase 25 U/l (Boehringer Mannheim, Mannheim, Germany), with an incubation at 65 °C for 7 min, followed by further incubation at 37 °C for 1 h. The reverse transcriptase was denatured at 90 °C for 5 min and the sample was cooled on ice. Sequences of the primers for the amplification were: a) for Eotaxin/CCL11, (sense) 5′-GCA GAG CTC CAC AGC GCT TC -3′, and (antisense) 5′-AGT CCT TGG GCG ACT GGT GT-3′; 399 bp, b) for JE/MCP-1/CCL2, (sense) 5′-CTC ACC TGC TGC TAC TCA TTC-3′, and (antisense) 5′-GCA TGA TGA GGT GGT TGT GAA AAA-3′; 320 bp c) for RANTES/CCL5, (sense) 5′-TGC CTC ACC ATA TGG CTC GG-3′, and (antisense) 5′-GGA CTA GAG CAA GCG ATG AC-3′; 245 bp, d) for TARC/ CCL17, (sense) 5′-CAG GAA GTT GGT GAG CTG GTA TA-3′, and (antisense) 5′-TTG TGT TCG CCT GTA GTG CAT A-3′), 301 bp. Reverse transcriptase reaction mixture was used in the polymerase chain reaction (PCR) in 20 µl final volume, 0.5 µl of each dNTP 100 mM, 2 µl of each primer 300 ng/l and 1.5 µl of Taq DNA polymerase 5 U/ml (Boehringer Mannheim). The mixture was incubated in a thermocycler using the following temperature profile: denaturation step at 94 °C for 4 min, followed by 35 cycles of denaturation at 94 °C for 45 s, annealing at 55 °C for 45 s, and extension at 72 °C for 45 s. The final extension step was 72 °C for 10 min. PCR samples were run on a 2% agarose gel stained with 10mg/ml ethidium bromide. Statistical analysis The data were analysed with the help of the Systat for Windows version 4 software, using factorial analysis of variance, and with the Tukey (HSD) correction for multiple comparisons between different treatments.

To define the optimal concentration of ovalbumin for inducing cell migration to the peritoneal cavity, both EWI mice and SHAM controls were used. Mice in each group were challenged, 15 days after surgery, with ovalbumin (3.3 µg, 10 µg or 30 µg) i.p. Cell numbers in the peritoneal lavage fluid were determined 48 h after challenge. As shown in Fig. 1, Panel A, a significant eosinophil migration was induced in EWI mice by challenge with 10 µg ovalbumin (P = 0.001, relative to SHAM controls). Challenge with 30 µg ovalbumin also induced significant eosinophil migration (P = 0.027). No significant difference in migration was observed between 10 and 30 µg ovalbumin (P = 0.069). However, the response of EWI mice to ovalbumin was significantly different between 10 and 3.3 µg (P = 0.008). By contrast, KLH induced no eosinophil migration, showing that the effects of challenge are specific for the sensitizing antigen. Therefore, for subsequent studies, we have used 10 µg ovalbumin, which was the lowest challenge dose that elicited a plateau response. To define the relationship between the duration of EWI-induced sensitization and the magnitude of eosinophil migration following challenge, both EWI and SHAM mice were challenged with ovalbumin at various time points after surgery (15, 21 and 30 days). As shown in Fig. 1, Panel B, eosinophil numbers in the peritoneal cavity were significantly higher in EWI mice (P b 0.001), relative to SHAM controls, at all three time points. Differences among EWI mice at these time points were not significant. Therefore, for subsequent studies, we have used 15 days after implantation as the earliest time point in which we had observed a plateau response in eosinophil migration. We have further evaluated the time course of eosinophil migration following challenge with the standardized dose. As shown in Fig. 1, Panel C, significantly increased eosinophil numbers were observed in the peritoneal cavity of EWI mice, relative to SHAM controls, 24, 48 and 72 h after challenge (P b 0.001 in all three cases). There was no significant difference between results at 48 and 72 h (which both differed significantly from results at 24 h), indicating that plateau levels were reached by 48 h, which was chosen as the observation time for the subsequent studies. Therefore, in these conditions (challenge with 10 µg per cavity, given 15 days after implant, and exudate collected 48 h later), ovalbumin induced significant eosinophil migration to the peritoneal cavity in EWI carriers, but not sham-implanted controls, providing evidence of recruitment through acquired immune mechanisms. By contrast, as shown in Table 1, the same protocol had no significant effect on the numbers of neutrophils attracted to the peritoneal cavity. The difference between eosinophils and neutrophils could not be accounted for by kinetic differences in migration, for, as shown in Table 2, no significant difference between ovalbumin-challenged EWI and SHAM mice with respect to the neutrophil numbers recruited to the peritoneal cavity was observed, at any of the time points. On the other hand, a significant difference (P b 0.01) was observed in mononuclear cell numbers, but only at 72 h. Hence, in this challenge protocol, out of the three leukocyte populations examined, only eosinophils were significantly increased following ovalbumin challenge, in EWI carriers relative to SHAM controls. Together, these data indicate that EWI induce long-lasting sensitization to ovalbumin, leading to selective eosinophil recruitment upon allergen exposure. Evidence for a role of specific immunity in eosinophil recruitment in this model was obtained in experiments with MHC II −/− mice, which have defective antigen-presenting cells, resulting in a lack of mature CD4+ T cells, along with suboptimal antibody production. As shown in Fig. 1, Panel D, there was no eosinophil recruitment following ovalbumin challenge in EWI-bearing MHC II −/− mice, in

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Fig. 1. Optimal conditions for eosinophil recruitment. Data are Mean ± SEM of the number of eosinophils in the peritoneal cavity of EWI and sham-implanted control mice. Panel A, mice were challenged 15 days post surgery with ovalbumin at the indicated doses, or with PBS or KLH (not shown). Cells were harvested 48 h after the challenge. Panel B, mice were challenged at the indicated times post surgery with 10 µg ovalbumin. Cells were harvested 48 h after challenge. Panel C, mice were challenged 15 days post surgery with 10 µg ovalbumin. Cells were harvested at the indicated times after challenge. In Panels A–C, ⁎indicates significant differences between EWI and sham-implanted control mice for the different challenge doses (A), sensitization times (B) and harvest times (C); differences among the control groups were not significant. Panel D, mice of the MH CII −/− strain or C57BL/ 6 controls received EWI and were injected i.p. with ovalbumin or PBS at day 15 after surgery. Cells were harvested 48 h after challenge. ⁎indicates significant differences between ovalbumin-challenged mice and the respective controls. Data are from groups of 6–9 (Panel A), 6–19 (Panel B), 4–8 (Panel C) and 5 (Panel D) mice.

contrast to control C57BL/6 mice, which fully responded to ovalbumin challenge (P = 0.001). An essential role for LTB4 To evaluate the contribution of inflammatory mediators to eosinophil migration induced by ovalbumin in the EWI model, EWI mice were treated, before ovalbumin challenge, with drugs targeting various inflammatory pathways. We evaluated the effects of inhibitors of the receptors for Bradykinin (Hoe-140) and Platelet Activating Factor (BN-52021), as well as with the inhibitors of NO production (aminoguanidine and nitroarginine). As shown in Fig. 2, there were no significant differences between animals treated with BN-52021 (P = 0.653), Hoe-140 (P = 0.883), Aminoguanidine (P = 0.936) or nitroar-

Table 1 Effect of ovalbumin challenge on migration of neutrophils and mononuclear cells to the peritoneal cavity of EWI and control mice

ginine (P = 0.303) and the respective controls treated with vehicle. Indomethacin was also ineffective in the same conditions (P = 0.332). By contrast, a set of drugs targeting leukotriene generation was very effective in blocking eosinophil recruitment by ovalbumin challenge: the 5-lipoxygenase activating protein inhibitor, MK-886; the LTB4 receptor antagonist, CP 105.696; and the 5-lipoxygenase blocker, BWA4C. Significant inhibition of eosinophil migration was observed after treatment with MK-886 (P = 0.001), BWA4C (P = 0.022) and with CP 105.696 (P = 0.017). These results suggest that eosinophil migration to the peritoneal cavity is dependent on leukotriene production. Because CP 105.696, a specific BLT1R antagonist (Kim et al., 2006), was effective, LTB4 is most likely to account for the observed effects. Because MK-886 was the most effective blocker among the various antileukotriene drugs tested, it was chosen for the subsequent experiments.

Table 2 Time course of the recruitment of neutrophils and mononuclear cells following challenge with ovalbumin Time after challenge

Ovalbumin dose

Neutrophils (×10− 6) Control EWI Mononuclears (× 10− 6) Control EWI

24 h

3.3 µg

10 µg

30 µg

0.33 ± 0.08 0.55 ± 0.19

0.45 ± 0.07 0.60 ± 0.15

0.69 ± 0.11 0.31 ± 0.04

4.78 ± 1.4 5.23 ± 0.81

4.66 ± 0.69 5.51 ± 1.03

5.81 ± 2.50 5.64 ± 0.51

Neutrophils (× 10− 6) Control EWI Mononuclears (×10− 6) Control EWI ⁎P b 0.01.

48 h

72 h

2.5 ± 0.26 3.9 ± 0.5

0.45 ± 0.07 0.60 ± 0.15

0.10 ± 0.03 0.9 ± 0.02

3.64 ± 0.31 4.59 ± 0.56

4.66 ± 0.69 5.51 ± 1.03

4.40 ± 0.44 6.8 ± 0.8⁎

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6 h (P = 0.02). By contrast, supernatants from CD4+-depleted cells stimulated with ovalbumin were ineffective (P = 0.913). Together, these observations imply that eosinophil accumulation in EWI carriers is initiated by CD4+ cells, depending on reexposure to the priming antigen and on a hemopoietic system able to provide a supply of mature eosinophils. Importantly, the contribution of CD4+ cells may be primarily to secrete soluble mediators, since these duplicate the effects of cell transfer. Requirement for LTB4 during CD4+ restimulation

Fig. 2. Effect of targeting inflammatory pathways on eosinophil recruitment following ovalbumin challenge. Data are Mean ± SEM of the number of eosinophils recovered from the peritoneal cavity of EWI mice or sham-implanted control mice, challenged with 10 µg of ovalbumin 15 days after surgery. EWI mice were pretreated with antiinflammatory drugs (as detailed in Methods), or with vehicle, as indicated. Cells were harvested 48 h after the challenge. Experimental groups included 4–20 animals. ⁎indicates significant differences between mice exposed to different drug treatments and the vehicle-treated control group.

As also shown in Fig. 2, dexamethasone very effectively (P = 0.001) inhibited eosinophil accumulation following allergen challenge. We have compared the effects of dexamethasone, which inhibits production of a number of inflammatory mediators, including leukotrienes and cytokines, with those of thalidomide, which selectively downregulates production of Tumor Necrosis Factor-α. Unlike dexamethasone, thalidomide was ineffective (P = 0.350), suggesting that synthesis of TNF-α is not required for eosinophil recruitment.

We have further explored the relationship between the effects of MK886 and the generation of eosinophil-recruiting mediators during restimulation of specific CD4+ cells, because: a) ovalbumin-specific CD4+ cells and their products recruit eosinophils after transfer; and b) MK886 and dexamethasone, which share the ability to suppress LTB4 production, inhibit ovalbumin-induced eosinophil accumulation. Peritoneal cells, cultured in medium alone for 1 h, released detectable amounts of LTB4; this production was significantly increased in the presence of ovalbumin (P b 0.05; Fig. 4, Panel A). As shown in Fig. 4, Panel B, both MK886 and dexamethasone, when present at this step, could prevent the subsequent eosinophil recruitment induced by transfer of ovalbumin-restimulated cells (P b 0.05). As further shown in Fig. 4, Panel C, when MK 886 was present in the culture only during the 6 h period of media conditioning (as opposed to the initial 1 h restimulation step), eosinophil recruitment into the peritoneal cavity in the recipients following transfer of the conditioned media was still significantly decreased, relative to the control supernatants (P = 0.047 for the

The role of antigen-specific CD4+ cells and their soluble products We have further analysed the role of the ovalbumin-specific immune response in the recruitment of eosinophils. To do so, we have collected peritoneal cells from EWI donors and cultured them for various periods of time in the presence or absence of ovalbumin. Ovalbumin-restimulated and -unstimulated cells were washed and: a) transferred into the peritoneal cavity of EWI carriers or of naive controls; b) further cultured for production of conditioned media, which were subsequently used for transfer to EWI recipients. As shown in Fig. 3, Panel A, when cells from EWI donors were transferred to EWI recipients, significant accumulation of eosinophils in the recipients' peritoneal cavities was induced by transfer of ovalbuminrestimulated donor cells, but not unstimulated control cells (P b 0.001 for the difference between these groups). Importantly, transfer of either cell population to naive control mice was ineffective, showing that the transferred cells can only act if the recipient has been primed by the implant to respond to this challenge by allowing eosinophils to accumulate in the challenge site. Furthermore, transfer of ovalbuminrestimulated, CD4+-enriched cells induced significant eosinophil accumulation in the peritoneal cavity (P b 0.001). By contrast, transfer of ovalbumin-restimulated, CD4+-depleted cells, or of CD4+-enriched cells cultured in medium alone (not shown), was ineffective (P = 0.601). As shown in Fig. 3, Panel B, these effects could be duplicated by transfer of conditioned media from the corresponding cell populations. Significant recruitment of eosinophils was induced by transfer of supernatants from ovalbumin-stimulated peritoneal cells from EWI donors, but not from unstimulated cultures (P b 0.001). No recruitment was observed after transfer of conditioned media to naive recipients. The effects of ovalbumin-stimulated CD4+-enriched cells could also be duplicated by their cell-free media, harvested after conditioning for

Fig. 3. Role of CD4+ cells and their products in eosinophil recruitment. Peritoneal exudate cells from EWI donor mice were cultured with ovalbumin (Ova), or Medium, for 1 h, washed, and either used for immediate transfer (Panel A) and further culture for 6 h (Panel B), or separated into CD4+-enriched (CD4+) and -depleted (CD4−) subpopulations before transfer (Panel A) or before further culture followed by transfer (Panel B). Cells were unseparated (Total), CD4+-depleted (CD4−) or CD4+-enriched (CD4+). Recipients of cells and conditioned media were either naïve (open bars) or EWI carriers (closed bars). Data are Means ± SEM of the number of eosinophils in the peritoneal cavity of recipient mice, 48 h after transfer of either cells or culture supernatants. Data are from groups of 4–9 animals (Panel A) and 4 animals (Panel B). ⁎indicates significant differences between each group and the medium-incubated EWI control.

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Fig. 4. Role of LTB4 during antigen restimulation. Peritoneal exudate cells from EWI donor mice were cultured with ovalbumin (−), or medium, for 1 h, washed, and either used for immediate transfer (Panel B) and further culture for 6 h (Panels A and C), or enriched in CD4+-cells before further culture (Panel D). Where indicated, MK886 or Dexamethasone was present during antigen restimulation (Panel B) or during subsequent culture (Panels C and D). ⁎indicates significant differences relative to the respective control cultures. Data are Means ± SEM of the concentration of LTB4 in conditioned medium (Panel A) or of the number of eosinophils in the peritoneal cavity of recipient mice, 48 h after transfer of either cells (Panel B) or culture supernatants (Panels C and D). Data are from groups of 5–6 animals.

difference between drug-treated and untreated cultures). Similar observations were made when dexamethasone was added to the cultures, instead of MK886 (P = 0.014 for the same comparison). Finally, as shown in Panel D of the same figure, when CD4+ cells were isolated after antigen restimulation, and incubated in the presence of MK886 during the 6 h period of medium conditioning, the ability of the conditioned media to recruit eosinophils upon transfer was significantly reduced, relative to the controls (P b 0.05). Inhibition of eosinophil migration in recipients of restimulated cells could not be accounted for by carryover of MK886, because it was eliminated by washing before cell transfer. In a separate control experiment (not shown), dexamethasone, if added only during the last hour of antigen restimulation ex vivo, failed to inhibit eosinophil recruitment (P = 1.000); this ruled out the carryover of dexamethasone as the cause of decreased eosinophil migration. Taken together, the data show that during ex vivo restimulation of peritoneal exudate cells with ovalbumin: a) LTB4 is produced; b) exposure to MK886 or dexamethasone is accompanied by a reduction in the ability of the drug-treated cells to recruit eosinophils upon transfer; c) MK886 inhibits the generation of the active conditioned media by both unseparated and CD4+-enriched cells, with comparable effectiveness. Chemokine generation following ovalbumin challenge and reduction of chemokine effects by MK886 Because eosinophil migration can be induced by a variety of chemokines, we evaluated whether ovalbumin challenge induced chemokine production in vivo. EWI carriers were injected with vehicle or ovalbumin and chemokines were quantified in the peritoneal lavage fluid. As shown in Fig. 5, both CCL2 (JE/MCP-1; Panel A) and CCL5 (RANTES; Panel B) were selectively induced by in vivo challenge. Because MK886 rendered ovalbumin challenge ineffective, we further

evaluated whether it interfered with eosinophil recruitment in response to chemokines. EWI carriers were pretreated with vehicle or with MK886, and injected with CCL11 (eotaxin; 100 ng/cavity), CCL2 (JE/MCP-1; 30 ng/cavity) or CCL5 (RANTES; 30 ng/cavity). Selective eosinophil accumulation was observed 48 h later, in response to all three chemokines. In all three cases, it was effectively prevented by MK886 pretreatment (P b 0.05), indicating that their in vivo activity is dependent on LTB4 production. We have also evaluated the alternative possibility that MK886 affected chemokine generation, as opposed to chemokine activity, by monitoring the expression of CC chemokines in peritoneal cells from EWI carriers by RT-PCR. Peritoneal cells (unseparated, CD4+ and CD4+depleted) were collected and cultured in the absence or in the presence of ovalbumin. Where indicated, MK886 was present during restimulation. As shown in Fig. 6, expression of mRNA for the 4 different chemokines tested was easily detectable in CD4+-enriched cells stimulated with antigen. By contrast, for 3 out of 4 (eotaxin/CCL11, JE/CCL2 and TARC/CCL17), expression was much weaker in CD4+depleted cells in the same conditions. This suggests that CD4+ cells are the main source of eosinophil-selective chemokines in the unseparated peritoneal exudate, with little or no contribution from the CD4− cells. The intensity of the signals observed in the unseparated cell population was consistent with this hypothesis, the strongest signals approaching those observed with the enriched population, for 3 out of 4 chemokines (JE/CCL2, RANTES/CCL5 and TARC/CCL17). Overall, however, the relationship between mRNA expression and antigen reexposure ex vivo was inconsistent. Eotaxin/CCL11 message was detectable only in antigen-restimulated CD4+-enriched cells. Message for TARC/CCL17 was present in antigen-restimulated cells, both unseparated and CD4+-enriched; by contrast, it was absent from medium-stimulated controls, as well as from CD4+-depleted cells stimulated with antigen. The expression of TARC/CCL17, unlike

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Fig. 5. Effect of MK 886 in chemokine-induced eosinophil accumulation. EWI carriers or naïve controls were injected with PBS or with ovalbumin and JE/CCL2 (Panel A) and RANTES/ CCL5 (Panel B) were quantified in the peritoneal lavage fluid. Panel C, EWI carrier mice were injected with PBS, or pretreated with MK886 or with vehicle, followed by i.p. administration of recombinant eotaxin/CCL11 (100 ng/cavity), JE/CCL2 (30 ng/cavity) or RANTES/CCL5 (30 ng/cavity). Data are Means ± SEM of chemokine concentrations (Panels A and B) or of the number of eosinophils in the peritoneal cavity of recipient mice, 48 h after administration of chemokines (Panel C). Symbols (⁎,⁎⁎) indicate significant differences relative to controls (PBS-injected or chemokine-injected, respectively). Data are from groups of 3 to 5 mice.

expression of eotaxin/CCL11, was inhibited when MK886 was present during restimulation. A third expression pattern was presented by mRNA for RANTES/CCL5 and JE/CCL2: these were clearly detectable in all samples, CD4+ or CD4+-depleted, regardless of antigen restimulation. Messages for these chemokines were not affected by the presence of MK886. Together, these observations support the hypothesis that MK886 interferes with eosinophil recruitment because LTB4 is required for the effectiveness of chemokines in vivo. By contrast, they do not support the idea that the effects of MK886 during antigen restimulation are due to a universal inhibition of chemokine gene expression.

Fig. 6. Chemokine mRNA in unseparated and CD4+ cells. Peritoneal exudate cells from EWI donor mice were cultured with ovalbumin (Ova), or medium (C, for control), for 1 h, washed, and used either immediately (left) or after enrichment/depletion in CD4+-cells (right) for isolation of mRNA, followed by RT-PCR analysis. Where indicated, MK886 was present during antigen restimulation (left). Data are from one experiment representative of 3. Message for the housekeeping gene for β-actin was monitored as an internal control.

Discussion The EWI model has features uniquely favourable for studying eosinophil production (Xavier Elsas et al., 2004) and migration (Siqueira et al., 1997) in allergic reactions. We have addressed the mechanisms involved in selective eosinophil migration to a challenge site, and obtained strong evidence of a role for cellular immunity in promoting eosinophil accumulation. Our arguments are: a) transfer of restimulated cells replaces direct challenge; b) CD4+ cells carry out the critical step; c) products secreted by CD4+ cells replace the latter in transfer protocols; d) the role of antigen is to activate the CD4+ cells and induce production of the relevant mediators, for in the absence of sensitization the carryover of antigen is irrelevant. Our observations are consistent with previous studies showing that eosinophil recruitment to delayed hypersensitivity sites was abolished by depletion of CD4+ cells (Teixeira et al., 2001), and that CD4+ cells promote eosinophilic, (but not neutrophilic), infiltration of the cornea, in experimental Onchocerca volvulus keratitis (Hall et al., 2000). We have gone beyond these previous studies, however, by transferring CD4+ cells and dissecting the contributions of different mediators released in response to antigen reexposure. Neither antigen challenge nor transfer of antigen-activated CD4+ cells were effective in naive hosts. This is most likely due to the lack of a sizeable pool of circulating eosinophils that can be recruited into the challenge site. In EWI carriers, unlike naïve controls, there is intense and long-lasting stimulation of eosinophilopoiesis, which provides the required eosinophil supply (Xavier Elsas et al., 2004). This is also consistent with the observation that upregulated eosinopoiesis is essential for eosinophil recruitment into delayed hypersensitivity reaction sites (Teixeira et al., 2001). The failure of B2 bradykinin receptor inhibitor Hoe-140 to suppress eosinophil recruitment in EWI carriers suggests that the mechanisms involved differ from those described in a more conventional model of allergic sensitization (Eric et al., 2003). Also, in contrast to our study, inhibition of the lipoxygenase pathway did not prevent eosinophilic

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infiltration in guinea pig lungs, while PAF receptor blockade was effective (Myou et al., 2001). Finally, indomethacin had no effect in the EWI model, ruling out the involvement of PGD2 described in a study with humans (Hirai et al., 2001). Together, these responses to pharmacological blockade set the EWI model apart from previously described systems, justifying further exploration. We have observed a critical contribution of LTB4 in this model, as shown by the coherent results obtained with a highly specific BLT1 receptor antagonist (Kim et al., 2006), a 5-lipoxygenase inhibitor, and a FLAP inhibitor. This is consistent with the pioneering work of Tager et al. (2000), who demonstrated BLTR-dependent eosinophil recruitment in vivo. In that study, it is remarkable that recruitment of neutrophils and macrophages was not significantly affected by BLTR inactivation, possibly due to the simultaneous operation of several chemoattractant pathways. Hence, the presence of BLTR in any leukocyte population was not the sole determinant of its migration in their model, just as in our study LTB4 was shown to be important, but not the sole determinant of eosinophil migration. Quite the opposite, as discussed below, its effects can only be understood with reference to other chemoattractant stimuli, especially chemokines. Using drug treatments in combination with transfer protocols, we showed that MK886 acts at an early step, to abort production of the relevant soluble mediators MK886 (and dexamethasone) is effective even if present only during the initial activation, being subsequently eliminated by washing. In medium-conditioning protocols, MK886 and dexamethasone were effective even after the initial activation had occurred in their absence. Neither drug acted in the recipient of cells or of conditioned media, however, as shown by a variety of carryover control experiments. Because the activated cells could be fully replaced by their secreted products, we assume the effects of MK886 to reflect the blockade of secretion of a critical soluble mediator, which again would be consistent with LTB4. In unseparated peritoneal cells, there are several cell types that produce LTB4 efficiently, including macrophages, mast cells and eosinophils. Because allergen challenge increases the amount of LTB4 produced, one possible interpretation is that antigen-activated CD4+ lymphocytes enhance LTB4 production by other cells, if they do not produce it themselves. Alternatively, in the presence of specific antibody, induced by the implant, ovalbumin might trigger LTB4 secretion by mast cells bearing cytophilic IgE and IgG1, or by macrophages and eosinophils which bind immune complexes. MK886 was effective when CD4+-enriched cells were cultured. This suggests that this population still contains enough LTB4-producing cells (CD4+ or CD4−) to account for the observed effect. Our findings are compatible with a model consisting of a few critical steps: 1. LTB4 is generated by some cell type other than CD4+ T cells at the challenge site; 2. CD4+ T cells are recruited to the challenge site by LTB4, presumably depending on their expression of BLT1R (Tager et al., 2003); 3. CD4+ T cells, as a result of being restimulated by ovalbumin in situ, secrete chemokines; 4. Chemokines attract eosinophils. However, this model should be refined to include contributions of both antigen and LTB4 at several steps, because: a) in our experiments, peritoneal cells were harvested from unchallenged EWI carriers and restimulated ex vivo. Therefore, the CD4+ cells we studied, which can recruit eosinophils when transferred to syngeneic recipients, must be present in the peritoneal cavity, even though the donors were not challenged with ovalbumin. This could be the case, if LTB4 production, and the resulting CD4+ T cell migration, had already taken place in vivo when peritoneal cells were harvested for ex vivo restimulation. This possibility is consistent with our observation that peritoneal cells did release LTB4 even without reexposure to ovalbumin. Importantly, however, this ex vivo LTB4 production was significantly enhanced by ovalbumin. Therefore, even if, in the transfer protocol, sufficient LTB4 may be generated in vivo to recruit CD4+ cells to the

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unchallenged peritoneum, in the directly challenged EWI carriers eosinophil recruitment may be enhanced by the effect of ovalbumin on LTB4 production, with a resulting increase in CD4+ T cell numbers at the challenge site. b) we have evidence of the involvement of LTB4 after CD4+ cells are recruited to the challenge site. In our transfer protocol, eosinophil recruitment is limited by the presence of ovalbumin to activate CD4+ cells. However, LTB4 is likely to play an important role at this step as well (Fig. 4, Panels B–D). Finally, LTB4 seems to be required for the full effectiveness of preformed, exogenous chemokines in recruiting eosinophils. We have shown that JE/CCL2 and RANTES/CCL5 are produced following allergen challenge of EWI carriers, and confirmed that mRNA for both ligands, and for eotaxin/CCL11, can be found in CD4+enriched peritoneal cells. Importantly, in the EWI model, JE/CCL2 did recruit eosinophils with an effectiveness comparable to that of eotaxin/CCL11. This is consistent with the expression of CCR2 in murine eosinophils (Oliveira et al., 2002) as well as the ability of antiCCR2 antibody to reduce eosinophil infiltration in vivo in nonhuman primates (Mellado et al., 2008). Similar effectiveness was demonstrated for RANTES/CCL5. Importantly, this result would not be predicted from the chemotactic response of murine eosinophils to either ligand in vitro (Borchers et al., 2002; Oliveira et al., 2002), despite the chemotactic responses to both chemokines described in humans (Borchers et al., 2002; Dunzendorfer et al., 2001). Detection of chemokine-induced eosinophil migration critically depends on experimental conditions, especially the presence of inflammatory cytokines (Oliveira et al., 2002). Because the effectiveness of RANTES/ CCL5, JE/CCL2 and eotaxin/CCL11 in the EWI model was strikingly reduced by MK886 pretreatment, our study confirms the importance of in vivo interactions between chemokines and other inflammatory mediators in eliciting eosinophil recruitment. One issue that needs further investigation in this model is the relationship between antigen challenge and chemokine production, as detected by molecular biological methods (as opposed to immunoassays). JE/CCL2 and RANTES/CCL5 were induced by antigen challenge in vivo, and their levels are undetectable in the unchallenged peritoneal cavity. This would suggest that actual chemokine secretion is strictly dependent on antigen stimulation. As a consequence, the presence of chemokine mRNA in the absence of allergen restimulation does not automatically imply secretion of the corresponding gene product in this condition. The very low eosinophil counts in unchallenged animals are consistent with the idea that eosinophil-recruiting chemokines are adaptively (not constitutively) released in the peritoneal cavity. Hence, at different steps, LTB4 may play different roles, namely: a) a direct chemoattractant effect on eosinophils (Tager et al., 2000); b) a synergic interaction with eosinophil-recruiting chemokines; or c) a permissive role for chemokine activity in vivo. A direct chemoattractant action of LTB4 is unlikely, because LTB4 is produced by the unchallenged peritoneal cells of EWI carriers, who produce large numbers of eosinophils (Xavier Elsas et al., 2004), but in the absence of challenge no significant eosinophil accumulation is detectable. Furthermore, on the other hand, synergism is more likely to occur when limiting amounts of the active chemoattractant are present. However, considerable amounts of JE/CCL2 and RANTES/CCL5 were induced by allergen challenge. Exogenous JE/CCL2, RANTES/CCL5 and eotaxin/CCL11 very effectively recruited eosinophils to the peritoneum. Nevertheless, in these conditions, in which LTB4 should not be required as a synergistic factor, MK886 effectively reduced recruitment. So, it is possible that LTB4 is permissive, for instance, by acting at the activation step that is required for migration through endothelial surfaces (Broide and Srimarao, 2001). Our findings raise the possibility that, during restimulation, both chemokines and LTB4 are induced, the latter acting as a permissive factor for the actions of the former.

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