* Immunopharmacology, Departamento de Bioquímica e Imunologia, ICB, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil;
Department of Pharmacology, Faculty of Medicine of Ribeirão Preto, São Paulo, Brazil; and
Department of Pathology, University of Michigan Medical School, Ann Arbor
Correspondence: Mauro Martins Teixeira, Departamento de Bioquímica e Imunologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Av. Antonio Carlos, 6627—Pampulha, 31270-901 Belo Horizonte MG, Brazil. E-mail: mmtex{at}icb.ufmg.br
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Key Words: chemokines • eotaxin (CCL11) • PAF • migration
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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There is much evidence suggesting an important role for eosinophils in the pathophysiology of allergic disease [5 6 7 ]. In allergic disease, such as asthma, eosinophils are a crucial source of cytotoxic proteins, lipid mediators, oxygen metabolites, and cytokines, which contribute to severity of disease [8 ]. Thus, an understanding of mechanisms involved in the recruitment of eosinophils may permit new therapies for eosinophil-associated diseases. Eosinophil recruitment in sites of allergic inflammation depends on the concerted action of a variety of molecules including the chemokines, such as CCL11 (eotaxin) and CCL5 [regulated on activation, normal T expressed and secreted (RANTES)], and lipid mediators, such as leukotriene B4 (LTB4) and platelet-activating factor (PAF) [9 10 11 12 13 14 ]. Recently, CCL22 was shown to be an activator of human eosinophil chemotaxis [15 ]. In this study, we have investigated the effects of CCL22 on the recruitment of murine eosinophils in vivo and in vitro and whether CCL22 works directly or indirectly to mediate eosinophil recruitment. Finally, the effects of anti-CCL22 antibodies on the recruitment of eosinophils in response to antigen challenge in sensitized mice were also investigated.
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Drugs and reagents
Recombinant murine (rm)CCL22 was purchased from Peprotech (London, UK). CCL22 was diluted in phosphate-buffered saline (PBS; pH 7.4) containing 0.01% bovine serum albumin (BSA) and was stored at -70°C until use. BSA, ovalbumin (OVA), and control rabbit serum were purchased from Sigma Chemical Co. (St. Louis, MO). The LTB4 antagonist CP105,696 and the specific and long-acting PAFR antagonist UK-74,505 were a gift from Pfizer Global Research and Development (Kent, UK). CP105,696 was dissolved in dimethyl sulfoxide and further diluted in PBS. UK-74,505 was dissolved in HCl 0.01N and further diluted in PBS. Control animals received drug vehicle.
Anti-CCL22 and anti-CCL11 antibodies
Rabbit anti-CCL22 antibody and anti-CCL11 antibody were prepared by multiple-site immunization of New Zealand white rabbits with rmCCL22 or CCL11 (R&D Systems, Minneapolis, MN) in complete Freund’s adjuvant. Polyclonal antibodies were titered by direct enzyme-linked immunosorbent assay (ELISA) and specifically verified by failure to cross-react to m interleukin (IL)-3, mIL-1
, m tumor necrosis factor (TNF-), CCL3 [m macrophage-inflammatory protein-1 (MIP-1)], IL-6, CCL2 [m homologue of monocyte chemoattractant protein-1 (MCP-1)], CCL8 (mMIP-1ß), CCL2 [human (h)MCP-1], CXCL8 (hIL-8), hRANTES, hMIP-1, hTNF. The immunoglobulin G (IgG) portion of the serum was purified over a protein A column and stored at -20°C in PBS until use.
Sensitization
Animals were immunized with OVA adsorbed to an aluminum hydroxide gel, as described previously [17
]. Briefly, mice were injected subcutaneously (s.c.) on days 1 and 8 with 0.2 ml solution containing 100 µg OVA and 70 µg aluminum hydroxide (Reheiss, Dublin, Ireland).
Leukocyte migration into the pleural cavity induced by CCL22 or antigen
CCL22 (10–100 ng/cavity) was injected intrapleurally (i.pl.) in naive WT or PAFR-/- mice, and animals were killed at 24 h after the i.pl. injection. Sensitized mice were challenged with antigen (OVA) or PBS. The cells present in the pleural cavity were harvested by injecting 2 ml PBS and total cell counts performed in a modified Neubauer chamber using Turk’s stain. Differential cell counts were performed on cytospin preparations (Shandon III) stained with May-Grumwald-Giemsa using standard morphologic criteria to identify cell types. The results are presented as the number of cells per cavity.
Anti-CCL22 or anti-CCL11 pretreatment
The role of endogenous chemokines on eosinophil recruitment induced by CCL22 or in the allergic pleurisy was investigated by using anti-CCL22, anti-CCL11, or control IgG. The IgG fraction of a rabbit polyclonal anti-CCL22 antibody or anti-CCL11 antibody or the IgG portion of serum of preimmune rabbits was prepared purified over a protein A column, as described previously [18
]. Anti-CCL22 was administered intraperitoneally (i.p.) at the dose of 100 µg/mouse 60 min before the i.pl. administration of CCL22 or OVA, and anti-CCL11 was administered i.p. at the dose of 100 µg/mouse 60 min before the i.pl. administration of CCL22. Control animals received preimmune IgG.
LTB4 receptor antagonist and PAFR antagonist pretreatment
To investigate the role of LTB4 and PAF on the eosinophil recruitment induced by CCL22, the LTB4 receptor antagonist CP105,696 (3 mg/Kg) or the PAFR antagonist UK-74,505 (1 mg/Kg) was administered i.p. 60 min before the stimulus. Control animals received drug vehicle.
Antigen elicited peritoneal eosinophil purification
Eosinophils were induced by the injection of thioglycolate plus soluble egg antigen (SEA) into the peritoneum of Schistosoma mansoni-infected mice. SEA was prepared in our laboratory by grinding isolated eggs from heavily infected S. mansoni-infected mice as described previously [19
]. This injection induces a pool of circulating eosinophils recruited into the peritoneum in an antigen-specific manner. After 48 h, the peritoneum of mice was lavaged, and cells were collected. The initial population that is isolated from the peritoneum is 
50% eosinophils with only 2–5% neutrophils and 35–45% mononuclear cells (lymphocytes and macrophages). Adherent cell populations were removed from the population by plastic adherence in tissue-culture dishes for 1 h. The nonadherent cells were washed and resuspended in PBS/BSA (90 µl PBS/BSA per 107 cells), and eosinophils were purified by negative immunomagnetic bead-coupled antibodies to exclude contaminating immune cells using the magnetic cell sorter (MACS) system. The antibodies used were anti-Thy1 (for T cells), anti-B220 (for B cells) and anti-class II (for antigen-presenting cells). After the plate adherence and MACS separation, the population of cells contained >97% eosinophils with contaminating neutrophils (1%) and mononuclear cells (1–2%).
In vitro chemotaxis
Eosinophil migration was quantitated by a modification of a Boyden chamber technique, as described previously [20
]. Purified eosinophils were suspended at 3 x 106 cells/ml in Dulbecco’s PBS plus 0.5% BSA and were placed in the top wells of the microchemotaxis chamber. Bottom wells were filled with CC chemokines (CCL11 and CCL22) in the final concentration of 10 and 100 ng/ml, respectively, or assay medium as negative control. A 5-µm pore-size polycarbonate filter separated the upper wells containing the cells from the control and chemokine samples in the bottom wells. The chambers were incubated for 1 h at 37°C in a 5% CO2 moist atmosphere, and the filters were then carefully scraped of nonmigrating cells, fixed with methanol, and stained with Diff-Quik. Eosinophil migration was quantitated by counting the number of eosinophils migrating completely through the matrix-coated filter in 10 high-powered fields (HPF) in triplicate samples. The data are expressed as the average number of countable adherent cells per HPF (±SEM).
Eosinophil degranulation in vitro and in vivo
To analyze eosinophil peroxidase (EPO) release in vitro, purified murine eosinophil cells were resuspended in phenol red-free Dulbecco’s modified Eagle’s medium and activated with chemokines (CCL11 and CCL22 in the final concentration of 10 and 100 ng/ml, respectively, or assay medium as negative control) for 4 h. The supernatant from the activated cells (2x106 eosinophils) was harvested, and the EPO level in the cell-free supernatant was determined as described previously [21
]. Eosinophil viability, as assessed by trypan blue exclusion, was consistently greater than 98% in control eosinophils or after stimulation by CCL11 or CCL22. To evaluate eosinophil degranulation in vivo, sensitized mice received an i.pl. injection of CCL22 or CCL11, 48 h after the challenge of the pleural cavity with antigen. After a further 2 h, cell-free supernatants were obtained from pleural cavity washes, and EPO levels were evaluated as described previously [21
]. Briefly, o-phenyldiamine (OPD) (10 mg) was dissolved into 5.5 ml dH2O, and 1.5 ml OPD solution was added to 8.5 ml of a Tris buffer (pH 8.0) followed by the addition of 7.5 µl H2O2. Using a 96-well plate, 100 µl substrate solution was added to a 50 µl sample. After 30 min, the reaction was quenched with 50 µl 4 M H2SO4, and the absorbance was read at 490 nm. The relative increase in samples was then compared. As a positive control, eosinophils (2x106 cells) were sonicated and measured as total EPO. A negative control was used by sonicating neutrophils (2x106).
Statistical analysis
All results are presented as the mean ± SEM. Normalized data were analyzed by one-way ANOVA, and differences between groups were assessed using Student-Newman-Keuls’ post-test. A P value <0.05 was considered significant.
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Figure 1. Dose-response (A) and time-course (B) effects of CCL22 on the recruitment of eosinophil to the pleural cavity of mice. For the dose-response experiments (A), CCL22 was administered at the indicated doses, and the number of infiltrating eosinophil was assessed after 24 h. For time-course experiments (B), 100 ng CCL22 was administered i.pl., and eosinophil recruitment was assessed 4, 24, 48, and 72 h after injection. The results are expressed as means ± SEM of five mice in each group. *, P < 0.05 when compared with controls.
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Table 1. Total and Differential Cell Counts (x105 Cells/Cavity) in Response to the i.pl. Injection of CCL22 in Naïve Micea
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Figure 2. Effects of the LTB4 receptor antagonist CP105,696 and PAFR antagonist UK-74,505 on the eosinophil recruitment induced by CCL22. Mice were pretreated with CP105,696 (CP; 3 mg/Kg) and UK-74,505 (UK; 1 mg/Kg) i.p. 60 min before the i.pl. injection of CCL22 (100 ng/cavity) in naïve mice, and the number of infiltrating eosinophil was assessed after 24 h. The control group of mice was injected with vehicle. The results are expressed as means ± SEM of five mice in each group. *, P < 0.05 when compared with PBS; #, P < 0.01 when compared with vehicle-treated animals.
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Figure 3. Effects of an anti-CCL11 polyclonal antibody on the recruitment of eosinophils induced by CCL22. Mice were pretreated with nonimmune IgG (400 µg, i.p.) or purified anti-CCL11 polyclonal antibody (400 µg, i.p.) 60 min before the i.pl. injection of CCL22 (100 ng/cavity) in naïve mice, and the number of infiltrating eosinophils was assessed after 24 h. The control group of mice was injected with PBS. The results are expressed as means ± SEM of six mice in each group. *, P < 0.01 when compared with PBS.
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Figure 4. Effects of anti-CCL22 pretreatment on the recruitment of eosinophil induced by antigen (Ova) challenge in sensitized mice or CCL22. Anti-CCL22 (hyperimmune Ig, 100 µg/animal) or nonimmune, purified Ig (100 µg/animal) was injected i.pl. 1 h before the i.pl. injection of CCL22 (100 ng/cavity) or OVA (1 µg/cavity), and the number of infiltrating eosinophils was assessed after 24 h injection of MDC and after 48 h injection of OVA. The results are expressed as means ± SEM of five mice in each group. *, P < 0.05 when compared with PBS; #, P < 0.01 when compared with animals injected with nonimmune IgG.
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(IFN-) and IL-5 in culture supernatants were measured by specific ELISA. Treatment with anti-CCL22 had little effect in the concentration of these cytokines in culture supernatant (IL-5, IgG control, 793.82±80.64 pg/ml; anti-CCL22, 832.93±39.78 pg/ml; IFN-, IgG control, 1018.03±96.12 pg/ml; anti-CCL22, 1837.95±178.4 pg/ml; P>0.05, n=4). Moreover, the antibody failed to cause any variation of IgG1 titers measured in the serum of these animals (IgG control, 0.144±0.02; anti-CCL22, 0.155±0.01, absorbance at 492 nm; P>0.05, n=4).
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Figure 5. Effects of treatment with anti-CCL22 antibodies during the sensitization phase on recruitment of eosinophil induced by OVA challenge in sensitized mice. Anti-CCL22 was injected 1 h before and 24 h after s.c. injection of OVA, and the number of infiltrating eosinophils was assessed after 48 h challenge with OVA. The results are expressed as means ± SEM of five mice in each group. *, P < 0.05 when compared with controls.
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Figure 6. Effects of CCL22 and CCL11 on the chemotaxis of antigen-elicited murine eosinophils. CCL22 and CCL11 were used at the concentrations of 10 or 100 ng/ml. Purified eosinophils were incubated for 1 h at 37°C in a 5% CO2 moist atmosphere with chemokines. Eosinophil migration was quantitated by counting the number of eosinophils migrating completely through the matrix-coated filter in 10 HPF. The results are expressed as the means ± SEM of triplicate determinations and are representative of at least three different experiments. *, P < 0.01 when compared with PBS.
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Figure 7. Effects of CCL22 and CCL11 on the degranulation of (A) antigen-elicited murine eosinophils in vitro and (B) in the allergic pleurisy model. (A) CCL22 and CCL11 were used at the concentrations of 10 or 100 ng/ml. Purified eosinophils were activated for 4 h with the chemokines, and degranulation was assessed by measuring the EPO levels in the cell-free supernatant. (B) Sensitized mice received an i.pl. injection of PBS, CCL22 (100 ng/cavity), or CCL11 (100 ng/cavity) 48 h after antigen challenge, and EPO levels in pleural cavity washes were evaluated after a further 2 h. The line represents background levels after injection of PBS in sensitized, unchallenged mice. The results are expressed as the means ± SEM of triplicate determinations and are representative of at least three different experiments. *, P < 0.01 when compared with PBS or eotaxin. O.D., Optical density.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Here, we demonstrated that the injection of CCL22 caused a dose- and time-dependent recruitment of eosinophils into the pleural cavity of mice. Maximal eosinophil recruitment occurred 24 h after injection of a dose of 100 ng/cavity of the chemokine. These effects of CCL22 were qualitatively similar to the shown effects of other chemokines, including CCL3, CCL5, and CCL11 [10 ]. Previous studies have demonstrated the involvement of lipid mediators, such as LTB4 and PAF, in the eosinophil recruitment induced by a range of inflammatory stimuli and following i.pl. administration of the chemokines [9 , 10 , 26 ]. Thus, we investigated whether CCL22 acted directly to induce eosinophil recruitment or did so by inducing the local release of lipid mediators. The treatment of mice with the LTB4 receptor antagonist CP105,696 had no significant effect on the eosinophil recruitment induced by CCL22. This is in contrast with the significant inhibitory effects of the treatment with a PAFR antagonist UK-74,505 and in agreement with other studies showing the involvement of PAF in the migration of eosinophils in vivo [9 , 27 28 29 ]. Thus, it appears that the ability of CCL22 to induce eosinophil recruitment is dependent on the release of PAF. More recently, we have shown that PAF-induced eosinophil recruitment is dependent on the release of CCL11 into the pleural cavity [9 ]. In agreement with these studies, pretreatment with anti-CCL11 antibody effectively inhibited the recruitment of eosinophils induced by CCL22. In the present study, we have been unable to detect CCL11 immunoreactivity in pleural cavity fluid of naïve animals after injection of CCL22 (data not shown). This is in contrast with the effects of the anti-CCL11 antibody in the system. However, we have previously shown that low concentrations of CCL11 may synergize with PAF to induce eosinophil recruitment [9 ]. It is thus possible that the amount of CCL11 present in the fluid is sufficient to cooperate with PAF to induce eosinophil recruitment but not detectable using our ELISA technique. Thus, the injection of CCL22 into the pleural cavity of naïve mice appears to trigger the release of PAF and sequential generation of CCL11, which ultimately induces eosinophil recruitment in our system. The inability of CCL22 to induce murine eosinophil chemotaxis in vitro supports the notion that intermediate mediators are needed for the in vivo effects of CCL22 on eosinophil recruitment.
As CCL22 induced eosinophil recruitment when injected exogenously and was capable of inducing PAF and CCL11, both important mediators during allergic inflammation [9 , 28 29 30 31 32 ], we evaluated the relevance of CCL22 for eosinophil recruitment in our allergic pleurisy model. Pretreatment with an anti-CCL22 polyclonal antibody, at a dose that effectively blocked the effects of CCL22 itself, had no inhibitory effect on the recruitment of eosinophils induced by antigen challenge of sensitized mice. This is qualitatively similar to the lack of effects of anti-CCL22 for the migration of eosinophils into the airway lumen in an allergic model of asthma [33 ]. In addition, mice that are deficient in CCR4, the receptor for CCL22, showed similar lung recruitment of eosinophils to WT controls [34 ]. In contrast, CCL22 was relevant for the initial but not late migration of eosinophils in an animal model of asthma [33 ]. Thus, although CCL22 can induce the recruitment of eosinophils in a PAF- and CCL11-dependent manner when added exogenously, it appears that endogenous release of CCL22 plays only a minor role in determining eosinophil recruitment in models of allergic inflammation.
As CCL22 acts on diverse cellular targets including DC, NK cells, and T cell subsets [1
], it may affect the sensitization phase of an allergic immune response. Treatment with the anti-CCL22 antibody during the sensitization phases had no significant effect on the eosinophil accumulation induced by antigen challenge. Moreover, we failed to observe any significant modification of the production of cytokines by spleen cell cultures, including IFN- and IL-5. In agreement with the lack of importance for CCL22 during the sensitization phase, CCR4-deficient mice were also immunized and responded normally in a model of allergic asthma [34
].
Although anti-CCL22 failed to affect eosinophil migration consistently, it was found to block the development of airway hyper-responsiveness in the murine model of asthma [33 ]. One possibility that stems from the latter observations is that CCL22 may activate eosinophils (and possibly other cell types), which have migrated to the tissues. In support of the latter possibility, we demonstrate here that elicited eosinophils degranulated in response to activation by CCL22 but not CCL11 in vitro and in vivo. Whether CCL22 activates human eosinophils in a similar manner is not known. Of note, Bochner et al. [15 ] have shown that CCL22 induced the chemotaxis of eosinophils purified from blood of allergic patients. The effect of CCL22 was independent on CCR3 (blocking antibody did not reverse the effects) or CCR4 (no mRNA for CCR4 on eosinophils) and thus, was dependent on a receptor distinct from CCR1 to 7 [15 ]. The inability of CCL22 to induce chemotaxis of murine eosinophils suggests that this latter, distinct chemokine receptor is absent on murine eosinophils or does not respond to CCL22 in this species. Similarly, although RANTES (CCL5) stimulates human eosinophils, it fails to activate murine eosinophils [12 ]. Thus, it is clear that in human eosinophils, CCL22 may not only activate eosinophil degranulation (as shown here for murine eosinophils) but also their chemotaxis, suggesting a role for this chemokine in human allergic diseases.
There are other CC chemokines, including CCL17/thymus and activation-regulated chemokine, CCL8/MIP-1ß, and CCL1/trichloroacetic acid-3, which are capable of preferentially inducing eosinophil activation rather than eosinophil migration (ref. [35 ]; and S. H. Oliveira et al., unpublished results). It is interesting that the opposite was observed with CCL11, which preferentially induced eosinophil migration over eosinophil activation, as assessed by degranulation. Thus, it appears that during the allergic process, specific chemokines may be important to induce eosinophil recruitment to sites of inflammation. Once there, eosinophils may come in contact with tissue matrix and locally produced mediators, become activated, express new chemokine receptors, and respond differentially to various chemokines [35 , 36 ]. Moreover, an important point not to be missed is the observation that two distinct chemokines acting on the same cell type trigger distinct functions, highlighting the complexity and cooperation of chemokines in in vivo systems. Whether differential signaling transduction pathways regulate the differential response of eosinophils to the various chemokines clearly deserves further investigation.
In conclusion, we show that although exogenously added CCL22 may induce eosinophil migration in vivo via the sequential release of PAF and CCL11, endogenous production of CCL22 does not drive eosinophil migration during allergic inflammation. However, CCL22 may be an important activator of eosinophils once these cells have migrated into tissue.
Received May 20, 2002; revised November 12, 2002; accepted November 19, 2002.
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