Eosinophil Recruitment in Type-2 Hypersensitivity Pulmonary Granulomas
2002; Elsevier BV; Volume: 161; Issue: 1 Linguagem: Inglês
10.1016/s0002-9440(10)64177-6
ISSN1525-2191
AutoresXiao-Zhou Shang, Bo‐Chin Chiu, Valerie R. Stolberg, Nicholas W. Lukacs, Steven L. Kunkel, Hedwig S. Murphy, Stephen W. Chensue,
Tópico(s)Parasites and Host Interactions
ResumoMonocyte chemotactic protein-3 (MCP-3/CCL7) has potent eosinophil chemoattractant properties. The present study determined its relative contribution to the formation of Th2 cytokine-mediated (type-2) eosinophil-rich interstitial lung granulomas induced by antigens of Schistosoma mansoni eggs. Both MCP-3 transcripts and protein levels were more strongly expressed in lungs with type-2 than with type-1 (mycobacterial antigen-elicited Th1-mediated) granulomas. In vivo treatment with neutralizing antibodies demonstrated that MCP-3 abrogated eosinophil accumulation in type-2 lesions by 40 to 50%. Immunohistochemical staining revealed that MCP-3 localized to vessels in or near granulomas suggesting that endothelial cells were an important in situ source of MCP-3. Maximal MCP-3 transcript expression was abrogated by anti-interleukin-4 treatment. Furthermore, cultured mouse lung endothelial cells displayed augmented MCP-3 production in response to interleukin-4. Together, these results suggest that MCP-3 contributes to a significant component of eosinophil recruitment in the type-2 interstitial granuloma formation and Th2 cytokines promote its production. Monocyte chemotactic protein-3 (MCP-3/CCL7) has potent eosinophil chemoattractant properties. The present study determined its relative contribution to the formation of Th2 cytokine-mediated (type-2) eosinophil-rich interstitial lung granulomas induced by antigens of Schistosoma mansoni eggs. Both MCP-3 transcripts and protein levels were more strongly expressed in lungs with type-2 than with type-1 (mycobacterial antigen-elicited Th1-mediated) granulomas. In vivo treatment with neutralizing antibodies demonstrated that MCP-3 abrogated eosinophil accumulation in type-2 lesions by 40 to 50%. Immunohistochemical staining revealed that MCP-3 localized to vessels in or near granulomas suggesting that endothelial cells were an important in situ source of MCP-3. Maximal MCP-3 transcript expression was abrogated by anti-interleukin-4 treatment. Furthermore, cultured mouse lung endothelial cells displayed augmented MCP-3 production in response to interleukin-4. Together, these results suggest that MCP-3 contributes to a significant component of eosinophil recruitment in the type-2 interstitial granuloma formation and Th2 cytokines promote its production. Because of their participation in hypersensitivity-related diseases, eosinophils and factors that direct their function have been the subject of extensive study.1Wardlaw AJ Brightling C Green R Woltmann G Pavord I Eosinophils in asthma and other allergic diseases.Br Med Bull. 2000; 56: 985-1003Crossref PubMed Scopus (172) Google Scholar A wide variety of factors can induce eosinophil chemotaxis and mobilization but recently investigative efforts have focused on the role of chemokines. It is recognized that chemokine ligands for C-C chemokine receptor 3 (CCR3) are important eosinophil chemotactic factors because this receptor is strongly expressed by eosinophils.2Gao JL Sen AI Kitaura M Yoshie O Rothenberg ME Murphy PM Luster AD Identification of a mouse eosinophil receptor for the CC chemokine eotaxin.Biochem Biophys Res Commun. 1996; 223: 679-684Crossref PubMed Scopus (72) Google Scholar, 3Heath H Qin S Rao P Wu L LaRosa G Kassam N Ponath PD Mackay CR Chemokine receptor usage by human eosinophils. The importance of CCR3 demonstrated using an antagonistic monoclonal antibody.J Clin Invest. 1997; 99: 178-184Crossref PubMed Scopus (442) Google Scholar, 4White JR Imburgia C Dul E Appelbaum E O'Donnell K O'Shannessy DJ Brawner M Fornwald J Adamou J Elshourbagy NA Kaiser K Foley JJ Schmidt DB Johanson K Macphee C Moores K McNulty D Scott GF Schleimer RP Sarau HM Cloning and functional characterization of a novel human CC chemokine that binds to the CCR3 receptor and activates human eosinophils.J Leukoc Biol. 1997; 62: 667-675Crossref PubMed Scopus (142) Google Scholar, 5Grimaldi JC Yu NX Grunig G Seymour BWP Cottrez F Robinson DS Hosken N Ferlin WG Wu XY Soto H O'Garra A Howard MC Coffman RL Depletion of eosinophils in mice through the use of antibodies specific for C-C chemokine receptor 3 (CCR3).J Leukoc Biol. 1999; 65: 846-853PubMed Google Scholar Reported ligands for this receptor include RANTES (CCL5), MCP-3 (CCL7), MCP-2 (CCL8), eotaxin-1 (CCL11), and MCP-4 (CCL13).6Zlotnik A Morales J Hedrick JA Recent advances in chemokines and chemokine receptors.Crit Rev Immunol. 1999; 19: 1-47Crossref PubMed Google Scholar, 7Zlotnik A Yoshie O Chemokines: a new classification system and their role in immunity.Immunity. 2000; 12: 121-127Abstract Full Text Full Text PDF PubMed Scopus (3341) Google Scholar We recently demonstrated the induction of several of these chemokines in a murine model of hypersensitivity-type, eosinophil-rich, type-2 pulmonary granuloma formation elicited by Schistosoma mansoni egg Ags.8Qiu B Frait KA Reich F Komuniecki E Chensue SW Chemokine expression dynamics in mycobacterial (type-1) and schistosomal (type-2) antigen-elicited pulmonary granuloma formation.Am J Pathol. 2001; 158: 1503-1515Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar Induction of CCL7, CCL8, and CCL11 ligands occurred during the eosinophil recruitment phase of the lesion and we undertook a systematic analysis to test the hypothesis that these chemokines might contribute to eosinophil recruitment. In a previous report analyzing the contribution of eotaxin-1 (CCL11), we showed that it did not seem to contribute significantly to local eosinophil recruitment within type-2 lung granulomas.9Ruth JH Lukacs NW Warmington KS Polak TJ Burdick M Kunkel SL Strieter RM Chensue SW Expression and participation of eotaxin during mycobacterial (type 1) and schistosomal (type 2) antigen-elicited granuloma formation.J Immunol. 1998; 161: 4276-4282PubMed Google Scholar In the present study, we similarly tested the potential of MCP-3 (CCL7) as an alternative mediator of eosinophil recruitment in this model. The findings support the notion that MCP-3 is at least one important eosinophil recruitment factor generated during hypersensitivity-type, T-cell-mediated, schistosomal egg Ag-elicited granuloma formation with endothelial cells appearing to be important local sources of this chemokine. Female, CBA/J mice were obtained from Jackson Laboratories, Bar Harbor, ME. Eotaxin −/− and eotaxin +/+ controls on a B6 × 129 background were kindly provided by Dr. R. Bravo (Department of Oncology and Experimental Pathology, Bristol-Myers Squibb, Princeton, NJ). All mice were maintained under specific pathogen-free conditions and provided food and water ad libitum. Type-1 and type-2 granulomas were generated as previously described.10Chensue SW Warmington K Ruth JH Lukacs N Kunkel SL Mycobacterial and schistosomal antigen-elicited granuloma formation in IFN-gamma and IL-4 knockout mice: analysis of local and regional cytokine and chemokine networks [published erratum appears in J Immunol 1999, 162:3106].J Immunol. 1997; 159: 3565-3573PubMed Google Scholar Briefly, mice were sensitized by subcutaneous injection of 20 μg of purified protein derivative (PPD) (Department of Agriculture, Veterinary Division, Ames, IA) of Mycobacteria bovis incorporated in 0.25 ml of completed Freund's adjuvant (product no. F-5881; Sigma, St. Louis, MO) or 3000 S. mansoni eggs suspended in 0.5 ml of phosphate-buffered saline (PBS). Fourteen to 16 days later PPD and schistosome egg-sensitized mice were respectively challenged by tail vein injection with 6000 Sepharose 4B beads (in 0.5 ml of PBS) covalently coupled to PPD or to soluble schistosome egg antigens (SEA). Polyclonal, anti-MCP-3 antibodies were generated in rabbits by multisite immunization as previously described.9Ruth JH Lukacs NW Warmington KS Polak TJ Burdick M Kunkel SL Strieter RM Chensue SW Expression and participation of eotaxin during mycobacterial (type 1) and schistosomal (type 2) antigen-elicited granuloma formation.J Immunol. 1998; 161: 4276-4282PubMed Google Scholar Preparations were used when titers reached 1:106 or greater. The anti-MCP-3 antibody (Ab) was highly specific, reacting only with MCP-3 when tested against the following panel of cytokines: eotaxin, MCP-1, MCP-3, MCP-5, MDC, MIP-1α, C10, RANTES, TARC, TCA3, KC, MIP-2, MIG, IP-10, 6Ckine, fractalkine, interleukin (IL)-4, interferon-γ, tumor necrosis factor-α, IL-1β. Neutralizing polyclonal anti-eotaxin and monoclonal anti-IL-4 antibodies were previously described.9Ruth JH Lukacs NW Warmington KS Polak TJ Burdick M Kunkel SL Strieter RM Chensue SW Expression and participation of eotaxin during mycobacterial (type 1) and schistosomal (type 2) antigen-elicited granuloma formation.J Immunol. 1998; 161: 4276-4282PubMed Google Scholar, 11Ruth JH Warmington KS Shang X Lincoln P Evanoff H Kunkel SL Chensue SW Interleukin-4 and -13 participation in mycobacterial (type-1) and schistosomal (type-2) antigen-elicited pulmonary granuloma formation: multiparameter analysis of cellular recruitment, chemokine expression and cytokine networks.Cytokine. 2000; 12: 432-444Crossref PubMed Scopus (36) Google Scholar Briefly, anti-murine eotaxin was prepared by multiple site immunization of rabbits with recombinant murine eotaxin. The antibody titer by direct enzyme-linked immunosorbent assay (ELISA) was 1:106 and other than eotaxin did not react with a panel of chemokines and cytokines as described above. In an in vitro chemotaxis assay, 150 μl of a 1:200 dilution of anti-eotaxin neutralized 300 ng of recombinant mouse eotaxin. Rat mAb with specificity for murine IL-4 was obtained from of the hybridoma line 11B11 (American Type Culture Collection, Rockville, MD) as previously described.9Ruth JH Lukacs NW Warmington KS Polak TJ Burdick M Kunkel SL Strieter RM Chensue SW Expression and participation of eotaxin during mycobacterial (type 1) and schistosomal (type 2) antigen-elicited granuloma formation.J Immunol. 1998; 161: 4276-4282PubMed Google Scholar 11B11 is of IgG isotype and has known neutralizing activity. For in vivo neutralization, mice were given an intraperitoneal injection of 10 mg of purified rabbit anti-murine eotaxin or anti-murine MCP-3 IgG in 1 ml of PBS at the time of bead challenge. Nonimmune rabbit IgG served as a control. For IL-4 depletion, 2 mg of rat anti-murine IL-4 antibodies were administered intraperitoneally in 1 ml of PBS. Nonimmune rat IgG served as a control. Four days after challenge, lungs were excised. One lobe was analyzed for chemokine transcript expression by real-time polymerase chain reaction (PCR) whereas remaining lobes were used for granuloma morphometry or dispersal for differential analysis. Four days after bead challenge, mice were euthanized and exsanguinated before lung harvest. After perfusion with cold RPMI, lungs, excluding the trachea and major bronchi, were excised. The left lower lung lobe of each mouse was postinflated with buffered formalin. The right lobe was snap-frozen in liquid nitrogen for immunohistochemical staining or mRNA isolation, and remaining lobes were used for protein extraction or granuloma dispersal. mRNA was isolated from frozen tissues using Poly(A) pure mRNA isolation kits (Ambion, Austin, TX). Purity of mRNA was confirmed by failure to detect gene PCR amplification in nonreverse-transcribed samples up to 37 amplification cycles. For protein extraction, snap-frozen lung lobes were suspended in 2 ml of PBS and homogenized for 20 seconds using a Tissue Tearor (Biospec Products, Inc., Bartlesville, OK). Next, 0.1 ml of fetal bovine serum was added as a protein stabilizer. The homogenate was centrifuged at 300 × g for 20 minutes and then the supernate was collected, aliquoted, and frozen at −80°C before ELISA. Chemokines were measured in the whole lung aqueous extracts by specific ELISA using commercial reagents (R&D Systems, Minneapolis, MN) as previously described.8Qiu B Frait KA Reich F Komuniecki E Chensue SW Chemokine expression dynamics in mycobacterial (type-1) and schistosomal (type-2) antigen-elicited pulmonary granuloma formation.Am J Pathol. 2001; 158: 1503-1515Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar Next, total lung protein concentration was determined in experimental and control samples, then chemokine levels were normalized to mg of lung protein after subtraction of fetal bovine serum protein component. Dispersed granulomas were prepared as previously described.10Chensue SW Warmington K Ruth JH Lukacs N Kunkel SL Mycobacterial and schistosomal antigen-elicited granuloma formation in IFN-gamma and IL-4 knockout mice: analysis of local and regional cytokine and chemokine networks [published erratum appears in J Immunol 1999, 162:3106].J Immunol. 1997; 159: 3565-3573PubMed Google Scholar, 12Chensue SW Warmington KS Ruth JH Lincoln P Kunkel SL Cytokine function during mycobacterial and schistosomal antigen-induced pulmonary granuloma formation. Local and regional participation of IFN-gamma, IL-10, and TNF.J Immunol. 1995; 154: 5969-5976PubMed Google Scholar A 200-cell differential analysis was performed on duplicate Wright-stained cytospin preparations of dispersed granulomas. As a further measure of eosinophil mobilization, direct morphometric analysis of eosinophils was performed on digitized images of five granuloma histological cross sections from each of the treated mice. A blinded observer counted six 50-square μm fields from each granuloma. Approximately 1 μg of mRNA was reverse-transcribed in a 20-μl reaction in a PCR reaction tube using Reverse Transcription System kits (Promega, Madison, WI). Five reactions were conducted to minimize variability between tubes. The products from each tube were pooled and subjected to real-time PCR using a TaqMan 7000 light cycler (Applied Biosystems, Foster City, CA.). For the current study the manufacturer's method (see User Bulletin no. 2, Perkin-Elmer) was adopted with GAPDH as endogenous reference. The generating PCR products of the target gene and GAPDH were monitored simultaneously in real-time in the reaction with a fluorescence amplification factor measured for each gene relative to GAPDH. Data are expressed as arbitrary units (AU), which are calculated from the fluorescence amplication factor as measured by the real-time PCR fluorescent detection unit. The original gene copy number (Co) is related to fluorescence of the generated signal as follows: Co = F × E−1 × I × 2−n, where F is an arbitrary conversion constant, E−1 is amplification efficiency constant (approximately = 1 for manufacturer's real-time primers sets), I is the fluorescent intensity reading, and n is the amplification cycle number. Hence, F × E−1 × I × 2−n constitutes an arbitrary measure of originally copy number that is directly related to the fluorescent product and inversely related to cycle number. For MCP-3 gene analysis, TaqMan predeveloped reaction kits (Perkin-Elmer) were used. Oligonucleotides for PCR primers were obtained from Operon Technologies (Alameda, CA.) and TaqMan probes were purchased from Applied Biosystems. In all cases, TaqMan Universal PCR Master Mix (Perkin-Elmer) was used, and the thermal cycling condition was programmed according to the manufacturer's instructions. Before being used in the actual mRNA expression analysis, each primer-probe set was pretested with an undiluted positive control sample and at 1:4 and 1:16 dilutions. Water served as a negative control. Five- to 7-μm-thick frozen tissue sections were mounted on poly-l-lysine-coated slides, fixed with acetone, then rehydrated in PBS. Sections were preblocked with avidin, biotin, and 2% fetal bovine serum. The sections were covered with 10 μg/ml of goat anti-mouse eotaxin or anti-mouse MCP-3 IgG (R&D Systems). Nonimmune goat IgG served as a control. Slides were incubated for 30 minutes at 37°C, then rinsed and overlaid with biotinylated anti-goat IgG (supersensitive reagent 1:20; Biogenex, San Ramon, CA). After a further 30-minute incubation at 37°C, the slides were rinsed, then overlaid with streptavidin-alkaline-phosphatase (1:20, Biogenex,). The slides were rinsed again in PBS and overlaid with alkaline-phosphatase substrate (Fast red) solution (Biogenex) for 30 minutes at room temperature. Finally, the sections were rinsed, counterstained with Gill's hematoxylin, and mounted with coverslips. Mouse lung endothelial cells were isolated and cultured as described by Murphy and colleagues.13Murphy HS Bakopoulos N Dame MK Varani J Ward PA Heterogeneity of vascular endothelial cells: differences in susceptibility to neutrophil-mediated injury.Microvasc Res. 1998; 56: 203-211Crossref PubMed Scopus (57) Google Scholar Briefly, strips of peripheral lungs from 4- to 5-week-old mice were removed, minced, and incubated in gelatin-coated tissue culture flasks in growth media consisting of RPMI-1640, supplemented with 20% fetal calf serum, penicillin-streptomycin, and endothelial cell growth supplement. After 65 hours, tissues were removed leaving cell monolayers that consisted uniformly of endothelial cells. Cells were used at 80 to 90% confluence at passage 1 for all experimental studies. Experiments described below were repeated on several separate isolates of endothelial cells. Cells were characterized as endothelial cells by their uniform cobblestone morphology and by the flow cytometric determination of a single population of cells with uniform uptake of fluorescently-labeled acetylated low-density lipoprotein (1,1′-diotadecyl-3,3,3′,3′-tetramethyl-indo-carbocyanine perchlorated) (DiI-Ac-LDL) (Biomedical Technologies Inc., Stoughton, MA). Human umbilical vein endothelial cells served as positive controls and dermal keratinocytes served as negative controls for low-density lipoprotein uptake studies. Cells demonstrated significant angiotensin-converting enzyme activity when compared to human serum with known angiotensin-converting enzyme activity using a spectrophotometric method using the hydrolysis of the synthetic peptide FAPGG by angiotensin-converting enzyme (Sigma Chemical Co.). The lung endothelial cells were cultured in 24-well plates and were stimulated with graded doses of recombinant cytokine. Triplicate wells were prepared for each dose in each experiment the experiment was repeated with fresh endothelial cells. The Student's t-test (two-tailed) was used to compare control with experimental groups. Values of P < 0.05 were considered to indicate significance. Initially, we compared MCP-3 protein and transcript levels in lungs with Th1-mediated type-1- (PPD) and Th2-mediated type-2 (SEA) bead granulomas. Type 1 lesions contain only a minor component of eosinophils, whereas the type-2 lesions are comprised of 20 to 30% eosinophils. As shown in Figure 1, although not exclusive to the type-2 response, both MCP-3 transcripts and protein levels were higher in the lungs of CBA mice with type-2 as compared to those with type-1 bead granulomas. To assess the potential contribution of MCP-3 to eosinophil recruitment, mice were administered Abs to MCP-3 at the time of type-2 granuloma elicitation. As additional controls, anti-eotaxin Abs either alone or in combination with anti-MCP-3 were administered to parallel groups of animals. At 4 days of granuloma development, lesion composition was assessed by dispersal and morphometric analysis. As shown in Table 1 and as we previously reported, in vivo eotaxin depletion did not significantly reduce eosinophil accumulation in type-2 lesions. In contrast, depletion of MCP-3 selectively reduced eosinophil recruitment in type-2 lesions (by 40 to 50% in three separate experiments) with no further reduction of eosinophils observed by combining anti-eotaxin and anti-MCP-3 treatment. In addition, we similarly tested eotaxin-1 knockout mice, which showed no reduction of eosinophils unless treated with anti-MCP-3 antibodies (Table 2).Table 1Effect of in Vivo Chemokine Depletion on Cell Composition of Dispersed Type-2 Granulomas in Wild-Type CBA MiceTreatmentLymphocytesMacrophagesEosinophilsNeutrophilsControl IgG45.0 ± 4.230.0 ± 1.421.0 ± 4.23.5 ± 0.7Anti-ETX41.0 ± 7.936.3 ± 2.519.0 ± 5.23.6 ± 1.5Anti-MCP-354.5 ± 3.529.0 ± 1.412.0 ± 4.2*P < 0.05 compared with control IgG group.3.5 ± 2.1Anti-ETX+ anti-MCP-348.5 ± 2.134.5 ± 4.912.0 ± 1.4*P < 0.05 compared with control IgG group.4.5 ± 0.7Values are mean ± SD of a representative experiment of three separate experiments with similar results; six to eight mice per group. Mice with type-2 pulmonary granuloma formation were treated with 10 mg of anti-chemokine antibodies or control IgG. On day 4, pulmonary granulomas were isolated, dispersed, and duplicate cytospin preparations were subjected to a 200-cell differential.* P < 0.05 compared with control IgG group. Open table in a new tab Table 2Effect of Eotaxin Knockout and MCP-3 Depletion on Cell Composition of Dispersed Type-2 GranulomasMouse strainTreatmentLymphocytesMacrophagesEosinophilsNeutrophilsEotaxin +/+None53.0 ± 5.623.0 ± 2.120.5 ± 0.83.5 ± 2.1Eotaxin−/−None49.5 ± 0.722.5 ± 0.727.0 ± 1.41.5 ± 0.8Eotaxin−/−IgG43.0 ± 4.027.0 ± 1.425.0 ± 2.06.0 ± 2.1Eotaxin−/−Anti-MCP-353.0 ± 2.124.0 ± 1.415.0 ± 2.0*P < 0.05 compared with control IgG group.5.0 ± 2.9Values are mean ± SD. Type-2 pulmonary granulomas were induced in wild-type (eotaxin +/+) and eotaxin knockout mice (eotaxin −/−). Additional knockout groups were treated with 10 mg of anti-chemokine antibodies or control IgG at the time of bead challenge. On day 4, pulmonary granulomas were isolated, dispersed, and duplicate cytospin preparations were subjected to a 200-cell differential. Five to six mice per group.* P < 0.05 compared with control IgG group. Open table in a new tab Values are mean ± SD of a representative experiment of three separate experiments with similar results; six to eight mice per group. Mice with type-2 pulmonary granuloma formation were treated with 10 mg of anti-chemokine antibodies or control IgG. On day 4, pulmonary granulomas were isolated, dispersed, and duplicate cytospin preparations were subjected to a 200-cell differential. Values are mean ± SD. Type-2 pulmonary granulomas were induced in wild-type (eotaxin +/+) and eotaxin knockout mice (eotaxin −/−). Additional knockout groups were treated with 10 mg of anti-chemokine antibodies or control IgG at the time of bead challenge. On day 4, pulmonary granulomas were isolated, dispersed, and duplicate cytospin preparations were subjected to a 200-cell differential. Five to six mice per group. Figure 2 shows the histological appearance of type-2 granulomas and the average eosinophil density for control and treated groups as determined by morphometric analysis. Similar to the dispersal analysis, anti-MCP-3 antibodies abrogated eosinophil accumulation in type-2 granulomas and again eotaxin depletion had no statistically significant effect. We and others have previously reported that IL-4 is an important mediator of schistosomal antigen-elicited granuloma formation.11Ruth JH Warmington KS Shang X Lincoln P Evanoff H Kunkel SL Chensue SW Interleukin-4 and -13 participation in mycobacterial (type-1) and schistosomal (type-2) antigen-elicited pulmonary granuloma formation: multiparameter analysis of cellular recruitment, chemokine expression and cytokine networks.Cytokine. 2000; 12: 432-444Crossref PubMed Scopus (36) Google Scholar, 14Chensue SW Terebuh PD Warmington KS Hershey SD Evanoff HL Kunkel SL Higashi GI Role of IL-4 and IFN-gamma in Schistosoma mansoni egg-induced hypersensitivity granuloma formation. Orchestration, relative contribution, and relationship to macrophage function.J Immunol. 1992; 148: 900-906PubMed Google Scholar, 15Cheever AW Williams ME Wynn TA Finkelman FD Seder RA Cox TM Hieny S Caspar P Sher A Anti-IL-4 treatment of Schistosoma mansoni-infected mice inhibits development of T cells and non-B, non-T cells expressing Th2 cytokines while decreasing egg-induced hepatic fibrosis.J Immunol. 1994; 153: 753-759PubMed Google Scholar We also previously reported reduced MCP-3 transcripts and eosinophil recruitment in IL-4 knockout mice.10Chensue SW Warmington K Ruth JH Lukacs N Kunkel SL Mycobacterial and schistosomal antigen-elicited granuloma formation in IFN-gamma and IL-4 knockout mice: analysis of local and regional cytokine and chemokine networks [published erratum appears in J Immunol 1999, 162:3106].J Immunol. 1997; 159: 3565-3573PubMed Google Scholar, 12Chensue SW Warmington KS Ruth JH Lincoln P Kunkel SL Cytokine function during mycobacterial and schistosomal antigen-induced pulmonary granuloma formation. Local and regional participation of IFN-gamma, IL-10, and TNF.J Immunol. 1995; 154: 5969-5976PubMed Google Scholar To further detect a possible link between IL-4 and MCP-3, we administered anti-IL-4 antibodies to wild-type mice and assessed effects on MCP-3 transcript expression. As shown in Figure 3, anti-IL-4 profoundly reduced levels of MCP-3 transcripts, which correlated with a significant reduction of eosinophils within lesions. These findings indicated that IL-4 was either directly or indirectly promoting MCP-3 production in vivo. The above studies indicated that MCP-3 contributes to eosinophil recruitment during type-2 cytokine-mediated granuloma formation. Therefore, it was of interest to determine the physical relationship of chemokine expression to the inflammatory lesion. To this end, we performed immunohistochemical staining for MCP-3, which was consistently associated with the luminal side of small vessels in or adjacent to type-2 granulomas (Figure 4; A, C, and E). Inflammatory cells were often found marginating and transmigrating these vessels, which included both terminal pulmonary arterioles and venules. Staining was not observed in normal lungs but was minimally detected in lungs challenged with PPD or non-Ag beads (Figure 4, G and H). Staining was also performed for eotaxin, another potential eosinophil chemotactic agent, and a different staining pattern was identified. Eotaxin was not observed on endothelia but was expressed in bronchoepithelial cells and focally in alveolar septal interstitial cells (Figure 5, A and C). The distribution of the latter was suggestive of interstitial macrophages. Surprisingly, eotaxin was also not identified in granulomas (Figure 5E). Interestingly, eotaxin was found in occasional bronchoepithelial cells within normal lungs and lungs challenged with PPD or non-Ag coated beads (Figure 5F), albeit to a lesser extent. The latter was fully consistent with reports of baseline constitutive eotaxin expression in mouse lungs.16Rothenberg ME MacLean JA Pearlman E Luster AD Leder P Targeted disruption of the chemokine eotaxin partially reduces antigen-induced tissue eosinophilia.J Exp Med. 1997; 185: 785-790Crossref PubMed Scopus (478) Google Scholar, 17Rothenberg ME Zimmermann N Mishra A Brandt E Birkenberger LA Hogan SP Foster PS Chemokines and chemokine receptors: their role in allergic airway disease.J Clin Immunol. 1999; 19: 250-265Crossref PubMed Scopus (79) Google Scholar These patterns of expression seemed to provide an explanation for the different contribution of eotaxin and MCP-3 to local eosinophil recruitment to type-2 granulomas noted in our chemokine depletion studies. Specifically, eotaxin was generally remote from granulomas, whereas MCP-3 was associated with vessels within and immediately adjacent to lesions.Figure 5Immunohistochemical localization of eotaxin. Frozen sections of lungs were prepared and stained with anti-eotaxin Abs as described in Materials and Methods. A: Bronchus from lung with type-2 (SEA) granulomas, eotaxin stain. B: Serial section of A, nonimmune Ab control. C: Lung parenchymal alveoli in lung with type-2 (SEA) granulomas, eotaxin stain. D: Lung parenchymal alveoli in lung with type-2 (SEA) granulomas, nonimmune Ab control; E, Type-2 (SEA) bead granuloma, eotaxin stain. F: Bronchus and nearby control bead lesion in lung challenged with antigen-free beads. Original magnifications: ×200 (A, C, F); ×400 (D, E).View Large Image Figure ViewerDownload Hi-res image Download (PPT) Our immunohistochemical studies suggested that vascular endothelial cells were potential sources of MCP-3. Human endothelial cells have been reported to produce MCP-3 in response to Th2 cytokines and are capable of binding chemokines to their surfaces by promiscuous membrane-binding receptors.18Polentarutti N Introna M Sozzani S Mancinelli R Mantovani G Mantovani A Expression of monocyte chemotactic protein-3 in human monocytes and endothelial cells.Eur Cytokine Netw. 1997; 8: 271-274PubMed Google Scholar, 19Hub E Rot A Binding of RANTES, MCP-1, MCP-3, and MIP-1alpha to cells in human skin.Am J Pathol. 1998; 152: 749-757PubMed Google Scholar To determine whether such a cytokine-chemokine network could occur in the mouse lung, we isolated mouse lung endothelial cells and tested their capacity to produce MCP-3 and eotaxin in response to graded doses of IL-4. As shown in Figure 6, IL-4 induced MCP-3 production by mouse pulmonary endothelial cells in a dose-response manner but did not elicit significant amounts of eotaxin. This study represents the first analysis of the role of MCP-3 (CCL7) in eosinophil recruitment during schistosomal Ag-elicited granuloma formation. MCP-3 is an agonist for the CCR3 chemokine receptor, a functional receptor expressed by eosinophils.2Gao JL Sen AI Kitaura M Yoshie O Rothenberg ME Murphy PM Luster AD Identification of a mouse eosinophil receptor for the CC chemokine eotaxin.Biochem Biophys Res Commun. 1996; 223: 679-684Crossref PubMed Scopus (72) Google Scholar, 3Heath H Qin S Rao P Wu L LaRosa G Kassam N Ponath PD Mackay CR Chemokine rece
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