Research Communication Mediators of Inflammation, 13(5/6), 349Á/355 (October/November 2004) THE aim of this study was to determine the role of CC Role of CC chemokine CCL6/C10 chemokine CCL6/C10 in acute inflammation. Intra- peritoneal injection of thioglycollate increased as a monocyte chemoattractant in peritoneal CCL6, which peaked at 4 h and remained elevated at 48 h. Neutralization of CCL6 significantly a murine acute peritonitis inhibited the macrophage infiltration (34 /48% reduction), but not other cell types, without decreas-Á ing the other CC chemokines known to attract Andrew M. LaFleur1,2, Nicholas W. Lukacs1, monocytes/macrophages. CCL6 was expressed in Steven L. Kunkel1 and Akihiro Matsukawa1,3,CA peripheral eosinophils and elicited macrophages, but not in elicited neutrophils. Peritoneal CCL6 level was not decreased in granulocyte-depleted mice where eosinophil influx was significantly impaired. 1Department of Pathology, University of Michigan Thus, CCL6 appears to contribute to the macrophage Medical School, Ann Arbor, Michigan, USA; infiltration that is independent of other CC chemo- 2Michigan State University, Kalamazoo Center for kines. Eosinophils pre-store CCL6, but do not release Medical Studies, Kalamazoo, Michigan, USA; and CCL6 in the peritoneum in this model of inflamma- 3Department of Pathology and Experimental tion. Medicine, Graduate School of Medical Sciences, Kumamoto University, 1-1-1 Honjo, Kumamoto Key words: Acute inflammation, CC chemokines, Eosino- 860-8556, Japan phils, Monocytes/macrophages, Neutrophils CACorresponding Author Tel: /81 96 373 5088 Fax: /81 96 373 5087 E-mail: [email protected] Introduction tonitis.12 Á15 These findings demonstrate an important role of CCL6 in chronic inflammation associated with The inflammatory response is characterized by leu- macrophage infiltration. However, the precise role of kocyte infiltration into inflammatory foci, which is CCL6 in acute inflammation remains to be elucidated. initiated and orchestrated by a variety of inflamma- The aim of the present study was to characterize tory mediators. Of these, chemokines are a group of the role of CCL6 in acute inflammation. For this cytokines that are chemotactic for specific types of purpose, we first examined the production kinetics of leukocyte populations.1,2 Chemokines have been CCL6 in a murine model of acute peritonitis induced divided mainly into two subfamilies, CXC and CC by thioglycollate injection. We then investigated chemokines, based on their sequence homology and whether CCL6 could contribute to the leukocyte the position of cysteine residues.3,4 CXC chemokines infiltration in this model by neutralizing the endo- are typically chemotactic for neutrophils, whereas CC genous CCL6 with antibodies against CCL6. Finally, chemokines attract and activate monocytes and we attempted to determine the major cellular sources of CCL6 using reverse transcription-polymerase chain lymphocytes. The role of CXC chemokine such as reaction (RT-PCR), enzyme-linked immunosorbent CXC8/IL-8 in a variety of inflammatory diseases has assay (ELISA) and immunohistochemistry. The pre- been extensively investigated.5 CC chemokines that sent data suggest that CCL6 is an important CC include CCL2/MCP-1, CCL3/MIP-1a and CCL22/MDC chemokine attracting monocytes/macrophages. are detected in an early phase of acute inflammation, Although CCL6 is pre-stored in circulating eosino- playing an essential role in the recruitment and phils, the CCL6 does not appear to be released in this activation of monocytes/macrophages.6Á 10 particular model. Murine CCL6/C10, a prototype CC chemokine, was initially identified as a transcript induced in bone marrow cells upon stimulation with gran- 11 ulocyteÁ/macrophage colony-stimulating factor. It Materials and methods has shown that CCL6 is present in a variety of chronic Mice inflammatory disorders including experimental de- myelinating diseases, allergic airway inflammation, Female CD-1 mice (6Á/8 weeks of age) were purch- bleomycin-induced lung fibrosis and chronic peri- ased from Jackson Laboratory (Bar Harbor, ME, USA) ISSN 0962-9351 print/ISSN 1466-1861 online – 2004 Taylor & Francis Ltd 349 DOI: 10.1080/09629350400014172 A. M. LaFleur et al. and housed in the animal care facility unit (University tran sedimentation, followed by Percoll gradient Laboratory of Animal Medicine). The animal use centrifugation and hypotonic lysis of erythrocytes. committee at the University of Michigan approved Diff-Quik staining of cytospin preparations showed all studies. that the purity of granulocytes and macrophages in each experiment was/92% and/94%, respectively. Resident peritoneal macrophages were harvested Peritonitis model from untreated mice, and the adherent cells were The mice received intraperitoneal (i.p.) injection of 1 used. Isolated cells were used for RT-PCR and ELISA. ml of a 2% thioglycollate broth (Difco Laboratories, For ELISA assay, cells were extracted with phosphate- Detroit, MI, USA). To neutralize CCL6, 0.5 ml of anti- buffered saline containing 1% Triton X-100 and murine CCL6 antiserum was i.p. injected 2 h prior to protease inhibitors (Roche Applied Science, Basel, thioglycollate injection. Polyclonal anti-murine CCL6 Switzerland). antiserum was raised by immunizing rabbits with recombinant murine CCL6 (R&D systems Inc., Min- RT-PCR neapolis, MN, USA), as previously described.13,16 The antibodies had a neutralizing activity against murine Total RNA was isolated from cells using Trizol recombinant CCL6 in vitro chemotaxis assay, and did reagent (Life Technologines, Grand Island, NY, not cross-react with a number of other murine USA) according to the manufacturer’s instructions. cytokines and chemokines available (data not First-strand cDNA was synthesized from 1 mgof shown). As a control, pre-immune rabbit serum (0.5 total RNA with oligo (dT)12 Á 18 as primers, and the ml) was used. At appropriate time points following first-strand cDNAs were then amplified by each PCR thioglycollate injection, mice were euthanized by in the presence of Taq polymerase (Life Technolo- cervical dislocation. Peritoneal lavages were collected gies) and specific primers. The primers were de- by washing the peritoneum with 2 ml of sterile saline, signed to amplify murine CCL6 referred to the cDNA were centrifuged, and cell-free peritoneal fluids were sequence from the National Center for Biotechnology stored at /208C for measurements of chemokines. Information database. The primers were as follows: Cell pellets were resuspended in saline, and the cell CCL6 sense, 5?-GACGTCATATGGGCCTCATACAAGA- numbers were counted in a hemocytometer. Differ- AATG-3? and antisense, 5?-TCGTGGATCCTGCCTGC- ential cell analysis was made after Diff-Quik staining CCTCCTTCTC-3?;GAPDH sense,5?-GGTGAAGGTCG- of cytospin preparations. GT-GTCAACGGATTT-3? and antisense, 5?-GATGC- CAAAGTTGTCATGGATGACC-3?. The PCR reaction was conducted at 35 cycles of 948C for 1 min, 558C for Granulocyte depletion 1 min, and 728C for 1 min. Ten microlitres of each PCR Granulocytes were depleted using RB6-8C5 mono- product was subjected to electrophoresis on a 2% clonal antibody directed against Ly-6G, previously agarose gel in the presence of ethidium bromide. known as Gr-1, an antigen on the surface of murine granulocytes that includes neutrophils and eosino- ELISA phils.17,18 A total of 100 mg of RB6-8C5 was i.p. administered 1 day prior to thioglycollate injection. Murine chemokines were quantitated using a stan- This resulted in peripheral blood granulopenia (B/50 dard method of sandwich ELISA as previously cells/ml) by days 1 and 3 after the administration, with described.13,16,21 The captured antibodies, detection a return of peripheral counts to pretreatment levels antibodies and the recombinant cytokines were by day 5.19 Due to the granulopenia, the treatment purchased from R&D Systems. The ELISAs employed effectively inhibited neutrophil and eosinophil infil- in this study did not cross-react with other murine tration in animal models of inflammation.19,20 Iso- cytokines, and constantly detected murine cytokines/ type-matched mouse IgG was used as a control. chemokines above 10 pg/ml. Isolation of granulocytes and macrophages Immunohistochemistry Elicited granulocytes and macrophages were isolated Cytospin preparations were fixed in 100% ethanol for from the peritoneal exudates after thioglycollate 10 min, and the endogenous neutrophil peroxidase injection by Percoll gradient centrifugation. For this, was blocked with 0.3% H2O2 in methanol. The slides exudates from five mice or two mice were combined were rehydrated in Tris-buffered saline (TBS) and in each experiment for granulocyte or macrophage blocked with 5% normal goat serum for 20 min at isolation, respectively. Six independent experiments room temperature. Staining for CCL6 was carried out were performed. Peripheral granulocytes were iso- with anti-CCL6 IgG or control rabbit IgG at 5 mg/ml in lated from heparinized untreated mice blood (10 TBSÁ/1% bovine serum albumin for 30 min at room mice each, three independent experiments) by dex- temperature. After washing with TBSÁ/Tween 20 350 Mediators of Inflammation × Vol 13 × 2004 Role of CC chemokine CCL6/C10 as a monocyte chemoattractant (0.1%), the slides were incubated with biotinylated control. The treatment did not affect the numbers of goat anti-rabbit IgG antibodies and rinsed. The slides infiltrating neutrophils, eosinophils, and lymphocytes were then incubated for 30 min with Vectastain Elite (Fig. 2).