Recruitment and Activation of Macrophages by Pathogenic CD4 T Cells in Type 1 Diabetes: Evidence for Involvement of CCR8 and CCL1 This information is current as of September 25, 2021. Joseph Cantor and Kathryn Haskins J Immunol 2007; 179:5760-5767; ; doi: 10.4049/jimmunol.179.9.5760 http://www.jimmunol.org/content/179/9/5760 Downloaded from References This article cites 36 articles, 19 of which you can access for free at: http://www.jimmunol.org/content/179/9/5760.full#ref-list-1 http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication by guest on September 25, 2021 *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2007 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology Recruitment and Activation of Macrophages by Pathogenic CD4 T Cells in Type 1 Diabetes: Evidence for Involvement of CCR8 and CCL11 Joseph Cantor and Kathryn Haskins2 Adoptive transfer of diabetogenic CD4 Th1 T cell clones into young NOD or NOD.scid recipients rapidly induces onset of diabetes and also provides a system for analysis of the pancreatic infiltrate. Although many reports have suggested a role for macrophages in the inflammatory response, there has been little direct characterization of macrophage activity in the pancreas. We showed previously that after migration to the pancreas, diabetogenic CD4 T cell clones produce a variety of inflammatory cytokines and chemokines, resulting in the recruitment of macrophages. In this study, we investigated mechanisms by which macrophages are recruited and activated by T cells. Analysis of infiltrating cells after adoptive transfer by the diabetogenic T cell clone BDC-2.5 Downloaded from indicates that large numbers of cells staining for both F4/80 and CD11b are recruited into the pancreas where they are activated to make IL-1␤, TNF-␣, and NO, and express the chemokine receptors CCR5, CXCR3, and CCR8. Diabetogenic CD4 T cell clones produce several inflammatory chemokines in vitro, but after adoptive transfer we found that the only chemokine that could be detected ex vivo was CCL1. These results provide the first evidence that CCR8/CCL1 interaction may play a role in type 1 diabetes through macrophage recruitment and activation. The Journal of Immunology, 2007, 179: 5760–5767. http://www.jimmunol.org/ doptive transfer of diabetogenic CD4 Th1 T cell clones contributions of donor T cells and recruited host cells through into young NOD or NOD.scid recipients rapidly induces analysis of cell surface phenotype and intracellular cytokine stain- A onset of diabetes and also provides a system for analysis ing. We have shown previously that diabetogenic CD4 T cell of the cellular components of the pancreatic inflammatory infiltrate clones produce a variety of inflammatory cytokines and chemo- (1, 2). These studies and others have established that there are kines after migration to the pancreas and, furthermore, that this ϩ ϩ large numbers of F4/80 and CD11b macrophages in the pan- activity results in the recruitment of large numbers of macrophages creatic infiltrates of NOD mice, both in spontaneous disease and in (6). These results have led us to hypothesize that recruitment and adoptive transfers of diabetogenic T cells (2, 3). The requirement activation of macrophages could be an important manifestation of 3 for macrophages in the pathogenesis of type 1 diabetes (T1D) has CD4 T cell effector function. We report here on the further char- by guest on September 25, 2021 been shown in studies in which disease was inhibited by their acterization of the macrophage component of the inflammatory depletion (4, 5). Although macrophages may be necessary for Ag infiltrate and on the analysis of the mechanisms by which macro- presentation to autoreactive T cells, other studies have suggested phages are recruited and activated by T cells. We show that mac- that they also function as effector cells in the destruction of islet ␤ rophages recruited and activated by pathogenic CD4 T cells ex- cells (5). There has been little evidence, however, to directly dem- press several important chemokine receptors involved in onstrate the effector function of macrophages in the diabetic pan- autoimmunity, including CCR8, which has recently been identified creas, and indeed, macrophage activity in the pancreatic infiltrate as a key molecule on activated microglia and macrophages in brain is almost completely uncharacterized at the single-cell level. lesions of patients with multiple sclerosis (7). Upon activation in Using an adoptive transfer system in which the activity of patho- the pancreas, macrophages are induced to make several inflamma- genic vs nonpathogenic T cell clones in young NOD or NOD.scid tory mediators, including chemokines that act as chemoattractants mice is compared, we have developed methods for the recovery for other immune cells. Our results include new and previously and ex vivo analysis of cells just before diabetes onset. These unreported findings on the properties of macrophages as effectors procedures allow us to investigate in an unequivocal manner the in ␤ cell destruction and establish these immune cells as key play- ers in pathogenesis of diabetes. Department of Immunology, University of Colorado Health Sciences Center, Denver, CO 80206 Materials and Methods Received for publication December 29, 2006. Accepted for publication August 21, 2007. Mice The costs of publication of this article were defrayed in part by the payment of page NOD and NOD.scid breeding mice were initially acquired from The Jack- charges. This article must therefore be hereby marked advertisement in accordance son Laboratory or the Barbara Davis Center for Childhood Diabetes and with 18 U.S.C. Section 1734 solely to indicate this fact. were housed in specific pathogen-free conditions at the University of Col- 1 This work was supported by National Institutes of Health Grant R01DK50561. orado Health Sciences Center for Laboratory Animal Care. NOD.scid mice 2 Address correspondence and reprint requests to Dr. Kathryn Haskins, Department of were housed in sterile isolation cages. Mice in NOD.scid litters (6–10 days Immunology, University of Colorado Health Sciences Center, National Jewish Med- old) were used as recipients in adoptive transfer experiments. Breeding ical & Research Center (NJMRC), 1400 Jackson Street, Denver, CO 80206. E-mail mice and experimental animals were monitored for development of disease address: [email protected] by urine glucose. The 6.9 TCR transgenic (TCR-Tg) mouse was produced 3 Abbreviations used in this paper: T1D, type 1 diabetes; Tg, transgenic. using TCR genes from a diabetogenic T cell clone, BDC-6.9 (8). All pro- cedures used were in accordance with institutional IACUC guidelines and Copyright © 2007 by The American Association of Immunologists, Inc. 0022-1767/07/$2.00 approved by the UCHSC Animal Care and Use Committee. www.jimmunol.org The Journal of Immunology 5761 Culture and expansion of T cell clones T cell clones were established from spleen and lymph nodes of diabetic NOD mice (9, 10) and were restimulated every 2 wk with a ␤ cell granule membrane fraction obtained from ␤ cell tumors as a source of Ag (11), irradiated NOD spleen cells as APCs, and EL-4 supernatant as a source of IL-2 in complete medium (CM). CM is DMEM supplemented with 44 mM sodium bicarbonate, 0.55 mM L-arginine, 0.27 mM L-asparagine, 1.5 mM L-glu- tamine, 1 mM sodium pyruvate, 50 mg/L gentamicin sulfate, 50 ␮M 2-ME, 10 mM HEPES, and 10% FCS. Cell numbers were expanded for transfer experiments by subculturing 3–6 ϫ 106 T cells 4 days after restimulation in a 5-fold volume of CM and additional IL-2. T cells were harvested, washed 3 times, and resuspended in HBSS for injection into young (Յ10 days of age) NOD.scid recipients. Assessment of cytokines and chemokines in vitro by intracellular staining Production of cytokines and chemokines by T cells in vitro was analyzed by intracellular cytokine staining, as described previously (6). In brief, T cells were stimulated in plates coated with 1 ␮g/ml anti-CD3 Ab for 24 h before Ab staining. The cells were surface stained in 50–100 ␮l of staining buffer (PBS, 0.5% BSA) containing rat anti-CD4 or isotype control Ab for 30–45 min, and then washed and fixed in 2% formaldehyde. Cells were Downloaded from resuspended in permeabilization buffer (staining buffer plus 0.5% saponin), containing an isotype control or specific Ab mix for intracellular cytokines/ FIGURE 1. Macrophage recruitment to the pancreas by a pathogenic vs chemokines. Polyclonal intracellular staining Abs used were obtained from ϩ R&D Systems and included polyclonal goat IgG anti-CCL1 (TCA-3), anti- a nonpathogenic T cell clone. A, The percentage of CD11b cells (mac- CCL3 (MIP-1␣), anti-CCL4 (MIP-1␤), anti-CCL5 (RANTES), anti-CCL6 rophages) in the pancreas was assessed at 6 to 7 days after transfer of a (C10), anti-CCL9/10 (MIP-1␥), and anti-CCL21 (SLC) as primary Abs, pathogenic T cell clone, BDC-2.5, or a nonpathogenic control, the Th1 T followed by FITC-rabbit anti-goat secondary Ab (Vector Laboratories). cell clone, BDC-2.4. Macrophage infiltration could be detected as early as http://www.jimmunol.org/ Monoclonal digoxigenin-MTAC-2 anti-lymphotactin and Cy5-anti- 2 days after T cell clone transfer, but was considered optimal at 6 days.