The Journal of Immunology T Cell Chemokine Receptor Expression in Aging1 Ruran Mo,* Jun Chen,* Yin Han,* Cecelia Bueno-Cannizares,* David E. Misek,† Pascal A. Lescure,† Samir Hanash,† and Raymond L. Yung2* Changes in chemokine receptor expression are important in determining T cell migration and the subsequent immune response. To better understand the contribution of the chemokine system in immune senescence we determined the effect of aging on CD4؉ T cell chemokine receptor function using microarray, RNase protection assays, Western blot, and in vitro chemokine transmi- ,gration assays. Freshly isolated CD4؉ cells from aged (20–22 mo) mice were found to express a higher level of CCR1, 2, 4, 5, 6 and 8 and CXCR2–5, and a lower level of CCR7 and 9 than those from young (3–4 mo) animals. Caloric restriction partially or completely restored the aging effects on CCR1, 7, and 8 and CXCR2, 4, and 5. The aging-associated differences in chemokine receptor expression cannot be adequately explained by the age-associated shift in the naive/memory or Th1/Th2 profile. CD4؉ cells from aged animals have increased chemotactic response to stromal cell-derived factor-1 and macrophage-inflammatory protein- 1␣, suggesting that the observed chemokine receptor changes have important functional consequences. We propose that the aging-associated changes in T cell chemokine receptor expression may contribute to the different clinical outcome in T cell chemokine receptor-dependent diseases in the elderly. The Journal of Immunology, 2003, 170: 895–904. he precise mechanisms linking the aging immune system (6–8). T cell-tropic HIV-1 isolates (X4 strains) preferentially use to diseases in the elderly are poorly understood. Studies in CXCR4 as a coreceptor for lymphocyte entry (9), whereas CCR5 T humans and animal models have demonstrated aging-as- acts as a cofactor by macrophage-tropic (R5 strains) and dual- sociated defects in selected inductive and effector T cell functions, tropic strains of HIV-1 (10–13). Recent data suggest that their most notably, decreased reactivity to Ag, impaired delayed hyper- level of expression is an important determinant of HIV-1 infectiv- sensitivity response to recall Ag, and diminished T cell-dependent ity and replication (14–16). In addition, the chemokine system is Ab response after vaccination (1–3). However, the notion that the extremely complex with significant redundancy. For example, aging-associated “decline” in immune response is the cause of di- some M-tropic strains of HIV can use CCR2 or 3 as coreceptor minished protective immunity and poor clinical outcome in the instead of CCR5. Ligands of chemokine receptors also affect their elderly is not supported by available evidence. With the aging function. CCR5-using chemokines (RANTES, macrophage-in- population there is an urgent need to better understand the changes flammatory protein (MIP)3-1␣, MIP-1␤) can block M-tropic HIV in immunity that occur with aging. Defining these changes will strains, whereas the CXCR4 ligand stromal cell-derived factor help to explain the susceptibility of the elderly to aging-related (SDF)-1 blocks T-tropic strains by down-regulating the receptor by guest on October 1, 2021. Copyright 2003 Pageant Media Ltd. diseases and to better predict the therapeutic response of older (14). The clinical courses of these T cell chemokine-dependent adults to biologics and immunization. diseases are different in the elderly compared with younger indi- Chemokines are a superfamily of small proteins important in viduals (17, 18). In the case of HIV-1 infection, it has been shown determining normal and pathological immune and inflammatory that older patients have a shorter observed AIDS-free interval and responses. They are classified according to the cysteine motif into shorter survival, along with more HIV and non-HIV related co- C, CC, CXC, and CX3C chemokines. In addition, at least 19 che- morbidity. This raises the possibility that aging-associated alter- mokine receptors have been identified (4, 5). These G protein- ation in chemokine response may play an important pathogenic coupled cell surface receptors possess a 7-transmembrane domain role in these diseases in older adults. http://classic.jimmunol.org and are widely distributed in lymphoid and nonlymphoid cells. Lymphocyte chemokine receptor expression has been shown to be Materials and Methods important in a number of disease processes. For example, CCR4, Mice CCR5, CXCR3, and CX3CR1 are critical in the recruitment and Young (3–4 mo), middle-aged (12–14 mo), and old (20–22 mo) C57BL/6 retention of leukocytes in the joints of rheumatoid arthritis patients and DBA/2, and caloric-restricted C57BL/6 old (20–21 mo) mice were obtained from the National Institute on Aging aged rodent colonies through Harlan Sprague Dawley (Indianapolis, IN).4 The caloric restriction proto- Downloaded from *Divisions of Geriatric Medicine and Rheumatology, Department of Internal Medi- col can be obtained through the National Institute on Aging (http://www. cine, and †Department of Pediatrics, University of Michigan, Ann Arbor, MI 48109 nia.nih.gov). Briefly, caloric restriction is initiated at 14 wk of age at 10% Received for publication August 26, 2002. Accepted for publication November restriction, increased to 25% restriction at 15 wk, and to 40% restriction at 5, 2002. 16 wk where it is maintained throughout the life of the animal. With the exception of the caloric-restricted mice that were sacrificed within 24 h of The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 3 Abbreviations used in this paper: MIP, macrophage-inflammatory protein; SDF, 1 This work was supported by Public Health Service Grants 1K08AR01977-01A1, stromal cell-derived factor; RPA, RNase protection assay. 1RO1 AI42753, 1RO1 HL61577, American Federation for Aging Research (Paul 4 Strain contamination of the C57BL/6 breeding colony in the National Institute on Beeson Physician Faculty Scholar Award), the University of Michigan Nathan Shock Aging contract aging mouse colony at Harlan Sprague Dawley with 129 or FVB was Center (AG13282), and the Geriatrics Research, Education, and Clinical Center of the recently discovered, affecting some aging mice with C57BL/6 background. The ev- Ann Arbor Veterans Affair Medical Center. idence indicates that the strain contamination first hit the aging colony with the spring 2 Address correspondence and reprint requests to Dr. Raymond L. Yung, Room 5312 or summer 2000 dates of birth. The mice used in this manuscript were born before the Cancer Center and Geriatrics Center Building, 1500 East Medical Center Drive, Ann estimated contamination date or were deemed to be of “low risk” by the National Arbor, MI 48109-0940. E-mail address: [email protected] Institute on Aging. Copyright © 2003 by The American Association of Immunologists, Inc. 0022-1767/03/$02.00 896 PRECISE MECHANISMS LINKING AGING IMMUNE SYSTEM TO DISEASE arrival, all mice were maintained in a pathogen-free environment provided stringent wash buffer (100 mM MES (pH 6.7), 0.1 M NaCl, 0.01% Tween by the Unit for Laboratory Animal Medicine at the University of Michigan 20) at 50°C, stained with streptavidin-PE (Molecular Probes, Eugene, OR), (Ann Arbor, MI) until they were used. washed again with 6ϫ SSPE, stained with biotinylated anti-streptavidin IgG, followed by a second staining with streptavidin-PE, and a third wash- CD4 cell isolation ing with 6ϫ SSPE. The arrays were scanned using the GeneArray scanner Careful inspection was done to exclude aged animals with cancer or lym- (Affymetrix). Data analysis was performed using GeneChip 4.0 software. ϩ ␮ ϳ phoma. CD4 T cells were then isolated by the MACS Microbeads tech- The 11k and the U74A chips contain 11,000 and 12,000 probe sets, nology (Miltenyi Biotec, Bergisch-Gladbach, Germany) according to the respectively, with each probe set representing a transcript. Each probe set manufacturer’s instructions. CD4ϩ cells were negatively selected using a typically consists of 20 perfectly complementary 25 base-long probes as combination of CD8a (Ly-2), CD11b (Mac-1), and CD19 Microbeads. Al- well as 20 mismatch probes that are identical except for an altered central ternately, CD4 cells were positively selected using CD4 (L3T4) Mi- base. We subtract the mismatch probe values from the perfect match values crobeads. Purity of the isolated cells was confirmed by flow cytometric and average the middle 50% of these differences as the expression measure analysis. Briefly, a single cell suspension was prepared by gentle teasing of for that probe set. A quantile normalization procedure was used to adjust the spleen with sterile forceps. Density gradient centrifugation was done for differences in the probe intensity distribution across different chips. We using Ficoll-Paque Plus (Amersham Pharmacia Biotech, Piscataway, NJ). then applied a monotone linear spline to each chip that mapped quantiles The live splenic mononuclear cells were then magnetically labeled with the 0.02 up to 0.98 (in increments of 0.02) exactly to the corresponding median ϩ ϩ ϩ appropriate Microbeads (10 ␮l/107 total cells) and passed through the LS quantiles for all the samples. Then, the transform log(100 max(X 100; separation column three times while placed in the magnetic field of a Midi- 0)) was applied to the data from each chip. The chemokine receptor results are then calculated as changes relative to the expression levels of unstimu- MACS separator (Miltenyi Biotec). The selected cells were then removed ϩ from the column by washing three times with 3 ml of LSϩ/VSϩ buffer lated young CD4 lymphocytes. away from the magnetic field. Purity of the isolated cells was determined by staining with the FITC-conjugated anti-CD4 RM4–5 and control IgG2a RNA protection assays (RPAs) Abs (both from BD PharMingen, San Diego, CA) and was consistently between 94–99%.