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The Journal of Immunology, 2003, 170: 895-904.
Copyright © 2003 by The American Association of Immunologists

T Cell Chemokine Receptor Expression in Aging1

Ruran Mo*, Jun Chen*, Yin Han*, Cecelia Bueno-Cannizares*, David E. Misek{dagger}, Pascal A. Lescure{dagger}, Samir Hanash{dagger} and Raymond L. Yung2,*

* Divisions of Geriatric Medicine and Rheumatology, Department of Internal Medicine, and {dagger} Department of Pediatrics, University of Michigan, Ann Arbor, MI 48109


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
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 transmigration 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{alpha}, 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.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The precise mechanisms linking the aging immune system to diseases in the elderly are poorly understood. Studies in humans and animal models have demonstrated aging-associated defects in selected inductive and effector T cell functions, most notably, decreased reactivity to Ag, impaired delayed hypersensitivity response to recall Ag, and diminished T cell-dependent Ab response after vaccination (1, 2, 3). However, the notion that the aging-associated "decline" in immune response is the cause of diminished protective immunity and poor clinical outcome in the elderly is not supported by available evidence. With the aging population there is an urgent need to better understand the changes in immunity that occur with aging. Defining these changes will help to explain the susceptibility of the elderly to aging-related diseases and to better predict the therapeutic response of older adults to biologics and immunization.

Chemokines are a superfamily of small proteins important in determining normal and pathological immune and inflammatory responses. They are classified according to the cysteine motif into C, CC, CXC, and CX3C chemokines. In addition, at least 19 chemokine receptors have been identified (4, 5). These G protein-coupled cell surface receptors possess a 7-transmembrane domain and are widely distributed in lymphoid and nonlymphoid cells. Lymphocyte chemokine receptor expression has been shown to be important in a number of disease processes. For example, CCR4, CCR5, CXCR3, and CX3CR1 are critical in the recruitment and retention of leukocytes in the joints of rheumatoid arthritis patients (6, 7, 8). T cell-tropic HIV-1 isolates (X4 strains) preferentially use CXCR4 as a coreceptor for lymphocyte entry (9), whereas CCR5 acts as a cofactor by macrophage-tropic (R5 strains) and dual-tropic strains of HIV-1 (10, 11, 12, 13). Recent data suggest that their level of expression is an important determinant of HIV-1 infectivity and replication (14, 15, 16). In addition, the chemokine system is extremely complex with significant redundancy. For example, some M-tropic strains of HIV can use CCR2 or 3 as coreceptor instead of CCR5. Ligands of chemokine receptors also affect their function. CCR5-using chemokines (RANTES, macrophage-inflammatory protein (MIP)3-1{alpha}, MIP-1{beta}) can block M-tropic HIV strains, whereas the CXCR4 ligand stromal cell-derived factor (SDF)-1 blocks T-tropic strains by down-regulating the receptor (14). The clinical courses of these T cell chemokine-dependent diseases are different in the elderly compared with younger individuals (17, 18). In the case of HIV-1 infection, it has been shown that older patients have a shorter observed AIDS-free interval and shorter survival, along with more HIV and non-HIV related comorbidity. This raises the possibility that aging-associated alteration in chemokine response may play an important pathogenic role in these diseases in older adults.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice

Young (3–4 mo), middle-aged (12–14 mo), and old (20–22 mo) C57BL/6 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 protocol can be obtained through the National Institute on Aging (http://www.nia.nih.gov). Briefly, caloric restriction is initiated at 14 wk of age at 10% restriction, increased to 25% restriction at 15 wk, and to 40% restriction at 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 arrival, all mice were maintained in a pathogen-free environment provided by the Unit for Laboratory Animal Medicine at the University of Michigan (Ann Arbor, MI) until they were used.

CD4 cell isolation

Careful inspection was done to exclude aged animals with cancer or lymphoma. CD4+ T cells were then isolated by the MACS Microbeads technology (Miltenyi Biotec, Bergisch-Gladbach, Germany) according to the manufacturer’s instructions. CD4+ cells were negatively selected using a combination of CD8a (Ly-2), CD11b (Mac-1), and CD19 Microbeads. Alternately, CD4 cells were positively selected using CD4 (L3T4) Microbeads. Purity of the isolated cells was confirmed by flow cytometric analysis. Briefly, a single cell suspension was prepared by gentle teasing of the spleen with sterile forceps. Density gradient centrifugation was done using Ficoll-Paque Plus (Amersham Pharmacia Biotech, Piscataway, NJ). The live splenic mononuclear cells were then magnetically labeled with the appropriate Microbeads (10 µl/107 total cells) and passed through the LS+ separation column three times while placed in the magnetic field of a MidiMACS separator (Miltenyi Biotec). The selected cells were then removed from the column by washing three times with 3 ml of LS+/VS+ buffer 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 Abs (both from BD PharMingen, San Diego, CA) and was consistently between 94–99%.

T cell culture and stimulation with mAbs

All the mAbs used were from BD PharMingen (San Diego, CA) unless otherwise stated. Combined anti-CD3 and anti-CD28 mAbs were used to provide maximum TCR/costimulation to the CD4 cells. Briefly, anti-CD3e (2.5 µg/ml final concentration) was diluted in PBS and immobilized to the individual wells of 6-well flat-bottom tissue culture plates (Corning Glass, Corning, NY) in a final volume of 6 ml overnight. The plates were then washed with PBS twice. Purified CD4 cells (1 x 106) were then cultured in 6 ml of media containing RPMI 1640 medium supplemented with 10% FBS, 2-ME, and anti-CD28 (2.25 µg/ml final concentration) in a humidified atmosphere at 5% CO2 at 37°C for the indicated time period. RNAs from unstimulated and anti-CD3/anti-CD28-stimulated cells were isolated by TRIzol LS reagent (Life Technologies, Grand Island, NY), and a second cleanup step was performed using the Qiagen RNeasy total RNA isolation kit (Valencia, CA). Intracellular proteins were isolated from the phenol-ethanol supernatant with isopropyl alcohol after precipitation with ethanol, as per standard protocol.

RNA expression by GeneChip microarrays

Chemokine receptor gene expression of young and old, unstimulated and mAb-stimulated CD4 cells was initially screened using the Affymetrix GeneChip microarray gene expression system (Santa Clara, CA) as before (19). To minimize individual variability, pooled RNAs from the splenic CD4+ T lymphocytes of 5 and 15 animals were used for the first (µ11k) and second (U74A) experiments, respectively. Total RNA was isolated using TRIzol reagent (Life Technologies), followed by cleanup on a RNeasy spin column (Qiagen), then used to generate cRNA probes. Preparation of cRNA, hybridization, and scanning of the mouse genome µ11K and U74A arrays were performed according to the manufacturer’s protocol (Affymetrix). Briefly, 5 µg of total RNA was converted into double-stranded cDNA by reverse transcription using a cDNA synthesis kit (Superscript Choice system; Life Technologies) with an oligo(dT)24 primer containing a T7 RNA polymerase promoter site added 3' of the poly T (Genset, La Jolla, CA). Following second-strand synthesis, labeled cRNA was generated from the cDNA sample by an in vitro transcription reaction supplemented with biotin-11-CTP and biotin-16-UTP (Enzo Diagnostics, Farmingdale, NY). The labeled cRNA was purified by using RNeasy spin columns (Qiagen). Fifteen micrograms of each cRNA was fragmented at 94°C for 35 min in fragmentation buffer (40 mM Tris-acetate (pH 8.1), 100 mM potassium acetate, 30 mM magnesium acetate) and then used to prepare 300 µl of hybridization mixture (100 mM MES, 1 M NaCl, 20 mM EDTA, 0.01% Tween 20) containing 0.1 mg/ml of herring sperm DNA (Promega, Madison WI), 500 µg/ml acetylated BSA (Life Technologies), and a mixture of control cRNAs for comparison of hybridization efficiency between arrays and for relative quantitation of measured transcript levels. Before hybridization, the mixtures were heated to 94°C for 5 min, equilibrated at 45°C for 5 min, then clarified by centrifugation (16,000 x g) at room temperature for 5 min. Aliquots of each sample (10 µg of fragmented cRNA in 200 µl of hybridization mixture) were hybridized to mouse genome µ11k or U74A arrays at 45°C for 16 h in a rotisserie oven set at 60 rpm. The arrays were then washed with nonstringent wash buffer (6x standard saline citrate phosphate/EDTA (SSPE)) at 25°C, followed by stringent wash buffer (100 mM MES (pH 6.7), 0.1 M NaCl, 0.01% Tween 20) at 50°C, stained with streptavidin-PE (Molecular Probes, Eugene, OR), washed again with 6x SSPE, stained with biotinylated anti-streptavidin IgG, followed by a second staining with streptavidin-PE, and a third washing with 6x SSPE. The arrays were scanned using the GeneArray scanner (Affymetrix). Data analysis was performed using GeneChip 4.0 software. The µ11k and the U74A chips contain ~11,000 and 12,000 probe sets, respectively, with each probe set representing a transcript. Each probe set typically consists of 20 perfectly complementary 25 base-long probes as well as 20 mismatch probes that are identical except for an altered central base. We subtract the mismatch probe values from the perfect match values and average the middle 50% of these differences as the expression measure for that probe set. A quantile normalization procedure was used to adjust for differences in the probe intensity distribution across different chips. We then applied a monotone linear spline to each chip that mapped quantiles 0.02 up to 0.98 (in increments of 0.02) exactly to the corresponding median quantiles for all the samples. Then, the transform log(100 + max(X + 100; 0)) was applied to the data from each chip. The chemokine receptor results are then calculated as changes relative to the expression levels of unstimulated young CD4+ lymphocytes.

RNA protection assays (RPAs)

Aging-related changes in T cell chemokine receptor and cytokine expression were confirmed by RPAs. Pooled RNAs from equal numbers of purified CD4 cells from mice in groups of 4–6 animals were used to minimize individual variability. The probes were synthesized by modification of the manufacturer’s protocol. Briefly, GACU nucleotide pool and [{alpha}-32P]UTP, RNasin, T7 RNA polymerase were added to the multiprobe template set mCK-1 (IL-2, 4, 5, 6, 10, 13, 15, IFN-{gamma}), mCR-5 (CCR1–5), mCR-6 (CXCR2, 4, and 5) or a custom-made probe set (CCR6–9 and CXCR3) (all from BD PharMingen) and placed on a heat block at 37°C for 1 h. The reaction was terminated by adding DNase and incubated at 37°C on a heat block for 30 min. Appropriate volumes of EDTA, Tris-saturated phenol, chloroform:isoamyl alcohol (50:1), and yeast tRNA were then added to the mixture, as suggested by the manufacturer. The aqueous layer was extracted by chloroform:isoamyl alcohol, then pelleted by adding a 1:5, 4 M ammonium acetate and ice-cold 100% ethanol mixture. Five micrograms of total RNA from each T cell sample were used for hybridization. The protected probes were then allowed to be resolved by electrophoresis using a 5% acrylamide gel, exposed to a phosphor screen, and quantified by a PhosphorImager using Image Quaint software (Molecular Dynamics).

Western immunoblotting

Proteins from young, middle-age, and old CD4 cells were resolved on 10% SDS-polyacrylamide gels and transferred to nitrocellulose-1 membrane (Life Technologies, Gaithersburg, MD). The membrane was blocked in PBS containing 5% nonfat dry milk and 0.05% Tween 20, and subsequently incubated with anti-mouse CCR4, CXCR4 (Torrey Pines Biolabs, San Diego, CA), or CCR5 (BD PharMingen) followed by HRP-conjugated anti-rabbit and anti-rat IgG F(ab')2 (Amersham Life Science, Arlington Heights, IL). Detection was performed using the ECL system (Amersham Life Science). The membranes were then stripped and reprobed with anti-mouse-{beta}-actin Abs (Sigma-Aldrich, St. Louis, MO) to confirm equal protein loading.

In vitro chemotaxis assay

Dual-chamber chemotactic assays were performed to compare the SDF-1 and MIP-1{alpha} response of CD4 cells from young and old animals. Briefly, freshly isolated 4 x 105 CD4 cells in 100 µl of RPMI 1640 medium supplemented with 0.5% BSA were placed in Transwell Clear culture inserts with 5-µm pores (Corning-Costar, Cambridge, MA). The inserts were then placed in a 24-well tissue culture plate (Corning-Costar) containing 600 µl of the indicated concentrations of murine SDF-1 or MIP-1{alpha} (PeproTech, Rocky Hill, NJ) in RPMI 1640 medium supplemented with 0.5% BSA for 5 h in a humidified incubator at 37°C. Cells from the top and bottom chambers were then harvested and counted with a Beckman Coulter counter (Fullerton, CA).

Cytokine detection by ELISAs

Release of IL-10 and IFN-{gamma} by young and old CD4+ T cells into culture supernatants at 24, 48, and 72 h post anti-CD3/anti-CD28 stimulation were detected using the OptEIA mouse IL-10 and IFN-{gamma} ELISA kits (BD PharMingen), following the manufacturer’s instructions.

RNA stability assay

Freshly purified CD4 cells from young and old C57BL/6 mice were treated with actinomycin D (2 mg/ml final concentration) for the indicated time points. Chemokine receptor transcript levels were then examined by RPAs as above.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Screening of chemokine receptor expression by microarray

Microarray gene expression array analyses were initially done to screen for changes in CD4+ T cell chemokine receptor expression in young and old C57BL/6 mice. The chemokine receptor gene expression profile of unstimulated and anti-CD3/anti-CD28-stimulated (24–72 h) CD4+ cells were determined using both the Mu11k (included CCR1, 2, 4, 5, and 7) and the U74A (included CCR1, 2, 4, 5, 6, 7, 8 and 10) array. The signal intensity of some of the genes (CCR1, 4, 6, 8) was below the background fluorescence in at least one of the two experiments suggesting that their level of expression is beyond the sensitivity of the assay. Therefore, these results were excluded from the final analysis. The results of the microarray analysis showed that freshly isolated CD4 cells from old animals have significantly higher expression of CCR2, CCR5, and CXCR5 and lower expression of CCR7 compared with young CD4 cells (Fig. 1). Anti-CD3/anti-CD28 stimulation resulted in decreased expression of CCR2, CCR5, and CXCR5 in both young and old cells. To minimize statistical bias, all chemokine receptor genes were preselected before data analysis. Nevertheless, because a large number of genes were examined it remains possible that some of the observed changes represent chance occurrences by this screening approach. Chemokine receptor gene expression determinations were therefore confirmed and extended using RPAs.



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  		FIGURE 1. Screening of chemokine receptor expression by microarray. Microarray analyses were done on pooled RNAs of freshly isolated unstimulated (0 h) and anti-CD3/CD28 stimulated (72 h) splenic CD4+ cells from young (3–4 mo) and old (20–22 mo) C57BL/6 mice. The first experiment was performed using the µ11k array (five young and five old mice) and the second experiment was performed using the U74Av2 array (15 young and 15 old mice). A, Hierarchical clustering of chemokine receptor genes and selected controls (TCR genes, GAPDH and {beta}-actin) (94 ). The color of each cell represents the fold change above (red) and below (green) the reference samples. B, Histogram showing selected gene expression relative to unstimulated CD4+ cells (=1) from young animals. The results represent mean ± SEM of two experiments.

 
Chemokine receptor expression by RPAs

The effect of aging on CD4 T cell chemokine receptor gene expression was examined by RPA using the three probe sets described. The results showed that aging is associated with significant increased expression of CCR1, 2, 4, 5, 6, 8, CXCR2–5, and decreased expression of CCR7 and 9 in freshly isolated CD4 cells (Fig. 2). Gene expression of most chemokine receptors including CCR1–3, 5, 9, and CXCR2–5 decreases following anti-CD3/CD28 stimulation to a level comparable to stimulated young CD4+ cells. In contrast, CCR4 and CCR8 expression increase with anti-CD3/anti-CD28 stimulation similar to what has previously been reported (20).



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  		FIGURE 2. Effect of aging on T cell chemokine receptor expression as measured by RPAs. CCR1–9 and CXCR2–5 gene expression of pooled CD4+ cells from unstimulated (0 h) and anti-CD3/CD28 stimulated (24, 48, and 72 h) young (3–4 mo), middle-aged (12–14 mo), and old (20–22 mo) C57BL/6 mice were measured. Autoradiograph of representative RPAs showing the effect of aging and anti-CD3/anti-CD28 stimulation on CD4+ cell CCR1–5 (A), CXCR2, 4, and 5 (B), CCR6–9 and CXCR3 (C). Lanes 1–3 are unstimulated CD4+ cells. Lanes 4–6 are cells after 24 h stimulation. Lanes 7–9 are cells after 48 h stimulation. Lanes 10–12 are cells after 72 h stimulation. Young = lanes 1, 4, 7, and 10; middle-age = lanes 2, 5, 9, and 11; old = lanes 3, 6, 9, and 12. D, The results represent the mean ± SEM of three to eight independent experiments and each experiment has four to six mice in each age group. Gel loading is corrected with L32 expression. *, p < 0.05, the difference between the means of young and old was compared using Student’s t test.

 
Stability of chemokine receptor mRNA

Changes in chemokine receptor gene expression in aged T cells may be secondary to the altered rate of RNA transcription or posttranscriptional degradation. To exclude the latter possibility, transcription was arrested by the addition of actinomycin D and CCR1–5 mRNA levels were measured over time by RPA. The results showed that the rate of degradation of CCR1–5 mRNA is faster in aged CD4 cells than young CD4 cells, suggesting that the increased expression of these chemokine receptors is likely due to the increased rate of gene transcription (Fig. 3).



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  		FIGURE 3. RNA stability assay. A, RPAs were performed using RNAs extracted from an equal number of freshly isolated CD4+ T cells treated with actinomycin D (2 mg/ml) for 0 (lanes 1 and 5), 45 (lanes 2 and 6), 90 (lanes 3 and 7), and 135 (lanes 4 and 8) min. The intensity of the bands from young (lanes 1–4) and old (lanes 5–8) was quantified using a PhosphorImager. Gel loading was corrected for L32 expression. The results show faster rate of CCR1–5 RNA degradation in old vs young CD4+ cells.

 
Western blots

We next determined whether the changes in chemokine receptor gene expression result in altered protein production. Western blot analyses were done because mAbs suitable for flow cytometric testing of murine chemokine receptors were not available. The results showed excellent correlation with the RNA data, with increased CCR4, CCR5, and CXCR4 protein levels in CD4+ lymphocytes from old animals (Fig. 4). Increases in CCR4 and decreases in CCR5 and CXCR4 protein levels following anti-CD3/anti-CD28 stimulation were also observed, similar to the RNA results obtained by RPAs.



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  		FIGURE 4. Western blot analyses of selected chemokine receptors. Protein levels of CCR4, CCR5, and CXCR4 of unstimulated and anti-CD3/CD28-stimulated (24, 48, and 72 h) CD4 cells from young (lanes 1, 4, 7, and 10), middle-age (lanes 2, 5, 8, and 11), and old (lanes 3, 6, 9, and 12) C57BL/6 mice were quantified by Western blot analyses. Protein loading was corrected using {beta}-actin as controls. Each result represents the mean ± SEM of two independent experiments. *, Values of p <0.05, the difference between means of young and old was compared using the Student t test.

 
Chemotactic assay

Dual-chamber chemotactic assays were done using freshly isolated, unstimulated CD4+ T cells from young and old C57BL/6 mice to determine whether the observed increased C-C and C-X-C chemokine receptor expression in aged CD4+ cells have functional consequences. The results show that aged CD4 cells transmigrate to a significantly greater extent than young cells in response to SDF-1 (CXCR4) and MIP-1{alpha} (CCR1, 4, 5, 9; Fig. 5), thus confirming a functional role of increased chemokine receptor expression in CD4+ T cells from old animals.



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  		FIGURE 5. Effect of aging on T cell chemotactic response. The indicated concentrations of SDF-1 (A) or MIP-1{alpha} (B) were placed in the lower chamber of a Costar Transwell system and allowed to equilibrate for at least 2 h. Equal numbers of freshly isolated CD4 cells from young and old mice were then placed in the upper chamber of a Transwell insert with a 5-µm pore size membrane. The cells in the top and bottom chambers were then harvested after 5 h and counted using a Coulter counter. The results are expressed as the percent of cells transmigrated to the lower chamber and represent the mean ± SD of triplicate determinations.

 
Th1/Th2 cytokine production of CD4 cells from young and old mice

Chemokine receptors have been reported to be variably expressed in Th1 (CCR5, CXCR3, CXCR6) and Th2 (CCR3, 4, 8) cells. Skewing toward a Th2 profile and increased IFN-{gamma} production have also been reported in aging CD4+ lymphocytes. Therefore, we determined the Th1/Th2 cytokine RNA (microarray, RPA) and protein (ELISA) levels in the isolated young and old CD4+ T cells (Fig. 6). Our data is consistent with what has been reported in the literature, with old CD4+ cells producing higher amounts of IFN-{gamma} and IL-10 (21, 22).



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  		FIGURE 6. Cytokine production of young and old CD4 cells. A, Initial screening was done by examining Th1 and Th2 cytokine gene expression by microarray analyses. The results are expressed relative to CD4 cells from young C57BL/6 mice (=1). B, Cytokine gene expression of anti-CD3/CD28-stimulated (72 h) CD4 cells pooled from five young and five old C57BL/6 mice was by examined by RPA. The results are expressed relative to cells from the young mice (=1). C, IL-10 and IFN-{gamma} levels in the supernatants of young and old anti-CD3/CD28-stimulated CD4 cells were measured by ELISAs. Results represent the means ± SD of triplicate determinations.

 
Chemokine receptor expression in young and old CD4+ cells from DBA/2 mice

A recent report suggested that the chemokine receptor expression profile of spleen cells from different strains of normal mice may be different (23). To exclude the possibility that the aging-associated chemokine receptor changes detected are strain-specific, pooled CD4+ T cells were isolated from five young (3–4 mo) and five old (20–22 mo) DBA/2 mice. RNAs were isolated as before and chemokine receptor expression was determined by RPAs (Fig. 7). The DBA/2 results are similar to those obtained from CD4 cells from C57BL/6 mice, suggesting that the observed aging-related changes are not unique to C57BL/6 mice.



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  		FIGURE 7. T cell chemokine receptor expression in DBA/2 mice. CC and CXC chemokine receptor gene expression of pooled unstimulated (0 h) and anti-CD3/CD28-stimulated (72 h) splenic CD4+ cells of five young (3–4 mo) and five old (20–22 mo) DBA/2 mice were examined by RPAs. Lane 1 = young unstimulated; lane 2 = old unstimulated; lane 3 = young 72 h after stimulation; lane 4 = old 72 h after stimulation.

 
Chemokine receptor expression in CD4+ cells from caloric restricted aged mice

Caloric restriction has been shown to reverse many features of immune senescence in mice and some primates (24). However, its effect on chemokine function is unclear. To determine whether caloric restriction will reverse the T cell chemokine receptor changes in aged animals, we compared the chemokine receptor profiles of aged C57BL/6 mice fed ad-lib and caloric-restricted aged C57BL/6 mice (Fig. 8). Caloric restriction down-regulated the expression of selected chemokine receptors including CCR1, 8, and CXCR2 to levels similar to CD4+ lymphocytes from young animals. In addition, the level of CCR7 expression was increased to that seen in lymphocytes from young mice. Caloric restriction also partially reversed the aging-associated changes in CCR9, CXCR4, and 5 expression although these changes did not reach statistical significance.



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  		FIGURE 8. Effect of caloric restriction (CR) on T cell chemokine receptor expression in aging. Freshly isolated splenic CD4+ cells were pooled from five old caloric restricted as well as five young and five old C57BL/6 mice that have been fed ad lib throughout their lives. Expression levels of CC and CXC chemokine receptors were then determined by RPAs. Lane 1 = CD4+ cells from young mice; lane 2 = CD4+ cells from old mice; lane 3 = CD4+ cells from old caloric-restricted mice. The results represent mean ± SD of two independent experiments (total 10 mice in each condition). *, Values of p <0.05 comparing old mice fed ad lib to caloric-restricted old mice using the Student t test.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Aging has often been associated with a "decline" in immune functions. However, attempts to correlate age-related changes in immunity with specific disease susceptibility or outcome have been largely unsuccessful (21, 22). Multiple large human studies have found, at the most, a small decline in the number of CD3, CD4, or CD8 cells of doubtful clinical significance. In contrast, an age-associated shift from naive to memory phenotype (25, 26), presumably due to lifelong antigenic stimulation, has been observed consistently and may account for many of the functional changes seen in T cells in aging. Murine studies have also suggested that aging may be accompanied by a shift to a predominantly Th2 cytokine profile (21, 22, 27). However, other investigators have reported conflicting results and attempts to reproduce the findings in humans have resulted in more controversy than answers (21, 22).

Very little is currently known about the effect of aging on the chemokine system. GRO/CINC-1 (IL-8-like chemokine) gene expression and production are increased in the nasal mucosa of old (~18 mo) compared with young rats (28). Serum level and T cell production of IL-8 are also increased in elderly humans (29). Others have showed age-related increased production of peripheral blood cell IL-8, monocyte chemoattractant protein-1, MIP-1{alpha}, and RANTES with or without stimulation with anti-CD3 mAb and LPS (30, 31). Similar increase in selected chemokine expression has been demonstrated in the brain of aged rodents (32). However, there is very little information on the effect of aging on T cell chemokine receptor expression. Tarazona et al. (33) showed decreased CCR7 expression on NK cells in elderly individuals, and suggested that this is due to the accumulation of memory T cells with aging. Carramolino et al. (34) recently examined CCR9 expression during development and showed that thymocyte CCR9 is expressed at all stages of T cell maturation in mice, with maximum expression in CD4+CD8+ cells. Expression of thymic CCR9 maintains or decreases slightly during the first year of rodent life. The investigators also found that the CD4+ cells of the secondary lymphoid organ lose, but CD8+ cells maintain, CCR9 expression in the neonatal period. These studies suggest that age and development affect leukocyte chemokine receptor expression. However, because the oldest animals examined were only 12 mo old, the effect of aging on chemokine receptor expression is unknown.

The current study demonstrated that aging is associated with important changes in T cell chemokine receptor expression. Specifically, aging is associated with the increased expression of CCR1, 2, 4, 5, 6, and 8, CXCR2–5 and decreased expression of CCR7 and 9 in CD4+ T cells. We further demonstrated that these T cell changes correlate to increase protein expression and greater chemotactic responses to SDF-1 and MIP-1{alpha}. Anti-CD3 plus anti-CD28 stimulation down-regulates the expression of multiple chemokine receptors, including CCR1–3, 5, 9, and CXCR2–5, in CD4+ cells from old animals to levels comparable to stimulated young CD4+ cells. Others have also found that stimulation of T cells with mitogens, phorbol ester, anti-CD3, or anti-CD3/anti-CD28 decreases selected chemokine receptors including CCR5 and CXCR4 (20, 35, 36, 37). However, in some studies the chemokine receptor changes following stimulation are less significant and the nature of the in vitro stimulation may affect the T cell chemokine receptor response (38, 39). Finally, caloric restriction has been shown to retard many aspects of aging-associated decline in immune functions including mitogen-induced T cell proliferation and IL-2 production (24, 40). Our data show that long-term caloric restriction can restore the expression of selected chemokine receptors (CCR1, 7, 8, and CXCR2) to levels similar to those seen in young CD4+ lymphocytes.

A number of studies have found that T cells from aged hosts have diminished proliferation and IL-2 response to mitogens and to anti-CD3 stimulation (41, 42, 43, 44, 45). Some, though not all, of these abnormalities can be linked to the replacement of naive by memory T cells in aging. Others have also reported that this age-associated decline in T cell activation can be rescued by a concomitant CD28 signal (46, 47, 48). Although this is consistent with our results showing that the age differences in CD4+ T cell chemokine receptor expression largely disappear following maximal anti-CD3 and anti-CD28 stimulation, whether submaximal stimulation with anti-CD3 alone or with mitogen will have the same effect is unclear. Finally, enhanced IFN-{gamma} and IL-4 production in aged CD4+ and CD8+ T cells following combined anti-CD3 and anti-CD28 stimulation, similar to what we have found, has also been reported (49).

The underlying mechanism for the observed aging-associated changes in chemokine receptor expression and function is unclear. Increased mRNA levels are likely the mechanisms contributing to increased chemokine receptor protein production in T cells in aging. Our RNA degradation data suggest that the increased mRNA levels are due to an increased rate of RNA transcription. However, translocation of intracellular protein stores to the cell surface (14) may provide an additional level of regulation that is not examined in the current study. Genetic polymorphism of the chemokine receptor promoter can affect chemokine receptor cell surface expression in individuals (50) but cannot explain any aging-related changes. A number of transcription factors are involved in the regulation of chemokine receptor expression. The transcription factor C/EBP {beta} (NF-IL-6) activates CCR5 gene expression in Jurkat T cells (51) and the gene regulation of C/EBP is dependent on interactions with other transcription factors such as NF-{kappa}B (52). C/EBP binding sites have been identified in other chemokine receptors including CCR2 (53). NF-{kappa}B is also an important regulator of a number of chemokine receptor genes (54, 55, 56, 57). However, whether changes in transcription factors are responsible for the observed aging-associated increase in chemokine receptor expression is unclear because activities of many transcription factors including NF-{kappa}B are diminished in aged T cells (58, 59). More recently, chromatin remodeling has been implicated in the regulation of CCR3 expression in T cells (60). Many chemokine receptor genes are located on the same chromosomes. For example, CCR1–5 and 8 are clustered in a relatively small region (p21.3-p24) of chromosome 3 (61). Therefore, the epigenetic mechanism that can affect chromatin structure can potentially affect expression of multiple chemokine receptor genes. However, it is currently unknown whether such changes are involved in the regulation of chemokine receptor expression in aging.

A shift in memory/naive profile in aging may account for some of the observed aging-associated changes in chemokine receptor expression. As mentioned previously, an increased memory T cell subset is known to occur with aging. Chemokine receptor expression has also been reported to differentiate naive and memory T cells. For example CCR4 (62), CCR5, and CXCR3 (62, 63) expressions are increased in memory cells and CXCR4 (63) are expressed primarily in naive cells. Interestingly, CCR7 expression in CD4 and CD8 cells has recently been shown to correlate with homing to secondary lymphoid organs (64). A number of investigators have also reported decreased leukocyte splenic homing with aging (65, 66, 67, 68, 69). Therefore, our finding that aged animals have decreased CD4+ T cell CCR7 expression may provide an explanation for the age difference in T cell trafficking. We also found that CD44 expression is increased in freshly isolated aged CD4 cells (2.9- and 3-fold increased CD44 expression in old compared with young cells in two separate microarray experiments) consistent with the reported higher memory cell population in aging. However, both naive (e.g., CXCR4) and memory (e.g., CCR4, CCR5) chemokine receptors are increased with aging, suggesting that the observed differences are not solely due to a shift in naive and memory subsets. Chemokine receptor expression in naive and memory young and old T cell subsets will need to be examined to definitively address this issue.

Results from a number of studies have led to the belief that expression of selected chemokine receptors correlates with Th1 and Th2 polarization. Th1 cells tend to express CCR5 (70, 71), CXCR3 (62, 63, 71), and CXCR6 (72) whereas Th2 cells are more likely to express CCR3 (71, 73), CCR4 (62, 63, 71, 74), and CCR8 (75). Anti-CD3/CD28 stimulation increased CCR1, CCR4, CCR5 expression and decreased CXCR3 and CXCR4 in human cloned Th1 cells (76). Similar activation increased CCR4, CXCR4 and decreased CCR2, CCR3, CCR5 expression in Th2 cells. As mentioned before, whether aging is linked to a more prominent Th2 response is not clear. In the current study, we found that both Th1- and Th2-associated chemokine receptors are increased in aged T cells. Therefore, an aging-associated shift in the Th1/2 dichotomy alone also cannot account for the observed aging differences in T cell chemokine receptor expression. Nevertheless, cytokines are known to play an important role in the regulation of chemokine receptor expression (77, 78) and it remains possible that changes in cytokine milieu in aging are important determinants of T cell chemokine receptor expression in aging. A recent report showed that polarized effector and nonpolarized memory T cells display a diverse combination of chemokine receptors with likely heterogeneous trafficking properties (79). Therefore, these are other recent data that highlight the potential roles multiple chemokine receptors play in the multistep process of T cell migration and function.

The chemokine system has been shown to play a critical role in the pathogenesis of many diseases important to the elderly. Our findings of an aging-related increase in T cell chemokine receptor expression and function may help explain the high prevalence and severe clinical course of these diverse diseases in the elderly. For example, the Centers for Disease Control reports that individuals 50 years or older constitute at least 10% of new AIDS cases, representing the fastest growing HIV-infected group (80). Studies have shown that older patients have a shorter observed AIDS-free interval and shorter survival, along with more HIV and non-HIV related comorbidity (81, 82, 83). HIV-1 viral strains preferentially use a number of chemokine receptors including CXCR4, CCR2, 3, and 5 as coreceptors for viral entry into lymphocytes and macrophages (11, 84). It has recently been shown that the level of chemokine receptor expression may predict susceptibility to the development of AIDS (14, 16, 85). Rheumatoid arthritis is another disease that preferentially affects the elderly (86, 87, 88). Elderly onset rheumatoid arthritis (age >60 years) is also a distinctly different disorder from younger-onset rheumatoid arthritis (age 30–50 years), with more abrupt onset of disease, increased likelihood of large joint involvement, and seronegativity (18). An important role of leukocyte chemokine receptor expression in rheumatoid arthritis has recently been established (6, 89). These studies identified CCR4, CCR5, CXCR3, and CX3CR1 as the critical chemokine receptors in the recruitment and retention of T cells and monocytes in the rheumatoid joint. Thus, it is possible that the age-associated increased susceptibility and different clinical course of HIV-1 infection and rheumatoid arthritis may be in part due to the age-associated increase in T cell chemokine receptor expression and function. Atherosclerosis is now viewed as a chronic inflammatory disease. T cells accumulate early in atheroma formation and persist at sites of lesion growth and rupture. T cell-activating chemokines are produced by atheroma-associated endothelial cells, smooth muscle cells, and macrophages, and are believed to play an important role in both early and late plaque formation (90, 91, 92, 93). The increased T cell chemokine response may provide a previously unexplored mechanism for this disease in the elderly.

In summary, we have provided the first evidence that aging is associated with important changes in T cell chemokine receptor expression and function. Improving our understanding of the chemokine biology of aging should lead to a better understanding of both normal and pathologic responses in the elderly.


   Footnotes
 
1 This work was supported by Public Health Service Grants 1K08AR01977-01A1, 1RO1 AI42753, 1RO1 HL61577, American Federation for Aging Research (Paul Beeson Physician Faculty Scholar Award), the University of Michigan Nathan Shock Center (AG13282), and the Geriatrics Research, Education, and Clinical Center of the Ann Arbor Veterans Affair Medical Center. Back

2 Address correspondence and reprint requests to Dr. Raymond L. Yung, Room 5312 Cancer Center and Geriatrics Center Building, 1500 East Medical Center Drive, Ann Arbor, MI 48109-0940. E-mail address: ryung{at}umich.edu Back

3 Abbreviations used in this paper: MIP, macrophage-inflammatory protein; SDF, stromal cell-derived factor; RPA, RNase protection assay. Back

4 Strain contamination of the C57BL/6 breeding colony in the National Institute on Aging contract aging mouse colony at Harlan Sprague Dawley with 129 or FVB was recently discovered, affecting some aging mice with C57BL/6 background. The evidence indicates that the strain contamination first hit the aging colony with the spring or summer 2000 dates of birth. The mice used in this manuscript were born before the estimated contamination date or were deemed to be of "low risk" by the National Institute on Aging. Back

Received for publication August 26, 2002. Accepted for publication November 5, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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