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* Northern Ireland Centre for Food and Health, University of Ulster, Coleraine, Northern Ireland; The Waltham Centre for Pet Nutrition, Waltham-on-the-Wolds, Melton Mowbray, Leicestershire, UK; and ** Department of Pharmaceutical Sciences, University of Nottingham, University Park, Nottingham, UK
3 To whom correspondence should be addressed. Email: dj.campbell{at}ulster.ac.uk.
KEY WORDS: • aging • feline • T cell • oxidative stress • apoptosis • proliferation
There is a substantial body of evidence to suggest that the mammalian immune system becomes altered with age, resulting in increased susceptibility to infectious illness (1–4). An altered peripheral T-cell pool is characteristic of aging in numerous mammalian species (5–12) and is believed to result in a loss of immune competence. The onset of these changes, collectively known as immune senescence, is likely to be a multifaceted and interactive process, with thymic atrophy, antigen exposure, and replicative senescence contributing to a differentiated and progressively indolent T-cell pool, resulting is a general loss of adaptive immune responsiveness.
Although there is some evidence for functional impairment at the level of the individual T cell with age, it remains unclear whether this contributes substantially to immune senescence other than in the very old (6). The ability to regulate both cell-cycle progression and apoptosis, however, is central to the maintenance of immune homeostasis. Any impairment of these processes within the aging T-cell compartment could exacerbate an already limited capacity to respond to novel antigens, driving the adaptive immune response below a threshold of competence and predisposing the host to opportunistic infections and cancer.
A reduced capacity of peripheral lymphocytes to respond to mitogenic stimuli in aging humans or animals has been reported regularly in addition to alterations in apoptotic pathways (13,14). Having established previously that there are extensive alterations to the T-cell compartment in healthy cats aged 10–14 y compared with young adults (11), our goal was to determine to what extent the processes of mitogenesis and oxidatively induced apoptosis were affected in cells from this particular age group.
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MATERIALS AND METHODS |
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Experimental design. Cats were assigned to 1 of 2 sex-matched age groups: a young control group, adult (age 2–5 y), and a senior group (age 10–14 y). A 5-mL blood sample was collected from 36 cats (adult, n = 14, senior, n = 22) to obtain lymphocytes for ex vivo culture, assessment of proliferative responses, and enumeration of T-cell populations. All samples were collected in sterile tubes, coated or uncoated with lithium-heparin or EDTA.
Differential white blood cell analysis. Lymphocyte numbers were determined after the measurement of total and differential white blood cell (WBC)4 counts, conducted on an automated hematology analyzer (SE-9500; Sysmex).
T-cell phenotyping by flow cytometry. A panel of commercially available monoclonal antibodies for feline lymphocytes was used to phenotype CD5+ pan T cells in feline whole blood by flow cytometry as described previously (11).
Preparation and culture of peripheral blood lymphocytes (PBL). For lymphocyte isolation, heparinized peripheral blood was diluted 1:3 with sterile PBS without Ca2+ and Mg2+ (Invitrogen) and layered over Ficoll-Histopaque (density 1.077 g/mL, Sigma-Aldrich). Samples were centrifuged at 400 x g for 30 min. The peripheral blood mononuclear cells (PBMC) that collected at the interface were transferred to a fresh tube, washed twice with PBS, and resuspended at a concentration of 5 x 105 cells/mL in AIM-V serum-free lymphocyte culture media with 50 mg/L gentamicin (Invitrogen). PBMC were cultured in tissue culture flasks (Nunclon, Nunc) at 37°C in a 5% CO2 atmosphere for 24 h before assay to allow time for monocytes to adhere to the flask surfaces. Cell suspensions consisted of >90% PBL with >95% viability.
Assessment of proliferative response.
DNA synthesis in PBL in culture and in cultured whole blood was assessed in the presence of, optimized concentrations of T-cell mitogen concanavalin A (Con A; 10 mg/L) or phytohemagglutinin (PHA; 9 mg/L) as previously described (14,15). Cell were separated and set up in triplicate wells at a concentration of 2.5 x105 cells/mL in AIM-V medium; whole blood was diluted 1:10 in RPMI medium (Invitrogen). Methyl-[3H]-thymidine (0.5 µCi/well; Amersham-Pharmacia Biotech) was added to each well for the final 16 h of culture and the activity of each well recorded as counts/min (cpm). T-cell proliferative responses to T-cell mitogens Con A and PHA were calculated for whole blood and data (cpm) were expressed per 2.5 x 105 cells (as below) using a method described previously (16–18).
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Induction of apoptosis in PBL. Cells were incubated for 24 h (37°C/5% CO2) in 96-well plates in AIM-V serum-free medium before further incubation for 16 h in 10 mmol/L 2-deoxy-D-ribose (dRib) (Sigma-Aldrich). Spontaneous apoptosis was measured by incubating cells in medium alone.
Cytofluorometric detection of apoptosis by Annexin V/7-amino-actinomycin D (7-AAD) staining. For the assessment of apoptosis, cells were washed once, resuspended in Annexin-V binding buffer stained with phycoerythrin-conjugated Annexin-V followed by the vital dye, 7-amino-actinomycin D (7AAD) (BD). Flow cytometric analysis of percentages of apoptotic cells was carried out on a FACScalibur cytometer (BD) using Cellquest software. Annexin-V positive/7AAD negative are considered to be early apoptotic cells.
Statistical analysis.
Differences between the adult and senior groups were analyzed by unpaired t test if the data were normally distributed, and by Mann-Whitney rank-sum test if they were skewed. Analyses were conducted using SPSS 9.0.1 software for Windows (SPSS). Differences were considered significant if P 
0.05. Data are expressed as means ± SEM.
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RESULTS |
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TOPMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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0.01) than in the adult group (2.47 ± 0.34 x 109cells/L).
Proliferative responses to T-cell mitogens in adult and senior cats.
Ex vivo cultures of PBL from the adult cats had a higher T-cell proliferative response to both Con A and PHA (P 0.01) than lymphocytes from the older cats (Fig. 1). Whole blood from adult cats incubated in the presence of Con A also had a higher response than that of the older cats (17.03 x 103 ± 2.3 vs. 11.8 x 103 ± 1.6 cpm; P 0.05) although the response to PHA did not differ. When values from the whole-blood assays where adjusted for T-cell concentration, however, the adult and senior cats did not differ in their proliferative responses to either of the T-cell mitogens (Fig. 2).
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0.001). Spontaneous apoptosis in PBL cultures derived from either the adult or senior cats did not differ, nor was there an observable effect of age on dRib-induced apoptosis in the adult or senior group of cats after detection of Annexin V positive/7AAD negative early apoptotic cells (Fig. 3). However, the groups differed (P 0.05) in the percentage of double positive (+/+) cells, which are regarded as late apoptotic or necrotic, with the younger cats having higher proportions of +/+ cells.
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DISCUSSION |
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TOPMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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In the current study, we investigated the functional status of T lymphocytes derived from a 10- to14-y-old group of cats by assessing the proliferative response to T-cell mitogens in addition to spontaneous and induced apoptosis. Assessment of the proliferative response to Con A and PHA was conducted using 2 separate assays to make comparisons with previous studies. Responses to the T-cell mitogens Con A and PHA were significantly lower in PBL from the older cats, and these findings agree with those of earlier studies employing similar methods in PBMC from aging dogs (7,8) and feline immunodeficiency virus seropositive cats (22). Initially, an attenuated response to Con A was also observed in the whole-blood assays. In these assays, however, it was possible to make an accurate assesment of the T-cell concentration, and when proliferation data from whole-blood assays were normalized for T-cell numbers, a restoration of the proliferation index resulted. This would suggest that the large differences in cpm observed in the purified PBL cultures are more likely attributable to lower proportions of responsive T cells than to impairment of T-cell mitosis. Findings similar to those of the current study were described in lymphocytes derived from 10-y-old Labrador retriever dogs where it was found that although lymphocyte numbers do decline significantly with age, the residual population remains fully responsive to stimulation (23,24).
In conclusion, data from the current study do not suggest that cells from 10- to 14-y-old cats show increased susceptibility to spontaneous or dRib-induced apoptosis nor is there evidence of an impaired proliferative response to T-cell mitogens when responses in these cats are compared on the basis of T-cell numbers. These findings suggest that some early observations of reduced proliferative response to T-cell mitogens in aging mammals may be attributable to lower T-cell proportions in PBMC cultures rather than to replicative senescence or other cellular dysfunction. Thus, when making life-stage associated assessments of functional status, it is important to make these observations in homogenous T-cell populations.
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FOOTNOTES |
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2 Author disclosure: This work was funded by the WALTHAM Centre for Pet Nutrition.
4 Abbreviations used: 7AAD, 7-amino-actinomycin D; Con A, concanavalin A; dRib, 2-deoxy-D-ribose; PBL, peripheral blood lymphocytes; PBMC, peripheral blood mononuclear cells; PHA, phytohemagglutinin.
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LITERATURE CITED |
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TOPMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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