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The Journals of Gerontology Series A: Biological Sciences and Medical Sciences 61:907-917 (2006)
© 2006 The Gerontological Society of America

Whole Blood Analysis of Phagocytosis, Apoptosis, Cytokine Production, and Leukocyte Subsets in Healthy Older Men and Women: The ZENITH Study

Clare F. Hodkinson, Jacqueline M. O'Connor, H. Denis Alexander, Ian Bradbury, Maxine P. Bonham, Bernadette M. Hannigan, William S. Gilmore, J. J. Strain and Julie M. W. Wallace

1 Northern Ireland Centre for Food and Health (NICHE), University of Ulster, Coleraine, Northern Ireland.
2 Department of Haematology, Belfast City Hospital, Northern Ireland.
3 School of Biology, Chemistry and Health Science, Manchester Metropolitan University, United Kingdom.

Address correspondence to Julie M. W. Wallace, PhD, Senior Research Fellow, Northern Ireland Centre for Food and Health (NICHE), University of Ulster, Coleraine, Northern Ireland, BT52 1SA. E-mail: j.wallace{at}ulster.ac.uk


   Abstract
Top
 Abstract
Methods
 Results
 Discussion
 References
 
Few studies to date have examined age-related changes in markers of immune status in healthy older individuals. The immune status of 93 healthy individuals aged 55–70 years was assessed by two- and three-color flow cytometry and biochemical analysis. There were significant age effects (p ≤.05) on monocyte phagocytic activity and cluster of differentiation (CD) 3/human leukocyte antigen-D-related (HLA-DR) late-activated T lymphocytes (% expression). There was a significant (p ≤ 0.1) Age x Sex interaction in absolute counts (x 109/L) of CD3/CD8 total cytotoxic T lymphocytes (CTL), the CD4 T- helper to CD8 CTL ratio, the CD3/CD4/CD45RA naïve T helper to CD3/CD4/CD45RO memory T helper lymphocyte ratio, and interleukin (IL)-1ß (% expression) by activated monocytes. The study shows that alterations in markers of immune status occur between 55 and 70 years, and provides reference values for the lymphocyte measures in healthy men and postmenopausal women in this age group. The study further highlights the need for sex-specific reference ranges for such markers.

DESPITE considerable research, the mechanisms underlying immunosenescence remain largely unclear. Involution of the thymus, which has been observed as early as the first year of life (1), and continues throughout life (2), may coincide with the establishment of fully functioning innate and adaptive immune responses in the infant (3). By age 40 years, the thymus may have undergone 90% involution (4), resulting in diminished replenishment of the peripheral T lymphocyte pool by naïve (CD3+/CD45RA+) T lymphocytes; such diminished replenishment with age is a hallmark of immunosenescence (5–7). Immunosenescence, however, is not simply a product of thymic involution but is in fact a multifaceted, complex phenomenon that is the consequence of numerous molecular and biochemical alterations.

Several other mechanisms, in addition to thymic involution, have been suggested to contribute to the development of immunosenescence. Reduced naïve T lymphocyte output with age is believed to contribute to the contraction of T lymphocyte T-cell receptor (TCR) repertoire (8), thus reducing the efficiency of response to novel antigens (9). Decreased telomerase activity in lymphocyte subpopulations with age is believed to result in replicative senescence, reducing cell proliferation within the periphery (10). Also, the oligoclonal expansions of effector memory (CD3+/CD8+/CD45RO+) cytotoxic T lymphocytes (CTL) with age may inhibit the production of naïve T lymphocytes (11–13). In addition, alterations in lymphocyte susceptibility to apoptosis may result in the loss of specific immune cells and expansion of other subpopulations. Together, such changes may contribute to dysfunction of the immune system with age (14,15).

Studies conducted predominantly in individuals >70 years of age have demonstrated that, although the most striking age-related changes have been observed in the cell-mediated arm of the immune system (16), radical age-associated restructuring of the whole immune system occurs as a consequence of the upregulation of some aspects of immunity and diminished function of others (4,17,18). Consequently, immunosenescence contributes to increased susceptibility to infection, autoimmune disease, and cancer, leading to higher morbidity and mortality. It is hypothesized that successfully aged individuals, such as healthy centenarians, currently represent a model of the aged immune system that has adapted appropriately with age (19).

To date, there is a distinct lack of studies that have investigated the age-related changes in the immune status of late-middle-aged (55–70 years) individuals. In addition, many studies investigating immune status have been based on selected samples of aging volunteers or clinically based samples, with the most detailed studies belonging to the SENIEUR project of EURAGE [e.g., (20)]. The elucidation of early alterations in immune status within late-middle-aged individuals may help to identify potential targets for therapeutic intervention prior to the onset of multiple deficiencies common in adaptive immunity from the seventh decade of life.

The current study aimed to assess the immunological status of free-living, apparently healthy late-middle-aged individuals, aged 55–70 years and to demonstrate the sex-specific age-associated changes in immune status that result in a differing pattern of immunological aging in men and women.


   METHODS
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 Abstract
 Methods
Results
 Discussion
 References
 
Participants
A total of 147 late-middle-aged individuals (77 women, 70 men) were recruited from across Northern Ireland through media coverage, leaflets, as well as national and local organizations with members spanning this age group. Of the 147 participants recruited, 101 apparently healthy individuals were invited to take part in the study based on defined exclusion criteria, including: body mass index (BMI) (<20 or >33 kg/m2); abnormal hematology, liver, and kidney function tests; unusual dietary habits (e.g., vegetarians and vegans); acute or chronic disease; poor neuropsychological performance; premenopause; use of hormone replacement therapy, immune-modulating medications, habitual use of vitamin and/or mineral supplements in the last 6 months; alcohol (>30 g/d for men and >20 g/d for women); and >10 cigarettes, cigars, or pipes/d. Subsequently, some 93 apparently healthy late-middle-aged individuals (48 women, 45 men), aged 55–70 years, participated in the study. The University of Ulster Research Ethical Committee granted approval for the study. All volunteers gave written informed consent in accordance with the declaration of Helsinki.

Experimental Protocol
Following an overnight (>12 hours) fast, participants were asked to attend the research center at 8:30 AM on the study day. Anthropometric measurements were determined, and blood was collected immediately for hematological and biochemical analysis.

Anthropometric Measurements
The anthropometric parameters body weight and height were measured. BMI was calculated as body weight (kg) divided by height (m) squared.

Collection of Peripheral Blood and Hematological Measures
Fasting, venous blood (55 ml) was collected in K3EDTA, lithium–heparin, sodium–heparin, and serum vacutainers, between 8:30 and 9:00 AM. K3EDTA whole blood was used for determination of immune status using the fluorescence-activated cell sorter (FACS) FACSCalibur flow cytometer (BD Biosciences, Oxford, U.K.) within 4 hours of collection. The fluorochrome-conjugated-monoclonal antibodies (mAb) used for FACS analysis can be seen in Table 1. K3EDTA anticoagulated whole blood was also used for assessment of full blood profiles, which was conducted at the Causeway Laboratory, Causeway NHSS Trust, Coleraine, Northern Ireland.


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  		Table 1. Antihuman Conjugated Monoclonal Antibodies Used in FACS Analysis of Peripheral Blood Leukocyte Subpopulations in Healthy Individuals, Aged 55–70 Years.

 
Biochemical Analysis
Aliquots of serum were stored at –80°C and analyzed for C-reactive protein (CRP; high sensitivity), complement protein-3 (C3), and complement protein-4 (C4), using commercially available kits on a Hitachi 912 analyzer (Roche Diagnostics, Lewes, U.K.). Dietary intakes were assessed using a 4-day food diary. Dietary records were analyzed using standard food portion sizes (21) and nutrient intake calculated using the Weighed Intake analysis Software Package (WISP) 3.0 for Windows (Tinuviel Software, Warrington, U.K.).

Leukocyte Immunophenotyping
Immunophenotyping was performed as previously described by Hodkinson and colleagues (22).

Apoptosis
The determination of early lymphocyte apoptosis by two-color flow cytometry was conducted using the Annexin V-fluorescein isothiocyanate (FITC) Apoptosis Detection Kit I (BD Biosciences). Briefly, peripheral blood mononuclear cells (PBMCs) were separated by density gradient centrifugation from sodium–heparin anticoagulated whole blood using Accuspin System-Histopaque-1077 tubes (Sigma-Aldrich, Dorset, U.K.). PBMCs were washed twice in fresh sterile filtered phosphate-buffered saline and then resuspended in buffer. PBMCs were stained using Annexin V-FITC and propidium iodide (PI). Lymphocytes were gated using a forward-scatter (FSC) versus side-scatter (SSC) dotplot. Lymphocytes staining positive for Annexin-V and negative for PI were determined using fluorescence channel (FL)-1 height (H) versus FL-2 H dotplots. The percentage of lymphocytes undergoing early apoptosis was obtained from FACS analysis, and absolute counts were calculated using the lymphocyte white blood cell differential (x 109/L).

Phagocytosis
The quantification of phagocytic capacity and activity of granulocytes and monocytes was determined using a PHAGOTEST kit (ORPEGEN Pharma, Heidelberg, Germany). Sodium–heparin anticoagulated whole blood (100 µL) was incubated with opsonized Escherichia coli-FITC. Monocytes and granulocytes were gated using FSC versus SSC dotplot. SSC versus E. coli-FITC dotplots were used to measure phagocytic capacity and activity for both cell types. Percentage of E. coli-FITC-positive cells determined phagocytic capacity, and mean fluorescence intensity (MFI) was used as a quantitative measure of the number of E. coli ingested per cell; this measure was used as a determinant of phagocytic activity.

Determination of Intracellular Cytokine Production by Activated Monocytes
To determine intracellular cytokine production by activated monocytes, 1 mL of sodium–heparin anticoagulated whole blood was incubated with lipopolysaccharide (LPS) at 1 µg/mL and brefeldin A (BFA) at 10 µg/mL (Sigma-Aldrich) for 4 hours at 37°C with 5%–7% CO2. After activation, 100 µL of activated blood was incubated with either 10 µL of immunoglobulin G (IgG) 2{alpha} FITC (isotype control) or CD14 FITC (BD Pharmingen, Oxford, U.K.) for 30 minutes in the dark at room temperature. Erythrocytes were lysed by incubation with 100 µL of Fixation medium A (Caltag, Invitrogen, Paisley, U.K.) for 30 minutes in the dark at room temperature. Cells were washed using cell wash solution (1 L of sterile phosphate-buffered saline containing 0.5% bovine serum albumin and 0.1% NaN3), and were centrifuged at 300 x g for 5 minutes at 4°C. Supernatant was removed, and the cell pellet resuspended and incubated with 100 µL of permeabilizing medium B (Caltag, Invitrogen) for 15 minutes in the dark at room temperature. Cells were then incubated with either 10 µL of IgG1- phycoerythrin (PE; isotype control), interleukin (IL)-1 ß PE, or IL-6 PE for 30 minutes in the dark at room temperature. Cells were washed as previously described, and fixed with 500 µL of 1X Cell Fix solution (BD Biosciences). Samples were analyzed immediately.

Monocytes were gated using FSC versus SSC dotplots, and percentages of cytokine-positive CD14+ cells were obtained from FL-1 H versus FL-2 H dotplots. Quantification of intracellular cytokine production, as determined by the antibody binding capacity (ABC), was achieved using PE QuantiBrite beads (BD Biosciences) for standardization of PE MFI.

Data Analysis
Statistical analysis was performed using a Statistical Package for Social Sciences (SPSS) version 11.0 (SPSS Inc., Chicago, IL, USA). Analysis of data on CRP, C3, C4, immunophenotyping, apoptosis, phagocytosis, and cytokine production revealed a skewed distribution and, consequently, values were transformed prior to statistical analysis to approximate normal distribution. Two-way analysis of variance was used to analyze the effect of age (p ≤.05), sex (p ≤.05), and Age x Sex interactions (p ≤.1) on immune status (23,24) in the sample population (n = 93). For presenting the data, reference intervals were estimated as 2.5 and 97.5 percentiles with the median as a measure of central tendency (25), and displayed as age-based subgroups according to sex. Associations between phenotypic and functional markers of immunity in the whole sample were assessed by partial correlation, with age as a controlling factor.


   RESULTS
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 Methods
 Results
Discussion
 References
 
At inclusion, all volunteers were considered to be healthy, defined as not suffering from any recent episode of infection, inflammation, malignancy, or particular condition that could influence the immune response, as evidenced by self-report and measurement of full blood profile, liver, and kidney function tests. Volunteers who participated in the current study were also considered to be unaffected by clinical depression and dementia.

Participant Characteristics
Participants had a mean (± standard deviation) age of 62.4 (4.48) years and a BMI of 26.9 (3.22) kg/m2. There were no significant differences in age or BMI between men and women. As determined by 4-day food diary, all participants were observed to have adequate nutritional status, comparable with intakes reported previously for a similar population (26–28).

Markers of Inflammation
The adult reference ranges for CRP, C3, and C4 are < 0.5 mg/dL, 0.9–1.8 g/L, and 0.1–0.4 g/L, respectively. Median (2.5–97.5 intervals) serum CRP, C3, and C4 for all participants were 0.10 (0.00–1.36) mg/dL, 1.25 (0.96–1.86) g/L, and 0.26 (0.15–0.37) g/L, respectively. There was no significant Age x Sex interaction observed on these indices of inflammation. However, 10% of participating individuals had subclinical inflammation, as indicated by elevated serum CRP concentration (>0.5 mg/dL).

Leukocyte Immunophenotyping
The percentage and absolute counts of total, T- helper lymphocytes, and CTL in peripheral blood were based on the expression of CD3, CD4, and CD8, respectively. The percent expression and absolute count of leukocyte subpopulations were within the normal range.

The reference ranges and significant Age x Sex interactions of leukocyte subpopulations can be seen in Tables 2 and 3. A significant interaction between age and sex was seen for percent expression of CD3+ T lymphocytes (p =.053); women demonstrated significantly higher percent expression of T lymphocytes than did men and, in addition, a small decrease in T lymphocytes in men was observed with age. Although men demonstrated a small decline in the absolute count of CD3+ T lymphocytes with advancing age, there was no significant Age x Sex interaction. A significant Age x Sex interaction was demonstrated for the percent expression of CD3–/CD(16+56)+ natural killer (NK) cells (p =.081). The percent expression of NK cells was seen to increase significantly with age in men, and also in women, albeit to a lesser degree. Men demonstrated higher percent expression of NK cells compared to women; however, although this trend was reflected by the absolute count of NK cells, it was not statistically significant. In men and women aged 55–70 years analyzed together, there was an increase in the number of eosinophils with age; however, this did not reach statistical significance (p =.059). A significant increase in the percent expression of CD3+/human leukocyte antigen-D related (HLA–DR)+ late-activated T lymphocytes with age (p =.027) was observed. Increases in the absolute count of late-activated T lymphocytes were seen with age; these increases approached statistical significance (p =.058).


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  		Table 2. Significant Interactions Between Age and Sex, and Percent Expression of Peripheral Blood Leukocyte Subpopulations in Healthy Men and Women, Aged 55–70 Years.

 

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  		Table 3. Significant Interactions Between Age and Sex, and the Absolute Count of Peripheral Blood Leukocyte Subpopulations in Healthy Men and Women, Aged 55–70 Years.

 
Total T-helper lymphocyte (CD3+/CD4+) percent expression demonstrated a significant Age x Sex interaction (p =.095), where men showed a small increase and women a decrease in T-helper lymphocytes with age. In women, although the absolute count of total T-helper lymphocytes displayed a trend similar to that of the percent expression, the result did not reach statistical significance. There was a significant Age x Sex interaction for total (CD3+/CD8+) CTL (percent expression p =.048, absolute count p =.039). Men demonstrated a significant decline in the overall percent expression and absolute count of total CTL with age, whereas women showed a marked increase in total CTL with age. The Age x Sex interaction for percent expression of naïve T-helper lymphocytes (CD3+/CD4+/CD45RA+) was p =.065. Whereas naïve T-helper lymphocyte percent expression remained relatively stable with age in men, naive T-helper lymphocytes significantly declined with age in women. A trend towards decreased absolute count of naïve T-helper lymphocytes was also observed in women, but this decrease did not reach statistical significance.

These findings were reflected by a significant Age x Sex interaction for the ratio of total T-helper lymphocytes to total CTL (CD4:CD8, p =.042). This ratio increased with age in men, but declined with age in women. There was also a significant Age x Sex interaction for the ratio of naïve T-helper to memory T-helper lymphocytes (CD3+/CD4+/CD45RA+:CD3+/CD4+/CD45RO+; p =.036); this ratio remained relatively unchanged with age in men, whereas there was a significant decrease with age observed in women. The ratio of naïve T-helper to memory T-helper lymphocytes was significantly higher in men compared to women.

Apoptosis
There was no significant Age x Sex interaction in the percent expression of (Annexin-V+/PI–) early lymphocyte apoptosis (Table 2); however, there was a significant Age x Sex interaction (p =.095) observed in the absolute count, with women demonstrating higher early lymphocyte apoptosis in the age groups 61–65 years and 66–70 years compared to men (Table 3).

Phagocytosis
Table 4 shows the effect of Age, Sex, and Age x Sex interactions on monocyte and granulocyte phagocytic capacity and activity. There were no significant Age x Sex interactions observed in either granulocyte or monocyte phagocytic capacity (the ability to engulf bacteria). Whereas there was no significant Age x Sex interaction observed in granulocyte phagocytic activity (mean number of bacteria engulfed per cell), there was a significant increase in monocyte phagocytic activity with age in all individuals (p =.014).


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  		Table 4. Significant Interactions Between Age and Sex, and Phagocytic Capacity (%) and Activity (MFI) in Healthy Men and Women Aged 55–70 Years.

 
Determination of Intracellular Cytokine Production by Activated Monocytes
There was a significant Age x Sex interaction for the percent expression of IL-1ß (p =.013; see Table 5). There was a decline in the percent expression of IL-1ß by activated monocytes (CD14+/IL-1ß+) in men from the age of 55 to 65 years, and a significant increase in percent expression of IL-1ß with age in women. A similar nonsignificant trend was observed for the percent expression of IL-6 by activated monocytes. There was no significant Age x Sex interaction observed for cytokine production (ABC), as shown in Table 6.


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  		Table 5. Significant Interactions Between Age and Sex, and Cytokine Percent Expression in Healthy Men and Women Aged 55–70 Years.

 

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  		Table 6. Significant Interactions Between Age and Sex, and Cytokine Antibody Binding Capacity (ABC) in Healthy Men and Women Aged 55–70 Years.

 
Associations Between Phenotypic and Functional Markers of Immunity
Associations between phenotypic markers of immunity and the functional markers of immunity (early lymphocyte apoptosis, phagocytic capacity and activity, and intracellular cytokine production) were assessed in all participants after controlling for age. Significant positive associations were observed between early lymphocyte apoptosis and the total lymphocyte count, total T lymphocytes (x109/L), natural killer-like T (NKT) cells (x109/L), late-activated T lymphocytes (x109/L), CTLs (% and x109/L), total memory T lymphocytes (x109/L), memory T-helper lymphocytes (x109/L), and naïve CTLs (x109/L); a significant inverse association was observed with total T-helper lymphocytes (%), naïve T-helper lymphocytes (%), total T-helper to total CTL ratio, and naïve T-helper to memory T-helper lymphocyte ratio (see Table 7).


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  		Table 7. Significant Associations Between Early Lymphocyte Apoptosis and Phenotypic Markers of Immunity in 36 Healthy Individuals Aged 55–70 Years*.

 
Table 8 shows the significant associations between phenotypic markers of immunity and phagocytic capacity and activity. Granulocyte phagocytic capacity showed a significant inverse association with memory CTL (% and x109/L). Granulocyte phagocytic capacity and activity demonstrated a significant positive association with lymphocyte count, but a significant negative association with B-lymphocyte numbers (x109/L). Monocyte phagocytic capacity was significantly associated with increased T lymphocyte percent expression, and monocyte phagocytic activity was significantly associated with increased eosinophil count.


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  		Table 8. Significant Associations Between Phagocytic Capacity and Activity and Phenotypic Markers of Immunity in 93 Healthy Individuals Aged 55–70 Years*.

 
The significant associations between the percent expression and production (ABC) of intracellular cytokines by activated monocytes and phenotypic markers of immunity can be seen in Table 9. IL-1ß (percent expression and ABC) showed a significant inverse association with neutrophil count. IL-1ß percent expression demonstrated a significant inverse association with NK cells (%) and a significant positive association with NKT cells (% and x109/L). IL-6 percent expression demonstrated a significant positive association with T lymphocytes (%), NKT cells (% and x109/L), and late-activated T lymphocytes (%). Furthermore, a significant positive association (r = 0.285, p =.007) was observed between IL-6 percent expression and T lymphocyte IL-2 receptor density (MFI). IL-6 production (ABC) showed a significant inverse association with the related functional markers of immunity, granulocyte (r = –0.224, p =.037) and monocyte (r = –0.251, p =.019) phagocytic activity (results not shown).


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  		Table 9. Significant Associations Between Intracellular Cytokine Production and Phenotypic Markers of Immunity in 93 Healthy Individuals Aged 55–70 Years*.

 

   DISCUSSION
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 Discussion
References
 
There are a limited number of studies that have reported reference values for peripheral blood lymphocyte subpopulations for individuals included in the age group 55–70 years (29–34). Furthermore, these studies failed to differentiate between the sexes, ascertain menopausal state in women, and focus on a broader age group. Therefore, the current study provides important reference values specifically for men and for (postmenopausal) women aged 55–70 years.

Overall, the ranges reported here are comparable to those reported by McNerlan and colleagues (31), who examined a similar population from Northern Ireland, and by Bisset and colleagues (33), who assessed immune status in healthy 19- to 70-year-olds in Switzerland. However, we report that between the ages of 55 and 70 years, alterations in immune status are evident in both the innate and adaptive immune systems. Furthermore, these changes appear to be sex specific. Increased monocyte phagocytic activity was associated with increased age, regardless of sex. Although the mean number of E. coli engulfed (phagocytic activity) by monocytes increased with age, it is not known whether the intracellular killing ability of these cells is altered with age. Increased levels of components of the inflammatory response, such as CRP and fibrinogen, have been shown to be associated with increasing age (35,36), notably in women (37). Consequently, the inflammatory response may be higher and active for prolonged periods in older individuals. However, we did not find any association between subclinical inflammation (as measured by CRP, C3, or C4) and age in this age group (data not shown). Furthermore, there is no clear consensus whether innate immunity is decreased, unaffected, or upregulated during aging (38–42). Therefore, we are unable to conclude whether the changes in the innate immune system observed in the current study are the result of low-grade inflammation or immune remodeling with age.

Within the adaptive immune system, although there were significant Age x Sex interactions observed for the percent expression of a number of lymphocyte subpopulations, not all of these interactions were duplicated in the absolute counts, a measure which may be of greater biological relevance. We observed that the absolute count of (CD3+/CD8+) CTLs was positively associated with age in women between the ages of 61 and 70 years and inversely associated with age in men. Higher cell numbers in women do not appear to be the result of increased memory CTLs with age, as might have been expected given the reported increased oligoclonal expansions of memory CTL with age (12,43). Nor was there any significant association between naïve CTL number and age. It has been reported that the number of CTL that express receptors common to NK cells, that is, CD16 and/or CD56, is positively associated with increasing age (44). Although not statistically significant, women aged 66–70 years appeared to have higher CD3+/CD(16+56)+ NKT cells than did women aged 55–65 years and men aged 55–70 years. NKT cells may comprise a subset of CD8+ CTL, and any increase may at least partly account for the increase in total CTL of women aged 66–70 years. Expansion of the CD8+ CTL subset expressing NKT receptors may be an adaptive mechanism to compensate for contracting TCR repertoires with age, and may provide a pathway for evoking killing activity independently from TCR signaling (45). This association between total CTL numbers and age in women is also reflected in a decreased ratio of T-helper lymphocytes to CTL (CD4:CD8).

Thymopoietic potential to generate de novo naïve T lymphocytes declines with age [see reviews (2,6)]. Despite maintaining a constant export rate of 1%–2% of thymocytes per day throughout life, the number of thymocytes exported in aged individuals is insufficient to replace naïve T lymphocytes lost daily in the periphery (5). Although both apparently healthy men and women, aged 55–70 years, demonstrate increased percent expression of late-activated T lymphocytes with age (suggestive of antigen-induced immune stimulation), the results reported here show that the percent expression of naïve T-helper lymphocytes and the ratio of naïve to memory T-helper lymphocytes is significantly decreased with age in women only. This observation may indicate that the accelerated transformation of (peripheral) naïve to memory T-helper lymphocytes, through the process of cumulative antigen exposure in adulthood (29,46,47), has a greater influence on T-lymphocyte subpopulation numbers in women compared to men in this age group. Subsequently, in men at least, peripheral expansion of mature naïve T-helper lymphocytes may be sufficient to maintain total naïve T lymphocyte numbers with age; albeit at the expense of a diverse TCR repertoire.

Currently, it is unclear what contribution apoptosis plays in immune dysfunction with age; however, it is thought that lymphocyte apoptosis increases with ageing (48). Our findings show that there is a small Age x Sex interaction in the absolute count of lymphocytes in early apoptosis between the ages of 55 and 70 years. Men demonstrated relatively unchanged early lymphocyte apoptosis with age and, though not systematic, women showed a significant positive association between early lymphocyte apoptosis and age.

At present, there is no definitive understanding of the alterations in IL-1ß and IL-6 production with age [see review (49)]. Here, we observed an Age x Sex interaction in the percent expression of IL-1ß, whereby women showed an increase in percent expression of IL-1ß with age. However, although there was a trend toward increased percent expression of IL-6 and IL-6 production, there was no significant Age x Sex interaction for either parameter. This finding may at least partly explain why CRP levels were not associated with increasing age, as the production of this acute phase protein is stimulated by IL-6.

The current study observed a number of significant associations between the phenotypic and functional markers of immunity, which to our knowledge has not been previously reported in healthy individuals. As perhaps would be expected, increased total lymphocyte count was associated with an increase in the proportion of lymphocytes in early apoptosis. In addition, expression of the phenotypic markers of activation (HLA-DR and CD45RO) on lymphocyte subpopulations showed strong positive associations with early lymphocyte apoptosis. We speculate that this most likely reflects the sensitivity of activated T lymphocytes to undergo apoptosis after repeated TCR-stimulation (50). Total and naïve absolute counts of CTL also demonstrated a strong positive association with early lymphocyte apoptosis; however, this effect was not seen for T-helper cells. Our observed association between T-lymphocyte phenotype and apoptosis, together with our observation of increased early lymphocyte apoptosis with age in women, may be at least partly explained by the reported increase in Fas death receptor expression on naïve and memory T lymphocytes with ageing (48).

We observed an inverse association between granulocyte phagocytic capacity and activity and decreased B lymphocyte and memory CTL numbers in this study population. B lymphocytes, like memory CTLs, may also be sensitive to apoptosis after immune stimulation. Phagocytes have previously been reported to play a role in clearance of apoptotic cells from circulation (51–53), which may explain our observed associations.

The current study also observed an inverse association between IL-1ß and IL-6 percent expression and production with neutrophil count and phagocytic activity in both monocytes and granulocytes, suggesting that high intracellular IL-1ß and IL-6 production may downregulate phagocytic activity. In vitro evidence strongly supports such a mechanism (54). Our observation of a positive association between monocyte pro-inflammatory cytokine production with T-lymphocyte numbers and T-lymphocyte IL-2 receptor density is supported by previous in vitro work which reported the contention that activation of T lymphocytes by pro-inflammatory cytokines only occurs when the cells are cocultured with monocytes (55,56).

We also observed a positive association between IL-1ß and IL-6 production and increased NKT cell numbers. To our knowledge, the effect of these pro-inflammatory cytokines on NKT cell proliferation has not been examined. It is possible that these cytokines may play a role in NKT cell biology.

Conclusion
This study has shown that there are a number of alterations in immune status in apparently healthy 55- to 70-year-olds, and that several of these changes appear to be sex specific. Even minor alterations observed in immune status are of significance given the relatively narrow age range of 15 years of the population studied. Comparison of these individuals with a younger age group would most likely have demonstrated alterations of a much greater magnitude. Currently, standard laboratory reference values do not normally take sex differences into account. Based on the findings of the current study and together with similar observations reported previously (30), we believe that all such studies assessing immune status in adults should allow for sex as well as age effects in their analysis, as sex differences may be an important factor in influencing immune status in adults, particularly later in life. Furthermore, the current study reports associations between phenotypic and functional markers of immunity, evidence that may contribute to our understanding of immunoregulation.


   Acknowledgments
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Support for this research was provided by the European Commission "Quality of Life and Management of Living Resources" Fifth Framework Program (Contract QLK1-CT-2001-00168) and by the Department of Education and Learning (DELNI), U.K.

We thank to Dr. Charles Coudray, INRA Centre de Clermont-Ferrand, France, as project coordinator of the ZENITH study and to Mary Kelly, University of Ulster, and Dr. Liz Simpson, School of Psychology, University of Ulster, for their role in volunteer recruitment and management. We also thank the Causeway Laboratory, Causeway NHSS Trust, Coleraine, Northern Ireland for their assistance with blood analysis, and to all the study volunteers that participated.

CFH was responsible for study execution, data analysis, and preparation of the manuscript. HDA provided expertise in method development and assisted in the preparation of the manuscript. IB provided statistical advice. JMO'C, MPB, BMH, JJS, and JMWW were responsible for study conceptualization and design, and assisted with study execution, data analysis, and preparation of the manuscript. WSG assisted with preparation of the manuscript.


   Footnotes
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Decision Editor: Huber R. Warner, PhD

Received January 5, 2006

Accepted March 2, 2006


   References
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 Journals of Gerontology Series A: Biological Sciences and Medical SciencesHome page
C. F. Hodkinson, M. Kelly, H. D. Alexander, I. Bradbury, P. J. Robson, M. P. Bonham, J. M. O'Connor, C. Coudray, J. J. Strain, and J. M. W. Wallace
Effect of Zinc Supplementation on the Immune Status of Healthy Older Individuals Aged 55-70 Years: The ZENITH Study
J. Gerontol. A Biol. Sci. Med. Sci., June 1, 2007; 62(6): 598 - 608.
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