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Supplement: Waltham International Symposium

Assessing Age-Related Changes in Peripheral Blood Leukocyte Phenotypes in Labrador Retriever Dogs Using Flow Cytometry

Waltham Centre for Pet Nutrition, Leicestershire, UK

²To whom correspondence should be addressed. E-mail: paul.heaton{at}eu.effem.com.

KEY WORDS: • age • leukocytes • flow cytometry • dogs

EXPANDED ABSTRACT

The classical view of "immune senescence" is one of a generalized, age-related unidirectional decline in immune responses (1). However, recent studies indicate that nearly every component of the immune system undergoes dramatic age-associated restructuring, leading to changes that include enhanced as well as diminished function (2). Age-related physiological changes in the immune system are considered to be one of the main contributing factors that influence life expectancy, morbidity and mortality in all species (3,4).

To date, most age-related changes in immune status have focused on human and rodent studies that indicate a general reduction in protective immunity, including reduced cell numbers, proliferative capacity and cellular dysfunction with increasing age (3,5,6). Such age-related alterations in immune function have been linked with increased incidences of infection and degenerative disorders such as cancer and arthritis (6). Nutritional supplementation studies in elderly human subjects suggest that modest increases in micronutrient intakes improve immunity and reduce the risk of infection in relation to placebo controls (7–9), indicating that reduced/suppressed immune function in relation to age may in part be alleviated through nutrition.

Before nutritional intervention can be used as a means of potentially reducing the detrimental effects of age-related canine immune dysfunction, relevant immunological data have to be collated. Preliminary studies to date have suggested that age-related changes similar to humans do occur in dogs (10,11), a finding supported by a recent Waltham study that demonstrated that total peripheral blood leukocyte counts decrease significantly with age in both Beagles and Labrador retrievers (Harper, E. J. & Heaton, P. R., unpublished observations, 2000).

No attempts have been made to try to define life-stage groups using immunological profiles as a parameter. Studies in humans (5,13,14) divide individuals into infant, adult and senior life-stage groups without a clear definition of how these divisions are made. Likewise, in canine immune senescence studies, Kearns et al. (15) define young Fox Terriers and Labrador retriever dogs with a mean age of 1–2 y and adult as 9–12 y old, whereas Strasser et al. (12) define German Shepherd dogs as young (2–4 y), adult (4.5–7.5 y) and old (8–13 y) with no clear guidelines of how these groups were defined.

In the present cross-sectional aging study, blood samples were analyzed for a variety of leukocyte populations (relative levels of white blood cells: lymphocytes, monocytes and granulocytes) and percentage leukocyte subgroups using fluorescence-activated cell-sorting (FACS) techniques to characterize changes in specific populations of peripheral blood leukocytes in relation to age. This information was used to define immunological life stages for Labrador retriever dogs.

	MATERIALS AND METHODS

TOP MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Whole blood samples were taken from 122 Labrador retrievers, 71 males and 51 females ranging from 6 mo to 14 y 2 mo of age. All dogs had been vaccinated for canine adenovirus, canine parvovirus and canine distemper virus and deemed clinically healthy before commencement of the study. All animals were fed commercially available, complete diets (Pedigree^®, Masterfoods, Melton Mowbray, UK) throughout the study period and were housed at the Waltham Centre for Pet Nutrition (Leicestershire, UK), where they were housed in purpose-built, environmentally enriched facilities (16) and treated in accordance with the Centre’s research ethics and UK Home Office regulations.

Samples were analyzed using lysed whole blood staining and triple-color flow cytometric analysis. Commercially available monoclonal antibodies were used to identify cell surface markers for T-cells (CD3, CD4, CD8), B-cells (CD21-like) and monocytes (CD14). Relative levels of lymphocytes, monocytes and granulocytes were also calculated.

Values were expressed as percentages of cellular populations for each individual animal. Age-related trends were assessed by linear regression analysis. Discriminant analysis and independent-sample t-test were used to identify cellular populations for defining life-stage groupings. The data were cross-validated using the leave-one-out classification method.

	RESULTS

TOP MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Linear regression analysis identified significant trends in the data obtained. Analysis identified a significant increase in CD3 (R² = 0.06, P < 0.008) (data not shown), a significant decrease in CD4 (R² = 0.03, P < 0.05) (data not shown), a significant increase in CD8 (R² = 0.24, P < 0.001) (Fig. 1), with a corresponding decrease in the CD4:CD8 ratio (R² = 0.15, P < 0.001) (Fig. 2) with increasing age. No significant differences were identified with any of the other populations analyzed (data not shown).

View larger version (13K): [in this window] [in a new window]   FIGURE 1 Age-related increase in relative percentage levels of CD8 in a Labrador retriever population (n = 122), R2 = 0.24, P < 0.001.

View larger version (13K): [in this window] [in a new window]   FIGURE 2 Age-related decline in CD4:CD8 in a Labrador retriever population (n = 122), R2 = 0.15, P < 0.001.

	DISCUSSION

TOP MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Our investigations have demonstrated significant age-related increases in the relative percentages of CD3 and CD8 T-cells, and decreases in CD4 T-cells and the CD4:CD8 ratio in peripheral blood from the Labrador retriever population used in the present study. These results are similar to those of other canine studies (10,11) and those obtained from human, rodent and feline studies (5,17–20). Likewise, with the feline study by the same authors (20), it was not unexpected that the R² values of certain parameters were low, given that the data represent the natural variability seen in a population when taking independent samples of healthy Labrador retrievers over an age range of 6 mo to 14 y 2 mo of age.

The CD3 T-cell marker, which defines total T-cells (encompassing both the CD4 and CD8 T-cell subgroups), demonstrated a significant increase in relative percentage levels with age. This may partly be a result of the relative percentage increase in CD8 T-cells being greater than the relative percentage decrease in CD4 T-cells. Studies in humans, also demonstrating increasing levels of CD3 T-cells with age, suggest they correspond to immature T-cells that are unable to attain their full mature functional status because of age-associated thymic involution processes (21). When compared to data from other canine and human studies, the increase in percentage of total T-cells may also be apparent because the absolute numbers of the T-cell subsets decline at a lower rate (5,10,12).

As highlighted in the feline study (20), the functional implications of CD4 and CD8 T-cells are very distinct from each other. The significant increase in relative percentage of canine CD8 T-cells observed could form part of the memory T-cell population that allows the individual to respond more quickly to an infection if that particular antigen is encountered again, as well as representing the normal outcome of proliferative responses to a variety of potential antigens throughout the life of the individual (22). Similar findings have been observed in a variety of other species (5,18,23).

Although the overall reduction in relative percentage levels of CD4 T-cells may be caused by increased maturation rates of naïve to mature cells (24), the major functional impact would be within the TH1 and TH2 subgroups of CD4 T-cell populations. A reduction in TH1 T-cells would reduce the potential for monocyte/macrophage populations to control bacterial infections, resulting in elderly subjects being more susceptible to infections (25). Reducing levels of TH2 T-cells could reduce/alter activation of B-cells to produce antibody and the type of antibody produced, suggesting that the humoral immune response would become less adapted with age, which is demonstrated by the reduction with age of vaccine-induced immunity to influenza in humans (26). Further studies will have to be conducted to determine the role that TH1/TH2 subgroups play in the canine immune system in relation to aging, but may contribute toward increased disease susceptibility observed in older dogs.

Other factors have been implicated with changing CD4 and CD8 T-cell profiles with age, such as compensatory homeostatic responses to reduced numbers of naïve cells and increased numbers of memory cells, changes in rates of apoptosis and specific effects of the aged environment, which actually promote the appearance and dominance of memory T-cells over naïve T-cells (24,27). The distribution shift in CD4 and CD8 T-cell populations also accounts for the inversion of the CD4:CD8 ratio observed in the present study, a parameter used to indicate levels of immunocompetence in relation to aging in other species (18,22).

Although there were decreases in relative percentage levels of the remaining parameters with age, the differences were not significant. Increasing the number of Labrador retriever dogs sampled from the present study may show significant differences. For example, the study by Greeley et al. (10) demonstrated a significant reduction in the percentage of B-cells, potentially leading to less-adapted antibody responses with age, whereas markers such as CD14, monocytes and granulocytes that are part of the innate immune response may not be affected by the aging process (20,28).

It is clear that significant changes do occur, which could lead to modulation of canine immune status with age. Using discriminant analysis on the scatterplot information for each of the leukocyte groups, CD3 and CD8 markers defined two statistically distinct life-stage groups, adult dogs below 8 y of age and senior dogs above 8 y of age (P < 0.008 and P < 0.001, respectively). Although the CD4:CD8 ratio and CD21 demonstrated significant differences at the same age point, the classification level was lower. Increasing the sample size may improve the classification data.

Although using components of the immune system to define life-stage classifications in dogs is an important step toward accurately defining physiological differences between puppy, adult and senior animals from an immunological standpoint, care would to be taken if trying to apply the same criteria across all dog breeds, particularly at the older end of the age scale where it is difficult to determine life stages between different breeds (29).

The present data suggest that immunological status does change according to age in the Labrador retriever dog, in line with the remodeling theory proposed by Franceschi et al. (30), which potentially alters the functional status of the aging immune system. While providing the basic platform to monitor immunological development and senescence, the data also indicate it is imperative that age be considered in any canine study where the interpretation of leukocyte subset data is utilized.

	FOOTNOTES

 
¹ Presented as part of the Waltham International Symposium: Pet Nutrition Coming of Age held in Vancouver, Canada, August 6–7, 2001. This symposium and the publication of symposium proceedings were sponsored by the Waltham Centre for Pet Nutrition. Guest editors for this supplement were James G. Morris, University of California, Davis, Ivan H. Burger, consultant to Mars UK Limited, Carl L. Keen, University of California, Davis, and D’Ann Finley, University of California, Davis.

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