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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 • cats
EXPANDED ABSTRACT
Recent studies indicate that as an animal ages, every component of the immune system undergoes age-associated changes, suggesting that immune senescence is a net result of the continuous adaptation of the body to the overall deterioration occurring over time (1,2), which is considered to be one of the main contributing factors that influence morbidity and mortality (3). Most age-related changes in the immune response have focused on human and rodent studies, indicating there is a general reduction in protective immunity, including reduced cell numbers, proliferative capacity and cellular dysfunction with increasing age (3–5). Such age-related alterations in immune function have been linked with increased incidences of infection and degenerative disorders such as cancer (5).
In a previous study we revealed significant differences in relative and absolute values for leukocyte populations and lymphocyte subsets between adult and senior cats (6). Campbell et al. (6) observed dramatic changes associated with T-lymphocytes with age, but the innate immune profile remained relatively intact in the senior group of cats. Despite the significance of this finding it was based on two discrete age populations, a factor common to other studies of feline immune status (7–9), and human immune status (4). What we do not know is how the profile of the adapting immune system alters as cats go through their various life stages. Herein lies an anomaly, however, because the definition of what constitutes a kitten, adult or senior cat is not based on any known physiological criteria. Consequently, the aim of the present study was to characterize changes in specific populations of peripheral blood leukocytes in a large population of cats and relate this to age. A second objective was to use this information to define immunological life stages for domestic shorthaired cats.
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MATERIALS AND METHODS |
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 TOP MATERIALS AND METHODS RESULTSDISCUSSIONLITERATURE CITED |
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Samples were analyzed using lysed whole blood staining and two-color flow cytometric analysis. Commercially available monoclonal antibodies were used to identify cell surface markers for T-cells (CD5, CD4, CD8), B-cells (CD21-like), monocytes (CD14) and granulocytes (CD11b). 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 and the data cross-validated by using the leave-one-out classification method.
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RESULTS |
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TOPMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Discriminant analysis of the CD4:CD8 ratio data allowed identification of two statistically distinct groups of cats, kittens (2–8 mo) and adults (8+ mo), with an overall correct classification of 77% (cross-validated).
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DISCUSSION |
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TOPMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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The significant increase in relative percentage of feline CD8 T-cells observed could form part of what is termed the "memory" T-cell population. Production of memory T-cells indicates that an individual has reacted to an antigenic stimulus and mounted a response to suppress or eliminate that particular infection. Over time, these same physiological responses lead to a progressive accumulation of clones of memory T-cells that allow the individual to respond more quickly to an infection if that particular antigen is encountered again. This supports similar findings in other feline, human and canine studies (4,6,14,16–19). These changes could also represent the possible outcome of normal and appropriate proliferative responses to a variety of potential antigens throughout the life of the individual (20).
Although CD4 T-cells contribute to the formation of the memory cell population, the predominance of the expansion within the CD8 population vs. CD4 probably relates to the stronger, more persistent nature of the antigenic drive for CD8 T-cells responding to intracellular pathogens, in contrast to the more discrete localization of extracellular pathogens recognized by CD4 T-cells (21). It is also conceivable that maturation of naïve CD4 T-cells may, in fact, be induced more rapidly in the aged environment (21), or the role of CD4 changes from developmental/selection costimulating molecule in the young developing immune system to that of a positive effector signal for antigen recognition/immunological memory in the mature immune system (22). Either way, these mechanisms may account for the relative percentage decrease of CD4 observed.
A reduction in relative percentage of CD4 T-cells could also have an impact on its two functional subgroups, T-helper 1 (TH1) and TH2 CD4 T-cells. A reduction in TH1 T-cells would reduce the potential for monocyte/macrophage populations to control bacterial infections, a characteristic common in AIDS patients and elderly subjects that have lower levels of CD4 T-cells who are more susceptible to infections that healthy younger subjects are able to combat (23).
Reducing levels of TH2 T-cells would 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 (24).
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 of memory T-cells over naïve T-cells (21).
The distribution shift in CD4 and CD8 T-cell populations also accounts for the inversion of the CD4:CD8 ratio with age observed, a parameter used to indicate levels of immunocompetence in relation to aging in other species (19). Other feline studies have demonstrated similar profiles, with kittens < 10 wk of age having the highest CD4:CD8 ratio, which decline to adult levels at approximately 1 y of age. The primary driver for the changing ratio in those studies was an increase in the CD8 T-cell population (11,14,18).
Although the reductions in relative percentages of CD21 (a marker for B-cell populations) were significant with age, the R2 value was low. Increasing the number of cats sampled may show greater reductions. The outcome of this reduction in B-cell percentage is thought to contribute to two changes in antibody production from B-cells. First, it leads to an increase in production of autoantibodies with age, which has been linked with the development of degenerative autoimmune diseases, such as arthritis (25). Second, antibodies produced to foreign antigens may increase but, because of the anti-idiotypic nature of the antibodies, will lead to the production of antibodies with a lower antigenic affinity that are less able to protect against infection (26). Thus, although antibody production may increase with age, the specificity and affinity of secreted antibodies may be reduced, leading to less-adapted antibody responses that may account for the reduction in vaccine-induced immunity in elderly subjects (24).
Linking in with changing profiles of the T- and B-cell populations, a significant decrease in relative levels of lymphocytes with age was also observed. The significant decreases observed in the CD4 and CD21 populations could account for this decrease in lymphocytes. Although there was a significant increase in the CD8 population with age, this may not have been sufficient to counteract the overall relative percentage decrease in the lymphocyte population.
Significant increases in the relative percentages of granulocytes and CD11b, and the negligible changes in relative percentages of monocytes and CD14 cells give an indication of how certain parameters of the innate immune system change with age. These data suggest that the "primitive" immune system acting as a first line of defense toward infection is less affected by the aging process or even might try to compensate for the decline in acquired immunity observed.
It is clear that significant changes do occur, which leads to modulation of feline immune status with age. Using discriminant analysis on the scatter-plot information for each of the leukocyte groups, the CD4:CD8 ratio defined two statistically distinct life-stage groups, kittens below 8 mo of age and adult cats above 8 mo of age. Using components of the immune system to define life-stage classifications in cats is an important step toward accurately defining physiological differences between kitten, adult and senior animals from an immunological point of view.
George et al. (12) demonstrated that aged cats (7.5–11.5 y) developed more severe disease than young adult cats (8–12 mo) when experimentally infected with feline immunodeficiency virus (FIV). Our data indicate that the observed significant changes in immune status at 8 mo of age also fall into the same age range as the young adult cats that showed reduced disease severity in the study by George et al. (12), suggesting this may be the most optimal period of immune performance during the life span of the cat.
Thus, in contrast to the idea of a systematic decline (27), the data from the present study and those from other feline, canine, human and rodent studies suggest that components of the immune system undergo gradual age-related shifts in cell populations, conforming to the paradigm of age-related remodeling of the immune system (1). Although restructuring of the immune system with increasing age is primarily a result of past antigenic exposure and adapting for future responses, other independent factors such as oxidative stress and nutrition (28) may have a major influence over immunological development, progression and senescence. Together, this information would provide the basic platform from which to monitor the plasticity of the immune system, maintenance of immunity over time and regulation of compensatory functions. Undoubtedly this will help develop novel strategies to cope with such stressors as infection, cancer and arthritis during old age.
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FOOTNOTES |
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LITERATURE CITED |
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TOPMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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