Acute aerobic exercise in humans increases cytokine expression in CD27− but not CD27+ CD8+ T-cells
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► Acute exercise primes late differentiated CD8+ T-cells to produce cytokines concurrently with their extravasation from the blood compartment into the tissues.
Introduction
It is generally accepted that regular bouts of moderate intensity exercise improve some aspects of immunity (Simpson et al., 2012), while frequent bouts of vigorous exercise may depress the immune system and increase infection risk, particularly among athletes (Walsh et al., 2011). Although the mechanisms that underpin such immune enhancement/depression have yet to be fully ascertained, one pathway by which exercise may alter immunity is through the actions of cytokines (Steensberg et al., 2001). These cell-signaling protein molecules, primarily although not exclusively secreted by cells of the immune system, are critical to the development of pro- and anti-inflammatory immune responses following infection or injury. While some cytokines (e.g., IFN-γ) trigger predominantly type I immune responses, other cytokines (e.g., IL-4) are involved in the activation of type II immune responses (Mosmann and Sad, 1996). It has been postulated that exercise causes a shift in the balance between type I and type II cytokine expression and that altered cytokine profiles could have implications for both enhanced and depressed immunity with exercise (Ibfelt et al., 2002, Lancaster et al., 2004, Ogawa et al., 2003, Steensberg et al., 2001, Zaldivar et al., 2006). This shift is particularly evident in CD8+ T-cells, where decreases in intracellular IFN-γ expression have been reported in response to a single bout of exercise, without concomitant changes in type II cytokines (Ibfelt et al., 2002, Lancaster et al., 2004, Steensberg et al., 2001).
The cytokine profile of a particular cell type is often established by quantifying the percentage of cells that expresses the cytokine following mitogen stimulation. This approach has shown shifts in cytokine production in CD8+ T-cells after acute exercise, whereby the percentage of CD8+ T-cells expressing IL-2, IL-4, IL-6, IFN-γ, TNF-α has been reported to change (Ibfelt et al., 2002, Lancaster et al., 2004, Starkie et al., 2001, Steensberg et al., 2001, Zaldivar et al., 2006). A potential confounder to this method is that the total CD8+ T cell pool consists of several subsets that vary considerably in the amount and type of cytokines they produce in response to activation. A common way to identify such functionally distinct CD8+ T cell subsets is on the basis of cell-surface expression of the markers CD45RA, CD27, and CD28 (Table 1) (Hamann et al., 1997, Plunkett et al., 2007, Sallusto et al., 2004, Tomiyama et al., 2002). Naïve (NA; CD45RA+/CD27+/CD28+) T-cells mainly produce IL-2 in response to in vitro stimulation, while central-memory (CM; CD45RA−/CD27+/CD28+/−) and effector-memory (EM; CD45RA−CD27−/CD28−) T-cells are more responsive to stimulation and readily produce IL-2, IL-4, IFN-γ, and TNF-α (Hamann et al., 1997, Tomiyama et al., 2002). EM T-cells have a decreased IL-2 production and increased IFN-γ production relative to CM T-cells (Hamann et al., 1999). Finally, EMRA (CD45RA+/CD27−/CD28−) are rapid-acting effector-memory cells that can produce copious amounts of IFN-γ and TNF-α, but do not express IL-2 or IL-4 when stimulated with the mitogen phorbol myristate acetate (PMA) (Appay et al., 2002, Hamann et al., 1997).
Acute exercise evokes a rapid mobilization of lymphocytes into the bloodstream resulting in a 2- to 3-fold increase in the blood lymphocyte count (Simpson, 2011). This response is largely due to an influx of NK-cells and CD8+ T-cells, while CD4+ T-cells and B-cells are mobilized in relatively fewer numbers (Gannon et al., 2001, Shek et al., 1995). During the early stages of exercise recovery, there is a rapid extravasation of the mobilized cells that often results in a transient lymphocytopenia (Simpson, 2011, Walsh et al., 2011). The aforementioned CD8+ T cell subsets also differ in the magnitude of their response to exercise, whereby EM and EMRA CD8+ T-cells, as compared to NA and CM cells, show a greater ingress into peripheral blood (lymphocytosis) during exercise and a greater egress (lymphopenia) post exercise (Campbell et al., 2009, Shek et al., 1995, Simpson et al., 2007). This ultimately increases the proportions of EM and EMRA subsets among total CD8+ T-cells immediately after exercise, while the proportions of EM and EMRA cells are substantially lowered during exercise recovery. Unfortunately, previous studies that have quantified CD8+ T-cells expressing cytokines in response to exercise have failed to consider the effects of shifts in the proportions of discrete CD8+ T cell subsets known to have vastly different cytokine expression profiles (Lancaster et al., 2004, Starkie et al., 2001, Steensberg et al., 2001, Zaldivar et al., 2006). It is therefore unknown if exercise veritably alters CD8+ T cell cytokine expression (i.e., on an individual cell basis), or if this effect is secondary to an altered composition of the CD8+ T cell pool. Identifying if specific CD8+ T cell subsets are responsible for the cytokine responses to acute exercise will therefore aid the interpretation of prior data and further our understanding of how exercise modulates immunity.
The aim of this study was to determine if shifts in the proportions of early/intermediate (CD27+) and late (CD27−) differentiated CD8+ T cell subsets with exercise account for changes in total CD8+ T cell cytokine expression. As late differentiated CD27− EM and EMRA subsets show a more robust mobilization and egress in response to exercise, and express different cytokines produced at a lower stimulus threshold, we hypothesized that an exercise-induced shift in the proportions of CD27− cells would account for the changes in the percentage of all CD8+ T-cells expressing cytokines after exercise.
Section snippets
Participants
Sixteen trained male cyclists (Mean ± SD: Age: 33 ± 5 yrs, Height: 179 ± 0.08 cm, Mass: 79.11 ± 7.79 kg) volunteered for this study. Participants were recruited from local cycling clubs and teams. Written informed consent and medical history were obtained from each volunteer after the procedures, benefits, and risks were explained verbally and provided in writing. The Committee for the Protection of Human Subjects (CPHS) at the University of Houston granted ethical approval for the study. Participants
Exercise elicits a preferential mobilization of differentiated (CD27−) CD8+ T-cells
Phenotypes used to define the various cell types are shown in Table 1. Replicating previous findings (Campbell et al., 2009, Simpson et al., 2007, Turner et al., 2010), exercise induced an acute leukocytosis in all participants, with a significant increase in whole blood lymphocyte (+59.3%) and CD8+ T cell numbers (+25.1%) (Table 2). These cell counts returned to baseline values 1 h post-exercise. As expected (Campbell et al., 2009), the numbers of EMRA cells showed the largest relative (+145%)
Discussion
This study examined the effects of an acute cycling exercise bout on intracellular CD8+ T cell cytokine expression in trained participants. The aim was to understand how exercise-induced shifts in the proportions of early/intermediate (CD27+) and late (CD27−) differentiated CD8+ T-cells affect the pattern of cytokine expression in the total pool of CD8+ T-cells. We found that exercise increased the expression of both type I (IL-2, IL-6, IFN-γ, TNF-α) and type II (IL-4, IL-10) cytokines in total
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