Heat shock proteins (HSP) represent cell-protective and antioxidant systems that may be induced by reactive oxygen species, cytokines, and hyperthermia. In the present study, we evaluated the influence of heavy endurance exercise and training on HSP27 and HSP70 in peripheral leukocytes of 12 athletes (before and at 0, 3, and 24 h after a half-marathon) and 12 untrained controls on protein and mRNA levels by flow cytometry and RT/PCR, respectively. HSP transcripts increased significantly immediately after acute exertion accompanied by elevated levels of corresponding proteins. HSP protein expression remained high until 24 h postexercise. Significant increases of plasma interleukin-8, myeloperoxidase, and creatine kinase occurred after exercise. Basal HSP expression was usually lower in trained compared with untrained subjects. Applying in vitro heat shock to resting blood samples of all subjects significantly stimulated HSP mRNA, showing higher increases in trained individuals. The exercise-induced alterations indicate that immunocompetent cells became activated. In addition to heat stress, other exercise-associated stress agents (oxidants, cytokines) may have also participated in stimulation of HSP expression in leukocytes. The expression pattern of HSP due to training status may be attributed to adaptive mechanisms.

Our investigations revealed an increased expression of iNOS at the transcriptional and translational level in response to vigorous exercise. This reflects an inflammatory response and may contribute to an exercise-induced rise of endogenous nitric oxide production. It remains unclear if these effects serve an in-vivo immunoregulatory or cell-damaging role.

Previous research revealed an increased expression of HSP72 in leukocytes after vigorous endurance exercise. We questioned whether more intensive but shorter exercise also induces leukocyte HSP72 synthesis. To delineate the role of reactive oxygen species (ROS) in exercise-related HSP72 induction, we additionally examined the effect of RRR-alpha-tocopherol (alpha-toc) on HSP72 expression using a double-blind placebo (P) controlled cross-over design. After supplementation with alpha-toc (500 I.U. daily) or P for 8 days, 9 male subjects performed a combined exhaustive treadmill protocol (total duration 29.4 +/- 2.0 min). HSP72 was assessed on mRNA (RT-PCR) and protein levels (flow cytometry). HSP72 mRNA rose 3 h after exercise only in the P group, but individual differences (alpha-toc - P) did not reveal significant treatment effects. A moderate but significant rise of HSP72 protein occurred in granulocytes up to 48 h after exercise. Three hours post-exercise, granulocyte HSP72 protein was lower when subjects received alpha-toc, but this effect vanished 24 and 48 h post-exercise. Exhaustive treadmill exercise augments HSP72 mRNA in leukocytes and induced a moderate but prolonged response of granulocyte HSP72 protein. These exercise effects are lower when compared to earlier findings obtained after vigorous endurance exercise. ROS seem to be involved, but do not play the major role in the induction of granulocyte HSP72 synthesis after exhaustive exercise.

Organ graft rejection is caused by the recognition of allogeneic MHC molecules by recipient T cells by two different pathways. The indirect pathway of alloreactivity requires the presentation of MHC peptides from the graft by autologous APC, as with conventional antigen. The direct pathway, on the other hand, requires the recognition of foreign MHC on foreign cells. The regulatory mechanisms for this component of alloreactivity have not been extensively studied. We show here that the T cell response activated by alloantigens in the direct pathway is similarly constrained and modulated by cytokines, as has been shown for classic antigen presentation. Thus, the inclusion of IL-2 or TGF-beta in MLC performed with purified responder T cells resulted in outgrowth of cells secreting IL-2 and IFN-gamma, whereas addition of IL-4, IL-10, or anti-TGF-beta encouraged outgrowth of cells secreting IL-4 and IL-10. T cells alloactivated via the direct pathway and then cloned in IL-2 alone secreted IL-4 and IL-10 as well as IFN-gamma and IL-2 (Th0 phenotype). Established clones remained susceptible to cytokine modulation, such that IL-4 and IL-10 decreased their secretion of IL-2 and IFN-gamma, whereas TGF-beta suppressed IL-4 and IL-10 secretion. The first alterations of Th0 toward Th1 or Th2 phenotypes could already be observed after only a very brief exposure to cytokines of 48 hr, followed by extended culture with IL-2 alone. These results confirm that human T cells with Th1 and Th2 phenotypes, recognizing alloantigen via the direct pathway, derive from the same IL-2-secreting precursor and can be manipulated by cytokines in an analogous fashion to conventional antigen-reactive cells. These findings may have implications for manipulating the direct pathway of alloantigen recognition in human organ transplantation.