Immune system functional testing of athletes at University of Stellenbosch

Carine Smith, Kathy Myburgh

Because of the ever-increasing high standards set in the sporting world today, athletes have to keep increasing their training efforts in order to excel at their chosen sport. This increased stress to the body (both physical and psychological) may affect many aspects of the normal functioning of the body, including the immune system. Based on epidemiological data (reviewed in Calabrese et al. 1996 and Nieman 1998) , it seems that while moderate exercise may serve as a booster to the immune system, excessive exercise may result in an increased risk to the athlete of suffering upper respiratory tract infections. As a result, the Exercise Science Division of the Department of Physiological Sciences at the University of Stellenbosch became interested in investigating the physiological effects of exercise on the immune system.

When we investigate the status of the immune system, the first tests we do are a full blood count and differential white cell count. These tests tell us whether there are enough white blood cells of each required type present. However, this does not tell us how efficiently these cells will respond to a pathogenic challenge. Therefore the numbers of the different types of white blood cells are not always a good indicator of immune ability and the numbers of immune cells available should always be looked at in conjunction with their capacity to react to a challenge.

There is more than one way in which to determine a white blood cell's reaction capacity. One can stain the cells for cell markers that indicate an activated status - these are molecules expressed on the cell surface whenever a cell commits itself to react to a challenge. Alternatively, one can measure the concentrations of the substances the cells secrete in response to a pathogen or stimulus.

We employed the latter method for several studies. We were interested in investigating the release of the cytokine interleukin-6 (IL-6) by immune cells of athletes. IL-6 is released by many cells in the body, such as immune cells, endothelial cells, fibroblasts, and contracting muscle cells. Of the immune cells, the monocytes and lymphocytes, the so-called mononuclear immune cells, are the main secretors of IL-6. IL-6 has several functions in the systemic immune response, which include a major role in stimulating the release of acute phase proteins from the liver, and inducing fever. It also plays a role in the endocrine response to stress, including indirectly stimulating cortisol release by increasing ACTH release, and directly stimulating the adrenal cortex to produce cortisol. Therefore, IL-6 release could be a valid marker of immune ability in athletes who are training hard and are under a lot of competitive stress.

However, since so many cells will secrete IL-6, it is difficult to determine the source of the cytokine in vivo. Therefore, a method which allows for testing of the function of the cells of interest outside the body (in vitro), may give a clearer indication of the ability of those specific cells, as it eliminates interference from other possible sources. We used a technique developed earlier with which we can measure the in vitro interleukin-6 secretion in whole blood cultures in response to stimulation by a bacterial endotoxin (E.coli lipopolysaccharide [LPS]).

The testing procedure consists of two parts: In part one, a whole cell culture is prepared by adding whole blood to two test tubes, with added nutrients to ensure that the cells remain viable. Then the stimulus (E.coli LPS) is added to the one tube, but not to the other. The test tubes, which now contain a "cell culture", is then incubated at 37 °C (body temperature) for a few hours, to allow the immune cells to react to the pathogen. The second part of the process is to measure the amount of IL-6 released into the culture tubes. This is done by a simple ELISA technique. The reason for having one tube with, and one without E.coli LPS added, is that some immune cells may already be activated in the body and others may become activated spontaneously during the testing procedure. The tube without added E.coli LPS will indicate how many cells spontaneously secreted IL-6 in the absence of an added pathogenic challenge. We then subtract this amount from the concentration of IL-6 in the tube with added E.coli LPS, to get the concentration of pathogen-induced IL-6. Performing this test on different athletes, or at different times of an athlete's training season, can give us an indication of how stressful the exercise is to the immune system, and whether or not the immune cells can still react to a different challenge despite this stress.

The research group at the Department of Physiological Sciences at the University of Stellenbosch, used this assay in a cross-sectional study of the functional ability of immune cells from rugby players, triathletes and sedentary people (accepted for publication in the European Journal of Physiology, 2002). In short, we found that the immune cells of professional rugby players and highly trained triathletes responded more poorly to the E.coli LPS challenge than those of the sedentary subjects. 

There is more than one possible explanation for this. Firstly, if the body is exposed to the stress of exercise for a long time, the immune cells may become less sensitive to stressors (which may be exercise or a pathogen). An alternative explanation is that the cells are reacting to the stress of exercise chronically, so that they are unable to respond further when a real pathogenic challenge occurs.

If the first explanation is true, the cells would still be able to respond well to a new challenge if the challenge was great enough. But, if the second explanation is true, the implication is that the response could be inadequate to defend the athlete against developing illness. We are currently testing these two possibilities using an actual ultra-endurance race as the challenge. We suspect that the subjects will show a poor response to E.coli LPS before the race, because of all the training done in preparation for the race. During ultra-endurance races, other researchers have shown that E.coli LPS actually leaks into the circulation from the gut. Thus the race itself can act as a pathogenic challenge which may influence the response to an additional pathogenic challenge added to the blood in vitro after the race.

In conclusion, being able to determine the functional capacity of the immune cells rather than simply the numbers of the different sub-types, opens doors to many new questions.

More Information

Akira S, Taga T, Kishimoto T (1993) Interleukin-6 in biology and medicine. Adv Immunol 54 : 1-78

Calabrese LH and Nieman DC (1996) Exercise, immunity, and infection. J Am Osteopath Assoc 96(3) : 166-176 Cox G, Gauldie J (1992) Structure and function of interleukin-6. In: S L Kukel and D G Remick (eds) Cytokines in Health and Disease. Dekker, New York, pp97-120

Jeukendrup AR, Vet-Joop K, Sturk A, Stegen JH, Senden J, Saris WH, Wagenmakers AJ (2000) Relationship between gastro-intestinal complaints and endotoxaemia, cytokine release and the acute-phase reaction during and after a long-distance triathlon in highly trained men. Clin Sci 98 : 47-55

Lyson K, McCann SM (1992) Induction of adrenocorticotrophic hormone release by interleukin-6 in vivo and in vitro. Ann N Y Acad Sci 15, 650: 182-185

Nieman DC (1998) Exercise and resistance to infection. Can J Physiol Pharmacol 76(5) : 573-580 Path G, Scherbaum WA, Bornstein SR (2000) The role of interleukin-6 in the human adrenal gland. Eur J Clin Invest. 30 (Suppl 3) : 91-95

Pool EJ, Bouic P (1999) The detection of pyrogens in sera from patients with symptoms of sepsis using an in vitro whole blood culture assay. J Immunoassay 20 : 1-11

Pool EJ, Johaar G, James S, Petersen I, Bouic P (1998) The detection of pyrogens in blood products using an in vitro whole blood culture assay. J Immunoassay 19 : 95-111


May 2002