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Basic Sciences: Original Investigations

In vivo cell-mediated immunity and vaccination response following prolonged, intense exercise

BRUUNSGAARD, HELLE; HARTKOPP, ANDREAS; MOHR, THOMAS; KONRADSEN, HELLE; HERON, IVER; MORDHORST, CARL H.; PEDERSEN, BENTE K.

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Medicine & Science in Sports & Exercise: September 1997 - Volume 29 - Issue 9 - p 1176-1181
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Abstract

Exercise induces alterations in the distribution and trafficking of peripheral mononuclear cells. During exercise especially natural killer(NK) cells, but also B and T cells, are recruited to the blood, and the total lymphocyte count increases (9). Following prolonged, intense exercise the number of lymphocytes in the blood are depressed below baseline, and the function of NK and B cells are impaired(9,14,23). The duration of this suppression depends on the intensity and the duration of the exercise(23). During the period of immunological suppression(often referred to as “the open window”) the host may be more susceptible to microorganisms (24). This hypothesis is supported by several epidemiological studies suggesting that intense acute exercise of long duration is associated with an increased risk of infections, and especially upper respiratory tract infections (17). Furthermore, epidemiological as well as experimental studies show that during the incubation period of an infection, depending on the pathogen, exercise may worsen the disease outcome (5). However, in vitro tests may not always provide accurate assessments of systemic immune responses, epidemiological studies may reflect concomitant conditions that have their own separate effects on the immune function, and procedures involving animals may not represent reasonable models of human exercise(1).

The purpose of the present study was to investigate whether an in vivo impairment of the immune system could be demonstrated after intense exercise of long duration. We chose to evaluate the response to a cellmediated hypersensitivity skin test and the level of specific antibodies after the performance of a vaccination program in triathletes having antigens introduced immediately after finishing a long, intense exercise program in the form of a training competition. Accordingly the application of the skin test and the immunizations were carried out when in vitro impairment of the immune system had been demonstrated. The vaccination program consisted of a pneumococcal polysaccharide vaccine that is generally considered to be T cell independent, and two toxoids (tetanus and diphtheritis) that are dependent on T cells (7), as activated T cells are important in helping B cells develop into immunoglobulin-secreting plasma cells. A difference in the response to the two types of immunizations may therefore reflect differences in the function of B and T lymphocytes following exercise.

MATERIALS AND METHODS

Protocol

The experimental protocol was approved by the ethical committee for the Copenhagen and Frederiksberg communities. All subjects were informed of the purposes and the risks of the study and their written informed consent was obtained.

Twenty-two male triathletes with a median age of 31 yr (range 21-53) performed a training competition including 3 km of swimming, 130 km of bicycling, and 21 km of running (group A). On another day maximal heart rate and oxygen uptake were measured (Ergo-Oxyscreen, Jaeger, Germany) during ergometer running, Table 1. So that we could evaluate the intensity of the work performed by the triathletes, they were equipped with a portable heart rate monitor (Polar Sport Tester, Finland) with a microcomputer which was programmed to record and store heart rate data at 15-s intervals. After the training session these values were interfaced directly into a computer port for analysis (with Polar Sport tester software). The median and maximal heart rate during biking and running were calculated. The accuracy of the heart rate monitor has previously been demonstrated(11).

The work intensity was expressed in percent of maximum heart rate as follows:Equation

Working times, heart rates, maximum heart rates, and relative intensities are given in Table 2. The work intensity is based on minimal values because of several flat ties (more than 20) leading to 5 to 10 min of no pulse registration, but with registration of elapsed time.

Blood samples for measuring antibody titers were taken at rest before the exercise (day 0 morning), 30 min after exercise (day 0 afternoon), and at rest in the morning 2 wk after the exercise (day 14). Vaccinations were performed 30 min after exercise (after the blood samples had been taken). At the same time a skin test was applied on the anterior surface of the forearm, and the reaction was read 48 h later.

The training competition took place in the summer of 1994.

Eleven healthy non-exercising male triathletes with a median age of 26 yr(range 20-44) were included as control group (B). Twenty-two healthy moderately trained males with a median age of 31 yr (range 21-48) were evaluated as another control group (group C). Blood samples, vaccinations, and skin tests were performed at the same times during the day, with the same day intervals, and at the same time of the year as in group A. Furthermore, measurements of pulse and oxygen uptake were obtained (seeTable 1).

The individuals in the two triathlon groups (groups A and B) were allowed to do their normal training programs but not to participate in major competitions during the 2 wk between the day of the vaccinations and the day of the last blood sample. They did not perform any exercise 12 h prior to the blood samples. Group C was not allowed to perform any exercise in the study period. All subjects were free of infections.

Skin Test

The skin test (Multitest, Pasteur Mérieux, Serums and vaccines, Copenhagen, Denmark) was used for evaluation of cell-mediated immunity. The Multitest consisted of an acrylic resin applicator with eight heads, each fixed onto a base and loaded with a standard antigen. The antigens were as follows: Two toxoids (tetanus and diphtheria), three bacterial (streptococcus, tuberculin, and proteus), and two fungal (candida and trichophyton). A glycerin/saline diluent served as a negative control. The average diameter of induration was measured after 48 h for each antigen. A score consisting of the sum of average diameters expressed in mm of all positive reactions read at 48 h were calculated. Furthermore, the number of positive skin test spots was enumerated.

Vaccination procedure and vaccines. The vaccinations were performed subcutaneously with 0.5 ml of a mixture of diphtheria and tetanus toxoid (Di-Te, SSI, Copenhagen, Denmark) and 0.5 ml of a pneumococcal polysaccharide vaccine containing purified capsular polysaccharide from 23 pneumococcal types (Pneumovax 23, Merck Sharp and Dohme, West Point, PA).

Estimation of Antibody Titer

Antibody titers were determined in sera stored at -80°C.

Diphtheria and tetanus toxoids. Antibodies in sera were assessed with an enzyme-linked immunosorbent assay (ELISA), as described previously(8,27). Titers were expressed in international units and calculations using a parallel line assay allowed expression of serum content in international units per milliliter.

Pneumococcal antigens. Antibodies against the pneumococcal vaccine polysaccharide (type 1, 4, 7F, 14, 18c, and 19 were determined with an ELISA as described previously (10).

Statistical Analysis

Since the vaccination and skin test data did not have a normal distribution, they are presented as medians and 1st and 3rd quartiles. When the skin test data were evaluated, groups were compared using the Kruskal Wallis test. The antibody titers were log transformed and differences between groups were evaluated by an ANOVA for repeated measures. Groups were compared using the Mann-Whitney rank sum test for unpaired samples. Statistical comparisons were calculated using software from SYSTAT. Inc., version 5(Evanston, IL).

RESULTS

Cell-Mediated Immunity

The cumulative responses (sum of the diameters of indurations and number of positive skin test spots) were significantly lower in group A compared with both groups B and C, whereas no difference was found between groups B and C(Fig. 1). The delayed-type hypersensitivity responses to individual antigens are shown in Table 3. There were no significant differences in the skin test response between group B and C. Group A revealed a significantly lower response to the tetanus antigen compared with both groups B and C. Also, group A responded less to stimulation with diphtheritis antigen than group B and less to stimulation with tuberculin antigen than group C.

Vaccination Response

Antibody titers against diphtheria and tetanus toxoid and the mean response against six pneumococcal antigens are shown inTables 4, 5, and 6. There were no significant differences among the groups regarding the level of specific antibodies.

DISCUSSION

Injection of antigens into the skin induces a delayed hypersensitivity reaction characterized by erythema and induration. Histologically an exudation of mono- and polymorphonuclear cells is seen. The latter soon migrates out of the lesion again and a mononuclear cell infiltrate is left. Memory T-cells recognize the antigen on antigen presenting cells and are stimulated to blast cell transformation, proliferation, and secretion of lymphokines. Secondly these lymphokines mediate attraction and activation of cytotoxic T cells, NK cells, and macrophages if they belong to Th1, and of eosinophils if they belong to Th2(25). Thus, the delayed hypersensitivity reaction is a complex immunological reaction involving several different cell types and chemical mediators. The finding of a decreased cellular immunity following prolonged, intense exercise may be a result of a decrease in the accumulation of cells or a result of a functional impairment (anergy, decreased production of cytokines). The underlying mechanism is not elucidated by the present study. Regarding the function of T lymphocytes, a decreased production of IL-2 and IFN-gamma in whole blood culture supernatants has been described (21). This may be related to the presence of lymphopenia in the blood. In regard to the proliferative response to mitogens and antigens in the recovery time after exercise, conflicting observations have been reported(2-4,6,15,19,26,32,35,36). The most constant finding is a suppressed PHA response after intense exercise. However, this may simply be related to an altered composition of lymphocyte subpopulations that influence the in vitro assay in which a fixed number of cells are studied. A transient decrease in the NK cell activity following intense exercise has been related to a decreased blood level of NK cells (12), but it has also been explained by a suppression caused by prostaglandins produced by an increased number of monocytes and neutrophils (31). NK cells are thought to play an important role in the defense against virus infections(37). The blood concentrations of monocytes and neutrophils increase during and after exercise. Furthermore, moderate and intense exercise of long duration may be associated with no change in or with an increased capacity of phagocytic activity (22). It has been proposed that an increased phagocytic activity may coexist with an increased susceptibility of upper respiratory tract infections because the macrophages and neutrophils may mainly be involved in tissue repair following intense exercise (20).

The level of salivary IgA decreases following exercise(13,28,29). The mechanism of the decreased secretion of immunoglobulins has been studied in a plaque forming assay(30). A decreased number of IgG-, IgA-, and IgM- secreting cells were found during and 2 h after exercise with in vitro stimulation with pokeweed mitogen, interleukin-2, and Epstein-Barr virus (EBV). The number of secreting cells was back to normal after 1 d of recovery. It was concluded that this was a result of a functional impairment rather than a quantitative decrease in the B lymphocyte level. In the present study no significantly decreased B cell function was found in regard to the ability to generate a specific antibody secretory response following intense exercise of long duration. This is in line with the findings of Eskola(2), who gave tetanus toxoid to four runners immediately after the performance of a marathon. When evaluated 15 d later, the mean antibody titer for the group of runners was higher than that of a group of controls, and the conclusion of that study was that humoral immune functions are not impaired by a marathon; they may even be enhanced. No difference in the response to pneumococcal polysaccharide vaccine versus immunizations with toxoids was found in the present study. Thus, as described in some studies (2,15) but not in others(3,4,6,16,19,26,33,35,36), a hypothetical transient decrease in T lymphocyte transformation lasting less than 24 h does not seem to have any consequences for generating an in vivo antibody response during the following weeks. Therefore, transient decreased immune function may be of importance for the acute establishment of infections, whereas it has no effect on the generation of a specific immune response during the following days.

It is possible that the suppression of a delayed-type hypersensitivity response observed after exercise is related to secondary effects of exercise rather than anergy or leukopenia. As an example, an increase in peripheral circulation could clear the antigens faster from the site of application. Because the introduction of the Multitest and the vaccinations was intended to occur during the period correlating to in vitro immunosuppression, we chose to introduce the antigens 30 min after the exercise as a compromise between these two conflicting aims.

Another problem with our design was that the read of the Multitest was not blinded. We thought that blinding would not be practically possible, as the arms of the more muscular triathletes would easily be recognized from the arms of the moderately trained males. On the other hand, the differences between group A and the two other groups were too pronounced to be explained by a bias in observation alone.

Whether the immune system of moderately trained subjects can be compared with the immune system of triathletes is questionable. Resting levels of NK cell activity has been found to be elevated in elite cyclists compared with sex- and age matched controls (34), whereas others(18) have found that there is no difference between athletes and non-athletes. The reason for including a control group of moderately trained subjects was that only a limited number of triathletes were available for inclusion in the study. Since the main purpose of the study was to compare the effect of a triathlon race on a skin test and vaccination response, we wanted to have as many triathletes as possible in group A. As a consequence, we decided to have only 11 individuals in group B and to introduce the additional control group of moderately trained individuals. This compromise was justified by the finding of no difference between the immunological responses in group B and C.

In conclusion, in vivo cell-mediated immunity was impaired in the first days following prolonged, highintensity exercise, whereas there was no impairment of the in vivo antibody production measured 2 wk after the vaccination. The explanation may be that mainly unspecific acute immunity is influenced by exercise, whereas the function of specific acquired immunity is not altered. Alternatively, the time factor may be critical as the immune system may have had time to regenerate during the 2 wk intermission. Transient suppression of immunity during the first 24 h following exercise may be of importance in weakening the first line of defense and increasing the risk of acute infections as URTI, whereas it is insignificant in the later establishment and generation of specific immunity.

Figure 1-Cell mediated immunity. Cumulative response. Medians and quartiles of the test score (sum of diameters of indurated skin test areas) and the number of indurated spots are shown in group A (triathletes performing a triathlon race), group B (nonexercising triathletes), and group C(moderately trained individuals). *indicates significant difference between groups A and B. # indicates significant difference between groups A and C. Medians and quartiles are shown.
Figure 1-Cell mediated immunity. Cumulative response. Medians and quartiles of the test score (sum of diameters of indurated skin test areas) and the number of indurated spots are shown in group A (triathletes performing a triathlon race), group B (nonexercising triathletes), and group C(moderately trained individuals). *indicates significant difference between groups A and B. # indicates significant difference between groups A and C. Medians and quartiles are shown.

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Keywords:

PHYSICAL ACTIVITY; TRIATHLON RACE; TRAINING; LYMPHOCYTE; IMMUNE SYSTEM; PNEUMOCOCCAL POLYSACCHARIDE; DIPHTHERITIS; TETANUS; SKIN TEST

©1997The American College of Sports Medicine