Introduction

Clinical data indicate that patients with spinal cord injury (SCI) suffer from profound immunological impairment at rest,1 probably due to autonomic dysfunction, physical inactivity, and selected medications.1 Numerous studies have investigated the immune responses after exercise in able-bodied (AB) persons. It is well known that strenuous exercise suppresses, whereas moderate exercise activates various immune parameters.2 Natural killer (NK) cell cytotoxic activity (NKCA) is commonly measured to assess the immune response because of its sensitivity to physiological and psychological stress.3 Most researchers agree that the immediate post-exercise increase in NKCA reflects the recruitment of NK cells into the circulation, mediated by sympathetic outflow and catecholamine action.4 However, there is disagreement about the reasons for the transient NKCA decrease during recovery.5

Very few studies have examined the immunological changes after exercise in SCI.6, 7, 8, 9, 10 We reported previously that competitive wheelchair athletes with paraplegia show decreased peripheral NK cell numbers and NKCA immediately after a full marathon race and recovery to baseline values after just one night of rest.6 However, NKCA increased significantly in paraplegics immediately after a wheelchair half-marathon and remained so during the next day of recreational athletics.7 We hypothesized that the decrease in NK cell function immediately after a full marathon might reflect the immune response to prolonged and intensive exercise. However, there are no studies on NKCA during voluntary exercise specifically in persons with cervical SCI (CSCI).

Klokker et al.9 studied NK cells in six subjects with CSCI after 30 min of electrically stimulated cycling exercise. No NK changes were seen in the quadriplegic group. Blood adrenaline concentrations increased during exercise in all subjects, although the rise was more pronounced in paraplegic than in quadriplegic subjects, indicating a difference between the groups in sympathetic nervous system integrity.9 The authors concluded that sympathoadrenal activity resulted in the recruitment of NK cells to the circulation during exercise.9 However, it is possible that electrically stimulated cycling exercise does not activate the pyramidal tract and medullary cardiovascular center in persons with CSCI. Furthermore, the extent of the NKCA response depends on the intensity and duration of exercise.5, 11, 12 Thus, further studies need to be conducted under physiological and clinically relevant conditions. To investigate the role of sympathoadrenal activity in increased NKCA during voluntary exercise, the intensity and duration of the exercise must be tightly controlled to levels that increase blood adrenaline level in AB but not in CSCI.

In general, the higher the level of the SCI, the more profound the effects of sympathetic nervous dysfunction below the level of the injury.13 Therefore, we were interested in the immunological changes induced during and after exercise in CSCI. To our knowledge, no studies have examined these immunological parameters during voluntary exercise of well-controlled duration and intensity in subjects with dysfunction of the sympathetic nervous system. This study investigated the effect of cervical transection on activation of immune parameters induced by voluntary exercise. Specifically, we compared the NKCA and adrenaline responses to arm-crank ergometer exercise in subjects with chronic CSCI and AB.

Materials and methods

Subjects

The Research Ethics Committee of Kibikogen Rehabilitation Center for Employment Injuries approved the experimental protocol and all subjects provided written informed consent. The study included eight persons with CSCI who were involved in a regular physical training program and six AB subjects as controls. The selection criteria for the study were as follows: (1) male (no females to exclude the effects of menstrual cycle-related hormone changes), (2) chronic injury state (58–64 months), (3) complete CSCI (ASIA Impairment Scale A), and (4) excellent current health and on no medications that would affect the immune and endocrine responses. Table 1 lists the subjects’ characteristics. There were no differences between CSCI and AB with respect to age, height, and weight. The mean VO2max of control subjects was higher than CSCI. Of the eight CSCI persons studied, seven used clean intermittent catheterization and one used indwelling supra-pubic catheter.

Table 1 Anthropometric data of participating subjects
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Study protocol

In this study, we selected an arm-crank ergometer exercise because all subjects with CSCI suffered from complete tetraplegia. Two weeks before the scheduled start of the study, subjects performed a progressive VO2max test on an arm-crank ergometer (818E, Hand ergometer, Monark, Sweden). The test protocol required the subjects to maintain a target cadence of 60 r.p.m. After a 15-min rest period, subjects performed unloaded exercise for 3 min. The power output of the ergometer was subsequently increased by 10 W every minute. The test was terminated when the subjects reached exhaustion, or if the cadence fell below 60 r.p.m. O2 uptake and ventilation were measured using a respiratory metabolic cart (WLCU-5207A, Westron, Japan) and EKG was monitored continuously (BP unit, PB14-136, NEC, Japan).

All subjects indicated that they had avoided intensive exercise for at least 24 h before the test and they were healthy and free of symptoms associated with respiratory or urinary tract infections. The subjects had their regular breakfast before 0900 hours and then refrained from eating, but were allowed to drink tap water ad libitum until the end of experiment. They reported to the Human Performance Laboratory between 1000 and 1100 hours and were outfitted with electrodes for EKG recording. Before the resting period, the subject emptied the bladder to eliminate possible autonomic dysreflexia induced by bladder distension. After resting in a quiet room for 30 min, subjects started to exercise on an arm-crank ergometer for 20 min at intensities of 60% VO2max; the power output was increased progressively from 0 W to the desired level over 3 min.

Blood samples were collected from the antecubital vein into heparinized tubes and EDTA-2K-containing tubes before, immediately after, 1 h after, and 2 h after completion of exercise. Total blood volume in each sampling period was 15 ml. Six AB subjects performed the same experiment as the control group.

NKCA assays and measurement of NK cell number

NKCA was assayed as described earlier.6, 7 Three-color flow cytometry was used to determine the total number of NK cells (CD3−CD16+CD56+). Briefly, 50 μl of whole blood was incubated at room temperature for 20 min with 10 μl of antibody cocktail in a polypropylene tube. Antibodies were purchased from Becton-Dickinson (San Jose, CA, USA). Erythrocytes were then lysed with 1 ml FACSlyse and incubated for a further 9 min. Finally, the tube was centrifuged at 2500 r.p.m. for 3 min before the supernatant was removed, and cells were then resuspended in 250 μl 1% formalin until analysis. Samples were sent to a commercial pathology laboratory (Sullivan Nicolaides, Taringa, Queensland, Australia) for analysis by a FACScan flow cytometer (Becton-Dickinson). The flow cytometer was calibrated daily by using different microbeads (QC Windows and Quantum 1000 (Flow Cytometry Standards, San Juan, Puerto Rico) and CaliBRITE (Becton-Dickinson)), as per standard methods for quantifying cellular antigens by flow cytometry. A total of 20 000 events were collected and the data recorded as percentage of positive cells. The total number of lymphocytes determined from a full blood count was multiplied by the percentage of CD3−CD16+CD56+ cells.

Other blood tests

Total blood cell counts, hematocrit, the percentage of neutrophils, total lymphocytes, and monocytes, plasma catecholamines, and cortisol levels were measured using the methods described earlier.10

Statistical analysis

Data were expressed as mean±standard error of the mean (s.e.m.) and analyzed using a 2 × 4 repeated measures analysis of variance (ANOVA). When the results of ANOVA tests were significant (P<0.05), we used Scheffe's test to determine differences between pre-exercise levels and each time period, and between two groups (control and exercise). A P-value <0.05 denoted the presence of a significant difference between the two groups.

Results

Blood parameters

Table 2 lists the blood cell counts of participants at four different time points: before exercise, immediately after exercise, 1 h after, and 2 h after exercise. Red blood cell (RBC) counts, absolute leukocyte numbers, hemoglobin levels, and hematocrit did not change in CSCI, whereas in AB, all four values increased immediately after exercise compared with the pre-exercise measurements, and then returned to the baseline levels by 1 h after exercise. In both groups, the absolute number of peripheral neutrophils was higher at immediately, 1 and 2 h after exercise, compared with baseline, but reached significance only at 2 h post-exercise in the CSCI.

Table 2 Changes in blood cell count, hemoglobin, hematocrit, and leukocyte subpopulations in cervical spinal cord injury subjects (CSCI) and able-bodied subjects (AB) during 20 min arm ergometer exercise at 60% VO2max
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NK cell count and NKCA

Figure 1 displays the number of peripheral NK cells at the aforementioned time intervals in CSCI and AB. In CSCI, the absolute numbers of NK cells remained constant throughout the study, whereas in AB, NK cell counts increased immediately after exercise then returned to the baseline, pre-exercise level by 1 h after exercise. However, the absolute numbers of NK cells at all time points were significantly lower in CSCI than AB.

Figure 1
figure 1

Number of NK cells during physical exercise (arm ergometer, 60% of VO2max, 20 min) in cervical spinal cord injury subjects (CSCI) and AB subjects (AB). Data are expressed as mean±s.e.m. *P<0.01, compared with baseline (before exercise). ##P<0.01, compared with the control.

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Pre-exercise NKCA was not significantly different between AB and CSCI. In controls, NKCA increased immediately after exercise and decreased below the pre-exercise level 1 h after exercise, before returning to pre-exercise values by 2 h after exercise. However, NKCA in CSCI remained constant throughout the study (Figure 2).

Figure 2
figure 2

NK cells activity after acute physical exercise (arm ergometer, 60% of VO2max, 20 min) in cervical spinal cord injury subjects (CSCI) and able-bodied subjects (AB). Data are expressed as mean±s.e.m. **P<0.01, *P<0.05, compared with baseline (before exercise). #P<0.05, compared with the control.

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Cortisol and adrenaline

The mean pre-exercise concentrations of cortisol were not significantly different between AB and CSCI, but were higher in CSCI than in AB at 1 and 2 h after exercise (Figure 3). In both groups, the plasma cortisol remained constant throughout the study, with a trend toward an increase immediately after exercise (Figure 3).

Figure 3
figure 3

Plasma cortisol response to acute physical exercise (arm ergometer, 60% of VO2max, 20 min) in cervical spinal cord injury subjects (CSCI) and able-bodied (AB) subjects. Data are expressed as mean±s.e.m. #P<0.05, ##P<0.01, compared with the control.

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Plasma adrenaline concentrations were significantly and markedly lower in CSCI than AB before exercise. Exercise significantly increased these concentrations in AB, but no change was observed in CSCI throughout the study (Figure 4). The exercise-induced increase in plasma adrenaline was reversed to pre-exercise levels at 1 h after exercise in AB.

Figure 4
figure 4

Plasma adrenaline response to acute physical exercise (arm ergometer, 60% of VO2max, 20 min) in cervical spinal cord injury subjects (CSCI) and able-bodied (AB) subjects. Data are expressed as mean±s.e.m. **P<0.01, compared with baseline (before exercise). ##P<0.01, compared with the control.

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Discussion

This study is the first to examine changes in NKCA during voluntary arm-crank ergometer exercise for 20 min at 60% of VO2max in CSCI. We showed that exercise of well-controlled duration and intensity significantly increased plasma adrenaline concentrations and NKCA in AB, whereas these parameters did not change in CSCI either during or after exercise.

Patients with high-level CSCI have impaired sympathetic nervous system and thereby a reduced catecholamine response to exercise, compared with both normal healthy subjects and to some extent subjects with lower thoracic-level SCI.14 This study also showed that plasma adrenaline concentrations in AB increased significantly immediately after exercise, whereas that of CSCI did not change throughout the experiment. Exercise-induced factors other than adrenaline might be similar in CSCI and AB, as the duration and intensity of the exercise were fixed. An impaired sympathetic nervous system might therefore also have a major function in the unchanged adrenaline response in CSCI.

Both the number and activity of circulating NK cells fluctuate greatly during and after exercise, as well as in response to other stressors.4 NK cells are rapidly mobilized into the peripheral circulation most likely via multiple mechanisms including: a catecholamine-induced downregulation of adhesion molecule expression and shear stress due to a substantial increase in peripheral blood flow.15

NK cells derive mainly from the spleen and are recruited into the circulation by sympathetic outflow and catecholamine action.4 We speculated that the increases of RBC, absolute leukocyte numbers, hemoglobin levels, and hematocrit immediately after exercise in only AB would be induced by a blood shift from the spleen. NK cells attached to vascular endothelial cells in vitro detach after brief incubations (as short as 5 min) with catecholamines, in a dose-dependent manner.16 The addition of a selective β-2 adrenoreceptor antagonist is reported to block this catecholamine-dependent detachment of NK cells.17 Compared with noradrenaline, adrenaline was more effective in inducing detachment of the NK cells, which correlates with the higher affinity of β-2 adrenoreceptors for adrenaline.4 A more recent report has emphasized that exercise-induced catecholamines modulate the expression of adhesion molecules on NK cells, resulting in the mobilization of NK cells into the circulation.18 Furthermore, plasma catecholamines may mobilize leukocytes by increasing cardiac output and thus intravascular shear forces.19 In this study, persistent changes in adrenaline levels, NK cell numbers, and NKCA in CSCI were observed during voluntary exercise. These findings strongly suggest that the increased NKCA during exercise was inhibited in CSCI mainly due to impaired sympathetic nervous system.

In an earlier study, we compared the immune responses to 2 h of arm-crank ergometer exercise at 60% of VO2max between seven subjects with SCI at Th11-L4 and six AB persons.10 NKCA increased significantly in AB immediately after exercise, but decreased significantly in SCI immediately after exercise.10 Cortisol levels remained constant throughout the experiment in all subjects. In this study, with substantially shorter exercise duration, NKCA in CSCI did not decrease immediately after exercise.

Klokker et al.9 showed that the increase in circulating NK cells after 30 min of electrically stimulated cycling exercise was lower in subjects with cervical spinal cord injuries, whose spinal injuries were at higher levels of the spinal cord affecting the sympathetic outflow, compared with paraplegics. This study required subjects with SCI to perform voluntary arm-crank ergometer exercise, as electrically stimulated cycling exercise might not activate the pyramidal tract and medullary cardiovascular center. In addition, our aim was to study subjects under physiological conditions that are more clinically relevant. With the fixed duration and intensity of the described voluntary arm exercise, circulating concentrations of adrenaline and NKCA remained constant in subjects with CSCI. These subjects have transected sympathetic nerve activities at cervical lesions connecting the peripheral and central nervous systems instead of normal activations in AB with unimpaired sympathetic nerve function. Therefore, it seems that NKCA increases through sympathoadrenal activity, mainly by increasing adrenaline, during voluntary exercise in humans.

There are two reasons for selecting 20 min for exercise duration and 60% of VO2max for exercise intensity. First, as sympathetic innervation in CSCI is terminated at the cervical lesion, the peak heart rate of CSCI was limited and the subjects could not complete either greater intensity or longer duration exercise than those selected in this study. In a series of preliminary studies, we selected the maximal workload that could be completed during the exercise task by the majority of CSCI subjects because both the intensity and duration of the exercise seem to influence the scale of the NK mobilization. Second, earlier studies reported an increase in NKCA and the proportion of NK cells after 6-min cycling exercise at 55% of VO2max in AB persons,20 indicating that short-duration low-intensity exercise can significantly increase NK cell number and activity. Therefore, we expected an increase in NKCA in the selected exercise, which was in fact observed in our AB subjects.

Conclusion

NKCA in AB increases significantly during and after 20-min arm exercise conducted at 60% of maximum oxygen consumption, whereas CSCI did not show increased NKCA. These findings probably reflect an impaired sympathetic nervous system in CSCI.