Kazuo Sugawara

Circulating cytokines and hormones with immunosuppressive but neutrophil-priming potentials rise after endurance exercise in humans

Abstract To investigate the mechanisms of exercise-induced immune perturbations, we measured promising immunomodulatory hormones and cytokines in plasma of 16 male marathon runners before and after a competitive 42.195-km race. Interleukin 1-beta (IL-1β) and interferon gamma (IFN-γ) concentrations remained unchanged after the marathon. The cytokines IL-12, IFN-α and tumour necrosis factor alpha (TNF-α) could not be detected even using highly sensitive specific immunoassays, indicating at least that overshooting responses of these cytokines had not occurred after exercise. As mechanisms for the small changes in these cytokines, we demonstrated for the first time a significant rise in concentrations of inhibitory cytokine IL-10 in addition to the immunosuppressive hormone cortisol, although concentrations of IL-4 and transforming growth factor-beta (TGF-β) were unaffected by the race. Furthermore, concentrations of IL-1 receptor antagonist (IL-1ra) and IL-6, which are negative-feedback inhibitors of cytokine production, increased by more than 100 times. As for humoral mediators of neutrophil mobilization, concentrations of growth hormone (GH), cortisol and granulocyte colony-stimulating factor (G-CSF) increased significantly. In addition, concentrations of neutrophil-priming substances (IL-6, IL-8, G-CSF, GH and prolactin) also increased significantly and the induction of IL-8 and G-CSF with exercise was demonstrated for the first time in the present study. In contrast, IL-2 concentration decreased, by 32%, and this was correlated with the induction of nitric oxide (NO) production. Muscle damage, monitored using changes in concentrations of creatine kinase and myoglobin, was also observed. These results suggested that exercise-induced pathogenesis including previously reported immunosuppression and neutrophil hyper-reactivity might be attributed, at least partly, to the systemic dynamics of the above bioactive substances.

Impact of a competitive marathon race on systemic cytokine and neutrophil responses

PURPOSE To investigate whether cytokines and neutrophils mediate exercise-related pathogenesis, we examined their responses and possible association after exhaustive exercise. METHODS Plasma and urine samples were obtained from 10 male runners before and after a 42.195-km marathon race. Major cytokines and neutrophil activation markers [myeloperoxidase (MPO) and lactoferrin (LTF)] were measured by enzyme-linked immunosorbent assays. Functional modulation of standard neutrophils and monocytes by plasma was determined on their luminol-dependent chemiluminescence responses. RESULTS The race induced peripheral neutrophilia accompanied by an increase in band neutrophils and monocytosis. Plasma MPO and LTF concentrations increased significantly by 1.8 and 1.4 times after the race. There was a greater increase in urine concentrations of MPO and LTF, 12.3 and 3.5 times after exercise, respectively, suggesting that neutrophil activation occurred and that renal clearance exceeded the increase in plasma concentrations. Plasma interleukin (IL)-6, IL-8, IL-10, granulocyte colony-stimulating factor (G-CSF), macrophage CSF (M-CSF), and monocyte chemotactic protein 1 (MCP-1) increased significantly after the race, and urine IL-1beta, IL-6, G-CSF, M-CSF, and MCP-1 increased significantly. The plasma IL-6 responses correlated with the increases of band neutrophil count (r = 0.860, P < 0.01), suggesting IL-6-mediated bone marrow release of neutrophils. Furthermore, the increases in urine MPO concentration were correlated with increases in urine IL-6 (r = 0.868, P < 0.01) and G-CSF (r = 0.875, P < 0.01), suggesting that these cytokines promoted neutrophil activation. However, preincubation of neutrophils and monocytes with postexercise plasma could not cause priming responses, possibly because of the exercise-induced enhancement of plasma antioxidant activity. CONCLUSION Although many cytokines recruiting and priming neutrophils and monocytes were secreted and functional after exhaustive exercise, overwhelming antioxidant and antiinflammatory defenses were induced, preventing exercise-induced oxidative stress.

Analysis and assessment of the capacity of neutrophils to produce reactive oxygen species in a 96-well microplate format using lucigenin- and luminol-dependent chemiluminescence

The chemiluminescence (CL) assay has been used to measure the reactive oxygen species (ROS)-generating capacity of phagocytes. To achieve more optimal measurement conditions for a multi-channel microplate photon-counting CL analyzer with the cooled charge-coupled device (CCD) camera which offers enhanced sensitivity, we investigated factors affecting the variability in lucigenin-dependent CL (LgCL) measurement of human neutrophils stimulated with either opsonized zymosan (OZ) or phorbol myristate acetate (PMA). We obtained sensitive LgCL responses with good reproducibility and rapid data-acquisition using 50 microl neutrophils (3 X 10(6) cells/ml) and 50 microl of 0.5 mM lucigenin per well, in addition to either 100 microl of OZ (5 mg/ml) when zymosan was opsonized with 10-20% serum or 100 microl of PMA solution (1 X 10(-6) M) with automatic regular intervals of mixing and detection during the continuous measurement at 37 degrees C. Furthermore, we studied the contribution of various ROS to LgCL and luminol-dependent CL (LmCL) using modulators of ROS metabolism including superoxide dismutase (SOD), catalase, deferoxamine and sodium azide (NaN3). LgCL was inhibited by SOD but not by the other agents, whereas LmCL was inhibited by NaN3 and deferoxamine. Thus, it was demonstrated that LgCL detects the superoxide anion with high selectivity whereas the LmCL assay measures myeloperoxidase (MPO)-mediated formation of hypochlorous acid. Such microplate-based multiple measurements facilitate the accurate assessment of phagocytic function.

Effect of incorporating low intensity exercise into the recovery period after a rugby match

Background: The psychological and physiological condition of athletes affect both their performance in competitions and their health. Rugby is an intense sport which appears to impose psychological and physiological stress on players. However, there have been few studies of the most appropriate resting techniques to deliver effective recovery from a match. Objectives: To compare the difference in recovery after a match using resting techniques with or without exercise. Methods: Fifteen Japanese college rugby football players were studied. Seven performed only normal daily activities and eight performed additional low intensity exercise during the post-match rest period. Players were examined just before and immediately after the match and one and two days after the match. Blood biochemistry and two neutrophil functions, phagocytic activity and oxidative burst, were measured to assess physiological condition, and the profile of mood states (POMS) scores were examined to evaluate psychological condition. Results: Immediately after the match, muscle damage, decreases in neutrophil functions, and mental fatigue were observed in both groups. Muscle damage and neutrophil functions recovered with time almost equally in the two groups, but the POMS scores were significantly decreased only in subjects in the low intensity exercise group. Conclusions: Rugby matches impose both physiological and psychological stress on players. The addition of low intensity exercise to the rest period did not adversely affect physiological recovery and had a significantly beneficial effect on psychological recovery by enhancing relaxation.

Seasonal changes in mortality rates from main causes of death in Japan

Background: It is well recognized that the season of the year exerts an influence on some diseases and causes of death such as coronary heart diseases, stroke, infectious diseases and so on. Methods: We evaluated the influence of seasonal changes on diseases and causes of death in Japan using the Japan Vital Statistics from 1970 to 1999 and recorded weather data (mean temperature), by a Fourier decomposition in a log linear regression model.Results: Major influences of seasonal change with the highest rates in winter were seen on the following: the overall causes of death; infectious and parasitic diseases including tuberculosis; respiratory disease, including pneumonia and influenza; heart and cerebrovascular diseases; diabetes; and digestive diseases and accidents. Two peaks were seen in suicides, a large peak in April and a small peak in autumn. Cancer and homicides were little or not at all influenced by seasonality. There was no major difference in changes between the years studied, except for respiratory disease and tuberculosis, which showed a clear reduction in the seasonality effect from 1970 to 1999. Conclusions: To reduce the overall mortality rate and to prolong life expectancy in Japan, measures must be taken to reduce those mortality rates associated with seasonal differences, especially those causes of death which show a strong correlation with seasonal change: respiratory, heart, cerebrovascular, diabetes and infectious diseases.

Low-level laser irradiation attenuates production of reactive oxygen species by human neutrophils.

OBJECTIVE The aim of this study was to examine the effects of low-level laser therapy (LLLT) on production of reactive oxygen (ROS) species by human neutrophils. BACKGROUND DATA LLLT is an effective therapeutic modality for inflammatory conditions. MATERIALS AND METHODS The laser device used was the infrared diode laser (GaAlAs), 830-nm continuous wave (150 mW/cm(2)). After irradiation, ROS production by neutrophils was measured using luminol-dependent chemiluminescence (LmCL) and expression of CD11b and CD16 on neutrophil surface was measured by flow cytometry. RESULTS The LmCL response of neutrophils was reduced by laser irradiation at 60 min prior to the stimulation with opsonized zymosan and calcium ionophore. The attenuating effect of LLLT was larger in neutrophils of smokers than non-smokers, while the amount of produced ROS was larger in neutrophils of smokers. Expression of CD11b and CD16 on neutrophil surface was not affected by LLLT. CONCLUSION Attenuation of ROS production by neutrophils may play a role in the effects of LLLT in the treatment of inflammatory tissues. There is a possible usage of LLLT to improve wound healing in smokers.

Exercise training and energy restriction decrease neutrophil phagocytic activity in judoists.

PURPOSE To investigate the effects of weight reduction as the result of exercise training and energy restriction on neutrophil function. METHODS Eighteen male competitive college judoists participated in the study. In a whole blood assay, oxidative burst activity, phagocytic activity, expressions of Fc gamma receptor 3 (CD16), and complement receptor 3 (CD11b) of neutrophils were measured on a per cell basis by flow cytometry at day 20, 5, and 1 before and at day 7 after the competition. RESULTS The rate of neutrophil producing reactive oxygen species decreased before the competition, whereas the oxidative burst activity per cell increased significantly in all subjects, which resulted in a significant increase of the total oxidative burst activity. However, there were no significant effect of energy restriction on oxidative burst activity. The rate of neutrophils incorporating opsonized zymosan decreased significantly with energy restriction. The total phagocytic activity of 10,000 neutrophils and the phagocytic activity per cell also decreased significantly by severe energy restriction. The surface antigen expressions of CD11b and CD16 were unaffected by weight reduction. CONCLUSIONS The results suggest that with respect to the management of health conditions, weight reduction for judoists should be composed of exercise training and energy restriction should be moderate.

Adverse effects of energy restriction on myogenic enzymes in judoists

In this study, we examined the effects of weight reduction by restricting energy intake on muscle function and serum concentrations of myogenic enzymes in judoists. Twenty-seven male judoists aged 19.3±0.6 years (mean±s) were divided into three groups based on the extent of body weight reduction: high weight reduction (height 1.70±0.05 m, body mass 78.5 ±13.6 kg), low weight reduction (height 1.70±0.05 m, body mass 80.7±13.1 kg) and a group that maintained body weight while continuing to perform exercise training (height 1.78± 0.07 m, body mass 78.7±8.8 kg). Body composition, blood biochemistry, energy intake and anaerobic power were assessed on four occasions: 20 days (baseline data), 4 days and 1 day before and 7 days after competition. Compared with baseline, body mass and fat-free mass were significantly lower 1 day before competition in the high and low weight reduction groups (both P <0.01); these changes persisted for 7 days after competition in the high weight reduction group. Serum creatine kinase was significantly elevated 1 day before competition in all groups (all P <0.05), and remained higher 7 days after competition in both the high and low weight reduction groups. Compared with baseline, maximal anaerobic power was significantly lower 1 day before competition only in the high weight reduction group (P <0.01). Our results suggest that energy restriction in addition to intense exercise training before major competition has an adverse effect on anaerobic power and elevates serum creatine kinase concentration, leading to an impairment of muscular function and an increased susceptibility of muscle tissue to injury.

Effect of sodium butyrate on reactive oxygen species generation by human neutrophils.

BACKGROUND Short-chain fatty acids enema has been shown to be effective in the treatment of ulcerative colitis (UC). However, the mechanisms that lead to this response have not been well characterized. The aims of this study were to investigate the effect sodium butyrate has on reactive oxygen species (ROS) generation by human neutrophils, which are responsible for mucosal injury. METHODS Human neutrophils incubated with or without sodium butyrate were stimulated with opsonized zymosan (OZ) or phorbol myristate acetate (PMA). ROS generation was largely differentiated with flow cytometry assays of hydroethidine oxidation and dichlorofluorescein oxidation for superoxide anion and hydrogen peroxide respectively, and luminol-dependent chemiluminescence for myeloperoxidase-mediated oxidants. RESULTS Sodium butyrate (up to 50 mM) did not alter hydroethidine oxidation upon stimulation of the OZ or PMA. However, sodium butyrate at a concentration of 25 mM elevated dichlorofluorescein oxidation to 125 + 8% (P = 0.028) of control upon stimulation of OZ and to 191 +/- 30% (P = 0.0016) upon stimulation of PMA. Contrary to these results, sodium butyrate greatly inhibited chemiluminescence responses in a dose-dependent manner. The inhibition by 50 mM sodium butyrate was 61 +/- 6% upon OZ and 71 +/- 9% upon PMA, respectively. CONCLUSIONS These data indicate that sodium butyrate up-regulates hydrogen peroxide generation but down-regulates generation of myeloperoxidase-mediated oxidants, the latter being more potent in killing microorganisms and in inducing tissue injury. A possible mechanism is suggested whereby sodium butyrate may inhibit myeloperoxidase activity and hence attenuate the destructive activities of neutrophils in UC.Background: Short-chain fatty acids enema has been shown to be effective in the treatment of ulcerative colitis (UC). However, the mechanisms that lead to this response have not been well characterized. The aims of this study were to investigate the effect sodium butyrate has on reactive oxygen species (ROS) generation by human neutrophils, which are responsible for mucosal injury. Methods: Human neutrophils incubated with or without sodium butyrate were stimulated with opsonized zymosan (OZ) or phorbol myristate acetate (PMA). ROS generation was largely differentiated with flow cytometry assays of hydroethidine oxidation and dichlorofluorescein oxidation for superoxide anion and hydrogen peroxide respectively, and luminol-dependent chemiluminescence for myeloperoxidase-mediated oxidants. Results:

Effects of rugby sevens matches on human neutrophil-related non-specific immunity

Aims: To evaluate the influences of the accumulative effect of two consecutive rugby sevens matches (Sevens) on aspects of human neutrophil-related non-specific immunity. Methods: In seven players participating in the Japan Sevens, neutrophil reactive oxygen species (ROS) production capability and phagocytic activity were measured using flow cytometry, and serum opsonic activity (SOA) was assessed by measuring neutrophil ROS using the peak height of lucigenin-dependent chemiluminescence before and after two consecutive matches. Results: ROS showed no change immediately after the first match, and had significantly (P<0.05) increased 4 h later, but showed a decrease after the second match. Phagocytic activity showed no change immediately after the first match, but had significantly (P<0.01) decreased 4 h later, and showed a further decrease after the second match, although it was not significant. SOA significantly (P<0.01) increased after the first match, and still maintained its high 4 h later, but decreased after the second match. ROS production capability, phagocytic activity and SOA significantly (P<0.01) decreased after the second match. Conclusions: When rugby players play two consecutive Sevens matches, the exercise loading is thought to be hard, similar to that experienced during a marathon race and intensive or long training in a training camp, although the expected changes were not seen after the first match. Differences between after the first and the second matches may be due to the “cumulative effect”.