CD56dimCD16high and CD56brightCD16− cell percentages associated with maximum knee extensor strength and incidence of death in elderly
SpringerPlus volume 5, Article number: 244 (2016)
Physical fitness is an indicator of systemic well-being in humans. Little is known about the role of physical fitness for maintaining systemic health in the elderly. Here, we study elderly subjects to determine the relationships between physical fitness and CD56 and CD16 surface NK cell markers on peripheral blood lymphocytes, as well as to analyze the relationship between the surface markers and incidence of death. We selected 253 independent elderly subjects (122 female; 131 male) who were 79–80 years old. Subjects having a higher proportion of CD56dimCD16high within CD56+CD16+ cells, or ration of CD56dimCD16high and CD56dimCD16− cells had a significant positive correlation with maximum bilateral knee extensor strength/weight (kg) (r = 0.425; P < 0.0001 or r = 0.323; P < 0.0001). In contrast, an increased proportion of CD56brightCD16− cells within lymphocyte significantly negatively correlated with the maximum bilateral knee extensor strength/weight (kg) (r = −0.290; P = 0.0004); and these subjects had a significantly lower mortality during the 5 years following measurement of death. Therefore, we found that a synergistic effect of the right and left leg muscle strength was associated with proportion of matured NK and NKT cells and induced a low proportion of CD56brightCD16− cells within lymphocyte. Moreover, the low proportion of CD56brightCD16− cells was associated with incidence of death. In conclusion, measurements of physical fitness, the proportion of CD56dimCD16high within CD56+CD16+ cells, the ratio of CD56dimCD56high and CD56dimCD16− cells, and the proportion of CD56brightC16− cells in lymphocytes are important indicators to check elderly health.
In developed countries, the elderly population is increasing in concurrence with a low birth rate. This has led to an increase in the bed-ridden elderly who have few opportunities to perform daily physical activity. This decreased daily physical activity increases the risk of systemic diseases, e.g., aspiration pneumonia, cardiovascular diseases, hypertension, osteoporosis, type II diabetes and obesity (Chodzko-Zajko et al. 2009; Kohl 2001; Yoneyama et al. 2002). The decline in muscular strength may lead to a reduction in immune function and thus to the loss of resistance to infectious disease, e.g., aspiration pneumonia (Takata et al. 2007; Eickmeyer et al. 2012) and cancer development (Brown et al. 2012; Eickmeyer et al. 2012; Dimeo et al. 2004). Low levels of physical activity are postulated to be a substantial contributor to cancer-related fatigue (Eickmeyer et al. 2012); and interventions to increase physical activity in cancer patients are shown to reduce the severity of this symptom. Physical activity is found to play a role in the prevention of certain malignancies, including breast, colon, and other cancers (Brown et al. 2012).
In athletes, the peripheral blood natural killer (NK, CD16+/56+) cell numbers are generally normal, although NK cell cytotoxic activity may be higher at rest in athletes compared with non-athletes (Kusaka et al. 1992; Nieman et al. 1995). Others report enhanced in vivo NK cell cytotoxicity after exercise intervention in smaller studies (Nieman et al. 1995; Nieman 2000). Results from a 10-week study among 25 elderly nuns (mean age, 77 years old) where the intervention consisted of 20–50 min of brisk walking 3 days per week suggested improved NK cell cytotoxicity but no effect on in vitro T-lymphocyte proliferation (Fahlman et al. 2000). Additionally, it has been largely suggested that moderate exercise training has no effect on T-lymphocyte proliferation, as well as the numbers and percentage of NK cells, leukocytes, and lymphocytes (Nieman 2000; Scanga et al. 1998). In contrast, moderate-intense exercise is associated with conditions linked to immunity, including resistance to upper respiratory tract infection (Chubak et al. 2006). There is no consistent correlation observed in the various reports involving exercise and immunity (Moreira et al. 2009).
Physical fitness refers to a physiologic state of well-being allowing individuals to meet the demands of daily living or provides the basis for sport performance, or both. Physical fitness appears to be similar to physical activity in its relationship to morbidity and mortality (Blair and Brodney 1999; Erikssen 2001) but it is stronger in predicting health outcomes than increased physical activity (Blair et al. 2001; Myers et al. 2004; Williams 2001). The assessment of physical fitness is often not feasible or practical in a large population-based investigation. Therefore, additional studies as addressed here are required to determine if there is a correlation between physical fitness and immune cells in a large population.
When a microbe invades, cell-mediated immunity is the first defense mechanism for protection against invaders. With cell-mediated immunity, NK cells respond to cues from sentinel immune cells, including macrophages and pathogen-infected tissues (Biron et al. 1999); and play an important role in the defense against viral infections as well as in tumor surveillance (Lanier 2008). NK cells are also associated with the adaptive immune response through the production of cytokines (Lanier 2008). In humans, NK cells are usually defined as CD3−CD56+ cells (Lanier et al. 1986), and can be further subdivided based on CD56 expression. CD56dim NK cells constitute the majority (90 %) of peripheral blood NK cells (Cooper et al. 2001a, b) and express high levels of the low-affinity Fc receptor, CD16, and high levels of perforin (Cooper et al. 2001a, b; Ferlazzo et al. 2004). CD56dim NK cells are terminally differentiated cells. These cells express the CD16 marker and are involved in antibody-dependent cellular cytotoxicity. CD16 is associated with the activation of motif-containing adaptor proteins (Lanier 2008). Engagement of CD16 is sufficient to induce IFNγ and TNFα secretion in addition to chemokine secretion. NK cell maturation is characterized by the down-regulation of CD56 and the acquisition of CD16. Reports show NK cell activation is highest when CD56dimCD16+ activated cells are observed (Dulphy et al. 2008). Therefore, CD56dimCD16+ cells include the most mature cell type of NK cells (André et al. 2000; Nagler et al. 1989).
NKT (natural killer-like T) cells were originally described as a unique population of T cells with the co-expression of NK cell markers (Golden-Mason et al. 2007; Mercer et al. 2005; Tarazona et al. 2000). A consistent subset of T cells expresses bright CD56; however, CD16 is rarely expressed on T cells in healthy subjects (Lambert et al. 2006). CD3+CD16+CD56+ T cells are not classical invariant NKT cells but are a broader group of T cells matching the original definition of NKT cells (Golden-Mason et al. 2007; Mercer et al. 2005) and have cytotoxic activity (Forner et al. 1994).
Understanding NK and NKT effector functions may lead to the development of improved strategies for the treatment of many diseases. NK and NKT cells are innate immune cells that act as the first line of defense against infection and tumorigenesis. NK cells are essential for tumor rejection (Park et al. 2003; Herberman et al. 1975; Kiessling et al. 1975); and Subleski et al. report that NK cells are an important component for tumor regression in a murine liver tumor model (Subleski et al. 2006). Recent studies support that NK cells play a critical role in tumor control and eradication (Waldhouer and Steinle 2008). An 11-year follow-up study of 3625 of Japanese population was performed for cancer immune-surveillance for NK cells; low NK cell cytotoxicity correlated with an increased risk for cancer (Imai et al. 2000). Thus, NK cell-mediated anti-tumor immunity is required to overcome the development of cancer.
In this study, we found three characteristic cell types, CD56dimCD16high, CD56dimCD16− and CD56brightCD16− cells, within the overall lymphocyte population in peripheral blood cells. We performed a correlation analysis between these three cell types, and indicators of physical fitness with incidence of death during 5 years following the initial testing. To clear relationship between the maturation of CD56+CD16+ cells and physical fitness, the ratios of CD56dimCD16high and CD56dimCD16− cells were analyzed. We found that the proportion of CD56dimCD16high within CD56+CD16+ cells and the ratio of CD56dimCD16high and CD56dimCD16− cells were positive immune indicators when compared to physical fitness such as the maximum bilateral extensor strength; proportion of CD56brightCD16− cells within lymphocytes showed as a poor outcome indicator leading to increased incidence of death in the very elderly.
The elderly population for this study was drawn from the Niigata Study. Briefly, the Niigata Study is a prospective community-based study initiated in 1998 to evaluate the relationships between an individual’s diseases. Questionnaires were initially sent to all inhabitants (n = 4542) age 70 years based on a registry of residents in Niigata City, Japan; all recipients were informed of the purpose of this survey. Among the elderly subjects participating in the Niigata study (n = 600), 367 individuals were 79 years in 2007 and 80 years in 2008 and were involved in the study. We randomly selected 253 independent elderly subjects (122 female; 131 male) age 79–80 years old; this group was limited because our assay to measure the characteristics of CD56+CD16+ cells in a FACS analysis had a maximum capacity of 40 samples per experimental day. As controls, young people (n = 35) age 18–22 years old were randomly selected; and were students attending Kanoya University and Chiba College of Health Sciences. When more than 40 subjects presented on a sampling day, the first 40 subject’s blood was extracted and their NK cells observed. The participants agreed to have medical and dental examinations, and physical fitness tests; and signed an informed consent form showing the protocol was approved by the Ethics Committees of Niigata University Graduate School of Medical Dental Science, National Institute of Infectious Diseases, National Institute of Health Science, and Chiba College of Health Science. Follow-up surveys were performed every year during June using the same methods as in the initial survey. Body height and weight were recorded and the body mass index (BMI) was calculated in all elderly subjects. All subjects were in good general health and did not require special care. Data for males were similar to data for females and, therefore, mixed data from males and females were analyzed to determine the correlations in physical fitness, immunological parameters and biochemical parameters.
Physical fitness parameters
We conducted a medical examination followed by six physical fitness tests: maximum hand grip strength (Rantanen et al. 1998; Sonn et al. 1995), maximum unilateral and bilateral knee extensor strength (Rantanen et al. 1999; Sonn et al. 1995), maximum stepping rate for 10 s (Shindo et al. 1987), one-leg standing time with eyes open (Michikawa et al. 2009; Haga et al. 1986; Stones and Kozma 1987), and a 10-m maximum walking speed test (Shinkai et al. 2000). The maximum hand grip strength in both the dominant and non-dominant hand was measured using a Smedley hand dynamometer (DM-100s, Yagami, Inc., Nagoya, Japan). The score used was the best of the trials between both grip strengths. The equipment for the isometric leg extension and hand grip strength tests were determined and calibrated before beginning the test. The maximum unilateral and bilateral isometric knee extensor strength was determined using a portable chair incorporating a dynamometer connected to a load cell. The subject sat on a seat in a vertical position that was adjusted so that he or she sat comfortably with legs hanging vertically and the knees bent at 90°. The measurement of the maximum unilateral knee extension strength was performed twice on the right and left legs alternately, and thereafter the measurement of the maximum bilateral knee extension strength was performed once. The rest interval between contractions of the leg muscles was more than 2 min. The maximum step rate for 10 s was used as an index of agility using an industrial step rate counter (Stepping Counter, Yagami Inc). The subject was instructed to step as fast as possible for 10 s with each leg alternately while in a sitting position. The stepping rate for the left and right leg was summed for this analysis. The one-leg standing time with eyes open was measured. The static balance function was measured with the subject’s eyes open and arms out, standing on one foot with the other off the floor. The score was either the number of seconds when the non-preferred foot was raised and balance was lost (when the subject began to hop around or when the raised foot was lowered to the floor) or when 2 min had elapsed. The subjects performed one trial on each foot and the best score was recorded. The maximum walking speed was measured having the subjects walk at their fastest pace over a 10-m course. The fastest walking speed was used.
Lymphocyte separation and CD56 and CD16 cell staining
Whole blood (5–7 ml) was collected from subjects in sodium heparin tubes (10 ml VENOJECT; Terumo, Japan) between 10:00 A.M. and 1:00 P.M., before lunch and after resting from recent physical activity. The blood was sent immediately to the National Institute of Infectious Diseases, diluted with an equal volume of Hanks’ Balanced Salt Solution (HBSS) (Gibco Laboratories, Life Technologies; Paisley, UK); then, layered on a Ficoll-Conray density gradient separation solution (Lymphosepar I, Immuno-Biological Laboratories, Gunma, Japan); and centrifuged at 570×g for 30 min at room temperature. The peripheral blood mononuclear cell (PBMC) layer was removed and washed twice in HBSS. The PBMCs were stained for markers using PC5-conjugated CD56 and fluorescence isothiocyanate (FITC)-conjugated CD16 mAbs to observe natural NK cells. FITC-conjugated anti-human CD16 (clone 3G8) monoclonal antibodies (mAbs) were purchased from BD PharMingen (San Diego, CA). Phycoerythrin-cyanine 5 (PC5)-conjugated anti-human CD56 and FITC and PC5-conjugated mouse immunoglobulin G1 (IgG1) (clone 679.1Mc7) were purchased from Immunotech (Marseille, France). Separating NK and NKT cells was not performed to reduce the numbers of antibodies for FACS analysis (CD3 expression was not evaluated). For each subject, PC5- and FITC-conjugated IgG1 isotype controls were used. The PBMCs (1 × 105) aliquots were placed in v-bottomed 96-well plates (Coster, Cambridge, MA); incubated with 20 μl mAb in 1 % bovine serum albumin for 60 min at 4 °C; washed three times with 150 μl of HBSS; fixed with 150 μl of 5 % formalin/PBS solution; and stored at 4 °C until flow cytometry analysis was performed. The percentage of FITC- and PC5-positive cells was measured using a FACSCalibur flow cytometer (Becton–Dickinson; Franklin Lakes, NJ); and the data were analyzed using CellQuest Pro software (Becton–Dickinson). Lymphocytes were defined and separately gated on the basis of forward light scatter (FSC) and side light scatter (SSC) for further analysis. Furthermore, the proportions of the major subsets of cells stained by antibodies were determined using quadrant analysis and gating of individual area on the basis of the first-gated lymphocytes.
Two types of cell groups (CD56dimCD16high cells and CD56dimCD16low cells) were found inside two individual squares in upper and right square using quadrant analysis (cells at either higher or lower than the median fluorescence intensity and the same intensity level as the CD16-positive population on the CD56dim cells) (Fig. 1a–c). Further, the two types of cell groups (CD56dimCD16− cells and CD56brightCD16− cells) were also found inside two individual squares in upper and left square (Fig. 1a–c). The dots in the left square show the numbers of CD56dimCD16− cells and CD56brightCD16− cells, which may be the less mature NK and NKT subpopulations; the dots in the right squares show the numbers of CD56dimCD16bright cells, which may be the mature NK and NKT subpopulations in each subject (A–C). Variable levels of CD16 cells with CD56dim or CD56high expressions were found in the elderly subjects. The proportions of CD56+CD16+ cells, CD56dimCD16high cells, CD56dimCD16low cells, CD56dimCD16− cells or CD56brightCD16− cells within the gated lymphocytes in guardant analysis (CD56+CD16+/lymphocytes, CD56dimCD16high/lymphocytes, CD56dimCD16low/lymphocytes, CD56dimCD16−/lymphocytes or CD56brightCD16−/lymphocytes) were analyzed for each subjects using CellQuest Pro software. The proportions of CD56dimCD16high cells or CD56dimCD16low cells within the gated CD56+CD16+ cells in guardant analysis (CD56dimCD16high/CD56+CD16+ or CD56dimCD16low/CD56+CD16+) were calculated for each subject by division between proportions of CD56dimCD16high/lymphocytes or CD56dimCD16low/lymphocytes and CD56+CD16+/lymphocytes. The ratio of CD56dimCD16high cells and CD56dimCD16− cells (CD56dimCD16high/CD56dimCD16−) was calculated for each subject by division between proportions of CD56dimCD16high/lymphocytes and CD56dimCD16−/lymphocytes.
Health follow-up study
After the medical examination in 2007 and 2008, a questionnaire survey by telephone every year during 5 years following the initial tests was performed to determine whether the elderly subjects were alive or had died.
All data were analyzed using the Windows Statistic Package for SPSS (version 100, Chicago, IL). Correlations between variables (cell types of NK cells and physical fitness parameters) were tested using the Spearman’s rank correlation. Body weight contributes to muscle force. Investigators suggested that the muscle force output, when measured with a dynamometer, might better reflect a force-generation impairment when normalized against body weight (Bohannon 2007; Eriksrud and Bohannon 2003). Therefore, the normalization by body weight was performed in every physical fitness data. To analyze the association of bilateral maximum knee extensor strength/weight (kg) compared to NK cell maturation, logistic regression models were used. The dependent variables consisted of the proportions of CD56dimCD16high cells, CD56dimCD16− cells, and CD56highCD16− cells among the total CD56+CD16+ cells or lymphocytes. The BMI, smoking habits, drinking habits and medical history of each disease (cancer, diabetic mellitus, hypertension, cardiovascular diseases) and the bilateral maximum knee extensor strength/weight were used as independent variables for the analysis. A P value of 0.05 or less was considered statistically significant.
Body height, weight, and BMI were 158.0 ± 3.4 and 147.9 ± 5.5 cm, 59.7 ± 17.1 and 48.9 ± 7.3 kg, and 23.8 ± 5.3 and 22.4 ± 3.0 kg/m2 in males and females, respectively. The data are at normal levels for healthy, elderly Japanese people. No detected information about drug use or diet was available, which may have biased the results through residual confounding. It seems unlikely, however, that these factors would account for all of the associations observed for knee strength, CD56+CD16+ cells and mortality.
Correlation comparison for cell expression of CD56 and CD16 on lymphocytes and physical fitness
We correlated the relationship between the four types of cell subpopulations in Fig. 1 and physical fitness. In summary, all correlation data and statistical analyses showed a larger proportion of CD56dimCD16high/CD56+CD16+ cells having a significant positive correlation to the maximum right knee extensor strength/body weight (kg), the maximum bilateral knee extensor strength (kg) and the strength (kg)/body weight (r = 0.165, P = 0.027, 0.420 and 0.425, P < 0.0001) (Table 1). However, the proportion did not correlate with any other parameters. The proportion of CD56dimCD16high/lymphocytes and CD56+CD16+/lymphocytes correlated positively with the maximum hand grip strength (r = 0.179 and 0.167; P = 0.004 and 0.008, respectively) but other parameters did not. The proportion of CD56dimCD16−/lymphocyte population did not show significant correlations with any parameters. However, the ratio of CD56dimCD16high/CD56dimCD16− cells correlated positively with the maximum bilateral knee extensor strength (kg) and strength (kg)/body weight (r = 0.317 and 0.323; P < 0.0001 and 0.0001, respectively) but did not correlate with the other parameters. The proportion of CD56brightCD16−/lymphocytes correlated negatively with the maximum bilateral knee extensor strength (kg) and strength (kg)/body weight (kg) (r = −0.290, P < 0.0001 and r = −0.256, P = 0.0004). The data normalized by body weight in physical fitness parameters excepting for maximum knee extensor strength did not correlate with various parameters of CD56+CD16+/− cells. Other parameters for physical fitness, BMI, smoking habits, diabetes mellitus, hypertension, and cardiovascular disease did not show significant relationships with the various cell parameters. To analyze the association of maximum bilateral knee extensor strength/body weight with the proportion of CD56dimCD16high/CD56+CD16+ cells after adjustment with other confounding factors, logistic regression analyses were performed. Two logistic regression models showed maximum bilateral knee extensor strength/body weight to be independently associated with the proportion of CD56dimCD16high/CD56+CD16+ cells in both males and females (Table 2). The coefficient of variation for the unilateral knee extension strength on the right and left legs were respectively, 35.9 and 37.2 %, in the total elderly population. There was no significant difference between right and left leg strength. The proportion of CD56dimCD16high/CD56+CD16+ cells is useful as an indicator for the level of bilateral lower-muscle strength in all of the males and females.
Correlation comparison of expression of CD56 and CD16 on lymphocytes and physical fitness to mortality
Physical fitness has positive effects on the health of the elderly. It is therefore interesting to identify the relationship between physical fitness and NK cell parameters related to mortality. Elderly subjects were phoned yearly for 5 years to determine if they were alive following the medical and dental examinations in 2007 and 2008. Twenty seven elderly people (4 female and 23 male) died during the 5 years after the primary medical examination. The causes of death were 55.6 % (15/27) cancer (lung, gall bladder, stomach, colon, pancreas and prostatic), 0.7 % (2/27) pneumonia, 0.7 % (2/27) renal insufficiency, 0.4 % (1/27) malignant sarcoma, 0.4 % (1/27) cerebral hemorrhage, 0.4 % (1/27) heart failure, 0.4 % (1/27) emphysema, 0.4 % (1/27) acute coronary syndrome and 0.4 % (1/27) ureteral tumor. To determine if there was an association of death with NK cell surface marker or physical fitness, the proportion of the various types of cells and levels of physical fitness were compared between dead and alive groups.
The proportion of CD56brightCD16−/lymphocytes (0.177 ± 0.121 %) was significantly lower in the dead elderly compared to the living elderly (0.393 ± 0.345 %) but there were no significant differences in other cell types between the dead and living elderly. Therefore, it was considered that proportion of CD56brightCD16−/lymphocytes is a predictor for the risk of death. The correlation between CD56brightCD16−/lymphocytes and maximum bilateral knee extensor strength/body weight was investigated in the total female and male population. The proportion of CD56brghtCD16−/lymphocytes had a significant negative correlation to the maximum bilateral knee extensor strength (kg)/body weight in all of the elderly (r = −0.256, P = 0.0004) (Fig. 2). The negative correlation was higher in females (r = −0.319, P = 0.004) and lower in males (r = −0.200, P = 0.036) (Fig. 2). The proportion of CD56brightCD16−/lymphocytes was associated inversely with the maximum bilateral knee extensor strength (kg)/body weight and was stronger in males than females. Red points were individually plotted as a dead elderly male or female (Fig. 2b, c). These red points were located less than 0.5 in the proportion of CD56brightCD16−/lymphocytes but there were no tendencies with the maximum bilateral knee extensor strength (kg)/body weight in these red points in male (Fig. 2b). In contrast, the red points were few (only 3), located less than 0.5 in the proportion of CD56brightCD16−/lymphocytes and more than 1.2 in the maximum bilateral knee extensor strength (kg)/body weight in females (Fig. 2c). Both averages of the proportion of CD56brightCD16−/lymphocytes and the maximum bilateral knee extensor strength (kg)/body weight were higher in young people than those in the elderly overall (Fig. 2a, d). There were no significant correlation (r = 0.107, P = 0.5430) between the proportion of CD56brightCD16−/lymphocytes and the maximum bilateral knee extensor strength (kg)/body weight in young people (Fig. 2d). Negative correlation between CD56brightCD16−/lymphocytes and the extensor strength was not observed in the young people who the proportion of CD56brightCD16−/lymphocytes (0.59 ± 0.43 %) was significantly higher in young people than those (0.37 ± 0.43 %) in elderly (P = 0.003).
Generally, people with mobility impairments tend to have a high morbidity and mortality rate that is caused by the deterioration of lower and upper skeletal muscle strength from reduced walking, eating and using the toilet (Yoshida et al. 2010). However, little is known about the interaction between the systemic increased mortality and physical fitness, especially regarding NK cells associated with protection against infection and tumor cell control in the very elderly.
Here, we found that CD56dimCD16high cells included the mature NK and NKT cells were significant positive cellular immune indicators when compared to knee extensor and hand grip strength in very elderly subjects. The proportion of CD56dimCD16high in CD56+CD16+ cells and the ratio of CD56dimCD16high and CD56dimCD16− cells correlated positively with the maximum bilateral knee extensor strength and maximum bilateral knee extensor strength/body weight. Logistic regression analysis showed the maximum bilateral knee extension strength/body weight was independently associated with the percentage of CD56dimCD16high cells within the total CD56+CD16+ cells in males and females (Table 2). Various expression levels of CD16 were found on CD56 cells in the elderly. Reports show during maturation of the CD56bright (immature cells) into CD3−CD56dimCD16+ NK cells (mature cells), the expression of NKp46, NKG2D, Nkp30, and NKG2A decreases and CD16 expression is acquired (Moretta et al. 2002). Thus, the amount of CD16 expression on the cell surface correlates with the level of maturation of the NK cell. CD3+CD56+CD16+ NKT cells also have cytotoxic activity. The CD56dimCD16high cells have higher expression levels of CD16, which may be useful indicators of the maturation of the NK and NKT subpopulation cells in the elderly.
There are reports concerning the relationship between NK cell activity and physical activity. Kusaka et al. report that higher NK activity was found in individuals reporting beneficial life style practices who exercise once or more a week as a leisure-time activity (Kusaka et al. 1992). Further, moderate aerobic exercise training significantly increased NK cell activity in the elderly (Yan et al. 2001). This suggests that habitual moderate daily physical activity is beneficial for maintaining NK cell activity in older people who live independently (Senchina and Kohut 2007). Other reports show that daily physical activity is associated with muscle strength in very old people (Yoshida et al. 2010). This suggests that daily physical activity may be a factor contributing to the maintenance and/or increase of muscle strength in the very elderly. These studies partially support the association of NK cells with lower- and upper-limbs muscle strength observed in our present study, although CD16 expression-related NK cell activity was not associated with all muscle strength parameters. In particular, lower-muscle bilateral leg strengths are required for the relationship with the NK and NKT cell populations, but this association is not seen in other parameters such as one-leg standing time with eyes open, maximum stepping rate for 10 s and 10-m maximum walking speed test, etc. The bilateral knee extension strength is necessary to standing, which is one of physical activities in batteries of physical functioning (Eriksrud and Bohannon 2003). The combination and balance of right and left knee strength may be appropriate for physical activity resulting in the induction of differentiated NK and NKT cells.
In the correlation, NK parameters of immunity and muscle strength, the influence of immunity on the muscle strength is not considered because there are no significant data or reports on this thesis research. In contrast, the influence of the muscle strength on immunity is considered because that is supported by many reports. Therefore, the level of bilateral lower-muscle strength may be one factor that affects the proportion of NK and NKT cell such as CD56dimCD16− cells and CD56dimCD16high cells. This suggests that elderly people having reduced physical function, such as the bilateral knee extensor strength in the legs, tend to be susceptible to immunity-related diseases because they have less-mature NK and NKT cells (Renna et al. 2012; Small et al. 2008).
Cytolytic NK cells may control infected and tumor cells from infiltrating secondary lymphoid organs (Ferlazzo et al. 2004). The NK cells in secondary lymphoid organs can play a major role at enhancing patient resistance against malignancies (Ferlazzo and Münz 2004). Remagnan et al. suggest that CD56brightCD16− and CD56dimCD16+KIR;/− NK cells correspond to sequential steps of differentiation and support the hypothesis that secondary lymphoid organs may be sites for final NK cell maturation (Romagnani et al. 2007). Proportion of CD56brightCD16− cells within lymphocytes associated negatively with the incidence of death. However, these cells lack perforin and killer cell Ig-like receptors (KIRs) and exist mainly in lymph nodes (Ferlazzo et al. 2004). In contrast, proportion of CD56dimCD16high cells within lymphocytes did not significantly correlate with the incidence of death. CD56dimCD16+ cells express perforin, the natural cytotoxity receptor (NCRs) Nkp30 and Nkp46, as well as in-part KIRs (Ferlazzo et al. 2004; Jacobs et al. 2001; Romagnani et al. 2007). More than 95 % of the peripheral blood and 85 % of the spleen NK cells are CD56dimCD16+ cells (Ferlazzo et al. 2004). Exercise-induced increase in NK cells cytotoxicity is largely due to an increase in the absolute numbers and percentage of circulating blood NK cells (Walsh et al. 2011). IL-2 is required to induce natural cytotoxicity in these abundant NK cells. Cytokine-induced activation by IL-2 should be sufficient to induce natural cytotoxicity in these abundant NK cells harbored in solid tissues (Ferlazzo et al. 2004; Romagnani et al. 2007). On IL-2 secretion by T cells, NK cells up-regulate activating and inhibitory receptors as well as perforin and acquire the ability to kill NK-sensitive targets. However, subsequent NK cell activation by IL-2 stimulation was significantly impaired (Millard et al. 2013). Therefore, NK cells mobilized into the circulation in response to exercise have altered sensitivity to IL-2 stimulation (Millard et al. 2013). The circulating CD56dimCD16+ cells may also be altered and impaired in the subsequent activation by IL-2, and lose killing virus-infected and cancer cells. In contrast, CD56brightCD16− cells can convert in CD56dimCD16+ cells able to kill virus-infected and cancer cells (Ferlazzo et al. 2004). Circulating CD56brightCD16− cells may provide fresh cytotoxic cells into the tissues infected by viruses or tumors. Several reports demonstrate that antiviral treatment with interferon-α (IFN-α) increased the proportions of the CD56bright subset, while those of the CD56dim subset declined slightly (Fathy et al. 2010; Lee et al. 2010). They concluded that this may have advantages for patients because an increased secretion of IFNγ by the CD56bright subset has an antiviral effect. Yamagiwa et al. suggests that a significant imbalance between CD56bright and CD56dim NK cell subsets in the liver may contribute to the progression of recurrent chronic hepatitis C after liver transplantation (Yamagiwa et al. 2014). Therefore, the differentiation from CD56brightCD16− cells to CD56dimCD16high cells may be responsible for the infected and tumor cells infiltrating secondary lymphoid organs and may be a key event to prevent spread of the infected and tumor cells in the very elderly. Taken together, the proportion of CD56brightC16− cells/lymphocyte was found to be an important indicator of the incidence of death rather than CD56dimCD16high/CD56+CD16+ cells associated with the bilateral knee extensor strength in the legs in the very elderly.
Thus, bilateral knee extensor strength can predict differentiated NK and NKT cells to active cells forms. The susceptibility of the knee strength to the differentiation is higher in the elderly than that in young people. However, the activation by the knee strength did not associate with the mortality risk. The knee strength tends to decrease in CD56brightCD16− cells/lymphocytes relating with incidence of death. Therefore, undifferentiated NK and NKT cells such as CD56brightCD16− cells may be important to correspond to death by cancer, infection and inflammation, or be a usual indicator for mortality. Elderly should avoid consumption of undifferentiated NK and NKT cells by excessive exercise because its number is limited in elderly.
The level of bilateral lower-muscle strength may be a factor that affects the proportion of NK and NKT cell such as CD56dimCD16− and CD56dimCD16high cells. The proportion of CD56brightC16− cells within lymphocyte in peripheral blood is a predictor of incidence of death and correlated negatively with bilateral lower-muscle strength in the very elderly. Measurements of physical fitness, the proportion of CD56dimCD16high cells in CD56+CD16+ cells, the ratio of CD56dimCD56high and CD56dimCD16− cells, and the proportion of CD56brightC16− cells in lymphocytes are important indicators to check elderly health.
André P, Spertioni O, Guia S, Rihet P, Dignat-George F, Brailly H, Sampol J, Anderson PJ, Vivier E (2000) Modification of P-selectin glycoprotein ligand-1 with a natural killer cell-restricted sulfated lactosamine creates an alternate ligand for L-selectin. Proc Natl Acad Sci USA 97:3400–3405
Biron CA, Nguyen KB, Pien GC, Cousens LP, Salazar-Mather TP (1999) Natural killer cells in antiviral defense: function and regulation by innate cytokine. Annu Rev Immunol 17:189–220
Blair SN, Brodney S (1999) Effects of physical inactivity and obesity on morbidity and mortality: current evidence and research issues. Med Sci Sports Exerc 31:S646–S662
Blair SN, Chexng Y, Holder JS (2001) Is physical activity or physical fitness more important in defining health benefits? Med Sci Sports Exerc 33:S379–S399
Bohannon RW (2007) Knee extension strength and body weight determine sit-to-stand independence after stroke. Physiother Theory Pract 23:291–297
Brown JC, Huedo-Medina TB, Pescatello LS, Ryan SM, Pescatello SM, Moker E, LoCroix JM, Ferrer RA, Johnson BT (2012) The efficacy of exercise in reducing depressive symptom among cancer survivors: a meta-analysis. PLoS One 7:230955
Chodzko-Zajko WJ, Proctor DN, Fiatarone Singh MA, Minson CT, Nigg CR, Salem GJ, Slinner JS (2009) American College of Sports Medicine position stand. Exercise and physical activity for older adults. Med Sci Sports Exerc 41:1510–1530
Chubak J, McTiernan A, Sorensen B, Wener NH, Yasui Y, Velasquez M, Wood B, Rajan KB, Wetmore CM, Potter JD, Ulrich CM (2006) Moderate-intensity exercise reduces the incidence of colds among postmenopausal women. Am J Med 119:937–947
Cooper MA, Fehniger TA, Caligiuri MA (2001a) The biology of human natural killer-cell subsets. Trends Immunol 22:633–640
Cooper MA, Fehniger TA, Turner SC, Chen KS, Ghaheri BA, Ghayur T, Carson WE, Caligiuri MA (2001b) Human natural killer cells: a unique innate immune-regulatory role for the CD56brught subset. Blood 97:3146–3151
Dimeo FC, Thomas F, Raabe-Menssen C, Pröpper F, Mathias M (2004) Effect of aerobic exercise and relaxation training on fatigue and physical performance of cancer patients after surgery. A randomised controlled trial. Support Care Cancer 12:774–779
Dulphy N, Haas P, Busson M, Belhadj S, Peffault de Latour R, Robin M, Carmagnat M, Loiseau P, Tamouza R, Scieux C, Rabian C, Di Santo JP, Charron D, Janin A, Socié G, Toubert A (2008) An unusual CD56brightCD16low NK cell subset dominates the early posttransplant period following HLA-Matched hematopoietic stem cell transplantation. J Immunol 181:2227–2237
Eickmeyer SM, Gamble GL, Shahpar S, Do KD (2012) The role and efficacy of exercise in persons with cancer. PM R 4:874–881
Eriksrud O, Bohannon RW (2003) Relationship of knee extension force to independence in Sit-to-Stand performance in patients receiving acute rehabilitation. Phys Ther 83:544–551
Erikssen G (2001) Physical fitness and changes in mortality: the survival of the fittest. Sports Med 31:571–576
Fahlman MM, Boardley D, Flynn MG, Braun WA, Lambert CP, Bouillon LE (2000) Effects of endurance training on selected parameters of immune function in elderly women. Gerontology 46:97–104
Fathy A, Eldin MM, Metwally L, Eida M, Abdel-Rehim M, Esmat G (2010) Interferon therapy shifts natural killer subsets among Egyptian patients with chronic hepatitis C. Braz J Infect Dis 14:398–405
Ferlazzo G, Münz C (2004) NK cell compartments and their activation by dendritic cells. J Immunol 172:1333–1339
Ferlazzo G, Thomas D, Lin S-L, Goodman K, Morandi B, Muller WA, Moretta A, Münz C (2004) The abundant NK cells in human secondary lymphoid tissue require activation to express killer cell Ig-like receptors and become cytolytic. J Immunol 172:1455–1462
Forner MA, Collazos ME, Barriga C, De la Fuente M, Rodriguez AB, Ortega E (1994) Effect of age on adherence and chemotaxis capacities of peritoneal macrophages. Influence of physical activity stress. Mech Ageing Dev 75:179–189
Golden-Mason L, Castelblanco N, O’Farrelly C, Rosen HR (2007) Phenotypic and functional changes of cytotoxic CD56pos natural T cells determine outcome of acute hepatitis C virus infection. J Virol 81:9292–9298
Haga H, Shibata H, Shibata K, Matsuzaki T, Hatano S (1986) Falls in the institutionalized elderly in Japan. Arch Gerontol Geriatr 5:1–9
Herberman RB, Nunn ME, Holden HT, Lavrin DH (1975) Natural cytotoxic reactivity of mouse lymphoid cells against syngeneic and allogenic tumors II. Characterization of effector cells. Int J Cancer 16:230–239
Imai K, Matsuyama S, Miyake S, Suga K, Nakachi K (2000) Natural cytotoxic activity of peripheral-blood lymphocytes and cancer incidence: an 11-year follow-up study of a general population. Lancet 356:1795–1799
Jacobs R, Hintzen G, Kemper A, Beul K, Kempf S, Behrens G, Synkora K-W, Schmidt RE (2001) CD56bright cells differ in their KIR repertoire and cytotoxic features from CD56dim NK cells. Eur J Immunol 31:3121
Kiessling R, Klein E, Pross H, Wigzell H (1975) “Natural” killer cells in the mouse. II. Cytotoxic cells with specificity for mouse Moloney leukemia cells, characteristics of the killer cell. Eur J Immunol 5:117–121
Kohl HW III (2001) Physical activity and cardiovascular disease: evidence for a dose response. Med Sci Sports Exerc 33:S472–S483
Kusaka Y, Kondou H, Morimoto K (1992) Healthy lifestyles are associated with higher natural killer cell activity. Prev Med 21:602–615
Lambert C, Cristina I, Christian G (2006) Enumeration of peripheral lymphocyte subsets using 6 vs. 4 color staining: a clinical evaluation of a new flowcytometer. Cytom B Clin Cytom 70:29–38
Lanier LL (2008) Evolutionary struggles between NK cells and viruses. Nat Rev Immunol 8:259–268
Lanier LL, Phillips JH, Hackett J Jr, Tutt M, Kumar V (1986) Natural killer cells: definition of a cell type rather than a function. J Immunol 137:2735–2739
Lee S, Watson MW, Flexman JP, Cheng W, Hammond T, Price P (2010) Increased proportion of the CD56 (bright) NK cell subset in patients chronically infected with hepatitis C virus (HCV) receiving interferon-alpha and ribavirin therapy. J Med Virol 82:568–574
Mercer JC, Ragin MJ, August A (2005) Natural killer T cells: rapid responders controlling immunity and disease. Int J Biochem Cell Biol 37:1337–1343
Michikawa T, Nishiwaki Y, Takebayashi T, Toyama Y (2009) One-leg standing test for elderly populations. J Orhop Sci 14:675–685
Millard AL, Valli PV, Stussi G, Mueller N, Yung GP, Seebach JD (2013) Brief exercise increases peripheral blood NK cell counts without immediate functional changes, but impairs their responses to ex vivo stimulation. Front Immunol 4:125
Moreira A, Delgado L, Moreira P, Haahtela T (2009) Does exercise increase the risk of upper respiratory tract infections? Br Med Bull 20:111–131
Moretta A, Bottino C, Mingari MC, Biassoni R, Moreleta L (2002) What is a natural killer cell? Nat Immunol 3:6–8
Myers J, Kaykha A, George S, Abella J, Zaheer N, Lear S, Yamazaki T, Froelicher V (2004) Fitness versus physical activity patterns in predicting mortality in men. Am J Med 117:912–918
Nagler A, Lanier LL, Cwirla S, Phillips JH (1989) Comparative studies of human FcR III—positive and negative natural killer cells. J Immunol 143:3183–3191
Nieman DC (2000) Exercise immunology: future directions for research related athletes, nutrition, and the elderly. Int J Sports Med 21:S61–S68
Nieman DC, Buckley KS, Hensen DA, Warren BJ, Suffles J, Ahle JC, Sinmandle S, Fagoaga OR, Nehlsen-Cannarella S (1995) Immune function in marathon runners versus sedentary controls. Med Sci Sports Exerc 27:986–992
Park SH, Kyin T, Bendelac A, Carnaud C (2003) The contribution of NKT cells, NK cells and other gamma-chain-dependent non-T non-B cells to IL-12-mediated rejection of tumors. J Immunol 170:1197–1201
Rantanen T, Masaki K, Foley O, Izmirlian G, White L, Guralnik JM (1998) Grip strength changes over 27 yr in Japanese-American men. J Appl Physiol 85:2047–2053
Rantanen T, Guralnik JM, Sakari-Rantala R, Leveille S, Simonsick EM, Ling S, Fried LP (1999) Disability, physical activity, and muscle strength in older women: the Women’s Health and Aging Study. Arch Phys Med Rehabil 80:130–135
Renna MS, Figueredo CM, Rodriguez-Galan MC, Icely PA, Peralta Ramos JM, Correa SG, Sotomayer CE (2012) Abrogation of spontaneous liver tolerance during immune response to Candida albicans: contribution of NKT and hepatic mononuclear cells. Int Immunol 24:315–325
Romagnani C, Juelke K, Falco M, Morandi B, D’Agostino A, Costa R, Ratto G, Forte G, Carrega P, Lui G, Conte R, Strowig T, Moretta A, Münz C, Thiel A, Moretta L, Ferlazzo G (2007) CD56brightCD16− killer Ig-like receptor NK cells display longer telomeres and acquire features of CD56dim NK cells upon activation. J Immunol 178:4947–4955
Scanga CB, Verde TJ, Paolona AM, Andersen RE, Wadden TA (1998) Effects of weight loss and exercise training on natural killer cell activity in obese women. Med Sci Sports Exerc 30:1666–1671
Senchina DS, Kohut ML (2007) Immunological outcomes of exercise in older adults. Clin Interv Aging 2:3–16
Shindo M, Kumagai S, Tanaka H (1987) Physical work capacity and effect of endurance training in visually handicapped boys and young male adults. Eur Appl Physiol Occup Physiol 56:561–567
Shinkai S, Watanabe S, Kumagai S, Fujiwara Y, Amano H, Yoshida H, Ishizaki T, Yukawa H, Suzuki T, Shibata H (2000) Walking speed as a good predictor for the onset of functional dependence in a Japanese rural community population. Age Ageing 29:441–446
Small C-L, McCormick S, Gill N, Kugathasan K, Santosuosso M, Donaldson N, Heinrichs DF, Ashkar A, Xing Z (2008) NK cells play a critical protective role in host defense against acute extracellular Staphylococcus aureus bacterial infection in the lung. J Immunol 180:5558–5568
Sonn U, Frandin K, Grimby G (1995) Instrumental activities of daily living related to impairments and functional limitations in 70-year-olds and changes between 70 and 76 years of age. Scand J Rehabil Med 27:119–128
Stones MJ, Kozma A (1987) Balance and age in the sighted and blind. Arch Phys Med Rehabil 68:85–89
Subleski JJ, Hall VL, Back TC, Orlaldo JR, Wiltrout RH (2006) Enhanced antitumor response by divergent modulation of natural killer and natural killer T cells in the liver. Cancer Res 66:11005–11012
Takata Y, Ansai T, Akifusa S, Soh I, Yoshitake Y, Kimura K, Sonoki K, Fujisawa K, Awano S, Kagiyama S, Hamasaki T, Nakamichi I, Yoshida A, Takehara T (2007) Physical fitness and 4-year mortality in an 80-year-old population. J Gerontol A Biol Sci Med Sci 62:851–858
Tarazona T, DelaRosa O, Alonso C, Ostos B, Espejo J, Pefna J, Solana R (2000) Increased expression of NK cell markers on T lymphocytes in aging and chronic activation of the immune system reflects the accumulation of effector/senescent T cells. Mech Ageing Dev 121:77–88
Waldhouer I, Steinle A (2008) NK cells and cancer immunosurveillance. Oncogene 27:5932–5943
Walsh NP, Gleeson M, Shephard RJ, Gleeson M, Woods JA, Bishop NC, Fleshner M, Green C, Pedersen BK, Hoffman-Goetz L, Rogers CJ, Northoff H, Abbasi A, Simon P (2011) Position statement. Part one: immune function and exercise. Exerc Immunol Rev 17:6–63
Williams PT (2001) Physical fitness and activity as separate heart disease risk factors: a meta-analysis. Med Sci Sports Exerc 33:754–761
Yamagiwa S, Sato Y, Ichida T, Setsu T, Tominaga K, Kamimura H, Tsuchiya A, Takamura M, Matsuda Y, Aoyagi Y (2014) Inbalance between CD56+bright and CD56+dim matural killer cell subsets in the liver of patients with recurrent repatitis C after liver transplantation. Biomed Res 15:177–184
Yan H, Kuroiwa A, Tanaka H, Shindo M, Kiyonaga A, Nagayama A (2001) Effect of moderate exercise on immune senescence in men. Eur J Appl Physiol 86:105–111
Yoneyama T, Yoshida M, Ohrui T, Mukaiyama H, Okamoto H, Hoshiba K, Ihara S, Yanagisawa S, Ariumi S, Morita T, Mizuno Y, Ohsawa T, Akagawa Y, Hashimoto K, Sasaki H, Oral Care Working Group (2002) Oral care reduces pneumonia in older patients in nursing homes. J Am Geriatr Soc 50:430–433
Yoshida D, Nakagaichi M, Saito K, Wakui S, Yoshitake Y (2010) The relationship between physical fitness and ambulatory activity in very elderly women with normal functioning and functional limitations. J Physiol Anthropol 29:211–218
HS designed the protocol of all experiments, participated in experiments to analyze population of NK cells and described manuscript. HM coordinated the health system of the elderly and carried out to get approval of an ethics panel. AY coordinated the health system of elderly and participated in blood tests. SY participated in experiments to analyze population of NK cells. NN participated in experiments to analyze population of NK cells. TK participated in experiments to analyze population of NK cells. NN performed the measurement of physical fitness. MM coordinated the health system of the elderly and performed acquisition of approval from the ethics panel. AT performed the statistical analysis. GY performed the measurement of physical fitness. MS performed the measurement of physical fitness and to get approval of an ethics panel. MO coordinated the health system of elderly. MN performed the measurement of physical fitness and health check. YK carried out measurement of physical fitness. YY conceived of the study, participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.
This work was supported in-part by a Grant from the Ministry of Health, Labor and Welfare (H20—Circulatory organ (teeth)—General—002). This work was also supported in-part by a Grant-in-Aid for Scientific Research from the National Institute of Fitness and Sports in Kanoya (President’s Discretional Budget to Y. Yoshitake).
The authors declare that they have no competing interests.
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Senpuku, H., Miyazaki, H., Yoshihara, A. et al. CD56dimCD16high and CD56brightCD16− cell percentages associated with maximum knee extensor strength and incidence of death in elderly. SpringerPlus 5, 244 (2016). https://doi.org/10.1186/s40064-016-1884-3
- Physical fitness
- Maximum bilateral knee extensor strength
- NK cell
- CD56dimCD16high cell
- CD56brightCD16− cell