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Association of White Blood Cell Subfraction Concentration with Fitness and Fatness
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Br J Sports Med. Published Online First: 16 October 2008
Neil M. Johannsen 1, Elisa L. Priest 2, Vishwa D. Dixit 1, Conrad P. Earnest 1, Steven N. Blair 3 and Timothy S. Church 1*
1 Pennington Biomedical Research Center, United States
2 Institute for Health Care Research and Improvement, Baylor Health Care System, United States
3 Arnold School of Public Health, University of South Carolina, United States
"our results are consistent with the hypothesis that increased circulating WBC subsets may be a risk factor for inflammatory disorders."
"Total WBC, neutrophil, lymphocyte, and basophil concentrations were higher in the low-fit-high-fat group compared with both high-fitness groups. Total WBC counts were also higher in the low-fit-low-fat group compared with the high-fit-low-fat group"
Abstract
OBJECTIVE: To examine the association between fitness, BMI, and neutrophil, lymphocyte, monocyte, basophil, and eosinophil concentrations in apparently healthy, nonsmoking men.
DESIGN: Cross-sectional study of 452 men from the Aerobics Center Longitudinal Study examining the resting concentration of white blood cell subfractions across fitness (maximal METS during a treadmill exercise test) and fatness (BMI) categories after adjusting for age.
RESULTS: Fitness was inversely associated with all WBC subfraction concentrations. After further adjusting for BMI, only total WBC, neutrophil, and basophil concentrations remained significantly associated with fitness. BMI was directly associated with total WBC, neutrophil, lymphocyte, monocyte, and basophil concentrations and when fitness was added to the model, only monocytes lost significance.
CONCLUSION: Fitness (inversely) and fatness (directly) are associated with WBC subfraction populations.
"our results are consistent with the hypothesis that increased circulating WBC subsets may be a risk factor for inflammatory disorders."
"fatness is strongly associated with elevated circulating leptin levels. Leptin activates neutrophils40 and induces production of pro-inflammatory cytokines like IL-1b, IL-6 and TNFa"
"several studies demonstrate that habitual exercise is inversely related to both TNF-a and IL-6.42-44 For example, Smith et al found that a 6 month exercise programme reduced TNF-a (n=43, average age=49.0 years) and Taaffe et al reported an inverse relationship between accumulated moderate and strenuous activity with IL-6 in 880 adults aged 70 to 79 years"
"Our findings that both fitness and fatness contribute to total WBC and WBC subfraction concentrations may provide one more mechanism whereby higher levels of fitness and lower levels of fatness reduce the risk of a CVD.... Age-adjusted total WBC and all WBC subfractions were inversely associated with fitness. When BMI was added to the model, only total WBC, neutrophil, and basophil concentrations remained significant
a study of 22 marathon runners and 18 sedentary controls that noted lower neutrophil concentrations amongst the marathoners.16 In our current report, we found an inverse association between fitness and neutrophil and basophil concentrations. Our results suggest that the neutrophil-fitness association may contribute to the total WBC-fitness association seen in our previous report.13..... Atherosclerosis and CVD are generally accepted as being related to an inflammatory process whereby endothelial damage to coronary vessels results in chronic, low-grade inflammation.26 Research indicates that neutrophils may play an important role in endothelial tissue damage because they release cytotoxic factors such as free oxygen radicals and non-specific proteases.27 Neutrophils also release leukotrienes that amplify the inflammatory reaction and contribute to atherosclerotic progression. 28 29 Although the physiological mechanisms whereby higher levels of fitness confer such benefits are not fully understood,30-32 it is reasonable to hypothesise that any mechanism that reduces the availability of neutrophils may retard the atherosclerotic process. This and other previous reports demonstrate that moderate to high levels of cardiorespiratory fitness may protect against CVD and all-cause mortality.
CONCLUSIONS
Fitness (inversely) and fatness (directly) are associated with WBC subfraction populations. These findings provide one more potential mechanism whereby higher levels of fitness and lower levels of fatness reduce the risk of CVD.
SHORT SUMMARY
White blood cell (WBC) count is a strong and independent predictor of coronary heart disease morbidity and mortality. While research has determined the effect of fitness and fatness on total WBC count, no studies have determined the relationship between WBC subfraction concentration and fitness and fatness. The data from 452 non-smoking men from the Aerobics Center Longitudinal Study were used to determine the association between WBC subfraction concentration, fitness, and fatness. This manuscript significantly adds to the growing body of literature because we show that WBC subfraction concentrations vary independently by both fitness and fatness in a relatively healthy sample of men.
Funding: This study was supported in part by US Public Health Service research grant AG06945 from the National Institute on Aging and the Simmons Foundation.
Elevated white blood cell (WBC) count is a strong and independent risk factor for coronary heart disease morbidity and mortality.1-10 Total WBC count represents the sum of the WBC subfractions of neutrophils, lymphocytes, monocytes, basophils, and eosinophils. Recent evidence from large epidemiological studies suggests that elevation in neutrophil concentration, or total granulocyte concentration, may be the strongest predictor of coronary heart disease incidence, carotid arteriosclerosis, and death.1 9-12 The associations with greater neutrophil or total granulocyte concentrations are independent of age, past smoking behaviour, sex, and other cardiovascular disease (CVD) risk factors and indicators of inflammation (C-reactive protein).
We previously reported that fitness (inversely) and fatness levels (directly) are associated with total resting WBC count and suggested that this may partially account for the reduction in mortality risk associated with higher levels of fitness and lower fatness.13 However, our previous report did not examine the associations between WBC subfraction concentration and fitness and fatness.
While individual WBC subfraction concentration may vary with physical activity level and body weight status,14-20 the association of WBC subfraction
with both fitness and fatness remains unknown. The purpose of this project was to examine the relationship between cardiorespiratory fitness, fatness, and WBC subfraction concentrations of neutrophils, lymphocytes, monocytes, basophils, and eosinophils in a large group of apparently healthy, non-smoking men.
DISCUSSION
In this study, we found that both fitness (inversely) and fatness (directly) were associated with total and fractionated WBC concentrations in a large, non-smoking, apparently healthy sample of men. Specifically, fitness level was inversely associated with all age-adjusted WBC subfractions but only total WBC, neutrophil, and basophil concentrations remained significantly associated with fitness level after adjustment for BMI. BMI was directly associated with age-adjusted total WBC, neutrophil, lymphocyte, monocyte, and basophil concentrations, and only monocyte concentration was no longer significant after adjustment for fitness. Our findings that both fitness and fatness contribute to total WBC and WBC subfraction concentrations may provide one more mechanism whereby higher levels of fitness and lower levels of fatness reduce the risk of a CVD.1 9-12
Although a paucity of literature exists examining the chronic effects of physical activity on WBC subfraction concentration, several small cross-sectional studies do compare WBC subfraction concentration between athletes and sedentary controls.14-17 Overall, these studies fail to consistently note differences in WBC concentrations between the two groups despite the dichotomy of fitness levels among the participants. The one exception was a study of 22 marathon runners and 18 sedentary controls that noted lower neutrophil concentrations amongst the marathoners.16 In our current report, we found an inverse association between fitness and neutrophil and basophil concentrations. Our results suggest that the neutrophil-fitness association may contribute to the total WBC-fitness association seen in our previous report.13
Atherosclerosis and CVD are generally accepted as being related to an inflammatory process whereby endothelial damage to coronary vessels results in chronic, low-grade inflammation.26 Research indicates that neutrophils may play an important role in endothelial tissue damage because they release cytotoxic factors such as free oxygen radicals and non-specific proteases.27 Neutrophils also release leukotrienes that amplify the inflammatory reaction and contribute to atherosclerotic progression. 28 29 Although the physiological mechanisms whereby higher levels of fitness confer such benefits are not fully understood,30-32 it is reasonable to hypothesise that any mechanism that reduces the availability of neutrophils may retard the atherosclerotic process. This and other previous reports demonstrate that moderate to high levels of cardiorespiratory fitness may protect against CVD and all-cause mortality.
While BMI-associated leucocytosis has been established,18 20 most current research examines the effect of sustained weight loss on chronic subclinical inflammation.19 33-35 A recent study by Dixon and O'Brien showed that not only was higher BMI associated with greater neutrophil and lymphocyte counts, but significant weight loss (average weight loss=29.4 kg) 2 years after Lap-Band surgery resulted in 11.7% and 6.9% reductions in neutrophil and lymphocyte concentrations, respectively (p<0.001 for both).19 These results were independent of preoperative smoking status, diabetes diagnosis, hypertension, and BMI. This paper supports these results, indicating that even in a sample of fairly healthy non-smokers an increase in BMI results in an age and fitness-independent increase in neutrophils, lymphocytes, and basophils.
A variety of physiological pathways exist whereby regular exercise and chronic obesity could affect neutrophil concentration. Potential pathways include activation of proinflammatory cytokines (IL-1b, IL-6, and TNF-a) and the hypothalamic-pituitary-adrenal (HPA) axis (stress). TNF-a (tumour necrosis
factor-a) is a potent stimulator of IL-6 production, and IL-6 stimulates neutrophils directly and possibly through cortisol and the hypothalamic-pituitary axis.36 37 Both TNF-a and IL-6 are released from monocytes, macrophages, and multiple cells within adipose tissue.38 39 In addition, fatness is strongly
associated with elevated circulating leptin levels. Leptin activates neutrophils40 and induces production of pro-inflammatory cytokines like IL-1b, IL-6 and TNFa.41 Thus, our results are consistent with the hypothesis that increased circulating WBC subsets may be a risk factor for inflammatory disorders.
While acute exercise raises levels of inflammatory parameters, several studies demonstrate that habitual exercise is inversely related to both TNF-a and IL-6.42-44 For example, Smith et al found that a 6 month exercise programme reduced TNF-a (n=43, average age=49.0 years) and Taaffe et al reported an inverse relationship between accumulated moderate and strenuous activity with IL-6 in 880 adults aged 70 to 79 years.43 45 Obesity has also been linked to increases in IL-6 and TNF-a via an NF-kB (nuclear factor- kB) dependent mechanism indicating a persistent metabolic proinflammatory state46 also termed metainflammation.47 Regular exercise and chronic weight loss may condition the body to respond efficiently to and limit fluctuations in immune parameters. Thus, the effect of regular exercise and weight loss on leptin, TNF-a, and IL-6 levels may be responsible for the reduction of neutrophil concentrations
seen with improved fitness and reduced fatness.
Because our study was cross-sectional, it cannot establish causality between fitness and fatness levels and increased total WBC counts, neutrophil, lymphocyte, and basophil concentrations. In addition, diet may influence many or all of the WBC subfractions; participants with an increased fitness level may have healthier diets than participants with a lower level of fitness.48 However, insufficient data in this project prevented analysis of the influence of diet. Finally, the predominantly white, male, middle-to-upper-class study population limits the
generalisability of the results of our study, but reduces the likelihood of confounding socioeconomic factors.
RESULTS
Table 1 presents the participant characteristics and mean unadjusted leucocyte data for this sample from the ACLS database. Mean age, BMI, maximal METs during a treadmill test, and systolic and diastolic blood pressures were 51.2
(10.1) years, 27.2 (3.9) kg/m2, 11.7 (1.8) METs, and 124.9 (13.6) and 84.1 (9.3) mm Hg, respectively.
Table 2 contains participant characteristics across fitness and fatness categories. Less fit individuals tended to be older and have higher BMI and systolic and diastolic blood pressures (p<0.001 for all comparisons). Individuals with a higher BMI tended to be less fit and have higher resting systolic and diastolic blood pressures (p<0.001 for all comparisons). Age-adjusted (model 1) and age- and BMI- or MET-adjusted (model 2) mean total WBC and WBC subfraction concentrations across fitness and fatness categories are presented in
table 3. Age-adjusted total WBC and all WBC subfractions were inversely associated with fitness. When BMI was added to the model, only total WBC, neutrophil, and basophil concentrations remained significant. Age-adjusted total WBC, neutrophil, lymphocyte, monocyte, and basophil concentrations increased with an increase in level of fatness. However, age-adjusted eosinophil subfraction concentration was not associated with level of fatness (p=0.17). After further adjustment for maximal METs, total WBC, neutrophil, lymphocyte, and basophil concentrations remained associated with fatness.
Figure 1 demonstrates the combined effect of fitness and fatness on total and fractionated WBC counts after adjustment for age. Total WBC, neutrophil, lymphocyte, and basophil concentrations were higher in the low-fit-high-fat group
compared with both high-fitness groups. Total WBC counts were also higher in the low-fit-low-fat group compared with the high-fit-low-fat group. Interestingly, the effect of fatness was negligible when individuals were categorised as highfitness. Monocyte and eosinophil concentrations were similar for all fitness and fatness categories.
METHODS
Patient data
The Aerobics Center Longitudinal Study (ACLS) is an epidemiological study of patients who have received a preventive medical examination at The Cooper Clinic in Dallas, Texas. Participants included in these analyses are 452 men examined during 2001. The participants are predominantly non-Hispanic whites, US residents, and welleducated. In order to avoid confounding factors
that may influence systemic inflammation, we excluded individuals with a previous history of diabetes mellitus (self-report or fasting glucose >126), stroke, heart attack, or chronic disease such as bronchitis, asthma, emphysema, or arthritis. Due to the strong effects of smoking on WBC concentrations and difficulty in quantifying duration, amount, and intensity of inhalation, we examined only non-smoking individuals. All participants gave their informed consent to participate in the clinical examination and follow-up and to the use of their exam data for research purposes. The study protocol was approved annually by The Cooper Institute Institutional Review Board.
Clinical examination
Given the associations between serum cortisol and diurnal alterations in WBC trafficking, all clinical examinations including blood draws were performed in the mornings after an overnight fast. The participants were instructed to refrain from
exercise for at least 24 h prior to the examination. Examinations were administered by trained laboratory technicians, following protocol procedures
specified in the manual of operations, and supervised by clinic physicians.
The examination consisted of: a physical examination by a clinic physician, a blood sample obtained by antecubital venipuncture for blood chemistry analyses, blood pressure by standardized sphygmomanometry, anthropometry, completion
of an extensive questionnaire on demographic characteristics, health history, family medical history, and a health habit inventory; and a maximal treadmill exercise test. Height and weight were measured on a standard physician's balance beam scale and stadiometer. Body mass index (BMI), an index of body fatness, was calculated as weight in kilograms divided by height in metres
squared. Blood pressures were obtained using mercury manometers following the American Heart Association protocol.21
Routine complete blood counts (CBCs) were performed in The Cooper Clinic laboratory, which participates in and meets the quality control standards of the CLIA (Clinical Laboratory Information Act). Total WBC count and relative subfraction composition (percentiles of total WBC counts) were determined
using a Pentra 60 (ABX Diagnostics, France). The concentration (number of cells/ml) was calculated from the total WBC count and percentage of each subfraction.
Cardiorespiratory fitness was assessed by maximal treadmill test using a modified Balke protocol.22 Patients began walking at 88 m/min (3.3 mph) with no elevation. After the first minute, the incline was increased to 2% and was increased 1% each minute thereafter until the 25th minute. For the few participants still able to continue the test beyond 25 minutes, the elevation was maintained at 25%, and the speed was increased by 5.4 m/min (0.2 mph) each minute to the end of the test. The test was terminated when the participants were exhausted or if the physician stopped the test for medical reasons. Total time during a treadmill test with this protocol is highly correlated (r=0.92) with measured maximal oxygen uptake.23 Thus, cardiorespiratory fitness in this study is analogous to maximal aerobic power.
Individuals were categorised by fitness tertiles according to age-group and sex-specific predicted metabolic equivalents (MET) from their end time during the treadmill test. A previous report from this same cohort reported a strong correlation between level of fitness and average age-adjusted weekly leisure time energy expenditure.24 There is a general consensus that increasing physical activity results in improved fitness.25 Thus, it is reasonable to conclude that physical fitness is strongly associated with regular physical activity.
Statistical analyses
Individuals were categorised by tertiles of fitness according to estimated METs during a treadmill test (<10.9 METs, >10.9 to <12.6 METs, and >12.6 METs) and fatness according to BMI (<25.2 kg/m2, >25.2 to <28.2 kg/m2, and >28.2 kg/m2).
Descriptive statistics (mean (SD)) were computed across fitness and fatness categories for each variable with trends for effects determined by linear regression. Due to the strong influence of age on WBC concentration, age-adjusted mean neutrophil, lymphocyte, monocyte, basophil, and eosinophil concentrations were calculated using general linear models (mean (95% CI)).
Trends for fitness and fatness were tested separately for each WBC subfraction using multiple linear regression models with age as a covariate. Additionally, independent fitness and fatness effects were determined using age- and BMI- or age- and METadjusted linear models, respectively. Age- and MET- or BMI adjusted mean WBC subfraction concentrations were determined (mean (95% CI)) using general linear models. Further analyses using a median split into low and high fitness (low <11.66 METs, high >11.66 METs) and fatness (low
<26.7 kg/m2, high >26.7 kg/m2) categories were conducted to determine the combined effects of fitness and fatness on age-adjusted total WBC and WBC subfraction concentrations. If the fitness-fatness relationship to total WBC and WBC subfraction concentration was significant, follow-up analyses were run
using a Tukey-Kramer post hoc test.
For all statistical tests, the alpha level adopted for significance was p<0.05 with Bonferroni correction for multiple comparisons. Statistical analyses were performed using SAS version 9.2.
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