452
Acute Exercise and Immune Function Relationship between Lymphocyte Activity and Changes in Subset Counts S. Shinkai, S. Shore2, P N. Shek2' , R. ,J Shephard2'
Abstract
ship between Lymphocyte Activity and Changes in Subset
haemagglutinin (PHA) and pokeweed mitogen (PWM) declined significantly during exercise, returning to normal by 120 mm of recovery. The natural killer (NK) activity rose markedly during exercise, but decreased to almost half the pre-exercise level at 30 and 60mm of recovery, returning to
Counts. mt j Sports Med. Vol 13, No 6, pp 452—46 1, 1992.
baseline levels after 120 mm of recovery. Functional capability
S. Shinkai, S. Shore, R N. Shek and R. .J Shephard, Acute Exercise and Immune Function — Relation-
Accepted after revision: April 13, 1992 Twenty-one young male subjects exercised on
a cycle ergometer for 60 mm at 60% of O2max. Blood
correlated well with the percentage of each major responder subset in the assay, suggesting that the in vitro lymphocyte PHA- and PWM-responsiveness and the NK activity did not change significantly on a per cell basis.
samples collected every 30 mm throughout exercise and con-
tinuing to 120 mm recovery served for the immunological tests.
The analysis of lymphocyte marker antigen
density revealed that the CD3, CD4, CD8 and CDl9 lymphocytes mobilized into the circulation during exercise did
Exercise induced biphasic changes in the various leucocyte subsets. There was a granulocytosis, lymphocytosis and monocytosis during exercise, and a further granulocytosis and a slight monocytosis, but a lymphocytopenia during recovery. All lymphocyte subsets (CD3 , CD 19 ", CD4, CD8, and CDl6 cells) increased in number during exercise, were decreased 30 mm after exercise, and had not returned to baseline levels by 120 mm of recovery. The apparent lymphocyte responsiveness to the mitogens phyto-
Introduction
not express the respective CD3, CD4, CD8 and CDI9 molecules as strongly as did the subsets circulating at rest, whereas the expression of the CD 16 antigen on CDI 6 + lymphocytes remained unchanged.
Key words Acute exercise, lymphocyte subsets, biphasic change, immune function, lymphocyte marker antigen density
_______
phocyte subsets (16), in vitro lymphocyte responsiveness to
Acute exercise induces changes in the car-
mitogens (25,27), or NK activity (2,23,29,30). It is also still debated whether the immunocompetent cells mobilized by ex-
diovascular and endocrine systems, leading to perturbations of the immune system. One of the most obvious changes in the immune system is a transient redistribution of immunocom-
ercise have the same per-cell functional capability as previously circulating cells (23).
petent cells (32). Some reports have also suggested that the function of the various immune cells is modified by exercise. Many researchers have observed changes in the
number of immunocompetent cells and their function as reflected in peripheral blood specimens. Acute exercise affects not only the circulating number of total leucocyte, leucocyte and lymphocyte subsets (2,11,15,16,18,25,27,31,33), but modifies in vitro neutrophil (15,33), monocyte (3, 14) and lym-
phocyte functions (2,11,22,25,27,29,30,35,38). However, results have not been consistent with respect to changes in lymmt. J. Sports Med. 13 (1992) 452—461 Georg Thieme Verlag Stuttgart New York
Several experimental variables contribute to inter-study differences in results. The most obvious cause is the diversity of exercise type, duration and intensity relative to the initial condition of the subjects (17,18,23). Several studies have shown no significant difference of exercise-induced immunological changes between trained and untrained subjects at the same relative intensity of effort (5, 18). Others have found small differences (27). Therefore, a precise description of the exercise protocol and the fitness level of the subjects should be given in
future studies of exercise immunology to enable inter-study comparisons.
There are also practical and technical problems. Blood sampling time may be a critical variable. Hansen
Downloaded by: University of Pennsylvania Libraries. Copyrighted material.
'Department of Hygiene, Ehime University School of Medicine, Ehime, Japan 2 Programme in Exercise Sciences, Graduate Department of Community Health, University of Toronto, Toronto Defence and Civil Institute of Environmental Medicine, North York "School of Physical and Health Education, University of Toronto, Toronto, Canada
mt. J Sports Med. 13 (1992) 453
Acute Exercise and Immune Function
In this study, we firstly provide a detailed description of the change in the number of immunocompetent cells in peripheral blood, and an analysis of their in vitro response to mitogens and NK activity during and following a standardized bout of exercise (60 mm of cycle ergometer exercise at 60 % VO2max). Changes in lymphocyte subsets were compared between whole blood and an isolated mononuclear cell suspension, and an attempt was made to clarify whether an acute, sustained bout of exercise modified lymphocyte functions per responder cell.
Moreover, we measured the density of surface marker antigens on the circulating lymphocyte before and after exercise. If a change were to be detected, this would suggest that the lymphocyte subset mobilized during exercise differed from the previously circulating species in terms of surface marker antigen density.
Materials and Methods
Hemoglobin concentration The hemoglobin concentration in whole blood was determined by the standard cyanomethemoglobin method, adjusting for blood volume change according to the formula: BVA/BVO = Hbo/HbA, in which BVA and By0 represent
blood volumes, and HbA and Hb0 are hemoglobin concentrations at times A and 0 (the pre-exercise baseline), respectively (10).
Counts of total leukocytes and of leukocyte subsets The total number of leukocytes in unit volume of peripheral blood was determined by counting in a Coulter Counter (Model ZM, Coulter Electronics, Luton, England). The numbers of three leukocyte subsets (granulocytes, lymphocytes and monocytes) were calculated by multiplying the proportion of each kind of cells observed in a flow-cytometer (see below) by the total number of leukocytes per unit volume. All counts
were adjusted for blood volume changes, i.e., an adjusted countA = an observed countA x BVA/BVO.
Preparation of mononuclear cells from peripheral blood
Subjects
Five ml of heparinized blood was diluted with an equal volume of phosphate-buffered saline (PBS). It was then layered over 5 ml of Ficoll-Paque solution (Pharmacia
Twenty-one young male volunteers partici-
LKB, Uppsala, Sweden), and centrifuged at 400 x g for 30 mm
pated in this study under conditions approved by the University of Toronto Committee on the ethics of human experimentation.
at 20 'C. The mononuclear cell layer was removed and washed
They signed informed consent forms after they had been informed in detail of the purpose of the study and the possible
RPMI 1640 culture medium (Gibco, NY) supplemented with 10 % fetal calf serum (Gibco, NY) (10% FCS- RPMI 1640) at 330 x g for 10 mm at 20 'C. The resultant pellet was reconstituted to a desired cell concentration in 10% FCS-RPMI 1640.
risks involved. A clinical examination and questionnaires ascertained that the subjects did not exercise regularly but were fit to undertake a maximal cycle-ergometer test.
twice, firstly with lOmi of PBS and secondly with lOml of
Counting of lymphocyte subsets
Maximal cycle-ergometer test The maximal 'O2 was determined by a graded
Lymphocyte subsets were stained and counted, both as whole blood and as PBMC.
protocol (6), using a Cardionics cycle ergometer. Expired gases
were analyzed with a SensorMedics metabolic cart (System 4400, Anaheim, CA).
Protocol of sub-maximal exercise and blood sampling Subjects refrained from any type of moderate or heavy exercise for at least one day before testing. The test typically began at 9 a. m. with insertion of an indwelling catheter into an antecubital vein. After 15 minutes of warm-up at 60
watts, the work-rate was then adjusted to 60 % of the individual's VO2max, and exercise continued at this intensity for 60mm. At 27—29mm and 57—59mm of exercise, the oxygen consumption and heart rate were measured to ensure that subjects were actually working at 60% of their personal VO2max. Water was made available to all subjects ad libitum throughout the test. Prior to exercise, at 30 and 60 mm of the exercise, and at 30, 60 and 120 mm of recovery, 15m1 aliquots of blood were drawn from the heparin-locked catheter into a heparinized vacutamer tube. Samples were kept at room temperature and were analysed within two hours.
A. Whole blood method A volume of 100 j.tl of whole blood was mixed with lOj.tl of selected monoclonal antibodies (mAb) conjugated with fluorescein isothiocyanate (FITC) or phycoerythrin (PE) in
the following double staining combinations: anti-CD3 mAb (FITC)/anti-CD 19 mAb (PE), anti-CD4 mAb (PE) /anti-CD8 mAb (FITC), anti-CD 16 mAb (FITC) /anti-CD25 mAb (PE). All mAb were purchased from Becton Dickinson, CA. After a 30-mm incubation on ice in the dark, 2m1 of 1 x FACS lysing solution (Becton Dickinson) was added, and the vials were left for 10 mm at room temperature to lyse the red cells. Non-lysed cells were centrifuged for 6 mm at room temperature and 500 x g; they were then washed twice with 2 ml of cold PBS containing 0.1 % sodium azide (0.1 % NaN3-PBS) and centrifuged for
a further 6 mm at 4 'C and 500 x g. The resultant pellet was resuspended in 0.3 ml of cold 0.5 % paraformaldehyde in 0.1 %
NaN3- PBS and was stored in the dark at 4 'C until it was analysed in a flow-cytometer. More than 95 % of the original leukocytes in a given volume of whole blood were recovered in a final resultant suspension.
Downloaded by: University of Pennsylvania Libraries. Copyrighted material.
et a!. (15) demonstrated a biphasic change in peripheral blood leucocyte subsets in response to strenuous exercise, suggesting a need for multiple blood sampling. Methodology may also bias the quality and/or quantity of results. Some studies have used whole blood (8,20), but most have used isolated mononuclear cells (2,16,18,25,27,29—31) when enumerating the subsets of circulating immunocompetent cells.
454 mt. i Sports Med. 13 (1992)
A volume of 50 .d of PBMC, at a concentration of 5 x 106 cells/mi, was mixed with lOp1 of each mAb and was then incubated for 30 mm on ice in the dark. Cells were washed twice, each time mixing with 2m1 of cold 0.1% NaN3-PBS for 6 mm at 4 'C and 500 x g. The resultant pellet was resuspended in 0.3 ml of cold 0.5% paraformaldehyde in 0.1 % NaN3-PBS and was stored in the dark at 4 'C until analysis.
channel
64
CD3ce11s
Median channel
MFI=7.581
n Relative Sj.fl
FACScan flow-cytometer (Becton Dickinson) on the same days as the bout of sub-maximal exercise. The FACScan system was
An unstained cell sample was used to determine the proportions of three leukocyte subsets, based on cell size and granularity; on a dot plot of forward versus side light scatters.
Assay of lymphocyte proliferative response to mitogen Triplicates of 100 jfl PBMC at a concentration of 2 x 106 cells/mi were placed into a sterile 96 U-shaped well plate (Flow Lab. Inc., VIR). 100 p.1 aliquots of a predetermined optimal mitogen dose, 40 .tg/ml phytohaemagglutinin (PHA) or 20 p.g/ml pokeweed mitogen (PWM), were then added to the desired wells and incubated for 66 hours at 37 'C in a 5 % CO2 incubator. 1 p.Ci of 3H- Thymidine (specific activity I mCi/mi, New England Nuclear) in 20 p.1 of 10% FCS-RPMI 1640 was added to each well, and the plates were then incubated for an additional 6 hours. Cells were collected with an automated PHD cell harvester (Cambridge Technology Inc., MA), and the radioactivity was counted in a Beckman scintillation counter (model LS5801). The typical test/retest variation was less than 5 %. Natural killer activity The natural killer (NK) activity of PBMC was measured using K562 target cells and a 51Cr release assay (26).
The viability of the K562 cells was typically around 90%, as determined by the trypan blue exclusion test. Triplicates of 100 p.1 PBMC at concentrations of 2 x 106, 1 x 106, and 0.5 x 106 cells/ml were incubated with 100 p.1 suspensions of target cells at counts of I x iO cells/mi; the 96 U-shaped well plate was incubated at 37 'C in a CO2 incubator for 4 h. After incubation, the plate was centrifuged for 5 mm at 1500rpm and 4 'C. A 100 p.! aliquot of supernatant was withdrawn and the radioactivity was determined by a Packard gamma counter (Model 5002). The spontaneous release of 51Cr was determined by incubation of control wells with 100 p.1 of plain medium, and the maximum
fluorescence intensity
of anti -C03 antibody channel
,
64
calibrated and optimized for analysis each day, using in turn
of peripheral blood, this value being adjusted for blood volume changes.
=661 .85
U
Stained cell samples were passed through a
were expressed as the percentage of cells yielding a specific fluorescence in a gated lymphocyte region. The absolute lymphocyte subset count was derived by multiplying this percentage of cells by the total number of lymphocytes in unit volume
MFII=394.7
a.
C. Flow-cytometry
CaliBrite beads (Becton Dickinson), an isotype negative control and a CD4/CD8 double-stained sample. The data were acquired and analyzed with Consort30 and LYSIS softwares. The number of cells counted was usually 10,000 per blood sample. Findings
CD3tcells
Ii na
corcells
CD3cells
C
Median channel
MFI=29.26
MF553
=680. 10
a.
a C,
,gB jgl 122 Relative fluorescence 2OpI
intensity
of anti-C03 antibody
Fig. 1 An example showing the determination of fluorescence intensity due to specific binding. The fluorescence spectra, (A) and (B), were obtained by the incubation of PBMC with 5 i.tl and 20 tl of anti-CD3 mAb, respectively. The median channel for each assay was calculated from the following equation: median channel 255.75 x log (MFl- MFI1, where MFl and MF[ are the median fluorescence intensities for a positively stained cell population and an unstained cell population, respectively.
of target cells plus 100 p.1 of medium with a non-ionic detergent, 10% Triton. The NK activity was expressed as the percentage lysis, determined as release was assessed by incubation of 100 j.tl
Percentage lysis = 100 x (test- spontaneous) cpm/(maximumspontaneous) cpm.
The test/retest variability of this measure for the three PBMC concentrations was less than 5 %. Determination of the density of
lymphocyte surface marker antigens In 16 of the 21 participants, the density of lymphocyte surface marker antigens was measured for the PBMC samples from blood samples obtained prior to exercise and at the immediate end of exercise. Flow-cytometer data were obtained using fluorescence-conjugated mAb; anti-CD3 mAb (FITC), anti-CD19 mAb (PE), anti-CD4 mAb (PE), anti-CD8 mAb (FITC) and anti-CD 16 mAb (FITC) (Becton-Dickinson). A 50p.l aliquot of PBMC at a concentration of 2 x 106 cells/mI was mixed with 5, 10, 15 or 20p.l of each mAb, and this reaction mixture was adjusted to a final volume of 70 p.1, using 0.1 % NaN3-PBS. After 30 mm of incubation on ice in the dark, stained cells were processed as described above.
Downloaded by: University of Pennsylvania Libraries. Copyrighted material.
B. PBMC method
S. Shinkai, S. Shore, P N. Shek, R. J. Shephard
mt. J Sports Med. 13 (1992) 455
Acute Exercise and Immune Function Table 1 Physical Characteristics of the Subjects (all male) Mean±S. D.
Range
Table 2 Characteristics of the cycle ergometer exercise cycle ergometer for 60 mm
Type
Actual values
at 30 mm
at 60 mm
Work rate (watts)
122±15 148±13 25.6±2.3 77.4±5.8 62.0
123±15 154±13 26.4±2.9
21
N
Age (yrs) Height (cm) Body mass (kg) BMIa VO2max (mi/kg. mm) HRmaxb (beats/mm)
22.9±3.9 177.5±6.0 79.5±9.5 25.2±2.7 41.4 190.8±9.0
19—31 169.2— 186.9
68.3—106.0 20.6—30.3 34.0—48.6 167—206
HR(beats/min) V02 (mI/kg.min) % HRmax (%) %VO2max (%)
* Each value represents the mean
80.5±5.1 63.9±6.0
D. of data from 21 subjects.
a Body mass index; b Maximal heart rate.
Data were acquired and stored as list mode data
was transformed into a log scale, and was then stored as a corresponding channel value ranging from 0 to 1023 (Fig. 1). Thus, one channel value difference corresponded to almost a I % difference in Fl. The number of events acquired was 8,000 to 10,000 per sample, gating on lymphocytes visually. In combination with fluorescence-conjugated mAb, flow-cytometry indicated the Fl of individual cells and the Fl spectrum of a cell population. Specific binding sites on the cells become progressively saturated with mAb as the concentration of mAb in the medium was increased. The Fl at the saturation point provided information on the specific mAb-directed surface antigen den-
a in
aC, c0 0
sity on the cells. The median channel corresponding to the
ca
median log Fl was adopted as the representative value to char-
U
acterize the FT spectrum of a given cell population (28). In order
to make comparisons between samples, the median Fl (MFI) due to specific binding was calculated by subtracting the MFI for an unstained cell population (MFL) from the MFT on a positively stained cell population (MFI), the difference being
8
-J
C)
C
0
a
U
60(0)
Statistical analysis Two-way ANOVAs and paired t-tests were applied to data for the total leukocyte count, leukocyte subsets and lymphocyte subsets in peripheral blood and PBMC to assess the effect of sampling time, and to compare individual exercise and post-exercise values with the baseline values, respectively. The
Friedman two-way analysis of variance and the Wilcoxon signed rand-sum test were applied to data for the CD4/CD8 ratio, lymphocyte proliferative responses and NK activity, and the Wilcoxon signed rank-sum test was also used for detecting
a difference in the median channel of positively stained cell populations between PBMCs obtained before and at the end of exercise.
Results
Physical characteristics and exercise response
The physical characteristics of the subjects were typical of sedentary young men (Table!). They exercised for 60 mm at a work intensity of 122 to 123 watts, eliciting an average of 62—64% of their TO2max (Table2).
Blood volume change Blood volume decreased significantly during exercise (mean±SEM, 94.3±0.7% of baseline at 30mm, 96.0
-.
** .—
expressed as a channel. Fig. 1 shows an example. Enercise +
I 30 60 Recovery
I
90
I
20
'I
SmpUng Time(min) Fig. 2 Changes in total white blood cell, granulocyte, lymphocyte and monocyte counts with exercise. Each bar represents mean± SEM at given time. All data points are significantly different (p
Acute Exercise and Immune Function Relationship between Lymphocyte Activity and Changes in Subset Counts S. Shinkai, S. Shore2, P N. Shek2' , R. ,J Shephard2'
Abstract
ship between Lymphocyte Activity and Changes in Subset
haemagglutinin (PHA) and pokeweed mitogen (PWM) declined significantly during exercise, returning to normal by 120 mm of recovery. The natural killer (NK) activity rose markedly during exercise, but decreased to almost half the pre-exercise level at 30 and 60mm of recovery, returning to
Counts. mt j Sports Med. Vol 13, No 6, pp 452—46 1, 1992.
baseline levels after 120 mm of recovery. Functional capability
S. Shinkai, S. Shore, R N. Shek and R. .J Shephard, Acute Exercise and Immune Function — Relation-
Accepted after revision: April 13, 1992 Twenty-one young male subjects exercised on
a cycle ergometer for 60 mm at 60% of O2max. Blood
correlated well with the percentage of each major responder subset in the assay, suggesting that the in vitro lymphocyte PHA- and PWM-responsiveness and the NK activity did not change significantly on a per cell basis.
samples collected every 30 mm throughout exercise and con-
tinuing to 120 mm recovery served for the immunological tests.
The analysis of lymphocyte marker antigen
density revealed that the CD3, CD4, CD8 and CDl9 lymphocytes mobilized into the circulation during exercise did
Exercise induced biphasic changes in the various leucocyte subsets. There was a granulocytosis, lymphocytosis and monocytosis during exercise, and a further granulocytosis and a slight monocytosis, but a lymphocytopenia during recovery. All lymphocyte subsets (CD3 , CD 19 ", CD4, CD8, and CDl6 cells) increased in number during exercise, were decreased 30 mm after exercise, and had not returned to baseline levels by 120 mm of recovery. The apparent lymphocyte responsiveness to the mitogens phyto-
Introduction
not express the respective CD3, CD4, CD8 and CDI9 molecules as strongly as did the subsets circulating at rest, whereas the expression of the CD 16 antigen on CDI 6 + lymphocytes remained unchanged.
Key words Acute exercise, lymphocyte subsets, biphasic change, immune function, lymphocyte marker antigen density
_______
phocyte subsets (16), in vitro lymphocyte responsiveness to
Acute exercise induces changes in the car-
mitogens (25,27), or NK activity (2,23,29,30). It is also still debated whether the immunocompetent cells mobilized by ex-
diovascular and endocrine systems, leading to perturbations of the immune system. One of the most obvious changes in the immune system is a transient redistribution of immunocom-
ercise have the same per-cell functional capability as previously circulating cells (23).
petent cells (32). Some reports have also suggested that the function of the various immune cells is modified by exercise. Many researchers have observed changes in the
number of immunocompetent cells and their function as reflected in peripheral blood specimens. Acute exercise affects not only the circulating number of total leucocyte, leucocyte and lymphocyte subsets (2,11,15,16,18,25,27,31,33), but modifies in vitro neutrophil (15,33), monocyte (3, 14) and lym-
phocyte functions (2,11,22,25,27,29,30,35,38). However, results have not been consistent with respect to changes in lymmt. J. Sports Med. 13 (1992) 452—461 Georg Thieme Verlag Stuttgart New York
Several experimental variables contribute to inter-study differences in results. The most obvious cause is the diversity of exercise type, duration and intensity relative to the initial condition of the subjects (17,18,23). Several studies have shown no significant difference of exercise-induced immunological changes between trained and untrained subjects at the same relative intensity of effort (5, 18). Others have found small differences (27). Therefore, a precise description of the exercise protocol and the fitness level of the subjects should be given in
future studies of exercise immunology to enable inter-study comparisons.
There are also practical and technical problems. Blood sampling time may be a critical variable. Hansen
Downloaded by: University of Pennsylvania Libraries. Copyrighted material.
'Department of Hygiene, Ehime University School of Medicine, Ehime, Japan 2 Programme in Exercise Sciences, Graduate Department of Community Health, University of Toronto, Toronto Defence and Civil Institute of Environmental Medicine, North York "School of Physical and Health Education, University of Toronto, Toronto, Canada
mt. J Sports Med. 13 (1992) 453
Acute Exercise and Immune Function
In this study, we firstly provide a detailed description of the change in the number of immunocompetent cells in peripheral blood, and an analysis of their in vitro response to mitogens and NK activity during and following a standardized bout of exercise (60 mm of cycle ergometer exercise at 60 % VO2max). Changes in lymphocyte subsets were compared between whole blood and an isolated mononuclear cell suspension, and an attempt was made to clarify whether an acute, sustained bout of exercise modified lymphocyte functions per responder cell.
Moreover, we measured the density of surface marker antigens on the circulating lymphocyte before and after exercise. If a change were to be detected, this would suggest that the lymphocyte subset mobilized during exercise differed from the previously circulating species in terms of surface marker antigen density.
Materials and Methods
Hemoglobin concentration The hemoglobin concentration in whole blood was determined by the standard cyanomethemoglobin method, adjusting for blood volume change according to the formula: BVA/BVO = Hbo/HbA, in which BVA and By0 represent
blood volumes, and HbA and Hb0 are hemoglobin concentrations at times A and 0 (the pre-exercise baseline), respectively (10).
Counts of total leukocytes and of leukocyte subsets The total number of leukocytes in unit volume of peripheral blood was determined by counting in a Coulter Counter (Model ZM, Coulter Electronics, Luton, England). The numbers of three leukocyte subsets (granulocytes, lymphocytes and monocytes) were calculated by multiplying the proportion of each kind of cells observed in a flow-cytometer (see below) by the total number of leukocytes per unit volume. All counts
were adjusted for blood volume changes, i.e., an adjusted countA = an observed countA x BVA/BVO.
Preparation of mononuclear cells from peripheral blood
Subjects
Five ml of heparinized blood was diluted with an equal volume of phosphate-buffered saline (PBS). It was then layered over 5 ml of Ficoll-Paque solution (Pharmacia
Twenty-one young male volunteers partici-
LKB, Uppsala, Sweden), and centrifuged at 400 x g for 30 mm
pated in this study under conditions approved by the University of Toronto Committee on the ethics of human experimentation.
at 20 'C. The mononuclear cell layer was removed and washed
They signed informed consent forms after they had been informed in detail of the purpose of the study and the possible
RPMI 1640 culture medium (Gibco, NY) supplemented with 10 % fetal calf serum (Gibco, NY) (10% FCS- RPMI 1640) at 330 x g for 10 mm at 20 'C. The resultant pellet was reconstituted to a desired cell concentration in 10% FCS-RPMI 1640.
risks involved. A clinical examination and questionnaires ascertained that the subjects did not exercise regularly but were fit to undertake a maximal cycle-ergometer test.
twice, firstly with lOmi of PBS and secondly with lOml of
Counting of lymphocyte subsets
Maximal cycle-ergometer test The maximal 'O2 was determined by a graded
Lymphocyte subsets were stained and counted, both as whole blood and as PBMC.
protocol (6), using a Cardionics cycle ergometer. Expired gases
were analyzed with a SensorMedics metabolic cart (System 4400, Anaheim, CA).
Protocol of sub-maximal exercise and blood sampling Subjects refrained from any type of moderate or heavy exercise for at least one day before testing. The test typically began at 9 a. m. with insertion of an indwelling catheter into an antecubital vein. After 15 minutes of warm-up at 60
watts, the work-rate was then adjusted to 60 % of the individual's VO2max, and exercise continued at this intensity for 60mm. At 27—29mm and 57—59mm of exercise, the oxygen consumption and heart rate were measured to ensure that subjects were actually working at 60% of their personal VO2max. Water was made available to all subjects ad libitum throughout the test. Prior to exercise, at 30 and 60 mm of the exercise, and at 30, 60 and 120 mm of recovery, 15m1 aliquots of blood were drawn from the heparin-locked catheter into a heparinized vacutamer tube. Samples were kept at room temperature and were analysed within two hours.
A. Whole blood method A volume of 100 j.tl of whole blood was mixed with lOj.tl of selected monoclonal antibodies (mAb) conjugated with fluorescein isothiocyanate (FITC) or phycoerythrin (PE) in
the following double staining combinations: anti-CD3 mAb (FITC)/anti-CD 19 mAb (PE), anti-CD4 mAb (PE) /anti-CD8 mAb (FITC), anti-CD 16 mAb (FITC) /anti-CD25 mAb (PE). All mAb were purchased from Becton Dickinson, CA. After a 30-mm incubation on ice in the dark, 2m1 of 1 x FACS lysing solution (Becton Dickinson) was added, and the vials were left for 10 mm at room temperature to lyse the red cells. Non-lysed cells were centrifuged for 6 mm at room temperature and 500 x g; they were then washed twice with 2 ml of cold PBS containing 0.1 % sodium azide (0.1 % NaN3-PBS) and centrifuged for
a further 6 mm at 4 'C and 500 x g. The resultant pellet was resuspended in 0.3 ml of cold 0.5 % paraformaldehyde in 0.1 %
NaN3- PBS and was stored in the dark at 4 'C until it was analysed in a flow-cytometer. More than 95 % of the original leukocytes in a given volume of whole blood were recovered in a final resultant suspension.
Downloaded by: University of Pennsylvania Libraries. Copyrighted material.
et a!. (15) demonstrated a biphasic change in peripheral blood leucocyte subsets in response to strenuous exercise, suggesting a need for multiple blood sampling. Methodology may also bias the quality and/or quantity of results. Some studies have used whole blood (8,20), but most have used isolated mononuclear cells (2,16,18,25,27,29—31) when enumerating the subsets of circulating immunocompetent cells.
454 mt. i Sports Med. 13 (1992)
A volume of 50 .d of PBMC, at a concentration of 5 x 106 cells/mi, was mixed with lOp1 of each mAb and was then incubated for 30 mm on ice in the dark. Cells were washed twice, each time mixing with 2m1 of cold 0.1% NaN3-PBS for 6 mm at 4 'C and 500 x g. The resultant pellet was resuspended in 0.3 ml of cold 0.5% paraformaldehyde in 0.1 % NaN3-PBS and was stored in the dark at 4 'C until analysis.
channel
64
CD3ce11s
Median channel
MFI=7.581
n Relative Sj.fl
FACScan flow-cytometer (Becton Dickinson) on the same days as the bout of sub-maximal exercise. The FACScan system was
An unstained cell sample was used to determine the proportions of three leukocyte subsets, based on cell size and granularity; on a dot plot of forward versus side light scatters.
Assay of lymphocyte proliferative response to mitogen Triplicates of 100 jfl PBMC at a concentration of 2 x 106 cells/mi were placed into a sterile 96 U-shaped well plate (Flow Lab. Inc., VIR). 100 p.1 aliquots of a predetermined optimal mitogen dose, 40 .tg/ml phytohaemagglutinin (PHA) or 20 p.g/ml pokeweed mitogen (PWM), were then added to the desired wells and incubated for 66 hours at 37 'C in a 5 % CO2 incubator. 1 p.Ci of 3H- Thymidine (specific activity I mCi/mi, New England Nuclear) in 20 p.1 of 10% FCS-RPMI 1640 was added to each well, and the plates were then incubated for an additional 6 hours. Cells were collected with an automated PHD cell harvester (Cambridge Technology Inc., MA), and the radioactivity was counted in a Beckman scintillation counter (model LS5801). The typical test/retest variation was less than 5 %. Natural killer activity The natural killer (NK) activity of PBMC was measured using K562 target cells and a 51Cr release assay (26).
The viability of the K562 cells was typically around 90%, as determined by the trypan blue exclusion test. Triplicates of 100 p.1 PBMC at concentrations of 2 x 106, 1 x 106, and 0.5 x 106 cells/ml were incubated with 100 p.1 suspensions of target cells at counts of I x iO cells/mi; the 96 U-shaped well plate was incubated at 37 'C in a CO2 incubator for 4 h. After incubation, the plate was centrifuged for 5 mm at 1500rpm and 4 'C. A 100 p.! aliquot of supernatant was withdrawn and the radioactivity was determined by a Packard gamma counter (Model 5002). The spontaneous release of 51Cr was determined by incubation of control wells with 100 p.1 of plain medium, and the maximum
fluorescence intensity
of anti -C03 antibody channel
,
64
calibrated and optimized for analysis each day, using in turn
of peripheral blood, this value being adjusted for blood volume changes.
=661 .85
U
Stained cell samples were passed through a
were expressed as the percentage of cells yielding a specific fluorescence in a gated lymphocyte region. The absolute lymphocyte subset count was derived by multiplying this percentage of cells by the total number of lymphocytes in unit volume
MFII=394.7
a.
C. Flow-cytometry
CaliBrite beads (Becton Dickinson), an isotype negative control and a CD4/CD8 double-stained sample. The data were acquired and analyzed with Consort30 and LYSIS softwares. The number of cells counted was usually 10,000 per blood sample. Findings
CD3tcells
Ii na
corcells
CD3cells
C
Median channel
MFI=29.26
MF553
=680. 10
a.
a C,
,gB jgl 122 Relative fluorescence 2OpI
intensity
of anti-C03 antibody
Fig. 1 An example showing the determination of fluorescence intensity due to specific binding. The fluorescence spectra, (A) and (B), were obtained by the incubation of PBMC with 5 i.tl and 20 tl of anti-CD3 mAb, respectively. The median channel for each assay was calculated from the following equation: median channel 255.75 x log (MFl- MFI1, where MFl and MF[ are the median fluorescence intensities for a positively stained cell population and an unstained cell population, respectively.
of target cells plus 100 p.1 of medium with a non-ionic detergent, 10% Triton. The NK activity was expressed as the percentage lysis, determined as release was assessed by incubation of 100 j.tl
Percentage lysis = 100 x (test- spontaneous) cpm/(maximumspontaneous) cpm.
The test/retest variability of this measure for the three PBMC concentrations was less than 5 %. Determination of the density of
lymphocyte surface marker antigens In 16 of the 21 participants, the density of lymphocyte surface marker antigens was measured for the PBMC samples from blood samples obtained prior to exercise and at the immediate end of exercise. Flow-cytometer data were obtained using fluorescence-conjugated mAb; anti-CD3 mAb (FITC), anti-CD19 mAb (PE), anti-CD4 mAb (PE), anti-CD8 mAb (FITC) and anti-CD 16 mAb (FITC) (Becton-Dickinson). A 50p.l aliquot of PBMC at a concentration of 2 x 106 cells/mI was mixed with 5, 10, 15 or 20p.l of each mAb, and this reaction mixture was adjusted to a final volume of 70 p.1, using 0.1 % NaN3-PBS. After 30 mm of incubation on ice in the dark, stained cells were processed as described above.
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B. PBMC method
S. Shinkai, S. Shore, P N. Shek, R. J. Shephard
mt. J Sports Med. 13 (1992) 455
Acute Exercise and Immune Function Table 1 Physical Characteristics of the Subjects (all male) Mean±S. D.
Range
Table 2 Characteristics of the cycle ergometer exercise cycle ergometer for 60 mm
Type
Actual values
at 30 mm
at 60 mm
Work rate (watts)
122±15 148±13 25.6±2.3 77.4±5.8 62.0
123±15 154±13 26.4±2.9
21
N
Age (yrs) Height (cm) Body mass (kg) BMIa VO2max (mi/kg. mm) HRmaxb (beats/mm)
22.9±3.9 177.5±6.0 79.5±9.5 25.2±2.7 41.4 190.8±9.0
19—31 169.2— 186.9
68.3—106.0 20.6—30.3 34.0—48.6 167—206
HR(beats/min) V02 (mI/kg.min) % HRmax (%) %VO2max (%)
* Each value represents the mean
80.5±5.1 63.9±6.0
D. of data from 21 subjects.
a Body mass index; b Maximal heart rate.
Data were acquired and stored as list mode data
was transformed into a log scale, and was then stored as a corresponding channel value ranging from 0 to 1023 (Fig. 1). Thus, one channel value difference corresponded to almost a I % difference in Fl. The number of events acquired was 8,000 to 10,000 per sample, gating on lymphocytes visually. In combination with fluorescence-conjugated mAb, flow-cytometry indicated the Fl of individual cells and the Fl spectrum of a cell population. Specific binding sites on the cells become progressively saturated with mAb as the concentration of mAb in the medium was increased. The Fl at the saturation point provided information on the specific mAb-directed surface antigen den-
a in
aC, c0 0
sity on the cells. The median channel corresponding to the
ca
median log Fl was adopted as the representative value to char-
U
acterize the FT spectrum of a given cell population (28). In order
to make comparisons between samples, the median Fl (MFI) due to specific binding was calculated by subtracting the MFI for an unstained cell population (MFL) from the MFT on a positively stained cell population (MFI), the difference being
8
-J
C)
C
0
a
U
60(0)
Statistical analysis Two-way ANOVAs and paired t-tests were applied to data for the total leukocyte count, leukocyte subsets and lymphocyte subsets in peripheral blood and PBMC to assess the effect of sampling time, and to compare individual exercise and post-exercise values with the baseline values, respectively. The
Friedman two-way analysis of variance and the Wilcoxon signed rand-sum test were applied to data for the CD4/CD8 ratio, lymphocyte proliferative responses and NK activity, and the Wilcoxon signed rank-sum test was also used for detecting
a difference in the median channel of positively stained cell populations between PBMCs obtained before and at the end of exercise.
Results
Physical characteristics and exercise response
The physical characteristics of the subjects were typical of sedentary young men (Table!). They exercised for 60 mm at a work intensity of 122 to 123 watts, eliciting an average of 62—64% of their TO2max (Table2).
Blood volume change Blood volume decreased significantly during exercise (mean±SEM, 94.3±0.7% of baseline at 30mm, 96.0
-.
** .—
expressed as a channel. Fig. 1 shows an example. Enercise +
I 30 60 Recovery
I
90
I
20
'I
SmpUng Time(min) Fig. 2 Changes in total white blood cell, granulocyte, lymphocyte and monocyte counts with exercise. Each bar represents mean± SEM at given time. All data points are significantly different (p
