Aerobic exercise, training and modulation of the human immune system

(1)

THESES, SIS/LIBRARY R.G. MENZIES BUILDING N0.2 Australian National University Canberra ACT 0200 Australia

USE OF THESES

This copy is supplied for purposes of private study and research only. Passages from the thesis may not be copied or closely paraphrased without the

written consent of the author.

THE AUSTRALIAN NATIONAL UNIVERSITY

Telephone: -+B1 2 6125 4631 Facsimile: -+B1 2 6125 4063

(2)

A thesis submitted for the degree of

Doctor of Philosophy

of The Australian National University.

(3)

The

David B. Pyne PhD Candidate

to

15 September 1994.

ll

IS

or organisations:

on a 8.

on a

(4)

lS

respectively.

to

assess

on

as

11

as a

are followed by experiments

the same group of subjects

some

or on

AvvHUAH'-'OCU stress

to

The following

stress

As

(5)

Physiology

Kaylene Hood, Mr

subjects, especially the ACTAFL umpires and the swunmers,

Campbell,

Cole,

endured

exhaustive exercise bouts and numerous invasive tests throughout the various

experiments.

Dr Louise Burke for her personal support and the setting of an exceptional work

ethic and academic standard.

(6)

v

1

Telford,R. .

an intensive

swimmers on neutrophil oxidative Proceedings

The effects of an

swimmers on oxidative

(7)

as

or

Press. Boca 31-50.

Gray,A.B.

(8)

of Figures List of Tables Abbreviations

Chapter

V I

to

Exercise-Induced Activation of Neutrophils Rate and Neutrophil Oxidative Study

Exercise-induced Downregulation of the Conclusions References l ll v x 1 4 7 9 11

14

1 61 76 82

83

85

Review - Exercise-induced muscle damage and inflammation. 9 5

Summary 96

Introduction 97

Mechanical stress 99

Metabolic stress l 01

The nature of exercise-induced damage to muscle 102

Exercise-induced oxidative damage 105

Measurement of free radical generation 110

Delayed Onset Muscle Soreness 112

Cytokines - immunomediators of inflammation and tissue damage 114 Neutrophil regulation of the immunological and inflammatory

events after exercise : a theoretical model 118

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Methods Discussion References

Eccentric exercise induces ... ''"'"'' cytokines trained athletes.

Summary Introduction Methods Results Discussion References

damage but not

(10)

References

Summary Introduction Methods Results

References

39

265

270 272

285

30

314 L Abstract Med. Sci. Sports. Exerc 25:Sl02, 1993. 3

2. Abstract ASMF Conference, 1993. 3

3. Abstract ASMF Conference, 1994 316

(11)

to

mechanical stress on

stress

significant

and soreness.

mcrease

x

systemic

_ ... ,..., (metabolic

of

to on

~ .. ,, .... ,..., was associated was

uu0•-.nv damage

a significant

of lymphocytes,

cortisol (p<0.05) after only growth

hormone increased (p<0.05) after level and downhill running. There were no

significant changes in the plasma concentration of the inflammatory mediators

interleukin-lb and complement component C3a, nor in the acute phase protein

C-reactive protein, after strenuous or eccentrically-biased running. This suggests that

trained runners may sustain moderate and transient elevations markers of tissue

(12)

no

was

chemiluminescent '"'"'""c'"J

(ie.

neutrophil

a a

se

mechanical stress on

spectrophotometric

Chemiluminescence decreased after

after

Analysis of an intensive 16-week training cycle by elite swimmers showed reactive oxygen formation by neutrophils was lower (p<0.05) across the 16 week

period compared with that observed in cells from sedentary individuals, and declined

significantly during strenuous training prior to competition. There was no difference

the self-reported upper respiratory tract infection rate between the swimmers and

(13)

stresses of exercise and

can

necessarily

statement on

non~

an

to

before

(14)

Ll 1 3.1 3 3 3 l 4.2

120 4.3

150 5.1

the to

sequence of events the of

to

of oxygen-and

exerc1se-induced activation neutrophils that act to damage v v • . n a • u membranes

components including lipids, proteins and nucleic acids.

Theoretical model of immunological and inflammatory responses to

exercise and muscle damage showing the central roles of cytokines and

neutrophils in the repair of damaged tissue.

Mean oxygen uptake averaged during the final four minutes of each of

eight by five minute intervals of near-level, uphill and downhill treadmill running. Significance of the difference between the first and

(15)

1 5

1

178 6.3

1 8

197 7 .1

201 7 .2

202 7.3

214 8.1

an regions.

minute bout of moderate

release temporal variability.

superoxide

two

experimental

Changes the plasma concentration of lymphocyte subsets uphill, downhill and near-level treadmill running.

a

Plasma adrenaline concentration before and after 40 min of near-level, uphill and downhill running.

Plasma cortisol concentration at various time points before and after 40 min of downhill, near-level and uphill running.

(16)

xv

8 to

1

and

238 9.3

to

260 Capacity response to

in-vitro stimulation of

261 10.6 Capacity of neutrophils to produce hydrogen peroxide response to in-vitro stimulation (PMA) before and after bouts of treadmill running.

262 10. 7 Plasma concentration of elastase before and after different bouts of treadmill running.

263 10.8 Expression of complement receptor C3bi by neutrophils before and after bouts of treadmill running.

(17)

lL 11 12.1 2.1 2 3.1 1 5.1 5 5.3

174 1

175 6.2

176 6.3

a reserve.

ranges and subsets.

Changes in the circulating concentration of leucocyte and granulocytes

with two 40 minutes bouts of moderate intensity treadmill runs.

Changes in neutrophil oxidative activity with two bouts of moderate

intensity treadmill running assessed by the fluorescent probe DHR

(flow cytometric) to OZ- and PMA-stimulation.

Changes in neutrophil oxidative activity during 40 minutes of moderate

intensity cycling assessed by the fluorescent probe DHR (flow

(18)

7.1

2 8.1

235

1 The effect

component (C3a).

11.1

1 11

1

11

summer

288 1

running) and mechanical stress (downhill

297 12.2 Summary of changes indicators of neutrophil oxidative activity with

(19)

B

beats per

CD3+ Teen

cm

C-reactive protein coefficient of

DA dopamine

DCFH-DA dichloroflourescein diacetate

DHPG dihydroxyphenolglycol

DHR dihydrorhodamine

DOMS delayed onset muscle soreness

DOPAC 3,4-dihydroxyphenylacetic acid

Dopamine 3,4-dihydroxyphethylamine

EDTA ethylenediaminetetraacetic acid

FcR Fe receptor

Fig Figure

[image:19.596.145.409.99.766.2]

(20)

SG GTP h HBSS IFNg 1 IgG MDA mmHg MPO NA NADH NADPH NADPH oxidase

NK

NO water Hank's 1 constant

. . . ~VL~,_, . . . ,..~ activating

malondialdehyde minute

millilitres per kilogram per minute millimetres of mercury (pressure) myeloperoxidase

noradrenaline

nicotinamide adenine dinucleotide (reduced)

nicotinamide adenine dinucleotide phosphate (reduced) enzyme complex located on plasma membrane of neutrophil that produces reactive oxygen species natural killer cell

(21)

PAF PE PGE2 PKC PMA

POMC

max

xx

kinase C

phorbol myristate acetate u•RJuu"•'-'= leucocyte

(22)

1

IS

or competition.

scientist are ""'~~'""'""" on

cellular and humoral ~~·~r,..:;-_;::-.~ .. w

statements

nature

terms

the literature is

UH\.,C\/.:> lS

to

and

""""'~~"''"'"' ... ~.~~·~~ changes

to establish whether

on whether exercise

(1 1

to

One of key concepts of issue is so-called paradox between exercise

intensity and immune cell function. Moderate-intensity exercise is thought to be

largely immunopotentiating or beneficial to immune cell function (Nieman, 1993).

Many recreational athletes and fitness enthusiasts claim that regular participation in

this form of physical activity improves immunity, with an associated reduction in the

frequency and severity of upper respiratory tract infections. This position is

supported in laboratory studies that have examined the effects of moderate exercise

(23)

Nehlsen-A

premise

to

et 1).

suggest that it

experimentally.

2

on

individuals

be difficult to test their reliability and validity

Much of the previous work in the field of exercise immunology has adopted the

experimental approach outlined in Fig 1.1. Studies have examined, variously, the

physical and/or psychological stresses of exercise and training, the effects of these

stresses on the distribution and function of cellular and humoral components of the

(24)

3

Ll areas

~

(25)

to

can be considered to

(innate) of the .::nucuci'""

defence as

or

neutrophil, a key non-specific immune cell, is important for several reasons: (a)

it is the most abundant immune cell in the circulation; (b) it is mobilised rapidly to form the so-called "first-line-of-defence" against microbial attack; (c) it plays a

crucial effector role as a "professional" phagocyte in non-specific host defence by

expressing potent bactericidal and viricidal activity; (d) its functional deficiency can

lead to recurrent and life-threatening infections. More recently the ability of the

neutrophil to synthesize cytokines, the immunomodulatory proteins that regulate a

(26)

(Morel et

bone marow

a

sequence is

is a

are

there

cells

5

to

What are the effects of exercise and training on the

been

stem

variable

to

marginated cells are

a

(number)

functional state (function) of the neutrophil population? It is important to distinguish

between these two possibilities, as neutrophils may respond to exercise in different,

even opposite, directions (eg. an exercise-induced increase in the number of

neutrophils in the circulation may be accompanied by a decrease in functional

activity individual cell). The overall effect of these responses may be beneficial,

neutral or detrimental to immunocompetence. Acute bouts of exercise generally

(27)

6

exercise is

acute

over

m

to two separate

to an U A A H n A U • o U V

generally report an the neutrophil, increased expression to

receptors on the plasma membrane; initiation of phagocytosis; release of proteolytic

enzymes (degranulation); translocation of cytoplasmic proteins to plasma

membrane; and evidence of the formation of toxic reactive oxygen and nitrogen

species - the so-called respiratory burst may have been initiated in vivo - (Hack et aL

1992; Gray et al. 1993). In terms of the so-called "paradox" between exercise

intensity and immune cell function, only one preliminary report (Dziedzak, 1990)

has examined this relationship directly by measuring neutrophil oxidative activity in

(28)

targets

function or

tract

is to

an PV1"\PT"1

to

.. ,,. •. ,,. .. ,""'""' to as

to enter the VH'V~AMU'VAAo

(sigA) "'vu.vvu

after exercise

(Mackinnon et

AAA . . . L~A> the

1 by phagocytosis.

lS

1993)

a

sigA-opsonized

acute

not been examined.

on

""'""'',. nature of the changes

occur after a single episode of exercise, it is possible that the cumulative effects

repeated episodes are responsible for eliciting more permanent changes in immune

cell function. Information on whether significant fluctuations neutrophil number

and function are associated with alterations in the frequency of infections of the

upper respiratory tract, may help to establish whether the observed changes

represent normal fluctuations in immunity, or whether they are the expression of a

(29)

8

at a our

,~v=~·.,.n,~~ between exercise and endocrine

relatively and immune cell function observed after single

episodes of exercise could simply to temporal (circardian rhythms) or

experimental variability. Few studies have employed a parallel non-exercising

control group to differentiate between the effects of exercise per se and those of

normal temporal variation evident in immune responses. In his review, Cannon

(1993) has suggested that future studies should include appropriately matched

control groups to account for these temporal (circadian and seasonal) differences.

(30)

9

1. assess

to measure

2. effects

of exercise.

To assess lS a between of

3.

on

5. To examine relationship between exercise-induced mobilisation and

activation of neutrophils with systemic markers of and the

acute phase response in trained individuals.

6. To determine the differential effects of metabolic and mechanical stress of

exercise on the ability of neutrophils to respond to stimulation in vitro at the

(31)

season

(32)

11

1 to

L

(1

1

Gray,A.B. Telford,R.D. Collins,M. Baker,M.S. and Weidemann,M.J. (1993).

Granulocyte activation induced by intense interval running. Journal of

Leukocyte Biology 53: 591-597.

Hack,V. Strobel,G. Rau,J.P. Weicker,W. (1992). The effect of maximal

exercise on the activity of neutrophil granulocytes in highly trained athletes in

a moderate training period. European Journal of Applied Physiology 65:

(33)

(1

19

B

N ehlsen-Cannarella, S

Yang,H. Chritton,D.B.W. Lee,J.W. and Arabatzis,

elite

Balk-Lamberton,A.J. (1990a). The effects of moderate exercise training on natural killer cells and acute upper respiratory tract infections. International Journal of Sports Medicine 11: 467-473.

Nieman,D.C. Johanssen,L.M. and Lee,J.W. (1990b). Infectious episodes m

runners before and after the Los Angeles Marathon. Journal of Sports Medicine and Physical Fitness 30: 316-328.

(34)

(

Sports

(35)

Challenge to

of System

Illness Infection

Specific Acute Effects of Different Types of Exercise (i) Acute Moderate Aerobic Exercise

(36)

(37)

lS on

Nehlsen-Cannarella, l

1· , et et

review proposes a events

leucocytosis exercise, the between exercise

presents

two

defence.

monocytes

acute phase proteins, complement, lysozyme. These components,

which do not require prior contact with an infectious agent to be invoked, are capable

of acting alone or combination with the antigen-specific defences.

The specific system, which includes the and B-lymphocytes and the soluble

immunoglobulins, uses immunological memory to produce specific antibodies or

cytotoxic cells directed against infectious agents. In contrast to the non-specific

system, the specific defence mechanisms are activated after an initial exposure to

(38)

(39)

18

cause

variety

severe

~·~·U~H to ..,,,,, ... .,JL..:>Vo

or external means. Acute 0"•-~~·•A

common

tetanus

on

uu·~~···~u to . . ~•.uu.uw are

body contact sports, to superficial tissues internal

organs. The typical over-use syndrome manifest in injuries such as "swimmer's

shoulder" or "jumper's knee" may result damage to skeletal muscle tissue,

bone, tendons, ligaments and cartilage.

Psychological Modification of the Immune System.

One of the difficulties of interpreting the results of research into the effects of exercise

and training on the immune system is to distinguish between physical and

(40)

stress It a

or

stress

stress was associated,

an increased "n'r"'r,n.ru tract

to

It is

two [Ebbeling (1989) (

et al. (1991)] as events stimulate then mediate

exercise-induced damage to muscle tissue. Although originally proposed to describe the

sequence of events in the pathology of tissue damage, they can also be applied, in

broad terms, to categorise events which influence exercise-induced activation of the

immune system. A suggested schema for the exercise-induced changes in the

(41)

High intensity exercise (Concentric mode)

flow Decreased

[image:41.596.120.482.99.643.2]

activities)

(42)

of

same way

cause

21

absence of a pathogenic agent (Armstrong et

research effects

less been to the

effect) as a

et

over

1992). It is generally agreed

et

bruises)

see

the leucocytosis depends on the of the exercise Of

particular interest is the effect different types and intensities exercise on the

distribution of the key specific (mononuclear leucocytes) and non-specific

(polymorphonuclear leucocytes) immune cells (Table

There is normally an increase in the total number of circulating leucocytes after

exercise, with accompanying changes in the proportions of the main subpopulations

(lymphocytes, monocytes, granulocytes). The leucocytosis of exercise is initiated by

(43)

to

exercise. Exercise

phase)

in the plasma of

rate

et

is

marrow

opioid peptides may contribute to conflicting results (Mackinnon and Tomasi,

1988). The early lymphocytosis is related to the catecholamine-induced release of

lymphocytes from various storage sites and the walls of endothelial tissues (Crary et

al. 1983). The lymphopoenia which develops one hour after exercise is most likely

due to cortisol, which inhibits the further entry of lymphocytes into the circulation

and promotes the redistribution of the proportion already there to peripheral tissues.

Although the absolute number of cells in each of the lymphocyte subsets increases

(44)

to

The

to mitogenic

excess

are

et

have long-term on

Several studies have shown that exercise causes an increase the number of

circulating granulocytes (Edwards et al. 1984; Nieman et 1989; McCarthy et al.

1991; Fry et al. 1992; Gabriel et al. 1992). The granulocytosis (primarily

neutrophilia) observed after exercise reflects either a mobilisation of a phagocytic and

(45)

L

Leucocytes - 1

- 1

1 - 1

These reference values are based on results of immunophenotyping of

athletes (at rest) at the Sports Science and Sports Medicine Centre,

Australian Institute of Sport, Canberra, Australia, and previously

suggested by Nieman and Nehlsen-Cannarella (1992) and Lydyard

and Grosi (1989). Other laboratories should develop a normal range

(46)

or

increase

et al. (

count

on

marrow.

or an

at the same

measurements

or

and immune systems. Intense exercise (> 60% generally activate the sympathoadrenal pituitary-adrenocortical endocrine systems (Bunt, 1986),

leading to an increase the plasma concentrations of the catecholamine (e.g.

adrenaline and noradrenaline) and glucocorticoid (e.g. cortisol) hormones.

Adrenaline and cortisol are associated with the regulation of the immune response

during and after exercise (McCarthy and Dale, 1988). Research into the

endocrinological control of leucocytosis was given increased impetus by studies

(47)

vessel

lymph nodes,

the~~~~~·

axes.

a

on

et

leucocytes to

et promoting

inducing the release of norepinephrine as triggers

immunosuppressive responses observed with intensive exercise with the

increase in the plasma concentration of cortisol. The release of cortisol from the

adrenal gland is dependent upon the duration and intensity of exercise, with

.

significant increases occurring at workloads in excess of 60% V02 max (Farrell et al.

1983). Moderate aerobic exercise usually induces no change, or even a slight

decrease, the plasma cortisol concentration. The immunosuppressive effects of

cortisol are mediated partly through the inhibition of the synthesis of

(48)

marrow is

race

count a

There is a

may

have

leading to proposal of an intensity-dependent model for the

neuro-endocrine control of neutrophil function. It seems plausible that exercise may

influence the intensity of the non-specific immune response through the interaction of

hormones and other immunomediators with receptor-bearing immune cells.

Despite increased understanding of the importance of the interactions between

hormones, neuropeptides and cells of the immune system, the effect of exercise on

these relationships is unclear. At present it is unknown whether alterations in both

(49)

not

as an

different

It is a

acute

cancer

between acute exercise,

stress-related aspects of a

measurements

together

to

... ~,··~ .. on

... ~ ... ~ system is invaluable monitoring

and designing individual training programs to

optimise fitness without increasing susceptibility to illness.

The major clinical diagnoses are those of self-limiting viral infections of the

respiratory (e.g. the common cold and influenza) and gastrointestinal tracts, or

specific infections such as pharyngitis, pneumonia and the frequently-encountered

infectious mononucleosis (Heath et al. 1992; Hanson, 1984; Brenner et al. 1994).

(50)

Exercise

to

considered

et

or at or

to

A

one

case, an of infectious hepatitis polluted water resulted 90

out of 97 members the football team becoming affected, despite fact that no

other students or staff became ill drinking the same water (Morse et al. 1972).

Another case study reported that the incidence of aseptic meningitis in a high school

was twice as high, and the symptoms more severe, among members of the football

team than among students peers (Baron et al. 1982).

Other studies have examined the relationship between training load or volume and the

(51)

a

runners

Douglas and AA~ . . u~· . .

crew

tract

runners

tract

or race

a race

a 61

are

Athletes often expenence intense psychological stress and

international level sporting competitions. these situations it is difficult to determine whether any significant change in immunity and, consequently, in the incidence of

illness and infection is caused by physiological and/or psychological stressors.

Several researchers have suggested that these stressors may have a cumulative

immunosuppressive effect (Simon, 1984; Fitzgerald, 1988; Mackinnon, 1992). This

may be one explanation for the increased incidence of illness in elite athletes in highly

(52)

et

minutes at 60%

unexercised f ' A r • r r r .

example.

3

an

rate reserve, 5 days per week) was

an

more recent

to

an

a common

body-contact sports where external force on spleen and any violent collision,

may have more severe consequences if the organ is already susceptible. Simon (1987) advises that patients with mild upper respiratory tract infections need not

restrict their training schedules. However, those with a lower respiratory tract

infection, fever, arthralgia or myalgia should avoid strenuous training until recovery

is complete. The athlete, coach and physician should consider carefully the need to

(53)

are to

expectoration

c

tract et

a on

as been

this work was provided by Cannon Kluger (1984), concluded, from a review of data from animal experiments, that moderate exercise prior to contact with a pathogen may increase resistance to infection. Smith et al. (1990) showed that

.

moderate exercise (60 minutes of cycling at 60% V02 max) by human subjects produced an increase neutrophil oxidative burst that was sustained for six hours. Pyne et al. (1993) confirmed this finding by showing that 40 minutes of running at

.

(54)

at 2 to serum

on

'""""'~"""'" an

exercise provides stress.

et (

cyclists

were

to two

(ii) Exhaustive Aerobic Exercise

Several laboratory studies have indicated that moderate to maximum intensity aerobic

exercise may have a negative effect on some aspects of immune cell function. The

changes that may influence immunity are usually transient, and levels return to

(55)

hygienic Ensure ,_,,._,iuvuui hygiene

common

over

or

return to over

(56)

tract

acute

completed four randomly-ordered

max, at 35% max,

65%

to

an an

of on

at

were

runners was

examined by Berk et al. (1989). Ten experienced marathoners were exercised for 3

hours on a laboratory treadmill. Serial blood samples were taken during the first 21

hours of recovery and analysed for the total number of lymphocytes expressing

membrane antigens specific for natural killer cells. At 1.5 and 6 hours of recovery

natural killer cell activity decreased significantly compared with the baseline

measurement. Normal activity was restored by 21 hours post-exercise. With data

from the same study, Nieman et al. (1989) reported that a 3 hour endurance run was

(57)

(Pedersen et

monocytes is ~~,,A~,,~

oxygen

199 The

21

not return

is depressed

(2000m) long-term (10,000m) tests ten moderately-trained

male subjects. Chemiluminescence decreased by 18% after the 10,000m tests,

indicating an impaired ability to generate reactive oxygen species. These data

indicate that exhaustive exercise tends to suppress some cellular activities associated

(58)

3:

973-981.

stress

to common

61

Crary,B. Hauser,S Borysenko,M. and Hoban,C. (1983).

induced changes the distribution of lymphocytes subsets peripheral blood

of humans. Journal of Immunology 131: 1178-1181.

D'Alessio,D.J. Peterson,J.A. Dick,C.R. and Dick,E.C. (1976). Transmission of

experimental rhinovirus colds in volunteer married couples. Journal Infectious

of Diseases 133: 28-35.

Douglas,D. and Hanson,P. (1978) Upper respiratory infections in the conditioned

(62)

(1

to

on B. (1983 ).

to

system.

3

Fitzgerald,L. (1991). Overtraining increases the susceptibility to infection.

International of Sports Medicine 12: Supplement l:S6-8.

Foster,N.K. Martyn,J.B. Ragno,R.E. Hogg,J.C. and Hardy,R.L. (1986).

Leukocytosis of exercise: role of cardiac output and catecholamines. Journal of

Applied Physiology 61: 2218-2223.

Fry,R. Morton,A.R. and Keast,D. (1991). Overtraining in athletes - An update.

(63)

1

(

to

1

(1

(1 Effect long-term

on

reactivity: Similarity to spaceflight reactions. Aviation and Space Environmental Medicine 59: 146-151.

Good,R.A. and Fernandes,G. (1981). Enhancement of immunologic function and

resistance to tumor growth in BALB le mice by exercise. Federation

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Gray,A.B. Smart,Y.C. Telford,R.D. Weidemann,M.J. and Roberts,T.K. (1992).

Anaerobic exercise causes transient changes in leucocyte subsets and IL-2R

(64)

(1

1 L

to

tract

Hetherington,S.V. and Quie,P.G. (1985). polymorphonucelar leukocytes of

the bone marrow, circulation and marginated pool: function and granule protein

content. American Journal of Haematology 20: 235-246.

Heyden,S. and Fodor,G.J. (1988). Does regular exercise prolong life expectancy?

Sports Medicine 6: 63-71.

Jemmot,J.B. (1984). Psychosocial factors, immunologic mediation and human

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(65)

8

(

stress on

(

killer decrements

status HIV-1. 229-242.

Lerner,A.M. and Wilson,F.M. (1973). Virus myocardiopathy. Progress Medical

Virology 63.

Lewicki,R. Tchorzewski,H. Denys,A. Kowalska,M. and Golinska,A. (1987).

Effect of physical exercise on some parameters of immunity in conditioned

(66)

1

(1

J.

(

6: 333-363.

cells : is

Exercise

exercise: a

McCarthy,D.A. Grant,M. Marbut,M. Watling,M. Wade,A.M. McDonald,L

Nicholson,S. Melsom,R.D. and Perry,J.D. (1991). Brief exercise induces an

immediate and a delayed leucocytosis. British Journal of Sports Medicine 25:

191-195.

McCarthy,D.A. Macdonald,L Grant,M. Marbut,M. Watling,M. Nicholson,S.

Deeks,}. Wade,A.J. and Perry,J.D. (1992a). Studies on the immediate and

delayed leucocytosis elicited by brief (30-min) strenuous exercise. European

(67)

to an

levels.

(1

Nieman,D. C. Nehlsen-Cannarella,S.L. (199 The effects of acute and

exercise on immunoglobulins. Sports Medicine 11: 183-201.

Nieman,D.C. Nehlsen-Cannarella,S.L. Donogue,K.M. Chritton .B.

Haddock,B.L. Stout,R.W. and Lee,J.W. (1991). The effects of acute

moderate exercise on leukocyte and lymphocyte subpopulations. Medicine and

Science in Sports and Exercise 23: 578-585.

Nieman,D.C. and Nehlsen-Cannarella,S.L. (1992). Effects of endurance exercise on

the immune response. in Endurance in Sport. R.J.Shephard and P.-0.Astrand

(68)

tract

823-831.

(1 acute

(

129-131.

Pedersen,B.K. Tvede,N. Klarlund,K. Christensen,L.D. Hansen,F.R. Galbo,H.

Kharazm,A. and Halkjaer-Kristensen,J. (1990). Indomethacin in vitro and in

vivo abolishes post-exercise suppression of natural killer cell activity in

peripheral blood. International Journal of Sports Medicine 11: 127-131.

Pedersen,B.K. (1991). Influence of physical activity on the cellular immune system:

Mechanisms of action. International Journal of Sports Medicine 12: (Suppl

(69)

(1

(

1 ).

serum

acid related to delayed-onset muscle soreness? Physician

11: 124-131.

.

(

Sports

c

strenuous

Shephard,KJ. (1991). Physical activity and cancer. International Journal of Sports

Medicine 11: 413-420.

Shephard,R.J. Verde,T.J. Thomas,S.G. and Shek,P. (1991). Physical activity and

(70)

(

141.

cancer (Ed)

(1 Exercise,

a

onset

L

Soppi,E. Varjo,P. (1982).Effects of strenuous stress on

circulating lymphocyte number and function before

of Clinical Laboratory Immunology 8: 43-46.

after training. Journal

Spandoni,G. Spagnoli,A. Cianfarani,S. Del Principe,D. Menichelli,A. Di

Giulo,S. and Boscherini,B. (1991). Enhancement by growth hormone of

phorbol diester-stimulated respiratory burst in human polymorphonuclear

(71)

S. (1

system.

(72)

1

3

Neutrophils

The Exercise

(73)

recent

Degranulation releases proteolytic enzymes and other proteins involved the

microbicidal process into the phagolysosomal vacuole. These two processes,

together, result in the killing and digestion of the engulfed bacteria and, if prolonged,

the development of a localised inflammatory response. Although the respiratory

burst has been well studied, the mechanisms by which exercise may influence its

intensity are not well understood. Most studies undertaken to date show a transient

suppression of oxidative burst capacity immediately after exercise, although cycling

(74)

(75)

as stress or

arm

exercise

on

most

pools, or

within specific tissues Once released into the circulation,

neutrophils may remain mobile for up to 10 hours before undergoing re-margination.

the presence of chemoattractants, neutrophils are recruited in large numbers from

the blood stream to local inflammatory sites via the processes of diapedesis,

migration and chemotaxis (Fig 3.1 ). The neutrophil is one of the first cells to arrive

at sites of injury or infection. It releases a number of chemoattractants to amplify the

response through the recruitment of additional neutrophils and a variety of

(76)

structures.

or or

a

events

if a

exercise-induced damage to skeletal muscle

fragments from damaged myofibrils and components leaking from the extracellular

matrix may constitute a significant particulate stimulus to initiate phagocytosis

chemotactically-attracted neutrophils. A number of soluble stimuli are produced

simultaneously and these may contribute to the initiation of phagocytosis and the

degradation of tissue fragments by a combination of powerful chemical and

enzyme-catalyzed reactions (Weiss, 1989). The recruitment of additional neutrophils to the

(77)

L

Tissues

5

6

events

L

(78)

is not

More

release

The

et

as as

recognised

a

of exercise

has studied extensively during the twenty years. A number of reviews have

examined various aspects of the relationship between exercise and the distribution of

leucocytes (McCarthy and Dale, 1988; Keast et al. 1988; Shephard et al. 1991;

Nieman and Nehlsen-Cannarella, 1992). The experimental studies have shown,

collectively, an increase in the circulating leucocyte count after exercise, with the

magnitude of the leucocytosis reflecting the intensity and duration of the workload.

The leucocytosis is biphasic in nature, consisting of an immediate and transient

(79)

same source

rncrease

plasma cortisol after a hour delay, to release newly-differentiated

neutrophils from the bone marrow. Further work by McCarthy et al. (1992)

supported the model with findings that post-exercise increases in plasma cortisol

correlated with the magnitude of the delayed neutrophilia. However, contradictory

results have been cited by Hansen et al. (1991) who found no correlation between the

plasma concentration of cortisol and the number of circulating neutrophils.

(80)

state

an

resprnrise to

exposure

account some

states a

to

acetate

The mechanism of priming is not clear, but it is thought to relate to coordinated

changes a number of related intracellular parameters, which include

concentration of cytosolic Ca2+, the number and state of plasma membrane receptors,

the organisation of various components of the NADPH oxidase complex and the

orientation of the intracellular granules. Priming is thought to be influenced by the

presence of such factors as bacterial lipopolysaccharide (LPS), platelet activating

(81)

act as

et

subsequently,

to

a

et

phorbol esters.

is one common

to

1 are

an ennm1ce:u n""r""""''u,1·" to

were

To confirm the proposed role of growth hormone, studies of the effects of

recombinant GH supplementation on neutrophil function in vivo are required. With

the wide range of known physiological and metabolic effects of growth hormone,

some work has been done on the effects of GH supplementation on

non-immunological parameters. Rudman et al. (1990) showed that a six month period of

dietary supplementation of elderly men with recombinant human growth hormone

(82)

of enzyme

state. events

VAJ"UUi>'-' is

Stimulation/Recognition Phase

is normally metabolically "dormant"

its non-mitochondrial oxidative metabolism by a

are

is

it is stimulated to accelerate

of 50-100 to deliver reactive

products needed for its phagocytic and cytotoxic actions. Physiological stimuli at the

site of local inflammation include opsonized and IgG-coated microorganisms and

tissue fragments, the complement fragment C5a, various N-formylated methionyl

peptides, bacterial endotoxins, platelet activating factor (PAF), leukotriene B4

(83)

Final assembly

oxidase on plasma membrane. Activation of NADPH oxidase

to produce intra-and extra-cellular release of reactive oxygen species (ROS).

events

ROS

(84)

the neutrophilic movement triggered et are to are a nature chemotaxis

occurs the onset

not

are

lS

related to the properties of Fe receptors on the cell surface. receptors on

monocytic and granulocytic cells mediate responses initiated by immune complexes

of IgG-coated targets (Petroni et al. 1988). Human neutrophils possess two classes

of receptor for the constant region of IgG. There are approximately 10,000 - 20,000

Fe receptor type II (FcRII) and 100,000 - 300,000 Fe receptor type III (FcRIII)

molecules expressed on the plasma membrane of each neutrophil. Huizinga et

(1990) have shown that occupation of FcRII activates both the NADPH oxidase and

(85)

release

target

stimulation

undergoes a

a

lS

to

translocation of protein kinase C to

the inner of the plasma membrane; degranulation of enzymes and oxidase

components; and, ultimately, assembly and activation of the NADPH oxidase

complex (Omann et al. 1987; Edwards, 1991 ). The GN protein is located within the

specific granules and is translocated to the plasma membrane during degranulation

and activation (Retrosen et al. 1990). It appears that this is a common mechanism

involved in signal transduction irrespective of the origin of the stimulus. Wilde et al.

(86)

to stores 2+

Activated protein kinase C phosphorylates specific cytosolic proteins (e.g p47phox) which then translocate to the membrane to form the

NADPH oxidase.

(87)

not an

a common

1

are

In

c

events to

After the initiation of signalling process, a series intermediary steps takes place

that involve the production of secondary messengers. One of the most important

sequences is the turnover of phosphoinositides (Sandborg and Smolen, 1988) which

leads, initially, to the activation and translocation of protein kinase C from the cytosol

to the plasma membrane where it catalyses protein phosphorylation. It is generally agreed that signal transduction involves the stimulation of a

phosphatidylinositol-4,5-bis phosphate-(PtdlnsP2)-specific phospholipase C through a

(88)

stores

superoxide

1991).

to

more recent

it

to

et 1

are

an

it

enzyme release (Lipschitz et

Although the roles of phosphotidyl inositol hydrolysis, free cytosolic calcium

concentration and PKC are well documented for some stimuli, it is claimed by the

group of Rossi and co-workers that these changes are neither essential nor unique

steps sequence of events that brings about activation of the respiratory burst

and phagocytosis in neutrophils (Rossi et al. 1989; Della Bianca et al. 1990).

(89)

one

oxygen

a 'V\;J_r-_LLV'-'

components oxidase are located cytosol

(90)

et nor

a

been

one

to

lS to

monophosphate shunt metabolism. molecule of oxygen accepts a single

electron, resulting in the generation one molecule of 02- anion, according to the

reaction (see also Fig 3.5).

(91)

rac

NADPH Binding Unit

rac

on

(92)

a more

shown

Or to is

1

to

- to

+ +

peroxide

is It

the the vacuole due to dismutation

Subsequent acidification of the vacuole (which causes the pH to

(93)

on

+

(94)

it

+ +

a

et

summary, the effects of NADPH oxidase activation can be either beneficial and/or

harmful. Beneficial effects occur when species released within the phagolysornal

area or the immediate extracellular environment cause death to pathogenic

microorganisms. Harmful effects of NADPH oxidase activation include damage to

the neutrophil itself and to surrounding host tissues. Exercise-induced activation of

NADPH oxidase may have destructive effects on skeletal muscle fibres that

(95)

amounts

of

as

sources of

can

as

et

or

ensures a

enzyme-complex) was measured eight ran for a

significant elevation (p<O.O 1) was noted, with peak values being reached

immediately after exercise.

Elastase, contained within the primary granules of neutrophils, consists of a mixture

of four isoenzymes that act directly by degrading bacterial cell walls and indirectly by

potentiating the activity of lysozyme (Thorne et al. 1976) and cathepsin G (Odeberg

and Olsson, 197 6). The activities of these enzymes may facilitate the penetration of

(96)

to contents

et (

et

an

et

to

is most

(Estensen et

decreases the number of lactoferrin-positive vesicles the area they

occupy. Flow cytometric analysis showed degranulation was accompanied by

an overall decrease in cell size. Lactoferrin released from secondary granules

response to a chemotactic stimulus may contribute to the regulation of neutrophil

adhesiveness. Since neutrophils are thought to be the exclusive source of

intravascular lactoferrin, measureable changes in the plasma concentration of this

(97)

an

now been experiments

to

on

assessment

changes. that have examined "'""'"!".'~"

most have adopted the following systematic approach (see 3.1):

blood sampling before and after a bout of exercise, use of whole blood or isolated

preparations of pure neutrophils, and then assessment of one or more of the

following processes: (a) migration; (b) adherence; (c) expression of various receptors

on the plasma membrane; (d) phagocytic activity; (e) release of cytoplasmic enzymes

such as elastase and myeloperoxidase; (f) decrease in flow cytometric side scatter (as

(98)

to

means,

et

of

even

literature.

The differential responses are highlighted by Lewicki et aL (1987) who measured

adherence and phagocytic activity in neutrophils from 20 trained cyclists and 19

untrained male control subjects undertaking progressive cycle ergometer exercise to

exhaustion. In the trained cyclists, the adherence of neutrophils to vascular

endothelial tissues decreased whilst phagocytic activity remained unchanged. In

untrained subjects, the adherence of the neutrophils remained unchanged despite an

(99)

an H H I J U H

exercise of more

prolonged,

seven ""'"u"''

ten

IS

after

(100)

L

.

V02max

Gray et 1993 1 intervals at

'

100% V02max Ortega et al 1993a 1 h cycling at 50%

.

V02max Pyne et al. 1993a 40 min running

.

50% V02max

.

90% V02max Pyne et al. 1993b 16 weeks of

sw1mrmn training

Burst Oxidative Burst Oxidative Burst De granulation Phagocytic function Oxidative Burst Oxidative Burst

Pre and Post Decreased

Pre and Post

50% max

80% max

Pre and Post

Post, 1, Increased

6, 24 h post

Pre, Post and Increased

15 min post

Pre, Post, 1, 6

and 24 h post Increased

Decreased

(101)

was

at

status et

findings the

at rest was

speculated episodes of may

increase resistance to infection by "priming" microbicidal activity neutrophils at

low stimulus concentration, whilst regular intensive training may lead to a more

prolonged increase in susceptibility to infection by diminishing this activity.

Rodrigeuz et al. (1991) also examined neutrophil activity after physical exercise.

Eleven young, healthy but untrained male and female subjects were required to

until a state of exhaustion'. The precise nature of this exercise challenge, apart from

(102)

6-81

rates

were

not

made of

was

on

Although these studies exercise as a factor that affects aspects of the

phagocytic and microbicidal functions of circulating neutrophils, the variability in

age, sex and initial fitness levels of the subjects studied, the intensity and duration of

the exercise protocols used, and the different methodological procedures employed,

have made it difficult to form a clear picture of the relationship. Further well-designed studies are required to identify the nature of the factors and mechanisms that

(103)

It

a

experiments

tract

a to

enter

substantial number of demonstrating that exercise may impact negatively on

lymphoid cell distribution and function (reviewed by Cannon, 1993; Nieman, 1993;

Shephard et al. 1991). The large number of high intensity training sessions that elite

athletes undertake each week may leave a significant proportion of their circulating

neutrophils in a chronically refractory state. This may be one explanation for the

observation that elite athletes, as a group, are more prone to upper respiratory tract

(104)

to

to a

as

It is or

responses subsequent

contradictory results, moderate exercise appears to a delayed

enhancement of neutrophil activity whilst intensive exercise may suppress

et

women

to

some

Further

studies are required to determine the effects of acute exercise and prolonged training

on more discrete aspects of neutrophil activity, including attachment, phagocytosis

and the respiratory burst.

An understanding of these aspects of exercise, training, exercise-induced muscle

damage and associated immunological and inflammatory processes is important in

(105)

H'V<VAUUUGH.hY are more 01.,.v'V,.UV

(106)

1 L

Cannon,J.G. (1993). Exercise

Physiology 74: 973-981. S.

(

as

energy

resistance to infection.

Casimir,C. Chetty,M. Bohler,M-C. Garcia,R. and Fischer,A. (1991). Identification

of the defective NADPH-oxidase component in chronic granulomatous disease:

a study of 57 European families. European Journal of Clinical Investigation

(107)

Edwards,S (Ed)

1). Regulation of

Oxygen

Press. Cambridge. pp 35-76.

(

1

a

on

Cambridge University

Esaguy,N. Aguas,A.P. Vilanova,M. and Silva,M.T. (1991). Activation of human

neutrophils by phorbol ester decreases the cytoplasm compactness and the

lactoferrin content of the granulocytes. Journal of Leukocyte Biology 50:

(108)

(

phagocytes by 2 1

Goldstein,LM. Hoff stein,S Weissman,G. (1975). Mechanisms of

lysosomal enzyme release from human polymorphonuclear leukocytes.

of Cellular Biology 66: 647.

Gray,A.B. Telford,R.D. Collins,M. Baker,M.S. and Weidemann,M.J. (1993).

(109)

5

strenuous

1 1.

1

s

180.

Keast,D. Cameron,K. and Morton,A.R. (1988). Exercise

Sports Medicine 5: 248-267.

L

the immune response.

Kokot,K. Schaefer,R.M. Teschner,M. Gilge,U. and Plass R. (1988). Activation of

leucocytes by prolonged physical exercise. Advances in Experimental Medicine Biology 240: 57-63.

Lash,J.A. Coates,T.D. Lafuze,J. Baehner,R.L. and Boxer,L.A. (1983). Plasma

(110)

Macha,M. Schlafer,M. and Kluger,M.J. (1990). peroxide generation following physical exercise.

and Physical Fitness 30: 412-419.

(

. (1988).

on

neutrophil hydrogen

of Sports Medicine

McCarthy,D.A. and Dale,M.M. (1988). The leucocytosis of exercise: a review and model. Sports Medicine 6: 333-363.

McCarthy,D.A. MacDonald,!. Grant,M. Marbut,M. and Watling,M. (1992). Studies

on the immediate and delayed leucocytosis elicited by brief (30-min) strenuous

(111)

s

to acute

tract

(1

~·~·,·~ .. of sedentary men

acute moderate exercise. Physiology 66:

Ortega,E. Barriga,C. and De Fuente,M. (1993b). Study of the phagocytic process

in neutrophils from elite sportswomen. European Journal of Applied

Physiology 66: 37-42.

(112)

91

s

( 8).

exocytosis to

(1

(

phagocyte V"-'.u"''"'

265: 8745-8750.

Rodrigeuz,A.B. Barriga,C. and De la Fuente,M. (1991). Phagocytic function

blood neutrophils in sedentary young people after physical exercise.

International Journal of Sports Medicine 12: 276-280.

Rosales,C. and Brown,E.J. (1991). Two mechanisms for IgG Fe-receptor-mediated

phagocytosis by human neutrophils. The Journal of Immunology 146:

(113)

events

(1

phagocytic

granulomatous disease.

centers

et

( on

(1

molecular pathology

83: 1785-1793.

Shephard,R.J. Verde,T.J. Thomas,S.G. and Shek,P. (1991). Physical activity and

the immune system. Canadian Journal of Sports Sciences 16: 163-185.

Smith,J.A. and Weidemann,M.J. (1990a). The exercise and immunity paradox: a

neuro-endocrine/cytokine hypothesis. Medical Science Research 18: 749-753.

Smith,J.A. and Weidemann,M.J. (1990b). Effect of human growth hormone men

(114)

(1

Acta

Novotny,M.M. (1989).

Evidence for a defect hereditary and acquired myeloperoxidase deficiency. Blood 73: 1980-1985.

Uhlinger,D.J. Tyagi,S.R. Inge,K.L. and Lambeth,J.D. (1993). The respiratory

burst oxidase of human neutrophils. The Journal of Biological Chemistry 268: 8624-8631.

Uings,I.J. Thompson,N.T. Randall,R.W. Spacey,G.D. and Bonser,R.W. (1991).

Tyrosine phosphorylation is involved in receptor coupling to phospholipase D

(115)

(116)

Mechanical

(117)

cause

to

measurement of

as

measurement

(118)

to

are

as

restore

to

a

These be a common sequence of

immunological events leading to reversible damage to muscle

fibres (Appell et al. 1992), removal of injured tissue, promotion of growth and

repair, and, ultimately, restoration of normal physiological function (Fig 4.1). This

sequence can be divided, according to the model of Armstrong et al. (1991), into

three phases; (i) the auto genie phase where the loss of membrane integrity initiates a phagocytic response (2-6 hours post-injury); (ii) the inflammatory phase, characterised by the infiltration of active macrophages into the perimyosium (hours

(119)

L

1).

Inflammatory

(Macrophage Response)

(hours - days)

3. Regenerative Phase

(Tissue Repair)

(days - weeks)

stress

a common

(120)

skeletal

measurement

process are be

are not

IS

damaging to animals et humans

(Schwane et 1983a,b; Newham et 1988). Novel or unaccustomed exercise,

particularly eccentrically-biased exercise such as downhill running, specific

plyometric exercises and negative repetition work during resistance training, may

result in significant muscle damage and soreness. Schwane et al. (1983a) found that

oxygen uptake and the accumulation of blood lactate as indicators of metabolic cost,

were significantly lower during eccentric (downhill running) compared with

concentric exercise (level running), yet the former produced significantly greater

(121)

to an rate

were to

cross-1 The

cause some focal

a

disorganisation occurred in eccentrically-contracted leg. a subsequent study,

which also used the step test protocol, Newham et al. (1986) found muscles

expose to eccentric work developed tenderness and discomfort, accompanied by

changes in membrane permeability that allowed accumulation of technectium

pyrophosphate (isotope uptake) and efflux of cytoplasmic enzymes. Newham

(1988) concluded that, despite considerable evidence that muscle damage occurs

after eccentric contractions, the actual cause of delayed onset muscle pain, and the

role of the implied relationship between enzyme release and muscle fibre

(122)

as enzyme efflux - were

studied _three~~'""'"'A two

was greatest

progressively bouts.

the recovery rates

second bouts.

is to

occur causes

waste

AAAU~AAA~A•VAU rate

cause these

ATP synthesis not ATP hydrolysis so insufficient

ATP is available to maintain contraction specific compartments of muscle.

Animal experiments have shown that experimental manipulation of calcium-sensitive

ATPase activity, leading to decreased steady-state levels of ATP, causes rapid and

dramatic damage to the ultrastructural components of muscle (Duncan and Jackson,

1987). Evidence of ultrastructural damage after temporary incomplete ischaemia is

characteristic of myopathies of ischaemic origin (Sjostrom et al. 1982).

O'Reilly et al. (1987) suggested that metabolic stress - in the form of muscle

(123)

occur or together,

exact nature of

to

exercise can

to

were

was no

u•;-,•u··-exercise can

depending on

to

the of myofibre, sarcomeres sarcoplasmic

reticulum, or a combination of these (Friden et 1984; Stauber, 1989). Animal

studies have shown that exercise-induced muscle damage may initiate the release of

filament fragments whose peptides are capable of being depicted as chemotactic

signals for leucocytes (Michna, 1989). This work provides a mechanism linking

muscle damage with mobilisation of an immune and inflammatory response.

Damage to muscle fibres is normally assessed by measuring the efflux of specific

cytosolic enzymes into the circulation, combined with histological techniques or

(124)

are

1

more

streaming or

the content

et

a

as a AAAVCH~·~· ~~'""~ ... et

is found exclusively and so most

exercise-induced muscle damage have included its measurement (Hortobagyi and Denahan,

1989). The time course of its efflux after exercise has been studied extensively

(Noakes, 1987; Clarkson et al. 1988; Hortobagyi and Denahan, 1989). Peak CK

efflux is delayed until well after the cessation of exercise, with the extent of the

delay dependent upon the type of exercise undertaken. Although there may be

detectable increase in activity immediately after exercise, its peak is usually reached

between 24 and 72 hours post-exercise (Lijnen et al. 1988). Tiidus and Ianuzzo

(125)

more

It is

free

to a

general hypothesis be

mediated a rise free

has been on the basis of

membrane to

was

been referred to as overload phase'

Armstrong et al. (199 in their model of exercise-induced damage to skeletal

muscle. A rise in free cytosolic calcium may also be related, independently, to the

activation of the respiratory burst in phagocytic cells (Rossi, 1986), which suggests

that parallels may exist between the mechanisms involved in exercise-induced tissue

damage and the activation of cells involved in non-specific immune responses.

Exercise-induced Oxidative Damage to Muscle.

Much attention has been paid to free radicals and their putative role as mediators of