S9
Immunologic Mediators as Parameters of the Reaction to Strenuous Exercise H. Northof! andA. Berg2 1DRK-Blutspendezentrale and Dept. of Transfusion Medicine, University of Ulm, Germany 2Dept. of Physical Performance Medicine, University of Freiburg, Germany
H. Northoff and A. Berg, Immunologic Mediators as Parameters of the Reaction to Strenuous Exercise. IntJSportsMed,Vol 12,Suppl l,ppS9—S15, 1991. Both the unspecific and the specific branch of the immune system are triggered and governed by contact and by a set of cytokines, including interleukin-l (IL-l), interleukin-6 (IL-6), and tumor necrosis factor (TNF). These mediators, which are produced by activated macrophages and other cells, have also multiple (pleiotropic) effects on
different cells and organs. While TNF and IL-I have strongly proinflammatory effects and seem to play a critical role in clinical situations such as septic shock, IL-6 has more
restorative effects by being the main inducer of the acute phase response of the liver. The monokines also induce fever and release of ACTH in the brain. Strenuous exercise leads to a significant elevation of cytokines in the serum thereby eliciting an acute phase response. Analysis of systemic cytokines in the serum of marathon runners by the 7TD I cell line assay revealed that the observed activity is very likely IL-6.
Key words
Exercise, strenuous exercise, cytokines, mediators, monokines, interleukin-l, interleukin-6
clonal
+ pension B and T cells
kines tissue of lamation
unspecific immune response
+ differentiation into T effectors or regulators and plasma cells
specific cellular and humoral immune response with memory
Fig. 1 The role of cytokines in the immune response. The specific and the unspecific immune response are governed by a set of cytokines which induce enzyme production, killing, growth, or differentiation. Tu = tumor cell; NK = natural killer cells; Mø = macrophages; B = lymphocytes; T = T lymphocytes.
producing plasma cells (Fig. 1). Action and function of both
branches of the immune system are triggered and governed by
contact (antigen-cell; cell-cell) and by the action of a set of polypeptides — mostly glycosylated and 15—25 kDa by molecular weight — which are known as mediators, cytokines, lym-
phokines, monokines, or interleukins (21, 45, 57, 58). They all Basic Structure of the Immune System and the Role of Mediators
The immune system of mammals disposes of two lines of defense against invading organisms such as bacteria, parasites, or viruses and possibly tumor cells: the unspecific line, which involves unspecific recognition as foreign
followed by instant phagocytosis and/or killing and which comprises the action of macrophages, granulocytes, or natural killer (NK) lymphocytes, and the specific line, which involves specific recognition of antigen by T and B lymphocytes, which then poliferate and differentiate into cytolytic T effector cells or regulator cells (T helper, Th; T supressor, Ts) or antibody-
Int.J.SportsMed. 12(1991)S9—S15 GeorgThieme Verlag StuttgartNew York
are active in extremely low concentrations and function by triggering their correspondent receptor on their target cells. The role of cytokines in the specific immune response is shown in Fig. 2. To trigger T cells and most B cells, antigen has to be
picked up, processed, and presented together with molecules of the own MHC II complex (HLA-DR) by macrophages or other antigen presenting cells. The latter supply IL-n-I, which serves as a second signal for T helper (Th) cells to produce IL2. IL-2 is the true mitogen which drives the clonal expansion of antigen-specific T effector and helper cells. Other mediators such as interferon , IL-4, -5, and-6 and tumor necrosis factor (TNF) are obligatory or potential regulators of growth, differentiation, and effector functions of T and B cells at various stages of their development. Others such as macrophage activating factor (MAF) and IL-I or chemotaxins attract and activate the cells of the unspecific defense. Several of the factors can also induce one or more of the colony-stimulating factors
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bacteria, viruses parasites, tumor cells
Abstract
mt. J. Sports Med. 12(1991)
H. Nort hoff and A. Berg
Pleiotropic Effects of Monokines It is now known that a host of different nonlym-
phatic cells such as endothelial cells and keratinocytes or mesangial cells can produce cytokines including IL-l, TNF, and IL-6 (10, 20, 39, 41, 60, 61). Still, it is widely accepted that
the major producer of the latter mediators are the monocytes
and macrophages. IL-l, TNF, and IL-6 are produced concomitantly (along with several other activities) when the cells
are stimulated by a variety of infectious or noninfectious agents. The effects of these monokines are by no means limited to cells of the immune system (Table 1). Indeed, they refer to many cells and organs. IL-I (31) has been shown to express adhesion molecules on endothelial cells and fibroblasts; to lead
to bone resorption and destruction of cartilage; to stimulate growth of fibroblasts, mesangial cells, synovial cells, and epithelial cells; to induce prostaglandin and enzyme synthesis in man; to kill certain cell lines; to be catabolic for muscle cells
(although this has been challenged recently) (34, 50); to induce part of the acute phase response in liver cells; to induce fever and to release ACTH. Finally, IL-I and TNF both induce IL-6(13, 39).
44 A
Fig. 2 The role of cytokines in the specific immune response. Different cytokines are active at different stages of development and activation. G=granulocyte; MHC=major histocompatibility complex; T rest= resting I cell; Th= helper T cell; Te= cytolytic effector T cell; S=stem cells; PL= plasma cell; IL-2R=IL-2 receptor. Other symbols, see Fig. 1.
TNF (17) was named after its ability to destroy some tumors by obliterating their vessels or by direct attack on the cells. TNF affects virtually the same range of cellular systems as IL- 1. In many cases the effects are identical or synergis-
tic. In addition, TNF is a potent inducer of IL-l in macro-
(G-CSF, M-CSF) or directly interfere with hemopoiesis. Some
substances, e. g, the lipid A part of bacterial lipopolysaccharide (LPS, endotoxin) are extremely potent stimulators of cytokine productin (IL-l, TNF, and IL-6) without being necessarily antigenic. This can have positive, adjuvant effects. It can, however, also be detrimental (see below).
phages and endothelial cells (22, 53). It is probably justified to say that TNF has the most prominent proinflammatory effects of all cytokines, while its role in the specific immune response is more limited. IL-6 (28, 63), finally, shares some but not all of the effects of 11-1 and TNF. Apart from its important functions as growth and differentiation factor in specific immunity and hematopiesis, its most pronounced effects are on the liver and on the central nerve system (CNS) (18f 28, 30, 59, 63, 70). One
can probably say that the overall effects of IL-6 are more restorative than proinflammatory. Some tumors such as myelomas seem to feed on an autocrine loop of IL-6 (40). Tab. 1 Pleuotropic effects of monokines
ILTNF skin
vessels
joints
muscle
conocect. kidney
liner
CNS
tissue growth of melanocytes
fever, somnolence, anorexia
collagen synthesis
[elial growth
L ACTH
induction of coagulation
acute phase reaction
j and extravauatjon loss of tonus angloge
tesis
jatocvtes
destruction of cartilage I inhibition of collagen synthesis I proliferation of synovial cells I and fibroblasi,
cell proliferation
[1esorPt ion proteolysin IlL-I = catabolin) cachenia
proliferation Iwound healingi induction of PC and other mediators synthesis/mobilization of triglycerides ITNF = cachectin)
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Sb
Int.J.SportsMed. 12 (1991) Si!
Immunologic Mediators as Parameters of the Reaction to Strenuous Exercise Endotoxin ILPS)
Trauma
infection (Endotoxin & Co.l
NoninfectiouS
irritants
1
IL-6, IL-1,TNF
TNF, IL-i (IL-61
TNF
N ///
AC?
cortisone
L fever
anorexia cachexia
tachycardia warm hypotension disseminated intravascular coagu
somnolenc
Septic shock
Fig. 3 The role of cytokines in septic shock. Endotoxin activates macrophages, which respond with massive production of monokines. These can induce all the symptoms of septic shock. Injection of recombinant cytokine has the same effects.
Role of Monokines in Some Clinical Settings
To demonstrate how the described actions of the mediators translate into participation in concrete clinical settings, two clinical situations may briefly be discussed. The first is the septic shock which is caused by endotoxin (lipopoly-
saccharide, LPS), the major toxic principle of gram-negative bacteria (Fig. 3). Minute amounts of LPS (pg/mi range!) induce massive production of cytokines by monocytes and macrophages (55, 56). The cytokines in turn can produce all the characteristic symptoms such as fever, somnolence, warm hypotension, diarrhea, chachexia, and disseminated intravascular coagulation. TNF and IL-i can both cause the whole range of symptoms upon injection (48, 65, 66). Other investigations confirm the critical role of monokines in this syndrome (27). The same mediators which fulfill indispensable tasks in the lokal defense against mircroorganisms and in the immune response can also cause the death of the body when the system is flooded. The second setting is the acute phase reaction. Anything which causes major induction of monokines — infections, trauma, or noninfectious irritants — will ulitmately lead to a massive change of liver cell metabolism, which is called the acute phase response (Fig. 4). It consists in increased produc-
tion of many proteins mostly transport proteins and protease inhibitors — and concomitantly decreased production of other proteins such as albumin and transferrin. A visible sign of the massive shift in serum proteins is an accelerated blood sedimentation rate. The main task of the acute phase response is obviously to restore lost functional proteins (such as C3 and
— decreased production of
increased production of
albumin and transferrin (Fe; ZNfl antitrypsine haptoglobin c reactive protein ICAPI fibrinogen C3
a5 acid glycoprotein serum amyloid A (SAAI ceruloplasmin factor B of complement Acute phase response
Fig. 4 The role of cytokines in the acute phase response. Monokines induce the whole spectrum of the acute phase response of liver cells. These need permissive amounts of cortisone, which can also be induced via ACTH by the monokines.
haptoglobin) and to counter the adverse side effects of the pri-
mary inflammatory reaction. Like inflammation itself, the acute phase response is also triggered and governed by the monokines. Here, however, IL-6 is clearly in the lead (2—4, 8, 18, 19, 30, 55, 62). TNF and IL-i both can only induce aminor
spectrum of acute phase proteins. Induction of acute phase proteins by liver cells is dependent on permissive amounts of corticoids (8). Interestingly, IL-i and IL-6 both can induce the pituitary gland to release ACTH, thereby generating the required cortisone level as soon as a systemic reaction is staged (69). Other diseases where a critical role for the monokines has
been implied are rheumatic fever, mononucleosis, and myeloma. Detection of Cytokines in Serum
General Aspects There are many ways to detect cytokines. They comprise bioassays in vivo and in vitro, and radio or enzymelinked immunoassays (RIA, ELISA). Many of the bioassays use cells which are dependent on cytokine for proliferation. Others measure prostaglandin production, differentiation, or cytotoxicity. Many of the assays detect several cytokines, some
are, however, quite specific (Table 2). To these belong the 7TD1 assay for IL-6 (1), the CTLL assay for IL-2, and the EL4/CTLL assay for IL-i. Most of the bioassays can be quantitated by titration and are very sensitive (29, 32). RIA and ELISA seem to be less complicated at first glance; they can, however, be too insensitive, detect inactive degradation products, or be corrupted by unspecific reactions. They are also prohibitively expensive where large series have to be tested. In
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rh IL-i rh
S12 mt. J. Sports Med. 12(1991)
H. Nort hoff andA. Berg
Table 2 Some bioassays fo r cytokines and th eir specific ity.
ports of systemic cytokines include mildly elevated levels of interferon-a (IFN a) after immunizations or hepatitis (7, 11).
IL-i
IL-6
TNF
IL-2
In vivo fever induction in rats or rabbits
+
+
+
?
C3HHeJ mouse thymocyte
+
+
—
+
—
—
—
+
assay
IL-2 dependent T cell line )CTLL) growth
+
—
—
(+)
7TDI hybridoma growth
—
+
—
—
L-929 cytotoxicity
—
—
+
—
EL-4/CTLL (1L-2 induction)
tissue, lympliokines may be detected by demonstration of mRNA or by immunofluorescense. Detection of cytokines in serum presents many problems. A major one is caused by the existence of so-called inhibitors, which may themselves be or not be true regulatory lymphokines such as the IL-i inhibitor described by Arend et a!. and Roux-Lombard et al. (6, 64). Work on that particular
field has left scores of frustrated investigators who found
Detection of Systemic Cytokines after Strenuous Exercise To our knowledge, the first evidence that an acute phase response similar to that which follows trauma or infection can be induced by strenuous exercise dates some 20 years back (37, 38). Cytokines — although their mere existence was still fiercely denied by a solid majority of immunologists — were soon discussed as possible cause (36, 43). Since 1979, im-
proved semiquantitative assays (51), techniques of purification, and later cloning have quickly removed the doubts. Since ELISAs have not yet been available or too insensitive, substantial efforts had to be made to overcome the technical problems
of bioassays as discussed above. Convincing evidence that mono-/cytokines are indeed systemically present after strenuous exercise was presented in 1983, using fever induction in rats (5, 16). The authors later confirmed their findings using column fractionation of serum and subsequent analysis by the thymocyte assay (14). They also report that a "suppressor" activity eluted in the 20—50 kDa range. According to the state of knowledge at that time, the detected cytokine activity was attributed to IL- 1 by the authors and some pain was taken to dissociate it from IL-2. From today's point of view, however, we
have to acknowledge that the observed cytokine might also have been IL-6 or a mixture of IL-l and IL-6 (TNF is inactive in
themselves purifying to homogeneity what turned out to be an artefact, (e. g., thymidine kinase, which cleaves off the label from 3 H-thymidine).
the thymocyte assay and therefore excluded). There is also a possibility that a different, hitherto undefined mediator may be involved.
So far the true nature of the serum-borne inhibitor of IL-i, IL-6, and TNF bioassays can only be labeled "un-
Recently a further cytokine, IFN-ct, has been found elevated in plasma after exercise (67). So far we are not aware of any further reports. Another way to investigate the possible involvement of lympho-monokines in exercise would
known", even if anecdotal reports claim that they could pinpoint the activity to a 20—50 kDa moiety (14). There is, however, no doubt that these inhibitors exist, active up to 1: 100 dilution, and that sytemic lymphokines can only be detected by most bioassays if they are active in still higher dilutions, unless the inhibitor can be removed or knocked out. Recently we
have found that simple treatment with 56 °C can destroy the serum-borne inhibitor of the 7TD 1 assay for IL-6, leaving the IL-6 activity itself essentially intact. This finding was also con-
firmed by personal communication from Dr. v. Snick, who kindly provided the 7TD1 cell line. How far inhibitors can in-
be to monitor the in vitro production of cytokines by cells drawn before and after exercise. This has been investigated recently (42). The experiments showed that LPS-induced mono-
kine production by blood monocytes was significantly increased after running, pointing to an activation of the macrophage system by exercise. A concomitant decrease in PHA-induced production of IL-2 by T cells, on the other hand, can be exhaustively explained by the exercise-induced shift in the T4/T8 ratio (T4 cells are far more efficient producers of IL-2).
terfere with RIA and ELISA is unknown at present.
Beyond reasonable doubt, lymphokines can be detected systemically in certain situations (9, 23, 33, 47, 52,
68). Transient elevation can be demonstrated regularly by ELISA in patients with polytrauma or sepsis. IL-I is definitely
more difficult to demonstrate. Reports of circulating IL-i which use thymocyte assay or fever induction must be interpreted with caution. While there is no doubt that these assays measure "real" cytokine activity, it is uncertain that the cytokine detected was IL-i. Both assays are also reactive to IL-a. Using an assay which is to our knowledge quite specific for IL-
6, it could be shown that the serum-borne cytokine activity, which was elevated in severely burned patients, was indeed IL-
6 (54) and that the IL-6 levels nicely correllated with fever. Since IL-6 was only identified as a separate monokine in late 1987 (28), reports of IL-i activity up to then and perhaps later should be cautiously interpreted as IL-l and/or IL-6. Other re-
Macrophage activation was also shown measuring increased phagocytic and enzymatic activity (24, 25). Activated macrophages could also be demonstrated invading muscle tissue after strenuous exercise (49). Intriguingly, a recent report has been able to demonstrate membrane-bound and perivascular IL-i IEI in muscle up to 5 days after exercise (15). This could also be extracted and was active in a bioassay.
TNF or IL-la could not be demonstrated. Taken together, available data from the literature clearly show that the response to strenuous exercise shows striking similarities with the response to infection. Several investigations point to an activation of the macrophage system which is followed by monokine production up to a systemically elevated and relevant level.
The remaining question is what induces the activation of the macrophages. Beside LPS and specific antigen,
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Specific for
Assay
mt. J. Sports Med. 12(1991) S13
Immunologic Mediators as Parameters of the Reaction to Strenuous Exercise fever ..— (hypothalamus)
culated in arbitrary units and compared. In 15 of the 17 runners, there was a significant increase of IL-6 measurable immediately after exercise, which was in most cases back to pre-run values 24 h later. At the same time we found no detectable IL-i
by an ELISA (detection limit 15 pg/mI) for IL1-3. IL-i was (hypophysis)
cortisone
also not found in the thymocyte assay. No attempts were made to remove the inhibitor which was present in 1:40—1: 100 dilution. Our present view of the involvement of monokines in the
reaction to exercise is elucidated in Fig. 5. IL-i TNFand IL6 are released from macrophages locally in the muscle and possibily in other organs. IL-6, which may be induced by the
primary stimulus and by IL-i, is generated in sufficient amount to reach detectable levels in the blood where it is prob-
ably transported in active form and sheltered from degrada______________
acute phase
inflaormatI1I4 44 44 4 response
Fig. 5 Possible role of monokines in exercise-induced acute phase response. Tissue injury and possibly LPS released from the gut activate monocyte/macrophages which produce IL-i, TNF, IL-6, and other inflammatory mediators. IL-i and TNF also induce IL-6 production in epithelial cells. lL-6 reaches systemic levels sufficient to induce the acute phase and to be detected by the 7TD1 bioassay.
of the acute phase response in the liver. Thus, we believe that IL-6 is the main systemic mediator after exercise causing the acute phase response. Even so, a bit of caution about the identity of the presumed IL-6 seems appropriate until a molecular weigth estimate and neutralization by specific antibody have been obtained. So far we could not do these experiments for lack of material. We also could not yet test the 7TD 1 cell line with IL-7, a newly described cytokine which seems to be active in the thymocyte assay, too. Finally, we cannot exclude that sensitive and specific assays might pick up some IL-i if the inhibitor is removed.
a host of nonspecific irritants, including phagocytosis per Se,
have been reported to stimulate IL-i production (60). Part of these observations may have been due to contamination of reagents, material, or media with LPS, a virtually ubiquitious agent. We were unable to induce IL-I by lysates of erythrocytes or muscle or organ extracts (unpublished) and so were others (14). Immunophagocytosis in our hands was likewise ineffective. Still, the possibility that detritus from destroyed cells induces the reaction in vivo cannot be ruled out by these experiments.
Another possibility is that LPS which is released from the gut under exercise (12, 44) may cause the acti-
vation. LPS is effective in very low concentrations, and the LPS-neutralizing capacity of serum lipoproteins (26) may be overrun. So far we have to note that the true reason for the macrophage activation remains unclear.
If we accept that exercise leads to production of cytokines, we face the question if this may be good or bad for our health. At present we feel unable to say if a given parame-
ter, e. g., elevation or suppression of one or more cytokines, has to be viewed as a good sign or as a warning. Maybe the future will provide correlations making the measurement of cytokines a useful diagnostic tool. So far, our speculation is that cytokines may well work in both directions. It is known that animals which are raised under sterile conditions are underdeveloped and prone to infections. Moderate but regular exposure to antigen and regular exercise might both provide cytokines necessary to keep up a "trained" immune system. On the other hand, chronic strenuous, exhaustive exercise may overexpose or exhaust the immune system thereby causing the immunodeficiencies which can occur in athletes with corresponding training programs. References
IL-6 Is Probably the Major Systemic Cytokine after Strenuous Exercise, Causing the Acute Phase Response
As pointed out above, the data from the literature convincingly showed that systemic elevation of cytokines occurs in serum after strenuous exercise. The identity of the observed cytokine is, however, uncertain. Since the full spectrum of the acute phase response is induced in the liver cells and since IL-6 is the main inducer which can do this, we feel that IL-6 must be involved in the reaction at some point. One might argue that IL-6 may be induced in the liver sinusoids by systemic IL-I. On the other hand, IL-6 itself (± IL-i) might be the systemic mediator. This question has recently been tested in our lab on 17 participants of a marathon run (manuscript in preparation). Blood was drawn before, immediately after, and 24 h after the run. The serum was tested using the 7TD 1 assay, which is very sensitive for IL-6 but is definitely negative for all other known cytokines discussed so far. The results were cal-
2
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63 Ritchie D. G., Fuller G. M.: Hepatocyte-stimulating factor: a monocyte derived acute-phase regulatory protein. Ann NY Acad Sci408:490—502, 1983. 64 Roux-Lombard P., Modoux C., Dayer J.-M.: Long-term cultures of purified human peripheral blood monocytes produce an interleukin-1 (IL-I) inhibitor of 23 kD which blocks IL-i binding and activity. Abstract, 7th mt. Congress of Immunology, 1989. 65 Tracey K. J., Beutler B., Lowry S. F., Merryweather J., Wolpe S., Milsark I. W., Hariri R. J., Fahey T. J. III, Zentella A., Albert J. D., Shires G. T., Cerami A.: Schock and tissue injury induced by recombinant human cachectin. Science 234:470—474, 1986. 66 TraceyK.J.,FongY.,HesseD.G.,ManogueK.R.,LeeA.T.,Kuo G. C., Lowry S. F., Cerami A.: Anti-cachectin/TNF monoclonal antibodies prevent septic shock during lethal bacteremia. Nature 330:662—664,1987. 67 Viti A., Muscettola M., Paulesu L., Bocci V., Almi A.: Effect of exercise on plasma interferon levels. JApplPhysiol 59 (2): 426—428, 1985. 68 Waage A., Haistensen A., Espevik T.: Association between tumor necrosis factor in serum and fatal outcome in patients with meningococcal disease. Lancetl: 355—7, 1987. 69 Woloski B. M. R. N. J., Smith E. M., Meyer W. Jill, Fuller G. M.,
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Int.J.SportsMed. 12 (1991) S15
Friv.-Doz. Dr. H. Northoff
DRK-Blutspendezentrale Helmholtzstrafie 10 W-7900 Ulm Germany
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Immunologic Mediators as Parameters of the Reaction to Strenuous Exercise
Immunologic Mediators as Parameters of the Reaction to Strenuous Exercise H. Northof! andA. Berg2 1DRK-Blutspendezentrale and Dept. of Transfusion Medicine, University of Ulm, Germany 2Dept. of Physical Performance Medicine, University of Freiburg, Germany
H. Northoff and A. Berg, Immunologic Mediators as Parameters of the Reaction to Strenuous Exercise. IntJSportsMed,Vol 12,Suppl l,ppS9—S15, 1991. Both the unspecific and the specific branch of the immune system are triggered and governed by contact and by a set of cytokines, including interleukin-l (IL-l), interleukin-6 (IL-6), and tumor necrosis factor (TNF). These mediators, which are produced by activated macrophages and other cells, have also multiple (pleiotropic) effects on
different cells and organs. While TNF and IL-I have strongly proinflammatory effects and seem to play a critical role in clinical situations such as septic shock, IL-6 has more
restorative effects by being the main inducer of the acute phase response of the liver. The monokines also induce fever and release of ACTH in the brain. Strenuous exercise leads to a significant elevation of cytokines in the serum thereby eliciting an acute phase response. Analysis of systemic cytokines in the serum of marathon runners by the 7TD I cell line assay revealed that the observed activity is very likely IL-6.
Key words
Exercise, strenuous exercise, cytokines, mediators, monokines, interleukin-l, interleukin-6
clonal
+ pension B and T cells
kines tissue of lamation
unspecific immune response
+ differentiation into T effectors or regulators and plasma cells
specific cellular and humoral immune response with memory
Fig. 1 The role of cytokines in the immune response. The specific and the unspecific immune response are governed by a set of cytokines which induce enzyme production, killing, growth, or differentiation. Tu = tumor cell; NK = natural killer cells; Mø = macrophages; B = lymphocytes; T = T lymphocytes.
producing plasma cells (Fig. 1). Action and function of both
branches of the immune system are triggered and governed by
contact (antigen-cell; cell-cell) and by the action of a set of polypeptides — mostly glycosylated and 15—25 kDa by molecular weight — which are known as mediators, cytokines, lym-
phokines, monokines, or interleukins (21, 45, 57, 58). They all Basic Structure of the Immune System and the Role of Mediators
The immune system of mammals disposes of two lines of defense against invading organisms such as bacteria, parasites, or viruses and possibly tumor cells: the unspecific line, which involves unspecific recognition as foreign
followed by instant phagocytosis and/or killing and which comprises the action of macrophages, granulocytes, or natural killer (NK) lymphocytes, and the specific line, which involves specific recognition of antigen by T and B lymphocytes, which then poliferate and differentiate into cytolytic T effector cells or regulator cells (T helper, Th; T supressor, Ts) or antibody-
Int.J.SportsMed. 12(1991)S9—S15 GeorgThieme Verlag StuttgartNew York
are active in extremely low concentrations and function by triggering their correspondent receptor on their target cells. The role of cytokines in the specific immune response is shown in Fig. 2. To trigger T cells and most B cells, antigen has to be
picked up, processed, and presented together with molecules of the own MHC II complex (HLA-DR) by macrophages or other antigen presenting cells. The latter supply IL-n-I, which serves as a second signal for T helper (Th) cells to produce IL2. IL-2 is the true mitogen which drives the clonal expansion of antigen-specific T effector and helper cells. Other mediators such as interferon , IL-4, -5, and-6 and tumor necrosis factor (TNF) are obligatory or potential regulators of growth, differentiation, and effector functions of T and B cells at various stages of their development. Others such as macrophage activating factor (MAF) and IL-I or chemotaxins attract and activate the cells of the unspecific defense. Several of the factors can also induce one or more of the colony-stimulating factors
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bacteria, viruses parasites, tumor cells
Abstract
mt. J. Sports Med. 12(1991)
H. Nort hoff and A. Berg
Pleiotropic Effects of Monokines It is now known that a host of different nonlym-
phatic cells such as endothelial cells and keratinocytes or mesangial cells can produce cytokines including IL-l, TNF, and IL-6 (10, 20, 39, 41, 60, 61). Still, it is widely accepted that
the major producer of the latter mediators are the monocytes
and macrophages. IL-l, TNF, and IL-6 are produced concomitantly (along with several other activities) when the cells
are stimulated by a variety of infectious or noninfectious agents. The effects of these monokines are by no means limited to cells of the immune system (Table 1). Indeed, they refer to many cells and organs. IL-I (31) has been shown to express adhesion molecules on endothelial cells and fibroblasts; to lead
to bone resorption and destruction of cartilage; to stimulate growth of fibroblasts, mesangial cells, synovial cells, and epithelial cells; to induce prostaglandin and enzyme synthesis in man; to kill certain cell lines; to be catabolic for muscle cells
(although this has been challenged recently) (34, 50); to induce part of the acute phase response in liver cells; to induce fever and to release ACTH. Finally, IL-I and TNF both induce IL-6(13, 39).
44 A
Fig. 2 The role of cytokines in the specific immune response. Different cytokines are active at different stages of development and activation. G=granulocyte; MHC=major histocompatibility complex; T rest= resting I cell; Th= helper T cell; Te= cytolytic effector T cell; S=stem cells; PL= plasma cell; IL-2R=IL-2 receptor. Other symbols, see Fig. 1.
TNF (17) was named after its ability to destroy some tumors by obliterating their vessels or by direct attack on the cells. TNF affects virtually the same range of cellular systems as IL- 1. In many cases the effects are identical or synergis-
tic. In addition, TNF is a potent inducer of IL-l in macro-
(G-CSF, M-CSF) or directly interfere with hemopoiesis. Some
substances, e. g, the lipid A part of bacterial lipopolysaccharide (LPS, endotoxin) are extremely potent stimulators of cytokine productin (IL-l, TNF, and IL-6) without being necessarily antigenic. This can have positive, adjuvant effects. It can, however, also be detrimental (see below).
phages and endothelial cells (22, 53). It is probably justified to say that TNF has the most prominent proinflammatory effects of all cytokines, while its role in the specific immune response is more limited. IL-6 (28, 63), finally, shares some but not all of the effects of 11-1 and TNF. Apart from its important functions as growth and differentiation factor in specific immunity and hematopiesis, its most pronounced effects are on the liver and on the central nerve system (CNS) (18f 28, 30, 59, 63, 70). One
can probably say that the overall effects of IL-6 are more restorative than proinflammatory. Some tumors such as myelomas seem to feed on an autocrine loop of IL-6 (40). Tab. 1 Pleuotropic effects of monokines
ILTNF skin
vessels
joints
muscle
conocect. kidney
liner
CNS
tissue growth of melanocytes
fever, somnolence, anorexia
collagen synthesis
[elial growth
L ACTH
induction of coagulation
acute phase reaction
j and extravauatjon loss of tonus angloge
tesis
jatocvtes
destruction of cartilage I inhibition of collagen synthesis I proliferation of synovial cells I and fibroblasi,
cell proliferation
[1esorPt ion proteolysin IlL-I = catabolin) cachenia
proliferation Iwound healingi induction of PC and other mediators synthesis/mobilization of triglycerides ITNF = cachectin)
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Sb
Int.J.SportsMed. 12 (1991) Si!
Immunologic Mediators as Parameters of the Reaction to Strenuous Exercise Endotoxin ILPS)
Trauma
infection (Endotoxin & Co.l
NoninfectiouS
irritants
1
IL-6, IL-1,TNF
TNF, IL-i (IL-61
TNF
N ///
AC?
cortisone
L fever
anorexia cachexia
tachycardia warm hypotension disseminated intravascular coagu
somnolenc
Septic shock
Fig. 3 The role of cytokines in septic shock. Endotoxin activates macrophages, which respond with massive production of monokines. These can induce all the symptoms of septic shock. Injection of recombinant cytokine has the same effects.
Role of Monokines in Some Clinical Settings
To demonstrate how the described actions of the mediators translate into participation in concrete clinical settings, two clinical situations may briefly be discussed. The first is the septic shock which is caused by endotoxin (lipopoly-
saccharide, LPS), the major toxic principle of gram-negative bacteria (Fig. 3). Minute amounts of LPS (pg/mi range!) induce massive production of cytokines by monocytes and macrophages (55, 56). The cytokines in turn can produce all the characteristic symptoms such as fever, somnolence, warm hypotension, diarrhea, chachexia, and disseminated intravascular coagulation. TNF and IL-i can both cause the whole range of symptoms upon injection (48, 65, 66). Other investigations confirm the critical role of monokines in this syndrome (27). The same mediators which fulfill indispensable tasks in the lokal defense against mircroorganisms and in the immune response can also cause the death of the body when the system is flooded. The second setting is the acute phase reaction. Anything which causes major induction of monokines — infections, trauma, or noninfectious irritants — will ulitmately lead to a massive change of liver cell metabolism, which is called the acute phase response (Fig. 4). It consists in increased produc-
tion of many proteins mostly transport proteins and protease inhibitors — and concomitantly decreased production of other proteins such as albumin and transferrin. A visible sign of the massive shift in serum proteins is an accelerated blood sedimentation rate. The main task of the acute phase response is obviously to restore lost functional proteins (such as C3 and
— decreased production of
increased production of
albumin and transferrin (Fe; ZNfl antitrypsine haptoglobin c reactive protein ICAPI fibrinogen C3
a5 acid glycoprotein serum amyloid A (SAAI ceruloplasmin factor B of complement Acute phase response
Fig. 4 The role of cytokines in the acute phase response. Monokines induce the whole spectrum of the acute phase response of liver cells. These need permissive amounts of cortisone, which can also be induced via ACTH by the monokines.
haptoglobin) and to counter the adverse side effects of the pri-
mary inflammatory reaction. Like inflammation itself, the acute phase response is also triggered and governed by the monokines. Here, however, IL-6 is clearly in the lead (2—4, 8, 18, 19, 30, 55, 62). TNF and IL-i both can only induce aminor
spectrum of acute phase proteins. Induction of acute phase proteins by liver cells is dependent on permissive amounts of corticoids (8). Interestingly, IL-i and IL-6 both can induce the pituitary gland to release ACTH, thereby generating the required cortisone level as soon as a systemic reaction is staged (69). Other diseases where a critical role for the monokines has
been implied are rheumatic fever, mononucleosis, and myeloma. Detection of Cytokines in Serum
General Aspects There are many ways to detect cytokines. They comprise bioassays in vivo and in vitro, and radio or enzymelinked immunoassays (RIA, ELISA). Many of the bioassays use cells which are dependent on cytokine for proliferation. Others measure prostaglandin production, differentiation, or cytotoxicity. Many of the assays detect several cytokines, some
are, however, quite specific (Table 2). To these belong the 7TD1 assay for IL-6 (1), the CTLL assay for IL-2, and the EL4/CTLL assay for IL-i. Most of the bioassays can be quantitated by titration and are very sensitive (29, 32). RIA and ELISA seem to be less complicated at first glance; they can, however, be too insensitive, detect inactive degradation products, or be corrupted by unspecific reactions. They are also prohibitively expensive where large series have to be tested. In
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rh IL-i rh
S12 mt. J. Sports Med. 12(1991)
H. Nort hoff andA. Berg
Table 2 Some bioassays fo r cytokines and th eir specific ity.
ports of systemic cytokines include mildly elevated levels of interferon-a (IFN a) after immunizations or hepatitis (7, 11).
IL-i
IL-6
TNF
IL-2
In vivo fever induction in rats or rabbits
+
+
+
?
C3HHeJ mouse thymocyte
+
+
—
+
—
—
—
+
assay
IL-2 dependent T cell line )CTLL) growth
+
—
—
(+)
7TDI hybridoma growth
—
+
—
—
L-929 cytotoxicity
—
—
+
—
EL-4/CTLL (1L-2 induction)
tissue, lympliokines may be detected by demonstration of mRNA or by immunofluorescense. Detection of cytokines in serum presents many problems. A major one is caused by the existence of so-called inhibitors, which may themselves be or not be true regulatory lymphokines such as the IL-i inhibitor described by Arend et a!. and Roux-Lombard et al. (6, 64). Work on that particular
field has left scores of frustrated investigators who found
Detection of Systemic Cytokines after Strenuous Exercise To our knowledge, the first evidence that an acute phase response similar to that which follows trauma or infection can be induced by strenuous exercise dates some 20 years back (37, 38). Cytokines — although their mere existence was still fiercely denied by a solid majority of immunologists — were soon discussed as possible cause (36, 43). Since 1979, im-
proved semiquantitative assays (51), techniques of purification, and later cloning have quickly removed the doubts. Since ELISAs have not yet been available or too insensitive, substantial efforts had to be made to overcome the technical problems
of bioassays as discussed above. Convincing evidence that mono-/cytokines are indeed systemically present after strenuous exercise was presented in 1983, using fever induction in rats (5, 16). The authors later confirmed their findings using column fractionation of serum and subsequent analysis by the thymocyte assay (14). They also report that a "suppressor" activity eluted in the 20—50 kDa range. According to the state of knowledge at that time, the detected cytokine activity was attributed to IL- 1 by the authors and some pain was taken to dissociate it from IL-2. From today's point of view, however, we
have to acknowledge that the observed cytokine might also have been IL-6 or a mixture of IL-l and IL-6 (TNF is inactive in
themselves purifying to homogeneity what turned out to be an artefact, (e. g., thymidine kinase, which cleaves off the label from 3 H-thymidine).
the thymocyte assay and therefore excluded). There is also a possibility that a different, hitherto undefined mediator may be involved.
So far the true nature of the serum-borne inhibitor of IL-i, IL-6, and TNF bioassays can only be labeled "un-
Recently a further cytokine, IFN-ct, has been found elevated in plasma after exercise (67). So far we are not aware of any further reports. Another way to investigate the possible involvement of lympho-monokines in exercise would
known", even if anecdotal reports claim that they could pinpoint the activity to a 20—50 kDa moiety (14). There is, however, no doubt that these inhibitors exist, active up to 1: 100 dilution, and that sytemic lymphokines can only be detected by most bioassays if they are active in still higher dilutions, unless the inhibitor can be removed or knocked out. Recently we
have found that simple treatment with 56 °C can destroy the serum-borne inhibitor of the 7TD 1 assay for IL-6, leaving the IL-6 activity itself essentially intact. This finding was also con-
firmed by personal communication from Dr. v. Snick, who kindly provided the 7TD1 cell line. How far inhibitors can in-
be to monitor the in vitro production of cytokines by cells drawn before and after exercise. This has been investigated recently (42). The experiments showed that LPS-induced mono-
kine production by blood monocytes was significantly increased after running, pointing to an activation of the macrophage system by exercise. A concomitant decrease in PHA-induced production of IL-2 by T cells, on the other hand, can be exhaustively explained by the exercise-induced shift in the T4/T8 ratio (T4 cells are far more efficient producers of IL-2).
terfere with RIA and ELISA is unknown at present.
Beyond reasonable doubt, lymphokines can be detected systemically in certain situations (9, 23, 33, 47, 52,
68). Transient elevation can be demonstrated regularly by ELISA in patients with polytrauma or sepsis. IL-I is definitely
more difficult to demonstrate. Reports of circulating IL-i which use thymocyte assay or fever induction must be interpreted with caution. While there is no doubt that these assays measure "real" cytokine activity, it is uncertain that the cytokine detected was IL-i. Both assays are also reactive to IL-a. Using an assay which is to our knowledge quite specific for IL-
6, it could be shown that the serum-borne cytokine activity, which was elevated in severely burned patients, was indeed IL-
6 (54) and that the IL-6 levels nicely correllated with fever. Since IL-6 was only identified as a separate monokine in late 1987 (28), reports of IL-i activity up to then and perhaps later should be cautiously interpreted as IL-l and/or IL-6. Other re-
Macrophage activation was also shown measuring increased phagocytic and enzymatic activity (24, 25). Activated macrophages could also be demonstrated invading muscle tissue after strenuous exercise (49). Intriguingly, a recent report has been able to demonstrate membrane-bound and perivascular IL-i IEI in muscle up to 5 days after exercise (15). This could also be extracted and was active in a bioassay.
TNF or IL-la could not be demonstrated. Taken together, available data from the literature clearly show that the response to strenuous exercise shows striking similarities with the response to infection. Several investigations point to an activation of the macrophage system which is followed by monokine production up to a systemically elevated and relevant level.
The remaining question is what induces the activation of the macrophages. Beside LPS and specific antigen,
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Specific for
Assay
mt. J. Sports Med. 12(1991) S13
Immunologic Mediators as Parameters of the Reaction to Strenuous Exercise fever ..— (hypothalamus)
culated in arbitrary units and compared. In 15 of the 17 runners, there was a significant increase of IL-6 measurable immediately after exercise, which was in most cases back to pre-run values 24 h later. At the same time we found no detectable IL-i
by an ELISA (detection limit 15 pg/mI) for IL1-3. IL-i was (hypophysis)
cortisone
also not found in the thymocyte assay. No attempts were made to remove the inhibitor which was present in 1:40—1: 100 dilution. Our present view of the involvement of monokines in the
reaction to exercise is elucidated in Fig. 5. IL-i TNFand IL6 are released from macrophages locally in the muscle and possibily in other organs. IL-6, which may be induced by the
primary stimulus and by IL-i, is generated in sufficient amount to reach detectable levels in the blood where it is prob-
ably transported in active form and sheltered from degrada______________
acute phase
inflaormatI1I4 44 44 4 response
Fig. 5 Possible role of monokines in exercise-induced acute phase response. Tissue injury and possibly LPS released from the gut activate monocyte/macrophages which produce IL-i, TNF, IL-6, and other inflammatory mediators. IL-i and TNF also induce IL-6 production in epithelial cells. lL-6 reaches systemic levels sufficient to induce the acute phase and to be detected by the 7TD1 bioassay.
of the acute phase response in the liver. Thus, we believe that IL-6 is the main systemic mediator after exercise causing the acute phase response. Even so, a bit of caution about the identity of the presumed IL-6 seems appropriate until a molecular weigth estimate and neutralization by specific antibody have been obtained. So far we could not do these experiments for lack of material. We also could not yet test the 7TD 1 cell line with IL-7, a newly described cytokine which seems to be active in the thymocyte assay, too. Finally, we cannot exclude that sensitive and specific assays might pick up some IL-i if the inhibitor is removed.
a host of nonspecific irritants, including phagocytosis per Se,
have been reported to stimulate IL-i production (60). Part of these observations may have been due to contamination of reagents, material, or media with LPS, a virtually ubiquitious agent. We were unable to induce IL-I by lysates of erythrocytes or muscle or organ extracts (unpublished) and so were others (14). Immunophagocytosis in our hands was likewise ineffective. Still, the possibility that detritus from destroyed cells induces the reaction in vivo cannot be ruled out by these experiments.
Another possibility is that LPS which is released from the gut under exercise (12, 44) may cause the acti-
vation. LPS is effective in very low concentrations, and the LPS-neutralizing capacity of serum lipoproteins (26) may be overrun. So far we have to note that the true reason for the macrophage activation remains unclear.
If we accept that exercise leads to production of cytokines, we face the question if this may be good or bad for our health. At present we feel unable to say if a given parame-
ter, e. g., elevation or suppression of one or more cytokines, has to be viewed as a good sign or as a warning. Maybe the future will provide correlations making the measurement of cytokines a useful diagnostic tool. So far, our speculation is that cytokines may well work in both directions. It is known that animals which are raised under sterile conditions are underdeveloped and prone to infections. Moderate but regular exposure to antigen and regular exercise might both provide cytokines necessary to keep up a "trained" immune system. On the other hand, chronic strenuous, exhaustive exercise may overexpose or exhaust the immune system thereby causing the immunodeficiencies which can occur in athletes with corresponding training programs. References
IL-6 Is Probably the Major Systemic Cytokine after Strenuous Exercise, Causing the Acute Phase Response
As pointed out above, the data from the literature convincingly showed that systemic elevation of cytokines occurs in serum after strenuous exercise. The identity of the observed cytokine is, however, uncertain. Since the full spectrum of the acute phase response is induced in the liver cells and since IL-6 is the main inducer which can do this, we feel that IL-6 must be involved in the reaction at some point. One might argue that IL-6 may be induced in the liver sinusoids by systemic IL-I. On the other hand, IL-6 itself (± IL-i) might be the systemic mediator. This question has recently been tested in our lab on 17 participants of a marathon run (manuscript in preparation). Blood was drawn before, immediately after, and 24 h after the run. The serum was tested using the 7TD 1 assay, which is very sensitive for IL-6 but is definitely negative for all other known cytokines discussed so far. The results were cal-
2
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Int.J.SportsMed. 12 (1991) S15
Friv.-Doz. Dr. H. Northoff
DRK-Blutspendezentrale Helmholtzstrafie 10 W-7900 Ulm Germany
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Immunologic Mediators as Parameters of the Reaction to Strenuous Exercise
