Thermogenic Effects of Cytokines: Methods and Mechanisms

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Thermogenic Effects of Cytokines: Methods and Mechanisms Nicholas J. Busbridge and Nancy J. Rothwell

Introduction Thermogenesis, literally defined as heat production, is an important physiological variable as well as a normal by-product of metabolic processes. Increased thermogenesis is a common feature of the acute-phase response and can be observed following injury, inflammation, infection, physical or emotional stress, and in certain chronic diseases such as malignancy. Thermogenesis is also a primary effector of thermorégulation in homeotherms, and an important mediator of fever. Cytokines have been proposed to mediate many aspects of the acute-phase response, including activation of thermogenesis and fever. Experimental studies have now demonstrated potent effects and probable mechanisms of action of a number of cytokines on thermogenesis.

Thermogenesis: Physiological Importance and Mechanisms Total heat production (also known as metabolic rate) in homeotherms comprises basal metabolic rate (required for obligatory processes), energy costs of physical activity and growth, and regulatory thermogenesis. The latter component, often (although not strictly correctly) referred to simply as thermogenesis, may be stimulated by exposure to cold, arousal from hibernation, hyperphagia or modification of dietary composition, and stress or disease. Thermoregulatory thermogenesis occurs at environmental temperatures below the thermoneutral range, and can be subdivided into two categories; shivering thermogenesis, where heat is produced by muscular contraction, and nonshivering thermogenesis (NST), which is dependent on involuntary processes activated largely by the sympathetic nervous system. In small mammals, NST is due largely to heat production in brown adipose tissue (BAT), which is stimulated by noradrenaline release from its rich sympathetic nervous supply, and interaction with a /33-adrenoceptor on brown adipocytes (1, 2). Thus total thermogenic capacity can be assessed from the increase in metabolic rate induced by peripheral injection of a maximal dose [—0.5 mg/kg 96

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intraperitoneal (ip) in rodents] of noradrenaline. The remarkable thermogenic capacity of BAT (which can cause a doubling of total metabolic rate in small animals) arises from a unique mitochondrial proton conductance pathway allowing controlled uncoupling of oxidative phosphorylation (2,3). The quantitative importance of these mechanisms in large mammals (including humans) is uncertain, although adult humans possess functional BAT, which may be activated in disease states (4). Increases in heat production that are not accompanied by appropriate changes in heat loss result in an elevation of body temperature. A passive rise in temperature, for example during heat gain from the environment or because of failure of compensatory heat loss mechanisms, results in hyperthermia. In contrast, fever is defined as a regulated increase in body temperature caused by an increase in the set point for temperature control, and is achieved by reductions in heat loss (e.g., vasoconstriction, huddling, piloerection), and increases in heat production (shivering and NST). Increasing heat production (thermogenesis) is well documented under experimental and clinical conditions associated with injection of pyrogens (e.g., bacterial endotoxin, cytokines), infection, inflammation, or injury (5, 6). However, these are not always accompanied by fever, and under some circumstances (e.g., during injury) thermogenesis may be activated independently of a change in body temperature. Studies on experimental animals (e.g., mice, rats, and rabbits) indicate that in most of the conditions described above, thermogenesis is due to sympathetic activation of brown adipose tissue (5, 6). This conclusion has been derived from observations that the thermogenic responses (increase in metabolic rate) to acute or chronic administration of endotoxin or cytokines, injury, bacterial infection, or inflammation can be inhibited by injection of/3-adrenoceptor antagonists (e.g., propranolol), and are associated with increased activity (determined ex vivo) of BAT (measurement of mitochondrial purine nucleotide binding) (see Effects of Cytokines on Thermogenesis; also Refs. 5, 6). However, shivering or gross changes in physical activity may also contribute to these thermogenic responses, particularly when NST is inhibited (7). Thermogenesis results from heat-producing mechanisms in the periphery, but is under direct control by the central nervous system (CNS). Thus activation of thermogenesis by peripherally administered pyrogens or by disease states results from afferent signals to the brain, and subsequent stimulation of sympathetic outflow (5, 6, 8). The central mechanisms involved in the control of thermogenesis have been discussed in detail elsewhere (9), and there is now extensive evidence that thermogenic actions of cytokines are also due to direct effects on the brain (10, 11).

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Measurement of Thermogenesis Thermogenesis can be determined from measurements of metabolic rate either directly, by determining heat production (direct calorimetry), or indirectly from oxygen consumption and carbon dioxide production (indirect calorimetry). Direct calorimeters are notoriously complex and expensive, and suffer the disadvantage that during rapid changes in body temperature (e.g., development of fever) short-term measurements of heat production may be erroneous. Indirect calorimetry has therefore been more widely employed in both experimental animals and humans. Heat production can be accurately calculated from rates of oxygen consumption (Vo2) and C0 2 production (Vco2). However, provided that marked changes in respiratory quotient do not occur, reliable estimates of thermogenesis can be achieved from determination of Vo2 alone (12). A number of commercially available indirect calorimeters exist for small mammals, usually based on accurate measurements of air flow through a small chamber and the difference in oxygen content (determined by paramagnetic analysis) of ingoing and outgoing air. However, a simple technique for measurement of Vo2 involves removal of C0 2 and water by inclusion of Carbasorb and silica gel within the circuit produced by the animal and replacement (and simultaneous measurement) of the amount of oxygen used. As oxygen is used and C0 2 and water are absorbed a small drop in pressure will occur inside the calorimeter, which is detected by microdifferential pressure switches, causing fixed volumes of oxygen to be pumped into the calorimeter until the original pressure is restored (13). For small animals (e.g., rats and mice) the volume of such calorimeters should be small (i.e., <2 liters) and the volume of replacement less than 1 ml in order to achieve a rapid and accurate measurement of Vo2. Because metabolic rate is highly dependent on environmental temperature, maintenance of a constant temperature (ideally close to or slightly below the thermoneutral range, i.e., 24-30°C for rodents) is essential. It is, however, difficult to exclude the contribution of physical activity, muscle tone, or shivering from values for metabolic rate. Anesthesia (e.g., sodium pentobarbitone) inhibits these processes, but may also modify the mechanisms responsible for activating NST. Short-term measurements (several hours) are usually sufficient to obtain reproducible baseline values and responses to acute stimuli such as injection of pyrogens. However, sustained periods of measurement should take account of diurnal variation, sleep-wake cycles, and the amount and type of diet consumed (12). The contribution of NST to total metabolic rate can be estimated from the inhibitory effect of ß-adrenoceptor blockade (e.g., by peripheral injection of propranolol), which is minimal in normal resting animals at environmental

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temperatures close to thermoneutrality. However, this can yield underestimates of NST, because during /3-blockade other forms of heat production (e.g., shivering) maybe substituted for NST (7). More indirect, and therefore less reliable, assessments of thermogenesis include simultaneous estimates of core temperature (e.g., rectal) and skin temperature, or of BAT activity. The latter can be determined in vitro, by removal of the tissue postmortem and measurement of the specific binding of radiolabeled purine nucleotides (e.g., GDP) to isolated mitochondria (14).

Effects of Cytokines on Thermogenesis The availability of purified recombinant cytokines has greatly facilitated the study of their biological actions, but has also led to a plethora of data, from which it is difficult to distinguish pharmacological and physiological effects. Kluger (10) has reviewed in detail the criteria necessary to establish a physiological role of any cytokine on fever. The same criteria can readily be applied to thermogenesis. First, administration of highly purified recombinant material by appropriate routes should elicit dose-dependent and reversible changes in thermogenesis in normal animals (or humans), which occur over a time course consistent with physiological responses to other thermogenic stimuli. For studies on fever and thermogenesis, it is essential that the cytokine preparation not contain significant quantities of endotoxin, and that control experiments (e.g., heat destruction to abolish biological activity) are undertaken. Such thermogenic responses have indeed been demonstrated for a number of cytokines in experimental animals. Interleukin 1/3 (IL-1/3) is one of the most potent activators of fever and thermogenesis. In the rat, intravenous injection of IL-Iß provokes a rapid increase in oxygen consumption (determined by indirect calorimetry at 24°C), which is apparent within 15 min. The response to peripheral injection of IL-1/3 is sometimes biphasic, reaching a maximal value after approximately 60 min; this is sustained for some time, even though the half-life of IL-1 in circulation is quite short, on the order of several minutes. These increases in Vo2 closely parallel the development of fever (which usually follows 5-10 min later); they can be observed in anesthetized rats, are not associated with increased physical activity (in fact, IL-1 is somnogenic), and are inhibited by ß-adrenoceptor blockade (by injection of propranolol, 20 mg/kg ip; Ref. 15). These observations, together with increased BAT activity, which has been reported in animals injected with IL-1/3 (15), indicate that this cytokine stimulates metabolic rate by activating NST. Injections of human, murine, or rat recombinant IL-1/3 from a number of different sources (A. Shaw, Glaxo, Geneva, Switzer-

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land; I. Otterness, Pfizer, New York, NY; British Biotechnology, London, England) elicit comparable increases in Vo2, and similar (although slightly delayed) thermogenic responses occur after intraperitoneal or subcutaneous injections. In the rat, maximal increases in Vo2 and body temperature occur in response to a peripheral dose of approximately 1 ^g of IL-l/3/kg [intravenous (iv), ip, or subcutaneous (sc); Fig. 1]. In these studies body temperature (fever) was determined from the change in core temperature determined by insertion of a plastic-coated thermocouple (Comark, London, England) approximately 5 cm beyond the rectum in lightly restrained rats. Core temperature can also be determined by remote radio telemetry, which involves less stress to the animal. In contrast, thermogenic and pyrogenic responses to intracerebroventricularly injected IL-1/3 occur at much lower doses (~5 ng; Fig. 2). Direct injection of cytokines into specific brain regions (e.g., preoptic and anterior hypothalamus) stimulates fever at low doses (<1 ng; Ref. 8). However, because such injections may cause local tissue damage, administration into the cerebral ventricles could be more appropriate, particularly in cases in which the specific site of action is unknown. This procedure can be readily achieved by prior stereotaxic implantation of guide cannulas into the third or lateral ventricles, under anesthesia. Guide cannulas (either purchased, or made from cut-down Luer needles) are lowered into the

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FIG. 1 Dose-response curves for the effects of subcutaneous injection of recombinant murine IL-1 (IL-Ια, I. Otterness, Pfizer; IL-1/3, A. Shaw, Glaxo) on the increase in colonie temperature and resting oxygen consumption (Vo2) of conscious, male Sprague-Dawley rats. Temperature was determined as the difference between colonie temperatures measured 2 hr after and immediately before injection of IL-1. Vo2 was measured by indirect calorimetry at 24°C in individual closed-circuit respirometers, for 2 hr before and 2 hr after injection of IL-1. Peak increases in Vo2 (% above baseline) occurred 90-120 min after treatments. Mean values ± SEM (n = 8).

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FIG. 2 Dose-response curves for the effects of intracerebroventricular (icv) injections of IL-1 (for methodological details, see caption to Fig. 1). Intracerebroventricular injections were administered to conscious, hand-held rats via guide cannulas stereotaxically implanted in the third ventricle of the brain at least 3 days previously under sodium pentobarbitone anesthesia, in volumes of less than 3 μ,Ι. Mean values ± SEM (n = 8).

brain after craniectomy and held in place with two or three screws and dental acrylic cement. After several days for recovery from implantation, studies can be conducted on conscious animals. Injections of small volumes (—1-3 μΐ) into the ventricles in lightly restrained animals allow substances to enter and diffuse through cerebrospinal fluid (CSF), gaining access to many brain regions. Cannula positions are subsequently verified by histological examination. Comparison of d o s e - r e s p o n s e curves for the effects of a number of recombinant cytokines [e.g., IL-Ια, IL-lß, IL-6, and tumor necrosis factor a(TNF-a)] on fever or thermogenesis reveals that all are more effective (by 50 to 2000-fold) when injected intracerebrally, indicating a central site of action (11, 16). However, although the brain appears to be the major site of action of cytokines on thermogenesis, and several cytokines have been identified in CSF or brain tissue (17), the means by which they enter the CNS from the periphery or even their ability to gain direct access to the brain have not yet been established (8). Circulating cytokines could act on areas of the brain that lack a substantial blood-brain barrier, such as the organum vasculosum of the lamina terminalis (OVLT; 8), or may be synthesized within the brain in response to peripheral signals (11, 17). The presence of IL-1/3, TNF-α, and IL-6, and their mRNAs, have been demonstrated within the brain, where they appear to be synthesized by both nerves and glial cells (17).

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Chronic administration of cytokines can be achieved by infusions or repetitive injections. In the rat, continuous infusion of recombinant IL-1/3 via osmotic minipumps (Charles River, Kent, England) implanted intraperitoneally or subcutaneously (1 ^g/rat/day) causes sustained increases in colonie temperature and Vo2, as well as reductions in body weight that persist for 3-5 days (N.J. Busbridge, M. J. Dascombe and N. J. Rothwell, unpublished data). Thereafter, the animals apparently develop tolerance to the infused IL-1 (i.e., no fever or weight loss is observed), but are able to respond normally to bolus injection of a maximal dose (1 /xg, ip) or IL-1/3 (N. J. Busbridge, M. J. Dascombe, and N. J. Rothwell, unpublished data). However, some caution must be exercised in the interpretation of data from such experiments because, although IL-1/3 in the minipumps remains active (retransplantation into naive rats elicits marked fever and increased thermogenesis), scar tissue forming around the pump may inhibit delivery of IL-1 (18, 19). Repetitive daily injections of IL-1/3 (1 ^g/kg ip or 5 ng/rat icv via indwelling guide cannulas) elicit transient increases in Vo2 and temperature that are maintained for several hours after each injection over the 7-day period of study (N. J. Busbridge, M. J. Dascombe, and N. J. Rothwell, unpublished data), and no tolerance is observed. The development of tolerance has been reported in animals treated with a number of cytokines (18, 19), but this may reflect use of inappropriate recombinant material (i.e., not native sequence) or routes and timing of administration. In an elegant study, Oliff et al. (20) demonstrated that implantation of tumors that produced excessive TNF (due to insertion of the TNF gene) in mice resulted in dramatic and prolonged weight loss. This effect could not simply be ascribed to hypophagia, indicating that this procedure for chronic administration of TNF caused sustained increases in thermogenesis. The effects described above indicate that several recombinant cytokines stimulate thermogenesis in experimental animals. However, such effects may reflect pharmacological responses to cytokines, for example, due to local inflammation or irritation. Two major criteria that should be established in order to assume a physiological role of a cytokine are that it is produced by the host in response to an appropriate stimulus, and that inhibition of the action of the endogenous cytokine blocks physiological responses (10). Attempts to correlate circulating concentrations of cytokines with development of fever and thermogenesis have proved notoriously difficult. In the case of IL-1 this problem is exacerbated by differences between bioassays (e.g., proliferation assays) and immunoassays (RIA or ELISA), and the presence of endogenous inhibitors of IL-1 activity (21, 22). Numerous studies have reported only modest or no increases in circulating IL-1 concentrations in animals or humans exhibiting fever and increased metabolic rate (23-27). However, in rats chronically infused with IL-1 (as described above), which

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show markedly increased body temperatures and metabolic rates, only modest (approximately twofold) increases in circulating IL-1 concentrations are observed (determined by sensitive bioassay D10 proliferation assay; N. J. Busbridge, S. J. Hopkins, and N. J. Rothwell, unpublished data). Cytokines such as IL-1 may be produced locally, and activate CNS mechanisms via release of other humoral factors (such as other cytokines) by producing neural afferent signals, or by traveling in the lymph system. These stimuli may in turn cause synthesis of endogenous IL-1 in the brain. For example, peripheral injection of bacterial endotoxin in the rat (which elicits marked increased in body temperature and metabolic rate) also induces expression of mRNA for IL-lß within the CNS (17; N. J. Busbridge and N. J. Rothwell, unpublished data), and in IL-1/3 itself in the brain (28), although (as mentioned above) circulating IL-1 concentrations often fail to increase. In contrast, circulating concentrations of IL-6 (measured by B9 bioassay) increase rapidly and dramatically following injection of pyrogens, and both plasma and CSF concentrations correlate with the development of fever (24, 25). In human volunteers infused intravenously with endotoxin (4-8 ng/kg, iv), circulating concentrations of IL-6 (determined by B9 bioassay) often exceed 20 ng/ml (M. Horan, personal communication). Thus, IL-6 which can be induced by IL-1, may act as a circulating pyrogen, although the means by which it gains access to the CNS remains unknown. For other thermogenic cytokines the picture is also unclear and although some of these (e.g., TNF-α) do appear in plasma during fever (24, 27), their sites of entry, synthesis, and action in the CNS are unknown. Selective inhibition of the actions of endogenous cytokines has been limited by the absence of specific receptor antagonists (an exception to this for IL-1 is discussed below). Biological activity of endogenous peptides can, however, be modified by passive immunization by injection of high-affinity neutralizing antibodies (29). Peripheral or central (intracerebroventricular) injection of neutralizing anit-IL-1/3 antibodies (obtained from H. Humphrey, Glaxo, Greenford, England, or from S. Poole, National Institute for Biological Standards and Controls, Potters Bar, UK) markedly attenuates the pyrogenic and thermogenic responses to endotoxin in the rat (30, 31). In both cases anti-murine IL-1/3 antibodies were used (approximately 0.5 ml iv or 3 μΐ icv), but because of the close sequence homology between mouse and rat IL-1/3, antimurine antibodies often neutralize rat cytokines. For these experiments it is important to establish neutralization of the appropriate cytokine by the antibody or antiserum either in vivo (by coinjection of cytokine and antibody) or in in vitro assays. Interestingly, antibodies to IL-la were ineffective in both cases (30, 31). These data indicate that endogenous IL-1/3, but not IL-la, mediates fever and thermogenesis in the rat. An important consideration in such studies is not only the affinity of the antibody,

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which will determine the amount to be injected (ideally less than 1 ml peripherally or 3 μΐ intracerebroventncularly), but also the rate of association of the antibody with the antigen (see Ref. 29 for discussion of methodology). In the brain, where endogenous peptides may act locally, the antibody must neutralize the peptide before it reaches and binds to the receptor. We have demonstrated that central injection (icv) of anti-rat IL-6 antiserum (3 μΐ, provided by J. Gauldie, McMaster University, Hamilton, Ontario, Canada) attenuates the pyrogenic and thermogenic effects of peripherally administered endotoxin or centrally injected IL-1/3 in the rat (32; Fig. 3). However, this antiserum did not completely abolish responses, possibly because of its relatively low affinity, limitation of the injection volume, and effects of central injection of a large protein per se. Studies using anti-TNF-α antibodies have yielded variable results. Nagai et al. (33) reported that TNF-α antibody inhibits endotoxin-induced fever, whereas Kluger's group has observed potentiation of endotoxin fever in the rat, and suggested that TNF-α may act as endogenous antipyretic agent (10, 30). The studies reported to date have generally used polyclonal antibodies or unpurified antisera to specific cytokines. More reliable data may be obtained as monoclonal antibodies to rodent cytokines became available, although it is less likely that monoclonal antibodies will successfully neutralize endogenous cytokine action. An endogenous IL-1 receptor antagonist protein (IL-Ira) has been identified and cloned (34). This antagonist (provided by Synergen, Boulder, CO) inhibits many actions of IL-1 (35), including peripheral effects of IL-1 on fever (36; Fig. 4) at doses 100 to 500-fold greater than IL-1 itself. However, central injection of a high doses (2000-fold excess over IL- Iß) of this antagonist fails to modify the pyrogenic and thermogenic actions of centrally injected IL-1/3 in the rat (37; Fig. 4). These data, together with published observations on differential effects and mechanisms of action of IL-la and ß (36), indicate that central activation of fever and thermogenesis by IL-1 in the rat are not mediated by interaction with a type I IL-1 receptor (11). Our studies indicate that more recently identified type II IL-1 receptor may be involved, because we have demonstrated that a monoclonal antibody to this receptor (2 μΐ, injected icv) obtained from P. Ghiara, (Sclaro Institute, Seeria, Italy) markedly inhibits central effects of IL-1/3 on fever and thermogenesis in the rat (Luheshi, N. J. Rothwell, and P. Ghiara, unpublished data).

Mechanisms of Thermogenic Actions of Cytokines Data discussed above suggest that increases in heat production induced by cytokines in experimental animals result from sympathetic stimulation of brown adipose tissue. Afferent signals whereby peripherally synthesized

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FIG. 3 Effects of icv injection (3 μΐ) of nonimmune serum (open bars) or anti-rat IL-6 antiserum (hatched bars) (J. Gauldie, McMaster University) on Vo2 (a) and colonie temperature responses (b) to peripheral injection of a maximal thermogenic dose of bacterial endotoxin (0.5 mg/kg, ip, Escherichia coli, 0127 :B8, Sigma, St. Louis, MO) or icv injection of a maximal dose (5 ng) of recombinant murine IL-/3. Nonimmune or antiserum was injected immediately prior to administration of endotoxin or IL-1. Mean values ± SEM (n = 8-10). *p < 0.005 vs respective group treat with nonimmune serum. cytokines might activate the CNS are largely unknown, but recent studies have provided some insight into their central mechanisms of action. The pyrogenic and thermogenic effects of most of the cytokines discussed here [IL-la, IL-1/3, TNF-a, IL-6, interferon y (IFN-γ)] are prevented by cyclooxygenase inhibitors (e.g., indomethacin, flurbiprofen, ibuprofen; 6, 8, 10, 11), and have been ascribed to synthesis of prostaglandins (particularly PGE 2 ),

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both in the periphery and at specific sites in the brain (8, 10). In contrast, effects of the lower molecular weight (Mr 8000) cytokines, IL-8 and MIP-1, are not modified by inhibitors of prostanoid synthesis (38, 39). Further evidence of multiple pathways for cytokine-induced fever and thermogenesis derive from studies on corticotropin-releasing factor (CRF; 37, 40). This peptide is synthesized, and can act directly within, the CNS independently of its effects on the pituitary, and is a potent thermogenic agent that stimulates metabolic rate and BAT activity (40). Inhibition of the actions of endogenous CRF by central injection of a receptor antagonist (25-50 ^g of a-helical CRF9_41; Sigma, St. Louis, MO) or neutralizing monoclonal or polyclonal anti-CRF antibodies (3 μΐ; Ε. Linton, Oxford, England; F. Tilders, Amsterdam) markedly inhibits the thermogenic and pyrogenic responses to centrally administered IL-1/3, IL-6, or IL-8 in the rat (11, 16, 40). These data indicate that CRF mediates the central actions of these cytokines on fever and thermogenesis. In contrast, the central actions of IL\a or TNF-α do not appear to depend on CRF synthesis and/or release, because they are not influenced by pretreatment with CRF antagonist or antibody (11, 16, 40). The implication from these data, that two separate pathways of cytokine action exist in the CNS, is supported by observations on the effects of lipocortin-1 (annexin-1). This endogenous phospholipidbinding protein is present in the brain and we have demonstrated that a recombinant fragment of human lipocortin-1 (amino acids 1-188, 1.2 μg icv) (provided by F. Carey, ICI Pharmaceuticals, Macklesfield, UK) potently inhibits the thermogenic actions of IL-1/3, IFN-γ, IL-6, IL-8, and CRF, when injected intracerebrally in the rat, but does not affect responses to IL-la or TNF-a (41, 42). Antiinflammatory and antipyretic effects of lipocortin-1 have been ascribed to suppression of arachidonic acid release, resulting in reduced prostanoid synthesis (43). However, the data presented above are not consistent with this mechanism and indicate that effects of lipocortin-1 on fever and thermogenesis may be due to inhibition of the actions of CRF rather than suppression of eicosanoid synthesis (42). Thus, several pathways for the central actions of cytokines on thermogenesis appear to exist, depending on the involvement of prostaglandins and/or CRF.

Clinical Implications of Thermogenic Effects of Cytokines The biological value of increases in thermogenesis in disease states is uncertain, and may depend on the severity and time course of the responses, and on associated clinical conditions. Banet (44) has argued that the thermogenic response itself (independently of fever) offers survival value during infections. However, increased body temperature is severely detrimental to neu-

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ronal survival after brain injury or ischemia (45). The increased oxygen demand required to support thermogenesis may also be detrimental when respiratory capacity or tissue perfusion is limited, for example, during hypotension, local tissue damage is ischemia. Sustained increases in metabolic rate, commonly observed following major trauma (46) or during chronic disease states, also impose increased energy demands on the host (6). This, together with the reduced food intake or absorption that usually accompany severe illness or injury, results in mobilization of body energy stores (fat and protein), leading to general wasting (cachexia). Thus, selective inhibition of thermogenesis offers considerable clinical advantages. Interventions to modify cytokine action directly will influence many aspects of the acutephase response, such as immune activation, fever, and endocrine responses, the value of which is questionable. A more selective approach, for example, by inhibition of peripheral thermogenic mechanisms, could therefore be preferable.

Summary and Conclusions Data obtained largely in exerimental animals, by a variety of techniques, demonstrate that a number of cytokines stimulate thermogenesis, probably by a central action. Although results are not yet conclusive, evidence exists to support the hypothesis that some cytokines act as endogenous thermogenic agents that mediate the metabolic responses to disease and injury. Increased availability of receptor antagonists, specific high-affinity antibodies, and production of transgenic animals will be necessary to validate these studies and to confirm the sites and mechanisms, and physiological importance, of the actions of cytokines on thermogenesis.

References 1. J. R. S. Arch, A. T. Ainsworth, M. A. Cawthorne, M. V. Piercy, M. V. Sennitt, V. E. Thody, C. Wilson, and S. Wilson, Nature {London) 309, 163 (1984). 2. N. J. Rothwell and M. J. Stock, Recent Adv. Physiol. 10, 349 (1984). 3. D, G. Nicholls and R. Locke, Physiol. Rev. 64, 1 (1984). 4. A. Bianchi, J. Bruce, A. L. Cooper, C. Childs, M. Kohli, I. D. Morris, P. MorrisJones, and N. J. Rothwell, Horm. Metab. Res. 21, 587 (1989). 5. N. J. Roth well, in "Circulating Regulatory Factors and Neuroendocrine Function," (J. C. Porter and D. Jeiovâ, eds.), p. 371. Plenum, New York, 1990. 6. N. J. Rothwell, in "Hormones and Nutrition in Obesity and Cachexia" (M. J. Müller, E. Danforth, A. G. Burger, and U. Siedentopp, eds.), p. 77. SpringerVerlag, Berlin, 1990.

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