Monokines in Growth and Development KIRK C. KLASING and BARBARA J. JOHNSTONE Department of Avian Sciences, University of California, Davis, California 95616 (Received for publication August 5, 1990)
1991 Poultry Science 70:1781-1789 INTRODUCTION
Most important physiological processes are closely regulated by the participation of both positive and negative mediators. In the case of growth, the balance of anabolic and catabolic regulatory substances determines the rate of cellular hypertrophy and hyperplasia. Regulatory factors generally fall into two main categories: hormones and cytokines. Hormones are classically defined as substances produced at a discrete location (i.e., a gland) that enter the blood and act systemically. Cytokines are substances produced by widely dispersed and often dissimilar cells and may act either locally in an autocrine or paracrine fashion or systemically in an endocrine-like manner. Key anabolic factors such as growth hormone, insulin-like growth factors, and thyroid hormones have been the topic of considerable investigation because the potential exists to manipulate them to augment growth beyond the genetic potential of the animal. Catabolic regulatory factors include corticosteroids released due to most stresses and a variety of leukocytic cytokines released during the immune response. Two main classes of leukocytic cytokines have been described, monokines and lymphokines. Monokines and lymphokines are hormone-like peptides released by the macrophage-monocyte lineage
of cells and by the lymphocytes, respectively. Study of these catabolic factors has received considerably less attention by animal and poultry scientists than study of anabolic factors, probably because their manipulation may at best result in "optimal" growth rates at the animal's genetic potential. Paradoxically, the role of leukocytic cytokines is among the most intensely studied areas in the medical and biological sciences. This large body of research in laboratory rodents and humans demonstrates that monokines are released as an integral part of the immune response and orchestrate a variety of metabolic, cellular, and behavioral changes that are antagonistic toward growtih. IMMUNOLOGICAL STRESS AND GROWTH
It is well established that stimulation of the immune system results in a decreased rate of growth, whether or not the challenge is of infectious or uninfectious etiology. Cells of the mononuclear phagocytic system are the first line of defense in the immune system and thus are uniquely suited to identify immunogens, respond against immunogens, and provide information to the rest of the body, via monokines, that an immune response is occurring. The complex sequelae responsible for impaired growth have been well defined in
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ABSTRACT Stimulation of the immune system results in a series of metabolic changes that are antagonistic toward growth. Monokines, including interleukin-1, tumor necrosis factor, and interleukin-6, are released from cells of the monocyte-macrophage lineage after recognition of immunogens. They appear to mediate homeorhetic response, which alters the partitioning of dietary nutrients away from growth and skeletal muscle accretion in favor of metabolic processes which support the immune response and disease resistance. These alterations include 1) decreased skeletal muscle accretion due to increased rates of protein degradation and decreased protein synthesis; 2) increased basal metabolic rate resulting in increased energy utilization; 3) use of dietary amino acids for gluconeogenesis and as an energy source instead of for muscle protein accretion; 4) synthesis by the liver of acute phase proteins; 5) redistribution of iron, zinc, and copper within the body due to the hepatic synthesis of metallothionein, ferritin, and ceruloplasmin; 6) impaired accretion of cartilage and bone; and 7) release of hormones such as insulin, glucagon, and corticosterone. These monokines also influence the differentiation of cells. Tumor necrosis factor suppresses the differentiation of myoblasts and adipocytes whereas the chicken monokine myelomonocytic growth factor induces the differentiation of granulocytes. (Key words: monokines, interleukin-1, tumor necrosis factor, growth, metabolism)
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It has become apparent that most of the metabolic changes brought about by stimulation of the immune system are mediated by monokines. Data from use of chicks, as well as many investigations using rats and mice strongly implicate monokines with interleukin1 (IL-1), tumor necrosis factor-a (TNF) and IL-6 activities as mediators (reviewed by Beutler and Cerami, 1988; Klasing, 1988; Dinerello, 1988). The circulating concentrations of these monokines increase following most immune responses. These monokines act directly on responding tissues, or indirectly by altering the hormonal milieu, to cause the metabolic changes characterized as immunologic stress (Table 1). In many cases, the monokines BL-1, IL-6, and TNF have identical effects on tissues in laboratory mammals. The extreme redundancy of action and complex regulation of monokine release demonstrates the integral importance of this stress response. Each of the monokines also has a specific role in the regulation of the immune response, including controlling the recruitment and rates of proliferation of stimulated leukocyte populations and consequently mediating the initia-
TABLE 1. Overlapping regulatory roles of monokines in growth-related metabolism Response General Decreased voluntary food intake Increased resting energy expenditure Increased body temperature Glucose metabolism Increased glucose oxidation Increased gluconeogenesis Lipid metabolism Decreased lipoprotein lipase activity Increased lipolysis in adipocytes Increased hepatic triglyceride synthesis Increased adipocyte triglyceride synthesis Protein metabolism Increased acute phase protein synthesis Increased muscle protein degradation Bone and mineral metabolism Increased cartilage resorption Increased bone resorption Increased metallothionein synthesis Increased hepatic ceruloplasmin synthesis Hormone release Increased corticosteroid release Decreased thyroxin release Increased insulin and glucagon release
Chicks
Rodents
Murine cytokines responsible
X2 X X
X X X
IL-1, TNF IL-1, TNF IL-1, TNF, IL-6
? ?
X X
IL-1, TNF IL-1
X ?
-
X X X
X
-
IL-1, TNF, IL-6 IL-1, TNF TNF ?
X X
X X
IL-1, IL-6 IL-1, TNF
X 7 X X
X X X X
IL-1, TNF
X X ?
X X X
IL-1, TNF, IL-6 IL-1 IL-1, TNF
IL-1, IL-6 IL-1, TNF
IL-1 = interleukin-1; TNF = tumor necrosis factor; n>6 = interleukin-6. X indicates that a monokine induces the physiological response, - indicates that monokines do not induce a response, ? indicates that no data is available.
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laboratory mammals and sufficient work in chickens allows extrapolation of this information to Aves with some modification. The metabolic changes represent a homeorhetic response that alters the partitioning of dietary nutrients away from growth and skeletal muscle accretion in favor of metabolic processes that support the immune response and disease resistance. They form the basis for impaired growth, feed utilization, and altered nutrient requirements in chicks. These alterations include 1) decreased skeletal muscle accretion due to increased rates of protein degradation and decreased protein synthesis; 2) increased basal metabolic rate resulting in increased energy utilization; 3) a switch from fatty acids to glucose as a preferred energy source by many tissues; 4) use of dietary amino acids for gluconeogenesis and as an energy source instead of for muscle protein accretion; 5) synthesis by the liver of acute phase proteins; 6) redistribution of iron, zinc, and copper within the body due to the hepatic synthesis of metallothionein, ferritin, and ceruloplasmin; 7) impaired accretion of cartilage and bone; and 8) release of hormones such as insulin, glucagon, and corticosterone.
SYMPOSIUM: AVIAN GROWTH AND DEVELOPMENT
Feed Intake One of the most important growth-related changes induced by an immune response is die suppression of appetite and voluntary feed intake. The anorexigenic effect of infection as well as vaccination is appreciated by poultry producers and can easily be demonstrated in the laboratory. Partially purified IL-1 preparations mimic the anorexigenic effect of immunogens including Escherichia coli, lipopolysaccharide, and Sephadex (Klasing et al., 1987). In mammals IL-1, IL-6, and TNF are all anorexigenic. The anorexigenic activity of these monokines can account for most of the reduced growth rate observed during an immune response; however, a portion of the reduced growth is not related to the decreased feed intake, but rather is due to changes in metabolism. Energy Metabolism Fever is one of the hallmarks of infection or an immune response to defined immunogens. Partially purified IL-1 induces fever in chicks that is similar in magnitude to mat induced by E.
coli lipopolysaccharide (Klasing et al., 1987). This increased body temperature represents an increase in basal metabolic rate of 10 to 15%/1 C (Beisel, 1977). In rats, similar increases in basal metabolic rate have been found by direct measurement of energy expenditure following infusions of BL-1 i.v. or intracerebroventricularly (Dascombe et al, 1989). The TNF, interleukin-2, and IL-6 are also hypermetabolic, and IL-1 and TNF have synergistic effects on resting energy expenditure (Flores et al., 1989). In rodents, it is clear that monokines increase the rate of gluconeogenesis from amino acids, amino acid oxidation, and glucose utilization (Meszaros et al, 1987; Tredget et al, 1988; Warren et al, 1988). Skeletal Muscle Immune responses elicited by a variety of immunogens or infections decrease the rate of skeletal muscle protein accretion in growing chicks. Chicks infected with coccidia have decreased fractional rates of protein synthesis compared with uninfected chicks (Symons and Jones, 1977). Lipopolysaccharide and sheep red blood cells decrease rates of protein synthesis and increase degradation in chick skeletal muscle (Klasing and Austic, 1984a,b). Hentges et al. (1984) demonstrated that administration of Newcastle disease and infectious bronchitis vaccines results in decreased skeletal muscle protein synthesis and decreased protein accretion. In chicks and rats, the mechanism of the decreased protein synthesis involves decreased translational activity as indicated by decreased rate of synthesis per unit of RNA (Jepson et al, 1986) and decreased numbers of ribosomes translating mRNA (Klasing and Austic, 1984a). The impaired skeletal muscle protein accretion is apparently the result of cytokines elaborated by stimulated leukocytes. Incubation of skeletal muscle preparations from chick wings with partially purified EL-1 results in increased rates of protein degradation with no influence on the rate of protein degradation (Klasing etal, 1987). Interestingly, injections of IL-1-like preparations into the intact chick result in decreased skeletal muscle protein synthetic rates accompanied by a disaggregation of polysomes (Klasing and Austic, 1984a). Presumably, the in vivo results are due to the fact mat IL^l induces corticosterone release in chicks, and glucocorticoids reduce protein synthetic rates (Klasing et al, 1987). Thus,
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tion and length of an immune response. Thus, IL-1, TNF, and IL-6 function significantly to regulate the immune response to foreign immunogens, to alert the rest of the body to the penetration of immunogens into the body, and to orchestrate changes in intermediary metabolism to maximize immunocompetence. To date, research in chickens has generally utilized crude or partially purified preparations of monokines. The preparations utilized are usually enriched via a combination of purification processes based on a specific bioactivity, e.g., thymocyte comitogenesis for IL-1. Because these preparations are not known to be completely homogeneous, it is never certain which monokine is the active component. Consequently the name chicken IL-1 will be used to signify preparations purified based on thymocyte comitogenic activity, the term TNF will be used to signify preparations purified based on tumor cytolytic activity, and the term IL-6 is used to indicate purification based on hepatic acute phase protein synthesis. Progress in the monokine field is hampered by the lack of cloning and characterization of the respective genes and the availability of scrupulously purified proteins produced in quantity by recombinant DNA techniques.
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Lipid Metabolism In response to an immunogen such as endotoxin, plasma triglycerides, cholesterol/and phospholipids are reduced from control levels when fasting is used to equalize intake (Curtis et al, 1980). Plasma levels of very low density lipoproteins (VLDL) are generally reduced in comparison to fed controls (Griffin and Butterwith, 1988). This effect is in contrast to mammals, in which hyperlipidemia due to the accumulation of VLDL occurs rapidly after an injection of endotoxin and persists for several hours. Monokines (TNF, IL-1) mediate the hyperlipidemia in mammals through a coordinate increase in hepatic lipid synthesis with a reduction in lipoprotein clearance. The inhibition of lipoprotein lipase (LPL) results in decreased fatty acid uptake from circulating
triglycerides into tissues for utilization and storage. Tissue LPL activities in birds are reported to be less responsive to the nutritional state than in mammals (Husbands, 1972). However, chicken adipose, leg muscle, and heart LPL levels are significantly reduced by endotoxin injections in comparison with fasted controls (Griffin and Butterwith, 1988). Crude chicken monokine preparations, as well as preparations enriched for chicken IL-1, inhibit LPL activity in murine 3T3-L1 adipocytes (Klasing and Peng, 1990). The TNF reduces LPL activity by an almost complete suppression of LPL synthesis corresponding to a decrease in LPL mRNA (Cornelius et al, 1988), whereas IL-1 inhibits LPL synthesis without a reduction in mRNA (Zechner et al, 1988). Both cytokines also show a marked synergistic effect in suppressing LPL activities (Ogawa et al, 1989). In vitro studies indicate that monokines are involved in the LPL inhibition of avian adipose tissue. Incubating chicken adipocytes with conditioned medium from an LPS-stimulated avian macrophage cell line (HD-11) results in an almost complete suppression of LPL, indicating that monokines may be involved. However, recombinant human TNF and IL-1 have no effect on avian adipose LPL in vitro (Butterwith and Griffin, 1989). In mammals, the rapid hepatic synthesis and secretion of triglycerides contributes significantly to the monokine-induced hypertriglyceridemia (Feingold and Grunfeld, 1987). In rats, TNF acutely regulates hepatic fatty acid synthesis in vivo by raising hepatic levels of citrate, an allosteric activator of the rate-limiting enzyme acetyl-coenzyme A (CoA) carboxylase. Several hours later acetyl-CoA carboxylase and fatty acid synthetase are increased and the allosteric inhibitor fatty acyl-CoA is reduced (Feingold et al, 1989). Although there is little known about the effect of monokines on avian lipogenesis in vivo, the injection of endotoxin in chickens did not change hepatic fatty acid synthetase activity in contrast to fasted controls (Griffin and Butterwith, 1988). Incubation of chick hepatocytes with conditioned medium from LPS-stimulated HD-11 macrophage cells or human TNF does not significantly alter lipogenesis. However, high levels of human TNF does decrease the rate of hepatic lipogenesis. Interestingly, incubation of chicken adipocytes with LPS-stimulated HD-11 supernatants increases lipogenesis 200% (Butterwith and Griffin, 1989). The monokine-induced increase
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impaired skeletal muscle accretion results from both the direct effects of monokines to increase the rate of protein degradation and the indirect effects via corticosterone to decrease the rate of protein synthesis. Corticosterone by itself does not influence the rate of skeletal muscle protein degradation and does not augment induction by IL-1. Surprisingly, the anabolic effect of insulin on skeletal muscle is abrogated by IL-1, suggesting that IL-1 induces a state of insulin resistance in skeletal muscle. In mammals, both IL-1 and TNF induce protein degradation in skeletal muscle and their activities are synergistic (Flores et al, 1989). The second messenger responsible for inducing augmented proteolysis is not well defined. Fagen and Goldberg (1985) have demonstrated that prostaglandin E2 mediates the enhanced protein degradation induced by partially purified IL-1 preparations. In chick muscle preparations, the increased rate of protein degradation induced by chick monokines is not prostaglandindependent as indicated by the inability of ibuprofin to block this event (Klasing, unpublished observations). In addition to influencing muscle hypertrophy via regulating the accumulation of protein, monokines may also influence the ability of skeletal muscle to recruit new DNA to support growth. For example, TNF blocks the in vitro differentiation and fusion of human satellite cells taken from juvenile muscle (Miller et al, 1988). The TNF specifically blocks the expression of myosin heavy chain and alphaskeletal actin in myoblasts but not in fully differentiated myotubes.
SYMPOSIUM: AVIAN GROWTH AND DEVELOPMENT
in chicken adipose lipogenesis is in contrast to murine 3T3-L1 adipocytes whereby TNF or conditioned medium from endotoxin-stimulated macrophages inhibits lipogenesis (Pekala et al, 1983; Patton et al., 1986). Although the adipose tissue is not considered to be a major lipogenic site in the bird, increased lipogenic activity in this tissue may contribute to alterations in whole body composition. Hepatic Synthesis of Acute Phase Proteins
Bone and Mineral Metabolism A variety of evidence indicate the catabolic nature of monokines in bone and cartilage. Highly purified chicken IL-1 preparations induce the resorption of cartilage as indicated by proteoglycan release in vitro (Klasing and Peng, 1990). In rodents, IL-1 and TNF induce a catabolic state in cartilage as indicated by enhanced secretion of metalloproteinases, and impaired chondrocyte proliferation (Schnyder et al, 1987; Iwamoto etal, 1989). Also in rodents, IL-1 promotes bone resorption by stimulating osteoclasts and acts synergistically with TNF and parathyroid hormone (Dewhirst et al, 1987; Stashenko et al, 1987). The IL-1 desensitizes bone to the action of vitamin D (Evans et al, 1989). The net effect of IL-1 and TNF in
mammals is to decrease bone formation and enhance bone resorption, resulting in reduced bone mass and hypercalcemia. Leukocyte supernatants induce bone resorption by chick osteoclasts as indicated by calcium and proline release from test preparations in vitro (de Vermejoul et al, 1988). Thus, the high levels of circulating monokines present following an immune response are likely to prevent maximal rates of bone growth in chicks. Changes in divalent cation metabolism are one of the most sensitive indications of an immune response and elevated monokine levels. Changes in metabolism include decreased circulating concentrations of iron and zinc due to hepatic sequestering and increased copper associated with the acute phase protein ceruloplasmin (Laurin and Klasing, 1987). In the chick, these changes in levels of circulating trace minerals and hepatic metallothionein can be duplicated by injections of partially purified IL-1 preparations (Klasing, 1984). INTERACTIONS BETWEEN MONOKINES AND THE ENDOCRINE SYSTEM
Endocrinologists have long recognized that circulating concentrations of most metabolically important hormones are altered by an immune response. Elevated glucocorticoids, glucagon, insulin, and growth hormone, as well as decreased thyroxin, occurs following a challenge with most immunogens (Beisel, 1977). In rats, these same changes are induced by IL-1 or TNF (reviewed by Blalock, 1989). Many of these hormones also affect the release of monokines, forming a classical feedback loop. The best characterized interaction is the immuno-endocrine loop between IL-1 and glucocorticoids in which each hormone effects the level of the other. The IL-1 induces the release of glucocorticoids by inducing the release of corticotropin-releasing factor and adrenocorticotropin hormone. Completing the regulatory loop, glucocorticoids inhibit the release of IL-1, BL-6, and TNF from macrophages. In chicks, injections of partially purified IL-1 preparations, or endotoxin, result in substantial increases in serum glucocorticoids and decreases in triiodothyronine and thyroxine concentrations (Klasing, 1987). It is likely that the altered hormonal milieu induced by monokines contributes to the metabolic scenario responsible for impaired growth rates.
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Injections of immunogens into growing chicks results in an anabolic response in the liver as evidenced by increased fractional rates of protein synthesis (Klasing and Austic, 1984a). In particular, the rates of transcription of mRNA in the membrane-bound fraction, representing largely secreted protein, is increased. Among the secreted proteins, the acute phase proteins are synthesized at a much greater rate, whereas albumin synthesis is decreased (Greininger et al., 1989). An EL-6-like factor appears to be the active monokine augmenting the synthesis of acute phase proteins in the chick (Amrani et al., 1986). The acute phase proteins in the chick include C-reactive protein, hemopexin, fibrin, fibrinogen, ceruloplasmin, fibronectin, avidin, cci-acid glycoprotein, transferrin, ai-globulin M, a2-macroglobulins, and metallothionein. Acute phase proteins have immunomodulatory roles, functioning to enhance specific aspects of the immune response, limit the damage that results from the elaboration of noxious substances by leukocytes and aiding in the repair of tissues.
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GROWTH AND CARCASS COMPOSITION
MONOKINES AND DEVELOPMENT
Monokines have recently been shown to be powerful regulators of the developmental process. A murine monokine has been shown to signal initial events necessary for gastrulation and organization of polarity (Sokol et al., 1990). This monokine, designated PIF, induces presumptive ectodermal cells to form anterior neural and mesodermal tissues, including the brain and eye. No other factor has been shown to have such wide-reaching regulatory action on these critical early developmental steps. Several monokines also influence the acquisition of specialized phenotype of specific cell types. The TNF inhibits myoblast differentiation and formation of myotubes (Miller et al., 1988) in a manner similar to transforming growth factor p. In particular, the expression of myosin genes are inhibited. The TNF also inhibits the differentiation of pre-adipocytes into adipocytes and can cause adipocytes to de-differentiate. Again, this property is similar to the macrophage product, transforming growth factor p (Torti et al., 1989). In mice,
PRACTICAL IMPLICATIONS
If one assumes that minimizing circulating monokine levels is conducive for optimal growth, then minimizing the state of activity of the immune system would be a goal in poultry production. The frequency and vigor with which a bird's immune system is activated is related to the level of sanitation in its environment. Pathogenic bacteria in particular have the capacity to gain entrance and then multiply in the host, potentially invoking a vigorous immune response of long duration. Generally, animals are vaccinated against most highly virulent pathogens and management techniques are implemented to preclude problems with other pathogens so that clinical diseases are kept to a minimum in the flocks. Nevertheless, poor sanitation requires the animal's immune system to be frequently called upon to dispose of the large numbers of nonpathogenic microbes that arrive at the various epithelia. The relationship between level of sanitation and growth performance is well documented in the literature. For example, chicks housed in a germ-free environment grow 15% faster than those raised in conventional environments (Coates et al., 1963) and chicks housed in clean, disinfected quarters grow faster and more efficiently than those in less sanitary conditions even when no clinically identifiable diseases or pathogenic agents are present (Hill et al., 1952; Lillie et al., 1952; Libby and Schaible, 1955). It is well known that the improvements in production resulting from antibiotic use are related to the degree of sanitation practice, with the largest improvements seen in the environments with the poorest sanitation. Feeding antibiotics results in little or no improvements in growth rate or feed efficiency in clean environments (Hill et al., 1952). Preliminary data indicate that a mechanism of action for antibiotics is to decrease the number and severity of microbial interactions with the animal (Klasing, 1989). It is possible that by
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Three types of evidence strongly implicate high levels of circulating monokines as a cause of impaired growth rates of chicks. First, the elevation of monokine concentrations caused by invoking a chronic immune response decreases rates of growth (Klasing et al, 1987). Second, injecting crude monokine preparations into chicks decreases their growth rates as does injections of pure TNF or IL-1 in rodents (Fong et al., 1989). Third, monokines act directly on tissues to induce a metabolic state which is not conducive for maximal growdi. Certainly, the catabolic action of monokines on skeletal muscle and bone would indicate slower rates of accretion in these tissues, although accretion in the liver is increased. On a whole body basis, IL-1 and TNF induce a more negative nitrogen balance, indicating a lower rate of whole body accretion (Flores et al., 1989). Decreased intake coupled with increased basal metabolic rate would suggest decreased availability of dietary energy to support growth. It is not clear whether lean tissue and adipose tissue accretion rates are affected similarly by monokines. Chronic immunologic challenges result in changes in carcass moisture indicative of increased carcass fat content (Klasing et al., 1987).
several monokines influence the differentiation of leukocytes, including IL-1 and IL-6. The chicken monokine, myelomonocitic growth factor, induces stem cells in the bone marrow to form macrophage and granulocyte colonies (Leutz et al., 1989). This monokine is distinct from mammalian equivalents of TNF, IL-6, and IL-1.
SYMPOSIUM: AVIAN GROWTH AND DEVELOPMENT
The present review has focused on the often deleterious effect of monokines on growth. Strategies to obstruct the release or activity of monokines via pharmacologic agents may seem like a good target for improving growth rates. However, it should be recognized that these same regulators act locally, at the site of their release, to coordinate and amplify many aspects of the immune response. Thus, monokines are necessary and beneficial from the point of view of preventing infection in the animal. Pharmacologically blocking their action may be deleterious unless those physiological processes involved in growth can be selectively blocked without interfering with the immune response. Currently, reducing the
stimuli for monokine release is a better strategy for maximizing growth than administering pharmacological agents that block the release or activity of monokines. REFERENCES Amrani, D. L., D. Mauzy-Melitz, and M. W. Mosesson, 1986. Effect of hepatocyte-stimulating factor and glucocorticoids on plasma fibronectin levels. Biochem. J. 238:365-371. Beisel, W. R., 1977. Metabolic and nutritional consequences of infection. Pages 125-143 in: Advances in Nutritional Research. H. H. Draper, ed. Volume 1. Plenum, New York, NY. Beutler, B., and A. Cerami, 1988. The history, properties and biological effects of cachecrin. Biochemistry 27: 7575-7582. Blalock, J. E., 1989. A molecular basis for bidirectional communication between the immune and neuroendocrine systems. Physiol. Rev. 69:1-32. Butterwith, S. C, and H. D. Griffin, 1989. The effects of macrophage-derived cytokines on lipid metabolism in chicken (Gallus domesticus) hepatocytes and adipocytes. Comp. Biochem. Physiol. 94A:721-724. Coates, M. E., R. Fuller, G. F. Harrison, M Lev, and S. F. Suffolk, 1963. A comparison of the growth of chicks in the Gustafsson germ-free apparatus and in conventional environment, with and without dietary supplements of penicillin. Br. J. Nutr. 17:141-150. Cornelius, P., S. Enerback, G. Bjursell, T. Olivecrona, and P. H. Pekala, 1988. Regulation of lipoprotein lipase mRNA content in 3T3-L1 cells by tumor necrosis factor. Biochem. J. 249:765-769. Curtis, M. J., B. W. Scott, and E. J. Butler, 1980. Effect of Escherichia coli 078 endotoxin on plasma lipids in the domestic fowl. Res. Vet. Sci. 29:133-134. Dascombe, M. J., N. J. Rothwell, B. O. Sagay, and M. J. Stock, 1989. Pyrogenic and thermogenic effects of interleukin-11} in the rat. Am. J. Physiol. 256:E7-11. de Vernejoul, M.-C, M. Horowitz, J. Demignon, L. Neff, and R. Baron, 1988. Bone resorption by isolated chick osteoclasts in culture is stimulated by murine spleen cell supernatant fluids (osteoclast-activating factor) and inhibited by calcitonin and prostaglandin E2. J. Bone Miner. Res. 3:69-80. Dewhirst, F. E., J. M. Ago, W. J. Peros, and P. Stashenko, 1987. Synergism between parathyroid hormone and interleukin-1 in stimulating bone resorption in organ culture. J. Bone Miner. Res. 2:127-134. Dinarello, C. A., 1988. Biology of interleukin 1. Fed. Am. Soc. Exp. Biol. J. 2:108-115. Evans, D. B., R.A.D. Bunning, J. Van Damme, and R.G.G. Russell, 1989. Natural human IL-jJ exhibits regulatory actions on human bone-derived cells in vitro. Biochem. Biophys. Res. Commun. 159:1242-1348. Fagan, J. M, and A. L. Goldberg, 1985. Muscle protein breakdown, prostaglandin E2 production, and fever following bacterial infection. Pages 201-210 in: Progress in Leukocyte Biology. Volume 2. The Physiological, Metabolic, and Immunologic Actions of Interleukin-1. M. J. Kluger, J. J. Oppenheim and M. C. Powanda, ed. Liss, New York, NY. Feingold, K. R., and C. Grunfeld, 1987. Tumor necrosis factor-alpha stimulates hepatic lipogenesis in the rat in vivo. J. Clin. Invest. 80:184-190.
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decreasing the number of microbe-host interactions, antibiotics decrease the degree of activity of the immune system. Thus, antibiotics and good sanitation may decrease the levels of IL-1 and other monokines and normalize endocrine parameters that otherwise would decrease protein accretion, increase the metabolic rate, and result in poor feed conversion. The metabolic changes orchestrated by monokines that culminate in slower growth also influence the nutrient requirements of broiler chicks. For example, the methionine and lysine requirements for maximal growth decrease when chicks are subjected to chronic immunostimulation (Klasing and Barnes, 1987). The requirement is apparently decreased due to a lowered need for amino acids to support growth and tissue accretion, which is quantitatively larger than the increased use for gluconeogenesis or oxidation as an energy source. These results are relevant in practical animal husbandry in that the lysine and methionine requirements estimated in clean, low stress environments of research institutions may not underestimate the amino acid requirements of growing chicks raised in suboptimal sanitation during those periods in which the immune system must vigorously respond. It should be emphasized that these studies examine growth and efficiency of feed conversion as indices of the amino acid requirement, whereas other physiological processes, such as optimal immunocompetence, may require higher dietary amino acid levels for optimal function (Tsiagbe et al., 1987). Chicks undergo a period of compensatory growth subsequent to terminating immunologic stress, and amino acid requirements may well be increased during this period.
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KLASING AND JOHNSTONE in chicks: influence of feed intake, corticosterone and interleukin-1. J. Nutr. 117:1629-1637. Klasing, K. C, and R. K. Peng, 1990. Monokine-like activities released from a chicken macrophage line. Anim. Biotechnol. 1:107-120. Laurin, D. E., and K. C. Klasing, 1987. Effects of repetitive immunogen injections and fasting versus feeding on iron, zinc, and copper metabolism in chicks. Biol. Trace Elem. Res. 14:153-165. Leutz, A., K. Damm, E. Sterneck, E. Kowenz, S. Ness, R. Frank, H. Gausepohl, Y.-C. E. Pan, J. Smart, M Hayman, and T. Graf, 1989. Molecular cloning of the chicken myelomonocytic growth factor (cMGF) reveals relationship to interleukin 6 and granulocyte colony stimulating factor. Eur. Mol. Biol. Organ. J. 8:175-181. Libby, D. A., and P. J. Schaible, 1955. Observations on growth response to antibiotics and arsenic acids in poultry feeds. Science 121:733-734. Lillie, R. J., J. R. Sizemore, and H. R., Bird, 1952. Environment and stimulation of growth of chicks by antibiotics. Poultry Sci. 32:466-475. Meszaros, K., C. H. Lang, G. J. Bagby, and J. J. Spitzer, 1987. Tumor necrosis factor increases in vivo glucose utilization of macrophage-rich tissues. Biochem. Biophys. Res. Comm. 149:1-6. Miller, S. C, H. Ito, H. M Blau, and F. M. Torti, 1988. Tumor necrosis factor inhibits human myogenesis in vitro. Mol. Cell. Biol. 8:2295-2301. Ogawa, H., S. Nielsen, and M. Kawakami, 1989. Cachectin-tumor necrosis factor and interleukin-1 show different modes of combined effect on lipoprotein lipase activity and intracellular lipolysis in 3T3-L1 cells. Biocbim. Biophys. Acta 1003: 131-135. Patton, J. S., H. M. Shepard, H. Wilking, G. Lewis, B. B. Aggarwal, T. E. Eessalu, L. A. Gavin, and C. Grunfeld, 1986. Interferons and tumor necrosis factors have similar catabolic effects on 3T3 LI cells. Proc. Natl. Acad. Sci. USA 83:8313-8317. Pekala, P. H., M. Kawakami, C. W. Angus, M. D. Lane, and A. Cerami, 1983. Selective inhibition of synthesis of enzymes for de novo fatty acid biosynthesis by an endotoxin-induced mediator from exudate cells. Proc. Natl. Acad. Sci. USA 80: 2743-2747. Schnyder, J., T. Payne, and C. A. Dinarello, 1987. Human monocyte or recombinant interleukin-1's are specific for the secretion of a metalloproteinase from chondrocytes. J. Immunol. 138:496-503. SokoL S., G. G. Wong, and D. A. Melton, 1990. A mouse macrophage factor induces head structures and organizes a body axis in Xenopus. Science 249: 561-564. Stashenko, P., F. E. Dewhirst, W. J. Peros, R. L. Kent, and J. M. Ago, 1987. Synergesteric interactions between interleukin-1, tumor necrosis factor and lymphotoxin in bone resorption. J. Immunol. 138:1464-1468. Symons, L.E.A., and W. O. Jones, 1977. Protein metabolism TV. Altered protein synthesis in hosts infected with Eimeria tenella or bacteria compared to synthesis in hosts infected with intestinal nematodes. Exp. Parasitol. 42:194-202. Tsiagbe, V. K., M. E. Cook, A. E. Harper, and M L. Sunde, 1987. Enhanced immune responses in broiler chicks fed methionine-supplemented diets. Poultry Sci. 66:1147-1154.
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Feingold, K. R., M. Soued, I. Staprans, L. A. Gavin, M. E. Donahue, B.-J. Huang, A. H. Moser, R. Gulli, and C. Grunfeld, 1989. Effect of tumor necrosis factor (TNF) on lipid metabolism in the diabetic rat. J. Clin. Invest. 83:1116-1121. Flores, E. A., B. R. Bistrian, J. J. Pomposelli, C. A. Dinarello, G. L. Blackburn, and N. W. Istfan, 1989. Infusion of tumor necrosis factor/cachectin promotes muscle catabolism in the rat J. Clin. Invest. 83: 1614-1622. Fong, Y., L. L. Moldawer, M. Marano, H. Wei, A. Barber, K. Manogue, K. J. Tracey, G. Kuo, D. A. Fischman, A. Cerami, and S. F. Lowry, 1989. Cachectin/TNF or rL-loc induces cachexia with redistribution of body proteins. Am. J. Physiol. 256:R659-R665. Grieninger, G., C. Oddoux, L. Diamond, L. Weissbach, and P. W. Plant, 1989. Regulation of fibrinogen synthesis and secretion by the chicken hepatocyte. Ann. N. Y. Acad. Sci. 557:257-271. Griffin, H. D., and S. C. Butterwith, 1988. Effect of Escherichia coli endotoxin on tissue lipoprotein lipase activities in chickens. Br. Poult. Sci. 29: 371-378. Hentges, E. J., D. N. Marple, D. A. Roland, Sr., and J. F. Pritchett, 1984. Muscle protein synthesis and growth of two strains of chicks vaccinated for Newcastle disease and infectious bronchitis. Poultry Sci. 63: 1738-1741. Hill, D. C, H. D. Branion, S. J. Sliuger, and G. W. Anderson, 1952. Influence of environment on the growth response of chicks to penicillin. Poultry Sci. 32:464-466. Husbands, D. R., 1972. The distribution of lipoprotein lipase in tissues of the domestic fowl and the effects of feeding and starving. Br. Poult. Sci. 13:85-90. Iwamoto, M., T. Koike, K. Nakashima, K. Sato, and Y. Kato, 1989. Interleukin 1: a regulator of chondrocyte proliferation. Immunol. Lett. 21:153-156. Jepson, M. M., J. M. Pell, P. C. Bates, and D. J. Millward, 1986. The effects of endotoxaemia on protein metabolism in skeletal muscle and liver of fed and fasted rats. Biochem. J. 235:329-336. Klasing, K. C , 1984. Effect of inflammatory agents and interleukin 1 on iron and zinc metabolism. Am. J. Physiol. 247:R901-R904. Klasing, K. C , 1987. Avian interleukin-1: immunological and physiological functions. Pages 82-84 in: Proc. 36th Western Poultry Disease Conference. Davis, CA. Klasing, K. C, 1988. Nutritional aspects of leukocytic cytokines. J. Nutr. 118:1436-1446. Klasing, K. C , 1989. Nutritional implications of an immune response. Pages 193-202 in: Proc. 10th Western Nutrition Conference. Vancouver, BC, Canada. Klasing, K. C , and R. E. Austic, 1984a. Changes in protein synthesis due to an inflammatory challenge. Proc Soc. Exp. Biol. Med. 176:285-291. Klasing, K. C , and R. E. Austic, 1984b. Changes in protein degradation in chickens due to an inflammatory challenge. Proc. Soc. Exp. Biol. Med. 176: 292-296. Klasing, K. C , and D. M. Barnes, 1988. Decreased amino acid requirements of growing chicks due to immunologic stress. J. Nutr. 118:1158-1164. Klasing, K. C, D. E. Laurin, R. K. Peng, and D. M. Fry, 1987. Immunologically mediated growth depression
SYMPOSIUM: AVIAN GROWTH AND DEVELOPMENT Torti, F. M., S. V. Torti, J. W. Lamcfc, and G. M. Ringold, 1989. Modulation of adipocyte differentiation by tumor necrosis factor and transforming growth factor beta. J. Cell Biol. 108:1105-1113. Tredget, E. E., Y. M. Yu, S. Zhong, R. Burini, S. Okusawa, J. A. Gelfand, C. A. Dinarello, V. R. Young, and J. F. Burke, 1988. Role in interleukin-1 and tumor necrosis factor on energy metabolism in rabbits. Am. J. Physiol. 255:E760-768. Warren, R. S., H. F. Starnes, Jr., N. Alcock, S. Calvano,
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and M. F. Brennan, 1988. Hormonal and metabolic response to combinant human rumor necrosis factor in rat: in vitro and in vivo. Am. J. Physiol. 255: E206-E212. Zechner, R., T. C. Newman, B. Sherry, A. Cerami, and J. L. Breslow, 1988. Recombinant human cachectin/ tumor necrosis factor but not interleukin-la downregulates lipoprotein lipase gene expression at the transcriptional level in mouse 3T3-L1 adipocytes. Mol. CeU. Biol. 8:2394-2401.
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1991 Poultry Science 70:1781-1789 INTRODUCTION
Most important physiological processes are closely regulated by the participation of both positive and negative mediators. In the case of growth, the balance of anabolic and catabolic regulatory substances determines the rate of cellular hypertrophy and hyperplasia. Regulatory factors generally fall into two main categories: hormones and cytokines. Hormones are classically defined as substances produced at a discrete location (i.e., a gland) that enter the blood and act systemically. Cytokines are substances produced by widely dispersed and often dissimilar cells and may act either locally in an autocrine or paracrine fashion or systemically in an endocrine-like manner. Key anabolic factors such as growth hormone, insulin-like growth factors, and thyroid hormones have been the topic of considerable investigation because the potential exists to manipulate them to augment growth beyond the genetic potential of the animal. Catabolic regulatory factors include corticosteroids released due to most stresses and a variety of leukocytic cytokines released during the immune response. Two main classes of leukocytic cytokines have been described, monokines and lymphokines. Monokines and lymphokines are hormone-like peptides released by the macrophage-monocyte lineage
of cells and by the lymphocytes, respectively. Study of these catabolic factors has received considerably less attention by animal and poultry scientists than study of anabolic factors, probably because their manipulation may at best result in "optimal" growth rates at the animal's genetic potential. Paradoxically, the role of leukocytic cytokines is among the most intensely studied areas in the medical and biological sciences. This large body of research in laboratory rodents and humans demonstrates that monokines are released as an integral part of the immune response and orchestrate a variety of metabolic, cellular, and behavioral changes that are antagonistic toward growtih. IMMUNOLOGICAL STRESS AND GROWTH
It is well established that stimulation of the immune system results in a decreased rate of growth, whether or not the challenge is of infectious or uninfectious etiology. Cells of the mononuclear phagocytic system are the first line of defense in the immune system and thus are uniquely suited to identify immunogens, respond against immunogens, and provide information to the rest of the body, via monokines, that an immune response is occurring. The complex sequelae responsible for impaired growth have been well defined in
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ABSTRACT Stimulation of the immune system results in a series of metabolic changes that are antagonistic toward growth. Monokines, including interleukin-1, tumor necrosis factor, and interleukin-6, are released from cells of the monocyte-macrophage lineage after recognition of immunogens. They appear to mediate homeorhetic response, which alters the partitioning of dietary nutrients away from growth and skeletal muscle accretion in favor of metabolic processes which support the immune response and disease resistance. These alterations include 1) decreased skeletal muscle accretion due to increased rates of protein degradation and decreased protein synthesis; 2) increased basal metabolic rate resulting in increased energy utilization; 3) use of dietary amino acids for gluconeogenesis and as an energy source instead of for muscle protein accretion; 4) synthesis by the liver of acute phase proteins; 5) redistribution of iron, zinc, and copper within the body due to the hepatic synthesis of metallothionein, ferritin, and ceruloplasmin; 6) impaired accretion of cartilage and bone; and 7) release of hormones such as insulin, glucagon, and corticosterone. These monokines also influence the differentiation of cells. Tumor necrosis factor suppresses the differentiation of myoblasts and adipocytes whereas the chicken monokine myelomonocytic growth factor induces the differentiation of granulocytes. (Key words: monokines, interleukin-1, tumor necrosis factor, growth, metabolism)
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It has become apparent that most of the metabolic changes brought about by stimulation of the immune system are mediated by monokines. Data from use of chicks, as well as many investigations using rats and mice strongly implicate monokines with interleukin1 (IL-1), tumor necrosis factor-a (TNF) and IL-6 activities as mediators (reviewed by Beutler and Cerami, 1988; Klasing, 1988; Dinerello, 1988). The circulating concentrations of these monokines increase following most immune responses. These monokines act directly on responding tissues, or indirectly by altering the hormonal milieu, to cause the metabolic changes characterized as immunologic stress (Table 1). In many cases, the monokines BL-1, IL-6, and TNF have identical effects on tissues in laboratory mammals. The extreme redundancy of action and complex regulation of monokine release demonstrates the integral importance of this stress response. Each of the monokines also has a specific role in the regulation of the immune response, including controlling the recruitment and rates of proliferation of stimulated leukocyte populations and consequently mediating the initia-
TABLE 1. Overlapping regulatory roles of monokines in growth-related metabolism Response General Decreased voluntary food intake Increased resting energy expenditure Increased body temperature Glucose metabolism Increased glucose oxidation Increased gluconeogenesis Lipid metabolism Decreased lipoprotein lipase activity Increased lipolysis in adipocytes Increased hepatic triglyceride synthesis Increased adipocyte triglyceride synthesis Protein metabolism Increased acute phase protein synthesis Increased muscle protein degradation Bone and mineral metabolism Increased cartilage resorption Increased bone resorption Increased metallothionein synthesis Increased hepatic ceruloplasmin synthesis Hormone release Increased corticosteroid release Decreased thyroxin release Increased insulin and glucagon release
Chicks
Rodents
Murine cytokines responsible
X2 X X
X X X
IL-1, TNF IL-1, TNF IL-1, TNF, IL-6
? ?
X X
IL-1, TNF IL-1
X ?
-
X X X
X
-
IL-1, TNF, IL-6 IL-1, TNF TNF ?
X X
X X
IL-1, IL-6 IL-1, TNF
X 7 X X
X X X X
IL-1, TNF
X X ?
X X X
IL-1, TNF, IL-6 IL-1 IL-1, TNF
IL-1, IL-6 IL-1, TNF
IL-1 = interleukin-1; TNF = tumor necrosis factor; n>6 = interleukin-6. X indicates that a monokine induces the physiological response, - indicates that monokines do not induce a response, ? indicates that no data is available.
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laboratory mammals and sufficient work in chickens allows extrapolation of this information to Aves with some modification. The metabolic changes represent a homeorhetic response that alters the partitioning of dietary nutrients away from growth and skeletal muscle accretion in favor of metabolic processes that support the immune response and disease resistance. They form the basis for impaired growth, feed utilization, and altered nutrient requirements in chicks. These alterations include 1) decreased skeletal muscle accretion due to increased rates of protein degradation and decreased protein synthesis; 2) increased basal metabolic rate resulting in increased energy utilization; 3) a switch from fatty acids to glucose as a preferred energy source by many tissues; 4) use of dietary amino acids for gluconeogenesis and as an energy source instead of for muscle protein accretion; 5) synthesis by the liver of acute phase proteins; 6) redistribution of iron, zinc, and copper within the body due to the hepatic synthesis of metallothionein, ferritin, and ceruloplasmin; 7) impaired accretion of cartilage and bone; and 8) release of hormones such as insulin, glucagon, and corticosterone.
SYMPOSIUM: AVIAN GROWTH AND DEVELOPMENT
Feed Intake One of the most important growth-related changes induced by an immune response is die suppression of appetite and voluntary feed intake. The anorexigenic effect of infection as well as vaccination is appreciated by poultry producers and can easily be demonstrated in the laboratory. Partially purified IL-1 preparations mimic the anorexigenic effect of immunogens including Escherichia coli, lipopolysaccharide, and Sephadex (Klasing et al., 1987). In mammals IL-1, IL-6, and TNF are all anorexigenic. The anorexigenic activity of these monokines can account for most of the reduced growth rate observed during an immune response; however, a portion of the reduced growth is not related to the decreased feed intake, but rather is due to changes in metabolism. Energy Metabolism Fever is one of the hallmarks of infection or an immune response to defined immunogens. Partially purified IL-1 induces fever in chicks that is similar in magnitude to mat induced by E.
coli lipopolysaccharide (Klasing et al., 1987). This increased body temperature represents an increase in basal metabolic rate of 10 to 15%/1 C (Beisel, 1977). In rats, similar increases in basal metabolic rate have been found by direct measurement of energy expenditure following infusions of BL-1 i.v. or intracerebroventricularly (Dascombe et al, 1989). The TNF, interleukin-2, and IL-6 are also hypermetabolic, and IL-1 and TNF have synergistic effects on resting energy expenditure (Flores et al., 1989). In rodents, it is clear that monokines increase the rate of gluconeogenesis from amino acids, amino acid oxidation, and glucose utilization (Meszaros et al, 1987; Tredget et al, 1988; Warren et al, 1988). Skeletal Muscle Immune responses elicited by a variety of immunogens or infections decrease the rate of skeletal muscle protein accretion in growing chicks. Chicks infected with coccidia have decreased fractional rates of protein synthesis compared with uninfected chicks (Symons and Jones, 1977). Lipopolysaccharide and sheep red blood cells decrease rates of protein synthesis and increase degradation in chick skeletal muscle (Klasing and Austic, 1984a,b). Hentges et al. (1984) demonstrated that administration of Newcastle disease and infectious bronchitis vaccines results in decreased skeletal muscle protein synthesis and decreased protein accretion. In chicks and rats, the mechanism of the decreased protein synthesis involves decreased translational activity as indicated by decreased rate of synthesis per unit of RNA (Jepson et al, 1986) and decreased numbers of ribosomes translating mRNA (Klasing and Austic, 1984a). The impaired skeletal muscle protein accretion is apparently the result of cytokines elaborated by stimulated leukocytes. Incubation of skeletal muscle preparations from chick wings with partially purified EL-1 results in increased rates of protein degradation with no influence on the rate of protein degradation (Klasing etal, 1987). Interestingly, injections of IL-1-like preparations into the intact chick result in decreased skeletal muscle protein synthetic rates accompanied by a disaggregation of polysomes (Klasing and Austic, 1984a). Presumably, the in vivo results are due to the fact mat IL^l induces corticosterone release in chicks, and glucocorticoids reduce protein synthetic rates (Klasing et al, 1987). Thus,
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tion and length of an immune response. Thus, IL-1, TNF, and IL-6 function significantly to regulate the immune response to foreign immunogens, to alert the rest of the body to the penetration of immunogens into the body, and to orchestrate changes in intermediary metabolism to maximize immunocompetence. To date, research in chickens has generally utilized crude or partially purified preparations of monokines. The preparations utilized are usually enriched via a combination of purification processes based on a specific bioactivity, e.g., thymocyte comitogenesis for IL-1. Because these preparations are not known to be completely homogeneous, it is never certain which monokine is the active component. Consequently the name chicken IL-1 will be used to signify preparations purified based on thymocyte comitogenic activity, the term TNF will be used to signify preparations purified based on tumor cytolytic activity, and the term IL-6 is used to indicate purification based on hepatic acute phase protein synthesis. Progress in the monokine field is hampered by the lack of cloning and characterization of the respective genes and the availability of scrupulously purified proteins produced in quantity by recombinant DNA techniques.
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Lipid Metabolism In response to an immunogen such as endotoxin, plasma triglycerides, cholesterol/and phospholipids are reduced from control levels when fasting is used to equalize intake (Curtis et al, 1980). Plasma levels of very low density lipoproteins (VLDL) are generally reduced in comparison to fed controls (Griffin and Butterwith, 1988). This effect is in contrast to mammals, in which hyperlipidemia due to the accumulation of VLDL occurs rapidly after an injection of endotoxin and persists for several hours. Monokines (TNF, IL-1) mediate the hyperlipidemia in mammals through a coordinate increase in hepatic lipid synthesis with a reduction in lipoprotein clearance. The inhibition of lipoprotein lipase (LPL) results in decreased fatty acid uptake from circulating
triglycerides into tissues for utilization and storage. Tissue LPL activities in birds are reported to be less responsive to the nutritional state than in mammals (Husbands, 1972). However, chicken adipose, leg muscle, and heart LPL levels are significantly reduced by endotoxin injections in comparison with fasted controls (Griffin and Butterwith, 1988). Crude chicken monokine preparations, as well as preparations enriched for chicken IL-1, inhibit LPL activity in murine 3T3-L1 adipocytes (Klasing and Peng, 1990). The TNF reduces LPL activity by an almost complete suppression of LPL synthesis corresponding to a decrease in LPL mRNA (Cornelius et al, 1988), whereas IL-1 inhibits LPL synthesis without a reduction in mRNA (Zechner et al, 1988). Both cytokines also show a marked synergistic effect in suppressing LPL activities (Ogawa et al, 1989). In vitro studies indicate that monokines are involved in the LPL inhibition of avian adipose tissue. Incubating chicken adipocytes with conditioned medium from an LPS-stimulated avian macrophage cell line (HD-11) results in an almost complete suppression of LPL, indicating that monokines may be involved. However, recombinant human TNF and IL-1 have no effect on avian adipose LPL in vitro (Butterwith and Griffin, 1989). In mammals, the rapid hepatic synthesis and secretion of triglycerides contributes significantly to the monokine-induced hypertriglyceridemia (Feingold and Grunfeld, 1987). In rats, TNF acutely regulates hepatic fatty acid synthesis in vivo by raising hepatic levels of citrate, an allosteric activator of the rate-limiting enzyme acetyl-coenzyme A (CoA) carboxylase. Several hours later acetyl-CoA carboxylase and fatty acid synthetase are increased and the allosteric inhibitor fatty acyl-CoA is reduced (Feingold et al, 1989). Although there is little known about the effect of monokines on avian lipogenesis in vivo, the injection of endotoxin in chickens did not change hepatic fatty acid synthetase activity in contrast to fasted controls (Griffin and Butterwith, 1988). Incubation of chick hepatocytes with conditioned medium from LPS-stimulated HD-11 macrophage cells or human TNF does not significantly alter lipogenesis. However, high levels of human TNF does decrease the rate of hepatic lipogenesis. Interestingly, incubation of chicken adipocytes with LPS-stimulated HD-11 supernatants increases lipogenesis 200% (Butterwith and Griffin, 1989). The monokine-induced increase
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impaired skeletal muscle accretion results from both the direct effects of monokines to increase the rate of protein degradation and the indirect effects via corticosterone to decrease the rate of protein synthesis. Corticosterone by itself does not influence the rate of skeletal muscle protein degradation and does not augment induction by IL-1. Surprisingly, the anabolic effect of insulin on skeletal muscle is abrogated by IL-1, suggesting that IL-1 induces a state of insulin resistance in skeletal muscle. In mammals, both IL-1 and TNF induce protein degradation in skeletal muscle and their activities are synergistic (Flores et al, 1989). The second messenger responsible for inducing augmented proteolysis is not well defined. Fagen and Goldberg (1985) have demonstrated that prostaglandin E2 mediates the enhanced protein degradation induced by partially purified IL-1 preparations. In chick muscle preparations, the increased rate of protein degradation induced by chick monokines is not prostaglandindependent as indicated by the inability of ibuprofin to block this event (Klasing, unpublished observations). In addition to influencing muscle hypertrophy via regulating the accumulation of protein, monokines may also influence the ability of skeletal muscle to recruit new DNA to support growth. For example, TNF blocks the in vitro differentiation and fusion of human satellite cells taken from juvenile muscle (Miller et al, 1988). The TNF specifically blocks the expression of myosin heavy chain and alphaskeletal actin in myoblasts but not in fully differentiated myotubes.
SYMPOSIUM: AVIAN GROWTH AND DEVELOPMENT
in chicken adipose lipogenesis is in contrast to murine 3T3-L1 adipocytes whereby TNF or conditioned medium from endotoxin-stimulated macrophages inhibits lipogenesis (Pekala et al, 1983; Patton et al., 1986). Although the adipose tissue is not considered to be a major lipogenic site in the bird, increased lipogenic activity in this tissue may contribute to alterations in whole body composition. Hepatic Synthesis of Acute Phase Proteins
Bone and Mineral Metabolism A variety of evidence indicate the catabolic nature of monokines in bone and cartilage. Highly purified chicken IL-1 preparations induce the resorption of cartilage as indicated by proteoglycan release in vitro (Klasing and Peng, 1990). In rodents, IL-1 and TNF induce a catabolic state in cartilage as indicated by enhanced secretion of metalloproteinases, and impaired chondrocyte proliferation (Schnyder et al, 1987; Iwamoto etal, 1989). Also in rodents, IL-1 promotes bone resorption by stimulating osteoclasts and acts synergistically with TNF and parathyroid hormone (Dewhirst et al, 1987; Stashenko et al, 1987). The IL-1 desensitizes bone to the action of vitamin D (Evans et al, 1989). The net effect of IL-1 and TNF in
mammals is to decrease bone formation and enhance bone resorption, resulting in reduced bone mass and hypercalcemia. Leukocyte supernatants induce bone resorption by chick osteoclasts as indicated by calcium and proline release from test preparations in vitro (de Vermejoul et al, 1988). Thus, the high levels of circulating monokines present following an immune response are likely to prevent maximal rates of bone growth in chicks. Changes in divalent cation metabolism are one of the most sensitive indications of an immune response and elevated monokine levels. Changes in metabolism include decreased circulating concentrations of iron and zinc due to hepatic sequestering and increased copper associated with the acute phase protein ceruloplasmin (Laurin and Klasing, 1987). In the chick, these changes in levels of circulating trace minerals and hepatic metallothionein can be duplicated by injections of partially purified IL-1 preparations (Klasing, 1984). INTERACTIONS BETWEEN MONOKINES AND THE ENDOCRINE SYSTEM
Endocrinologists have long recognized that circulating concentrations of most metabolically important hormones are altered by an immune response. Elevated glucocorticoids, glucagon, insulin, and growth hormone, as well as decreased thyroxin, occurs following a challenge with most immunogens (Beisel, 1977). In rats, these same changes are induced by IL-1 or TNF (reviewed by Blalock, 1989). Many of these hormones also affect the release of monokines, forming a classical feedback loop. The best characterized interaction is the immuno-endocrine loop between IL-1 and glucocorticoids in which each hormone effects the level of the other. The IL-1 induces the release of glucocorticoids by inducing the release of corticotropin-releasing factor and adrenocorticotropin hormone. Completing the regulatory loop, glucocorticoids inhibit the release of IL-1, BL-6, and TNF from macrophages. In chicks, injections of partially purified IL-1 preparations, or endotoxin, result in substantial increases in serum glucocorticoids and decreases in triiodothyronine and thyroxine concentrations (Klasing, 1987). It is likely that the altered hormonal milieu induced by monokines contributes to the metabolic scenario responsible for impaired growth rates.
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Injections of immunogens into growing chicks results in an anabolic response in the liver as evidenced by increased fractional rates of protein synthesis (Klasing and Austic, 1984a). In particular, the rates of transcription of mRNA in the membrane-bound fraction, representing largely secreted protein, is increased. Among the secreted proteins, the acute phase proteins are synthesized at a much greater rate, whereas albumin synthesis is decreased (Greininger et al., 1989). An EL-6-like factor appears to be the active monokine augmenting the synthesis of acute phase proteins in the chick (Amrani et al., 1986). The acute phase proteins in the chick include C-reactive protein, hemopexin, fibrin, fibrinogen, ceruloplasmin, fibronectin, avidin, cci-acid glycoprotein, transferrin, ai-globulin M, a2-macroglobulins, and metallothionein. Acute phase proteins have immunomodulatory roles, functioning to enhance specific aspects of the immune response, limit the damage that results from the elaboration of noxious substances by leukocytes and aiding in the repair of tissues.
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GROWTH AND CARCASS COMPOSITION
MONOKINES AND DEVELOPMENT
Monokines have recently been shown to be powerful regulators of the developmental process. A murine monokine has been shown to signal initial events necessary for gastrulation and organization of polarity (Sokol et al., 1990). This monokine, designated PIF, induces presumptive ectodermal cells to form anterior neural and mesodermal tissues, including the brain and eye. No other factor has been shown to have such wide-reaching regulatory action on these critical early developmental steps. Several monokines also influence the acquisition of specialized phenotype of specific cell types. The TNF inhibits myoblast differentiation and formation of myotubes (Miller et al., 1988) in a manner similar to transforming growth factor p. In particular, the expression of myosin genes are inhibited. The TNF also inhibits the differentiation of pre-adipocytes into adipocytes and can cause adipocytes to de-differentiate. Again, this property is similar to the macrophage product, transforming growth factor p (Torti et al., 1989). In mice,
PRACTICAL IMPLICATIONS
If one assumes that minimizing circulating monokine levels is conducive for optimal growth, then minimizing the state of activity of the immune system would be a goal in poultry production. The frequency and vigor with which a bird's immune system is activated is related to the level of sanitation in its environment. Pathogenic bacteria in particular have the capacity to gain entrance and then multiply in the host, potentially invoking a vigorous immune response of long duration. Generally, animals are vaccinated against most highly virulent pathogens and management techniques are implemented to preclude problems with other pathogens so that clinical diseases are kept to a minimum in the flocks. Nevertheless, poor sanitation requires the animal's immune system to be frequently called upon to dispose of the large numbers of nonpathogenic microbes that arrive at the various epithelia. The relationship between level of sanitation and growth performance is well documented in the literature. For example, chicks housed in a germ-free environment grow 15% faster than those raised in conventional environments (Coates et al., 1963) and chicks housed in clean, disinfected quarters grow faster and more efficiently than those in less sanitary conditions even when no clinically identifiable diseases or pathogenic agents are present (Hill et al., 1952; Lillie et al., 1952; Libby and Schaible, 1955). It is well known that the improvements in production resulting from antibiotic use are related to the degree of sanitation practice, with the largest improvements seen in the environments with the poorest sanitation. Feeding antibiotics results in little or no improvements in growth rate or feed efficiency in clean environments (Hill et al., 1952). Preliminary data indicate that a mechanism of action for antibiotics is to decrease the number and severity of microbial interactions with the animal (Klasing, 1989). It is possible that by
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Three types of evidence strongly implicate high levels of circulating monokines as a cause of impaired growth rates of chicks. First, the elevation of monokine concentrations caused by invoking a chronic immune response decreases rates of growth (Klasing et al, 1987). Second, injecting crude monokine preparations into chicks decreases their growth rates as does injections of pure TNF or IL-1 in rodents (Fong et al., 1989). Third, monokines act directly on tissues to induce a metabolic state which is not conducive for maximal growdi. Certainly, the catabolic action of monokines on skeletal muscle and bone would indicate slower rates of accretion in these tissues, although accretion in the liver is increased. On a whole body basis, IL-1 and TNF induce a more negative nitrogen balance, indicating a lower rate of whole body accretion (Flores et al., 1989). Decreased intake coupled with increased basal metabolic rate would suggest decreased availability of dietary energy to support growth. It is not clear whether lean tissue and adipose tissue accretion rates are affected similarly by monokines. Chronic immunologic challenges result in changes in carcass moisture indicative of increased carcass fat content (Klasing et al., 1987).
several monokines influence the differentiation of leukocytes, including IL-1 and IL-6. The chicken monokine, myelomonocitic growth factor, induces stem cells in the bone marrow to form macrophage and granulocyte colonies (Leutz et al., 1989). This monokine is distinct from mammalian equivalents of TNF, IL-6, and IL-1.
SYMPOSIUM: AVIAN GROWTH AND DEVELOPMENT
The present review has focused on the often deleterious effect of monokines on growth. Strategies to obstruct the release or activity of monokines via pharmacologic agents may seem like a good target for improving growth rates. However, it should be recognized that these same regulators act locally, at the site of their release, to coordinate and amplify many aspects of the immune response. Thus, monokines are necessary and beneficial from the point of view of preventing infection in the animal. Pharmacologically blocking their action may be deleterious unless those physiological processes involved in growth can be selectively blocked without interfering with the immune response. Currently, reducing the
stimuli for monokine release is a better strategy for maximizing growth than administering pharmacological agents that block the release or activity of monokines. REFERENCES Amrani, D. L., D. Mauzy-Melitz, and M. W. Mosesson, 1986. Effect of hepatocyte-stimulating factor and glucocorticoids on plasma fibronectin levels. Biochem. J. 238:365-371. Beisel, W. R., 1977. Metabolic and nutritional consequences of infection. Pages 125-143 in: Advances in Nutritional Research. H. H. Draper, ed. Volume 1. Plenum, New York, NY. Beutler, B., and A. Cerami, 1988. The history, properties and biological effects of cachecrin. Biochemistry 27: 7575-7582. Blalock, J. E., 1989. A molecular basis for bidirectional communication between the immune and neuroendocrine systems. Physiol. Rev. 69:1-32. Butterwith, S. C, and H. D. Griffin, 1989. The effects of macrophage-derived cytokines on lipid metabolism in chicken (Gallus domesticus) hepatocytes and adipocytes. Comp. Biochem. Physiol. 94A:721-724. Coates, M. E., R. Fuller, G. F. Harrison, M Lev, and S. F. Suffolk, 1963. A comparison of the growth of chicks in the Gustafsson germ-free apparatus and in conventional environment, with and without dietary supplements of penicillin. Br. J. Nutr. 17:141-150. Cornelius, P., S. Enerback, G. Bjursell, T. Olivecrona, and P. H. Pekala, 1988. Regulation of lipoprotein lipase mRNA content in 3T3-L1 cells by tumor necrosis factor. Biochem. J. 249:765-769. Curtis, M. J., B. W. Scott, and E. J. Butler, 1980. Effect of Escherichia coli 078 endotoxin on plasma lipids in the domestic fowl. Res. Vet. Sci. 29:133-134. Dascombe, M. J., N. J. Rothwell, B. O. Sagay, and M. J. Stock, 1989. Pyrogenic and thermogenic effects of interleukin-11} in the rat. Am. J. Physiol. 256:E7-11. de Vernejoul, M.-C, M. Horowitz, J. Demignon, L. Neff, and R. Baron, 1988. Bone resorption by isolated chick osteoclasts in culture is stimulated by murine spleen cell supernatant fluids (osteoclast-activating factor) and inhibited by calcitonin and prostaglandin E2. J. Bone Miner. Res. 3:69-80. Dewhirst, F. E., J. M. Ago, W. J. Peros, and P. Stashenko, 1987. Synergism between parathyroid hormone and interleukin-1 in stimulating bone resorption in organ culture. J. Bone Miner. Res. 2:127-134. Dinarello, C. A., 1988. Biology of interleukin 1. Fed. Am. Soc. Exp. Biol. J. 2:108-115. Evans, D. B., R.A.D. Bunning, J. Van Damme, and R.G.G. Russell, 1989. Natural human IL-jJ exhibits regulatory actions on human bone-derived cells in vitro. Biochem. Biophys. Res. Commun. 159:1242-1348. Fagan, J. M, and A. L. Goldberg, 1985. Muscle protein breakdown, prostaglandin E2 production, and fever following bacterial infection. Pages 201-210 in: Progress in Leukocyte Biology. Volume 2. The Physiological, Metabolic, and Immunologic Actions of Interleukin-1. M. J. Kluger, J. J. Oppenheim and M. C. Powanda, ed. Liss, New York, NY. Feingold, K. R., and C. Grunfeld, 1987. Tumor necrosis factor-alpha stimulates hepatic lipogenesis in the rat in vivo. J. Clin. Invest. 80:184-190.
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decreasing the number of microbe-host interactions, antibiotics decrease the degree of activity of the immune system. Thus, antibiotics and good sanitation may decrease the levels of IL-1 and other monokines and normalize endocrine parameters that otherwise would decrease protein accretion, increase the metabolic rate, and result in poor feed conversion. The metabolic changes orchestrated by monokines that culminate in slower growth also influence the nutrient requirements of broiler chicks. For example, the methionine and lysine requirements for maximal growth decrease when chicks are subjected to chronic immunostimulation (Klasing and Barnes, 1987). The requirement is apparently decreased due to a lowered need for amino acids to support growth and tissue accretion, which is quantitatively larger than the increased use for gluconeogenesis or oxidation as an energy source. These results are relevant in practical animal husbandry in that the lysine and methionine requirements estimated in clean, low stress environments of research institutions may not underestimate the amino acid requirements of growing chicks raised in suboptimal sanitation during those periods in which the immune system must vigorously respond. It should be emphasized that these studies examine growth and efficiency of feed conversion as indices of the amino acid requirement, whereas other physiological processes, such as optimal immunocompetence, may require higher dietary amino acid levels for optimal function (Tsiagbe et al., 1987). Chicks undergo a period of compensatory growth subsequent to terminating immunologic stress, and amino acid requirements may well be increased during this period.
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KLASING AND JOHNSTONE in chicks: influence of feed intake, corticosterone and interleukin-1. J. Nutr. 117:1629-1637. Klasing, K. C, and R. K. Peng, 1990. Monokine-like activities released from a chicken macrophage line. Anim. Biotechnol. 1:107-120. Laurin, D. E., and K. C. Klasing, 1987. Effects of repetitive immunogen injections and fasting versus feeding on iron, zinc, and copper metabolism in chicks. Biol. Trace Elem. Res. 14:153-165. Leutz, A., K. Damm, E. Sterneck, E. Kowenz, S. Ness, R. Frank, H. Gausepohl, Y.-C. E. Pan, J. Smart, M Hayman, and T. Graf, 1989. Molecular cloning of the chicken myelomonocytic growth factor (cMGF) reveals relationship to interleukin 6 and granulocyte colony stimulating factor. Eur. Mol. Biol. Organ. J. 8:175-181. Libby, D. A., and P. J. Schaible, 1955. Observations on growth response to antibiotics and arsenic acids in poultry feeds. Science 121:733-734. Lillie, R. J., J. R. Sizemore, and H. R., Bird, 1952. Environment and stimulation of growth of chicks by antibiotics. Poultry Sci. 32:466-475. Meszaros, K., C. H. Lang, G. J. Bagby, and J. J. Spitzer, 1987. Tumor necrosis factor increases in vivo glucose utilization of macrophage-rich tissues. Biochem. Biophys. Res. Comm. 149:1-6. Miller, S. C, H. Ito, H. M Blau, and F. M. Torti, 1988. Tumor necrosis factor inhibits human myogenesis in vitro. Mol. Cell. Biol. 8:2295-2301. Ogawa, H., S. Nielsen, and M. Kawakami, 1989. Cachectin-tumor necrosis factor and interleukin-1 show different modes of combined effect on lipoprotein lipase activity and intracellular lipolysis in 3T3-L1 cells. Biocbim. Biophys. Acta 1003: 131-135. Patton, J. S., H. M. Shepard, H. Wilking, G. Lewis, B. B. Aggarwal, T. E. Eessalu, L. A. Gavin, and C. Grunfeld, 1986. Interferons and tumor necrosis factors have similar catabolic effects on 3T3 LI cells. Proc. Natl. Acad. Sci. USA 83:8313-8317. Pekala, P. H., M. Kawakami, C. W. Angus, M. D. Lane, and A. Cerami, 1983. Selective inhibition of synthesis of enzymes for de novo fatty acid biosynthesis by an endotoxin-induced mediator from exudate cells. Proc. Natl. Acad. Sci. USA 80: 2743-2747. Schnyder, J., T. Payne, and C. A. Dinarello, 1987. Human monocyte or recombinant interleukin-1's are specific for the secretion of a metalloproteinase from chondrocytes. J. Immunol. 138:496-503. SokoL S., G. G. Wong, and D. A. Melton, 1990. A mouse macrophage factor induces head structures and organizes a body axis in Xenopus. Science 249: 561-564. Stashenko, P., F. E. Dewhirst, W. J. Peros, R. L. Kent, and J. M. Ago, 1987. Synergesteric interactions between interleukin-1, tumor necrosis factor and lymphotoxin in bone resorption. J. Immunol. 138:1464-1468. Symons, L.E.A., and W. O. Jones, 1977. Protein metabolism TV. Altered protein synthesis in hosts infected with Eimeria tenella or bacteria compared to synthesis in hosts infected with intestinal nematodes. Exp. Parasitol. 42:194-202. Tsiagbe, V. K., M. E. Cook, A. E. Harper, and M L. Sunde, 1987. Enhanced immune responses in broiler chicks fed methionine-supplemented diets. Poultry Sci. 66:1147-1154.
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Feingold, K. R., M. Soued, I. Staprans, L. A. Gavin, M. E. Donahue, B.-J. Huang, A. H. Moser, R. Gulli, and C. Grunfeld, 1989. Effect of tumor necrosis factor (TNF) on lipid metabolism in the diabetic rat. J. Clin. Invest. 83:1116-1121. Flores, E. A., B. R. Bistrian, J. J. Pomposelli, C. A. Dinarello, G. L. Blackburn, and N. W. Istfan, 1989. Infusion of tumor necrosis factor/cachectin promotes muscle catabolism in the rat J. Clin. Invest. 83: 1614-1622. Fong, Y., L. L. Moldawer, M. Marano, H. Wei, A. Barber, K. Manogue, K. J. Tracey, G. Kuo, D. A. Fischman, A. Cerami, and S. F. Lowry, 1989. Cachectin/TNF or rL-loc induces cachexia with redistribution of body proteins. Am. J. Physiol. 256:R659-R665. Grieninger, G., C. Oddoux, L. Diamond, L. Weissbach, and P. W. Plant, 1989. Regulation of fibrinogen synthesis and secretion by the chicken hepatocyte. Ann. N. Y. Acad. Sci. 557:257-271. Griffin, H. D., and S. C. Butterwith, 1988. Effect of Escherichia coli endotoxin on tissue lipoprotein lipase activities in chickens. Br. Poult. Sci. 29: 371-378. Hentges, E. J., D. N. Marple, D. A. Roland, Sr., and J. F. Pritchett, 1984. Muscle protein synthesis and growth of two strains of chicks vaccinated for Newcastle disease and infectious bronchitis. Poultry Sci. 63: 1738-1741. Hill, D. C, H. D. Branion, S. J. Sliuger, and G. W. Anderson, 1952. Influence of environment on the growth response of chicks to penicillin. Poultry Sci. 32:464-466. Husbands, D. R., 1972. The distribution of lipoprotein lipase in tissues of the domestic fowl and the effects of feeding and starving. Br. Poult. Sci. 13:85-90. Iwamoto, M., T. Koike, K. Nakashima, K. Sato, and Y. Kato, 1989. Interleukin 1: a regulator of chondrocyte proliferation. Immunol. Lett. 21:153-156. Jepson, M. M., J. M. Pell, P. C. Bates, and D. J. Millward, 1986. The effects of endotoxaemia on protein metabolism in skeletal muscle and liver of fed and fasted rats. Biochem. J. 235:329-336. Klasing, K. C , 1984. Effect of inflammatory agents and interleukin 1 on iron and zinc metabolism. Am. J. Physiol. 247:R901-R904. Klasing, K. C , 1987. Avian interleukin-1: immunological and physiological functions. Pages 82-84 in: Proc. 36th Western Poultry Disease Conference. Davis, CA. Klasing, K. C, 1988. Nutritional aspects of leukocytic cytokines. J. Nutr. 118:1436-1446. Klasing, K. C , 1989. Nutritional implications of an immune response. Pages 193-202 in: Proc. 10th Western Nutrition Conference. Vancouver, BC, Canada. Klasing, K. C , and R. E. Austic, 1984a. Changes in protein synthesis due to an inflammatory challenge. Proc Soc. Exp. Biol. Med. 176:285-291. Klasing, K. C , and R. E. Austic, 1984b. Changes in protein degradation in chickens due to an inflammatory challenge. Proc. Soc. Exp. Biol. Med. 176: 292-296. Klasing, K. C , and D. M. Barnes, 1988. Decreased amino acid requirements of growing chicks due to immunologic stress. J. Nutr. 118:1158-1164. Klasing, K. C, D. E. Laurin, R. K. Peng, and D. M. Fry, 1987. Immunologically mediated growth depression
SYMPOSIUM: AVIAN GROWTH AND DEVELOPMENT Torti, F. M., S. V. Torti, J. W. Lamcfc, and G. M. Ringold, 1989. Modulation of adipocyte differentiation by tumor necrosis factor and transforming growth factor beta. J. Cell Biol. 108:1105-1113. Tredget, E. E., Y. M. Yu, S. Zhong, R. Burini, S. Okusawa, J. A. Gelfand, C. A. Dinarello, V. R. Young, and J. F. Burke, 1988. Role in interleukin-1 and tumor necrosis factor on energy metabolism in rabbits. Am. J. Physiol. 255:E760-768. Warren, R. S., H. F. Starnes, Jr., N. Alcock, S. Calvano,
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