Progress 10/01/03 to 09/30/09
Outputs
OUTPUTS: A large body of evidence has accumulated during the last decade to demonstrate that the immune and endocrine systems interact to regulate important physiological processes, ranging from cell growth and metabolism to animal behavior. It has been well documented that the major growth-promoting endocrine hormones, growth hormone (GH) and insulin-like growth factor-I (IGF-I), can modulate a plethora of immune events. The surprising recent development is the flip side of this interaction: cytokines from the innate immune system exert profound effects on the endocrine system. Many cytokines and hormones share intracellular biochemical substrates and transcription factors, which provides a molecular basis for intracellular crosstalk between the endocrine and immune systems. In response to inflammatory processes such as tissue injury, low grade chronic infections or stress, pro-inflammatory cytokines not only reduce feed intake but they also antagonize cellular responses to the major anabolic growth-promoting hormones, insulin, GH and IGF-I. The latter event leads to a state of cytokine-induced hormone resistance. Some antibiotics have now been shown to exhibit anti-inflammatory properties, which may antagonize hormone resistance. This possibility is particularly relevant considering the common practice of feeding sub-therapeutic prophylactic levels of antimicrobial products to intensively-reared production animals. With the growing public concern about antimicrobial resistance caused by feeding such large amounts of antibiotics to farm animals, the global health ramifications of this practice continue to be fiercely debated. This research project demonstrated the potential impact of inflammation-induced hormone resistance on muscle growth and development and discovered a biochemical mechanism that is responsible proinflammatory cytokines induces resistance to the major growth factor, IGF-I. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Muscle mass of meat-producing animals is increased primarily by IGF-I, which is regulated by GH. In the absence of antibiotics, pathogens more easily overwhelm and activate the innate immune system, leading to an increase in proinflammatory cytokines such as IL-1β and TNF-α. These cytokines act in two major ways to reduce animal growth: (a) they cause a reduction in food intake; and (b) they interfere with the ability of the major growth promoting hormones, GH, insulin and IGF-I, to promote protein accretion in skeletal muscle. This phenomenon is a result of hormone resistance.
Publications
- O'Connor, J.C., McCusker, R.H., Johnson, R.W., Dantzer, R. and Kelley, K.W. 2009. Antibiotics, inflammation and animal growth: Cytokine-induced hormone resistance. Encylopedia of Animal Science, Second Edition. Taylor and Francis (In Press).
- Kelley, K.W., Curtis, S.E. and Dantzer, R. 2009. Disease-environment interactions: Another contribution of Louis Pasteur, 1878. http://www.brainimmune.com/mockup/index.phpoption=com_content&view=a rticle&id=35:active-link-r-disease-environment-interact.
- Strle, K., McCusker, R.H., Tran, L., King, A., Johnson, R.W., Freund, G.G., Dantzer, R. and Kelley, K.W. 2007. Novel activity of an anti-inflammatory cytokine: IL-10 prevents TNFα-induced resistance to IGF-I in myoblasts. J. Neuroimmunology 188:48-55.
- Kelley, K.W., Weigent, D.A. and Kooijman, R. 2007. Protein hormones and immunity. Brain, Behavior, and Immunity 21:384-392.
- McCusker, R.H., Strle, K., Broussard, S.R., Dantzer, R., Bluthe, R.M. and Kelley, K.W. 2006. Crosstalk between insulin-like growth factors and proinflammatory cytokines. In: R. Ader, R. Dantzer, R. Glaser, C. Heijnen, M. Irwin, D. Padgett and J.Sheridan (eds.) Psychoneuroimmunology. Fourth Edition. Elsevier, San Diego. pp. 171-192.
- Bluthe, R.M., Kelley, K.W. and Dantzer, R. 2006. Effects of insulin-like growth factor-I on cytokine-induced sickness behavior in mice. Brain, Behavior, and Immunity 20:57-63.
- McCusker, R.H., McCrae, K., Zunich, S., Dantzer, R., Broussard, S.R., Johnson, R.W. and Kelley, K.W. 2006. Insulin-like growth factor-I enhances the biological activity of brain-derived neurotrophic factor on cerebrocortical neurons. J. Neuroimmunology 179:186-190.
- Strle, K., Broussard, S.R., McCusker, R.H., Shen, W.H., LeCleir, J., Johnson, R.W., Freund, G.G., Dantzer, R. and Kelley, K.W. 2006. C-jun N-terminal kinase mediates TNFα suppression of differentiation in myoblasts. Endocrinology 147:4363-4373.
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Progress 01/01/08 to 12/31/08
Outputs
OUTPUTS: Prolonged and excessive inflammation is implicated in resistance to the biological actions of IGF-I and contributes to the pathophysiology of neurodegenerative, metabolic and muscle-wasting disorders. The transcription factor and stress kinase, c-jun N-terminal kinase, is critically involved in the signaling pathway of many inflammatory proteins. IL-10 is an important anti-inflammatory cytokine that restrains inflammatory responses in macrophages and T cells by inhibiting cytokine and chemokine synthesis and reducing expression of their receptors. We have demonstrated that IL-10 plays a protective role in non-hematopoietic cells by suppressing the ability of exogenous IL-1 to inhibit IGF-I-induced myogenin and myosin heavy chain expression in myoblasts. This action of IL-10 is not caused by impairing IL-1-induced synthesis of IL-6 or the ability of IL-1 to activate two members of the MAPK family, ERK1/2 or p38. Instead, this newly-defined protective role of IL-10 occurs by specifically reversing IL-1 activation of the JNK kinase pathway. IL-10 blocks IL-1-induced phosphorylation of JNK, but not ERK1/2 or p38, indicating that only the JNK component of IL-1-induced MAPK signaling pathway is targeted by IL-10. Collectively, these data demonstrate that IL-10 acts in a novel, non-classical, protective manner in non-hematopoietic cells to inhibit the IL-1 receptor-induced JNK kinase pathway, resulting in prevention of IGF-I resistance. JNK can also be activated by another major proinflammatory cytokine known as tumor necrosis factor (TNF). This protein is now known to elicit a number of physiological changes and behavioral changes that are mediated in the brain. The molecular mechanisms by which TNF induces sickness behavior in the central nervous system are still unknown. The biological activity of TNF is mediated by a 60 kDa (TNFR1) and an 80 kDa (TNFR2) receptor. TNF binding induces receptor trimerization, ultimately resulting in activation of the JNK and induction of downstream transcription factors. We hypothesized that TNF-induced sickness behavior requires JNK activation. To test this idea, we determined if sickness behavior induced by central TNF could be inhibited by pre-treatment with a fusion protein consisting of a protein transduction domain fused to a novel peptide inhibitor of JNK kinase activity, D JNKI 1. C57BL/6J mice were pretreated i.c.v. with two concentrations of D-JNKI-1 (5 or 10 ng/mouse) or the control peptide with the protein transduction domain, D-TAT (10 ng/mouse), and then centrally injected with an optimal amount of TNF (50 ng/mouse) to induce sickness behavior. Sickness was assessed as a loss of body weight, depression in social exploration of a novel juvenile and the increase in duration of immobility. D-JNKI-1 (10 ng/mouse), but not D-TAT, prevented all indices of sickness induced by central TNFα. These data thereby directly implicate brain-derived JNK in TNF-induced sickness behavior. The findings also suggest that inhibition of central JNK kinase activity may serve as a potential therapeutic tool for treating cytokine-induced sickness behavior. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Acute disease causes animals to become sick, reduce food intake, and lose body mass. Here we discovered that a stress kinase known as JNK is a critical downstream signaling component by which proinflammatory cytokines like TNF induces sickness behavior. A new mechanism of action was also discovered for the anti-inflammatory cytokine IL-10, which we have now established to directly reduce the ability of proinflammatory cytokines to activate JNK. Inhibition of JNK during inflammation may promote animal productivity.
Publications
- Dantzer, R., O'Connor, J.C., Freund, G.G., Johnson, R.W. and Kelley, K.W. 2008. From inflammation to sickness and depression: When the immune system subjugates the brain. Nature Reviews Neuroscience 9:46-57. doi:10.1038/nrn2297.
- Strle, K., McCusker, R.H., Johnson, R.W., Zunich, S.M., Dantzer, R. and Kelley, K.W. 2008. The prototypical anti-inflammatory cytokine IL-10 prevents loss of IGF-I-induced myogenin protein expression caused by IL-1β. Am. J. Physiol. Endocrinol. Metab. 294:709-718. doi:10.1152/ajpendo.00662.2007 0193-1849/08.
- Palin, K., McCusker, R.H., Strle, K., Moos, F., Dantzer, R. and Kelley, K.W. 2008. Tumor necrosis factor-α-induced sickness behavior is impaired by central administration of an inhibitor of c-jun N-terminal kinase. Psychopharmacology 197:629-635. doi 10.1007/s00213-008-1086-y.
- O'Connor, J.C., McCusker, R.H., Strle, K., Johnson, R.W., Dantzer, R. and Kelley, K.W. 2008. Regulation of IGF-I function by proinflammatory cytokines: At the interface of immunology and endocrinology. Cellular Immunology doi:10.1016/j.cellimm.2007.09.010.
- Moreau, M., Andre, C., O'Connor, J.C., Dumich, S.A., Woods, J.A., Kelley, K.W., Dantzer, R., Lestage, J. and N. Castanon. 2008. Inoculation of Bacille Calmette-Guerin to mice induces an acute episode of sickness behavior followed by chronic depressive-like behavior. Brain, Behavior, and Immunity 22: doi:10.1016/j.bbi.2008.04.001.
- Andre, C., O'Connor, J.C., Kelley, K.W., Lestage, J., Dantzer, R. and Castanon, N. 2008. Spatio-temporal differences in the profile of murine brain expression of proinflammatory cytokines and indoleamine 2,3-dioxygenase in response to peripheral lipopolysaccharide administration. J. Neuroimmunology 10.1016/j.neuroim.2008.06.011.
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Progress 01/01/07 to 12/31/07
Outputs
A number of observations and discoveries over the past 20 years support the concept of important physiological interactions between the endocrine and immune systems. The best known pathway for transmission of information from the immune system to the neuroendocrine system is humoral in the form of cytokines, although neural transmission via the afferent vagus is well documented also. In the other direction, efferent signals from the nervous system to the immune system are conveyed by both the neuroendocrine and autonomic nervous systems. Communication is possible because the nervous and immune systems share a common biochemical language involving shared ligands and receptors, including neurotransmitters, neuropeptides, growth factors, neuroendocrine hormones and cytokines. This means that the brain functions as an immune-regulating organ participating in immune responses. A great deal of evidence has accumulated and confirmed that hormones secreted by the
neuroendocrine system play an important role in communication and regulation of the cells of the immune system. Among protein hormones, this has been most clearly documented for prolactin (PRL), growth hormone (GH), and insulin-like growth factor-1 (IGF-I), but significant influences on immunity by thyroid stimulating hormone (TSH) have also been demonstrated. Here we review evidence obtained during the past 20 years to clearly demonstrate that neuroendocrine protein hormones influence immunity and that immune processes affect the neuroendocrine system. New findings highlight a previously undiscovered route of communication between the immune and endocrine systems that is now known to occur at the cellular level. This communication system is activated when inflammatory processes induced by proinflammatory cytokines antagonize the function of a variety of hormones, which then causes endocrine resistance in both the periphery and brain. IL-10 is an anti-inflammatory cytokine that
suppresses synthesis of proinflammatory cytokines and their receptors. We tested the possibility that TNFa-induced hormone resistance in myoblasts might be overcome by IL-10. We found that IL-10 restores myogenesis by suppressing the ability of exogenous TNFa to inhibit IGF-I-induced myogenin. This protection occurs without decreasing global activity of TNF receptors since IL-10 does not impair TNFa-induced IL-6 synthesis or ERK1/2 phosphorylation. Instead, IL-10 acts to prevent TNFa-induced phosphorylation of JNK. These findings demonstrate that IL-10 serves a previously unrecognized protective role in muscle progenitors by overcoming TNFa-induced resistance to IGF-I. Homeostasis during inflammation is achieved by a balance between cytokines and endocrine hormones.
Impacts
A new mechanism of action of the anti-inflammatory cytokine IL-10 was discovered. This cytokine was shown to reverse the ability of TNFa to induce phosphorylation of the stress kinase, c-jun-N-terminal kinase, and thereby restore IGF-I-induced myogenesis in muscle progenitor cells. These new data establish the protective activity of IL-10 in reversing resistance to IGF-I in developing muscle.
Publications
- Strle, K., McCusker, R.H., Tran, L., King, A., Johnson, R.W., Freund, G.G., Dantzer, R. and Kelley, K.W. 2007. Novel activity of an anti-inflammatory cytokine: IL-10 prevents TNFalpha-induced resistance to IGF-I in myoblasts. J. Neuroimmunology 188:48-55.
- Ader, R. and Kelley, K.W. 2007. A global view of twenty years of Brain, Behavior, and Immunity. Brain, Behavior, and Immunity 21:20-22.
- Dantzer, R. and Kelley, K.W. 2007. Twenty years of research on cytokine-induced sickness behavior. Brain, Behavior, and Immunity 21:153-160.
- Kelley, K.W., Weigent, D.A. and Kooijman, R. 2007. Protein hormones and immunity. Brain, Behavior, and Immunity 21:384-392.
- Palin, K., Bluthe, R.M., McCusker, R.H., Moos, F., Dantzer, R. and Kelley, K.W. 2007. TNFa-induced sickness behavior in mice with functional 55 kD TNF receptors is blocked by central IGF I. J. Neuroimmunology 187:55-60.
- McCusker, R.H., Strle, K., Broussard, S.R., Dantzer, R., Bluthe, R.M. and Kelley, K.W. 2006. Crosstalk between insulin-like growth factors and proinflammatory cytokines. In: R. Ader, R. Dantzer, R. Glaser, C. Heijnen, M. Irwin, D. Padgett and J.Sheridan (eds.) Psychoneuroimmunology. Fourth Edition. Elsevier, San Diego. pp. 171-192.
- Dantzer, R., Bluthe, R.M., Castanon, N., Kelley, K.W., Konsman, J.P., Laye, S., Lestage, J. and Parnet, P. 2006. Cytokines, sickness behavior, and depression. In: R. Ader, R. Dantzer, R. Glaser, C. Heijnen, M. Irwin, D. Padgett, and J.Sheridan (eds.) Psychoneuroimmunology. Fourth Edition. Elsevier, San Diego. pp. 281-318.
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Progress 01/01/06 to 12/31/06
Outputs
Inflammation associated with infections is well known to reduce animal growth, and proinflammatory cytokines are now recognized to be integral components of inflammation. We have explored the possibility that one of the major proinfammatory cytokines, tumor necrosis factor (TNF), adversely affects the function of insulin-like growth factor-I (IGF-I). Growth hormone, IGF-I and their interaction control nearly 80% of postnatal growth. The stress kinase C-jun N-terminal kinase (JNK) is a major component of TNF receptor signaling, but its potential role in regulating inflammatory events in skeletal muscle has not been explored. We used a novel, JIP-derived JNK peptide inhibitor to establish that JNK suppresses the biological activity of IGF-I in skeletal muscle progenitor cells. In these myoblasts, TNF and its downstream receptor substrates, N-SMase and C2-ceramide, induce JNK kinase activity in a time-dependent manner. Consistent with these results, TNF induces JNK
binding to IRS-1 but is unable to inhibit IGF-I-induced IRS-1 tyrosine phosphorylation in myoblasts that are treated with the JNK peptide inhibitor. More importantly, JNK activation induced by TNF, C2-ceramide and N-SMase is associated with reduced expression of the critical muscle transcription factor, myogenin, as well as the differentiation marker, MHC. The JNK peptide inhibitor, but not the control peptide, completely reverses this inhibition of both myogenin and MHC. In the absence of IGF-I, TNF, C2-ceramide, N-SMase and the JNK inhibitor are inactive, as shown by their inability to affect IRS tyrosine phosphorylation and protein expression of myogenin and MHC. These results establish that the resistance of muscle progenitor cells to IGF-I, which is caused by inflammatory stimuli, is mediated by the JNK stress kinase pathway.
Impacts
The stress kinase transcription factor C-jun N-terminal kinase (JNK) is activated in muscle cells by proinflammatory cytokines released as a result of infectious diseases. JNK prevents IGF-I from acting properly in muscle progenitor cells. This phenomenon of hormone resistance is becoming increasingly accepted to be involved in major disease processes.
Publications
- Strle, K., Broussard, S.R., McCusker, R.H., Shen, W.H., LeCleir, J., Johnson, R.W., Freund, G.G., Dantzer, R. and Kelley, K.W. 2006. C-jun N-terminal kinase mediates TNF alpha suppression of differentiation in myoblasts. Endocrinology 147:4363-4373.
- Bluthe, R.M., Kelley, K.W. and Dantzer, R. 2006. Effects of insulin-like growth factor-I on cytokine-induced sickness behavior in mice. Brain, Behavior, and Immunity 20:57-63.
- McCusker, R.H., McCrae, K., Zunich, S., Dantzer, R., Broussard, S.R., Johnson, R.W. and Kelley, K.W. 2006. Insulin-like growth factor-I enhances the biological activity of brain-derived neurotrophic factor on cerebrocortical neurons. J. Neuroimmunology 179:186-190.
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Progress 01/01/05 to 12/31/05
Outputs
Growth hormone and IGF-I control over 80% of postnatal growth. We recently established that TNFa impairs the ability of IGF-I to increase protein synthesis and promote expression of myogenin in myoblasts. Here we extend these results by showing that ceramide, a second messenger in both TNFa and IL-1b receptor signaling pathways, is a key downstream sphingosine-based lipid that leads to IGF-I resistance. A cell permeable ceramide analog, C2-ceramide, inhibits IGF-I-induced protein synthesis by 65% and blocks the ability of IGF-I to increase expression of two key myogenic factors, myogenin and MyoD. Identical results were obtained with both TNFa and IL-1b (1 ng/ml). Consistent with these data, neutral sphingomyelinase (N-SMase), an enzyme that catalyzes formation of ceramide from sphingomyelin, blocks IGF-I-induced protein synthesis and expression of both myogenin and MyoD. The possibility that cytokine-induced ceramide production is required for disruption of IGF-I
biologic activity was confirmed by treating C2C12 myoblasts with inhibitors of all three ceramide-generating pathways. A N-SMase inhibitor, GSH, as well as an acidic sphingomyelinase (A-SMase) inhibitor, D609, reverse the cytokine-inhibition of IGF-I-induced protein synthesis by 80% and 45%, respectively. Likewise, an inhibitor of de novo ceramide synthesis, FB1, causes a 50% inhibition. Similarly, all three inhibitors significantly impair the ability of both TNFa and IL-1b to suppress IGF-I-driven expression of myogenin. These experiments establish that ceramide, derived both from sphingomyelin and de novo synthesis, is a key intermediate by which proinflammatory cytokines impair the ability of IGF-I to promote protein synthesis and expression of critical muscle-specific transcription factors.
Impacts
The growth hormone-induced peptide, IGF-I, interacts with growth hormone to control over 80% of animal growth. Proinflammatory cytokines are synthesized by the immune system in a variety of infectious diseases. These experiments establish a biological mechanism by which proinflammatory cytokines impair the action of IGF-I in muscle cells.
Publications
- Strle, K., Broussard, S.R., McCusker, R.H., Shen, W.H., Johnson, R.W., Freund, G.G., Dantzer, R. and Kelley, K.W. 2004. Proinflammatory cytokine impairment of IGF-I-induced protein synthesis in skeletal muscle myoblasts requires ceramide. Endocrinology 145:4592-4602.
- O'Connor, J.C., Satpathy, A., Hartman, M.E., Horvath, E.M., Kelley, K.W., Dantzer, R., Johnson, R.W. and Freund, G.G. 2005. IL-1b-mediated innate immunity is amplified in the db/db mouse model of type 2 diabetes. J. Immunology 174:4991-4997.
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Progress 01/01/04 to 12/31/04
Outputs
Discoveries in the physiology of immunology have increased at an increasing rate during the past two decades, as summarized in a recent review. It is now recognized that the immune system is just another physiological system that regulates, and is regulated by, other physiological systems such as the brain. These advances make it clear that recent findings in genomic biology must be interpreted in the context of the environment in which animals and humans live. Lack of a strong genetic basis for significant human mental health disorders, such as major depression, points to the critical importance of interactions. Several examples of environmental x genetic x disease interactions were presented. Regulation of cells of the hematopoietic lineage by two genes that control over 80% of postnatal growth, growth hormone and IGF-I, were then highlighted. The reciprocal relationship of how proinflammatory cytokines from the immune system regulate the growth hormone/IGF-I axis
was also summarized. Particular emphasis was placed upon TNF-induced IGF-I resistance in neurons, muscle cells and epithelial cells. This cytokine regulation of hormone action may ultimately be more important for human and animal health than direct effects of growth hormone and IGF-I on hematopoietic cells. We recently demonstrated that TNF impairs the ability of IGF-I to promote protein synthesis in muscle precursor cells. We extended those findings by showing that low concentrations of IL-1 impair IGF-I-dependent differentiation of myoblasts, as assessed by expression of the muscle specific protein, myosin heavy chain. In the absence of exogenous IGF-I, IL-1 (1 ng/ml) did not impair muscle cell development. However, in the presence of IGF-I, 100-fold lower concentrations of IL-1 (0.01 ng/ml) significantly suppressed myoblast differentiation, protein synthesis and myogenin expression. Increasing IL-1 to 1 ng/ml completely blocked the anabolic actions of IGF-I in murine C2C12
myoblasts. Similarly, IL-1 inhibited IGF-I-stimulated protein synthesis in primary porcine myoblasts. IL-1 impaired the actions of IGF-I at a point distal to the IGF receptor, and this was not due to IL-1-induced cell death. Instead, IL-1 inhibited the ability of IGF-I to phosphorylate tyrosine residues on both of its down-stream docking proteins, insulin receptor substrate (IRS)-1 and IRS-2. These data establish that physiological concentrations of IL-1 block the ability of IGF-I to promote protein synthesis, leading to reduced expression of the myogenic transcription factor, myogenin, and the subsequent development of more mature differentiated cells that express myosin heavy chain. Collectively, the results are consistent with the notion that very low concentrations of IL-1 significantly impair myogenesis, but they are unable to do so in the absence of the growth factor IGF-I.
Impacts
The practical implication of these findings is that the synthesis of proinflammatory cytokines that is initiated in many economically-important infectious diseases of livestock may act to reduce the growth factor-induced accretion of protein in progenitor muscle cells.
Publications
- Kelley, K.W. 2004. Norman Cousins Lecture: From hormones to immunity: The physiology of immunology. Brain, Behavior, and Immunity 18:95-113.
- Shen, W.H., Yin, Y., Broussard, S.R., McCusker, R.H., Freund, G.G., Dantzer, R. and Kelley, K.W. 2004. Tumor necrosis factor inhibits cyclin A expression and retinoblastoma hyperphosphorylation triggered by insulin-like growth factor-I induction of new E2F-1 synthesis. J. Biological Chemistry 279:7438-7446.
- Broussard, S.R., McCusker, R.H., Novakofski, J.E., Strle, K., Shen, W.H., Johnson, R.W., Dantzer, R. and Kelley, K.W. 2004. IL-1 impairs insulin-like growth factor I-induced differentiation and downstream activation signals of the insulin-like I growth factor receptor in myoblasts. J. Immunology 172:7713-7720.
- Shen, W.H., Zhou, J.H., Broussard, S.R., Johnson, R.W., Dantzer, R. and Kelley, K.W. 2004. Tumor necrosis factor inhibits insulin-like growth factor-I-induced hematopoietic cell survival and proliferation. Endocrinology 145:3101-3105.
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Progress 01/01/03 to 12/31/03
Outputs
Tumor necrosis factor (TNF) is elevated following damage to skeletal muscle. We provided evidence that TNF acts on muscle cells to induce a state of IGF-I receptor resistance. We established that TNF inhibited IGF-I-stimulated protein synthesis in primary porcine myoblasts. Similar results were observed in C2C12 murine myoblasts, where as little as 10 pg/ml TNF significantly inhibited protein synthesis induced by IGF-I. TNF also impaired the ability of IGF-I to induce expression of a key myogenic transcription factor, myogenin. The inhibition by TNF of IGF-I-induced protein synthesis and expression of myogenin was not due to direct killing of myoblasts by TNF. Although IGF-I induced an 19-fold induction in tyrosine phosphorylation of the b chains of its receptor, TNF did not inhibit this autophosphorylation. Instead, TNF significantly reduced by 50% IGF-I-stimulated tyrosine phosphorylation of two of the major downstream receptor docking molecules, insulin receptor
substrate (IRS)-1 and IRS-2. These results established that as little as 100 picogram/ml of TNF acts on both porcine and murine myoblasts to impair tyrosine phosphorylation of both IRS-1 and IRS-2, but not the receptor itself. These data are consistent with the notion that very low, physiological concentrations of TNF interfere with both protein synthesis and muscle cell development by inducing a state of IGF-I receptor resistance. IGF-I can act like some non-proinflammatory cytokines, like IL-3 and IL-4, to promote survival of progenitor myeloid cells. We demonstrated that IGF-I, IL-3 and IL-4 all significantly blocked activation of caspase-3 in promyeloid cells following growth factor deprivation. However, only IL-3 and IGF-I increased enzymatic activity and phosphorylation of the survival-promoting kinase Akt. IGF-I failed to reduce caspase-3 activity and cell death in the presence of the PI 3-kinase inhibitors, wortmannin and LY294002, whereas these blockers did not affect the
ability of IL-3 to maintain cell survival. IL-4 inhibited caspase-3 activity and promoted promyeloid cell survival by a substrate for PI 3-kinase that was not Akt. These data established that IGF-I inhibits activation of caspase-3 and promotes promyeloid cell survival through a PI 3-kinase dependent pathway, whereas IL-3 does not. It therefore appears that signal transduction pathways for all three receptors converge upstream of caspase-3 to prevent apoptosis of progenitor myeloid cells, but their receptors differ in the intracellular substrates that are used to promote cell survival.
Impacts
These data establish that IGF-I promotes survival of progenitor myeloid cells by a mechanism that is different than IL-3 and IL-4. The results also show that proinflammatory cytokines from an activated immune system impair the ability of IGF-I to promote protein synthesis in porcine and murine myoblasts.
Publications
- Kelley, K.W. 2003. Inaugural Editorial: A new look for brain, behavior, and immunity. Brain, Behavior, and Immunity 17:1-2.
- Kelley, K.W., Bluthe, R.M., Dantzer, R., Zhou, J.H., Shen, W.H., Johnson, R.W. and Broussard, S.R. 2003. Cytokine-induced sickness behavior. Brain, Behavior, and Immunity 17:S112-S118.
- Burgess, W., Jesse, K., Tang, Q., Broussard, S.R., Dantzer, R. and Kelley, K.W. 2003. Insulin-like growth factor-I and the cytokines IL-3 and IL-4 promote survival of progenitor myeloid cells by different mechanisms. J. Neuroimmunology 135:82-90.
- Broussard, S.R., McCusker, R.H., Novakofski, J.E., Strle, K., Shen, W.H., Johnson, R.W., Freund, G.G., Dantzler, R. and Kelley, K.W. 2003. Cytokine-hormone interactions: Tumor necrosis factor a impairs biologic activity and downstream activation signals of the IGF-I receptor in myoblasts. Endocrinology 144:2988-2996.
- Deszo, E., Kelley, K.W. and Freund, G.G. 2003. IL-4-dependent CD86 expression requires JAK/STAT6 activation and is negatively regulated by PKCd. Cellular Signalling (In Press).
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Progress 01/01/02 to 12/31/02
Outputs
Sickness in humans and animals resulting from infection with microbial pathogens leads to a perturbation in immune homeostasis that is characterized by fever, inactivity, and reduced appetite. This response to immune challenge is mediated by the induction of proinflammatory cytokines, such as tumor necrosis factor alpha (TNFa) and interleukin-1 (IL-1). These proinflammatory cytokines are the major reason that immunologically challenged animals fail to eat. We summarized a new pathway by which proinflammatory cytokines might reduce animal growth and productivity. We proposed that activation of receptors for proinflammatory cytokines affect the somatotropic axis, as they do for the insulin receptor, by reducing sensitivity of GH receptors in the liver and that of IGF-I receptors in target tissue. Proinflammatory cytokines induce resistance of hepatic GH receptors, leading to a significant reduction in plasma IGF-I. Resistance of the IGF-I receptor at target tissues has
been shown by TNFa inhibiting the ability of IGF-I to promote protein synthesis in human myoblasts. The mechanism by which proinflammatory cytokines induce resistance of the IGF-I receptor occurs by cytokine and hormone receptor cross talk on a single cell. This hypothesis is supported by our recent findings that established TNFa as a potent inhibitor of IGF-I receptor signaling in neurons, which leads to the silencing of survival signals (SOSS). The molecular mechanism by which TNFa impairs sensitivity of the IGF-I receptor is by decreasing the capacity of IGF-I to tyrosine phosphorylate its downstream docking molecule, insulin receptor substrate-2 (IRS-2). This action of TNFa occurs at concentrations of <1 ng/ml. The action of TNF reduces biological activity of IGF-I, thereby leading to receptor resistance. It is likely that the reduction in growth and productivity that occurs in diseased animals is also the result of cytokine and hormone receptor cross talk, resulting in a decrease
in GH-induced hepatic IGF-I synthesis (GH receptor resistance) and a reduction in responsiveness of target tissues to IGF-I stimulation (IGF-I receptor resistance). Sickness behavior refers to the coordinated set of behavior changes that develop in sick individuals during the course of an infection. At the molecular level, these changes are due to the effects of proinflammatory cytokines as interleukin-1 on the brain. In a review article, we briefly described the notion of sickness behavior and summarized the distribution of IL-1Rs in the central nervous system of the human, mouse and rat. We then presented arguments in favor of the functionality of the various subtypes of receptors and evaluated the nature of the signaling pathways activated by brain IL-1Rs to initiate central modifications leading to symptoms of sickness. Finally, modulation of IL-1 action on its receptor by various opposing factors including glucocorticoids and anti-inflammatory cytokines was discussed.
Impacts
These data establish that proinflammatory cytokines are active within the central nervous system where they reduce animal growth and productivity. These cytokines, particularly TNFa, also act in the periphery in skeletal muscle to reduce protein synthesis.
Publications
- Parnet, P., Kelley, K.W., Bluthi, R.M. and Dantzer, R. 2002. Expression and regulation of interleukin-1 receptors in the brain. Role in cytokines-induced sickness behavior. J. Neuroimmunology 125:5-14.
- French, R.A., Broussard, S.R., Meier, W.A., Minhall, C., Arkins, S., Zachary, J.F., Dantzer, R. and Kelley, K.W. 2002. Age-associated loss of bone marrow hematopoietic cells is reversed by GH and accompanies thymic reconstitution. Endocrinology 143:690-699.
- Strle, K., Zhou, J.H., Broussard, S.R., Venters, H.D., Johnson, R.W., Freund, G.G., Dantzer, R. and Kelley, K.W. 2002. IL-10 promotes survival of microglia without activating Akt. J. Neuroimmunology 122:9-19.
- Shen, W.H., Zhou, J.H., Broussard, S.R., Freund, G.G., Dantzer, R. and Kelley, K.W. 2002. Proinflammatory cytokines block growth of breast cancer cells by impairing signals from a growth factor receptor. Cancer Research 62:4746-4756.
- Strle, K., Zhou, J.H., Shen, W.H., Broussard, S.R., Johnson, R.W., Freund, G.G., Dantzer, R. and Kelley, K.W. 2001. Interleukin-10 in the brain. Critical Reviews in Immunology 21:427-449.
- Broussard, S.R., Zhou, J.H., Venters, H.D., Bluthi, R.M, Freund, G.G., Johnson, R.W., Dantzer, R. and Kelley, K.W. 2001. At the interface of environment-immune interactions: Cytokine and growth factor receptors. J. Anim. Sci. 79 (E. Suppl.):E268-E284.
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Progress 01/01/01 to 12/31/01
Outputs
The first studies carried out on the mechanisms by which peripheral immune stimuli signal the brain to induce fever, activation of the hypothalamic-pituitary-adrenal axis and sickness behavior in animals emphasized the importance of fenestrated parts of the blood-brain barrier. These areas of the brain are known as circumventricular organs, and they allow blood-borne proinflammatory cytokines to regulate brain functions. The discovery in the mid-1990s that subdiaphragmatic sectioning of the vagus nerves attenuates the brain effects of systemic cytokines, together with the demonstration of an inducible brain cytokine compartment, shifted the attention from circumventricular organs to neural pathways in the transmission of the immune message to the brain. Since then, neuroanatomical studies have confirmed the existence of a fast route of communication from the immune system to the brain via the vagus nerves. This neural pathway is complemented by a humoral pathway that
involves cytokines produced at the level of the circumventricular organs and the choroid plexus and at the origin of a second wave of cytokines produced in the brain parenchyma. Depending on their source, these locally produced cytokines can either activate neurons that project to specific brain areas or diffuse by volume transmission into the brain parenchyma to reach their targets. Activation of neurons by cytokines can be direct or indirect, via prostaglandins. The way the neural pathway of transmission interacts with the humoral pathway remains to be elucidated. However, one potential advance in this area has been made from studies devoted to learning whether an endogenous interleukin-1 receptor antagonist is expressed in the brain. This protein is the natural inhibitor of the biological effects of interleukin-1 during inflammation. Interleukin-1 receptor antagonist refers to three isoforms: one secreted and two intracellular forms (types I and II). We therefore investigated the
expression of interleukin-1 receptor antagonist isoforms in the rat brain in vivo in response to an i.p. injection of lipopolysaccharide. The interleukin-1 receptor antagonist was studied at the messenger and protein levels by reverse transcription-polymerase chain reaction and western blot analysis, respectively. Interleukin-1 receptor antagonist messenger RNA was constitutively expressed in the brain and its expression increased in response to lipopolysaccharide. The three interleukin-1 receptor antagonist protein isoforms were up-regulated after lipopolysaccharide treatment in a time-dependent manner. Their relative expression differed according to the isoform and brain region studied. Double immunofluorescence staining revealed interleukin-1 receptor antagonist positive neurons and microglia in hippocampus 24 hours after lipopolysaccharide stimulation. These results demonstrate for the first time that brain cells are able to produce interleukin-1 receptor antagonist isoforms in
response to a peripheral immune challenge with a predominance of the secreted over intracellular forms.
Impacts
These data establish that both a neural pathway involving the afferent vagus nerve and a humoral pathway that regulates cytokine synthesis in the brain controls clinical symptoms of sickness during disease. Regulation of the IL-1 receptor antagonist in the brain may be controlled by both systems.
Publications
- DANTZER, R., KONSMAN, J.P., BLUTHE, R.M. and KELLEY, K.W. 2000. Neural and humoral pathways of communication from the immune system to the brain: Parallel or convergent? Autonomic Neuroscience: Basic and Clinical 1:60-65.
- PALIN, K., POUSSET, F., VERRIER, D., DANTZER, R., KELLEY, K.W., PARNET, P. and LESTAGE, J. 2001. Characterization of interleukin-1 receptor antagonist isoform expression in the brain of lipopolysaccharide-treated rats. Neuroscience 103:161-169.
- KELLEY, K.W. 2001. Presidential Address: Its time for psychoneuroimmunology. Brain, Behavior and Immunity 15:1-6.
- REBEIZ, N., ARKINS, S., KELLEY, K.W., DURACK, G. and REBEIZ, C.A. 2001. Modulator of heme biosynthesis induces apoptosis in leukemia cells. J. Clinical Laser Medicine and Surgery 19:59-67.
- DESZO, E.L., BRAKE, D.K., CENGEL, K.A., KELLEY, K.W. and FREUND, G.G. 2001. CD45 negatively regulates monocytic cell differentiation by inhibiting PMA-dependent activation and tyrosine phosphorylation of PKCd. J. Biological Chemistry 276:10212-10217.
- ARKINS, S., JOHNSON, R.W., MINSHALL, C., DANTZER, R. and KELLEY, K.W. 2001. Immunophysiology: The interaction of hormones, lymphohemopoietic cytokines and the neuroimmune axis. In B.S. McEwen (Ed.) Coping with the Environment: Neural and Endocrine Mechanisms, Handbook of Physiology, Section 7, The Endocrine System, Vol. 4. American Physiological Society. Oxford University Press. New York, NY. pp. 469-495.
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Progress 01/01/00 to 12/31/00
Outputs
Insulin receptor substrate-2 (IRS-2) is phosphorylated on tyrosine by a number of cytokine receptors and is implicated in the activation of phosphatidylinositol 3'-kinase (PI 3-kinase). We have demonstrated that induction of granulocytic differentiation of human promyeloid HL-60 cells leads to an increase in the amount of IRS-2 that is phosphorylated in response to IGF-I. Although PI 3-kinase is often activated following interaction with IRS-1, we did not detect IRS-1 protein, IRS-1 mRNA or IRS-1-precipitable PI 3-kinase enzymatic activity. However, PI 3-kinase activity that was co-immunoprecipitated with either anti-phosphotyrosine or anti-IRS-2 following IGF-I stimulation was increased 100-fold. Heightened tyrosine phosphorylation of IRS-2 during granulocytic differentiation was not caused by an increase in expression of the tyrosine kinase insulin-like growth factor-I receptor, as measured by the amount of both the ?- and ?-subunits. Instead, immunoblotting
experiments with an antibody to IRS-2 revealed that induction of granulocytic differentiation caused a large increase in IRS-2, and this occurred in the absence of detectable IRS-1 protein. These IRS-2 positive cells could not differentiate into more mature myeloid cells in serum-free medium unless IGF-I was added. These data are consistent with a model of granulocytic differentiation that requires at least two signals, the first of which leads to an increase in the cytoplasmic pool of IRS-2 protein and a second molecule that acts to tyrosine phosphorylate IRS-2 and enhance granulocytic differentiation. In a second series of experiments, we evaluated the potential role of interleukin-1beta (IL-1beta) in the pituitary-adrenal (HPA) axis during stress. Mature IL-1beta derives from a 31-kD precursor (pro-IL-1beta) that is processed by IL-1beta-converting enzyme (ICE). Mice in which the ICE gene was deleted by homologous recombination were used to investigate the role of IL-1beta in the
HPA axis response. Plasma levels of corticosterone and adrenocorticotropic hormone (ACTH) in response to an intraperitoneal injection of 5 micrograms lipopolysaccharide (LPS) were similar in ICE-deficient mice and wild-type (WT) controls. In contrast, the plasma ACTH response to restraint or to 200 ng of rat recombinant IL-1beta (rrIL-1beta) was higher in ICE-deficient mice as compared to WT animals. This hyperreactivity of the HPA axis in ICE knockout mice appears not to be related to the production of plasma IL-1beta or IL-6, which was similar to that of WT mice after rrIL-1beta injection. After LPS, ICE-deficient mice exhibited a smaller increase in plasma-immunoreactive IL-1beta and IL-6 as compared to WT controls. After restraint stress, neither an increase in plasma IL-1beta nor IL-6 was observed. The mechanisms responsible for the increased reactivity of the HPA axis in ICE-deficient mice may result from a higher sensitivity of the HPA axis to inflammatory cytokines or to
cleavage products of pro-IL-1beta processed by non-ICE proteases.
Impacts
These results establish that a major growth-promoting peptide, IGF-I, can also promote development of cells of the immune system. Conversely, the immune system regulates stress hormone secretion because ICE-deficient mice display increased ACTH responses to restraint.
Publications
- Schacher, D.H., VanHoy, R.W., Liu, Q., Arkins, S., Dantzer, R., Freund, G.G. and Kelley, K.W. 2000. Developmental expression of insulin receptor substrate-2 during dimethylsulfoxide-induced differentiation of human HL-60 cells. J. Immunology 164:113-120.
- Liege, S., Moze, E., Kelley, K.W., Parnet, P. and Neveu, P.J. 2000. Activation of the hypothalamic-pituitary-adrenal axis in IL-1beta-converting enzyme-deficient mice. Neuroimmunomodulation 7:189-194.
- Laye, S., Gheusi, G., Cremona, S., Combe, C., Kelley, K.W., Dantzer, R. and Parnet, P. 2000. Endogenous brain IL-1 mediates LPS-induced anorexia and hypothalamic cytokine expression. Amer. J. Physiol. (Regul. Integr. Comp. Physiol.) 279:R93-R98.
- Venters, H.D., Dantzer, R. and Kelley, K.W. 2000. A new concept in neurodegeneration: TNFalpha is a silencer of survival signals. Trends in Neurosciences 23:175-180.
- Pousset, F., Dantzer, R., Kelley, K.W. and Parnet, P. 2000. IL-1 signaling in mouse astrocytes involves Akt: A study with IL-4 and IL-10. European Cytokine Network 11:427-434.
- Dantzer, R., Gawlick, U., Heusmann, G. and Kelley, K.W. 2000. Immunology. In A.E. Kazdin (Ed) Encyclopedia of Psychology. American Psychological Association, Washington DC and Oxford University Press-USA. New York, NY. Eight Volume Set, 4128 pp.
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Progress 01/01/99 to 12/31/99
Outputs
Cyclin-dependent kinase inhibitors have recently been shown to lead to cellular differentiation by causing cell cycle arrest, but it is unknown whether similar events occur in differentiating promyeloid cells. Here we show that the classical growth factor, insulin-like growth factor-I (IGF-I), potently promotes vitamin D3-induced macrophage differentiation of promyeloid cells as early as 24 h following initiation of terminal differentiation. Addition of IGF-I to vitamin D3-differentiating cells also leads to an early increase in expression of cyclin E, phosphorylation of the retinoblastoma tumor suppressor protein and a doubling of cell number. Early expression of CD11b (24 h) is simultaneously accompanied by inhibition in the expression of p27KIP1. Cell cycle analysis with propidium iodide revealed that CD11b expression at 24 h following initiation of differentiation occurs at all phases of the cell cycle instead of only those cells arrested in G0/G1. Similarly,
development of a novel double-labeling intra- and extracellular flow cytometric technique demonstrated that single cells expressing the mature leukocyte differentiation antigen CD11b can also incorporate the thymidine analog, bromodeoxyuridine. Likewise, expression of the intracellular DNA polymerase delta cofactor (PCNA) at 24 h is also simultaneously expressed with the surface marker CD11b, indicating that these cells continue to proliferate early in their differentiation program. Finally, at 24 h following induction of differentiation, IGF-I promoted a 4-fold increase in the uptake of [3H] thymidine by purified populations of CD11b-expressing cells. These data demonstrate that the initial steps associated with terminal macrophage differentiation occur concomitantly with progression through the cell cycle and that these very early events do not require the accumulation of p27KIP1. In a second series of experiments, the combined effects of co-stimulation of both IGF-I and TNF
receptors on granule neurons was studied. Conventional roles for these two proteins are neuroprotection by IGF-I and neurotoxicity by TNF-alpha. Although the mechanisms of action for IGF-I and TNF-alpha in the central nervous system originally were established as disparate and unrelated, we hypothesized that the signaling pathways of these two cytokines may interact during neurodegeneration. Here we show that concentrations of TNF-alpha as low as 10 pg/ml markedly reduce the capacity of IGF-I to promote survival of primary murine cerebellar granule neurons. TNF-alpha suppresses IGF-I-induced tyrosine phosphorylation of insulin receptor substrate 2 (IRS-2) and inhibits IRS-2-precipitable phosphatidylinositol 3'-kinase activity. These experiments indicate that TNF-alpha promotes IGF-I receptor resistance in neurons and inhibits the ability of the IGF-I receptor to tyrosine-phosphorylate the IRS-2 docking molecule and to subsequently activate the critical downstream enzyme
phosphatidylinositol 3'-kinase. This intracellular crosstalk between discrete cytokine receptors reveals a novel pathway that leads to neuronal degeneration whereby a proinflammatory cytokine inhibits receptor signaling by a survival peptide.
Impacts
Insulin-like growth factor-I (IGF-I) is one of the most important proteins that regulate the growth and development of both farm animals and humans. These results show that IGF-I is also important for the development of cells in both the immune and central nervous system. This information is important for helping to better understand why sick animals and humans fail to grow and thrive.
Publications
- POUSSET, F., CREMONA, S., DANTZER, R., KELLEY, K.W., and PARNET, P. 1999. Interleukin-4 and interleukin-10 regulate IL-1b induced mouse primary astrocyte activation: A comparative study. Glia 26: 12-21.
- BLUTHE, R.M., CASTANON, N., POUSSET, F., BRISTOW, A., BALL, C., LESTAGE, J., MICHAUD, B., KELLEY, K.W., and DANTZER, R. 1999. Central injection of IL-10 antagonizes the behavioural effects of lipopolysaccharide in rats. Psychoneuroendocrinology 24: 301-311.
- DANTZER, R., GHEUSI, G., JOHNSON, R.W., and KELLEY, K.W. 1999. Central administration of insulin-like growth factor-I inhibits lipopolysaccharide-induced sickness behavior in mice. NeuroReport 10: 289-292.
- MINSHALL, C., ARKINS, S., DANTZER, R., FREUND, G., and KELLEY, K.W. 1999. Phosphatidylinositol 3' kinase, but not S6-kinase, is required for insulin-like growth factor-I and IL-4 to maintain expression of Bcl-2 and promote survival of myeloid progenitors. J. Immunology 162: 4542-4549.
- DANTZER, R., AUBERT, A., BLUTHE, R.M., GHEUSI, G., CREMONA, S., LAYE, S., KONSMAN, J.P., PARNET, P., and KELLEY, K.W. 1999. Mechanisms of the behavioral effects of cytokines. Adv. Exp. Med. Biol. 461: 83-105.
- KONSMAN, J.P., KELLEY, K.W., and DANTZER, R. 1999. Temporal and spatial relationships between lipopolysaccharide-induced expression of Fos, interleukin-1b and inducible nitric oxide synthase in rat brain. Neuroscience 89: 535-548.
- LIU, Q., VANHOY, R.W., ZHOU, J.H., DANTZER, R., FREUND, G., and KELLEY, K.W. 1999. Elevated cyclin E levels, inactive retinoblastoma protein and suppression of the p27KIP1 inhibitor characterize early development of promyeloid cells into macrophages. Molecular and Cellular Biology 19: 6229-6239.
- YAO, J.H., YE, S.M., BURGESS, W., ZACHARY, J.F., KELLEY, K.W., and JOHNSON, R.W. 1999. Mice deficient in interleukin-1b converting enzyme resist anorexia induced by central lipopolysaccharide. Amer. J. Physiol. (Regul. Integr. Comp. Physiol.) 277: R1435-R1443.
- VENTERS, H.D., TANG, Q., LIU, Q., VANHOY, R.W., DANTZER, R., and KELLEY, K.W. 1999. A new mechanism of neurodegeneration: A proinflammatory cytokine inhibits receptor signaling by a survival peptide. Proc. Natl. Acad. Sci. 96: 9879-9884.
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Progress 10/01/98 to 12/31/98
Outputs
Interleukin-1beta (IL-1beta) mediates many of the behavioral responses to infection and inflammation, and IL-1beta converting enzyme (ICE) processes intracellular IL-1beta leading to its maturation and secretion. Here we demonstrate that intracerebroventricular (ICV) injections of LPS produced a greater reduction in both food intake and food-motivated behavior in wild type (WT) as compared to ICE-deficient (ICE-/-) mice. This defect occurred even though ICE-/- mice were able to fully respond to ICV injections of IL-1beta. In contrast, ICE-/- mice remained fully responsive to intraperitoneal (IP) injections of LPS. These results indicate that brain, but not peripheral, IL-1beta plays a critical role in the depression in food intake that occurs during inflammation. In another project, we have investigated the role of IL-1 receptors in the brain. To study their functionality, mice were centrally treated with neutralizing monoclonal antibody (MAb) directed against IL-1RI
(35F5, 1 microgram) or against IL-1RII (4E2, 2 micrograms) and were centrally injected with recombinant rat IL-1 beta at a dose (2 ng) that decreased social exploration. Only 35F5 was effective in abrogating the behavioral effect of IL-1 beta. Moreover, 4E2 (1 microgram i.c.v.) did not potentiate the behavioral response to a subthreshold dose of IL-1 beta (1 ng i.c.v.). To examine the ability of brain IL-1RI to mediate the effects of endogenous IL-1 beta, mice were centrally treated with 35F5 (4 micrograms) and peripherally injected with IL-1 beta (1 microgram). Like the IL-1 receptor antagonist (4 micrograms i.c.v.), 35F5 abrogated the effects of IL-1 beta. These results suggest that brain IL-1RI mediates the behavioral effects of IL-1beta in mice. Finally, we have been examining the role of TNF on neurons. We previously demonstrated TNF toxicity, at high TNF doses or in the presence of actinomycin D, in the N1E-115 neuronal cell line (N1Es) (which expresses only the 55 kDa TNF
receptor (TNFR)). To determine whether the presence of 75 kDa TNFR increases N1E sensitivity to TNF toxicity, cells were transfected with a 75 kDa TNFR expression construct. However, 75 kDa TNFR protein expression was undetectable in stably transfected N1Es. Further investigation revealed endogenous membrane-associated TNF in this neuronal line. Co-transfection with beta-galactosidase and the 75 kDa TNFR or empty vector (pcDNA3)indicated cell loss in the 75 kDa TNFR-transfected population relative to vector-transfected populations, while inhibition of membrane-associated TNF with a neutralizing antibody led to increased 75 kDa TNFR expression in transiently transfected N1Es. We conclude that neutralization of membrane-associated TNF inhibits its interaction with the introduced 75 kDa TNFR, increasing neuronal survival and promoting 75 kDa TNFR expression. Induced 75 kDa TNFR expression in the presence of membrane-associated TNF and the 55 kDa TNFR results in lymphocyte cell death.
These data demonstrate that membrane-associated TNF and the 75 kDa TNFR similarly contribute to neuronal cell death.
Impacts
(N/A)
Publications
- Cremona, S.; Goujon, E; Kelley, K.W.; Dantzer, R. and Parnet, P. 1998. Brain type I but not type II IL-1 receptors mediate the effects of IL-1 beta on behavior in mice. Amer. J. Physiol. (Regul. Integr. Comp. Physiol.) 274: R735-R740.
- Godbout, J.P.; Cengel, K.A.; Cheng, S.L.; Minshall, C.; Kelley, K.W. and Freund, G.G. 1998. Insulin activates caspase-3 by a phosphatidylinositol 3' kinase dependent pathway. Cellular Signalling (In Press).
- Burgess, W.; Gheusi, G.; Yao, J.; Johnson, R.W.; Dantzer, R. and Kelley, K.W. 1998. Interleukin-1 converting enzyme deficient mice resist central but not systemic endotoxin-induced anorexia. Amer. J. Physiol. (Regul. Integr. Comp. Physiol.) 274: R1829-R1833.
- Liu, Q.; Ning, W.; Dantzer, R.; Freund, G.G. and Kelley, K.W. 1998. Activation of protein kinase c-zeta and phosphatidylinositol 3' kinase and promotion of macrophage differentiation by insulin-like growth factor-I. J. Immunology 160: 1393-1401.
- Finck, B.N.; Kelley, K.W.; Dantzer, R. and Johnson, R.W. 1998. In vivo and in vitro evidence for the involvement of tumor necrosis factor-alpha in the induction of leptin by lipopolysaccharide. Endocrinology 139: 2278-2283.
- Sipe, K.J.; Dantzer, R.; Kelley, K.W. and Weyhenmeyer, J.A. 1998. Expression of the 75 kDA TNF receptor and its role in contact-mediated neuronal cell death. Molecular Brain Research 62: 111-121.
- Kelley, K.W.; Meier, W.A.; Minshall, C.; Schacher, D.H.; Liu, Q.; VanHoy, R.; Burgess, W. and Dantzer, R. 1998. Insulin-like growth factor-I inhibits apoptosis in hematopoietic progenitor cells: Implications in thymic aging. New York Academy of Sciences. New York, NY 840: 518-524
- Dantzer, R.; Bluthe, R.M.; Laye, S.; Bret-Dibat, J.L.; Parnet, P. and Kelley, K.W. 1998. Cytokines and sickness behavior. New York Academy of Sciences. New York, NY 840: 586-590.
- Dantzer, R.; Bluthe, R.M.; Gheusi, G.; Cremona, S.; Laye, S.; Parnet, P. and Kelley, K.W. 1998. Molecular basis of sickness behavior. New York Academy of Sciences. New York, NY 856: 132-138.
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Progress 01/01/97 to 12/31/97
Outputs
We have investigated the possibility that peripheral neural afferent pathways are involved in transmitting inflammatory signals from the periphery to the central nervous system. Vagal afferents represent one of the main pathways of communication from the abdominal cavity to the brain. To investigate the hypothesis that neural transmission of the peripheral immune signal through vagal afferents from abdominal organs is critical for the induction of sickness, we tested the ability of subdiaphragmatic vagotomy to block the behavioral effects of peripherally-injected LPS. Three weeks following surgery, rats were injected i.p. with LPS or physiological saline immediately after a baseline session of social exploration. LPS significantly depressed social exploration in sham-vagotomized but not in vagotomized rats. Furthermore, using food-motivated behavior as an end-point, we have also observed that subdiaphragmatic vagotomy attenuates the suppressing effects of both IL-1
and LPS injected i.p. into mice. We have also observed that surgical vagotomy does not affect IL-1 responses when IL-1 is given via the i.v. or s.c. routes. These results establish that subdiaphragmatic vagotomy can block the behavioral effects of intraperitoneal immune stimuli.
Impacts
(N/A)
Publications
- Liu, Q, D. Schacher, C. Hurth, G.G. Freund, R. Dantzer and K.W. Kelley. 1997. Activation of phosphatidylinositol 3'-kinase by insulin-like growth factor-I rescues promyeloid cells from apoptosis and permits their differentiation into granulocytes. J. Immunology 159:829-837.
- Minshall, C., S. Arkins, J. Straza, J. Connors, R. Dantzer, G.G. Freund and K.W. Kelley. 1997. IL-4 and insulin-like growth factor-I inhibit the decline in Bcl-2 and promote the survival of IL-3-deprived myeloid progenitors. J. Immunology 159:1225-1232.
- Johnson, R.W., S. Arkins, R. Dantzer and K.W. Kelley. 1997. Hormones, lymphohemopoietic cytokines and the neuroimmune axis. Comparative Biochemistry and Physiology 116:183-201.
- Li, Y.M., D.H. Schacher, Q. Liu, S. Arkins, N. Rebeiz, R.H. McCusker, R. Dantzer and K.W. Kelley. 1997. Regulation of myeloid growth and differentiation by the insulin-like growth factor-I receptor. Endocrinology 138:362-368.
- Johnson, R.W., G. Gheusi, S. Segreti, R. Dantzer and K.W. Kelley. 1997. C3H/HeJ mice are refractory to lipopolysaccharide in the brain. Brain Res. 752:219-226.
- Bret-Dibat, J.L., C. Cremion, J.Y. Couraud, K.W. Kelley, R. Dantzer and S. Kent. 1997. Systemic capsaicin pretreatment fails to block the decease in food-motivated behavior induced by lipopolysaccharide and interleukin-1beta. Brain Res. Bull. 42:443-449.
- Segreti, J., G. Gheusi, R. Dantzer, K.W. Kelley and R.W. Johnson. 1997. Defect in interleukin-1beta secretion prevents sickness behavior in C3H/HeJ mice. Physiol. Behav. 61:873-878.
- Bluthe, R.M., B. Michaud, K.W. Kelley and R. Dantzer. 1997. Cholecystokinin receptors to not mediate the behavioral effects of lipopolysaccharide in mice. Physiol. Behav. 62:385-389.
- Kelley, K.W., K. Hutchison, R. French, R.M. Bluthe, P. Parnet, R.W. Johnson and R. Dantzer. 1997. Central IL-1 receptors as mediators of sickness. In P.M. Moore, Ed. Neuropsychiatric Manifestations of Systemic Lupus Erythematosus. New York Academy of Sciences. New York, NY. 823:234-246.
- Dantzer, R., S. Laye, E. Goujon, R.M. Bluthe, J.P. Konsman, P. Parnet and K.W. Kelley. 1997. Mechanisms of action of cytokines on the central nervous system: Interaction with glucocorticoids. In G.A. Rook and S. Lightman, Eds. Steroid Hormones and the T-Cell Cytokine Profile. Springer-Verlag. London, Great Britain. pp 1-13.
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Progress 10/01/95 to 09/30/96
Outputs
Interleukin-1 Beta (IL-Beta) is a cytokine released by activated macrophages andmonocytes, which mediates many of the responses to inflammation. Interleukin-1 Beta induces anorexia in rats when administered peripherally or centrally. An endogenous antagonist for the IL-1 type I receptor has been characterized and cloned (IL-1ra). We have used this protein to ascertain the site of action for the anorexic effects of IL-1 Beta. Male rats were food restricted and trained on an operant schedule for food reinforcement. Administration of recombinant human IL-1 Beta (4 ug IP or 40 ng ICV) induced profound deceases in operant responding, with maximal effects 1-4 h post-injection. Interleukin-1ra pretreatment (2.4 mg IP or 24 ug ICV) completely blocked these effects when administered by the same route. In contrast, ICV IL-1ra only partially blocked the effects of IP IL- 1 Beta, and IP IL-1ra was unable to block the effects of ICV IL-1 Beta. Interleukin-1ra did not affect
responding by itself. These results suggest that IL-1 Beta acts at both peripheral and central IL-1 receptors to reduce food-motivated behavior. To determine the central site of action of IL-1 Beta, small quantities of IL-1 Beta (5 and 30ng) were infused into the ventromedial hypothalamus of male rats. Both doses produced profound decreases in responding. These results suggest that the VMH may serve as a central site of action for the depressive effects of IL-1 Beta on food intake.
Impacts
(N/A)
Publications
- GOUJON, E., PARNET, P., LAYE, S., COMBE, C., KELLEY, K.W. and DANTZER, R. 1995. Stress downregulates lipopolysaccharide-induced expression of proinflammatory cytokines in the spleen, pituitary and brain of mice. Brain, Behavior and Immunity LAYE, S., et al. 1995. Subdiaphragmatic vagotomy blocks the induction of IL-1 Beta mRNA in mice brain in response to peripherally administered LPS. Amer. J. Physiol. 268 (Regulatory Integrative Comp. Physiol. 37) R1327-R1331.
- BRET-DIBAT, J.L., BLUTHE, R. M., KENT, S., KELLEY, K.W. and DANTZER, R. 1995. Lipopolysaccharide and interleukin-1 depress food-motivated behavior in mice by a vagal-mediated mechanism. Brain, Behavior and Immunity 9:242-246.
- KENT, S., BLUTHE, R. M., GOODALL, G., KELLEY, K.W. and DANTZER, R. 1994. Central nervous system control of sickness behavior. In N.J. Rothwell and F. Berkenbosch(Eds.) Brain Control of Responses to Trauma. Cambridge University Press. pp. 15.
- KENT, S.P., BRET-DIBAT, J. L., KELLEY, K.W. and DANTZER, R. 1996. Mechanisms of sickness-induced decreases in food-motivated behavior. Neurosci. Biobehavioral Rev. 20:171-175.
- BLUTHE, R. M., BEAUDU, C., KELLEY, K.W. and DANTZER, R. 1995. Differential effects of IL-1ra on sickness behavior and weight loss induced by IL-1 in rats. Brain Research 677:171-176.
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Progress 10/01/94 to 09/30/95
Outputs
Not so long ago, it was believed that the brain was totally devoid of immunologic reactions, that cytokines derived from activated leukocytes serve only as communication molecules between leukocytes and that the immune system is regulated solely by intrinsic mechanisms. These traditional views have fallen by the wayside as neuroscientists, endocrinologists and pharmacologists have begun to explore immunology. The old view was that the immune system is autonomous because it neither affects nor is it affected by other physiologic systems. The new view is that cells of the immune system are inextricably linked with other physiological systems, including the neuroendocrine, cardiovascular, reproductive and central nervous systems. Changes in one system evoke changes in the other, and it is likely that communication loops have evolved between cells of the immune system and those of other tissues to coordinate and regulate functional activities aimed at preserving
homeostasis during inflammation. The integrated view of immunophysiologists that cells of the immune system interact with the entire body, rather than existing as a separate physiologic system that operates autonomously, should help to unravel a number of mysteries in immunoregulation, such as the well-recognized redundant and pleiotrophic properties of cytokines. A number of new discoveries provide insights into how leukocytes discriminate between self and non-self by enlisting the aid of the neuroendocrine and central nervous system.
Impacts
(N/A)
Publications
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Progress 10/01/93 to 09/30/94
Outputs
Immunologists and neuroscientists are starting to understand why sick animals become anoretic. The old idea that anorexia is caused by the secretion of "toxic" substances, such as enterotoxins, by infectious pathogens remains valid. Toxins which are produced by pathogenic bacteria and viruses cannot entirely explain why sick animals do not eat normally. Toxins and a variety of other pathogenic agents induce cells of the immune system to synthesize and release protein molecules called cytokines. These proinflammatory cytokine molecules act in a variety of ways to amplify the ongoing immune response and activate physiological disease defense mechanisms such as the hepatic synthesis of acute phase proteins. Some of these cytokines induce important physiological and behavioral adaptations in sick animals that are aimed at preserving homeostasis and furthermore, alter normal functioning of certain aspects of the neuroendocrine system. For example, the
hypothalamic-pituitary-adrenal system can be activated, and the secretion of important growth promoting hormones such as somatotropin and insulin-like growth factor-I (IGF-I) can be affected. In addition to these indirect effects mediated by the neuroendocrine system, many cytokines have direct effects on hepatic, muscle and adipose tissue and can therefore play a critical role in intermediary metabolism. In newborn pigs the regulation and secretion of these cytokines could assume utmost importance.
Impacts
(N/A)
Publications
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Progress 10/01/92 to 09/30/93
Outputs
Physiologic responses to infection include nonspecific symptoms of sickness, such as anorexia, depressed activity, and loss of interest in usual activities. Increasing evidence suggests that these symptoms are part of an organized defense response to antigenic challenge and that they are mediated by the neural effects of cytokines such as interleukin-1. An understanding of the mechanisms involved in these effects should permit development of new strategies aimed at decreasing sickness or promoting recovery processes. In particular, interleukin 1(beta) (IL-1) is a cytokine released during immune activation that mediates the host's response to infection and inflammation. Peripheral and central injections of IL-1 induce fever and sickness, including decreased food motivation and reduced interest in social activities. To determine the receptor mechanisms responsible for these effects, rats were injected with IL-1 receptor antagonist (IL-Ira), an endogenous cytokine that
acts as a pare antagonist of IL-1 receptors, IL-Ira blocked the increased body temperature and oxygen consumption induced by injection of recombinant human IL-1 only when both cytokines were administered i.p. In contrast, i.p. or intracerebroventricular administration if IL-Ira blocked the depressive effect of IL-1 on food-motivated behavior and social exploration when this cytokine was administered by the same route as the antagonist.
Impacts
(N/A)
Publications
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Progress 01/01/92 to 12/30/92
Outputs
Infection, particularly with gram-negative bacteria, results in physiological and metabolic alterations including fever, anorexia, hormonal changes, tissue damage, hypotension and death. The toxicity caused by gram-negative bacteria has been attributed to lipopolysaccharide (LPS), which is well known to induce the synthesis of cytokines such as interleukin-1 (IL-1) and tumor necrosis factor(alpha)(TNF(alpha)) by macrophages. We first demonstrated that both the behavioral changes and body weight loss caused in IL-1(alpha) and IL-1(beta) can be blocked by pretreatment with a 1,000 fold excess of a specific antagonist of IL-1 receptors. We then demonstrated that TNF(alpha) also decreases social exploration and induces weight loss in mice in a dose- and time-dependent manner. To assess the role of IL-1 in these effects, mice pretreated with the IL-1 receptor antagonist were injected with TNF(alpha). Pretreatment with this antagonist inhibited the depressive effects of
TNF/A. on behavior but only partially attenuated the weight loss induced by this cytokine. These results suggest that TNF(alpha)-induced sickness is mediated mainly by endogenously released IL-1 whereas metabolic changes are dependent on the release of other additional cytokines.
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Progress 10/01/90 to 09/30/91
Outputs
One half of pituitary-intact or sham-operated rats survive infection with 10 colony forming units of Salmonella typhimurium, whereas rats without a pituitary gland all die within a few days. When the dose S. typhimurium is reduced 603 fold, 15% to 25% of the hypophysectomized rats survive, and the survival rate is significantly enhanced by administration of tetracycline, recombinant interferon-gamma (IFN-gamma) or recombinant growth hormone (GH). The protective effect of GH is abolished by heat inactivation or with an antibody to GH. Spleens from both normal and hypophysectomized rats treated with tetracycline, IFN-gamma orGH have 59% to 99% fewer bacteria five days after infection as compared to controls rats. Peritoneal macrophages from hypophysectomized rats that are infected in vitro with S. typhimurium kill as many extracellular bacteria as compared to pituitary-intact rats, and this bactericidal capacity is significantly augmented 75% to 95% by either GH or
IFN-gamma. These data establish that the pituitary gland is essential for homeostasis during an infectious episode, and that GH plays an important role in host resistance by augmenting the ability of macrophages to kill S. typhimurium.
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Progress 01/01/90 to 12/30/90
Outputs
Newcastle disease virus (NDV) has been used to induce regression of tumors in human cancer patients. In this report, we examined the effects of inactivation of NDV and a serine protease inhibitor, N-1-tosylamide-2-phenylethyl-chloromethyl ketone (TPCK), upon viral enhancement of TNF cytotoxicity. Virulent NDV, but not heat- or UV-inactivated NDV, induced a one-hundred fold increase in the sensitivity of L929 cells to recombinant human TNF-(alpha) (rHuTNF-(alpha); 20 pg vs 2000 pg), rHuTNF-(beta) (8 pg vs 800 pg), and recombinant murine TNF-(alpha) (rMuTNF-(alpha); 34 pg vs 3,400 pg). Unlike TNF-(alpha) and TNF-(beta), several other cytokines (rHuIL-1(beta), rMuIL-4, murine IFN-(alpha), rMuIFN-(gamma) rRaIFN-(gamma)) did not have cytotoxic activities toward NDV-treated L929 cells. Specificity for TNF killing of NDV-treated L929 cells was also confirmed by antibody inhibition experiments. TPCK, which is an inhibitor of chymotrypsin-like proteases, blocked between 42% and
93% of the cytolytic activity of rMuTNF-(alpha), rHuTNF-(alpha), and rHuTNF-(beta) toward NDV-treated L929 cells. Similarly, TPCK abrogated 62% of the cytotoxicity of rMuTNF-(alpha) toward actinomycin D (ActD)-treated L929 cells. These results show that virulent, but not inactivated NDV, induces susceptibility of tumor cells to the lytic effects of TNF.
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Progress 10/01/88 to 09/30/89
Outputs
The oncolytic strain 73-T of Newcastle Disease Virus (NDV) has been reported to be beneficial in the treatment of cancer patients, but little is known about the mechanism of action. In this study, NDV strain 73-T and a wild-type isolate of NDV were found to be potent inducers of tumor necrosis factor (TNF) production by both human peripheral blood mononuclear cells (PBMC) and rat splenocytes. Antibody inhibition experiments identified TNF-variation as the major species of TNF induced by NDV in PBMC. The effect of recombinant human TNF-variation (rHuTNF)-variation) on human cancer cells was then examined. Neither rHuTNF-variation nor supernatants from NDV-stimulated PBMC were cytotoxic toward TNF-resistent MEL-14 human malignant melanoma cells. However, when MEL-14 cells were infected with NDV-73-T, both rHuTNF- variation and supernatants from NDV-stimulated PBMC killed48% and 55%, respectively, of these tumor cells. Infection with NDV also conferred TNF
susceptibility to the TNF-resistant MEL-21 human malignant melanoma and K562 human myelogenous leukemia cells. In contrast to its enhanced cytotoxicity towards NDV-treated cancer cells, rHuTNF-variation had no effect on NDV-treated normal human PBMC proliferating in response to concanavalin A. These results suggest two important mechanisms for the antineoplastic activity of NDV: (1) induction of TNF-variation secretion by human PBMC and (b) enhancement of the sensitivity of neoplastic cells to the cytolytic effects of TNF-variation.
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Progress 10/01/87 to 09/30/88
Outputs
This study was designed to determine whether the sex of an individual affects various parameters of the immune system. It is known that several aspects of T cell-mediated immune responses decline with age, but it is not known how gender affects this decline. Using 3-and 26-month-old male and female Fischer 344 rats, we examined the effects of sex and age on four different immune events that normally decline during aging: antibody synthesis to a T-dependent antigen, lectin-induced proliferative responses, IL-2 synthesis, and natural killer activity. We found that all these responses decreased with age. Spleen cells from aged females had higher spontaneous, phytohemagglutinin (PHA), and concanavalin A (Con A)-induced proliferative responses, and a two-fold increase in IL-2 synthesis than aged males, although no differences in these responses were evident between young males and females. Both natural killer (NK) activity and the ability to generate plaque-forming cells to
sheep erythrocytes (SRBC) declined with age, but there were no differences between males and females for these responses in either age group. These data indicate that sex-associated differences in IL-2 synthesis and spontaneous and lectin-induced proliferative responses that are not detected in young animals become evident wih advancing age.
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Progress 10/01/86 to 09/30/87
Outputs
An inhibitor of lectin-induced splenocyte proliferation from serum of normal chickens has been characterized. This suppressive factor, found in both serum and plasma and at concentrations as low as 3%, causes a 50% inhibition in proliferative responses to T-cell lectins of autologous and heterologous lymphoid cells. The inhibitor in serum also dramatically suppresses murine IL-2 synthesis, proliferation of murine spleen cells stimulated with PHA, and synthesis of DNA in xenogeneic-transformed mammalian lymphoblastoid cell lines. Serum does not block binding of the lectin to lymphoid cells and the suppressive activity cannot be overcome by any dose of lectin. The inhibitor of DNA synthesis is destroyed by pepsin, NH(4(2)) SO(4) (50%) and TCA (15%) treatments both precipitate the suppressor factor, which further indicates that the suppressive factor is a protein. A 330-fold purification of the inhibitory protein from serum was obtained when boiled serum was passed
over a Sepharose 6B and then a DEAE-Sephacel column which was washed at pH 3.0 and cluted with 0.2 M NaCl. SDS-PAGE with silver staining revealed a nonreduced protein with an apparent molecular weight of 61 kDa. Less than 2 mu g of the protein thus obtained caused a 50% inhibition in the proliferation of chicken lymphoid cells to Con A.
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Progress 01/01/86 to 09/30/86
Outputs
Thymic size and T-cell function decrease with age, and it has not yet been possible to totally reverse this thymic atrophy and completele restore T-cell-dependent immune functions. In this study, GH(3) pituitary adenoma cells, which secrete growth hormone and prolactin, were implanted subcutaneously into 16- and 22-month old female Wistar-Furth rats and the rats were sacrificed approximately two months later. Only thymic remnants were detected in aged, non-implanted rats, but thymus glands were found in both the 18- and 24-month old rats that had been implanted with GH(3) cells. Thymus glands from the GH(3)-implanted, 18 month-old rats contained distinct cortical thymocytes and medullary epithelial cells. Depending on the concentration of phytohemagglutinin or concanavalin A, T cell proliferative responses of splenocytes from these implanted rats were 2- to 5-fold greater than those of 18-month-old controls. At the optimal concentration of mitogen, proliferative
responses to either lectin could be restored to those levels observed in splenocytes from 3-month-old Wistar-Furth females. These data show that it is possible to regenerate normal thymic tissue in situ and reverse the natural loss of cell-mediated immunity that occurs in aging. These data also provide evidence that either hormone or prolactin may be able to augment immune function even in young domestic animals.
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Progress 01/01/85 to 12/30/85
Outputs
Infection with Newcastle disease virus (NDV) has been shown to augment plasma glucocorticoid concentrations in hypophysectomized mice. Indirect fluorescence showed that splenocytes from NDV-infected mice stained positive with an antibody directed against ACTH, which suggested that splenocytes might be the extrapituitary source of ACTH. Pro-opiomelanocortin (POMC) is the precursor molecule for production of adrenocorticotropin (ACTH), alpha-, beta- and gamma-melanotropin, beta-endorphin and beta- and gamma-lipotropin. We now show that NDV-infected murine splenocytes express POMC-like RNA as determined by dot blot hybridization. Total cellular RNA was isolated by the quanidinium/hot phenol method from murine splenocytes that were stimulated for 18 hr with concanavalin A (Con A) or NDV. Control cells were incubated with RPMI alone. Cellular RNA from the mouse ACTH-secreting pituitary tumor cell line (AtT-20) and a murine IL-2 dependent cell line were also tested.
Increasing concentrations of denatured RNA were blotted onto Gene Screen transfer membranes and hybridized overnight with the recombinant plasmid, pMKSU16, that was labeled with alpha-PdCTP. This POMC probe contains a 932-base pair insert comprising the entire POMC-coding sequence which was constructed from the mouse AtT-20/D16v cell line.
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