Kirk R. Manogue

Detection of Circulating Tumor Necrosis Factor after Endotoxin Administration

Cytokines, products of stimulated macrophages, are thought to mediate many host responses to bacterial infection, but increased circulating cytokine concentrations have not been detected consistently in infected patients. We measured plasma concentrations of circulating tumor necrosis factor alpha (cachectin), interleukin-1 beta, and gamma interferon, together with physiologic and hormonal responses, in 13 healthy men after intravenous administration of Escherichia coli endotoxin (4 ng per kilogram of body weight) and during a control period of saline administration. Eight additional subjects received ibuprofen before receiving endotoxin or saline. Plasma levels of tumor necrosis factor were generally less than 35 pg per milliliter throughout the control period, but increased 90 to 180 minutes after endotoxin administration to mean peak concentrations of 240 +/- 70 pg per milliliter, as compared with 35 +/- 5 pg per milliliter after saline administration. Host responses were temporally associated with the increase in circulating tumor necrosis factor at 90 minutes, and the extent of symptoms, changes in white-cell count, and production of ACTH were temporally related to the peak concentration of tumor necrosis factor. Ibuprofen pretreatment did not prevent the rise in circulating tumor necrosis factor (mean peak plasma level, 170 +/- 70 pg per milliliter) but greatly attenuated the symptoms and other responses after endotoxin administration. Concentrations of circulating interleukin-1 beta and gamma interferon did not change after endotoxin administration. We conclude that the response to endotoxin is associated with a brief pulse of circulating tumor necrosis factor and that the resultant responses are effected through the cyclooxygenase pathway.

Metabolic effects of cachectin/tumor necrosis factor are modified by site of production. Cachectin/tumor necrosis factor-secreting tumor in skeletal muscle induces chronic cachexia, while implantation in brain induces predominantly acute anorexia.

We have developed a murine model of wasting by injecting intracerebrally cells which continuously secrete h-cachectin/TNF (CHO-TNF) to: (a) determine the effects of cachectin/TNF produced continuously in the central nervous system (CNS), and (b) compare the metabolic effects of cachectin/TNF-secreting tumor in the brain to the cachexia caused by CHO-TNF tumor in peripheral tissue (IM). Intracerebral CHO-TNF tumors produced increased serum h-cachectin/TNF levels with lethal hypophagia and weight loss (mean survival time of 11 d); these changes were not observed in association with nonsecretory control brain tumors. The metabolic consequences of intracerebral cachectin/TNF production were indistinguishable from acute, lethal starvation: whole-body lipid content was decreased significantly but protein was conserved. Although intramuscular cachectin/TNF-secreting tumors caused similar increases of serum h-cachectin/TNF levels, profound anorexia did not develop; wasting developed after a longer period of tumor burden (50 d) with classical signs of cachexia (i.e., anemia and depletion of both protein and lipid). These studies provide a reproducible animal model of site-specific cytokine production and suggest that, regardless of serum levels, cachectin/TNF produced locally in brain influences both the rate of development of wasting and its net metabolic effects.

Cachectin (Tumor Necrosis Factor)

Invasive stimuli, including parasitic, bacterial, or viral infection and neoplastic disease, precipitate catabolic changes in cellular metabolism and pathological alterations in the physiology of their mammalian hosts. These disruptions of normal homeostasis, left unresolved, can lead to the depletion of host energy stores advancing to wasting (cachexia), tissue damage, multiple organ system failure, shock, and death. Although such symptomatic patterns are all too familiar to clinicians, the underlying causes of this progressive decline have remained obscure. Because these patterns are frequently attributed to the metabolic demands or direct actions of the pathogen or its products, treatment has quite naturally focused on aggressive elimination of the invading agent and reactive support of failing host physiology. It now appears that invasive stimuli provide a trigger for the release of host-secreted cytokines, and the combined actions of these endogenous mediators elicit most or all of the biologic responses culminating in the pathophysiology of cachexia and shock. This new appreciation for the essential role of host-derived inflammatory mediators has provided a focus for new therapeutic approaches to a broad spectrum of invasive diseases.

Human decidua: a source of cachectin-tumor necrosis factor

Cachectin-tumor necrosis factor (TNF-alpha) has been implicated as a possible signal for the initiation of human parturition in the setting of infection. These studies were conducted to determine whether human decidua can produce TNF-alpha in response to bacterial lipopolysaccharide (LPS). Decidual explants from women undergoing elective cesarean sections were incubated with and without Escherichia coli LPS (25 ng/ml) for 20 h. TNF-alpha concentration in the conditioned media was measured with an enzyme-linked immunoassay and bioassay (L929 bioassay). While conditioned media from unstimulated decidual explants contained either undetectable or low levels of TNF-alpha, conditioned media from LPS stimulated decidua contained TNF-alpha (mean = 2.6 pmol/mg protein per 20 hours, SEM +/- 1.03). There was a strong correlation between the immunoreactive and bioactive TNF-alpha (Spearman rank correlation r = 0.76, P less than 0.001). We conclude that human decidua in vitro can produce TNF-alpha in response to LPS.

Interleukin-2 initiates metabolic responses associated with critical illness in humans.

The cytokine interleukin-2 is a primary modulator of the immune response that occurs after infection, trauma, and transplant rejection, yet its role as a mediator of associated metabolic changes in surgical illness is unknown. We studied clinical and metabolic responses in eleven tumor-bearing humans with normal renal and hepatic function receiving bolus intravenous (I.V.) interleukin-2 (30,000 U/kg). Additional subjects (n = 6) were pretreated with the cyclooxygenase inhibitor, ibuprofen (1600 mg, orally), before interleukin-2 administration. Serial measurements were made of vital signs, symptoms, hematology, and plasma concentrations of pituitary and stress hormones and selected cytokines. Administration of interleukin-2 resulted in fever, tachyacardia, “flu-like” symptoms, and neurohormonal elaboration. The responses observed were quantitatively similar to those that occurred after endotoxin administration in healthy subjects (n = 13), but differed in the following manner: 1) the onset of fever and endocrine changes occurred after a longer latent interval (180–240 minutes vs. 60–90 minutes after endotoxin), 2) peak responses after the administration of interleukin-2 also occurred later, 3) no increased circulating tumor necrosis factor was detected after administration of interleukin-2 (peak plasma concentration was <35 pg/ml vs. 270 ± 70 pg/ml after endotoxin administration), and 4) administration of interleukin-2 but not of endotoxin was associated with increased circulating concentrations of gamma interferon (peak plasma concentration 1.7 ± 0.2 NIH U/ml vs. < 0.1 NIH U/ml after endotoxin administration). Fever and neurohormonal responses after interleukin-2 administration were greatly attenuated by ibuprofen administration. Interleukin-2 induces other cytokines that exert their effects largely through the cyclooxygenase pathway. Interleukin-2 may be an important signal, initiating the integrated host responses to infection and injury.

Cachectin/TNF-mediated lactate production in cultured myocytes is linked to activation of a futile substrate cycle

The cytokine cachectin/TNF induces a rapid increase in lactate production and in glucose metabolism in L6 myocytes in culture; glucose uptake was maximal after 17 h, while elevated glucose utilization and lactate production persisted for up to 32 h. These increases are suggestive of increased glycolytic activity, and were associated with a 10% decrease in cellular oxygen consumption and a comparable decrease in the production of 14C-labelled CO2 from 14C-labelled glucose. This decrease in aerobic metabolism, however, could account for only a small fraction of the energetic requirement for increased glycolytic activity. Furthermore, maximal stimulation of pyruvate dehydrogenase (PDH) by dichloroacetate (DCA) treatment in conjunction with cachectin/TNF abolished lactate production, but increased glucose uptake persisted. Taken together, this suggests that the primary effect of cachectin/TNF on myocyte carbohydrate metabolism is to increase glycolysis. Correspondingly, we postulated that cachectin/TNF must activate one or more ATP-depleting cellular processes to account for the lack of feed-back inhibition on glycolysis by the ATP produced. This led to the identification of a futile substrate cycle between fructose 6-phosphate and fructose 1,6-bisphosphate as a novel energy sink that is activated by cachectin/TNF. Cachectin/TNF treatment led to increased activity of both phosphofructokinase (PFK) and fructose bisphosphate phosphatase (FBP) in myocytes in culture, detectable after 1 h of incubation and persisting for up to 16 h. The possible role of cachectin/TNF-mediated futile substrate cycling in increased glycolytic activity, increased energy expenditure, heat production and tissue wasting during bacterial infections is discussed.

Selective brain stem transections affecting reproductive behavior of female rats: The role of hypothalamic output to the midbrain

Abstract In estrogen-treated, ovariectomized rats, selective transections were used to interrupt, together or separately, the medial and lateral pathways by which efferent fibers from the ventromedial nucleus (VMN) of the hypothalamus reach the lower brain stem. Dorsal hemisections placed to interrupt both projections reduced or eliminated lordotic behavior. Transections placed to intercept all of the medially descending fibers, but spare the lateral pathway, did not reduce lordosis in mating or manual stimulation tests. The lateral pathway was interrupted at two different locations. Parasagittal transections at the level of the VMN showed that the lateral pathway, as a whole, was not required for lordosis when the medial pathway was left intact. Also, no particular subset of fibers assuming a lateral trajectory from the VMN to the brain stem was required for the display of lordosis. However, the fibers running through the lateral brain stem do play some role in the expression of the reflex, and more caudal bilateral lateral transections did reduce lordotic behavior. The absence of lordosis in mating tests was not a result of a systematic increase in rejecting behaviors. The observation of intermittent lordotic responses, or improved lordotic behavior following additional treatment with estrogen and/or progesterone revealed that these laterally transected animals were still able to produce the motor pattern of lordosis. The deficits seen were attributed to the interruption of fibers mediating the control of lordosis by the hypothalamus. This role of the ventromedial nucleus can be described as a tonic, estrogen-dependent facilitation of supraspinal mechanisms which control lordosis and are located more caudally in the brain stem. The laterally descending VMN efferents may play a quantitatively more important role in the control of lordosis than the medially descending fibers.

Respiratory gating of activity in the avian vocal control system

Abstract Electrical stimulation of brain nuclei within the vocal control pathway of budgerigals (Melopsittacus undulatus) or zebra finches (Poephila guttata) evoked neural activity in the hypoglossal nerves innervating the vocal organ, the syrinx. This activity was gated within the hypoglossal motor nucleus such that stimulation recruited more hypoglossal motor neurons to fire during expiration than during inspiration. This respiratory gating mechanism represents a novel solution to the problem of shared control of motor neurons during different behaviors, and may contribute to the patterning of neural activity during song production.