The influence of substrate background on the acute metabolic response to epinephrine and cortisol.

The acute metabolic effects of epinephrine and cortisol, as well as the influence of substrate background on the effects of these two hormones were investigated in normal subjects. While receiving a hypocaloric dextrose feeding (50 ng/kg/h) (DEX), the subjects received a 6-hour continuous intravenous infusion of epinephrine (30 ng/kg-min) (EPI), followed by a 6-hour infusion of a combination of epinephrine (30 mg/kg-min) and cortisol (3.0 microgram/kg-min) (EC). The hormone infusion was repeated 1 week after a continuous intravenous feeding regimen (TPN) was begun with a caloric content of 1.5 times the measured metabolic rate. Under both DEX and TPN, EPI produced increased energy expenditure, hyperglycemia, hyperlactic acidemia, and hypoaminoacidemia. Except for a further increase in circulating glucose levels during the DEX condition, these variables were not altered by the addition of cortisol. Epinephrine under both feeding conditions increased lactate efflux from the extremity without changes in peripheral oxygen or glucose uptake. The hypoaminoacidemic response to EPI in the DEX condition was associated with a decrease in extremity efflux of amino acids (-654 +/- 89 nmol/min/100 cm3 tissue at baseline vs -330 +/- 86 nmol/min/100 cm3 for EPI, p less than 0.05). No change in extremity amino acid flux was noted in response to EPI during total parenteral nutrition. Even with addition of cortisol no significant efflux of amino acids above baseline levels was noted in either feeding condition. We therefore conclude that (1) total parenteral nutrition cannot abolish the hypermetabolic or hyperglycemic response to epinephrine and cortisol; (2) increased extremity lactate efflux and lactic acidosis can occur in response to epinephrine without evidence of diminished oxygen delivery to the extremity; and (3) these two hormones are not primary mediators of acute extremity nitrogen loss.

Influence of enterectomy on peripheral tissue glutamine efflux in critically ill patients.

Glutamine and alanine are dominant nitrogen carriers from skeletal muscle stores to splanchnic organs. In addition, these amino acids may also serve as a primary energy source for the gastrointestinal tract during injury. To investigate these contributions, we studied extremity amino acid efflux during hypocaloric dextrose feedings and during total parenteral nutrition in a population of normal volunteers (NL VOL) (n = 9), a group of patients with sepsis who had undergone laparotomy without bowel resection and were in the intensive care unit (ICU) (n = 7), and patients with sepsis after laparotomy (PT) (n = 2) who had recently undergone greater than 80% bowel resection. Circulating alanine and glutamine levels were significantly lower in the patients compared with NL VOL under both feeding conditions. The peripheral output of alanine was higher in the ICU group than in the NL VOL during hypocaloric feedings. Glutamine efflux, however, was independent of either the counterregulatory hormone or substrate background. By contrast, enterectomy was associated with a marked decrease of extremity glutamine efflux compared with NL VOL or the ICU patients who did not undergo enterectomy (-62 +/- 9 nmol/min/dl tissue in the PT vs -265 +/- 32 nmol/min/dl tissue in the NL VOL and -311 +/- 58 nmol/min/dl tissue in the ICU group) during the dextrose feedings; this difference persisted during subsequent total parenteral nutrition (+12 +/- 13 nmol/min/dl tissue in PT vs -178 +/- 56 nmol/min/dl tissue in the NL VOL and -287 +/- 81 nmol/min/dl tissue in the ICU group). These data suggest that distinct mechanisms regulate peripheral alanine and glutamine balance and that the gastrointestinal tract provides a feedback signal to peripheral tissues to maintain glutamine mobilization under both nonstressed and stressed conditions.

Antibodies to cachectin/tumor necrosis factor reduce interleukin 1 beta and interleukin 6 appearance during lethal bacteremia.

Cytokines secreted in response to invading micro-organisms are important mediators of detrimental hemodynamic and metabolic changes in the host. To test whether cachectin/TNF plays a role in triggering release of other cytokines in the setting of infection, anesthetized baboons were passively immunized against systemic cachectin/TNF before infusion of a LD100 dose of live Escherichia coli. Bacteremia led to significant increases in circulating levels of cachectin/TNF, IL-1 beta, and IL-6. Although bacterial endotoxin/lipopolysaccharide is a potent stimulus for the synthesis and release of IL-1 and IL-6 in vitro, specific neutralization of cachectin/TNF in vivo with mAb pretreatment significantly attenuated both the IL-1 beta and the IL-6 responses despite fulminant overwhelming bacteremia. These data suggest that cachectin/TNF is essential for the initiation or amplification of IL-1 and IL-6 release during lethal gram-negative septic shock syndrome.

Intravenous refeeding blocks growth hormone (GH)-provoked rises in serum free fatty acids and blunting of somatotroph response to GH-releasing hormone in normal men.

We measured the serum GH responses to GHRH (1 micrograms/kg) in six normal men who had been rendered hyperinsulinemic and hypolipidemic by 10 days of total parenteral nutrition (TPN subjects) with a 25% dextrose-amino acid solution. The men underwent GHRH testing after 3 h of infusion of NaCl or Met-human (h) GH (2 micrograms/kg.h). The results of these tests were compared with those of five men tested in the post-absorptive state (PA subjects). The serum GH response to GHRH during NaCl infusion was significantly lower in the TPN subjects than in the PA subjects. During the Met-hGH infusion, the serum GH response to GHRH in the PA subjects was significantly lower than that after the NaCl infusion, whereas in the TPN subjects the response was similar to that during the NaCl infusion. The mean integrated areas under the GH response-time curve after GHRH treatment were 3963 +/- 2086 min/micrograms.L following NaCl infusion and 413 +/- 64 min/micrograms.L following Met-hGH infusion in PA subjects; they were 1127 +/- 500 min/micrograms.L following NaCl infusion and 1456 +/- 682 min/micrograms.L during Met-hGH infusion in the TPN subjects. The Met-hGH infusions resulted in a significant increase in serum FFA concentrations in the PA, but not the TPN, subjects. These results suggest that hyperalimentation induces a metabolic background which inhibits GH secretion, as manifested by a diminished serum GH response to GHRH administered after NaCl infusion. The absent FFA response to Met-hGH infusion in the TPN subjects may explain why the Met-hGH infusion in them did not result in a reduced serum GH response to GHRH as occurred in the PA subjects. Hence, FFA may play an important role in the effects of short term Met-hGH infusion on GH secretion.

Peripheral blood leukocyte kinetics following in vivo lipopolysaccharide (LPS) administration to normal human subjects. Influence of elicited hormones and cytokines.

Lipopolysaccharide (LPS, endotoxin) administration to human subjects elicits significant elevations in plasma cachectin/TNF, epinephrine, and cortisol. This study examined the temporal relationship between changes in blood leukocyte subsets and plasma mediators following endotoxin administration to normal human subjects. A five-minute intravenous infusion of purified LPS (20 units/kg Escherichia coli) was administered to 12 healthy volunteers. Blood samples were obtained at varying intervals after infusion and analyzed for differential cell counts and lymphocyte subsets (CD2, CD3, CD4, CD8, CD20, and HLA-DR) by flow microfluorimetry, and also assayed for plasma cachectin/TNF, epinephrine, and cortisol. Plasma cachectin/TNF was significantly elevated at 75 and 90 minutes after infusion with a peak concentration of 261 +/- 115 pg/ml noted 75 minutes after infusion. A significant plasma epinephrine elevation of 181 +/- 75 pg/ml was demonstrated one hour after infusion, while significant elevations in plasma cortisol were noted from one to five hours after infusion with a peak level of 34 +/- 3 micrograms/dl three hours after infusion. A profound monocytopenia (p less than 0.01) was noted one hour after infusion. Temporally associated with the rise in plasma cortisol was a reversal of the early granulocytopenia to a significant granulocytosis (p less than 0.01 versus preinfusion mean), whereas a marked lymphocytopenia (p less than 0.01) was observed from one to six hours after infusion. During the period of hypercortisolemia, CD2, CD3, and CD4 lymphocyte percentages were decreased (p less than 0.01) while CD20 and HLA-DR lymphocyte percentages were increased (p less than 0.01). There was a small percentage decrease in CD8 lymphocytes from one to 24 hours after infusion (p less than 0.01), although relative to the one-hour nadir, there was a significant rise in the percentage during the time of elevated plasma cortisol concentrations. A six-hour infusion of epinephrine (30 ng/kg/min) administered to six healthy volunteers resulted in a monocytosis (p less than 0.05) and granulocytosis (p less than 0.01) without a change in lymphocyte number or lymphocyte subset percentage. Previous reports have shown that in vivo corticosteroid infusion causes a prominent granulocytosis, monocytopenia, and lymphocytopenia with a decrease in the percentages of CD3 and CD4 lymphocytes. The peripheral blood leukocyte dynamics documented in the current study are similar to patterns observed following in vivo corticosteroid administration. This study suggests that the acute adrenocortical response to endotoxemia primarily mediates the subsequent changes in leukocyte subsets.

Recombinant growth hormone enhances muscle myosin heavy-chain mRNA accumulation and amino acid accrual in humans.

A potentially lethal complication of trauma, malignancy, and infection is a progressive erosion of muscle protein mass that is not readily reversed by nutritional support. Growth hormone is capable of improving total body nitrogen balance, but its role in myofibrillar protein synthesis in humans is unknown. The acute, in situ muscle protein response to an infusion of methionyl human growth hormone was investigated in the limbs of nutritionally depleted subjects during a period of intravenous refeeding. A 6-hr methionyl growth hormone infusion achieved steady-state serum levels comparable to normal physiologic peaks and was associated with a significant increase in limb amino acid uptake, without a change in body amino acid oxidation. Myosin heavy-chain mRNA levels, measured by quantitative dot blot hybridization, were also significantly elevated after growth hormone administration. The data indicate that methionyl growth hormone can induce intracellular amino acid accrual and increased levels of myofibrillar protein mRNA during hospitalized nutritional support and suggest growth hormone to be a potential therapy of lean body wasting.

Cachectin/tumor necrosis factor production by fetal and newborn rat hepatic macrophages.

The capacity of fetal and newborn hepatic macrophages to produce cachectin/tumor necrosis factor (TNF) has not been previously examined. The role that TNF plays in newborn infection is also unknown. In the present report, fetal and newborn rat hepatic macrophages have been maintained in short-term tissue culture, and are shown to produce TNF in response to in-vitro endotoxin stimulation. Cells obtained are morphologically and antigenically consistent with macrophages, and are depleted of accessory cells and lymphocytes. In response to increasing doses of endotoxin, fetal and newborn hepatic macrophages produce TNF, and this production is suppressed fully by pretreatment with dexamethasone. The results demonstrate that fetal and newborn hepatic macrophages possess the capability to produce TNF in vitro. We propose that TNF may play a role in the response of the fetus and newborn to bacterial infection.

Differential neutrophil activation before and after endotoxin infusion in enterally versus parenterally fed volunteers.

This study was done to determine whether or not increased susceptibility to infection seen in enterally versus parenterally fed patients was caused by altered neutrophil (PMN) responsiveness. To determine the differential effects of route of feeding on human PMN activation, plasma C3a levels, circulating PMN counts, PMN migration to leukotriene B4 (LTB4), the peptide, N-formyl-methionyl-leucyl-phenylalanine (FMLP) and zymosan activated serum (ZAS) and generation of LTB4 were assayed before and after and infusion of endotoxin. Nine normal volunteers were enterally (n=4) or parenterally (n=5) fed a diet sufficient to maintain body weight for seven days prior to a standard challenge of endotoxin. Samples were taken prior to the infusion and hourly thereafter for six hours. Prior to the injection of endotoxin, significant differences were seen in the two feeding groups. Plasma C3a levels, absolute circulating PMN counts and chemotaxis to LTB4 were all significantly (p less than 0.02) elevated in the enterally fed group. Generation of LTB4 was higher in the intravenously fed group at base line than the orally fed group (p less than 0.05). Plasma C3a levels rose in the enterally fed group, but not in the intravenously fed group, at two hours after infusion. Neutrophil counts rose in both feeding groups after endotoxin infusion; but the change in percentage was greater in the enterally fed group than in the intravenously fed group. Chemotaxis to FMLP and ZAS was not different during the study and did not differ between the two feeding can have significant impact on neutrophil function and that parenteral nutrition may impair host responsiveness.

Tumor necrosis factor-enhanced leukotriene B4 generation and chemotaxis in human neutrophils.

In an in vivo study of five normal volunteers infused with endotoxin (20 U/kg of US reference endotoxin lot EC-5), increased neutrophil (PMN) generation of leukotriene B4 and chemotaxis to leukotriene B4 were found concomitantly with elevated plasma tumor necrosis factor (TNF) levels. To clarify the role of TNF in PMN activation, neutrophil responsiveness after in vitro treatment with TNF was examined. Neutrophils from seven normal subjects were incubated with TNF for 30 minutes and tested for chemotaxis to leukotriene B4, formyl-methionyl-leucyl-phenylalanine and zymosan-activated serum, or the calcium ionophore A23187 to assess leukotriene B4 generation. A range of 10(-13) to 10(-9) mol/L of TNF was used for these assays. When 10(-9) mol/L of TNF was used, the amount of leukotriene B4 that was produced was significantly greater than in control cells. The effect of TNF on PMN chemotaxis was uniformly inhibitory for the three stimuli at 10(-10) mol/L compared with untreated cells. At a picomolar range, PMN migration to leukotriene B4, but not to zymosan-activated serum or formyl-methionyl-leucyl-phenylalanine, was significantly increased over that of PMNs not exposed to TNF. This suggests that TNF has a specific facilitatory effect on PMN responsiveness for both leukotriene B4 production and chemotaxis to leukotriene B4 and may be the same signal for this phenomenon in endotoxemic patients.

Influence of substrate background on peripheral tissue responses to growth hormone.

To evaluate the effects of substrate and hormonal background on the peripheral response to methionyl human growth hormone (met-hGH), metabolic measurements were performed in 10 normal subjects before and at the end of a 6-hr infusion of met-hGH. Five subjects were studied in the postabsorptive state and five were studied on the 10th day of intravenous repletion. Measurements of hormonal levels, serum amino acid levels, and free fatty acid levels, as well as extremity amino acid and free fatty acid fluxes, were performed. Met-hGH in the postabsorptive setting had no effect on extremity amino acid flux but significantly increased extremity free fatty acid efflux. In contrast, met-hGH in the background of intravenous feeding produced a significant extremity uptake of total amino acids but had no effect on free fatty acid flux. These data suggest a relationship between the substrate background and the actions of met-hGH on both peripheral protein and lipid metabolism. Therefore, the adequacy and composition of the feeding regimen may be important when considering growth hormone as an adjunct to nutritional therapy.

Submaximal exercise during intravenous hyperalimentation of depleted subjects.

The peripheral nitrogen wasting and loss of functional capacity caused by the malnutrition of disease and the immobilization of hospitalization may not be readily reversed by refeeding alone. In order to examine submaximal exercise as an adjunctive anabolic stimulus to intravenous refeeding (IVF) in depleted subjects, 14 volunteers were studied in the postabsorptive (PA) state, after 10 days of total starvation, and again after 10 days of nutritional repletion with I.V. feedings. The subjects were randomized to one group that received IVF alone and one group that performed 1 hour of submaximal (51% of VO2max) stationary bicycle exercise daily during IVF. The exercised group was not significantly different from the nonexercised group in urinary nitrogen balance, resting energy expenditure, extremity amino acid flux, or maximal oxygen consumption. Acute exercise did not induce significant derangements in electrolytes or counter-regulatory hormone concentrations. Ten days of submaximal exercise does not appear to be detrimental in this population recovering from moderate hospitalized malnutrition, but additional anabolic stimulae may be needed for improvements in protein accrual or functional capacity.

Cachectin/tumor necrosis factor induces cachexia, anemia, and inflammation.

Cachexia is a potentially lethal syndrome of unknown etiology characterized by anorexia, weight loss, and protein wasting that frequently complicates the treatment of chronic inflammation and cancer. Cachectin/TNF was isolated during the search for a humoral mediator of cachexia and found to stimulate the breakdown of energy stores from adipocytes and myocytes in vitro, but the chronic effects of the monokine in vivo are not known. Sublethal doses of recombinant human cachectin administered twice daily for 7-10 d caused cachexia in rats, as evidenced by reduced food intake, weight loss, and depletion of whole-body lipid and protein stores. Significant anemia is also observed and found to be the result of decreased red blood cell mass, not expanded plasma volume. Leukocytosis and histopathological evidence of tissue injury and inflammation are observed in several organs, including omentum, liver, spleen, and heart. These data suggests that the exposure of the normal host to cachectin is capable of inducing a pathophysiological syndrome of cachexia, anemia, and inflammation similar to that observed during inflammatory states or malignancy.

Cytokine appearance in human endotoxemia and primate bacteremia.

The results of recent work have demonstrated that endotoxin elicits the production of several immunopeptide cytokines that likely mediate the development of septic shock. Bolus injection of endotoxin (20 units per kilogram of body weight) to four volunteers resulted in peak serum cachetin/tumor necrosis factor (TNF) levels of 358 +/- 166 picograms per milliliter within 90 minutes after challenge (p less than 0.05 versus base line) and peak serum interleukin-1 levels of 2.14 +/- 0.89 units per milliliter within two hours after challenge. By contrast, the infusion of a lethal dose of live Escherichia coli to four baboons revealed peak serum cachectin/TNF levels of 20,500 +/- 9,890 picograms per milliliter within 90 minutes after bacteria were given (p less than 0.05 versus base line) and peak interleukin-1 levels of 14.2 +/- 10.1 units per milliliter three hours after bacterial challenge. No detectable monokine levels were observed in either model six hours after challenge. Interferon-gamma levels reached a peak of 2.67 +/- 1.66 nanograms per milliliter in baboon sera at eight hours after bacterial infusion and was no longer detectable by 12 hours. Interferon-gamma was not detected in the sera of humans. These results suggest that the transient release of cachectin/TNF, followed by interleukin-1 and interferon-gamma, may participate in the cascade of events noted in overwhelming bacterial invasion.

Macrophages secrete a novel heparin-binding protein with inflammatory and neutrophil chemokinetic properties.

We report the identification and purification of a new inflammatory monokine synthesized by the macrophage tumor cell line RAW 264.7 in response to endotoxin. This monokine, which we term "macrophage inflammatory protein" (MIP), is a doublet with an apparent molecular mass of approximately 8,000 daltons on SDS-PAGE but forms aggregates of greater than 2 x 10(6) daltons as assessed by gel filtration. Partial NH2-terminal amino acid sequence data reveal no significant homology with any previously described protein. Although the monokine is anionic under physiological conditions, it is one of two major macrophage-secreted proteins that bind to heparin at high salt concentrations. At 100 ng/ml or greater, MIP is chemokinetic for human polymorphonuclear cells and triggers hydrogen peroxide production. Subcutaneous injection of 10 ng or greater of MIP into footpads of C3H/HeJ mice elicits an inflammatory response, characterized by neutrophil infiltration. These findings suggest that MIP is an endogenous mediator that may play a role in the host responses that occur during endotoxemia and other inflammatory events.

Anti-cachectin/TNF monoclonal antibodies prevent septic shock during lethal bacteraemia.

Bacterial infection of the mammalian bloodstream can lead to overwhelming sepsis, a potentially fatal syndrome of irreversible cardiovascular collapse (shock) and critical organ failure. Cachectin, also known as tumour necrosis factor, is a macrophage-derived peptide hormone released in response to bacterial lipopolysaccharide, and it has been implicated as a principal mediator of endotoxic shock, although its function in bacterial sepsis is not known. Anaesthetized baboons were passively immunized against endogenous cachectin and subsequently infused with an LD100 dose of live Escherichia coli. Control animals (not immunized against cachectin) developed hypotension followed by lethal renal and pulmonary failure. Neutralizing monoclonal anti-cachectin antibody fragments (F(ab')2) administered to baboons only one hour before bacterial challenge protected against shock, but did not prevent critical organ failure. Complete protection against shock, vital organ dysfunction, persistent stress hormone release and death was conferred by administration of antibodies 2 h before bacterial infusion. These results indicate that cachectin is a mediator of fatal bacteraemic shock, and suggest that antibodies against cachectin offer a potential therapy of life-threatening infection.

Decreased T lymphocyte migration in patients with malignancy mediated by a suppressor cell population.

The migration and concentration of lymphocytes at sites of antigenic challenge are an integral part of the expression of delayed cutaneous hypersensitivity, as well as of tumor and graft rejection. In this study, we have analyzed the migration of T lymphocytes from patients with malignancy. We used casein and concanavalin A (Con A)-stimulated mononuclear cell supernatants to stimulate T cell locomotion. Peripheral blood T lymphocytes from 30 patients with established malignancy, 10 patients with indolent malignancy or benign tumor, and 42 normal adult controls were tested. Data are expressed as a migration index (MI), which represents the difference in micrometers between the distance migrated in response to a stimulus and the distance migrated in response to media alone. We observed a marked depression in casein-stimulated T lymphocyte migration in patients with established malignancy (mean MI +/- 1 SD = 17.0 +/- 9 microns) as compared with normal adult controls (mean MI +/- 1 SD = 35.3 +/- 10 microns). Similar results were observed with migration in response to Con A supernatants. T cells from patients with established malignancy had a mean MI of 5.8 +/- 4 microns to Con A supernatants as compared with 24.5 +/- 5 for controls. This depressed migration was apparent both in the distance that cells migrated and in the number of cells that migrated into the membrane. Of 10 patients with indolent malignancy or benign tumor, T cell migration in 8 was not significantly decreased as compared with controls. When we mixed equal concentrations of normal control T lymphocytes with T lymphocytes from patients with cancer and added the mixture directly to the upper compartment of the chemotaxis chamber, the response of the normal T cells to casein was inhibited by an average of 48%. We observed inhibition of this migration of normal cells when we added as little as 10% of patient cells to normal cells. When we mixed normal control T lymphocytes from different donors and added them directly to the upper compartment of the chemotaxis chamber, T lymphocyte migration in response to casein was not significantly altered. If T cells from patients with cancer were cultured overnight, the suppressive effect on lymphocyte locomotion was lost. Our results indicate that there is a population of T lymphocytes in patients with cancer that suppress normal T lymphocyte migration. This suppressor activity may partially explain the subversion of immunosurveillance in established neoplastic states, as well as the defective inflammatory reaction to intradermal injection of antigen observed in many patients with malignancy.