Leukotriene B4 stimulates human polymorphonuclear leukocytes to synthesize and release interleukin-8 in vitro.

Interleukin-8 (IL-8) is a potent chemotactic protein for polymorphonuclear leukocytes (PMN). Here we examine whether PMN synthesize and release IL-8 in response to stimulation by leukotriene B4 (LTB4). PMN isolated from normal heparinized peripheral human blood were incubated in RPMI culture medium at 37 degrees C in 5% CO2, with and without LTB4. The culture supernatants were tested for IL-8 bioactivity through chemotactic activity measurements with and without neutralizing anti-IL-8 serum. Immunoreactive IL-8 was quantified by ELISA, and de novo IL-8 synthesis was evaluated by metabolic labeling with [35S]cysteine followed by immunoprecipitation. LTB4 stimulated PMN to produce IL-8 in a dose- and time-dependent manner. The IL-8 concentrations reached maximal levels after 16 h of incubation with LTB4. Significant increases in IL-8 production occurred with LTB4 doses of 10 to 1,000 nM/ml. Immunoprecipitation of labeled IL-8 documented new synthesis of IL-8 by LTB4-treated PMN. Northern blot analysis of total RNA from PMN using a 30 mer oligonucleotide for IL-8 demonstrated increased mRNA expression in LTB4-stimulated PMN compared with untreated PMN. These data show that peripheral blood PMN can be stimulated by LTB4 to synthesize and secrete biologically active IL-8. PMN and other cells capable of producing LTB4 may induce IL-8 protein production by inflammatory PMN and thereby amplify or perpetuate the acute inflammatory response by recruiting additional PMN into an inflammatory site.

Acute inflammation in a sheep model of unilateral lung ischemia: the role of interleukin-8 recruitment of polymorphonuclear leukocytes.

Polymorphonuclear leukocytes (PMN) contribute to post-ischemic injury in many organs and in a variety of clinical situations. PMN accumulate in both lungs during unilateral lung ischemia in sheep, but the mechanism has not been defined. In this study, we tested the hypothesis that PMN accumulation is a response to chemotactic signals generated during lung ischemia. Chemotactic activity was measured in a modified Boyden chamber using normal sheep PMN as the responding cells. Increased chemotactic activity was observed in both plasma and lung lymph in a time-dependent manner after ischemia. These data indicate that a chemotactic substance immunoreactive to interleukin-8 antibody is formed as a result of unilateral lung ischemia in sheep in vivo and is a possible mediator of PMN inflammation in this model.

A sensible approach to the nutritional support of mechanically ventilated critically ill patients.

OBJECTIVES: The goal of this review is to educate physicians in the details of nutritional support of mechanically ventilated critically ill patients. DESIGN: The subtopics of this review include: introduction, goals of nutritional treatment, assessment of nutritional status, estimation of nutritional requirements, estimation of protein requirements, recommended approach to the initial nutritional regimen, route of nutrition, and monitoring the response to nutrition. SETTING: The information is primarily germane to the medical management of patients with acute respiratory failure superimposed on chronic lung disease and malnutrition. CONCLUSION: Malnutrition is prevalent in mechanically ventilated critically ill patients. Undernutrition is associated with respiratory muscle weakness and may contribute to ventilator dependency. Overnutrition may increase CO2 production and increase ventilatory demands. This review advocates a titrated approach to nutritional management based on protein balance. Careful monitoring is necessary to ensure a regimen which maintains or improves body protein composition. Preliminary data exists which indicates that careful nutritional support may improve clinical outcome but more information is needed to recommend a universal approach.

Granulocyte/macrophage colony-stimulating factor stimulates human polymorphonuclear leukocytes to produce interleukin-8 in vitro.

Interleukin-8 (IL-8) is a potent chemotactic factor for polymorphonuclear leukocytes (PMN). Here we examine whether PMN synthesize and release IL-8 in response to stimulation by selected inflammatory cytokines. PMN isolated from normal heparinized peripheral human blood were incubated in RPMI culture medium at 37 degrees C in 5% CO2, with and without granulocyte/macrophage colony-stimulating factor (GM-CSF). The culture supernatants were tested for chemotactic activity using a modified Boyden chamber. Immunoreactive IL-8 protein was measured by ELISA with a monoclonal antibody specific for IL-8. GM-CSF (0.01 to 50 ng/ml) stimulated PMN to produce chemotactic activity in a dose- and time-dependent manner. The amount of chemotactic activity reached maximal levels after 3 h of incubation with GM-CSF. Treatment of culture media supernatants with rabbit antiserum against IL-8 blocked the GM-CSF-induced chemotactic activity. IL-8 protein concentrations detected by ELISA closely paralleled the chemotactic bioactivity in both the dose-response and kinetic studies. Northern blot analysis of total RNA from PMN using a 30 mer oligonucleotide complementary to mRNA for IL-8 yielded a single 1.6-kb band. Its intensity increased 4-fold 2 h after treatment of PMN with GM-CSF. These data suggest that peripheral blood PMN can be stimulated by GM-CSF to synthesize and secrete bioactive IL-8. Since both IL-8 and GM-CSF accumulate in sites of acute inflammation, PMN may induce IL-8 gene expression in response to GM-CSF and thereby amplify the acute inflammatory response by recruiting additional PMN into inflammatory sites.

Effects of methoxamine and phenylephrine on left ventricular contractility in rabbits.

The end-systolic pressure-volume relation is employed to evaluate left ventricular contractility. In clinical studies, pharmacologic vasoconstriction is used to increase left ventricular systolic pressure to assess pressure-volume relations. However, the effect of vasoconstrictors on the ventricular contractile state is not well characterized. The effects of methoxamine and phenylephrine on systemic arterial pressure and left ventricular contractility in rabbits were studied with three protocols. In protocol 1, anesthetized rabbits (n = 10) were injected with incremental doses of methoxamine and phenylephrine intravenously. Methoxamine (4 mg) increased the mean arterial pressure by 50 +/- 12% (mean +/- SE) (n = 5, p = 0.001). Phenylephrine (0.2 mg) increased mean arterial pressure by 82 +/- 14% (n = 5, p = 0.004). In protocol 2, isolated blood-perfused hearts were injected with incremental doses of these drugs in the ascending aorta in amounts approximately equal to the concentrations injected in the intact rabbits. Methoxamine (2 mg) reduced isovolumic peak systolic left ventricular pressure by 43 +/- 9% (n = 7, p = 0.003), whereas phenylephrine (0.1 mg) increased the isovolumic pressure by 24 +/- 9% (n = 7, p less than 0.05). These responses indicated an enhanced contractile state with phenylephrine and a reduced contractile state with methoxamine. Pretreatment with propranolol blunted the effect of phenylephrine on isovolumic pressure (n = 6, p less than 0.02). In protocol 3, cross-circulation experiments allowed study of the effect of these drugs on isovolumic left ventricular pressure in the isolated heart and simultaneously on the systemic arterial pressure in the intact anesthetized rabbit (support rabbit).(ABSTRACT TRUNCATED AT 250 WORDS)

Hepatic 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase: phosphate dependence and effects of other oxyanions.

The effects of various oxyanions on the activities of rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (EC 2.7.1.105/3.1.3.46) were examined. No evidence could be found for an absolute dependence of the kinase activity on inorganic phosphate as was recently reported by M. Laloux, E. Van Schaftingen, and H.-G. Hers ((1985) Eur. J. Biochem. 148, 155-159). Rather, phosphate and arsenate activated the kinase by decreasing the enzyme's Km for fructose 6-phosphate without affecting its Km for ATP or Vmax. The Km of the kinase for fructose 6-phosphate in the presence of inorganic phosphate was found to be significantly lower (6 microM) than previously reported (30 microM) when the hydrolysis of fructose 2,6-bisphosphate by the concomitant bisphosphatase activity at low Fru 6-P concentrations was taken into account. The KA's for phosphate and arsenate activation of the kinase were 0.2 and 0.3 mM, respectively. A number of other oxyanions, including pyrophosphate, sulfate, tungstate, selenate, and molybdate all inhibited the kinase by increasing the Km for fructose 6-phosphate. The apparent Ki's for inhibition of the kinase were in the 0.5-1 mM range. In contrast, all of these oxyanions activated the bisphosphatase, with half-maximal effects requiring millimolar concentrations. Inorganic phosphate was the most potent activator with a KA of 1 mM. In contrast to the other oxyanions, vanadate and meta-periodate inhibited the kinase but had no effect on the bisphosphatase. Vanadate appeared to be a noncompetitive inhibitor since its effects were not overcome by Pi, ATP, or fructose 6-phosphate, and the species responsible was shown to be decavanadate. Like vanadate, meta-periodate had no effect on the bisphosphatase, though it was a potent inhibitor (I0.5 = 30 microM) of the kinase. Its effects were shown to be time-dependent and reversed by dithiothreitol, suggesting that it acted by an oxidative mechanism. These results augment the mounting body of evidence that the enzyme's two reactions are catalyzed at discrete active sites.

Pentobarbital-anesthesia decreases fructose 2,6-bisphosphate levels in rat liver.

Fructose 2,6-bisphosphate levels were assayed in freeze-clamped livers of anesthesized rats. All doses of pentobarbital that were effective in anesthesizing the rats caused a significant decrease in fructose 2,6-bisphosphate levels. Injection of pentobarbital also resulted in decreased 6-phosphofructo 2-kinase activity and increased fructose 2,6-bisphosphatase activity measured in dialyzed (NH4)2SO4-treated liver extracts but with no change in pyruvate kinase activity. It was concluded that the anesthesia-induced decrease in fructose 2,6-bisphosphate levels results at least in part from increased phosphorylation of 6-phosphofructo 2-kinase/fructose 2,6-bisphosphatase.