Judy A. Spitzer

Endotoxin and TNFα directly stimulate nitric oxide formation in cultured rat hepatocytes from chronically endotoxemic rats

We examined the effects of endotoxin on nitric oxide formation in isolated rat hepatocytes in primary culture. Endotoxin was administered either in vivo, by continuous infusion for 30 or 3 h, or in vitro, on cultured cells. The spontaneous production of nitrites in hepatocytes from in vivo ET-infused rats was lower than equivalent saline controls in the absence of added stimuli. However in vitro addition of endotoxin in culture to hepatocytes from 30 h ET-infused rats greatly enhanced production relative to saline controls. This effect was mimicked by TNF alpha, and activators of protein kinase C (PMA and Ca2+ ionophore A23187). The effects of ET were blocked by NMMA, dexamethasone and protein synthesis inhibitors Actinomycin D and cycloheximide. No in vitro effect of ET was observed in the 3 h infusion model. The results show that chronic exposure to sub-lethal levels of ET primes liver parenchymal cells for the production of nitric oxide, when exposed in vitro to ET or TNF alpha.

Attenuation of hepatic neutrophil sequestration by anti-CINC antibody in endotoxic rats.

Cytokine-induced neutrophil chemoattractant (CINC) is a member of the chemokine a subfamily. It is induced in rats by tumor necrosis factor-a (TNF-α), interleukin-1, and lipopolysaccharide and is implicated in neutrophil infiltration in response to inflammatory stimuli. We tested the hypothesis that pretreatment with anti-CINC antibody or by cobra venom factor attenuates hepatic neutrophil accumulation induced by a 90 min infusion of Escherichia coli endotoxin. Changes in the expression of CD11b/c and CD18 and in plasma TNF-α levels were also investigated. Cultured hepatocytes and Kupffer cells of endotoxic rats produced significantly more CINC than those of saline-infused controls. CINC generation by Kupffer cells was much lower than generation by hepatocytes. Pretreatment with anti-CINC antibody or cobra venom factor significantly reduced hepatic neutrophil sequestration, but did not affect the up- regulation of CD11b/c and CD18 expression on liver-sequestered neutrophils or plasma TNF-α levels. We conclude that CINC-mediated hepatic neutrophil accumulation may not be necessarily associated with up-regulation of neutrophil adhesion molecules or elevated circulating TNF-α levels. Attenuation of hepatic neutrophil sequestration by anti-CINC antibody is likely based on blocking of the chemotactic activity of CINC and thus diminishing the chemotactic gradient established in the liver.

Hepatic neutrophil sequestration in early sepsis: enhanced expression of adhesion molecules and phagocytic activity.

Abdominal sepsis was produced by cecal ligation and puncture (CLP) in rats to observe neutrophil (PMN) migration into the liver and assess the functional alteration in circulating PMNs, liver sequestered PMNs, and Kupffer cells. 7 h following CLP, rats demonstrated severe leukopenia and major amount of PMNs sequestered into the liver (17.0 ± 5.5 x 106 vs. 3.1 ± 1.6 x 106 in sham-operated rats, p < .01). Light microscopic evidence demonstrated the presence of such PMNs in the sinusoids and in the liver parenchyma. By 20 h following CLP, the number of PMNs in the liver was not different from sham controls. CD11b/c expression on circulating PMNs was significantly upregulated from 1.6 ± .3 to 7.8 ± .9 mean channel fluorescence (MCF) in 7 h CLP rats. Liver-sequestered PMNs showed further enhancement of CD11b/c expression to 10.4 ± 1.9 MCF than the circulating PMNs. However, in the late septic rats, CD 11b/c expression on circulating PMNs 2.9 ± .5 MCF returned to the control level of 1.9 ± 7 MCF. Liver-sequestered PMNs and Kupffer cells in septic rats exhibited remarkably enhanced phagocytic activities 53.0 ± 10.8 and 56.9 ± 7.7% phagocytosis, respectively, regardless of the suppression of phagocytosis in circulating PMNs (16.0 ± 5% phagocytosis). In 7 h CLP rats, liver-sequestered PMNs exhibited a significantly higher level of superoxide anion generation 21.8 ± 12.2 nmol/30 min/106 cells than did Kupffer cells (4.2 ± 3.0 nmol/30 min/106 cells). These results demonstrate that the liver is a target organ for neutrophil sequestration during the septic response. Early hepatic recruitment of PMNs may be important in host defense; however, these PMNs are also likely to be mediators for liver injury during sepsis.

Time course of changes in gluconeogenesis from various precursors in chronically endotoxemic rats

Rates of gluconeogenesis (GNG) from lactate and triosephosphate precursors were measured in hepatocytes isolated from rats that have received endotoxin or physiological saline by continuous infusion from an implanted pump. Six hours after the onset of infusion (day 2 postsurgery) GNG from lactate was significantly elevated in hepatocytes of endotoxemic (ET) animals. By 24 hours later, the gluconeogenic rate was depressed, compared to cells of NaCl-infused controls. However, providing ET cells with lactate at concentrations found in the in vivo milieu resulted in glucose production at rates not different from those of control cells incubated at their respective in vivo (lower) substrate levels. On day 2 postsurgery, ET rats were hyperglycemic and hyperlactacidemic; on day 3 the elevated blood lactate concentration was maintained, but the plasma glucose values were not different from those of NaCl controls. The glucagon-induced increment in glucose synthesis was depressed in cells of ET rats both on day 2 and day 3 postsurgery, although the total amount of glucose released was significantly less only on day 3. The pattern of norepinephrine stimulation was similar to that of glucagon, except for the increase above the basal rate of GNG on day 3 being the same for control and ET cells. GNG was also assessed from oxidized substrates (fructose (F) and dihydroxyacetone (DHA] and reduced substrates (sorbitol and glycerol) entering the pathway at the triosephosphate level. On day 2 both cell populations produced glucose from each of the four precursors at comparable basal rates.(ABSTRACT TRUNCATED AT 250 WORDS)

Functional characterization of peripheral circulating and liver recruited neutrophils in endotoxic rats

Neutrophil accumulation in tissues is a hallmark of inflammation and is associated with a variety of pathological conditions. In bacterial infection neutrophils are selectively attracted in large numbers to phagocytose and kill invading microorganisms. However, activated neutrophils can also cause injury to tissues. To investigate functional alterations in liver recruited neutrophils (PMNs), we studied the functional characteristics of circulating blood and liver sequestered PMNs in terms of host defense mechanisms, such as nitric oxide (NO) and superoxide (SO) generation, β 2 integrin expression, phagocytosis, and eicosanoid profile. Cells were isolated from rats infused with a nonlethal dose (320 μg/kg) of E. coli endotoxin (ET) or pyrogen‐free saline for 90 min. Liver PMNs produced significantly more NO both in the absence and in the presence of an in vitro endotoxin challenge than did blood PMNs. No significant difference was observed in phorbol myristate acetate‐stimulated SO generation. Endotoxin infusion significantly up‐regulated the expression of CD11b/c in circulating and even more so in liver PMNs. Phagocytosis was significantly enhanced by in vivo ET treatment in blood PMNs, and liver PMNs showed even greater phagocytic activity than blood PMNs or Kupffer cells. The percent distribution of prostaglandins D2 and E2 of total 14C‐eicosanoids was significantly higher and that of thromboxane B2 and 5‐, 12‐, and 15‐HETEs was significantly lower in liver than in blood PMNs. Our study demonstrates several functional differences between liver‐recruited and circulating PMNs in an acute endotoxic model. The implications of altered neutrophil function may extend to mechanisms of host defense and hepatotoxicity associated with sepsis and endotoxemia. J. Leukoc. Biol. 56: 166–173; 1994.

Glucose kinetics and development of endotoxin tolerance during long-term continuous endotoxin infusion

Alterations in glucose metabolism are seen following the acute administration of lethal doses (LD) and nonlethal doses of endotoxin, but relatively little information is available concerning glucose kinetics during long-term continuous endotoxin infusion. A nonlethal dose of endotoxin was administered intravenously to catheterized rats for up to 54 hours via a subcutaneously implanted osmotic pump; time-matched control animals were saline-infused. Glucose kinetics were assessed in vivo after 6, 30, and 54 hours of endotoxin by the constant infusion of [6-3H, U-14C]-glucose. The endotoxemic animals were hemodynamically stable throughout the experimental protocol and exhibited a febrile response at six and 30 hours of endotoxin infusion. Elevations in glucose turnover (50% and 42%) and recycling (140% to 150%) were seen after six and 30 hours of endotoxin infusion, but had returned to control values by 54 hours. A major portion (53% to 62%) of the increased glucose turnover in endotoxemic rats was accounted for by the elevated rate of recycling. The increased turnover appeared to be almost entirely due to enhanced gluconeogenesis and is consistent with the 160% to 170% elevation in plasma glucagon and increased lactate availability. After 54 hours, the plasma concentrations of glucose and lactate, glucose kinetics, and body temperature were not different between endotoxemic and control animals. In separate groups of rats, a bolus injection of endotoxin (LD 100) was administered to determine the presence of endotoxin tolerance. Endotoxin-infused animals showed improved survival after 30 and 54 hours (LD 20 and LD 0).(ABSTRACT TRUNCATED AT 250 WORDS)

MODULATION OF THE LUNG HOST RESPONSE BY GRANULOCYTE COLONY-STIMULATING FACTOR IN RATS CHALLENGED WITH INTRAPULMONARY ENDOTOXIN

The effects of granulocyte colony-stimulating factor (G-CSF) on the functional activities of circulating and lung-recruited neutrophils (PMNs) and alveolar macrophages (AMs) were studied in rats to further elucidate the mechanisms underlying G-CSF-enhanced pulmonary host defense. Animals received G-CSF or vehicle twice a day for 2 days, followed by an intratracheal challenge with endotoxin or saline. G-CSF up-regulated CD11b/c expression and mean channel fluorescence intensity of phagocytosis in circulating PMNs. G-CSF also enhanced phagocytic activities, reflected by both the percentage of phagocytosis and mean channel fluorescence intensity in lung-recruited PMNs and AMs in intratracheal endotoxin-challenged rats. The endotoxin-induced increase in pulmonary production of tumor necrosis factor-α and cytokine-induced neutrophil chemoattractant was not affected by G-CSF pretreatment. These data demonstrate that G-CSF-enhanced pulmonary recruitment of PMNs is primarily based on the effects of G-CSF on the PMNs themselves, rather than the generation of certain chemotactic stimuli, i.e., cytokine-induced neutrophil chemoattractant and tumor necrosis factor-α. The enhanced phagocytic activities of lung-recruited PMNs and AMs also augment pulmonary host defenses in G-CSF-pretreated animals.

Lipoprotein lipase activity in rat heart myocytes

Abstract Previous indirect evidence suggested that lipoprotein lipase originated in the parenchymal cells of the heart. In the present study extracts of heart myocytes isolated from maturing rats hydrolyzed triglycerides. The enzyme activity was inhibited by NaCl and protamine sulfate. The data provide direct evidence for the presence of lipoprotein lipase in heart cells.

Glucose and lactate kinetics in septic rats

Glucose turnover was measured in rats subjected to experimental peritonitis. Fifteen hours following cecal ligation and puncture or sham surgery, rats were anesthetized with pentobarbital and glucose metabolism was estimated by constant infusion tracer techniques. Septic rats demonstrated significantly elevated rates of glucose appearance without any elevation of plasma glucose concentration. There was also significant hyperlactacidemia in the cecally ligated rats. The elevated plasma lactate was not accompanied by a lowering of blood pH. Arterial blood pressure were not different between groups, however the blood pressure of septic rats did fall during the infusion experiment. Septic rats demonstrated significantly elevated initial heart rates and blood hematocrit. The turnover of lactate was also elevated in the septic rats. By calculation of the relative percentage of glucose production from labeled lactate, it is obvious that septic rats had elevated rates of gluconeogenesis. Determination of plasma immunoreactive insulin indicated that septic rats were not insulin resistant.

Eicosanoid production in nonparenchymal liver cells isolated from rats infused with E. coli endotoxin.

Continuous i.v. infusion of a nonlethal dose of Escherichia coll endotoxin induced an early (3‐h) accumulation of neutrophils in the rat liver followed by a later (30‐h) greater extravasation of mononuclear phagocytes (MNP). These inflammatory cells, recovered together by centrifugal elutriation, were analyzed for their potential capacity to metabolize [1‐14C]‐AA. Ca2+ ionophore A23187 (5 μM) stimulated the release of [1‐14C]‐AA from PC and PI both in cells from saline‐ and ET‐infused rats, the latter showing a higher capacity to further metabolize AA to eicosanoids. LTB4 and 5‐HETE were the major metabolites accumulated in cells from rats infused with ET for 3 h, while PGD2 played the main role in cells from saline‐infused rats. This could reflect [1‐14C]‐AA metabolism by PMNP and Kupffer cells, respectively. By 30 h of ET‐infusion, a shift from PGD2 to PGE2 release was observed. These results suggest that eicosanoids released by nonparenchymal cells (i.e., Kupffer and endothelial cells) and PMNP in the liver of ET‐infused rats may alter the normal intercellular information flow between parenchymal and nonparenchymal cells, contributing to the severe impairment in liver function and metabolism during endotoxicosis and sepsis.