Chikara Ueno

Mechanism of the inhibitory effect of protease inhibitor on tumor necrosis factor α production of monocytes

If the inflammatory response becomes excessive or uncontrolled by some stimuli, inappropriate inflammatory responses occur. Monocytes are extremely important cells for regulating the cytokine network and tumor necrosis factor alpha (TNFalpha) and interleukin- (IL) 10, which are mainly synthesized by monocytes, are representative cytokines that play a central role in the cytokine network. Protease inhibitors such as gabexate mesilate (GM) and ulinastatin (UTI) have been shown to have various beneficial effects by inhibiting the activation of leukocytes, but the mechanism for this has yet to be fully elucidated. In this study we investigated the mechanism of the inhibitory effect of protease inhibitors on the proinflammatory cytokine production of lipopolysaccharide- (LPS) stimulated monocytes. LPS-stimulated monocytes were treated with GM or UTI. The value of TNFalpha and IL-10 in the culture medium of monocytes was measured and each mRNA expression was assayed. The inhibitory effect of protease inhibitors on the activity of intracellular signal transduction pathways such as protein kinase C (PKC) and nuclear factor kappa B (NFkappaB) were also evaluated. GM decreased the TNFalpha production of LPS-stimulated monocytes as shown by the inhibition of mRNA expression and increased the IL-10 production of LPS-stimulated monocytes. GM also suppressed the NFkappaB activity of LPS-stimulated monocytes. UTI decreased the TNFalpha production of LPS-stimulated monocytes, but did not inhibit the TNFalpha mRNA expression. The present study shows that the inhibitory effect of GM on the TNFalpha production of activated human monocytes is mediated by the suppression of NFkappaB activation, while the mechanism of UTI inhibiting TNFalpha production of human monocytes may be due to the inhibition of either the translation or secretion of TNFalpha.

Severe sepsis induces deficient interferon-gamma and interleukin-12 production, but interleukin-12 therapy improves survival in peritonitis

BACKGROUND After severe sepsis, there is an increase of Th2 cytokine and a decrease in Th1 cytokine that may account for impaired cellular immunity. The aim of this study is to evaluate the Th1, Th2 cytokine balance in the serum, peritoneal lavage fluid (PLF) and liver mononuclear cells (MNC) of experimental peritonitis mice, and determine the effect of interleukin-12 (IL-12), a cytokine stimulating Th1 cytokine production, when administered to septic mice. METHODS Experimental bacterial peritonitis mice was induced by cecal ligation and puncture (21-gauge needle, mild peritonitis) or cut (5 mm, severe peritonitis). Serum and PLF levels and liver MNC production of interferon (IFN)-gamma, IL-10, and IL-12 were measured after the procedure. Mild and severe peritonitis mice were treated intraperitoneally with recombinant IL-12 (r-IL-12) either 6 hours before or 6 and 24 hours after the procedure. The survival rates were then compared with nontreated mice. RESULTS Serum and PLF IFN-gamma, IL-12 levels in severe peritonitis mice were significantly lower than those in mild peritonitis mice at 6 and 12 hours after the procedure. On the other hand, serum and PLF IL-10 levels in severe peritonitis mice were significantly higher than those in mild peritonitis mice at 6 hours after the procedure. Furthermore, liver MNC IFN-gamma production in severe peritonitis mice was significantly higher than that in mild peritonitis mice at 6 hours after the procedure, but liver MNC IL-12 production in severe peritonitis mice was significantly lower than that in mild peritonitis mice at 12 hours after the procedure. Severe peritonitis mice treated with r-IL-12 at 6 hours before the procedure improved survival rate, and mild peritonitis mice treated with r-IL-12 at 24 hours after the procedure showed significantly improved survival rates. CONCLUSIONS Change in the Th1, Th2 cytokine balance in peritonitis mice might induce a shift toward a Th2 dominant phenotype according to the severity of peritonitis, and the capacity to produce IFN-gamma and IL-12 by liver MNC is reduced. Therapies designed to augment the production of Th1 cytokines, such as IL-12, may thus prove to be beneficial in the treatment of severe sepsis after peritonitis.

Increased Monocyte Activation in Elderly Patients after Surgical Stress

Objective: To investigate age-related changes in the host response to surgical stress. The clinical course, serum interleukin-6 (IL-6) levels, monocyte production of tumor necrosis factor-α (TNF-α), and monocyte expression of CD11b/CD18 were used as markers of the systemic response. Methods: Patients with gastric cancer, undergoing distal gastrectomy were divided into 2 groups: >75 years of age (elderly group) and ≤75 years of age (young group). Serum IL-6 levels, TNF-α production and CD11b/CD18 expression by monocytes, and the postoperative clinical course were compared between the 2 groups. Results: TNF-α production by lipopolysaccharide-stimulated monocytes and CD11b/CD18 expression on monocytes after surgical stress were significantly higher in the elderly than in the young group. Moreover, serum IL-6 levels on the first postoperative day in the elderly group were significantly higher than those in the young group. The incidence and duration of systemic inflammatory response syndrome (SIRS) were significantly greater in the elderly than in the young group. Conclusions: The activation of monocytes and hypercytokinemia occur readily after surgical stress in the elderly and may therefore contribute to SIRS and increased susceptibility to postoperative complications.

Effects of L-Arginine Infusion During Ischemia on Gut Blood Perfusion, Oxygen Tension, and Circulating Myeloid Cell Activation in a Murine Gut Ischemia/Reperfusion Model

BACKGROUND Gut hypoperfusion is considered to be a mechanism for early multiple-organ failure after severe surgical insults. L-Arginine (ARG) may preserve gut microcirculation as a substrate of nitric oxide synthase, but simultaneously may enhance immune cell response. It remains unknown if ARG infusion during gut ischemia improves the outcome after gut ischemia-reperfusion (I/R). METHODS Male Institute of Cancer Research mice were randomized to control and ARG groups. After i.v. cannulation, mice underwent 90 (Exp. 1) or 60 (Exp. 2 and 3) minutes of gut I/R. Control mice received normal saline infusion at 1 mL/h for 60 minutes during ischemia, whereas the ARG group was given 1% ARG hydrochloride solution. In Exp. 1, survival was observed for 72 hours (n = 35). In Exp. 2, blood perfusion and oxygen tension of the small intestine were measured (n = 9). In Exp. 3, peripheral blood was obtained at 2 or 4 hours after reperfusion (n = 22). Reactive oxygen intermediate (ROI) production by myeloid cells with or without phorbol myristate acetate (PMA) stimulation and expression of CD11a and CD11b on myeloid cells were examined using flow cytometry. RESULTS Exp. 1: There was no significant difference in survival times (log rank test, p = .2). However, survival rates at 12 hours were 72% (13/18) for the control group and 35% (6/17) for the ARG group (p < .05 Fisher). Exp. 2: ARG infusion significantly improved gut blood perfusion ratio during ischemia but had no effect on oxygen tension. Exp. 3: In the ARG group, ROI production with PMA and CD11b expression at 4 hours were higher than those at 2 hours, whereas there were no significant changes in the control mice. CONCLUSIONS ARG infusion improves intestinal blood perfusion during ischemia but primes and activates circulating myeloid cells excessively. Consequently, i.v. infusion of ARG during ischemia reduces survival rate.

Glutamine infusion during ischemia is detrimental in a murine gut ischemia/reperfusion model

BACKGROUND Gut ischemia/reperfusion (I/R) frequently occurs in clinical settings as a result of disproportionate splanchnic hypoperfusion during shock. Glutamine (GLN) supplementation of total parenteral nutrition (TPN) before gut I/R improves survival after gut I/R compared with standard TPN. However, it is unknown whether GLN treatment after the occurrence of the insult is beneficial or not. The aims of this study were to examine effects of GLN infusion during gut ischemia on survival, myeloid cell (neutrophils + monocytes) activation, and vascular permeability in organs. METHODS Male Institute of Cancer Research (ICR) mice were randomized to control and GLN groups. After IV cannulation, mice underwent 90 (experiments 1 and 2) or 60 (experiment 3) minutes of gut I/R. Control mice received normal saline infusion at 1 mL/h for 60 minutes during ischemia, whereas the GLN group was given 3% GLN solution. In experiment 1, survival rates were monitored for 72 hours (n = 25). In experiment 2, peripheral blood was obtained at 2 or 4 hours after reperfusion (n = 17). Reactive oxygen intermediate (ROI) production by myeloid cells was determined by flow cytometry using dihydrorhodamine 123 with or without phorbol myristate acetate stimulation. Expression of CD11a and CD11b on myeloid cells was also measured. Myeloperoxidase (MPO) activity in the lung was evaluated. In experiment 3, vascular permeability in organs was measured using Evans blue at 2 or 4 hours. RESULTS In experiment 1, survival time in the GLN group was significantly reduced compared with the control group (p = .02, log-rank test). The survival rates were 92% (12/13) and 42% (5/12) for the control and GLN groups at 12 hours (p = .01) and 38% (5/13) and 0% (0/12) at 48 hours (p = .02), respectively. In experiment 2, ROI production was significantly higher in the GLN group than in the control group after PMA stimulation both at 2 and 4 hours. CD11b expression was significantly higher in the GLN group than in the control group at 4 hours. There was no difference in pulmonary MPO activity at either time point. In experiment 3, GLN infusion significantly increased hepatic vascular permeability compared with saline infusion at 4 hours. CONCLUSIONS GLN infusion during ischemia is detrimental for survival after gut I/R. A possible mechanism is excessive priming of myeloid cells caused by GLN infusion. Timing of GLN administration is critical for outcome after gut ischemic insult.

Intestinal polymeric immunoglobulin receptor is affected by type and route of nutrition.

BACKGROUND Secretory immunoglobulin A (SIgA) prevents adherence of pathogens at mucosal surfaces to prevent invasive infection. Polymeric immunoglobulin receptor (pIgR) is located on the basolateral surface of epithelial cells and binds dimeric immunoglobulin A (IgA) produced by plasma cells in the lamina propria. This IgA-pIgR complex is transported apically, where IgA is exocytosed as SIgA to the mucosal surface. Our prior work shows that mice fed intragastric (IG, an elemental diet model) and IV parenteral nutrition (PN) solution have reduced intestinal T and B cells, SIgA, and interleukin-4 (IL-4) compared with mice fed chow or a complex enteral diet (CED). Prior work also demonstrates a reduction in IgA transport to mucosal surfaces in IV PN-fed mice. Because IL-4 up-regulates pIgR production, this work studies the effects of these diets on intestinal pIgR. METHODS Male Institute of Cancer Research (ICR) mice were randomized to chow (n = 11) with IV catheter, CED (n = 10) or IG PN (n = 11) via gastrostomy and IV PN (n = 12) for 5 days. CED and PN were isocaloric and isonitrogenous. Small intestine was harvested for pIgR and IL-4 assays after mucosal washing for IgA. IgA and IL-4 levels were analyzed by enzyme-linked immunosorbent assay and pIgR by Western blot. RESULTS Small intestinal pIgR expression, IgA levels, and IL-4 levels decreased significantly in IV PN and IG PN groups. CONCLUSIONS Lack of enteral stimulation affects multiple mechanisms responsible for decreased intestinal SIgA levels, including reduced T and B cells in the lamina propria, reduced Th-2 IgA-stimulating cytokines, and impaired expression of the IgA transport protein, pIgR.

T Lymphocyte Numbers in Human Gut Associated Lymphoid Tissue Are Reduced Without Enteral Nutrition

BACKGROUND Clinically, in the absence of enteral nutrition, the morbidity of infectious complication is high. Although experiments using mice have shown alterations in gut-associated lymphoid tissue (GALT) to be an important mechanism underlying impaired host defense, there are no clinical studies on the effects of nutritional routes on GALT. METHODS A total of 27 colon cancer cases who underwent right colectomy or hemicolectomy were reviewed. Six patients did not receive enteral nutrition for 4 to 28 days before surgery because of bowel obstruction (parenteral nutrition [PNI group). Twenty-one patients were enterally fed before surgery (enteral nutrition [EN] group). The terminal ileum from resected specimens was examined microscopically. T-cell numbers in intraepithelial spaces (IE) and the lamina propria (LP) were determined immunohistochemically in blinded fashion. RESULTS There were no significant differences in baseline characteristics between the 2 groups. T-cell number in the LP was significantly lower in the PN group than in the EN group, with no difference in IE cell numbers. CONCLUSIONS Lack of enteral delivery of nutrients reduces GALT cell number in patients with colon cancer, as is the case in mice.

Nutritional route affects ERK phosphorylation and cytokine production in hepatic mononuclear cells.

Objective:To clarify the influence of nutritional route on hepatic immunity in a murine model. Summary Background Data:Parenteral nutrition is disadvantageous for preventing infectious complications in critically ill and/or severely injured patients as compared with enteral nutrition. To date, lack of enteral nutrition has been demonstrated to impair mucosal immunity, gut barrier function, and the peritoneal defense system. However, influences of nutritional route on hepatic immunity, another important defense system against infection, have not been well studied. Methods:Male ICR mice were randomized to 3 groups: ad libitum chow (chow), intravenous (IV)-TPN and intragastric (IG)-TPN groups. The TPN groups were given isocaloric and isonitrogenous TPN solutions. After the mice had been fed for 5 days, hepatic mononuclear cells (MNCs) were isolated. Hepatic MNC numbers and functions (cytokine production, intracellular signaling, and LPS receptor expression) were determined. Moreover, 1.0 × 107 Pseudomonas aeruginosa were delivered by intraportal injection. Survival and histology were examined. Results:Hepatic MNC numbers were significantly lower in the IV-TPN group than in the chow and IG-TPN groups, without subpopulation changes. As compared with enterally fed mice, cytokine production (TNF-α, IFN-γ, and IL-10) by hepatic MNCs in response to LPS was impaired in parenterally fed mice in association with blunted phosphorylation of ERK1/2, a MAPK. Hepatic MNCs from IV-TPN mice showed decreased expressions of CD14 and TLR4/MD2, as compared with enterally fed mice. Survival times were reduced in the IV-TPN group as compared with the chow and IG-TPN groups. Conclusion:Preservation of hepatic immunity with enteral feeding is important for prevention of infectious complications in severely injured and/or critically ill patients.

Reversal of parenteral nutrition-induced gut mucosal immunity impairment with small amounts of a complex enteral diet.

BACKGROUND Although parenteral nutrition (PN) prevents progressive malnutrition, lack of enteral nutrition (EN) during PN leads to gut associated lymphoid tissue (GALT) atrophy and dysfunction. Administering a small amount of EN with PN reportedly prevents increases in intestinal permeability. However, its effects on GALT remain unclear. We analyzed the minimum amount of EN required to preserve gut immunity during PN. METHODS Male Institute of Cancer Research mice underwent jugular vein catheter insertion and tube gastrostomy. They were randomized into four groups to receive isocaloric and isonitrogenous nutritional support with variable EN to PN ratios (EN 0, EN 33, EN 66, and EN 100). EN was provided with a complex enteral diet. After 5 days of feeding, the mice were killed and whole small intestines were harvested. GALT lymphocytes were isolated and counted. Their phenotypes were analyzed by flow cytometry. IgA levels of small intestinal washings were analyzed with enzyme-linked immunosorbent assay. RESULTS Body weight changes did not differ between any two of the groups. Peyers patch lymphocyte numbers increased in proportion to EN amount, whereas lamina propria lymphocyte numbers were significantly higher in the EN 100 than in the EN 0 group, with no marked increases in the EN 33 and EN 66 groups. Small intestinal IgA levels increased EN amount-dependently and reached a plateau at EN 66. CONCLUSIONS A small amount of EN partially reverses PN-induced GALT changes, suggesting beneficial but limited effects on gut mucosal immunity.

Arginine-Enriched Total Parenteral Nutrition Improves Survival in Peritonitis by Normalizing NFκB Activation in Peritoneal Resident and Exudative Leukocytes

Background:Enteral nutrition maintains peritoneal defense more effectively than parenteral nutrition, at least partly by preserving NF&kgr;B activation in peritoneal cells. We hypothesized that arginine (ARG)-enriched parenteral nutrition would normalize NF&kgr;B activation in peritoneal leukocytes, thereby improving the survival of peritonitis models. Methods:A total of 105 ICR mice were randomized to chow (n = 33), IV feeding of a standard (STD) total parenteral nutrition (STD-TPN) solution (ARG 0.3%) (n = 35), or 1% ARG-TPN solution (n = 37), and fed accordingly for 5 days.Experiment 1: Thirty mice were used for intranuclear NF&kgr;B measurement in peritoneal resident cells (PRCs). After incubation with lipopolysaccharide (LPS: 0, 1, 10 &mgr;g/mL) for 30 minutes, intranuclear NF&kgr;B activity was examined by laser scanning cytometry.Experiment 2: Fifty-one mice were injected with 2 mL of 1% glycogen intraperitoneally. Peritoneal exudative cells (PECs) were obtained at 2 or 4 hours after glycogen administration for NF&kgr;B measurement. Cytokine (TNF&agr;, IL-10) levels in peritoneal lavage fluid were also determined by ELISA.Experiment 3: After 5 days of feeding, 24 mice underwent cecal ligation and puncture. Survival was observed up to 5 days. Results:Experiment 1: Intranuclear NF&kgr;B levels in the ARG-TPN and chow groups increased dose-dependently after LPS stimulation, while the level in the STD-TPN group was unchanged.Experiment 2: Intranuclear NF&kgr;B level was significantly higher at 2 hours in the chow than in the STD-TPN group, whereas in the ARG-TPN mice the level was midway between those of the chow and STD-TPN groups. TNF&agr; and IL-10 levels of the chow group were significantly higher than those of STD-TPN mice at 2 hours. TNF&agr; was significantly higher in the ARG-TPN group than in the STD-TPN group, but the IL-10 level showed no recovery.Experiment 3: Survival times were significantly reduced in the STD-TPN as compared with the chow group, though ARG-TPN improved survival. Conclusion:ARG-enriched TPN is a surrogate for enteral feeding which maintains peritoneal defense by preserving NF&kgr;B activation in peritoneal resident and exudative leukocytes.