Mechanical Ventilation in Healthy Mice Induces Reversible Pulmonary and Systemic Cytokine Elevation with Preserved Alveolar Integrity: An In Vivo Model Using Clinical Relevant Ventilation Settings

Background: Mechanical ventilation (MV) may activate the innate immune system, causing the release of cytokines. The resulting proinflammatory state is a risk factor for ventilator-induced lung injury. Cytokine increase results from direct cellular injury but may also result from cyclic stretch alone as demonstrated in vitro: mechanotransduction. To study mechanotransduction in vivo, the authors used an animal MV model with clinically relevant ventilator settings, avoiding alveolar damage.

Methods: Healthy C57BL6 mice (n = 82) were ventilated (tidal volume, 8 ml/kg; positive end-expiratory pressure, 4 cm H2O; fraction of inspired oxygen, 0.4) for 30, 60, 120, and 240 min. Assigned animals were allowed to recover for 2 days after MV. Both pulmonary tissue and plasma interleukin (IL)-1[alpha], IL-1[beta], tumor necrosis factor [alpha], IL-6, IL-10, and keratinocyte-derived chemokine levels were measured. Histopathologic appearance of lung tissue was analyzed by light microscopy and electron microscopy.

Results: In lung tissue, all measured cytokines and keratinocyte-derived chemokine levels increased progressively with MV duration. Light microscopy showed increased leukocyte influx but no signs of alveolar leakage or albumin deposition. Electron microscopy revealed intact epithelial cell and basement membranes with sporadically minimal signs of partial endothelial detachment. In plasma, increased levels of IL-1[alpha], tumor necrosis factor [alpha], IL-6, and keratinocyte-derived chemokine were measured after MV. In the recovery animals, cytokine levels had normalized and no histologic alterations could be found.     

Mechanical ventilation in healthy mice induces reversible pulmonary and systemic cytokine elevation with preserved alveolar integrity: an in vivo model using clinical relevant ventilation settings

BACKGROUND: Mechanical ventilation (MV) may activate the innate immune system, causing the release of cytokines. The resulting proinflammatory state is a risk factor for ventilator-induced lung injury. Cytokine increase results from direct cellular injury but may also result from cyclic stretch alone as demonstrated in vitro: mechanotransduction. To study mechanotransduction in vivo, the authors used an animal MV model with clinically relevant ventilator settings, avoiding alveolar damage. METHODS: Healthy C57BL6 mice (n = 82) were ventilated (tidal volume, 8 ml/kg; positive end-expiratory pressure, 4 cm H2O; fraction of inspired oxygen, 0.4) for 30, 60, 120, and 240 min. Assigned animals were allowed to recover for 2 days after MV. Both pulmonary tissue and plasma interleukin (IL)-1alpha, IL-1beta, tumor necrosis factor alpha, IL-6, IL-10, and keratinocyte-derived chemokine levels were measured. Histopathologic appearance of lung tissue was analyzed by light microscopy and electron microscopy. RESULTS: In lung tissue, all measured cytokines and keratinocyte-derived chemokine levels increased progressively with MV duration. Light microscopy showed increased leukocyte influx but no signs of alveolar leakage or albumin deposition. Electron microscopy revealed intact epithelial cell and basement membranes with sporadically minimal signs of partial endothelial detachment. In plasma, increased levels of IL-1alpha, tumor necrosis factor alpha, IL-6, and keratinocyte-derived chemokine were measured after MV. In the recovery animals, cytokine levels had normalized and no histologic alterations could be found. CONCLUSIONS: Mechanical ventilation induces reversible cytokine increase and leukocyte influx with preserved tissue integrity. This model offers opportunities to study the pathophysiologic mechanisms behind ventilator-induced lung injury and the contribution of MV to the "multiple-hit" concept.     

Absence of toll-like receptor 4 (TLR4) signaling in the donor organ reduces ischemia and reperfusion injury in a murine liver transplantation model.

This study analyzes how toll-like receptor 4 (TLR4) signaling in the donor organ affects the ischemia and reperfusion injury (IRI) sequel following liver transplantation. Isogenic orthotopic liver transplantations (OLTs) with rearterialization were performed in groups of wild-type (WT) and TLR4 knockout (KO) mice after donor liver preservation in University of Wisconsin solution at 4 degrees C for 24 hours. Unlike WT OLTs, TLR4-deficient OLTs transplanted to either WT or TLR4 KO recipients suffered significantly less hepatocellular damage, as evidenced by serum alanine aminotransferase levels, and histological Suzuki's grading of liver IRI. Disruption of TLR4 signaling in OLTs decreased local neutrophil sequestration, CD4+ T cell infiltration, interferon (IFN)-gamma-inducible protein 10 (CXCL10) and an intercellular adhesion molecule (ICAM-1), as well as tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta, IL-2, and IFN-gamma, yet increased IL-4 and IL-10 expression. The well-functioning OLTs from TLR4 KO donors revealed attenuated activity of capase-3, and enhanced heme oygenase-1 (HO-1) expression, along with decreased levels of apoptotic endothelial cells/hepatocytes, as compared with WT OLTs with intact TLR4 signaling. Thus, the functional sentinel TLR4 complex in the donor organ plays a key role in the mechanism of hepatic IRI after OLT. Disruption of TLR4 pathway downregulated the early proinflammatory responses and ameliorated hepatic IRI. These results provide the rationale to locally modify innate TLR4 signaling in the donor organ to more efficiently control the adaptive posttransplantation IRI-dependent responses.     

Toll-Like Receptor and Heme Oxygenase-1 Signaling in Hepatic Ischemia/Reperfusion Injury

Ischemia/reperfusion injury (IRI) represents the major problem in clinical liver transplantation. We have shown that toll-like receptor 4 (TLR4) signaling is specifically required in initiating antigen-independent IRI leading to liver inflammation, whereas local induction of anti-oxidant heme oxygenase-1 (HO-1) is cytoprotective. This study analyzes in vivo interactions between HO-1 and sentinel TLR system in the pathophysiology of liver IRI. Using a 90-min lobar warm ischemia model, wild type (WT), TLR4 KO/mutant and TLR2 KO mice were first assessed for the severity of hepatocellular damage at 6 h postreperfusion. Unlike in WT or TLR2-deficient mice, disruption/absence of TLR4 pathway reduced IRI, as manifested by liver function (serum alanine aminotransferase levels), histology (Suzuki's scores), neutrophil infiltration (myeloperoxidase activity) and local/systemic TNF-alpha production (mRNA/protein levels). Moreover, defective TLR4 but not TLR2 signaling increased mRNA/protein HO-1 expression. In contrast, tin protoporphyrin-mediated HO-1 inhibition restored hepatic damage in otherwise IRI-resistant TLR4 mutant/KO mice. CoPP-induced HO-1 overexpression ameliorated hepatic damage in IRI-susceptible TLR2 KO mice, comparable with WT controls, and concomitantly diminished TLR4 levels. In conclusion, this study highlights the importance of cross talk between HO-1 and TLR system in the mechanism of hepatic IRI. Hepatic IRI represents a case for innate immunity in which HO-1 modulates proinflammatory responses that are triggered via TLR4 signaling, a putative HO-1 repressor.     

Mechanical Ventilation with Lower Tidal Volumes and Positive End-expiratory Pressure Prevents Pulmonary Inflammation in Patients without Preexisting Lung Injury

Background: Mechanical ventilation with high tidal volumes aggravates lung injury in patients with acute lung injury or acute respiratory distress syndrome. The authors sought to determine the effects of short-term mechanical ventilation on local inflammatory responses in patients without preexisting lung injury.

Methods: Patients scheduled to undergo an elective surgical procedure (lasting >=5 h) were randomly assigned to mechanical ventilation with either higher tidal volumes of 12 ml/kg ideal body weight and no positive end-expiratory pressure (PEEP) or lower tidal volumes of 6 ml/kg and 10 cm H2O PEEP. After induction of anesthesia and 5 h thereafter, bronchoalveolar lavage fluid and/or blood was investigated for polymorphonuclear cell influx, changes in levels of inflammatory markers, and nucleosomes.

Results: Mechanical ventilation with lower tidal volumes and PEEP (n = 21) attenuated the increase of pulmonary levels of interleukin (IL)-8, myeloperoxidase, and elastase as seen with higher tidal volumes and no PEEP (n = 19). Only for myeloperoxidase, a difference was found between the two ventilation strategies after 5 h of mechanical ventilation (P < 0.01). Levels of tumor necrosis factor [alpha], IL-1[alpha], IL-1[beta], IL-6, macrophage inflammatory protein 1[alpha], and macrophage inflammatory protein 1[beta] in the bronchoalveolar lavage fluid were not affected by mechanical ventilation. Plasma levels of IL-6 and IL-8 increased with mechanical ventilation, but there were no differences between the two ventilation groups.     

Interleukin-7 influences osteoclast function in vivo but is not a critical factor in ovariectomy-induced bone loss.

IL-7 is produced by stromal cells in bone marrow and is a major regulator of B and T lymphopoiesis. It is also a direct inhibitor of osteoclastogenesis in vitro. In this study we show that IL-7-deficient mice have increased OC and decreased trabecular bone volume compared with WT mice but mimic WT mice in the amount of trabecular but not cortical bone lost after ovariectomy. INTRODUCTION: Interleukin (IL)-7 is a potent regulator of lymphocyte development, which has significant effects on bone. Bone marrow cell cultures from IL-7 deficient (IL-7KO) mice produced significantly more TRACP(+) osteoclasts (OCs) than did cells from wildtype (WT) mice. A previous study found that treatment of mice with a neutralizing antibody to IL-7 blocked ovariectomy (OVX)-induced bone loss. We examined if differences exist between the bones of WT and IL-7KO mice and if OVX altered bone mass in IL-7KO mice. MATERIALS AND METHODS: Studies were in 2-month-old sham-operated (SHAM) and OVX female mice that were killed 4 weeks after surgery. IL-7KO mice and WT controls were in a C57BL/6 background. Both vertebrae (L(1)) and femora were evaluated by DXA, muCT, and histomorphometry. IL-7KO mice were confirmed as IL-7 deficient by their almost total lack of mature B cells in their bone marrow. RESULTS: There was significantly less trabecular bone volume in the vertebrae of IL-7KO mice than in WT mice. In addition, IL-7KO mice had significantly decreased (p < 0.05) trabecular number (13%) and increased trabecular spacing (15%). OVX decreased vertebral trabecular bone volume (TBV) by 21% (p < 0.05) in WT mice and by 22% (p < 0.05) in IL-7KO mice compared with SHAM. IL-7KO SHAM mice also had significantly less (30%) TBV (TA/TTA) in their femurs, as measured histomorphometrically, than did WT SHAM mice. Femurs from IL-7KO SHAM mice had significantly increased percent OC surface (23%) compared with WT SHAM. As in the vertebrae, OVX significantly decreased femoral TBV in both WT and IL-7KO mice by similar amounts (47% and 48%, respectively, p < 0.05 for both) compared with SHAM. However, OVX decreased cortical bone mass in WT but not in IL-7KO bones. We also examined bone marrow cells from WT and IL-7KO mice. Bone marrow cells from IL-7KO animals showed a significant increase in the number of TRACP(+) osteoclast-like cells (OCLs), which formed in cultures that were stimulated with macrophage-colony stimulating factor (M-CSF) and RANKL (both at 30 ng/ml). However, there was no significant difference in the number of OCLs that formed in B lymphocyte-depleted (B220(-)) bone marrow cell cultures from WT and IL-7KO mice. CONCLUSIONS: IL-7 deficiency in mice caused increased OC number in bone and decreased bone mass. OVX-induced bone loss in IL-7-deficient mice was selective and occurred in trabecular but not cortical bone.     

抑制低氧诱导因子-1α表达对小鼠肠缺血／再灌注所致肺损伤的影响

目的探讨通过低氧诱导因子-1α（hypoxiainduciblefactor-1α,HIF—1α）抑制剂YC-1预先抑制该基因表达对肠缺血,再灌注（ischemia／reperfusion,I／R）致急性肺损伤的影响。方法6周～8周龄健康雄性C57BL／6小鼠36只,采用随机数字表法随机分为3组（每组12只）：假手术组（S组）、I／R组和I／R±YC—I预处理组（YC—I组）。YC-1组于术前10min腹腔注入YC-1（1mg／kg）,采用夹闭C57BL／6小鼠肠系膜前动脉45min后再灌注6h的方法造成肠YR损伤模型,取小鼠肺标本称重后计算肺湿干重比,苏木素-伊红（hematoxylin-eosin,HE）染色后观察肺组织病理学改变,分光光度法测定髓过氧化物酶（myeloperoxidase,MPO）活性、酶联免疫吸附测定法检测肺组织肿瘤坏死因子-α（tumor necrosis factor-α,TNF-α）和白细胞介素（interleukin,IL）-1β表达,反转录-PCR法检测HIF-1α、Toll样受体4（toll—likereceptor4,TLR4）mRNA的表达。结果与I／R组比较,预先抑制HIFqct表达使肺实质水肿及中性粒细胞浸润聚集减少,肺组织病理学损伤减轻,肺湿干重比显著降低（RnOI）,MPO活性下调[（1．88±0．82）u／g],TNF-α[（187±20）ng／L）]、IL一1β[（536±54）ng／L）]、HIF-1α、TLR4mRNA的表达水平下降（P〈0．05）。结论YC-1预处理可使肺组织TLR4mRNA表达下调,抑制肠I／R肺组织中促炎细胞因子的释放,明显减轻小鼠肠I／R后急性肺损伤。     

Deficiency of Smad3 results in enhanced inducible nitric oxide synthase–mediated hypotension in lipopolysaccharide-induced endotoxemia

Background

Smad3 is a principal intracellular mediator of signaling for transforming growth factor β, a cytokine involved in pleiotropic pathophysiological processes including inflammation and immunity. The function of Smad3 in regulating inducible nitric oxide synthase (iNOS) expression and septic shock has not been characterized.

Methods

Smad3^−/−(referred hereafter as KO) and wild-type (WT) mice were injected intraperitoneally with lipopolysaccharide (LPS) to induce the septic hypotension. Mortality, blood pressure, and plasma levels of nitrite were measured. The iNOS messenger RNA and protein levels in lung, kidney, and spleen were also analyzed.

Results

Mice lacking functional Smad3 respond to LPS with greater mortality than their WT littermates. The high mortality of KO mice is accompanied by enhanced hypotension after intraperitoneal injection of LPS. Both KO and WT mice displayed an increase in plasma nitrite during the experimental period; however, LPS administration caused more dramatic changes in KO mice than WT mice. Likewise, the iNOS messenger RNA and protein levels in lung, kidney, and spleen were more strongly increased in KO mice than in WT mice after LPS administration.

Conclusions

Defects in the Smad3 gene may increase susceptibility to the development of septic hypotension because of enhanced iNOS production.     

TNF receptor I mediates chemokine production and neutrophil accumulation in the lung following systemic lipopolysaccharide.

BACKGROUND: Tumor necrosis factor (TNF)-alpha is a critical effector of lipopolysaccharide (LPS)-induced acute lung injury, and its effects are mediated by two structurally related receptors, RI and RII. Cellular adhesion molecules and C-X-C chemokines (Keratinocyte chemoattractant (KC) and macrophage inflammatory protein [MIP]-2) regulate tissue neutrophil polymorphonuclear neutrophil (PMN) accumulation in a multitude of inflammatory states. We hypothesized that TNFRI signaling dictates PMN accumulation in the lung via regulation of chemokine molecule production. Therefore, the purposes of this study were to (1) delineate LPS-induced lung TNF-alpha production and (2) characterize the contribution of both TNF receptors to lung chemokine production and neutrophil influx following systemic LPS. METHODS: Wild-type or TNFRI and TNFRII knockout (KO) mice were injected with vehicle (saline) or LPS (Escherichia coli 0.5 mg/kg intraperitoneally). After 2, 4, 6, or 24 h, lungs were analyzed for TNF-alpha and chemokine (KC and MIP-2) protein expression (enzyme-linked immunosorbent assay) and PMN accumulation (myeloperoxidase assay). RESULTS: There was an increase in total lung TNF-alpha (vehicle, 5.0 +/- 1.2 pg/mg total protein vs LPS, 950 +/- 318; P < 0.05) after LPS. Lung chemokine production and PMN accumulation were also increased compared to vehicle-injected mice. Lung chemokine production and PMN accumulation were significantly lower in TNFRI KO, but not TNFRII KO, mice, despite no difference in TNF-alpha production (TNFRI KO, 925 +/- 301 vs TNFRII KO, 837 +/- 267, P = 0.82). CONCLUSIONS: Acute lung injury following systemic LPS administration is characterized by increased lung (1) TNF-alpha production, (2) C-X-C chemokine production, and (3) neutrophil accumulation. The maximal effect of LPS-induced lung neutrophil accumulation appears to be dependent upon the TNFRI receptor but not the TNFRII receptor. .     

Isoflurane attenuates pulmonary interleukin-1β and systemic tumor necrosis factor-α following mechanical ventilation in healthy mice

Background: Mechanical ventilation (MV) induces an inflammatory response in healthy lungs. The resulting pro-inflammatory state is a risk factor for ventilator-induced lung injury and peripheral organ dysfunction. Isoflurane is known to have protective immunological effects on different organ systems. We tested the hypothesis that the MV-induced inflammatory response in healthy lungs is reduced by isoflurane.
Methods: Healthy C57BL6 mice ( n =34) were mechanically ventilated (tidal volume, 8 ml/kg; positive end-expiratory pressure, 4 cmH₂O; and fraction of inspired oxygen, 0.4) for 4 h under general anesthesia using a mix of ketamine, medetomidine and atropine (KMA). Animals were divided into four groups: (1) Unventilated control group; (2) MV group using KMA anesthesia; (3) MV group using KMA with 0.25 MAC isoflurane; (4) MV group using KMA with 0.75 MAC isoflurane. Cytokine levels were measured in lung homogenate and plasma. Leukocytes were counted in lung tissue.
Results: Lung homogenates: MV increased pro-inflammatory cytokines. In mice receiving KMA+ isoflurane 0.75 MAC, no significant increase in interleukin (IL)-1β was found compared with non-ventilated control mice.
Plasma: MV induced a systemic pro-inflammatory response. In mice anesthetized with KMA+ isoflurane (both 0.25 and 0.75 MAC), no significant increase in tumor necrosis factor (TNF)-α was found compared with non-ventilated control mice.
Conclusions: The present study is the first to show that isoflurane attenuates the pulmonary IL-1β and systemic TNF-α response following MV in healthy mice.     

AMPK-mediated AS160 phosphorylation in skeletal muscle is dependent on AMPK catalytic and regulatory subunits

AMP-activated protein kinase (AMPK) is a heterotrimeric protein that regulates glucose transport mediated by cellular stress or pharmacological agonists such as 5-aminoimidazole-4-carboxamide 1 beta-d-ribonucleoside (AICAR). AS160, a Rab GTPase-activating protein, provides a mechanism linking AMPK signaling to glucose uptake. We show that AICAR increases AMPK, acetyl-CoA carboxylase, and AS160 phosphorylation by insulin-independent mechanisms in isolated skeletal muscle. Recombinant AMPK heterotrimeric complexes (alpha1beta1gamma1 and alpha2beta2gamma1) phosphorylate AS160 in a cell-free assay. In mice deficient in AMPK signaling (alpha2 AMPK knockout [KO], alpha2 AMPK kinase dead [KD], and gamma3 AMPK KO), AICAR effects on AS160 phosphorylation were severely blunted, highlighting that complexes containing alpha2 and gamma3 are necessary for AICAR-stimulated AS160 phosphorylation in intact skeletal muscle. Contraction-mediated AS160 phosphorylation was also impaired in alpha2 AMPK KO and KD but not gamma3 AMPK KO mice. Our results implicate AS160 as a downstream target of AMPK.     

Interleukin-17A plays a pivotal role after partial hepatectomy in mice

Background

Liver regeneration after partial hepatectomy (PH) is regulated by tumor necrosis factor (TNF)-α derived from the Kupffer cell. Furthermore, it was reported from our laboratory that interleukin (IL)-17A enhances the production of TNF-α by the Kupffer cell, suggesting that IL-17A may play a role in liver regeneration.

Objective

The purpose was to determine the role of IL-17A and the spleen in liver regeneration after PH.

Methods

Two mouse models including the wild-type (WT) mice or the IL-17A knockout (KO) mice underwent PH. Animals were killed at the designated time points; liver tissues were harvested for further investigation. Proliferation of hepatocytes was evaluated. Furthermore, the messenger RNA and protein expression of TNF-α and IL-6 were measured in the liver. In another set of experiments, the two animal models underwent splenectomy before PH. In an in vitro study, CD4-positive lymphocytes in the spleen were isolated from mice, and the number of IL-17A–positive cells was investigated.

Results

Liver regeneration was significantly impaired in the KO mice compared with the WT mice. This was associated with suppression of cell proliferation assessed by cell proliferation markers in the KO mice. In the WT mice that underwent splenectomy, liver regeneration was significantly delayed compared with animals without splenectomy. In contrast, splenectomy did not affect liver regeneration in the KO mice. IL-17A–positive lymphocytes increased significantly in the spleen in the WT mice after PH.

Conclusions

These results indicate that IL-17A derived from CD4-positive lymphocytes in the spleen is a key regulator in liver regeneration after PH.     

A pathophysiologic role for T lymphocytes in murine acute cisplatin nephrotoxicity

Recent evidence supports a role for an inflammatory pathogenesis of cisplatin nephrotoxicity, but immune cell-mediated mechanisms in this disease are still largely unknown. The role for T lymphocytes on cisplatin-induced acute kidney injury was examined with C57BL/6 T cell-deficient (nu/nu) mice and CD4- or CD8-deficient mice and their wild-type (WT) littermates. All mice received a single dose of cisplatin at 40 mg/kg (intraperitoneally) and were followed up for 72 h. At 72 h after cisplatin administration, T cell-deficient mice had a marked attenuation in renal dysfunction (serum creatinine 3.2+/-0.5 versus 0.8+/-0.1 mg/dl; P=0.007), kidney tubular injury (scores 1.44+/-0.15 versus 0.22+/-0.08; P<0.0001), and survival. Adoptive transfer of T cells into nu/nu mice followed by cisplatin enhanced renal dysfunction and tubular injury. The increase in renal myeloperoxidase activity after cisplatin administration was blunted in nu/nu mice. Renal TNF-alpha, IL-1beta, and keratinocyte-derived chemokine protein expression was increased in WT mice but not in nu/nu mice after cisplatin administration. T cell levels significantly increased in kidneys of WT mice after cisplatin administration as early as at 1 h, peaked at 12 h, and declined by 24 h. CD4- and, to a lesser degree, CD8-deficient mice were relatively protected from cisplatin-induced mortality and renal dysfunction compared with WT mice. These data demonstrate that T lymphocytes are direct mediators of experimental cisplatin nephrotoxicity. Targeting T lymphocytes could lead to improved ways to administer cisplatin safely to cancer patients.     

小鼠全肝缺血再灌注时肺泡巨噬细胞TLR2的激活与肺损伤的机制

目的：探讨肺泡巨噬细胞Toll样受体2（TLR2）的激活机制及其在肝脏缺血再灌注（HIR）中肺损伤的意义。方法：用野生型小鼠C3h/Heouj和TLR4缺失小鼠C3h/Hej建立HIR动物模型。于再灌注1，6，12h后经支气管肺泡灌洗液获取肺泡巨噬细胞，采用荧光定量PCR方法检测TLR2/4mRNA的表达。同时检测支气管肺泡灌洗液中内毒素及肿瘤坏死因子（TNF）的水平，肺组织湿干重比值，肺组织髓过氧化物酶的浓度，并进行肺组织学评分。结果：C3h/Heouj组HIR缺血再灌后各时点肺泡巨噬细胞TLR2/4mRNA表达升高，TLR2mRNA表达持续升高，TLR4mRNA6h达到最高值。同时C3h/Heouj组HIR后支气管肺泡灌洗液中TNF水平明显升高，肺损伤加重，肺组织湿干重比值持续升高，肺组织髓过氧化物酶持续增加(P<0.05)。C3h/Hej组HIR后TLR2mRNA表达仅轻度升高，且支气管肺泡灌洗液中TNF水平低于C3h/Heouj组(P<0.05)，肺损伤轻于C3h/Heouj组(P<0.05)。结论：HIR可致肺泡巨噬细胞表面TLR4的激活，可上调TLR2的表达，从而可加重HIR时的肺损伤。     

Deficiency of receptor-interacting protein kinase 3 (RIPK3) attenuates inflammation and organ injury in neonatal sepsis

Introduction

Sepsis is the third leading cause of morbidity and mortality in neonates. Sepsis in neonates is characterized as the systemic inflammation owing to infection within the first 28 days after birth. The molecular mechanism causing the exaggerated inflammation phenotype in neonates has not been completely elucidated. Receptor interacting protein kinase 3 (RIPK3) is a protein identified as a mediator in programmed necrosis or necroptosis. We hypothesize that RIPK3 could be responsible for the inflammatory response in neonates and that deficiency in the RIPK3 protein attenuates inflammation and organ injury in neonatal sepsis.

Experimental autoimmune anti-glomerular basement membrane glomerulonephritis: a protective role for IFN-gamma

IL-12 and IFN-gamma play key roles in murine lupus and planted antigen models of glomerulonephritis. However, their roles in renal organ-specific autoimmunity are unknown. To establish the roles of endogenous IFN-gamma and IL-12 in experimental autoimmune anti-glomerular basement membrane (GBM) glomerulonephritis (EAG), EAG was induced in normal C57BL/6 mice (WT), IL-12p40-deficient (IL-12p40-/-) mice, and IFN-gamma-deficient (IFN-gamma-/-) mice by immunization with alpha3-alpha5(IV)NC1 heterodimers. At 13 wk, WT mice developed EAG with linear mouse anti-GBM antibody deposition, histologic injury, proteinuria, and mild tubulointerstitial disease. Compared with WT mice, IL-12p40-/- mice had decreased histologic injury and trends to decreased leukocyte infiltrates. In contrast, 40% (4 of 10) of IFN-gamma-/- mice developed significant crescent formation and focal or diffuse interstitial infiltrates (WT, 0 of 8). Compared with WT and/or IL-12p40-/- mice, IFN-gamma-/- mice developed increased injury: histologic injury, total glomerular cell numbers, leukocytes in glomeruli, and renal expression of P-selectin and intercellular adhesion molecule 1. All groups developed similar serum anti-alpha3-alpha5(IV)NC1 antibodies and glomerular Ig deposition, but IFN-gamma-/- mice had decreased anti-alpha3-alpha5(IV)NC1 IgG2a. Therefore, IFN-gamma-/- mice developed increased cellular reactants despite a potentially less damaging antibody response. Dermal delayed-type hypersensitivity was increased in alpha3-alpha5(IV)NC1 immunized IFN-gamma-/- mice and was suppressed by recombinant murine IFN-gamma. CD4+ cells from draining nodes of immunized IFN-gamma-/- mice showed increased proportions of proliferating CD4+ cells but similar numbers of apoptotic cells. These studies demonstrate that in renal organ-specific autoimmunity, IL-12 is pathogenetic but IFN-gamma is protective. They lend weight to the hypothesis that depending on the context/severity of the nephritogenic immune response IFN-gamma has different effects.     

Inosine exerts a broad range of antiinflammatory effects in a murine model of acute lung injury

OBJECTIVE: To investigate the effects of inosine on the acute lung inflammation induced by lipopolysaccharide (LPS) in vivo and on the activation and cytotoxicity elicited by proinflammatory cytokines on human lung epithelial (A549) cells in vitro. SUMMARY BACKGROUND DATA: Inosine is an endogenous purine recently shown to exert immunomodulatory and antiinflammatory effects. METHODS: Mice challenged with intratracheal LPS (50 microg) were treated after 1, 6, and 12 hours with inosine (200 mg/kg intraperitoneal) or vehicle. After 24 hours, bronchoalveolar lavage fluid was obtained to measure proinflammatory (tumor necrosis factor-alpha [TNF-alpha], interleukin [IL]-1beta, IL-6), and antiinflammatory (IL-10, IL-4) cytokines, chemokines (MIP-1alpha and MIP-2), myeloperoxidase activity and total cell counts, nitric oxide production, and proteins. Lung histology and immunohistochemical detection of 3-nitrotyrosine, a marker of nitrosative stress, were performed in inflated-fixed lungs. In vitro, cell viability and production of the chemokine IL-8 were evaluated in A549 cells stimulated with a mixture of cytokines in the presence or absence of inosine. RESULTS: Inosine downregulated the LPS-induced expression of TNF-alpha, IL-1beta, IL-6 and MIP-2 and tended to reduce MIP-1alpha, whereas it enhanced the production of IL-4. Total leukocyte counts, myeloperoxidase, nitric oxide production, and proteins were all significantly decreased by inosine. The purine also improved lung morphology and suppressed 3-nitrotyrosine staining in the lungs after LPS. Inosine attenuated the cytotoxicity and the expression of IL-8 induced by proinflammatory cytokines in A549 cells. CONCLUSIONS: Inosine largely suppressed LPS-induced lung inflammation in vivo and reduced the toxicity of cytokines in lung cells in vitro. These data support the proposal that inosine might represent a useful adjunct in the therapy of acute respiratory distress syndrome.     

Differences in arterial and mixed venous IL-6 levels: the lungs as a source of cytokine storm in sepsis

BACKGROUND: Several investigators have shown that blood levels of interleukin 6 (IL-6) correlate with the severity of illness in critically ill or injured patients. However, little is known about differential arterial and venous blood levels of the cytokine, especially across the lungs. METHODS: We measured differences in IL-6 levels in pulmonary and systemic arterial blood and then documented the production or elimination of IL-6 by the lungs in 19 patients with severe illness. Prospective data were obtained from multiple, simultaneous systemic arterial (ART) and mixed venous (MV) blood samples that were drawn for IL-6 analysis from systemic arterial and pulmonary artery catheters in 7 patients awaiting vascular operation and in 12 trauma patients being treated in the intensive care unit. RESULTS: A lung disorder was present in 5 patients (pneumonia [n = 1], lung trauma [n = 4]) and absent in the remaining 14 patients. The following data were obtained (mean +/- SD) from the highest MV IL-6 levels (pg/mL) in each patient. In patients with a lung disorder (n = 5) compared with those with no disorder (n = 14), ART IL-6 was 9309 +/- 12,521 versus 134 +/- 128 (P =.010), MV IL-6 was 5516 +/- 7420 versus 137 +/- 129 (P =.011), the absolute difference was 3793 +/- 5271 versus -3 +/- 15 (P =.011), and the percentage difference was 37.4% +/- 29.8% versus 1.5% +/- 12.3% (P =.001). The ART and MV IL-6 levels tended to be much higher in the 5 patients with pneumonia (n = 1) and lung injuries (n = 4) than in the patients without apparent pulmonary problems. In addition, the patients with a primary lung disorder demonstrated a net increase in IL-6 levels across the lungs, whereas there was no increase, but rather, a net reduction of IL-6 levels across the lungs in patients without a lung disorder. CONCLUSIONS: The lung appears to be a major producer of IL-6 in patients with an inflammatory lung process. There is a 39% increase in the level of IL-6 as it passes through inflamed lung, producing a marked difference in ART and MV IL-6 levels. Normal lung demonstrated little effect on either ART or MV IL-6 levels.