Thioredoxin-related mechanisms in hyperoxic lung injury in mice

Reduction of glutathione disulfide (GSSG) to glutathione (GSH) by glutathione reductase (GR) enhances the efficiency of GSH-dependent antioxidant activities. However, GR-deficient (a1Neu) mice are less susceptible to acute lung injury from continuous exposure to > 95% O(2) (96 h: 6.9 +/- 0.1 g right lung/kg body versus room air 3.6 +/- 0.3) than are C3H/HeN control mice (10.6 +/- 1.3 versus 4.2 +/- 0.3, P < 0.001). a1Neu mice have greater hepatic thioredoxin (Trx)1 and Trx2 levels than do C3H/HeN mice, suggesting compensation for the absence of GR. a1Neu mice exposed to hyperoxia for 96 hours showed lower levels of inflammatory infiltrates in lungs than did similarly exposed C3H/HeN mice. Pretreatment with aurothioglucose (ATG), a thioredoxin reductase (TrxR) inhibitor, exacerbated the effects of hyperoxia on lung injury in a1Neu mice (11.6 +/- 0.8, P < 0.001), but attenuated hyperoxic lung edema and inflammation in C3H/HeN mice (6.3 +/- 0.4, P < 0.001). No consistent alterations were observed in lung GSH contents or liver GSH or GSSG levels after ATG pretreatment. The data suggest that modulation of Trx/TrxR systems might provide therapeutically useful alterations of cellular resistance to oxidant stresses. The protective effects of ATG against hyperoxic lung injury could prove to be particularly useful therapeutically.     

Tibialis anterior muscles in mdx mice are highly susceptible to contraction-induced injury

Skeletal muscles of patients with Duchenne muscular dystrophy (DMD) and mdx mice lack dystrophin and are more susceptible to contraction-induced injury than control muscles. Our purpose was to develop an assay based on the high susceptibility to injury of limb muscles in mdx mice for use in evaluating therapeutic interventions. The assay involved two stretches of maximally activated tibialis anterior (TA) muscles in situ. Stretches of 40% strain relative to muscle fiber length were initiated from the plateau of isometric contractions. The magnitude of damage was assessed one minute later by the deficit in isometric force. At all ages (2–19 months), force deficits were four- to seven-fold higher for muscles in mdx compared with control mice. For control muscles, force deficits were unrelated to age, whereas force deficits increased dramatically for muscles in mdx mice after 8 months of age. The increase in susceptibility to injury of muscles from older mdx mice did not parallel similar adverse effects on muscle mass or force production. The in situ stretch protocol of TA muscles provides a valuable assay for investigations of the mechanisms of injury in dystrophic muscle and to test therapeutic interventions for reversing DMD. This revised version was published online in July 2006 with corrections to the Cover Date.     

Glutathione reductase targeted to type II cells does not protect mice from hyperoxic lung injury

Exposure of the lung epithelium to reactive oxygen species without adequate antioxidant defenses leads to airway inflammation, and may contribute to lung injury. Glutathione peroxidase catalyzes the reduction of peroxides by oxidation of glutathione (GSH) to glutathione disulfide (GSSG), which can in turn be reduced by glutathione reductase (GR). Increased levels of GSSG have been shown to correlate negatively with outcome after oxidant exposure, and increased GR activity has been protective against hyperoxia in lung epithelial cells in vitro. We tested the hypothesis that increased GR expression targeted to type II alveolar epithelial cells would improve outcome in hyperoxia-induced lung injury. Human GR with a mitochondrial targeting sequence was targeted to mouse type II cells using the SPC promoter. Two transgenic lines were identified, with Line 2 having higher lung GR activities than Line 1. Both transgenic lines had lower lung GSSG levels and higher GSH/GSSG ratios than wild-type. Six-week-old wild-type and transgenic mice were exposed to greater than 95% O2 or room air (RA) for 84 hours. After exposure, Line 2 mice had higher right lung/body weight ratios and lavage protein concentrations than wild-type mice, and both lines 1 and 2 had lower GSSG levels than wild-type mice. These findings suggest that GSSG accumulation in the lung may not play a significant role in the development of hyperoxic lung injury, or that compensatory responses to unregulated GR expression render animals more susceptible to hyperoxic lung injury.     

Septic mice are susceptible to pulmonary aspergillosis

Clinical data underscores the fact that subsequent high mortality rates occur in patients who survive acute septic episodes. Herein, we described a clinically relevant model of experimental sepsis that we believe will allow further investigation of the manner in which the pulmonary innate immune response is modulated after sepsis. C57BL/6 mice were subjected to cecal ligation and puncture (CLP) model, whereby the cecum was partially ligated and punctured nine times with a 21-gauge needle. This procedure was associated with 100% mortality at 3 days after surgery. In contrast, when mice subjected to CLP were treated with antibiotic beginning at 8 hours after surgery, and every 12 hours thereafter until 3 days, approximately 60% of the mice survived. Interestingly, CLP survivors quickly succumbed (100% mortality) to pulmonary infection when intratracheally challenged, at day 3 after CLP, with viable Aspergillus fumigatus conidia. No mortality was observed in conidia-challenged sham-operated mice. The defective innate immune response against A. fumigatus in CLP mice could not be explained by a failure of neutrophils to infiltrate the lungs. Instead, gene array analysis revealed that several components of the innate immune response, including the nuclear factor-kappaB signaling pathway, were down-regulated. Thus, we describe a system of sepsis-induced innate immune failure in the lungs of C57BL/6 mice.     

Humanized mice are susceptible to Salmonella typhi infection

Salmonella enterica serovar Typhi is a pathogen that only infects humans. Currently, there is no animal model for studying this pathogen. Recently, alymphoid RAG-2(-/-)/γ(c)(-/-) mice engrafted with human leukocytes, known as humanized mice, have been successfully utilized to develop experimental models for several human-specific viral infections, including HIV, human-like dengue fever and hepatitis C virus. Little is known about the usefulness and feasibility of the humanized mouse model for the study of human-specific bacterial pathogens, such as S. typhi. The aim of this study was to determine if Salmonella enterica serovar Typhi could establish productive infection in humanized mice. Here we report that intravenous inoculation of S. typhi into humanized mice, but not controls, established S. typhi infections. High bacterial loads were found in the liver, spleen, blood and bone marrow of mice reconstituted with human leukocytes, but not in the unreconstituted control mice. Importantly, S. typhi-infected humanized mice lost significant body weight, and some of the infected mice displayed neurological symptoms. Our data suggest, for the first time, that humanized mice are susceptible to S. typhi challenge and that this model can be utilized to study the pathogenesis of S. typhi to develop novel therapeutic strategies.     

IL-18-deficient mice are resistant to endotoxin-induced liver injury but highly susceptible to endotoxin shock

IL-18 is an IL-1-related cytokine which shares biological functions with IL-12. These include the activation of NK cells, induction of IFN-gamma production and Th1 cell differentiation. In this study we analyzed the effect of IL-18 deficiency on lipopolysaccharide (LPS)-induced liver injury and endotoxin shock in Propionibacterium acnes-primed mice. P. acnes-primed IL-18-deficient (IL-18KO) mice showed resistance to LPS-induced liver injury. Unexpectedly, P. acnes-primed IL-18KO mice were highly susceptible to LPS-induced endotoxin shock. Serum level of tumor necrosis factor (TNF)-alpha were markedly elevated (approximately 10-fold higher) within 1.5 h after LPS challenge in IL-18KO mice as compared with wild-type mice. Anti-TNF-alpha antibody administration to IL-18KO mice was significantly protective against endotoxin-induced lethality. P. acnes-primed IL-18KO macrophages produced approximately 6-fold more TNF-alpha protein than did P. acnes-primed wild-type control macrophages. Taken together, these findings demonstrate that IL-18 is responsible for the progression of endotoxin-induced liver injury as well as down-regulation of endotoxin-induced TNF-alpha production in P. acnes-primed mice.     

IL-13 gene-deficient mice are susceptible to cutaneous L. mexicana infection.

Recent studies have demonstrated that IL-13 mediates susceptibility to cutaneous L. major infection via IL-4-independent pathway. To determine whether IL-13 also plays a similar role in pathogenesis of cutaneous L. mexicana infection, we analyzed the course of L. mexicana infection in IL-13(-/-) and IL-4/IL-13(-/-) C57BL/6x129sv/Ev mice and compared with that in similarly infected wild-type mice. IL-13(-/-) mice were as susceptible as the wild-type mice to L. mexicana and developed rapidly progressing, large non-healing lesions following cutaneous L. mexicana infection. In contrast, similarly infected IL-4/IL-13(-/-) mice were highly resistant and developed either no lesions or small lesions containing few parasites that totally resolved by 12 weeks following infection. Throughout the course of infection IL-13(-/-) and the wild-type mice produced significantly more Th2-associated L. mexicana antigen (LmAg)-specific IgG1 than IL-4/IL-13(-/-) mice. All three groups produced comparable levels of Th1-associated IgG2a. At week 12 post infection, LmAg-stimulated spleen cells from L. mexicana-infected IL-4/IL-13(-/-) produced significantly higher levels of IL-12 and IFN-gamma as compared to those from similarly infected wild-type and IL-13(-/-) mice. Although both IL-13(-/-) and the wild-type spleen cells produced IL-4 following in vitro antigenic stimulation, the wild-type mice produced significantly more. These findings demonstrate that IL-13 is not involved in mediating susceptibility to L. mexicana. Moreover, they also indicate that IL-4 not IL-13 is a dominant cytokine involved in pathogenesis of cutaneous L. mexicana infection.     

Proliferative changes in the pulmonary arterial wall during short-term hyperoxic injury to the lung.

Injury to the lung during in vivo exposure to hyperoxia results in vascular restructuring and pulmonary hypertension. This study reports the pattern of cellular proliferation that occurs in proximal intrapulmonary arteries over time during vessel wall injury and adaptation to increased partial pressures of oxygen. Although the remodeling of the capillary bed has been emphasized particularly during oxygen injury to the lung, this report identifies significant proliferative changes within the vessel wall of proximal arterial segments isolated from rats exposed to 85% oxygen. An increased incorporation of 3H-thymidine by endothelial cells is the earliest and most dramatic vessel wall response. The labeling index of these cells is increased more than tenfold by the end of 7 days in hyperoxia. Proliferation of medial smooth muscle cells and adventitial fibroblasts is also significantly increased. The increased cell number within these compartments is noted especially for its contribution to the overall vessel wall hypertrophy observed in chronic hyperoxic pulmonary hypertension. This general proliferative response is accompanied by specific shifts in the relative percentages of different actin protein isoforms as identified by two-dimensional gel electrophoresis. Changes in the distribution of actin isoforms are discussed as potential markers of a phenotypic modulation among vascular smooth muscle cells that occurs during the progression of pulmonary vessel wall remodeling.     

Alveolar macrophage procoagulant activity is increased in acute hyperoxic lung injury.

Alveolar macrophage accumulation and interstitial fibrin deposition are prominent in adult respiratory distress syndrome and chronic interstitial lung diseases. The role of alveolar macrophages in the initiation of fibrin deposition and lung injury in these diseases is uncertain. Expression of procoagulant activity by these cells may provide evidence of macrophage activation and involvement in the initiation of lung fibrin deposition. An experimental model of hyperoxia-induced lung injury in rats was studied for assessment of the relationship of lung injury, fibrin deposition, and alveolar macrophage procoagulant activity. Lung injury was assessed histologically and functionally, and the accumulation of inflammatory cells was quantified by bronchoalveolar lavage. Pulmonary injury, manifested by increased capillary permeability, developed progressively during exposure to hyperoxia and was associated with significant augmentation of the procoagulant activity of alveolar macrophages early in the disease. This increase preceded the accumulation of polymorphonuclear leukocytes. Alveolar macrophage procoagulant activity had functional characteristics consistent with tissue factor. These studies provide evidence of early alveolar macrophage activation in acute hyperoxic lung injury in rats and suggest a role for procoagulant activity in the development of interstitial fibrin deposition.     

Interleukin-6-induced protection in hyperoxic acute lung injury

Hyperoxic lung injury is commonly encountered in patients who require treatment with high concentrations of inspired oxygen. To determine whether interleukin (IL)-6 is protective in oxygen toxicity, we compared the effects of 100% O(2) in transgenic mice that overexpress IL-6 in the lung and transgene (-) controls. IL-6 markedly enhanced survival, with 100% of transgene (-) animals dying within 72 to 96 h, 100% of transgene (+) animals living for more than 8 d and more than 90% of transgene (+) animals living longer than 12 d. This protection was associated with markedly diminished alveolar-capillary protein leak, endothelial and epithelial membrane injury, and lung lipid peroxidation. Hyperoxia also caused cell death with DNA fragmentation in the lungs of transgene (-) animals and IL-6 markedly diminished this cytopathic response. The protective effects of IL-6 were not associated with significant alterations in the activities of copper/ zinc superoxide dismutase (SOD) or manganese SOD. They were, however, associated with the enhanced accumulation of the cell-death inhibitor Bcl-2, but not the cell-death stimulator BAX, and with the heightened accumulation of the cell-death regulator tissue inhibitor of metalloproteinase-1 (TIMP-1). These studies demonstrate that IL-6 markedly diminishes hyperoxic lung injury and that this protection is associated with a marked diminution in hyperoxia-induced cell death and DNA fragmentation. They also demonstrate that this protection is not associated with significant alterations in SOD activity, but is associated with the induction of Bcl-2 and TIMP-1.     

脂蛋白脂酶基因缺陷的极度高甘油三酯血症小鼠胰腺炎的研

目的:利用一种特殊的脂蛋白脂酶（LPL）基因缺陷的极度高甘油三酯血症(HTG)小鼠，作为HTG性胰腺炎的实验动物模型，研究其胰腺炎发病特点。 方法:检测野生型及LPL缺陷的遗传性极度HTG小鼠血浆淀粉酶活性并观察胰腺的形态学改变，研究自发性胰腺炎的发生情况；通过腹腔注射左旋精氨酸（L-Arg）诱导小鼠发生急性胰腺炎（AP），研究HTG时胰腺炎易感性的变化。 结果:对胰腺组织的病理观察表明，LPL缺陷小鼠中约34.5%可发生自发性胰腺炎，特点主要为炎细胞浸润和腺泡破坏，有时伴纤维化和出血，病变严重程度与血浆TG水平呈正相关（r=0.604,P<0.05），血浆淀粉酶活性与对照组相比无明显差异。L-Arg诱导的LPL缺陷实验组小鼠胰腺炎病理损害明显重于野生型实验组（P<0.01），但两组间血浆淀粉酶活性无显著差异。 结论:LPL缺陷的HTG小鼠可发生自发性胰腺炎，对于L-Arg诱导的AP比野生型小鼠更加易感，为进一步研究HTG性胰腺炎的发病机制提供了理想的动物模型。     

IL-5-deficient mice are susceptible to experimental autoimmune encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE) is an animal model commonly used to investigate mechanisms involved in the activation of self-reactive T cells. Whereas auto-reactive T(h)1 cells are believed to be involved in the generation of EAE, T(h)2 cells can induce EAE in immunocompromised hosts. Since the T(h)2 cytokine IL-5 can influence the nature and severity of disease, we investigated the role of IL-5 in the EAE model. Wild-type C57BL/6J and IL-5(-/-) mice were immunized with myelin oligodendrocyte glycoprotein (MOG)(35-55) peptide and the development of EAE observed. Our results show that IL-5(-/-) mice developed EAE with a similar day of onset and comparable severity to wild-type mice. Primed T cells isolated from IL-5(-/-) mice proliferated equally to wild-type cells in response to antigen challenge with MOG(35-55). Antigen-specific T cells from IL-5(-/-) mice produced IFN-gamma and tumor necrosis factor-alpha, but no IL-4 or IL-10, indicating that a predominant T(h)1 environment was induced following immunization. No differences in the types of cells infiltrating into the central nervous system were observed between IL-5(-/-) and wild-type mice. Our results suggest that IL-5 is not directly involved in the initiation or effector phase of MOG(35-55)-induced EAE in immunocompetent C57BL/6J mice.     

Transgenic mice expressing C-reactive protein are susceptible to infection with Plasmodium yoelii sporozoites.

Human and rat C-reactive proteins, major acute-phase reactants, bind to sporozoites and inhibit their in vitro development in hepatocytes (A. Nussler, S. Pied, M. Pontet, F. Miltgen, L. Renia, M. Gentilini, and D. Mazier, Exp. Parasitol. 72:1-7, 1991, and S. Pied, A. Nussler, M. Pontet, F. Miltgen, H. Matile, P.-H. Lambert, and D. Mazier, Infect. Immun. 57:278-282, 1989). We show here that rabbit C-reactive protein has identical properties. Nevertheless, infection by Plasmodium yoelii sporozoites was not prevented in transgenic mice engineered to express rabbit C-reactive protein following induction of gluconeogenesis.     

Repair of chronic hyperoxic lung injury: changes in lung ultrastructure and matrix

We studied changes in lung ultrastructure, fibronectin, and collagen during repair of chronic hyperoxic lung injury induced by exposure of rats to 85% oxygen for 14 days. Morphologically, the most persistent changes were in the alveolar interstitium. After 28 days of repair, the extracellular matrix volume was still twofold normal. Total interstitial cell numbers also remained high and interstitial myofibroblast number actually doubled between Days 7 and 14. These changes contrast markedly with repair of acute lung injury induced by 100% oxygen (Thet et al. (1986) Exp. Lung Res. 11, 209-228) in which matrix volume and interstitial myofibroblast number increased initially but then returned to normal. Biochemically, tissue-associated fibronectin was high initially and peaked at 3 days before slowly declining. Tissue collagen content began to increase after the peak in fibronectin content and was over 150% of controls at 28 days; this correlated with an increase in visible collagen fibers. We conclude that changes in lung morphology and matrix after chronic hyperoxic lung injury are more persistent than after acute hyperoxic lung injury and result in a greater degree of chronic interstitial fibrosis.     

Migration-inhibitory factor gene-deficient mice are susceptible to cutaneous Leishmania major infection

To determine the role of endogenous migration-inhibitory factor (MIF) in the development of protective immunity against cutaneous leishmaniasis, we analyzed the course of cutaneous Leishmania major infection in MIF gene-deficient mice (MIF(-/-)) and wild-type (MIF(+/+)) mice. Following cutaneous L. major infection, MIF(-/-) mice were susceptible to disease and developed significantly larger lesions and greater parasite burdens than MIF(+/+) mice. Interestingly, antigen-stimulated lymph node cells from MIF(-/-) mice produced more interleukin-4 (IL-4) and gamma interferon (IFN-gamma) than those from MIF(+/+) mice, although the differences were statistically not significant. IFN-gamma-activated resting peritoneal macrophages from MIF(-/-) mice showed impaired macrophage leishmanicidal activity and produced significantly lower levels of nitric oxide and superoxide in vitro. The macrophages from MIF(-/-) mice, however, produced much more IL-6 than macrophages from wild-type mice. These findings demonstrate that endogenous MIF plays an important role in the development of protective immunity against L. major in vivo. Furthermore, they indicate that the susceptibility of MIF(-/-) mice to L. major infection is due to impaired macrophage leishmanicidal activity rather than dysregulation of Th1 and Th2 responses.