Heme-regulated eIF2alpha kinase modifies the phenotypic severity of murine models of erythropoietic protoporphyria and beta-thalassemia

Heme-regulated eIF2alpha kinase (HRI) controls protein synthesis by phosphorylating the alpha-subunit of eukaryotic translational initiation factor 2 (eIF2alpha). In heme deficiency, HRI is essential for translational regulation of alpha- and beta-globins and for the survival of erythroid progenitors. HRI is also activated by a number of cytoplasmic stresses other than heme deficiency, including oxidative stress and heat shock. However, to date, HRI has not been implicated in the pathogenesis of any known human disease or mouse phenotype. Here we report the essential role of HRI in 2 mouse models of human rbc disorders, namely erythropoietic protoporphyria (EPP) and beta-thalassemia. In both cases, lack of HRI adversely modifies the phenotype: HRI deficiency exacerbates EPP and renders beta-thalassemia embryonically lethal. This study establishes the protective function of HRI in inherited rbc diseases in mice and suggests that HRI may be a significant modifier of many rbc disorders in humans. Our findings also demonstrate that translational regulation could play a critical role in the clinical manifestation of rbc diseases.     

Neutrophil depletion impairs natural killer cell maturation, function, and homeostasis

Natural killer (NK) cells are bone marrow (BM)-derived granular lymphocytes involved in immune defense against microbial infections and tumors. In an N-ethyl N-nitrosourea (ENU) mutagenesis strategy, we identified a mouse mutant with impaired NK cell reactivity both in vitro and in vivo. Dissection of this phenotype showed that mature neutrophils were required both in the BM and in the periphery for proper NK cell development. In mice lacking neutrophils, NK cells displayed hyperproliferation and poor survival and were blocked at an immature stage associated with hyporesponsiveness. The role of neutrophils as key regulators of NK cell functions was confirmed in patients with severe congenital neutropenia and autoimmune neutropenia. In addition to their direct antimicrobial activity, mature neutrophils are thus endowed with immunoregulatory functions that are conserved across species. These findings reveal novel types of cooperation between cells of the innate immune system and prompt examination of NK cell functional deficiency in patients suffering from neutropenia-associated diseases.     

MIP-1alpha as a critical macrophage chemoattractant in murine wound repair.

At sites of injury, macrophages secrete growth factors and proteins that promote tissue repair. While this central role of the macrophage has been well studied, the specific stimuli that recruit macrophages into sites of injury are not well understood. This study examines the role of macrophage inflammatory protein 1alpha (MIP-1alpha), a C-C chemokine with monocyte chemoattractant capability, in excisional wound repair. Both MIP-1alpha mRNA and protein were detectable in murine wounds from 12 h through 5 d after injury. MIP-1alpha protein levels peaked 3 d after injury, coinciding with maximum macrophage infiltration. The contribution of MIP-1alpha to monocyte recruitment into wounds was assessed by treating mice with neutralizing anti-MIP-1alpha antiserum before injury. Wounds of mice treated with anti-MIP-1alpha antiserum had significantly fewer macrophages than control (41% decrease, P < 0. 01). This decrease in wound macrophages was paralleled by decreased angiogenic activity and collagen synthesis. When tested in the corneal micropocket assay, wound homogenates from mice treated with anti-MIP-1alpha contained significantly less angiogenic activity than control wound homogenates (27% positive for angiogenic activity versus 91% positive in the control group, P < 0.01). Collagen production was also significantly reduced in the wounds from anti-MIP-1alpha treated animals (29% decrease, P < 0.05). The results demonstrate that MIP-1alpha plays a critical role in macrophage recruitment into wounds, and suggest that appropriate tissue repair is dependent upon this recruitment.     

严重烧伤后小鼠腹腔巨噬细胞肿瘤坏死因子的变化及免疫功能机制

背景严重烫伤导致机体免疫系统各方面的功能紊乱,活化的巨噬细胞可分泌许多生物活性递质.烧伤后巨噬细胞功能紊乱与信号转导的关系目前尚不清楚.目的观察严重烫伤后不同时相点及运用特异性NF-κB抑制剂吡咯啉烷二硫代氨基甲酸盐(pyrrolidine dithiocarbamate,PDTC)后小鼠腹腔巨噬细胞内NF-κB活性、IκB-α的表达及肿瘤坏死因子(TNF-α)的变化,从信号转导的角度探讨巨噬细胞功能紊乱的机制.设计随机对照的实验研究.单位创伤、烧伤与复合伤国家重点实验室.材料实验于1999-01/06在第三军医大学烧伤研究所实验室(国家级)完成.实验动物为健康清洁级近交系昆明小白鼠30只.干预以小鼠常规烫伤模型造成体表面积15%Ⅲ度烫伤.实验按烫伤前及伤后不同时相随机分为6组,即0(正常对照组),2,6,12,24,48 h组.收集腹腔巨噬细胞.采用放射免疫法检测TNF-α的含量,电泳迁移率改变分析法测NF-κB的活性,免疫印迹法测IκB-α的表达,反转录-PCR测TNF-α mRNA的表达.主要观察指标①检测TNF-α的含量.②测定NF-κB的活性.③检测IκB-α的表达.④测TNF-α mRNA的表达.结果烫伤后巨噬细胞分泌TNF-α亢进,于伤后12 h达到高峰,为(1 085.65±122.99)ng/L,较正常对照组明显增高(t=14.92,P＜0.01).NF-κB活性于伤后明显活化,于2 h达到了高峰(t=13.31,P＜0.01),为(56.8±7.3)RDU,早于TNF-α的增多.与正常对照组相比,IκB-α的表达于烫伤后2 h显著下降(t=4.23,P＜0.01),达到0.632±0.086,以后上升,至24 h达到高峰(t=7.06,P＜0.01),为1.161±0.097,48 h稍降(t=4.82,P＜0.01),为1.149±0.167.以伤后12 h为调控点,予PDTC后NF-κB活性及TNF-α mRNA表达量均显著下降(P＜0.01).结论烫伤后NF-κB活性及TNF-α表达明显增强,IκB-α对NF-κB在高水平上维持着一种制约关系.烫伤后小鼠腹腔巨噬细胞内NF-κB信号途径参与TNF-α表达的调控.     

Tumor necrosis factor alpha maintains the viability of murine epidermal Langerhans cells in culture, but in contrast to granulocyte/macrophage colony-stimulating factor, without inducing their functional maturation

Freshly isolated murine epidermal Langerhans cells (LC) are weak stimulators of resting T cells but increase their stimulatory capacity 10-30-fold upon 2-3 d of culture together with other epidermal cells. This maturation of LC is mediated by two keratinocyte products. Granulocyte/macrophage colony-stimulating factor (GM-CSF) maintains viability and increases function. IL-1 alone does not keep LC alive, but when combined with GM-CSF further enhances their stimulatory activity. We have now searched for a cytokine that would keep LC in a viable, but functionally immature state. When LC (enriched to greater than 75%) were cultured in the presence of GM-CSF (2 ng/ml) or murine (TNF-alpha) (plateau effect at 62 U/ml), the recovery of viable LC after 72 h was identical. The LC cultured in murine TNF-alpha, however, were 10-30 times less active in stimulating resting T cells. A series of experiments demonstrated that this phenomenon was not due to the induction of insufficient amounts of GM-CSF, the induction of a suppressor factor, or a toxic effect of TNF-alpha. Interestingly, the observed TNF-alpha activity exhibited a species preference, as human TNF-alpha was not active at comparable doses. We have observed an unexpected effect of TNF-alpha on LC in vitro. Though we found that freshly prepared epidermal cells express TNF-alpha mRNA, further studies are needed to establish whether TNF-alpha plays a role in vivo by keeping resident LC in a viable, but functionally immature state.     

Myelopoietic enhancing effects of murine macrophage inflammatory proteins 1 and 2 on colony formation in vitro by murine and human bone marrow granulocyte/macrophage progenitor cells

Two recently identified and purified murine macrophage inflammatory proteins MIP-1 and MIP-2 were tested in vitro both alone, and in combination with purified recombinant (r) murine (mu) GM-CSF, natural (n)muCSF-1, or rhuman (hu)G-CSF, for effects on mouse marrow CFU-GM, in combination with erythropoietin for effects on mouse marrow BFU-E, and in combination with rhuGM-CSF or rhuG-CSF for effects on human marrow CFU-GM. MIP-1 and MIP-2 did not stimulate, but did enhance by up to threefold, colony formation of mouse CFU-GM co-stimulated by rmuGM-CSF and nmuCSF-1, but not by rhuG-CSF, in the absence or presence of serum. MIP-1 and MIP-2 were maximally active at concentrations greater than or equal to 100 ng/ml and the actions appeared to be initiated during the DNA synthetic portion of the cell cycle. Neither MIP-1 nor MIP-2 at up to 1 microgram/ml had any effect on mouse BFU-E, in the absence or presence of erythropoietin. Both MIP-1 and MIP-2 had direct acting effects on purified mouse CFU-GM. The cloning efficiency of 200 purified cells plated with 50 U muCSF-1 was 82% with and 43% without MIP; the cloning efficiency with 50 U rmuGM-CSF was 65% with and 35% without MIP. MIP effects were not mimicked by bacterial LPS, rhuIL-1 alpha, rhuIL-6, or rmuIL-4, and were neutralized by their respective specific antibodies. MIP-1 and MIP-2 also enhanced endogenously stimulated and rhuGM-CSF-, but not rhuG-CSF-, stimulated colony formation by human marrow CFU-GM. These results demonstrate a new role for MIP-1 and MIP-2 in vitro as myelopoietic enhancing activities for CFU-GM.     

Ironing out mechanisms of iron homeostasis and disorders of iron deficiency

Iron plays an important role in mammalian physiological processes. It is a critical component for the function of many proteins, including enzymes that require heme and iron-sulfur clusters. However, excess iron is also detrimental because of its ability to catalyze the formation of reactive oxygen species. As a result, cellular and systemic iron levels are tightly regulated to prevent oxidative damage. Iron deficiency can lead to a number of pathological conditions, the most prominent being anemia. Iron deficiency should be corrected to improve adult patients’ symptoms and to facilitate normal growth during fetal development and childhood. However, inappropriate use of intravenous iron in chronic conditions, such as cancer and heart failure, in the absence of clear iron deficiency can lead to unwanted side effects. Thus, this form of therapy should be reserved for certain patients who cannot tolerate oral iron and need rapid iron replenishment. Here, we will review cellular and systemic iron homeostasis and will discuss complications of iron deficiency.     

The IL-21 receptor augments Th2 effector function and alternative macrophage activation

The IL-21 receptor (IL-21R) shows significant homology with the IL-4R, and CD4+ Th2 cells are an important source of IL-21. Here we examined whether the IL-21R regulates the development of Th2 responses in vivo. To do this, we infected IL-21R-/- mice with the Th2-inducing pathogens Schistosoma mansoni and Nippostrongylus brasiliensis and examined the influence of IL-21R deficiency on the development of Th2-dependent pathology. We showed that granulomatous inflammation and liver fibrosis were significantly reduced in S. mansoni-infected IL-21R-/- mice and in IL-21R+/+ mice treated with soluble IL-21R-Fc (sIL-21R-Fc). The impaired granulomatous response was also associated with a marked reduction in Th2 cytokine expression and function, as evidenced by the attenuated IL-4, IL-13, AMCase, Ym1, and FIZZ1 (also referred to as RELMalpha) responses in the tissues. A similarly impaired Th2 response was observed following N. brasiliensis infection. In vitro, IL-21 significantly augmented IL-4Ralpha and IL-13Ralpha1 expression in macrophages, resulting in increased FIZZ1 mRNA and arginase-1 activity following stimulation with IL-4 and IL-13. As such, these data identify the IL-21R as an important amplifier of alternative macrophage activation. Collectively, these results illustrate an essential function for the IL-21R in the development of pathogen-induced Th2 responses, which may have relevance in therapies for both inflammatory and chronic fibrotic diseases.     

The liver-specific microRNA miR-122 controls systemic iron homeostasis in mice

Systemic iron homeostasis is mainly controlled by the liver through synthesis of the peptide hormone hepcidin (encoded by Hamp), the key regulator of duodenal iron absorption and macrophage iron release. Here we show that the liver-specific microRNA miR-122 is important for regulating Hamp mRNA expression and tissue iron levels. Efficient and specific depletion of miR-122 by injection of a locked-nucleic-acid-modified (LNA-modified) anti-miR into WT mice caused systemic iron deficiency, characterized by reduced plasma and liver iron levels, mildly impaired hematopoiesis, and increased extramedullary erythropoiesis in the spleen. Moreover, miR-122 inhibition increased the amount of mRNA transcribed by genes that control systemic iron levels, such as hemochromatosis (Hfe), hemojuvelin (Hjv), bone morphogenetic protein receptor type 1A (Bmpr1a), and Hamp. Importantly, miR-122 directly targeted the 3′ untranslated region of 2 mRNAs that encode activators of hepcidin expression, Hfe and Hjv. These data help to explain the increased Hamp mRNA levels and subsequent iron deficiency in mice with reduced miR-122 levels and establish a direct mechanistic link between miR-122 and the regulation of systemic iron metabolism.     

Haptoglobin polymorphisms and iron homeostasis in health and in disease

Haptoglobin (Hpt) is a plasma protein with hemoglobin-binding capacity. It is a well-known marker of hemolysis. Hpt is also an acute-phase protein that functions as a bacteriostatic agent, an inhibitor of prostaglandin synthesis and angiogenesis. However, the best-known biological function of Hpt is capture of hemoglobin (Hb). The identification of functional differences in haptoglobin molecules resulting from relatively common polymorphisms has further elucidated the importance of haptoglobin in iron homeostasis and in disease processes influenced by iron metabolism. In this review the effect of Hpt polymorphism on these different disease entities will be discussed.     

The AMP-activated protein kinase alpha2 catalytic subunit controls whole-body insulin sensitivity

AMP-activated protein kinase (AMPK) is viewed as a fuel sensor for glucose and lipid metabolism. To better understand the physiological role of AMPK, we generated a knockout mouse model in which the AMPKalpha2 catalytic subunit gene was inactivated. AMPKalpha2(-/-) mice presented high glucose levels in the fed period and during an oral glucose challenge associated with low insulin plasma levels. However, in isolated AMPKalpha2(-/-) pancreatic islets, glucose- and L-arginine-stimulated insulin secretion were not affected. AMPKalpha2(-/-) mice have reduced insulin-stimulated whole-body glucose utilization and muscle glycogen synthesis rates assessed in vivo by the hyperinsulinemic euglycemic clamp technique. Surprisingly, both parameters were not altered in mice expressing a dominant-negative mutant of AMPK in skeletal muscle. Furthermore, glucose transport was normal in incubated isolated AMPKalpha2(-/-) muscles. These data indicate that AMPKalpha2 in tissues other than skeletal muscles regulates insulin action. Concordantly, we found an increased daily urinary catecholamine excretion in AMPKalpha2(-/-) mice, suggesting altered function of the autonomic nervous system that could explain both the impaired insulin secretion and insulin sensitivity observed in vivo. Therefore, extramuscular AMPKalpha2 catalytic subunit is important for whole-body insulin action in vivo, probably through modulation of sympathetic nervous activity.     

2型糖尿病患者单核/巨噬细胞功能研究

目的 研究2型糖尿病(T2DM)患者单核/巨噬细胞分泌功能.方法 分析T2DM患者及健康者在葡萄糖耐量试验中白细胞介素(IL)-1、IL-6、肿瘤坏死因子(TNF)-α及胰岛素(Ins)的动态变化.结果 T2DM患者IL-1、IL-6、TNF-α、Ins分泌异常,空腹值偏高,服糖后分泌下降(P<0.05).结论 单核/巨噬细胞与胰腺β细胞分泌功能障碍与T2DM发病机制密切相关.     

The role of Rho kinase and extracellular regulated kinase-mitogen-activated protein kinase in alpha2-adrenoceptor-mediated vasoconstriction in the porcine palmar lateral vein

Alpha2-adrenoceptor-mediated vasoconstriction in the porcine palmar lateral vein is dependent upon activation of the extracellular signal-regulated kinase-mitogen-activated protein (ERK-MAP) kinase signal transduction pathway. Recent studies have shown that alpha2-adrenoceptor-mediated vasoconstriction in the rat aorta is also dependent upon activation of Rho kinase. The aim of this study was to determine whether Rho kinase and ERK-MAP kinase are part of the same signaling pathway. The Rho kinase inhibitor Y27632 (trans-4-[(1R)-1-aminoethyl]-N-4-pyridinylcyclohexanecarboxamide dihydrochloride) (10 microM) almost completely inhibited the contractile response to the alpha2-adrenoceptor agonist UK14304 (5-bromo-6-[2-imidazolin-2-ylamine]-quinoxaline bitartrate) in segments of porcine palmar lateral vein [maximum response 2.9 +/- 2.3% of 60 mM KCl response (mean +/- S.E.M.) in the presence of Y27632, compared with 64.9 +/- 7.1% in control tissues, n = 4]. However, Y27632 had no effect on alpha2-adrenoceptor-mediated ERK activation, as measured by Western blotting. Alpha2-adrenoceptor-mediated vasoconstriction was associated with an increase in phosphorylation of the myosin phosphatase-targeting subunit (MYPT) at Thr696 (the Rho kinase phosphorylation site). This phosphorylation was inhibited by 10 microM Y27632. In contrast, inhibition of ERK activation with the MAP kinase kinase inhibitor PD98059 (2-amino-3-methoxyflavone) (50 microM) had no effect on MYPT phosphorylation. Both Y27632 and PD98059 inhibited myosin light chain phosphorylation. These data indicate that alpha2-adrenoceptor-mediated vasoconstriction in the porcine palmar lateral vein is dependent upon both Rho kinase and ERK activation, although these are separate pathways. Rho kinase causes vasoconstriction through inhibition of myosin phosphatase and an increase in myosin light chain phosphorylation, whereas ERK causes vasoconstriction through a myosin phosphatase-independent pathway.     

Granulocyte/macrophage colony-stimulating factor and interleukin 1 mediate the maturation of murine epidermal Langerhans cells into potent immunostimulatory dendritic cells

Freshly isolated, murine epidermal Langerhans cells (LC) are weak accessory cells for primary T cell-dependent immune responses, but increase their stimulatory capacity at least 20-fold progressively over a 3-d culture with keratinocytes. We have studied the mediators of LC maturation. LC enriched from 12-h epidermal cultures by negative selection do not survive when cultured for 60 h in standard medium. LC survive and show increased stimulatory capacity for oxidative mitogenesis and the primary MLR when 30% keratinocyte-conditioned medium is included. Of the three cytokines that are known to be produced by keratinocytes, only granulocyte/macrophage CSF (GM-CSF) maintains viability and increases stimulatory capacity. IL-1 alone does not keep LC alive, but further enhances the stimulatory activity when combined with GM-CSF. IL-3 has no effect. The increase in LC stimulatory capacity is not due to increased Ia antigen expression, which does not change between 12 and 60 h. Function is not simply due to improved viability, as GM-CSF does not enhance the function of 12-h LC when added to the short-term oxidative mitogenesis assay. By generating LC with strong stimulating activity for resting T cells, GM-CSF and IL-1 may be critical in the sensitization phase of T cell-mediated immunity.     

Cloning and characterization of cDNAs for murine macrophage inflammatory protein 2 and its human homologues

A cDNA clone of murine macrophage inflammatory protein 2 (MIP-2) has been isolated from a library prepared from lipopolysaccharide (LPS)-stimulated RAW 264.7 cells and the nucleotide sequence determined. This cDNA was used to clone cDNAs for human homologues of MIP-2 from a library prepared from phorbol myristate acetate-treated and LPS-stimulated U937 cells. Two homologues were isolated and sequenced. Human MIP-2 alpha and MIP-2 beta are highly homologous to each other and to a previously isolated gene, human gro/melanoma growth-stimulating activity (MGSA). These three human genes, MIP-2 alpha, MIP-2 beta, and gro/MGSA, constitute a sub-family within the cytokine family represented by platelet factor 4 and interleukin 8.     

Autonomous maturation of alpha/beta T lineage cells in the absence of COOH-terminal Src kinase (Csk)

The deletion of COOH-terminal Src kinase (Csk), a negative regulator of Src family protein tyrosine kinases (PTKs), in immature thymocytes results in the development of alpha/beta T lineage cells in T cell receptor (TCR) beta-deficient or recombination activating gene (rag)-1-deficient mice. The function of Csk as a repressor of Lck and Fyn activity suggests activation of these PTKs is solely responsible for the phenotype observed in csk-deficient T lineage cells. We provide genetic evidence for this notion as alpha/beta T cell development is blocked in lck(-/)-fyn(-/)- csk-deficient mice. It remains unclear whether activation of Lck and Fyn in the absence of Csk uncouples alpha/beta T cell development entirely from engagement of surface-expressed receptors. We show that in mice expressing the alpha/beta TCR on csk-deficient thymocytes, positive selection is biased towards the CD4 lineage and does not require the presence of major histocompatibility complex (MHC) class I and II. Furthermore, the introduction of an MHC class I-restricted transgenic TCR into a csk-deficient background results in the development of mainly CD4 T cells carrying the transgenic TCR both in selecting and nonselecting MHC background. Thus, TCR-MHC interactions have no impact on positive selection and commitment to the CD4 lineage in the absence of Csk. However, TCR-mediated negative selection of csk-deficient, TCR transgenic cells is normal. These data suggest a differential involvement of the Csk-mediated regulation of Src family PTKs in positive and negative selection of developing thymocytes.