The macrophage stimulating protein/RON system: a potential novel target for prevention and treatment of endometriosis

Our recent DNA microarray analysis using tissue obtained by laser capture microdissection (LCM) identified up-regulation of RON (a tyrosine kinase receptor) during the late secretory phase in eutopic endometrial epithelial cells from patients with deep endometriosis compared with control endometrium from women with macroscopically normal pelvic cavities. In the present study, we further investigated mRNA expression of RON and its ligand, macrophage stimulating protein (MSP), in deep endometriotic lesions, eutopic endometrium from patients with deep endometriosis and control endometrium by using LCM and quantitative real-time RT-PCR. MSP mRNA expression in endometrial epithelial cells was significantly up-regulated in endometriosis patients during the late secretory phase compared with expression in controls. Furthermore, we detected up-regulation of MSP mRNA in ectopic endometrial epithelial cells compared with matched eutopic endometrial epithelial cells within the same patients regardless of the menstrual phase. MSP has an intrinsically dual functional nature through its receptor RON-it is a trophic cytokine preventing apoptosis and a scatter factor promoting invasion, both of which may be necessary for the initial development and growth of endometriosis. The present findings suggest that the MSP/RON system may be involved in the pathophysiology of endometriosis.     

巨噬细胞集落刺激因子及其受体mRNA在人卵泡颗粒细胞和胎盘及子宫内膜中的表达

目的： 观察巨噬细胞集落刺激因子（MCSF）及其受体（MCSFR）在人卵泡颗粒细胞、胎盘及子宫内膜细胞中的表达情况，进一步了解它们在生殖生理中的作用。方法： 收集人卵泡颗粒细胞、胎盘及子宫内膜细胞标本。采用逆转录-聚合酶链式反应（RT-PCR）及原位逆转录-聚合酶链式反应（in situ RT-PCR）技术观察MCSF及MCSFR mRNA的表达情况。结果： 在人卵泡颗粒细胞、胎盘及子宫内膜细胞中，RT-PCR显示有MCSF及MCSFR的表达，原位RT-PCR也进一步证实了这一点,并定位于细胞浆。结论： MCSF可能参与了人卵泡颗粒细胞、胎盘及子宫内膜细胞生长、发育的调节，在生殖生理过程中可能起一定的作用。     

粒细胞-巨噬细胞集落刺激因子治疗肺泡蛋白沉积症1例

收集1例肺泡蛋白沉积症(pulmonary alveolar proteinosis,PAP)患者临床资料并相关文献复习.患者因气短4个月,加重1个月入院,行纤维支气管镜活检.经过碘酸雪夫(Periodic acid-Schiff,PAS)染色阳性明确诊断为PAP,在经2次全肺灌洗治疗,病情反复后给予皮下注射粒细胞-巨噬细胞集落刺激因子,病情得到改善,提示对特发性PAP患者皮下注射粒细胞-巨噬细胞集落刺激因子是一种可行的治疗方法.     

Macrophage-stimulating protein and RON receptor tyrosine kinase: potential regulators of macrophage inflammatory activities

Macrophage-stimulating protein (MSP) is a serum protein belonging to the plasminogen-related growth factor family. The specific receptor for MSP is the RON (recepteur d'origine nantais) receptor tyrosine kinase - a member of the MET proto-oncogene family. Activation of RON by MSP exerts dual functions on macrophages. The stimulatory activities include the induction of macrophage spreading, migration and phagocytosis. However, MSP also inhibits lipopolysaccharide (LPS)-induced production of inflammatory mediators, including inducible nitric oxide and prostaglandins. These suppressive effects are mediated by RON-transduced signals that block LPS-induced enzymatic cascades that activate nuclear factor kappa-B (NFkappaB) pathways. Recent in vivo studies demonstrated that inactivation of the RON gene results in increased inflammatory responses and susceptibility to LPS-induced septic death in mice, suggesting that RON expression is required for attenuating the extent of inflammatory responses in vivo. Thus, MSP and RON are potential regulators that control macrophage activities during bacterial infection in vivo.     

Role of cysteinyl leukotrienes in human allergen-specific Th2 responses induced by granulocyte macrophage-colony stimulating factor

Background:  The pro-inflammatory cytokine, granulocyte macrophage-colony stimulating factor (GM-CSF), which is elevated in the lungs of atopic asthmatic patients, has been shown to enhance major histocompatibility class II expression of alveolar macrophages (AM). We hypothesized that exposure of AM and monocytes from atopic asthmatic patients to GM-CSF would enhance their antigen presenting function, and investigated putative mechanisms for this effect.
Methods:  Alveolar macrophages were purified from bronchoalveolar lavage by plastic adherence. Monocytes and CD4⁺ T cells were purified from peripheral blood by magnetic bead separation. Antigen-presenting cell (APC) were pretreated with GM-CSF, pulsed with allergen and cocultured with autologous T cells. T-cell proliferation was determined by tritiated thymidine incorporation and cytokine production by enzyme-linked immunosorbent assay.
Results:  Exposure of allergen-pulsed AM and peripheral blood monocytes to GM-CSF significantly increased allergen-specific T-cell proliferation and T helper 2 (Th2) cytokine production. The enhanced response was dependent on costimulation by CD86, but not CD80. Inhibition of the 5-lipoxygenase pathway abrogated GM-CSF-mediated upregulation by monocytes of allergen-specific interleukin-5 (IL-5) and IL-13 cytokine production. Blocking of the cysteinyl leukotriene receptor 1 (cysLT₁) receptor by a specific receptor antagonist inhibited allergen-specific IL-5 production in response to GM-CSF pretreatment.
Conclusion:  Exposure to GM-CSF enhanced the capacity of human APC from atopic asthmatic patients to induce allergen-specific Th2 responses by a mechanism involving cysLT. Novel immunotherapies, targeting production of GM-CSF or its actions on APC have the potential, therefore, to prove beneficial in treatment of patients with inflammatory airway disease.     

Ultrastructural effects of granulocyte colony stimulating factor (G-CSF) and macrophage colony stimulating factor (M-CSF) on rat neutrophils and monocytes

Human granulocyte colony stimulating factor (G-CSF) and macrophage colony stimulating factor (M-CSF) were administered intravenously to rats, and their effects on neutrophils and monocytes were examined by electron microscopy. G-CSF increased the number of cytoplasmic granules in neutrophils. It also enhanced maturation of the nuclear shape in the neutrophils, while chromatin condensation and peroxidase distribution remained immature. M-CSF induced proliferation of monocytes in peripheral blood and bone marrow, but did not affect morphology or distribution of peroxidase reactivity. This study was presented at the 25th Annual Meeting of the Clinical Electron Microscopy Society of Japan, Matsumoto, September 28–30, 1993.     

Therapeutic effect of surfactant protein D in allergic inflammation of mite-sensitized mice

BACKGROUND: Surfactant protein D (SP-D) is involved in the innate immunity within the lung and may have important roles in modulating the inflammatory process of asthma. OBJECTIVE: To examine the potential immunomodulating role of SP-D on the allergic response in mice, and its interaction with the alveolar macrophages (AMs) during allergic inflammation. METHODS: A recombinant 60 kDa fragment of human SP-D (rfh SP-D), Survanta, and budesonide were administrated, respectively, to Der p-sensitive BALB/c mice before or after allergen challenge (AC). Total and differential cell counts, levels of cytokines in bronchoalveolar lavage fluids(BALFs), and levels of Der p-specific IgE and IgG1 antibodies in sera, were assayed. The production of nitric oxide (NO), and inducible NO synthase (iNOS) expression, in AMs, were determined by ELISA and RT-PCR, respectively. RESULTS: Instillation of rfh SP-D to sensitized mice 6 h after AC (therapeutic), but not 24 h before AC (preventive), markedly reduced infiltration of eosinophils, and also reduced levels of IL-4, IL-5, eotaxin, and TNF-alpha but elevated levels of IFN-gamma in the BALF. These effects were comparable with those obtained with budesonide treatment, whereas Survanta did not have a suppressive effect, either before or after AC. There was significant inhibition of NO production in the rfh SP-D pre-treated AMs of allergen-sensitized mice, but not in naive mice. CONCLUSIONS: These results indicate that rfh SP-D has a therapeutic effect on allergen-induced bronchial inflammation, and that this might be because of its inhibitory effect on NO and TNF-alpha production by AMs, and it thus prevents the development of T-helper type 2 cytokine response.     

肾肿瘤中受体酪氨酸激酶RON的表达及意义

目的 探讨RON在肾肿瘤中的表达以及RON表达与肾肿瘤局部侵袭和分化程度的关系.方法 利用免疫组化法测定48例肾透明细胞癌、20例嫌色细胞癌、3例嗜酸性细胞腺瘤肿瘤组织RON表达.结果 RON在肾嫌色细胞癌中表达强度最高,透明细胞癌其次,而在嗜酸性细胞腺瘤中最低,但无统计学意义差异;透明细胞Fuhrman核分级3-4级阳性表达强度高于核分级1或2级(P<0.05);肿瘤直径>7 cm的RON表达强度大于<7 cm病例;RON在存在.肾盂侵袭的病例中有较高表达的趋势.结论 RON肾细胞癌的演进中可能起重要作用.     

骨调素和巨噬细胞集落刺激因子在糖尿病大鼠肾组织中的表达及霉酚酸酯的干预作用

目的： 研究骨调素(OPN)和巨噬细胞集落刺激因子（M-CSF)在糖尿病大鼠肾组织中的表达及免疫抑制剂霉酚酸酯（MMF）的干预作用，旨在探讨MMF对糖尿病肾病(DN)的保护作用及机制。方法: Wistar大鼠行右肾切除术2周后,随机分为右肾切除对照组(NC)、糖尿病组(DM)、霉酚酸酯治疗组(DM+MMF)。腹腔注射链脲佐菌素(STZ,65 mg/kg ) 诱发糖尿病模型,MMF15 mg·kg^-1·d^-1灌胃。检测各组8周末的左肾重/体重比值、24 h尿蛋白（Upro）、血糖(BG) 、血肌酐( Scr)，观察肾脏形态学变化，免疫组化检测肾组织中OPN、M-CSF及CD68表达，荧光实时定量PCR测定肾组织中OPN mRNA表达。结果: 与对照组相比，DM组大鼠血糖、Upro、肾重/体重比值均显著上升(P<0.01);肾间质纤维化面积扩大(P<0.01);肾组织内OPN、M-CSF、CD68表达及OPN mRNA的表达均显著上调(P<0.01)。MMF干预后，上述指标除血糖外均被明显抑制(P<0.05或P<0.01)。结论: MMF减少糖尿病大鼠肾组织中OPN、M-CSF、CD68及OPN mRNA的表达，降低蛋白尿，预防肾损伤。MMF明显抑制DN肾小管－间质损害，可能与其抑制巨噬细胞的趋化与增殖有关。     

Neutrophil apoptosis: impact of granulocyte macrophage colony stimulating factor on cell survival and viability in chronic kidney disease and hemodialysis patients

Introduction

Altered neutrophil apoptosis might be responsible for recurrent bacterial infections encountered in hemodialysis (HD) and chronic kidney disease (CKD) patients. This work was designed to assess the neutrophil apoptotic activity and the impact of implementation of granulocyte macrophage colony stimulating factor (GM-CSF), as a survival factor, on neutrophil apoptosis among these patients.

Material and methods

Twenty-five patients on regular HD along with 34 CKD patients on conservative treatment, as well as 15 healthy controls, were investigated for apoptotic rate via assessment of neutrophil expression of Annexin-V by flow cytometry, before and after 20 h culture in absence and presence of GM-CSF. Neutrophil viability was determined using light microscopy. The preservation of neutrophil activation in these patients was analyzed by flow cytometric CD18 neutrophil expression. Chronic inflammatory state was evaluated by estimating C-reactive protein (CRP) and soluble intercellular adhesion molecule-1 (sICAM-1). Obtained data were statistically analyzed.

Results

Compared to controls, both HD and CKD groups had a significant increase of Annexin-V and CD18 expression and significant decrease in neutrophil viability. Culture of their neutrophils with GM-CSF showed significant decrease of apoptosis accompanied by improvement of neutrophil viability compared to their cultured cells without GM-CSF. These patients also showed significant elevation of CRP and sICAM-1.

Conclusions

Granulocyte macrophage colony stimulating factor demonstrated an evident impact on improving in vitro neutrophil survival and viability in HD and CKD patients. Therefore, this may represent promising preventive and/or therapeutic strategies against infection frequently observed in these patients and causing morbidity and mortality.     

二甲双胍激活AMPK通路减轻饱和脂肪酸诱导的RAW264.7巨噬细胞炎性反应

目的 观察二甲双胍对饱和脂肪酸(SFA)诱导的RAW264.7巨噬细胞炎性反应中炎性因子的影响及探讨其可能机制.方法 SFA干预RAW264.7巨噬细胞建立体外炎性反应模型;实验分为对照组、SFA干预组、二甲双胍+ SFA干预组、AMPK抑制剂Compound C+二甲双胍+SFA干预组;实时定量PCR检测TNF-α和IL-6 mRNA的表达,ELISA法检测TNF-α和IL-6蛋白的分泌,Western blot分析腺苷酸活化蛋白激酶(AMPK)的磷酸化水平.结果 与对照组比较,SFA干预组RAW264.7巨噬细胞TNF-α、IL-6 mRNA表达及蛋白分泌水平均显著升高(P＜0.05);与SFA组比较,二甲双胍+SFA干预组TNF-α、IL-6 mRNA表达水平及蛋白分泌水平均降低(P＜0.05),而细胞AMPK的磷酸化水平增强(P＜0.05);与二甲双胍+SFA干预组比,Compound C+二甲双胍+SFA干预组TNF-α、IL-6 mRNA表达及蛋白分泌水平均升高,AMPK磷酸化水平降低(P＜0.05).结论 二甲双胍激活AMPK降低饱和脂肪酸诱导的RAW264.7巨噬细胞炎性因子TNF-α、IL-6的分泌.     

Methanol extract of Phellodendri cortex alleviates lipopolysaccharide-induced acute airway inflammation in mice

Background and aim: The effects of methanol extract of Phellodendri cortex on acute airway inflammation induced by intranasal administration of lipopolysaccharide (LPS, 300μg/kg) were investigated in female BALB/c mice.

Materials and methods: At 2?h after LPS exposure, mice were treated orally with methanol extract of Phellodendri cortex (100, 200 and 400?mg/kg). At the end of this study, bronchoalveolar lavage fluids (BALF) were collected and number of total cells, macrophages and neutrophils, protein concentration were analyzed. Tumor necrosis factor-alpha (TNF-α), macrophage inflammatory protein (MIP-2), IL-10 levels and nitric oxide (NO) production in BALF were also determined.

Results: Methanol extract of Phellodendri cortex dose-dependently alleviated LPS-induced acute airway inflammation via decreasing the infiltration of inflammatory cells and the release of inflammatory mediators.

Conclusion: The relief of airway inflammation provides a possible therapeutic application of Phellodendri cortex for the treatment of infectious pulmonary diseases.     

Granulocyte macrophage colony stimulating factor is elevated in alveolar macrophages from sheep naturally infected with maedi-visna virus and stimulates maedi-visna virus replication in macrophages in vitro

Infection by maedi-visna virus, a lentivirus of sheep, leads to chronic inflammatory reactions of various tissues. In this report we have analysed the role of specific cytokines in the disease process. A significant increase in expression of interleukin-6, interleukin-10, granulocyte macrophage-colony stimulating factor (GM-CSF) and transforming growth factor-beta1 mRNA was observed in alveolar macrophages isolated from the lungs of naturally infected animals when compared with lungs of seronegative controls. Levels of GM-CSF mRNA expression in alveolar macrophages correlated with the presence of lung lesions, but there was no correlation of interleukin-10, interleukin-6, tumour necrosis factor-alpha and transforming growth factor-beta1 mRNA levels in alveolar macrophages from animals with pulmonary lesions. In vitro investigation showed that GM-CSF in the range 0.1-10 ng/ml induced a significant increase in viral p25 production after 7 days in acutely infected blood monocyte-derived macrophages. The production of p25 peaked between 7 and 14 days exposure to 10 ng/ml of GM-CSF. Quantitative polymerase chain reaction showed that the level of viral DNA in monocyte-derived macrophages was dose-dependent following GM-CSF treatment in the range 0.1-100 ng/ml after 7 days. Viral mRNA expression was also enhanced. These findings indicate a role for GM-CSF in the pathogenesis of lymphoid interstitial pneumonia in infected animals.     

Divergent regulation of cell surface protease expression in HL-60 cells differentiated into macrophages with granulocyte macrophage colony stimulating factor or neutrophils with retinoic acid

Taking advantage of the recently demonstrated presence of N-aminopeptidasesand the serine protease dipeptidyi aminopeptidase IV (DPP IV)at the surface of human myeloblastic HL-60 cells, the regulationof these protease activities in HL-60 cell differentiation hasbeen assessed using combined spectrophotometric and flow cytometricassays. Addition of human recombinant granulocyte macrophagecolony stimulating factor (rHu-GM-CSF) to HL-60 cells to inducedifferentiation into macrophages led to a time and dose-dependentincrease in both cell surface N-aminopeptidase and DPP IV activities.Protease up-regulation was due to an enhancement in cell surfaceprotease number, associated with a slight rise in apparent affinitiesof the enzymes for their substrates. In contrast, in HL-60 cellsinduced to differentiate into neutrophils in the presenceofretinoic acid, expression of cell surface N-amlnopeptidaseswas almost completely abolished in a time-and dose-dependentfashion, and this down-regulation was accompanied by a weakbut significant decrease in affinity. However, no noticeabledifference was seen in serine DPP IV expression between retinoicacid-treated and untreated HL-60 cells. Retinoic acid treatmentalso reduced soluble protease activity in vitro indicating thatdown-regulation of membrane aminopeptldases was not due to theirproteolytic clip. No modulation in the activity of any of theenzymes tested was seen with human recombinant tumor necrosisfactor- or retinol which do not induce HL-60 cell differentiation.The up-regulation of cell surface protease expression in HL-60cells differentiated into macrophages was similar to that observedin monocytes isolated from peripheral blood: both DPP IV andN-aminopeptidase activities strictly increased on cells thatundergo macrophage maturation (up to 5-fold) and independentlyof the nature of the differentiation inducer. Thus, the distinctivepatterns of N-aminopeptidase and DPP IV expression that areseen in differentiating neutrophils and macrophages appear tobe relatedto differences in stage of myeloid maturation. Becausecell surface proteases are crucially involved in leukocyte functions,the data presented suggest that alterations in cell surfaceprotease expression are associated with events controlling thedifferentiation of immature cells.     

酪氨酸激酶受体RON在上皮性卵巢癌中的表达及其意义

目的:研究酪氨酸激酶受体 RON 在上皮性卵巢癌组织中的表达及其与临床病理学参数的关系.方法:采用逆转录聚合酶链反应和免疫组织化学方法分别检测 42 例上皮性卵巢癌新鲜组织 RON mRNA 及其对应 32 例石蜡组织中 RON蛋白的表达.结果:上皮性卵巢癌新鲜组织中 RON mRNA 阳性表达率为57.14%,对应的上皮性卵巢癌石蜡组织中 RON蛋白平均面密度值为(0.060 1±0.028 4),RON mRNA 和蛋白表达均与上皮性卵巢癌组织的临床分期、组织学分级及淋巴结转移有关.结论:RON 的过度表达与上皮性卵巢癌的进展、转移密切相关,检测 RON 的异常对判断肿瘤的临床进展及转移有一定的参考价值,RON 可能成为诊断治疗的新靶点.     

Macrophage inflammatory protein-1α influences eosinophil recruitment in antigen-specific airway inflammation

Allergic airway inflammation is characterized by peribronchial eosinophil accumulation within the submucosa of the airway of the lung. In the present study we have utilized a model of airway inflammation induced by intratracheal challenge with parasite (Schistosoma mansoni) egg antigen (SEA) in presensitized mice. The recruitment of neutrophils and eosinophils into the airway was found to be maximal at 8 and 48 h post challenge, respectively. Since macrophage inflammatory protein-1α (MIP-1α) has previously been found to be chemotactic for eosinophils, in vitro, we postulated that MIP-1α was involved in the airway inflammation and more specifically in eosinophil recruitment into the airway. Initial studies demonstrated an increase in MIP-1α mRNA expression at 8 h post-SEA challenge, as compared to vehicle-treated control mice. We next demonstrated a significant increase in MIP-1α protein in the lungs of SEA-challenged mice at 8 h compared to control challenged mice, correlating to the mRNA data. Immunohistochemical staining of lungs from SEA-challenged mice demonstrated MIP-1α protein expression in airway epithelial cells, alveolar macrophages and in recruited mononuclear cell populations. Immunolocalization of MIP-1α to cells within the bronchoalveolar lavage fluid demonstrated that macrophages and eosinophils stained positive for the protein. To determine the contribution of MIP-1α expression to eosinophil accumulation, SEA-challenged mice were passively immunized with either neutralizing MIP-1α antibodies or normal rabbit IgG, 3–4 h prior to the intratracheal SEA challenge. These studies demonstrated a > 50% decrease in eosinophil recruitment to the lungs and airway in animals receiving neutralizing MIP-1α antibodies with no effect on early neutrophil recruitment. These results suggest that the production of MIP-1α, induced by an antigen-specific response, plays an important role in recruitment of eosinophils in this airway model of inflammation.     

Altered expression of the RON receptor tyrosine kinase in various epithelial cancers and its contribution to tumourigenic phenotypes in thyroid cancer cells

Aberrant expression of the RON receptor tyrosine kinase has been implicated in the pathogenesis of epithelial tumours. The aim of this study was to determine RON expression in various normal epithelial cells and their corresponding tumours by immunohistochemistry. The role of RON in regulating tumourigenic phenotypes was also studied using thyroid cancer cells as a model. RON was almost exclusively expressed at variable levels in normal epithelial cells from the digestive track, lung, kidney, pancreas, liver, breast, bladder, skin, and others. Among 15 types of cancer studied, RON was overexpressed in significant numbers in cancers derived from breast (56%), colon (51%), lung (48), thyroid (42%), skin (37%), bladder (36%), and pancreas (33%). In contrast, limited RON overexpression was observed in cancers from stomach, kidney, brain, liver, ovary, and prostate. Detailed analysis of thyroid tissues showed that RON was hardly detected in normal thyroid cells, moderately expressed in adenoma samples, but overexpressed in about half of papillary and follicular cancer specimens. Overexpression correlated with advanced clinical stage and was associated with lymph node metastasis. In cultured thyroid cancer cells, RON was highly expressed, with constitutive phosphorylation. Activation of RON increased cell growth and migration via the MAP kinase and AKT pathways. Silencing RON expression significantly prevented cell growth and increased cell apoptotic death. These findings show that RON overexpression occurs in a particular group of epithelial cancers. The requirement for RON in sustaining tumourigenic phenotypes suggests that it is a potential target for therapeutic intervention.