Human monocyte interactions with non-enzymatically glycated collagen

Summary We have previously shown that receptors for advanced glycation end products are expressed on activated human monocytes. We now report that activated human monocytes exhibit increased adhesion to non-enzymatically glycated collagen substrates (+32%±1, p<0.001), and the increased adhesion can be competitively inhibited with non-enzymatically glycated albumin. Non-activated monocytes, which do not express receptors for advanced glycation end products, exhibit decreased adhesion (-16%±1, p<0.001). Similar results were observed with substrates of fibronectin and endothelial cell matrix proteins. As the presence of glycation adducts on collagen interferes with the normal binding of monocytes/macrophages, one possible role for advanced glycation adduct receptors on activated monocytes is to counterbalance such decreased adherence. Overcompensation for long periods of time may lead to pathological changes. Additionally, such receptors may play a role in monocyte-mediated remodelling of glycated matrix proteins, as we have observed increased degradation of nonenzymatically glycated collagen substrates by activated human monocytes at 2 h (+52%±11, p=0.01), 3 h(+49% ±10, p=0.01), and 4 h (+36%±6, p<0.01) after adding activated monocytes to ¹²⁵I-labelled substrates.     

Renoprotective effects of combination of angiotensin converting enzyme inhibitor with mycophenolate mofetil in diabetic rats

Objective and design Previously it was shown that blocking of the renin-angiotensin system (RAS) by angiotensin converting enzyme (ACE) inhibitors, or suppression of inflammatory responses by immunosuppressive drugs such as mycophenolate mofetil (MMF) could attenuate renal injury in diabetic rats. Whether RAS blockade combined with an immunosuppressive drug provides superior renoprotection against diabetic nephropathy has not been clearly delineated. Materials Diabetes was induced by injection of streptozotocin after uninephrectomy. Treatment Rats were randomly separated into five groups: control, diabetes, diabetes treated with enalapril (an ACE inhibitor, 10 mg/kg/d by gastric gavage), diabetes treated with MMF (10 mg/kg/d by gastric gavage), or diabetes treated with a combination of both agents and were followed for 8 weeks. Methods 24 h urinary albumin excretion rate (AER) was determined, renal injury was evaluated, immunohistochemistry for ED-1 macrophage marker, intercellular adhesion molecule-1 (ICAM-1) and monocyte chemoattractant protein-1 (MCP-1) were performed, and expression of transforming growth factor (TGF)-β1 protein was determined by Western blotting analysis. Results Diabetes was associated with a considerable increase in AER. Both enalapril and MMF retarded the increase in albuminuria, which was nearly completely abrogated by combination therapy. Increased glomerular volume and tubulointerstitial injury index in diabetic rats was attenuated by treatment with either enalapril or MMF and further reduced by the combination of the two. Elevated malondialdehyde levels in renal tissue were reduced by enalapril or MMF and, more effectively, by combined enalapril with MMF. Renal overexpression of ICAM-1 was not retarded by enalapril and attenuated by MMF or combined enalapril with MMF. Combination therapy was associated with a superior suppression of diabetes-induced macrophage recruitment and overexpression of MCP-1 and TGFβ1 compared to either monotherapy in renal tissue. Conclusion The combination of enalapril and MMF confers superiority over monotherapy in renoprotection, a mechanism which may be at least partly correlated with synergistic suppression of increased macrophage recruitment and overexpression of MCP-1 and TGF-β1 in renal tissue in diabetic rats. Received 26 July 2005; returned for revision 5 October 2005; returned for final revision 9 January 2006; accepted by M. Katori 23 January 2006     

Fibrinogen promotes monocyte adhesion via a protein kinase C dependent mechanism

The accumulation of blood monocytes at sites of predilection of the vessel wall is an early cellular event of atherogenesis. Proteins of the vessel wall may facilitate monocyte adhesion and thus promote their recruitment. It has been shown that the relative content of extracellular fibrinogen increases during lesion development, and this study investigated the contribution of immobilized fibrinogen to monocyte adhesion and the underlying mechanism. Freshly isolated human blood monocytes were cultivated in serum-free RPMI 1640 in tissue culture wells precoated with albumin, fibrinogen, or fibrin. After 16 h the plates were washed and adherent cells enumcrated. Immobilized fibrinogen enhanced monocyte adhesion more than 1.9-fold compared to immobilized albumin or fibrin (P<0.05). Concomitant addition of the protein kinase C (PKC) inhibitors staurosporine or H7 suppressed monocyte adherence to immobilized fibrinogen but exerted no significant effect upon adhesion to any other surface tested. Stimulation of monocytes using phorbol myristate acetate resulted in increased binding of monocytes on fibrinogen but not on bovine serum albumin. When PKC activity was reduced through prolonged incubation with PMA for 16h, a significant reduction of monocyte adhesion on fibrinogen was observed. Peptides containing RGD sequences, which have been demonstrated to be ligands for certain integrins, did not inhibit monocyte adhesion. The data suggest that fibrinogen promotes monocyte adhesion in vitro by a PKC-dependent mechanism. PKC appears to be important not only for the initial cell adhesion but also for sustained binding of monocytes to fibrinogen.Abbreviations BSA Bovine serum albumin - ECM Extracellular matrix - PKC Protein kinase C - PMA Phorbol myristate acetate     

云芝多糖B抑制ox-LDL诱导巨噬细胞株单核细胞趋化蛋白-1mRNA表达的调控机制

目的：探讨云芝多糖（CVPS）-CVPS-B对氧化修饰低密度脂蛋白（ox-LDL）诱导的巨噬细胞(RAW264.7)单核细胞趋化蛋白-1（MCP-1）mRNA表达的影响，及对核因子-κB（NF-κB）和细胞内谷胱甘肽（GSH）的调控作用。方法:应用反转录聚合酶链反应（RT-PCR）测定RAW264.7细胞MCP-1 mRNA的表达；凝胶滞留法（EMSA）测定NF-κB的结合活性；荧光分光光度法测定细胞内GSH的活性。结果:CVPS-B呈剂量依赖性抑制ox-LDL诱导的RAW264.7细胞MCP-1 mRNA的表达。应用GSH 特异性消耗剂buthionine-［S，R］-sulfoximine （BSO），ox-LDL不能诱导RAW264.7细胞MCP-1 mRNA的表达。CVPS-B抑制ox-LDL诱导RAW264.7细胞内GSH活性下降。CVPS-B呈剂量依赖性抑制 ox-LDL诱导的RAW264.7细胞NF-κB活性；在完全消耗GSH的RAW264.7细胞，ox-LDL不能诱导NF-κB的活性。结论:CVPS-B可抑制ox-LDL诱导RAW264.7细胞MCP-1 mRNA的表达；其抑制作用可能通过GSH/NF-κB的调控。     

LOX-1介导ox-LDL诱导的血管内皮细胞MCP-1的表达

目的：观察血凝素氧化低密度脂蛋白受体-1（LOX-1）对氧化LDL（ox-LDL）诱导的人脐静脉内皮细胞(human unbilical vein endothelial cells, HUVECs)表达单核细胞趋化蛋白（monocyte chemoattractant protein-1, MCP-1）基因及蛋白的影响。 方法： 用RT-PCR和Western blot的方法观察ox-LDL对培养的HUVECs表达LOX-1和MCP-1基因及蛋白的影响，然后用LOX-1的受体阻滞剂爱兰苔胶（carrageenan） 和聚肌苷酸［polyinosinic acid，poly(I)］与HUVECs预先作用后，再观察内皮细胞表达LOX-1和MCP-1基因和蛋白的变化。 结果： 用不同浓度的ox-LDL（0 mg/L、10 mg/L、20 mg/L 、50 mg/L、100 mg/L）与HUVECs培养24 h后，LOX-1和MCP-1的mRNA和蛋白的表达明显增加，呈浓度依赖性；用Carrageenan 和polyinosinic acid与HUVECs预先作用2 h后，再加入50 mg/L的ox-LDL培养24 h，与未加Carrageenan和polyinosinic acid相比，HUVECsLOX-1和MCP-1的mRNA和蛋白的表达明显减少。 结论： ox-LDL可以调节培养的HUVECsLOX-1和MCP-1基因和蛋白的表达，LOX-1作为ox-LDL的特异性受体，可能介导了ox-LDL诱导血管内皮细胞分泌MCP-1，从而在动脉粥样硬化的发生发展中起着重要的作用。     

Outer membrane protein A renders dendritic cells and macrophages responsive to CCL21 and triggers dendritic cell migration to secondary lymphoid organs

Outer membrane protein A (OmpA) is a class of bacterial cell wall protein that is immunogenic without adjuvant. As specific immune responses are initiated in the lymph nodes (LN, we analyzed the effect of the OmpA from Klebsiella pneumoniae (KpOmpA) onchemokine/ chemokine receptor expression by APC and on cell migration to the LN. Upon contact with KpOmpA, human immature DC and macrophages acquire CCR7 expression and responsiveness to CCL21. In parallel, CCR1 and CCR5 expression is down-regulated and CXCL8, CCL2, CCL3 and CCL5 production is up-regulated. Mice injected subcutaneously with KpOmpA present a transient inflammatory reaction at the site of injection accompanied by an enlargement of the draining LN with a higher proportion of DC and macrophages. Lastly, when exposed to KpOmpA prior injection, DC but not macrophages migrate to the draining LN. In conclusion, KpOmpA confers a migratory phenotype to DC and triggers their migration to the regional LN. This property contributes to explain how innate cells initiate adaptive immune response upon recognition of conserved bacterial components and also why OmpA is immunogenic in the absence of adjuvant.     

Human immunodeficiency virus type 1 Tat protein induces an intracellular calcium increase in human monocytes that requires DHP receptors: involvement in TNF-alpha production

HIV-1 Tat protein, acting at the cell membrane, stimulates the production by human monocytes of TNF-α, a cytokine implicated in both HIV-1 replication and pathogenesis. Here, we analyze, in primary human monocytes, the mechanisms involved in Tat-stimulated calcium mobilization and its relationship with TNF-α production. We show that the Tat protein induces a calcium signal by mobilizing calcium from extracellular stores. This calcium signal is totally blocked when cells are stimulated in the presence of DHP receptor inhibitors such as nimodipine or calcicludine, thus suggesting the implication of this L-type calcium channel. By using RT-PCR amplification, Western blot with antibodies directed against the α1D subunit, binding assays with specific agonists or antagonists, and inhibition with specific antisense oligonucleotides, we show that DHP receptors are expressed and functional in primary human monocytes. Interestingly, we demonstrate that Tat-induced calcium mobilization is tightly linked to TNF-α production, thus indicating that Tat-induced mobilization and TNF-α production are entirely mediated by DHP receptors, as shown by their total inhibition by nimodipine, calcicludine, or anti-α1D antisense oligonucleotides.     

白细胞介素1,肿瘤坏死因子α和脂多糖对人脐静脉内皮细胞表达 …

目的 观察细胞因子白细胞介素１（ＩＬ－１）β、肿瘤坏死因子（ＴＮＦ）α和脂多糖（ＬＰＳ）是否诱导人脐静脉内皮细胞表达单核细胞趋化蛋白１（ＭＣＰ－１）ｍＲＮＡ及蛋白。方法 选取生长汇合的人脐胸脉内皮细胞,在其培养基中分别加入终浓度为２ｎｇ／ｍｌ的ＩＬ－１β、２０ｎｇ／ｍｌ的ＴＮＦβ和１００ｎｇ／ｍｌ的ＬＰＳ,３７℃共育４ｈ后,按照一步法提取其总ＲＮＡ,用γ－＾２２Ｐ标记的寡核苷酸ｄｏｔ ｂｌｏｔ分析     

Dectin-2 is predominantly myeloid restricted and exhibits unique activation-dependent expression on maturing inflammatory monocytes elicited in vivo

Dectin-2 is a recently described dendritic-cell-associated receptor, suggested to be involved in the initiation and maintenance of UV-induced tolerance. To understand the physiological relevance of the proposed functions of this C-type lectin-like receptor, we have generated monoclonal antibodies against its extracellular domain and performed a detailed study of its expression. In naive mice, Dectin-2 has a novel distribution pattern compared with other myeloid markers, but is predominantly expressed by a wide variety of tissue macrophages. Its expression was limited on dendritic cells and notably absent from brain microglia and choroid plexus or meningeal macrophages. On peripheral blood monocytes, Dectin-2 expression was very low on the surface but was transiently and markedly up-regulated on induction of inflammation in vivo using a variety of stimuli. This change in Dectin-2 expression occurs on 'inflammatory' monocytes after arrival at the inflammatory lesion as demonstrated by adoptive cell-transfer studies, and is independent of whether the macrophages elicited by the stimuli ultimately expressed Dectin-2. These observations show Dectin-2 expression to be characteristic of monocyte activation/maturation at an inflammatory lesion and provide a new perspective on the interpretation of Dectin-2 function in vivo.     

核因子-κB活化参与Ox-LDL诱导人肾小球系膜细胞表达单核细胞趋化蛋白-1(英文)

目的：研究核因子-κB(NF-κB)在氧化低密度脂蛋白(Ox-LDL)诱导的体外培养的人肾小球系膜细胞表达单核/巨噬细胞趋化蛋白-1(MCP-1)中的作用。方法： 采用凝胶迁移率变动分析检测NF-κB的DNA结合活性变化，以免疫组化观测细胞内REL P65的核转位，用细胞ELISA法检测细胞内MCP-1及IκBα蛋白含量变化。结果： 不同浓度(10、25、50、100 mg/L)Ox-LDL刺激肾小球系膜细胞均可引起细胞NF-κB的DNA结合活性增强，50 mg/L Ox-LDL活化MCs效果最明显(8.50±1.14，P＜0.01 vs control； P＜0.05 vs 10, 25和100 mg/L Ox-LDL)。Ox-LDL刺激MCs 30-240 min均可以活化NF-κB，60 min时相点活性最强(11.0±2.11，P＜0.01 vs control； P＜0.05 vs 30 min or 240 min)。以50 mg/L Ox-LDL刺激MCs 1 h后，细胞内IκBα蛋白水平最低(0.050±0.006，n=5,P＜0.01 vs control)，作用24 h MCP-1表达水平最高(0.331±0.016， n=5,P＜0.01 vs control)。NF-κB活化的同时伴有REL P65核转位。上述效应可被NF-κB特异性抑制剂吡咯二硫氨基甲酸酯(PDTC)所抑制。结论： Ox-LDL刺激人肾小球系膜细胞产生MCP-1是由NF-κB调控，NF-κB参与了脂质肾损害的发病过程。