Role Of Monocytes In Immunopathology

单核细胞在免疫病理学中的作用

• 批准号: 7967042
• 负责人: LARRY M WAHL
• 金额: $ 81.01万
• 依托单位: NATIONAL INSTITUTE OF DENTAL & CRANIOFACIAL RESEARCH
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7967042
• 关键词: Affect; Animal Model; Antiplasmin; Area; Arthritis; Atherosclerosis; Biological; Biological Markers; Cell Line; Cell membrane; Cell surface; Cells; Clinical Research; Connective Tissue; Connective Tissue Alteration; Disease; Disorder by Site; Endopeptidases; Enzyme Inhibitor Drugs; Enzyme Inhibitors; Enzymes; Exposure to; Extracellular Matrix; Family; Family member; Gelatinase A; Gelatinase B; Gelatinases; Genetic Polymorphism; Goals; Granulocyte-Macrophage Colony-Stimulating Factor; Human; Inflammation; Inflammation Mediators; Inflammatory; Interferon-alpha; Interleukin-4; Interstitial Collagenase; Lesion; Lymphocyte Function; MAP Kinase Gene; MAPK11 gene; MAPK14 gene; Malignant Epithelial Cell; Malignant Neoplasms; Matrix Metalloproteinase Inhibitor; Matrix Metalloproteinases; Mediator of activation protein; Messenger RNA; Oral; Pathology; Periodontal Diseases; Plasmin; Plasminogen; Plasminogen Activator Inhibitor 1; Play; Process; Production; RNA; Regulation; Regulatory Pathway; Research; Rest; Risk Factors; Role; Serine Protease; Signal Pathway; Signal Transduction; Signal Transduction Pathway; Site; Squamous cell carcinoma; Staging; Stromal Cells; Stromelysin 1; Subgroup; Testing; Time; Tissues; Tumor Necrosis Factor-alpha; Zinc; base; cancer cell; cell type; cytokine; human TNF protein; immune function; immunopathology; inhibitor/antagonist; interest; keratinocyte; macrophage; matrix metalloproteinase 12; membrane-type matrix metalloproteinase; monocyte; neoplastic cell; tissue procollagenase; tumor progression; Affect; Animal Model; Antiplasmin; Area; Arthritis; Atherosclerosis; Biological; Biological Markers; Cell Line; Cell membrane; Cell surface; Cells; Clinical Research; Connective Tissue; Connective Tissue Alteration; Disease; Disorder by Site; Endopeptidases; Enzyme Inhibitor Drugs; Enzyme Inhibitors; Enzymes; Exposure to; Extracellular Matrix; Family; Family member; Gelatinase A; Gelatinase B; Gelatinases; Genetic Polymorphism; Goals; Granulocyte-Macrophage Colony-Stimulating Factor; Human; Inflammation; Inflammation Mediators; Inflammatory; Interferon-alpha; Interleukin-4; Interstitial Collagenase; Lesion; Lymphocyte Function; MAP Kinase Gene; MAPK11 gene; MAPK14 gene; Malignant Epithelial Cell; Malignant Neoplasms; Matrix Metalloproteinase Inhibitor; Matrix Metalloproteinases; Mediator of activation protein; Messenger RNA; Oral; Pathology; Periodontal Diseases; Plasmin; Plasminogen; Plasminogen Activator Inhibitor 1; Play; Process; Production; RNA; Regulation; Regulatory Pathway; Research; Rest; Risk Factors; Role; Serine Protease; Signal Pathway; Signal Transduction; Signal Transduction Pathway; Site; Squamous cell carcinoma; Staging; Stromal Cells; Stromelysin 1; Subgroup; Testing; Time; Tissues; Tumor Necrosis Factor-alpha; Zinc; base; cancer cell; cell type; cytokine; human TNF protein; immune function; immunopathology; inhibitor/antagonist; interest; keratinocyte; macrophage; matrix metalloproteinase 12; membrane-type matrix metalloproteinase; monocyte; neoplastic cell; tissue procollagenase; tumor progression

Research in the Immunopathology Section focuses on the biological mediators and signal transduction pathways involved in the modulation of human monocyte and lymphocyte functions that may contribute to the immunopathology associated with various inflammatory lesions. Monocytes/macrophages are prominent in many inflammatory diseases, such as periodontal disease, rheumatiod arthritis, atherosclerosis, and cancer. The pathology associated with these diseases involves alterations in the integrity of the connective tissue framework implicating a role for matrix metalloproteinases (MMPs). MMPs are comprised of a family of zinc dependent endopeptidases divided into major subgroups that include the interstitial collagenases, the gelatinases, stromelysins, membrane type MMPs and others. Collectively these enzymes are capable of degrading all the extracellular matrix components. Because MMPs and tissue inhibitors of MMPs (TIMPs) are believed to play a major role in the destruction and remodeling of connective tissue, a major emphasis has been placed on how these enzymes and inhibitors are regulated in the human monocyte/macrophage as well as in the reciprocal interaction between monocytes/macrophages and tumor cells. Interaction between monocytes/macrophages and tumor cells in the production and activation of MMPs Subsequent to entering an inflammation site monocytes differentiate into macrophages and exposure to stimulants such as IFN-alpha; or IL-4 result in their polarization into M1 (classically activated) or M2 (alternatively activated) macrophages, respectively. The reciprocal interaction between tumor cells and monocytes and monocyte-derived macrophages may play a significant role in the production of MMPs involved in the alteration of connective tissue leading to tumor progression. To assess this possibility and identify MMPs that are potentially involved in this process we are carrying out research in the following areas: (1) determine by real time PCR the MMP family members and TIMPs expressed by human monocytes, resting macrophages, M1 and M2 macrophages; (2) determine by real time PCR the MMP family members and TIMPs expressed by normal keratinocytes and two oral squamous carcinoma cell lines, HN-12 and HN-30; (3) examine the reciprocal interaction between monocytes/macrophages and tumor cells on MMP and TIMP expression; (4) study the mechanism of activation of pro-MMPs in the interaction between monocytes/ macrophages and cancer cells; and (5) determine the signal signal transduction pathways involved in the regulation of MMP production by squamous cell carcinomas. 1) Expression of MMPs and TIMPs in monocytes, macrophages, M1 macrophages and M2 macrophages Expression of MMPs was determined from RNA obtained from donor matched monocytes and macrophages. Major findings include: monocytes express significantly more MMP-1, -10, and -14 when activated than macrophages whereas monocytes express low levels of MMP-2 and TIMP-3 compared to macrophages; monocyte MMP expression is more responsive to LPS and TNF-alpha than macrophages; LPS induced higher levels of MMP-1, -7, and -10 in monocytes than TNF-alpha; and GM-CSF whereas the converse was the case for MMP-9; interestingly, LPS caused a significant decrease in TIMP-2 and TIMP-4 expression by monocytes; with the exception of MMP-1, -7, and -14, macrophage expression of MMPs was relatively unaffected by LPS or TNF-alpha; and GM-CSF; comparison of resting macrophages with M1 and M2 macrophages revealed that these subsets had lower levels of MMP-1 as well as MMP-2 with the greatest decrease occurring in MMP-2 expression in M2 macrophages. In contrast M2 macrophages had higher levels of MMP-12. 2) Real Time PCR analysis of MMPs and TIMPs produced by keratinocytes, HN12 and HN30 cell lines. We are currently analyzing 23 MMP family members and TIMPs in keratinocytes, HN12 and HN30 cell lines by qPCR. Western analysis has shown that there is a difference between HN12 and HN30 cells in MMP-1 production with HN12 cells producing low levels of MMP-1 whereas HN30 produce large amounts of MMP-1. It is anticipated that other differences in MMPs as well as TIMPs will be found which may affect the degree of invasiveness of these cell lines. 3) Effect of mediators from normal keratinocytes, HN12 and HN30 cells on MMP and TIMP production by monocytes and macrophages. In the first stage of the study on the reciprocal interaction of monocytes/macrophage with tumor cells we are testing the mediators found in the culture supernatants of normal keratinocytes and HN12 and HN30 cells on the expression of monocyte and macrophage MMPs and TIMPs by qPCR. The MMPs selected for analysis are those based on the qPCR analysis results described in item 1. 4) Conversion of HN30 pro-MMP-1 to active MMP-1 by monocytes. Interaction of stromal cells, such as monocytes/macrophages, with tumor cells is believed to be involved in the induction and activation of MMPs. We have previous found that MMP-1 in monocyte cultures is primarily in the active form as compared to macrophage cultures in which only pro-MMP-1 is detected. In our recent studies we have shown the squamous carcinoma cell line HN30 produces significant amounts of pro-MMP-1 which can be converted to active MMP-1 by exposure to cell membranes from monocytes but not macrophages. The ability of monocytes to activate tumor cell pro-MMP-1 appears to be due to cell surface serine protease activity. Plasminogen activator inhibitor-1 (PAI-1) or alpha 2-antiplasmin block this conversion indicating the role of cell surface associated plasminogen on monocytes in this process. PCR analysis has revealed low levels of plasminogen mRNA in monocytes. Current studies are determining if plasminogen synthesis is lost as monocytes mature into macrophages. These results indicate that one of the affects of monocytes on tumor progression is the activation of MMP-1. 5) Signaling pathways involved in the regulation of MMP production by squamous carcinoma cells. Examination of the regulatory pathways involved in the endogenous production of MMP-1 by HN30 cells revealed that the ERK1/2 MAPK is a positive regulator of MMP-1. In contrast, p38 MAPK functions as a negative regulator of MMP-1 as evidenced by a significant increase in MMP-1 production by HN30 cells when the action of phosphorylated p38 is inhibited. Of interest, NH12, which do not produce significant levels of MMP-1, have high levels of phosphorylated ERK1/2 and p38 in contrast to HN30 cells which have lower levels of phosphorylated ERK1/2 and p38. However, when HN12 cells are stimulated with plasmin they secrete detectable levels of MMP-1. Initial findings suggest that this may be due to a change to a higher ratio of phosphorylated ERK1/2 compared to p38.

免疫病理学部分的研究重点关注于调节人单核细胞和淋巴细胞功能的生物学介质和信号转导途径，这些途径可能有助于与各种炎症性病变相关的免疫病理学。单核细胞/巨噬细胞在许多炎症性疾病中很突出，例如牙周疾病，风湿性关节炎，动脉粥样硬化和癌症。 与这些疾病相关的病理涉及结缔组织框架完整性的改变，这暗示了基质金属蛋白酶（MMP）的作用。 MMP由锌依赖性内肽酶家族组成，分为主要亚组，其中包括间质胶原酶，明胶酶，曲折蛋白，膜型MMP等。这些酶能够降解所有细胞外基质成分。由于据信MMP（TIMP）的MMP和组织抑制剂在结缔组织的破坏和重塑中起主要作用，因此对这些酶和抑制剂在人类单核细胞/巨噬细胞中如何调节这些酶和抑制剂的主要重点是如何调节的。 MMP的产生和激活中单核细胞/巨噬细胞与肿瘤细胞之间的相互作用 在进入炎症部位后，单核细胞分化为巨噬细胞，并暴露于IFN-Alpha等兴奋剂；或IL-4分别使它们的极化分别为M1（经典激活）或M2（替代激活）巨噬细胞。 肿瘤细胞与单核细胞与单核细胞衍生的巨噬细胞之间的相互相互作用可能在参与结缔组织改变的MMP的产生中起重要作用，从而导致肿瘤进展。为了评估这种可能性并确定潜在参与此过程的MMP，我们正在以下领域进行研究：（1）通过实时确定人类单核细胞表达的MMP家族成员和TIMP，由人类单核细胞，静止的巨噬细胞，M1和M2巨噬细胞确定； （2）通过实时PCR确定由正常角质形成细胞和两个口服鳞状癌细胞系HN-12和HN-30表达的MMP家族成员和TIMP； （3）检查MMP和TIMP表达上的单核细胞/巨噬细胞与肿瘤细胞之间的相互相互作用； （4）研究单核细胞/巨噬细胞与癌细胞之间相互作用中促MMP的激活机制； （5）确定鳞状细胞癌调节MMP产生的信号信号转导途径。 1）在单核细胞，巨噬细胞，M1巨噬细胞和M2巨噬细胞中表达MMP和TIMP 从供体匹配的单核细胞和巨噬细胞获得的RNA确定MMP的表达。 主要发现包括：与巨噬细胞相比，单核细胞表达的MMP-1，-10和-14明显更多，而单核细胞与巨噬细胞相比表达低水平的MMP-2和TIMP-3。单核细胞MMP表达对LPS和TNF-Alpha的反应比巨噬细胞更敏感。与TNF -Alpha相比，LPS在单核细胞中诱导更高水平的MMP -1，-7和-10；和GM-CSF，而MMP-9是这种情况；有趣的是，LPS导致单核细胞的TIMP-2和TIMP-4表达显着降低。 除MMP -1，-7和-14外，MMP的巨噬细胞表达相对不受LPS或TNF -Alpha的影响。和GM-CSF； 与M1和M2巨噬细胞的静息巨噬细胞的比较表明，这些子集的MMP-1水平较低，而MMP-2水平较低，而M2巨噬细胞中MMP-2表达的降低最大。相反，M2巨噬细胞具有较高的MMP-12。 2）由角质形成细胞，HN12和HN30细胞系产生的MMP和TIMP的实时PCR分析。 我们目前正在通过QPCR分析角质形成细胞，HN12和HN30细胞系中的23个MMP家族成员和TIMP。 西方分析表明，在MMP-1产生中HN12和HN30细胞之间存在差异，而HN12细胞产生了低水平的MMP-1，而HN30产生了大量的MMP-1。预计将发现MMP和TIMP的其他差异可能会影响这些细胞系的侵入性程度。 3）正常角质形成细胞，HN12和HN30细胞对单核细胞和巨噬细胞产生MMP和TIMP的影响。 在研究的第一阶段，关于单核细胞/巨噬细胞与肿瘤细胞的相互作用相互作用，我们正在测试在正常角质形成细胞的培养上清液中发现的介质，HN12和HN30细胞在单核细胞和巨噬细胞MMPS和TIMP的表达上通过qPCR进行了测试。 选择用于分析的MMP是基于项目1中描述的QPCR分析结果。 4）单核细胞将HN30 Pro-MMP-1转换为主动MMP-1。 据信，基质细胞（例如单核细胞/巨噬细胞）与肿瘤细胞的相互作用与MMP的诱导和激活有关。 我们以前已经发现，与仅检测到pro-MMP-1的巨噬细胞培养物相比，单核细胞培养物中的MMP-1主要是活性形式。 在我们最近的研究中，我们显示了鳞状癌细胞系HN30产生大量的Pro-MMP-1，可以通过暴露于单核细胞而不是巨噬细胞中的细胞膜来转化为活性MMP-1。 单核细胞激活肿瘤细胞Pro-MMP-1的能力似乎是由于细胞表面丝氨酸蛋白酶活性。 纤溶酶原激活剂抑制剂-1（PAI-1）或α2-抗刺蛋白阻断这种转化，表明与细胞表面相关的纤溶酶原对单核细胞的作用。 PCR分析表明，单核细胞中的纤溶酶原mRNA水平较低。 当前的研究正在确定随着单核细胞成熟成巨噬细胞的成熟，纤溶酶原合成是否丢失。 这些结果表明，单核细胞对肿瘤进展的一种影响是MMP-1的激活。 5）与鳞状癌细胞调节MMP产生有关的信号通路。 对HN30细胞内源性产生MMP-1涉及的调节途径的检查表明，ERK1/2 MAPK是MMP-1的积极调节剂。 相反，当抑制磷酸化p38的作用时，p38 MAPK充当MMP-1的负调节剂，HN30细胞的MMP-1产生显着增加。 有趣的是，与具有较低水平的磷酸化ERK1/2和P38的HN30细胞相比，不产生高水平的MMP-1的NH12具有高水平的磷酸化ERK1/2和P38。 但是，当用纤溶酶刺激HN12细胞时，它们会分泌可检测到的MMP-1水平。 初步发现表明，这可能是由于与p38相比，磷酸化的ERK1/2的比例更高。