High serum levels of M-CSF and G-CSF in Kawasaki disease

To examine any role of macrophage colony-stimulating factor (M-CSF), in the immune responses in Kawasaki disease (KD), we serially assayed M-CSF and several related cytokines using ELISA. In 10 paediatric patients with KD the level of M-CSF was significantly higher in the acute phase than in the convalescent phase (1476.1 +/- 443.6 v 805.0 +/- 184.7 U/ml). Higher levels of serum granulocyte colony-stimulating factor (G-CSF) and interleukin-6 were also found in the acute phase. These results suggest that M-CSF, G-CSF and interleukin-6, derived from monocytes as monokines or derived from vascular endothelial cells, might play an important role in the acute phase of KD.     

Differential expression and regulation of protease-activated receptors in human peripheral monocytes and monocyte-derived antigen-presenting cells

Protease-activated receptors (PARs) are stimulated by proteolytic cleavage of their extracellular domain, unmasking a new N-terminus acting as tethered ligand. Whereas the role of PARs in platelets is well known, their presence and function in human monocytes and other antigen-presenting cells has not been characterized. Here it is demonstrated that human peripheral monocytes and monocyte-derived macrophages and dendritic cells differentially express PARs. Human monocytes express mainly PAR1 and less PAR3. Differentiation of monocytes into macrophages by either macrophage colony-stimulating factor (M-CSF) or granulocyte-macrophage colony-stimulating factor (GM-CSF) elicits enhanced expression of PAR1, PAR2, and PAR3. In contrast, dendritic cells differentiated from monocytes by GM-CSF and interleukin-4 (IL-4) strongly down-regulated PAR1, PAR2, and PAR3, both at the mRNA and the protein level. Down-regulation of the PAR expression was apparently due to IL-4, because treatment of macrophages with IL-4 caused down-regulation of PAR1, PAR2, and PAR3. PAR4 mRNA expression remained undetectable in any of the cell types investigated. Stimulation of PAR1, PAR2, and PAR3 with thrombin, trypsin, or established receptor-activating peptides (PAR-APs) triggered cytosolic Ca2+ responses, indicating functionally active PARs. Further, stimulation of monocytes or macrophages with thrombin or PAR1-AP, but not with PAR2-or PAR4-AP, triggers expression of monocyte chemoattractant protein-1 (MCP-1) both at the mRNA and the protein level. These data demonstrate that differentiation of human monocytes is associated with differential expression of functionally active PARs that mediate distinct regulatory functions in inflammation and atherogenesis.     

Lymphokine regulation of HLA-DR gene expression in human thyroid cell monolayers

Studies were conducted to examine the regulation of HLA class II gene expression in human thyroid cells in vitro. Normal human thyroid cells cultured in the absence of lectin or gamma-interferon stimulation lacked detectable HLA-DR cell surface antigen, although low levels of DR alpha-chain-specific mRNA were present. Cyclosporine A, known to inhibit lymphokine production, inhibited basal as well as lectin-mediated increases in levels of DR alpha-chain-specific mRNA and DR surface antigen expression on normal human thyrocytes. Cyclosporine had no effect on the induction of DR antigen gene expression by recombinant gamma-interferon. These data suggested that lectin enhancement of DR antigen expression in human thyroid cells may be mediated by a lymphokine(s) produced in primary human thyroid cell monolayers. This suggestion was confirmed by studies that demonstrated the abrogation of lectin responsiveness by antibody directed against gamma-interferon. Indirect immunofluorescence studies using flow cytometric analyses identified 1.6 +/- 0.2% (mean +/- SD) of cells in primary thyroid cultures as T lymphocytes, a potential source of lymphokine production. Cells derived from thyroid follicular adenomas and carcinomas demonstrated reduced lectin-mediated increases in DR antigen expression compared to normal thyroid cells. DR expression could be enhanced in these lectin-treated cells, however, by T cell coculture. Dose-response studies demonstrated that human thyroid cells were as sensitive to gamma-interferon induction of DR antigen expression as human monocyte/macrophages. These results indicate that human thyroid cell HLA-DR antigen gene expression is sensitive to low levels of lymphokines, such as gamma-interferon; an intrathyroidal T cell population, which may serve as a source of lymphokine(s), remains associated with thyroid epithelial cells in primary thyroid cultures; and lymphokine-thyroid cell interactions may be implicated in the immunopathology of human autoimmune thyroid disease.     

Downregulation of c-fms gene expression in human monocytes treated with phorbol esters and colony-stimulating factor 1

The colony-stimulating factor-1 (CSF-1) regulates survival, growth, and differentiation of monocytes by binding to a single class of high-affinity receptors. The CSF-1 receptor is identical to the product of the c-fms protooncogene. The present studies monitored the effects of TPA and CSF-1 on c-fms gene expression in human monocytes. The results demonstrate that TPA downmodulates the constitutive expression of c-fms mRNA to low but detectable levels. Treatment of human monocytes with TPA was similarly associated with decreases in levels of the 138- and 125-Kd c-fms-encoded proteins. However, the kinetics of c-fms protein downmodulation indicated independent effects of TPA on c-fms expression at the RNA and protein levels. Furthermore, c-fms protein levels subsequently recovered despite persistently low levels of c-fms mRNA. Although previous studies demonstrated that c-fms protein is down-regulated in the presence of CSF-1, the present results indicate that CSF-1 also downregulates levels of c-fms mRNA. Moreover, the results indicate that CSF-1 increases protein kinase C activity in the membrane fraction. Together, these findings suggest that c-fms gene expression is differentially regulated at both the RNA and protein levels after activation of protein kinase C in human monocytes treated with TPA and CSF-1.     

阿托伐他汀钙对原代成骨细胞OPG、RANKL、M—CSF基因表达的影响

目的观察不同浓度阿托伐他汀钙对体外培养原代成骨细胞增生和对OPG、RANKL、M—CSF基因表达的影响,探讨阿托伐他汀钙抗骨质疏松的分子机制,为临床用药提供实验依据。方法采用胰酶和I型胶原酶混合酶消化法分离获得原代成骨细胞;采用差速贴壁法进行纯化。光镜下观察细胞的形态结构和生长状况,通过碱性磷酸酶染色和I型胶原免疫组化SABC法对细胞进行鉴定。用不同浓度的阿托伐他汀钙（10^-9、10^-8、10^-7、10^-6和10^-5mol／L）干预成骨细胞,在24、48和72h后,用实时荧光定量方法检测OPGmRNA、RANKLmRNA、M—CSFmRNA的表达水平。结果镜下观察显示细胞具有成骨细胞的典型特征,细胞多为单核,呈三角、多边、长梭等形态,并伸有粗大伪足;碱性磷酸酶染色及I型胶原免疫组化染色呈阳性。实时荧光定量PCR结果显示：不同浓度的阿托伐他汀钙（10^-9、10^-8、10^-7、10^-6、10^-5mol／L）均可以促进OPGmRNA的表达,抑制RANKLmRNA的表达,对M—CSFmRNA基因的表达也具有促进作用,且与药物浓度及作用时间有关。结论采用胰酶和I型胶原酶混合酶消化法体外分离成骨细胞,采用多次“差速贴壁法”可以获得数量多、纯度高且活性好的成骨细胞。阿托伐他汀钙可以促进成骨细胞OPG基因mRNA的表达,抑制RANKL基因mRNA的表达,对M—CSF基因的表达也有一定的促进作用。     

Expression of ornithine decarboxylase gene in elderly human monocytes

The proliferative capacity of the immune system is impaired in elderly subjects and the expression of various genes involved in cell cycle progression is reduced in PHA stimulated lymphocytes during the aging process. Macrophages play a fundamental role in the immune system response. It has recently been demonstrated that the process of macrophage activation is accompanied by a rapid, transient rise of ornithine decarboxylase (ODC) mRNA levels. In fact, the ODC gene seems to be involved in macrophage activation and differentiation. The authors demonstrated that the steady-state levels of ODC mRNA and the correlated superoxide anion production are lower in the monocytes of elderly subjects with respect to those in young subjects used as control. These results confirmed the impaired immune function of the elderly.