Cytokine measurement in lymphocyte culture supernatant of inactive lepromatous leprosy patients

The aim of the present study was to determine the effects of stimulation of sonicated Mycobacterium leprae (MLS) extract and phorbol myristate acetate (PMA) on the pattern of cytokine production in peripheral blood mononuclear cells (PBMC) and to find out whether there is any difference between stimulation of MLS extract and PMA. Blood samples were collected and PBMC isolated from 43 inactive lepromatous leprosy patients. After culture for 24 hours, lymphocytes were stimulated with MLS extract and PMA. In the culture supernatant, IL-2, 4, 6, 8, TNF-alpha and TGF-beta levels were measured by using ELISA. M. leprae stimulated group IL-6, IL-8, TNF-alpha, TGF-beta levels were found significantly higher than PMA stimulated group (P < 0.05). However, there was no difference between the two groups for IL-4. Only IL-2 levels were higher in PMA stimulated group than M. leprae stimulated group. Sonicated M. leprae extract have a strong effect on cytokine levels in vitro. Our results suggest that antigens with varying specificities favour the production of distinct cytokine patterns following in vitro restimulation.     

Immunocytochemical localization of inducible nitric oxide synthase and transforming growth factor-beta (TGF-β) in leprosy lesions

Inducible nitric oxide synthase (iNOS) and TGF-β were localized by immunocytochemistry in skin lesions from patients across the leprosy spectrum, and from patients undergoing reversal reaction. iNOS expression was highest at the tuberculoid pole of the spectrum, and increased during reversal reaction. TGF-β was observed throughout the leprosy spectrum, but was highest at the lepromatous pole. Levels of TGF-β decreased during reversal reaction. Reduced levels of TGF-β may contribute to unregulated inflammatory responses during reactional episodes.     

Roles of tumour necrosis factor-alpha (TNF-alpha), transforming growth factor-beta (TGF-beta), and IL-10 in the modulation of intercellular adhesion molecule-1 (ICAM-1) expression by macrophages during mycobacterial infection

Profiles of ICAM-1 expression on cultured murine peritoneal macrophages infected with Mycobacterium avium complex (MAC) were examined, with special reference to modulating roles of TNF-alpha, TGF-beta, and IL-10. When macrophages were infected with MAC, ICAM-1 expression, measured by microscopic counting of ICAM-1+ macrophages stained with anti-ICAM-1 antibody, ELISA, and flow cytometric analysis, was rapidly increased, peaking at day 3 (early-phase up-regulation) due to endogenous TNF-alpha, and thereafter gradually declined to the normal level within 1 week or more (late-phase down-regulation). The late-phase ICAM-1 down-regulation was also seen in macrophages phagocytosing heat-killed MAC and those stimulated with lipopolysaccharide but not in macrophages phagocytosing latex beads. ICAM-1 mRNA expression was augmented markedly at day 1 after MAC infection and thereafter decreased. While TNF-alpha and IL-10 production by MAC-infected macrophages was observed during the first 3 days, TGF-beta production was initiated from day 3 and continued until day 14. Exogenously added TGF-beta strongly inhibited the early-phase increase in ICAM-1 expression by infected macrophages, and the blockade of endogenous TGF-beta with anti-TGF-beta antibody markedly inhibited late-phase ICAM-1 down-regulation. Moderate blocking effect was also observed for anti-IL-10 antibody. On the other hand, late-phase ICAM-1 down-regulation was not prevented by the addition of exogenous TNF-alpha. Therefore, TGF-beta and IL-10, especially the former, appear to play active roles in the late-phase down-regulation of ICAM-1 in MAC-infected macrophages during long-term cultivation.     

Low-density lipoproteins enhance transforming growth factor-beta 1 (TGF-beta 1) and monocyte chemotactic protein-1 (MCP-1) expression induced by cyclosporin in human mesangial cells.

Cyclosporin (CsA) is widely used in the treatment of renal disease and transplantation, which are often complicated by alterations of lipid metabolism. Both chronic administration of CsA and hyperlipidaemia have been shown to evoke an early macrophage influx and have progressively led to glomerular and interstitial sclerosis. MCP-1 is the major monocyte chemoattractant secreted by stimulated mesangial cells and TGF-beta 1 is a key mediator of fibrogenesis in chronic progressive renal fibrosis. Thus, the combined effect of CsA and low-density lipoprotein (LDL) on the gene and protein expression of MCP-1 and TGF-beta 1 in cultured human mesangial cells (HMC) was explored. Both agents induced an early and persistent increase of MCP-1 and TGF-beta 1 mRNA levels and protein release. The simultaneous addition of CsA and LDL did not display any additive effect on target gene expression, but it caused a synergistic effect on MCP-1 and TGF-beta 1 protein secretion into culture medium. On the other hand, CsA and LDL had different effects on cell proliferation: the latter increased DNA synthesis, whereas CsA inhibited both spontaneous and mitogen-stimulated mesangial cell growth. The study concludes that CsA and LDL display an additive effect on TGF-beta 1 and MCP-1 synthesis and release by HMC, thus possibly co-operating to induce an early macrophage influx and the subsequent mesangial expansion and increased extracellular matrix deposition. However, in contrast they seem to modulate HMC proliferation differently, which is a further critical event intimately involved in the development of glomerulosclerosis.     

转化生长因子β1对乳鼠心肌细胞转化生长因子结合蛋白2表达的影响

 目的：探讨转化生长因子β1（TGF-β1）在诱导心肌细胞表达转化生长因子结合蛋白2（LTBP2）中的作用及信号传导通路。 方法：培养乳鼠心肌细胞;实时定量聚合酶链式反应（Real-time PCR）、蛋白质印迹和免疫细胞化学方法检测不同时间和不同浓度的TGF-β1对大鼠乳鼠心肌细胞LTBP2基因及蛋白表达的影响;用TGF-β1相关信号通路阻断剂探讨TGF-β1调节LTBP2表达改变的信号传导机制。 结果：LTBP2基因表达随着TGF-β1浓度增加（0、2、5、10 ng/mL）而明显升高,在5 ng/mL时刺激最强（P < 0.05）;5 ng/mL的TGF-β1刺激下心肌细胞内LTBP2基因和蛋白表达的升高呈时间依赖性,均在12 h最高,24 h开始呈下降趋势（P < 0.05或P<0.01）;免疫细胞化学结果显示TGF-β1明显升高LTBP2的表达。信号传导通路研究显示TGF-β1在心肌细胞内主要通过ERK信号通路和PI3K信号通路诱导LTBP2的表达。 结论：TGF-β1在乳鼠心肌细胞内通过ERK信号通路和PI3K信号通路上调LTBP2的表达。     

Expression of transforming growth factor-beta isoforms and their receptors in chronic tendinosis

Chronic tendon lesions are degenerative conditions and may represent a failure to repair or remodel the extracellular matrix after repeated micro-injury. Since TGF-β is strongly associated with tissue repair, we investigated the expression of TGF-β isoforms (β1, β2 and β3) and their 2 signalling receptors (TGF-βRI and TGF-βRII) in normal and pathological Achilles tendons. In all tissues, all 3 TGF-β isoforms and the 2 receptors were present at sites of blood vessels. Cells in the matrix showed no staining for TGF-β1 or β3, while TGF-β2 was associated with cells throughout the normal cadaver tendon. Tissue from tendons with pathological lesions showed an increase in cell numbers and percentage TGF-β2 expression. TGF-βRII showed a wide distribution in cells throughout the tissue sections. As with TGF-β2, there was an increase in the number of cells expressing TGF-βRII in pathological tissue. TGF-βRI was restricted to blood vessels and was absent from the fibrillar matrix. We conclude that despite the presence and upregulation of TGF-β2, TGF-β signalling is not propagated due to the lack of TGF-βRI. This might explain why chronic tendon lesions fail to resolve and suggests that the addition of exogenous TGF-β will have little effect on chronic tendinopathy.     

High numbers of autoantigen-reactive mononuclear cells expressing interferon-gamma (IFN-gamma), IL-4 and transforming growth factor-beta (TGF-beta) are present in cord blood.

Umbilical cord blood of neonates and peripheral blood of healthy adults were analysed by in situ hybridization for numbers of mononuclear cells (MNC) expressing the cytokines IFN-gamma, TGF-beta and IL-4 mRNA without culture and after culture in the presence of acetylcholine receptor (AChR), myelin basic protein (MBP) and peripheral myelin protein P2. These antigens were chosen since they represent autoantigens in putatively immune-mediated neurological diseases. The numbers of cells expressing cytokine mRNA after 72 h culture in the presence of AChR, MBP and P2 were higher in cord blood than in peripheral blood of healthy adults. IFN-gamma, TGF-beta and IL-4 were always elevated in parallel. In cord blood there was a pronounced reactivity to several of the tested antigens, while such broad reactivity was not found in peripheral blood of healthy adults. No differences in cytokine mRNA expression were found between cord blood and peripheral blood of adults when cells were analysed without culture. The results show a capacity of cord blood cells to react to several autoantigens by the up-regulation of cytokine mRNA expression.     

Enhanced production of transforming growth factor-beta (TGF-beta) during autologous mixed lymphocyte reaction of systemic sclerosis patients.

Systemic sclerosis (SSc) is characterized by systemic fibrosis and microvascular lesions. As TGF-beta is suggested to be related to skin fibrosis, we examined the production of TGF-beta from peripheral mononuclear cells (MNC) of SSc patients. Since anti-TGF-beta neutralizing antibody improved the defective proliferative response in autologous mixed lymphocyte reaction (AMLR) of SSc patients, TGF-beta was thought to participate in the decreased AMLR of SSc patients. Greater amounts of TGF-beta in the active as well as in the latent forms were produced during AMLR of SSc patients than that of normal subjects. It was suggested that TGF-beta excessively produced from the MNC of SSc patients might play a major role in the fibrosis of the patients during AMLR-like in vivo responses.     

Transforming growth factor-beta type 1 receptor (ALK5) and Smad proteins mediate TIMP-1 and collagen synthesis in experimental intestinal fibrosis

Transforming growth factor β (TGF-β) is known to play a key role in intestinal fibrosis; however, the underlying mechanisms are not well understood. TGF-β signal transduction is through TGF-β receptors, including the TGF-β type 1 receptor. Most cell types contain a TGF-β type 1 receptor form known as activin receptor-like kinase 5 (ALK5), which propagates the signal to the nucleus through the phosphorylation of Smad2 and Smad3 proteins. Therefore, we assessed the effect of the disruption of TGF-β/ALK5/Smad signalling by an ALK5 inhibitor (SD-208) in two experimental animal models of intestinal fibrosis: anaerobic bacteria- and trinitrobenzensulphonic acid-induced colitis. In addition, isolated myofibroblasts were pretreated with SD-208 and exposed to recombinant TGF-β1. Finally, myofibroblasts were transfected with ALK5, Smad2, and Smad3-specific siRNA. Up-regulation of ALK5 and TIMP-1, phosphorylation of Smad2 and Smad3 proteins, and increased intestinal wall collagen deposition were found in both experimental animal models. These effects were decreased by SD-208. TGF-β1 treatment also induced phosphorylation of Smad2 and Smad3 and up-regulation of ALK5 protein, TIMP-1, and α2 type 1 collagen gene expression in isolated myofibroblasts. Again these effects were inhibited by SD-208. Also, ALK5, Smad2, and Smad3 siRNA abolished the induction of TIMP-1 and α2 type 1 collagen. Our findings provide evidence that the TGF-β/ALK5/Smad pathway participates in the pathogenesis of experimental intestinal fibrosis. These data show promise for the development of an effective therapeutic intervention in this condition.     

Remission of collagen-induced arthritis is associated with high levels of transforming growth factor-beta expression in the joint

Immunization of genetically susceptible strains of mice with heterologous type II collagen leads to the induction of a self-limiting polyarthritis that begins to subside around 10 days after onset of clinical disease. The aims of this study were to compare pro- and anti-inflammatory cytokine expression in the joints during the course of arthritis in order to identify cytokines involved in spontaneous remission of arthritis. DBA/1 mice were immunized with type II collagen and an immunohistochemical analysis of expression of proinflammatory cytokines [tumour necrosis factor (TNF)-alpha, interleukin (IL)-1beta and IL-6] and anti-inflammatory cytokines [IL-10, IL-1ra, transforming growth factor (TGF)-beta1, TGF-beta2 and TGF-beta3] in joints was carried out over the course of the disease. Both pro- and anti-inflammatory cytokines were found to be expressed in early arthritis. However, around 10 days after onset of arthritis, the level of expression of proinflammatory cytokines declined while the level of expression of anti-inflammatory cytokines, particularly TGF-beta1 and TGF-beta2, increased. Surprisingly, TNF-alpha continued to be expressed at low levels during the period of disease remission (30 days after onset). Blockade of TNF-alpha during the period of disease remission had no effect on TGF-beta expression. This study confirms that the level of inflammation in arthritis correlates strongly with the balance of pro- and anti-inflammatory cytokine expression in the joints. Of the anti-inflammatory cytokines studied, TGF-beta1 and TGF-beta2 predominate during the time of disease remission, suggesting that these cytokines are involved in regulating disease activity.     

Increased levels of interferon-gamma (IFN-gamma), IL-4 and transforming growth factor-beta (TGF-beta) mRNA expressing blood mononuclear cells in human HIV infection.

Evidence has been presented for the involvement of IFN-gamma, IL-4 and TGF-beta in AIDS. Measured plasma levels may, however, poorly reflect in vivo production, since cytokines act auto- and paracrinally and have very short half life in plasma. In situ hybridization with complementary DNA oligonucleotide probes was used to enumerate blood mononuclear cells expressing cytokine messenger RNA (mRNA). HIV-infected patients had elevated blood levels of cells expressing each of the cytokines, with predominance for cells expressing TGF-beta mRNA. All AIDS patients included had elevated numbers of IL-4 mRNA-expressing cells, and levels of cells expressing this cytokine correlated inversely with counts of CD4+ cells in blood, reflecting the involvement of Th2-like cells in later stages of HIV infection. The described approach should be useful in further studies of cytokines in HIV infection and other diseases.     

Detection of transforming growth factor-beta 1 in dermal lesions of different clinical forms of leprosy.

Immunohistochemical studies were performed to determine the presence and distribution of polypeptide transforming growth factor (TGF)-beta 1, a cytokine with macrophage-suppressing activity, in skin biopsies from 41 patients with different clinical forms of leprosy. We used an anti-TGF-beta 1 polyclonal antibody and the avidinbiotin-peroxidase (ABC complex) method. The results demonstrated that the lesions of the lepromatous and borderline lepromatous forms presented intense cytoplasm staining for TGF-beta 1 in the cells of the dermal infiltrate. A reaction of moderate intensity was observed in the cells of granulomas from borderline borderline cases, whereas no detectable immunoreaction was observed in granuloma cells from the tuberculoid and borderline tuberculoid forms. Considering that in the lepromatous leprosy form Mycobacterium leprae multiplies in the cytoplasm of macrophages and the lesions are diffuse and consist of poorly differentiated young macrophages, we believe that these alternations may be explained at least in part by the presence of TGF-beta 1 in the dermal infiltrate. Production of the cytokine may be induced by the presence of the bacillus itself and of its constituents, causing a mechanism of parasite evasion. Similarly, the absence of TGF-beta 1 in tuberculoid leprosy, which progresses with a specific immune response to M. leprae, may explain the intense differentiation of macrophage cells with the formation of well defined epithelioid granulomas capable of eliminating most of the bacilli.     

Analysis of the secretion pattern of monocyte chemotactic protein-1 (MCP-1) and transforming growth factor-beta 2 (TGF-beta2) by human retinal pigment epithelial cells.

Retinal pigment epithelial (RPE) cells, situated between the neurosensory retina and the vascularized choroid, form part of the blood-eye barrier and are important for homeostasis of the outer retina. These cells are able to produce a variety of cytokines which may play a role in the maintenance of the immunosuppressive milieu inside the eye and in intraocular inflammatory responses. In the present study, we investigated whether RPE cells secreted the anti-inflammatory cytokine TGF-beta2 and the proinflammatory cytokine MCP-1 in a polarized manner. Monolayers of human donor RPE cells were cultured on transwell filters. Secretion of TGF-beta2 and MCP-1 at either the apical or basal side of the RPE cell monolayers, that were not treated or stimulated with IL-1beta (200 U/ml), was analysed by ELISA. All three cell lines examined had a different TGF-beta2 secretion pattern. In two of the three donor RPE cell lines tested, TGF-beta2 secretion was polarized, but not in the same direction. TGF-beta2 secretion was not up-regulated by stimulation with IL-1beta. In contrast, IL-1beta strongly induced MCP-1 secretion preferentially into the basal compartment of all RPE monolayers tested. These data indicate that human RPE cells are able to secrete TGF-beta2 and MCP-1 in a polarized fashion. Our results suggest that MCP-1 can be secreted by RPE cells in the direction of choroidal vessels during inflammatory responses in the posterior part of the eye, which may limit damage to the neurosensory retina.     

转化生长因子-β1诱导大鼠毛乳头细胞转分化为成纤维细胞的研究

目的 研究大鼠触须毛乳头细胞在转化生长因子-β1（TGF-β1）诱导作用下转分化为成纤维细胞的可能性,从新的角度探讨增生性瘢痕的形成机制.方法 消化收集第4代毛乳头细胞,处理组以 TGF-β1（10 ng/mL）处理细胞4 d;对照组加入正常培养基（不含胎牛血清的DMEM/F12培养液）,每隔24 h观察两组细胞生长形态及生长方式;分别用实时一步法RT-PCR和流式细胞仪检测细胞α平滑肌肌动蛋白（α-SMA）、波形蛋白（Vimentin）的mRNA表达和蛋白表达,Western blotting检测成纤维细胞特异蛋白（FSP1）的表达.结果流式细胞仪检测TGF-β1诱导48 h、72 h后α-SMA表达明显低于对照组（P〈0.01）;TGF-β1诱导48 h、72 h、96 h后Vimentin表达明显高于对照组（P〈0.01）.实时一步法RT-PCR检测Vimentin的mRNA表达逐渐增强,α-SMA的mRNA表达在48 h后明显下降（P〈0.01）,但96 h后表达又有所增加;Western blotting法检测FSP1表达在诱导48 h后逐渐增加（P〈0.01）.结论 毛乳头细胞经TGF-β1诱导后,失去其典型的细胞形态及生长方式,而表现出成纤维细胞的细胞形态及生长方式;其相对特异性标记物α-SMA的表达下降,而成纤维细胞的相对特异性标记物Vimentin和FSP1表达增加.故TGF-β1可诱导大鼠毛乳头细胞转分化为成纤维细胞.     

Expression of MHC antigens by intestinal epithelial cells. Effect of transforming growth factor-beta 2 (TGF-beta 2).

The effect of interferon-gamma (IFN-gamma) and TGF-beta 2 on expression of MHC antigens by the human intestinal epithelial cell line HT-29 was examined by immunohistochemistry and flow cytometry. Untreated HT-29 cells constitutively expressed HLA-ABC but little HLA-DR. Expression of both molecules was increased by IFN-gamma (100 U/ml, 24 h). TGF-beta 2 at concentrations > or = 0.5 ng/ml given before or simultaneously with IFN-gamma, inhibited the IFN-induced expression of HLA-DR. Small increases in HLA-ABC expression by IFN-gamma were further increased by pretreatment with TGF-beta 2, while a strong induction of HLA-ABC was inhibited by the TGF-beta 2 pretreatment. Our results suggest that the inhibitory action of the TGF-beta 2 on both HLA-ABC and HLA-DR correlates with the degree of induction following IFN-gamma treatment. Since TGF-beta 2 is present in milk and is produced by gut epithelial cells, one of its possible functions may be to regulate expression of HLA antigens in the neonatal intestine and/or diseased intestine.     

Increased serum transforming growth factor-beta1 in human colorectal cancer correlates with reduced circulating dendritic cells and increased colonic Langerhans cell infiltration

Cancer-related cytokines may interfere with the differentiation and migration of dendritic cells (DCs) and with the associated up-regulation of co-stimulatory molecules in vitro. We determined whether cytokines affected the distribution and activation of DCs in patients with colorectal cancer by measuring the levels of serum cytokines [transforming growth factor (TGF)-beta1 and vascular endothelial growth factor (VEGF)], DC numbers and phenotype from peripheral blood and mesenteric lymph nodes draining the cancer, and the infiltration of DCs into colorectal cancer. A significant increase in the serum level of TGF-beta1 correlated with a significant reduction in the level of circulating DCs in cancer patients that was associated with an increased infiltration of Langerhans cells into colorectal mucosa. The prevalence but not intensity of co-stimulatory molecule expression in circulating and mesenteric lymph node DCs was reduced in patients with colorectal cancer compared to patients with inflammatory bowel conditions. There was no correlation between co-stimulatory molecule expression and serum TGF-beta1. Thus the circulating DC depletion in colorectal cancer could be explained by a TGF-beta1-related DC redistribution from the circulation into the colorectal cancer and adjacent mucosa where DC levels were increased. There was an impairment of DC activation within colorectal cancer that was not related to serum level of cytokines.