Immunohistochemical and ultrastructural studies of the effects of prednisolone on transformation of fibroblast to regenerated mesothelial cells

We have proposed in the past that chest wall fibroblasts are transformed to regenerated mesothelial cells. This study was conducted to investigate the effects of prednisolone on the differentiation and migration of fibroblasts in their transformation to mesothelial cells. Rat fibroblasts harvested from intercostal thoracic wall specimens were cultured in culture medium until cell spheroids were formed. An experimental cell spheroid group to whose culture medium prednisolone had been added and a control spheroid group with no addition of prednisolone were then subjected to immunohistochemical and ultrastructural studies of the changes in the fibroblasts with the passage of time. On days 1 and 2 of culture, the fibroblasts in each group were cytokeratin negative. However, on day 3 the control group became cytokeratin positive, and ultrastructural observations revealed formation of macula adherens and microvilli. In contrast, the experimental group fibroblasts remained cytokeratin negative even on day 3, but became cytokeratin positive on day 5 of culture. Macula adherens and microvilli also manifested on day 5. Prednisolone inhibited the differentiation and migration of fibroblasts, but it was surmised that fibroblasts that have resisted from the effects of prednisolone finally differentiate into mesothelial cells which have formed macula adherens.This study was presented at the 33rd annual meeting of the Clinical Electron Microscopy Society of Japan, Nagasaki, September 27, 2001     

包皮成纤维细胞作为饲养层对人胚胎生殖细胞生长的影响

目的 以人包皮成纤维细胞为饲养层体外培养人胚胎生殖细胞,观察饲养层对其生长的影响,并对人胚胎生殖细胞进行鉴定。 方法 分离培养4～5岁小儿包皮成纤维细胞,取P₃～P₃₀代细胞用丝裂霉素灭活后铺板备用,酶联免疫吸附测定（ELISA）双抗夹心法检测其分泌物成纤维细胞生长因子（FGF）的含量;分离培养5～11周人胚胎原始生殖细胞,在不添加任何细胞因子的人包皮成纤维细胞饲养层上培养人胚胎生殖细胞;细胞化学法检测人胚胎生殖细胞碱性磷酸酶的活性,免疫细胞化学法检测其胚胎表面特异性抗原SSEA-1及SSEA-4的表达,RT-PCR法检测Oct-4的表达。 结果 包皮成纤维细胞可以传60代以上,P₃～P₃₀代均适宜作饲养层;P₃～P₃₀代包皮成纤维细胞上清液中FGF含量在(172.09±2.66)pg/L～(245.25±1.66)pg/L之间波动。分离培养的人胚胎生殖细胞在饲养层上可形成典型的胚胎生殖细胞集落,体外连续培养超过3代,其碱性磷酸酶活性呈强阳性,集落未分化标志检测显示SSEA-l、SSEA-4呈阳性,Oct-4表达阳性。 结论 用人包皮成纤维细胞作为饲养层能支持人胚胎生殖细胞的生长并维持未分化状态。     

Growth characteristics of fibroblasts isolated from the body and limb of the Caspian miniature horse and the effect of hydrocortisone in vitro

The present study was designed to determine the in vitro effects of hydrocortisone on the growth of fibroblasts isolated from both the neck and distal limb of the Caspian miniature horse. Three grammes of subcutaneous tissue was harvested from the neck and metacarpi of four Caspian miniature horses and tissue from each site was placed in a separate culture medium. After separation of fibroblasts using explant culture method, a concentration of 10,000?cells/ml was placed into each well of a 24-well plate. Hydrocortisone was added in three different concentrations (control group: without hydrocortisone, treatment groups A, 10; B, 100 and C, 300?μg/ml) into the culture medium containing fibroblasts, 4?days later the cells were counted. The effects of hydrocortisone on fibroblasts growth and viability were evaluated. Results show that the mean fibroblast growth rate from neck-derived fibroblasts was significantly greater than those derived from the limb. There were no significant differences in fibroblast growth rate between the control group and group A in both neck and limb-derived fibroblast (P?>?0.05), but groups B and C suppressed fibroblast growth significantly in comparison with their related control groups (P?<?0.05). Cell viability decreased significantly in treatment groups B and C in comparison with the control group in both neck and limb-derived fibroblasts. The mean percentage of fibroblast growth inhibition from the neck and limb significantly decreased in treatment groups B and C in comparison with their related control groups. The effect of hydrocortisone on cultured fibroblast growth was dose-dependant.     

Colorectal cancer cells activate adjacent fibroblasts resulting in FGF1/FGFR3 signaling and increased invasion

Cancer-associated fibroblasts expressing fibroblast activation protein (FAP) have been implicated in the invasive behavior of colorectal cancer. In this study, we use FAP expression as a marker of fibroblast activation and analyze the effect of activated fibroblasts on colorectal cancer migration and invasion in experimental cell studies. We also investigated the expression pattern of FAP in cancer-associated fibroblasts during transformation from benign to malignant colorectal tumors. In immunohistochemical analyses, FAP was expressed in fibroblasts in all colorectal cancer samples examined, whereas all normal colon, hyperplastic polyps, or adenoma samples were negative. In in vitro studies, conditioned medium from colon cancer cells, but not adenoma cells, activated fibroblasts by inducing FAP expression. These activated fibroblasts increased the migration and invasion of colon cancer cells in Boyden chamber experiments and in a three-dimensional cell culture model. We identify fibroblast growth factor 1/fibroblast growth factor receptor 3 (FGF1/FGFR-3) signaling as mediators leading to the increased migration and invasion. Activated fibroblasts increase their expression of FGF1, and by adding a fibroblast growth factor receptor inhibitor, as well as an FGF1-neutralizing antibody, we reduced the migration of colon cancer cells. Our findings provide evidence of a possible molecular mechanism involved in the cross talk between cancer cells and fibroblasts leading to cancer cell invasion.     

Mesothelial cells produce a chemoattractant for lung fibroblasts: role of fibronectin

Pleural fibrosis may complicate several types of non-exudative pleural injury. Although the pathogenesis of such lesions is poorly understood, it is conceivable that mesothelial cells may recruit fibroblasts to sites of pleural damage. In order to test this possibility, conditioned medium from cultured rat mesothelial cells was tested for chemoattractant activity towards RL-87 rat lung fibroblasts. For this purpose, rat pleural or pericardial mesothelial cells were maintained in vitro for 6 to 96 h. Conditioned medium from each source was obtained at defined culture times and tested for chemotactic activity in a 48-well microchemotaxis assembly. A progressive, time-dependent increase in fibroblast chemoattractant activity was detected in both pleural and pericardial mesothelial cell conditioned medium samples. This effect was maximal in 96-h cultures. Checkerboard analysis revealed that the conditioned medium was truly chemotactic for lung fibroblasts. Characterization of the chemoattractant demonstrated that it was a nondialyzable (greater than 16 kD), thermolabile (100 degrees C for 15 min), acid-stable (pH 2.5), trypsin-sensitive, and pepsin-sensitive protein. The chemotaxin was shown to be fibronectin, since activity was abolished, in a dose-dependent manner, by treatment with anti-rat fibronectin antiserum as well as by passage through a gelatin agarose affinity column. This product consisted of two bands on sodium dodecyl sulfate polyacrylamide gel electrophoresis of apparent molecular masses 250 and 220 kD. The secretion of a mesothelial cell-derived fibroblast chemoattractant may play a role in the response of the pleura to injury and in the pathogenesis of pleural fibrosis.     

人胚胎成纤维细胞对人胚胎生殖细胞生长的作用

目的：研究人胚胎成纤维细胞对人胚胎生殖细胞（EG细胞）生长的作用。方法：采用组织块体外培养法体外培养人EG细胞,不添加任何细胞因子,利用源于胚胎组织自身的成纤维细胞作为饲养层,收集培养3d和9d的上清液,用抗体夹心ABC-ELISA法定量检测其白血病抑制因子（LIF）、干细胞生长因子（SCF）和碱性成纤维细胞生长因子（bFGF）的含量。培养的细胞用免疫细胞化学法进行SSEA-3、OCT-4的检测。结果：培养9d的上清液中3种因子均含量为LIF55．25pg／ml、SCF90．39pg／ml、bFGF26．06pg／ml．均高于培养3d相应的平均含量。培养的细胞SSEA-3、OC-4呈强阳性表达。结论：上清液中LIF、SCF和bFGF的含量与胚胎成纤维细胞的生长呈正比,胚胎成纤维细胞能分泌这些细胞因子,以维持EG细胞体外增殖并抑制其分化。     

Effects of Culture Medium on Cystic Fibrosis and Normal Fibroblasts Studied by X-Ray Microanalysis

The effect of culture medium from fibroblast cultures of cystic fibrosis (CF) patients and healthy controls on the elemental composition of fibroblasts was investigated by X-ray microanalysis. Exposure of normal fibroblasts to culture medium from CF fibroblasts caused an increase in calcium level. Exposure of CF fibroblasts to culture medium from normal cells caused an increase of the sodium content of CF cells to approximately normal levels; the calcium level of the CF fibroblasts, however, remained abnormally high. The results may indicate that CF fibroblasts lack a factor needed for the regulation of sodium transport. CF fibroblast medium apparently contains a factor that interferes with the regulation of calcium transport.     

Effects of Culture Medium on Cystic Fibrosis and Normal Fibroblasts Studied by X-Ray Microanalysis

The effect of culture medium from fibroblast cultures of cystic fibrosis (CF) patients and healthy controls on the elemental composition of fibroblasts was investigated by X-ray microanalysis. Exposure of normal fibroblasts to culture medium from CF fibroblasts caused an increase in calcium level. Exposure of CF fibroblasts to culture medium from normal cells caused an increase of the sodium content of CF cells to approximately normal levels; the calcium level of the CF fibroblasts, however, remained abnormally high. The results may indicate that CF fibroblasts lack a factor needed for the regulation of sodium transport. CF fibroblast medium apparently contains a factor that interferes with the regulation of calcium transport.     

新生大鼠心肌成纤维细胞的内皮细胞分化潜能

目的 探讨心肌成纤维细胞（CFs）向内皮细胞分化以及成血管潜能。方法 新鲜左心室组织,体外分离、培养、纯化CFs,利用内皮细胞诱导液对第3代 CFs诱导培养,连续诱导培养28 d后,换用内皮细胞培养基（ECM）,并消化传代扩增至第3代。观察诱导细胞生长情况和形态变化,利用免疫细胞化学法、流式细胞术和血管形成分析实验对诱导后细胞的内皮细胞标志物的表达和功能特性进行评价。结果 新生大鼠第3代CFs,呈三角形、梭形和多边形,增殖速度迅速;波形蛋白（vimentin）、盘状结构受体2（DDR2）均呈阳性表达。诱导第3天细胞开始汇合,21 d部分细胞形成串珠样连接,28 d出现细胞汇集成环状样形状;免疫组织化学标记vWF和CD31呈阳性表达;细胞免疫荧光标记vWF、CD34、CD105均呈阳性表达;流式细胞术检测诱导后细胞表面标志物CD31表达率为50.5%,对照组CD31表达率为5.82%。结论 血管内皮生长因子（VEGF）、碱性成纤维细胞生长因子（bFGF）体外定向诱导CFs可使其向血管内皮细胞分化,并表现其功能特征。     

Basic fibroblast growth factor (bFGF) contributes to the enlargement of brown adipose tissue during cold acclimation

The contribution of basic fibroblast growth factor to brown adipose tissue (BAT) enlargement during cold acclimation was investigated using rat brown adipocytes in primary culture. After cold exposure (at 5° C) for 28 days, the level of bFGF messenger ribonucleic acid (mRNA) in BAT of cold-acclimated rats was markedly increased with the increase in the BAT weight. In addition, the blood plasma from cold-acclimated rats considerably enhanced the expression of basic fibroblast growth factor mRNA in rat brown adipocytes. Likewise, the blood plasma from cold-acclimated rats significantly stimulated the growth of rat brown adipocyte precursor cells compared with that from warm-acclimated rats, whereas there was no difference of effect between the two blood plasmas on the growth of bovine capillary endothelial cells. Basic fibroblast growth factor, but not platelet-derived growth factor stimulated the growth of brown adipocyte precursor cells. The conditioned medium from brown adipocyte primary culture markedly stimulated the growth of bovine capillary endothelial cells and the effect was inhibited considerably by antibasic fibroblast growth factor antibody. These results suggest that some factors concerned with the growth of brown adipocyte precursor cells are present in the blood plasma from cold-acclimated rats, and that basic fibroblast growth factor produced by brown adipocytes may significantly contribute to BAT enlargement by autocrine mechanisms during cold exposure.     

Rat alveolar type II cells inhibit lung fibroblast proliferation in vitro

Fibroblasts stimulate alveolar type II epithelial cell differentiation and proliferation in vitro and during lung development. However, little is known about the effects of adult type II cells on fibroblasts. We investigated the effect of adult rat type II cells on proliferation of adult human lung fibroblasts. Fibroblasts were suspended within rat tail collagen which was gelled on a floating polycarbonate filter, and type II cells were cultured on Matrigel. In this coculture system, alveolar type II cells inhibited fibroblast proliferation and indomethacin blocked the inhibitory effect on fibroblast growth. Prostaglandin (PG) E2, the major PG secreted by type II cells, inhibited fibroblast proliferation and was increased during the period of inhibition of fibroblast proliferation. Incubation with arachidonate showed that most of the PGE2 in the coculture system was produced by the fibroblasts. In addition, we found that rat type II cells also inhibited rat fibroblasts and that inhibition of fibroblast growth by type II cells could be stimulated by keratinocyte growth factor. We conclude that in this coculture system, type II cells inhibit fibroblast proliferation by secreting a factor(s) that stimulates PGE2 production by fibroblasts, and that PGE2 directly inhibits fibroblast proliferation.     

Effect of chronic treatment with cystic fibrosis fibroblast medium on rat submandibular gland acinar cells

The effect of chronic treatment with cystic fibrosis (CF) fibroblast medium on rat submandibular gland and pancreas was investigated. Rats were injected for 8 days with conditioned medium from normal or CF fibroblasts. The elemental content of the acinar cells was measured by X-ray microanalysis of cryosections. A significant increase in cellular calcium, and a decrease in cellular sodium concentrations were found after treatment with CF medium. The ultrastructure of the submandibular acinar cells was not affected by the conditioned CF fibroblast culture medium. No effect of treatment with CF medium on ultrastructure and elemental content of pancreatic acinar cells could be demonstrated. The response to alpha-adrenergic, beta-adrenergic, cholinergic, and peptidergic stimulation in submandibular gland acinar cells of rats injected with normal or CF medium was investigated in vitro. With regard to changes in elemental composition after stimulation, no significant differences in response between the two groups could be found. Apparently, a factor in conditioned medium from cultured CF fibroblasts induces a net increase in calcium content of rat submandibular gland acinar cells. Possibly, this factor acts in a similar way in CF patients and may cause elevated calcium levels in CF cells.     

Leukotriene C4 induces TGF-beta1 production in airway epithelium via p38 kinase pathway

Cysteinyl leukotrienes (CysLTs) play an important role in the pathogenesis of airway remodeling. We investigated the interaction between epithelium and CysLTC4, and the contribution of this interaction to airway fibrosis. Human airway epithelial cells were grown on air-liquid interface culture inserts. CysLTC4 was employed to stimulate the cells. Conditioned medium following CysLTC4 stimulation was coincubated with human lung fibroblasts. Our results have demonstrated that CysLTC4 stimulates airway epithelial cells, through a p38 mitogen-activated protein kinase (MAPK) activation mechanism, to produce transforming growth factor beta1 (TGF-beta1), which results in fibroblast proliferation. The selective p38 MAPK inhibitor S203580 successfully inhibits p38 MAPK phosphorylation and subsequent TGF-beta1 production. CysLT1 receptor antagonist montelukast and corticosteroid inhibit TGF-beta1 production at the mRNA and protein levels. When treated with LTC4, the conditioned medium from epithelial cells enhances fibroblast proliferation, this mitogenic effect being attributed to TGF-beta1 and LTC4 remaining in the culture medium. In addition, LTC4 itself acts as a potential growth factor for lung fibroblasts. These data indicate that interactions between LTC4 and airway epithelial cells may contribute to the pathogenesis of airway remodeling. Early intervention to stop these processes may be useful in preventing airway fibrosis in chronic allergic inflammation.     

Effects of basic fibroblast growth factor and transforming growth factor-beta on maturation of human pediatric aortic cell culture for tissue engineering of cardiovascular structures

Optimal in vitro conditions are necessary for the development of a strong, well structured, and functional tissue engineered cardiovascular structure eventually designed for implantation. To further optimize in vitro conditions for cell proliferation and extracellular matrix formation in tissue engineering of cardiovascular structures, in this study, ascorbic acid and growth factors as additives to standard cell culture medium were evaluated for their effect on tissue development in vitro. Biodegradable polymer patches [polyglycolic acid (PGA) coated with poly-4-hydroxybutyrate (P4HB)] were seeded with human pediatric aortic cells and cultured for 7 and 28 days. Group A was cultured with standard medium (DMEM with 10% fetal calf serum and 1% antibiotics) supplemented with ascorbic acid; group B was cultured with standard medium plus ascorbic acid and basic fibroblast growth factor (bFGF); group C was cultured with standard medium adding ascorbic acid and transforming growth factor (TGF). Analysis of the cell seeded polymer constructs included DNA assay, collagen assay, and histologic and immunohistochemical examination for cell proliferation and collagen formation. After 7 and 28 days of culture, group B and group C showed a significantly higher DNA content compared with group A. The addition of bFGF (group B) led to a markedly higher collagen synthesis after 28 days of culture compared with the additives in groups C and A. The histologic and immunohistochemical examination also revealed a more dense, organized tissue development with pronounced matrix protein formation in the tissue engineered structures in group B after 28 days of culture. When seeded on to the polymeric scaffold, human vascular cells proliferate and form organized cell tissue after 28 days of culture. The addition of bFGF and ascorbic acid to the standard medium enhances cell proliferation and collagen synthesis on the biodegradable polymer, which leads to the formation of more mature, well organized tissue engineered structures.     

Oral fibroblasts produce more HGF and KGF than skin fibroblasts in response to co-culture with keratinocytes

The production of hepatocyte growth factor (HGF) and keratinocyte growth factor (KGF) in subepithelial fibroblasts from buccal mucosa, periodontal ligament, and skin was determined after co-culture with keratinocytes. The purpose was to detect differences between the fibroblast subpopulations that could explain regional variation in epithelial growth and wound healing. Normal human fibroblasts were cultured on polystyrene or maintained in collagen matrix and stimulated with keratinocytes cultured on membranes. The amount of HGF and KGF protein in the culture medium was determined every 24 h for 5 days by ELISA. When cultured on polystyrene, the constitutive level of KGF and HGF in periodontal fibroblasts was higher than the level in buccal and skin fibroblasts. In the presence of keratinocytes, all three types of fibroblasts in general increased their HGF and KGF production 2-3 times. When cells were maintained in collagen, the level of HGF and KGF was decreased mainly in skin cultures. However, in oral fibroblasts, induction after stimulation was at a similar level in collagen compared to on polystyrene. Skin fibroblasts maintained in collagen produced almost no HGF whether with or without stimulation. The results demonstrate that the secretion of KGF and HGF in both unstimulated fibroblasts and in fibroblasts co-cultured with keratinocytes is dependent on the type of fibroblasts. In general, the periodontal fibroblasts had the highest level of cytokine production. This high level of growth factor production may influence the proliferation and the migration of junctional epithelium and thereby influence the development of periodontal disease.     

碱性成纤维细胞生长因子基因转染优化脐带间充质干细胞的培养

背景：目前多采取重组腺相关病毒作为载体介导碱性成纤维细胞生长因子基因转染的方法,将外源性碱性成纤维细胞生长因子基因转入脐带间充质干细胞内并持续表达,调控细胞增殖及定向分化,取得高效持久的治疗作用。 目的：了解采用重组腺相关病毒作为载体介导碱性成纤维细胞生长因子基因转染对体外培养的脐带间充质干细胞增殖及细胞周期的影响。 方法：体外培养脐带间充质干细胞,经重组腺相关病毒作为载体介导碱性成纤维细胞生长因子基因转染,分为对照组、空载病毒组、碱性成纤维细胞生长因子转染组。用RT-PCR,Western blot检测脐带间充质干细胞在转染前后碱性成纤维细胞生长因子基因和蛋白的表达。应用细胞生长曲线、CCK-8观察细胞生长的优化作用,采用流式细胞术测定细胞周期分布的变化。 结果与结论：转染碱性成纤维细胞生长因子后,转染组与对照组、空载病毒组相比碱性成纤维细胞生长因子基因和蛋白水平均有表达,细胞的生长速度明显增快,细胞周期G0/G1期减少,S期细胞数增多,各组间差异有显著性意义(P < 0.05)。说明,通过重组腺相关病毒作为载体介导碱性成纤维细胞生长因子基因转染能促进体外培养的脐带间充质干细胞增殖,对其培养有优化作用。     

Interstitial pulmonary macrophages produce platelet-derived growth factor that stimulates rat lung fibroblast proliferation in vitro.

Alveolar macrophages from humans and several animal species produce factors in vitro that modulate fibroblast growth and have been proposed as mediators of interstitial pulmonary fibrosis. Pulmonary interstitial macrophages (IMs) have not been studied previously in this regard. Pulmonary IMs were isolated from prelavaged rat lungs by enzymatic digestion of tissue and subsequent differential adherence of cells to culture dishes. The ability of IMs to release modulators of fibroblast growth into the culture medium was assessed by measuring [3H]thymidine incorporation into DNA and/or nuclear labeling of early-passage rat lung fibroblasts exposed to medium conditioned by IMs. The percentages of nuclei labeled in fibroblast cultures exposed to interstitial macrophage-conditioned medium (IMCM) alone did not significantly differ from that observed in controls, but fibroblasts exposed to IMCM supplemented with 2% platelet-poor plasma showed a 2.6-fold increase in labeling, indicating that IMCM contains predominantly "competence" growth factor activity. Similar results were obtained using purified human platelet-derived growth factor (PDGF). The level of growth factor activity released by IMs increased in cells that had phagocytized iron spheres during the culture period. In addition, fractionation of IMCM by high-performance liquid chromatography demonstrated most of the growth factor activity at a relative molecular mass of about 35 kd. Subsequent quantitative analysis of the fractions by an enzyme immunoassay for PDGF demonstrated that IMCM contains a homologue of human PDGF. These results show that IMs are capable of producing a PDGF-like growth factor for autologous fibroblasts and that release of this factor is enhanced by exposure to an insoluble inorganic particle. Because PDGF is a potent growth factor for fibroblasts and is released by IMs, it is essential to ask in future studies whether this or similar macrophage products play a significant role in mediating fibroblast proliferation in vivo.     

Bioactive glass stimulates the secretion of angiogenic growth factors and angiogenesis in vitro

Neovascularization of tissue-engineered constructs remains a limiting factor for the engineering of larger tissue constructs. Attempts to stimulate neovascularization, using recombinant protein or gene transfer of angiogenic growth factors, have been proposed; however, these approaches have been associated with problems regarding the delivery and duration of exposure of the growth factor. This study was performed to determine the ability of biologically active glass to stimulate the secretion of angiogenic growth factors from human stromal cells and subsequent angiogenesis. CCD18Co human fibroblasts were cultured on tissue culture surfaces coated with specific quantities of 45S5 Bioglass particles. At 24-, 48-, and 72-h intervals the gene expression of vascular endothelial growth factor (VEGF) and the protein secretion of VEGF and basic fibroblast growth factor (bFGF) from fibroblasts were measured. The effect of conditioned medium collected from Bioglass-stimulated fibroblasts on human dermal microvascular endothelial cells was assessed using in vitro angiogenesis assays. Results showed that surfaces coated with Bioglass produced a significant increase in the secretion of VEGF and bFGF. Conditioned medium from stimulated fibroblasts significantly increased the proliferation of human dermal microvascular endothelial cells and induced a significant increase in the formation of anastomosed networks of human endothelial cell tubules. It is concluded that the ability of 45S5 Bioglass to stimulate the release of angiogenic growth factors and to promote angiogenesis provides a novel alternative approach for stimulating neovascularization of tissue-engineered constructs.     

Induction of fibroblast apoptosis by anti-CD44 antibody: implications for the treatment of fibroproliferative lung disease.

Fibroblast migration and proliferation within the alveolar wall and airspace after lung injury can lead to the development of fibrosis. Fibroblast cell surface CD44 is an adhesion receptor for provisional matrix proteins and mediates fibroblast invasion into fibrin matrices. Here we show that incubation of cultured fibroblasts with an anti-CD44 monoclonal antibody induces fibroblast detachment from the substratum and morphological changes compatible with apoptosis. In addition, we show that anti-CD44 monoclonal antibody rapidly induces fibroblast apoptosis within fibrin matrices. The effect of anti-CD44 antibody on induction of fibroblast apoptosis occurred within 8 hours and was dose dependent. Anti-CD44 antibody also induced fibroblast apoptosis in suspension. Furthermore, fibroblasts plated on anti-CD44-antibody-coated surfaces initially attached and spread on the antibody; however, after an 8-hour incubation time, many of the cells developed characteristic morphological features of apoptosis. Collectively, these data indicate that apoptosis did not result solely due to detachment from the substratum. Our results identify a new function for fibroblast cell surface CD44 related to the control of cell viability. We suggest this function may be important in fibroblast population control and could potentially be exploited in designing anti-fibrotic therapies.     

The Fibroblast Mitogenic Activity Released from Human Basophilic Cell Line KU812 is Separate from Tryptase and PDGF Expression

The human leukaemia cell line KU812 has previously been used to study basophil differentiation. In this study the authors analysed the capacity of KU812 to produce the mast cell proteinase tryptase and to synthesize factor(s) mitogenic for fibroblasts. KU812 cells were treated with tetradecanoyl-phorbol-13-acetate (TPA), conditioned medium from the human T-cell line Mo (Mo-CM), or cultured under serum free conditions. After 4 days the cells were analysed for cell growth, differentiation, content of tryptase, and secretion of fibroblast mitogenic activity. Mo-CM and serum starvation increased the expression while TPA treatment down-regulated the expression of FcεRI-α chain. An increase in tryptase content in cell extracts was detected after 4 days of culture in serum-free medium or in the presence of Mo-CM. KU812 conditioned media was found to have a baseline expression of mitogenic activity on normal human foreskin fibroblasts that was increased after serum starvation or after treatment with TPA. Mast cell-derived tryptase has previously been reported to be mitogenic for fibroblasts, but in this study the expression of tryptase did not correlate with the expression of fibroblast mitogenic activity in KU812 cells. Furthermore, affinity-purified lung tryptase did not show any mitogenic activity. Platelet-derived growth factor was also excluded. Although the factor(s) from KU812 cells stimulating fibroblast proliferation have not been identified, our results indicate that basophils may be potential producers of growth factors inducing fibroblast proliferation.