Enhancement by transforming growth factor-beta 1 (TGF-beta 1) of the proliferation of leukemic blast progenitors stimulated with IL-3.

We studied the effect of transforming growth factor-beta 1 (TGF-beta 1) on colony formation of leukemic blast progenitors from ten acute myeloblastic leukemia (AML) patients stimulated with granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3), interleukin-6 (IL-6), or interleukin-1 beta (IL-1 beta). These CSFs and interleukins by themselves stimulated the proliferation of leukemic blast progenitors without adding TGF-beta 1. G-CSF, GM-CSF, and IL-3 stimulated blast colony formation in nine patients, IL-6 stimulated it in five, and IL-1 beta stimulated in four. TGF-beta 1 significantly reduced blast colony formation stimulated by G-CSF, GM-CSF, or IL-6 in all patients. In contrast, TGF-beta 1 enhanced the stimulatory effect of IL-3 on blast progenitors from three cases, while in the other seven patients TGF-beta 1 reduced blast colony formation in the presence of IL-3. To study the mechanism by which TGF-beta 1 enhanced the stimulatory effect of IL-3 on blast progenitors, we carried out the following experiments in the three patients in which it occurred. First, the media conditioned by leukemic cells in the presence of TGF-beta 1 stimulated the growth of leukemic blast progenitors, but such effect was completely abolished by anti-IL-1 beta antibody. Second, the addition of IL-1 beta in the culture significantly enhanced the growth of blast progenitors stimulated with IL-3. Third, leukemic cells of the two patients studied were revealed to secrete IL-1 beta and tumor necrosis factor-alpha (TNF-alpha) constitutively; the production by leukemic cells of IL-1 beta and TNF-alpha was significantly promoted by TGF-beta 1. Furthermore, the growth enhancing effect of TGF-beta 1 in the presence of IL-3 was fully neutralized by anti-IL-1 beta antibody. These findings suggest that TGF-beta 1 stimulated the growth of blast progenitors through the production and secretion of IL-1 beta by leukemic cells.     

Effect of cytokines and growth factors on the macrophage colony-stimulating factor secretion by human bone marrow stromal cells

We have investigated the effect of growth factors, inflammatory and anti-inflammatory cytokines on the macrophage colony-stimulating factor (M-CSF) secretion by cultured human bone marrow stromal cells. Their production of M-CSF cultured in serum-free medium is enhanced in a time-dependent manner in response to tumour necrosis factor (TNF-)alpha and interleukin (IL-)4 but not to IL-1, IL-3, IL-6, IL-7, IL-10, SCF, granulocyte-macrophage colony-stimulating factor (GM-CSF), G-CSF, bFGF and transforming growth factor (TGF-)beta. The co-addition of IL-4 and TNF-alpha has a greater than additive effect on the secretion of M-CSF suggesting that they act synergistically. The anti-inflammatory molecules IL-10 and TGF-beta have no effect on the TNF-alpha-induced M-CSF synthesis by marrow stromal cells. In conclusion TNF-alpha and IL-4 are potent stimulators of the M-CSF synthesis by human bone marrow stromal cells, a result of importance regarding the role of M-CSF in the proliferation/differentiation of mononuclear-phagocytic cells and the role of marrow stromal cells as regulators of marrow haematopoiesis.     

Production of IL-10, TNF-alpha, IFN-gamma, TGF-beta1 by different populations of erythroid cells derived from human embryonal liver

It has previously been determined that erythroid cells of mice are capable of expressing such cytokines as interleukin (IL) 1 alpha and beta, IL-4, IL-6, interferon gamma (IFN-gamma), granulocyte-macrophage colony-stimulating factor (GM-CSF) and transforming growth factor beta (TGF-beta). It has been shown that glycophorin A(+) (GlA(+)) and antigen erythroblasts (AG-EB(+)) (both human erythroid cells of embryonic origin) are also capable of producing a series of cytokines such as IL-1 beta, IL-2, IL-4 and IL-6. The aim of this work was to study the capacity of erythroid cells from human embryonic liver to produce such cytokines as IFN-gamma, TGF-beta1, tumour necrosis factor alpha (TNF-alpha) and IL-10. The erythroid cells were isolated by means of antibodies specific to erythroblasts (GlA and AG-EB), as well as those from single erythroid colonies. The production level of some cytokines varies insignificantly under the action of erythropoietin (Epo) and quantitatively differs in GlA(+) and AG-EB(+) erythroid cells. Hence, the erythroid cells express IFN-gamma, TGF-beta1, TNF-alpha and IL-10. The erythroid cells could be involved through the production of these cytokines in the regulation of such processes as self-renewal, proliferation and differentiation of cells of other blood-forming sites.     

Temporal cytokine profiles in severely burned patients: a comparison of adults and children

A severe burn leads to hypermetabolism and catabolism resulting in compromised function and structural changes of essential organs. The release of cytokines has been implicated in this hypermetabolic response. The severity of the hypermetabolic response following burn injury increases with age, as does the mortality rate. Due to the relationship between the hypermetabolic and inflammatory responses, we sought to compare the plasma cytokine profiles following a severe burn in adults and in children. We enrolled 25 adults and 24 children who survived a flame burn covering more than 20% of total body surface area (TBSA). The concentrations of 22 cytokines were measured using the Linco multiplex array system (St. Charles, MO, USA). Large perturbations in the expression of pro- and anti-inflammatory cytokines were seen following thermal injury. During the first week following burn injury, IFN-gamma, IL-10, IL-17, IL-4, IL-6, and IL-8 were detected at significantly higher levels in adults compared with children, P < 0.05. Significant differences were measured during the second week post-burn for IL-1beta (higher in children) and IL-5 (higher in adults), P < 0.05. IL-18 was more abundant in children compared with adults during the third week post-burn, P < 0.05. Between post-burn d 21 and d 66, IL-1alpha was detected at higher concentrations in pediatric compared with adult patients, P < 0.05. Only GM-CSF expression was significantly different at all time points; it was detected at lower levels in pediatric patients, P < 0.05. Eotaxin, G-CSF, IL-13, IL-15, IP-10, MCP-1, and MIP-1alpha were detected at significantly different concentrations in adult compared with pediatric patients at multiple time points, P < 0.05. There were no differences in IL-12, IL-2, IL-7, or TNF levels in adult compared with pediatric burn patients at any of these time points. Following severe flame burns, the cytokine profiles in pediatric patients differ compared with those in adult patients, which may provide insight with respect to the higher morbidity rate in adults. Furthermore, the dramatic discrepancies observed in plasma cytokine detection between children and adults suggest that these two patient populations may benefit from different therapeutic interventions to achieve attenuation of the post-burn inflammatory response.     

Regulation by cytokines of extracellular superoxide dismutase and other superoxide dismutase isoenzymes in fibroblasts.

The influence of cytokines on extracellular superoxide dismutase (EC-SOD) expression by human dermal fibroblasts was investigated. The expression was markedly stimulated by interferon-gamma (IFN-gamma), was varying between fibroblast lines stimulated or depressed by interleukin-1 alpha (IL-1 alpha), was intermediately depressed by tumor necrosis factor-alpha (TNF-alpha), and markedly depressed by transforming growth factor-beta (TGF-beta). TNF-alpha, however, enhanced the stimulation by a high dose of IFN-gamma, whereas TGF-beta markedly depressed the stimulations given by IFN-gamma and IL-1 alpha. The ratio between the maximal stimulation and depression observed was around 30-fold. The responses were generally slow and developed over periods of several days. There were no effects of IFN-alpha, IL-2, IL-3, IL-4, IL-6, IL-8, granulocyte-macrophage colony-stimulating factor, human growth hormone, Escherichia coli lipopolysaccharide, leukotriene B4, prostaglandin E2, formylmethionylleucylphenylalanine, platelet-activating factor, and indomethacin. The cytokines influencing the EC-SOD expression are also known to influence superoxide production by leukocytes and other cell types, and the EC-SOD response pattern is roughly compatible with the notion that its function is to protect cells against extracellular superoxide radicals. The results show that EC-SOD is a participant in the complex inflammatory response orchestrated by cytokines. The CuZn-SOD activity of the fibroblasts was not influenced by any of the cytokines, whereas the Mn-SOD activity was depressed by TGF-beta. TNF-alpha, IL-1 alpha, and IFN-gamma stimulated the Mn-SOD activity, as previously known, and these responses were reduced by TGF-beta. The different responses of the three SOD isoenzymes illustrate their different physiological roles.     

Human first trimester forebrain cells express genes for inflammatory and anti-inflammatory cytokines

Using in situ hybridization and immunohistochemistry the induction of potential cytokines [interleukin 1beta (IL-1beta), IL-4, IL-6, IL-10, tumour necrosis factor alpha (TNF-alpha), interferon gamma (IFN-gamma) and transforming growth factor beta (TGF-beta)] in human embryonic forebrain cells at weeks 5, 7 and 10 of gestation was studied. The aims were to investigate the capacity of these cells to express cytokines, to determine the kinetics of induction and to display the type of cytokine expressing cells in the developing brain. Constitutive cytokine gene expression was recorded from week 5, augmented by about 50% at week 7 and by more than 100% at week 19 (except TGF-beta at week 10). Among other cytokines, IL-1beta exhibited the highest expression at week 10. TGF-beta showed maximum expression at week 7 and declined at week 10. Combined techniques revealed that glial cells are a major source of cytokines. The study show and present for the first time the kinetics of spontaneous cytokine expression in human embryonic forebrain cells. The increased mRNA expressions with age suggest an important role for cytokines in promotion of brain development. The capacity of inducing these cytokines may, however, be implicated in the immunopathogenesis of several brain diseases. The distinctive TGF-beta profile suggests a further role for TGF-beta on modulation of cytokine responses during development.     

The transcriptional control of TGF-beta in human osteoblast-like cells is distinct from that of IL-1 beta.

Transforming growth factor beta (TGF-beta) and interleukin 1 (IL-1) are among the most potent osteotropic cytokines. The expression of mRNA for both TGF-beta and IL-1 beta was studied in human osteoblast-like cells in vitro. These cells constitutively expressed TGF-beta but not IL-1 beta mRNA. Treatment of the cells with the systemic hormones 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] (10(-8) M) and parathyroid hormone (10(-7) M) induced an increase in TGF-beta mRNA but failed to stimulate the production of IL-1-beta mRNA. Retinoic acid (10(-8) M) had no effect on either mRNA species. The cytokines IL-1 alpha (200 pg/ml), tumour necrosis factor alpha (TNF-alpha) (17 ng/ml) and bacterial lipopolysaccharide (LPS) (500 ng/ml) stimulated the production of IL-1 beta mRNA after 6-8 hours. This was followed by an increase in protein production after 24 hours. In contrast, the production of TGF-beta mRNA remained constant after treatment with these agents. Treatment of the cells with hydrocortisone (10(-8) M) resulted in the suppression of both TGF-beta and IL-1 beta mRNA. However, when the stimulating agent 1,25-(OH)2D3 was added in conjunction with hydrocortisone the mRNA expression of TGF-beta mRNA returned to 70% of the stimulated level. In contrast, the addition of the stimulatory agent IL-1 alpha to hydrocortisone-treated cells resulted in no increase in IL-1 beta mRNA. In-situ hybridization demonstrated both TGF-beta and IL-1 beta mRNA at the cellular level.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of adenovirus-mediated overexpression of decorin on metalloproteinases, tissue inhibitors of metalloproteinases and cytokines secretion by human gingival fibroblasts.

Decorin is a small leucine-rich proteoglycan that plays a role in control of cell proliferation, cell migration, collagen fibrillogenesis and modulation of the activity of TGF-beta. In the present study, we investigated the effects of decorin on the production of metalloproteinases (MMP-1, -2, -3, -9 and -13), tissue inhibitors of metalloproteinases (TIMP-1, -2) and cytokines (TGF-beta, IL-1beta, IL-4 and TNF-alpha). Decorin was overexpressed in cultured human gingival fibroblasts using adenovirus-mediated gene transfer. Decorin infection resulted in decreased protein levels of MMP-1 and MMP-3 whereas MMP-2 and TIMP-2 secretion was increased. MMP-9, MMP-13 and TIMP-1 were not affected by decorin infection. Cytokine measurements by ELISA showed that decorin overexpression reduced TGF-beta and IL-1beta. In contrast, IL-4 and TNF-alpha levels were markedly increased in decorin-infected cells. These results suggest that decorin could modulate the expression of certain metalloproteinases and their inhibitors, as well as the production of cytokines. Altogether, our data suggest that decorin might play a pivotal role in tissue remodeling by acting on the balance between extracellular matrix synthesis and degradation.     

HepG2 human hepatoma cells express multiple cytokine genes

Although cytokines are known to be involved in the regulation of a variety of hepatocellular functions, hepatocytes themselves are generally considered only targets but not producers of these important mediators. In order to investigate whether cells of hepatocellular linages are a potential source of various regulatory cytokines we have estimated the multiple cytokine gene expression in the culture of well differentiated human HepG2 hepatoma cells using RT-PCR. Our findings demonstrate that HepG2 cells express mRNAs for interferon gamma (IFN-gamma), tumour necrosis factor alpha (TNF-alpha), transforming growth factor beta (TGF-beta), macrophage colony-stimulating factor (M-CSF), oncostatin-M (OSM), intercellular adhesion molecule (ICAM-1), interleukin 4 (IL-4), IL-5, IL-7, IL-10, IL-11, IL-12 and IL-6 receptor (IL-6R). At the same time the expression of IL-1, IL-2, IL-3, IL-6, CD40 ligand and IL-2R genes was not detected. It was concluded that hepatocytes are potential producers of a variety of cytokines, some of them being able to regulate hepatocellular functions directly, while others are important regulators of leukocyte activity. Thus, on the one hand, hepatocytes may express autoregulatory cytokines and on the other hand, influence the functions of other liver cells like Kupffer, Ito or endothelial cells. Due to their large amount, liver parenchymal cells could be an important source of sytemically acting pro- and anti-inflammatory and other regulatory cytokines.     

Induction of multiple cytokine secretion from RAW264.7 cells by alginate oligosaccharides

We investigated the cytokine-inducing activities of guluronate (G3-G6) and mannuronate (M3-M6) oligomers on RAW264.7 cells with the Bio-Plex assay system. Relatively high levels of tumor necrosis factor-alpha (TNF-alpha), granulocyte colony-stimulating factor (G-CSF), monocyte chemoattractant protein-1 (MCP-1), regulated upon activation normal T cell expressed and secreted (RANTES), granulocyte macrophage (GM)-CSF, and eotaxin were induced by alginate oligomers to different extents depending on the oligomer structures, and low but significant levels of interleukin (IL)-1alpha, IL-1beta, IL-6, IL-9, and IL-13 were also induced. Throughout all cytokines tested, M-oligomers tended to be more potent than G-oligomers in terms of cytokine induction, and this tendency was evident in differences between G3 and M3.     

Transforming growth factor-beta(1), -beta(2), -beta(3), basic fibroblast growth factor and vascular endothelial growth factor expression in keratinocytes of burn scars

Keratinocytes are increasingly recognized as key regulators of skin inflammation and remodeling, as they are capable of producing growth factors and cytokines that are important mediators in the wound healing process. We investigated the expression and distribution of TGF-beta 1 mRNA by mRNA in situ hybridization and of TGF-beta 1, TGF-beta 2, TGF-beta 3, bFGF and VEGF protein expression using immunohistochemistry in spontaneously healed, partial-thickness burns and compared this with the expression of these markers in matched unburned skin. This was done to assess their role in the remodeling phase of burn wound healing. Punch biopsies were taken from both partial-thickness burns after re-epithelialization and from matched, unburned skin. At 4 and 7 months post-burn, biopsies were taken of normotrophic and hypertrophic scars that had developed in these wounds. We observed a higher expression of all mentioned growth factors in keratinocytes in scars at 1 month post-burn compared with matched unburned skin. At 4 months, keratinocytes still displayed a higher expression of TGF-beta 3 and bFGF, but the expression of TGF-beta 1, TGF-beta 2 and VEGF was normalized. The expression of TGF-beta 3 in the epidermis of hypertrophic scars was slightly higher than in normotrophic scars. At 7 months post-burn, all growth factors studied showed a normal expression on keratinocytes. Our results suggest that keratinocytes are not only involved in re-epithelialization, but also in the scar maturation. The data support the idea that keratinocytes not only respond to cytokines and growth factors in an autocrine fashion, but also exert regulatory paracrine effects on contiguous cells.     

Interleukin-2 and alpha/beta interferon down-regulate hepatitis B virus gene expression in vivo by tumor necrosis factor-dependent and -independent pathways.

We have recently reported that administration of recombinant tumor necrosis factor alpha (TNF-alpha) to hepatitis B virus (HBV) transgenic mice reduces the hepatic steady-state content of HBV-specific mRNA by up to 80% in the absence of liver cell injury. In the current study, we analyzed the regulatory effects of several other inflammatory cytokines in the same transgenic model system. Hepatic HBV mRNA content was reduced by up to 90% following administration of a single noncytopathic dose (100,000 U) of interleukin 2 (IL-2). Comparable effects were produced by administration of alpha and beta interferons (IFN-alpha and IFN-beta), but only after multiple injections of at least 500,000 U per mouse. Importantly, the regulatory effect of IL-2 was completely blocked by the prior administration of antibodies to tumor necrosis factor alpha (TNF-alpha), which did not block the effect of IFN-alpha or IFN-beta. In contrast to these observations, recombinant IFN-gamma, IL-1, IL-3, IL-6, TNF-beta, transforming growth factor beta, and granulocyte-monocyte colony-stimulating factor were inactive in this system. These results suggest that selected inflammatory cytokines can down-regulate HBV gene expression in vivo by at least two pathways, one that is dependent on TNF-alpha and another that is not. These results imply that antigen-nonspecific products of the intrahepatic HBV-specific inflammatory response may contribute to viral clearance or persistence during HBV infection.     

Inflammatory cytokines augments TGF-beta1-induced epithelial-mesenchymal transition in A549 cells by up-regulating TbetaR-I

Epithelial-mesenchymal transition (EMT) is believed to play an important role in fibrosis and tumor invasion. EMT can be induced in vitro cell culture by various stimuli including growth factors and matrix metalloproteinases. In this study, we report that cytomix (a mixture of IL-1beta, TNF-alpha and IFN-gamma) significantly enhances TGF-beta1-induced EMT in A549 cells as evidenced by acquisition of fibroblast-like cell shape, loss of E-cadherin, and reorganization of F-actin. IL-1beta or TNF-alpha alone can also augment TGF-beta1-induced EMT. However, a combination of IL-1beta and TNF-alpha or the cytomix is more potent to induce EMT. Cytomix, but not individual cytokine of IL-1beta, TNF-alpha or IFN-gamma, significantly up-regulates expression of TGF-beta receptor type I (TbetaR-I). Suppression of TbetaR-I, Smad2 or Smad3 by siRNA partially blocks EMT induction by cytomix plus TGF-beta1, indicating cytomix augments TGF-beta1-induced EMT through enhancing TbetaR-I and Smad signaling. These results indicate that inflammatory cytokines together with TGF-beta1 may play an important role in the development of fibrosis and tumor progress via the mechanism of epithelial-mesenchymal transition.     

Maximal local and minimal systemic cytokine response to colorectal surgery: the influence of perioperative filgrastim

Thirty consecutive patients scheduled for elective colorectal surgery were prospectively randomized to receive either filgrastim [the recombinant human form of granulocyte colony-stimulating factor (r-metHu-G-CSF)] or placebo blindly. The levels of interleukin (IL-)1beta, tumour necrosis factor-alpha (TNF-alpha), IL-6, IL-8, transforming growth factor-beta (TGF-beta), and IL-10 were determined 5 and 24 h postoperatively from peripheral blood, peritoneal fluid, and wound fluid. The concentrations of all the measured cytokines were enormously higher locally at the operative site than in the systemic circulation. The only difference between the two medication groups was the lower concentration of IL-8 in peripheral blood in the filgrastim-treated patients. The present study shows abundant release of pro- and anti-inflammatory cytokines into the wound and the peritoneal cavity after abdominal surgery. The systemic response to surgery seems to be a secondary and minor reflection of local events. Filgrastim did not have any effect on the studied local cytokine levels at the operative site.     

Cytokine expression in the intestine of rainbow trout (Oncorhynchus mykiss) during infection with Aeromonas salmonicida

Gene expression of a number of cytokines in the intestine of rainbow trout (Oncorhynchus mykiss) was investigated after challenge with a pathogenic strain of Aeromonas salmonicida. Fish were exposed to A. salmonicida by immersion in a bacterial suspension (bath challenge) and tissue samples of the distal and proximal intestine were collected at days 0, 2, 4, 6 and 8 post-exposure. Head kidney tissue was also collected to assess the effect in a systemic immune tissue. A classic profile of pro-inflammatory cytokine upregulation was observed in the proximal intestine of fish infected by bath challenge, as determined by semi-quantitative RT-PCR. Expression of IL-1beta, IL-8, TNF-alpha and IFN-gamma was increased in the proximal intestine. TGF-beta was significantly decreased in the distal intestine. In the head kidney, infection with A. salmonicida by bath challenge caused decreased expression levels of IL-1beta, IL-8, TNF-alpha and TGF-beta. The results are discussed in the context of potential immune mechanisms in the gut to prevent infection.     

Regulation of rat pulmonary artery endothelial cell transforming growth factor-beta production by IL-1 beta and tumor necrosis factor-alpha.

Recent studies suggest that transforming growth factor-beta (TGF-beta) production is up-regulated at sites of tissue injury, inflammation and repair, or fibrosis. Endothelial cells represent a potentially important in vivo source of TGF-beta; however, the identity of endogenous modulators of TGF-beta production by these cells remains unclear. To address this issue, the effects of the cytokines, IL-1 beta, and TNF-alpha on TGF-beta production by rat pulmonary artery endothelial cells were examined. Conditioned media from cells treated with 0 to 20 ng/ml IL-1 beta and/or TNF-alpha were assayed for TGF-beta activity using a mink lung epithelial cell line. The results show that rat pulmonary artery endothelial cells secreted undetectable amounts of active TGF-beta in the absence of cytokines. However, upon acidification of the conditioned media before assay, a time-dependent increase in TGF-beta activity was noted in media from both untreated and cytokine-treated cells. However, both IL-1 beta and TNF-alpha treatment caused the secretion of significantly greater amounts of TGF-beta activity than control cells, in a dose-dependent manner, with maximal response obtained at cytokine doses of greater than 10 ng/ml. At equivalent doses of cytokine tested, the magnitude of the response was significantly greater with IL-1 beta. These responses were paralleled by increases in steady state mRNA levels for TGF-beta 1. Addition of both cytokines resulted in a synergistic response. Synergism with IL-1 beta was also noted with the fibrogenic agent bleomycin. Kinetic studies indicated that a minimum of 4 h of treatment with either IL-1 beta or TNF-alpha was required for detection of significant increases in either secreted TGF-beta activity or steady state TGF-beta 1 mRNA levels. Thus, endothelial cells could play a role in various TGF-beta-dependent processes in vivo, in situations wherein IL-1 beta and/or TNF-alpha may be present at comparable concentrations.