Cytokine production by a megakaryocytic cell line

Summary The regulation of megakaryopoeisis by cytokines is not yet well understood. It is possible that autocrine loops are established during megakaryocyte growth and differentiation, aiding in the maturation of these cells. The CHRF-288-11 human megakaryoblastic cell line has been examined for cytokine production in growing cells and cells stimulated to differentiate by the addition of phorbol esters. It has been demonstrated that these cells produce RNA corresponding to the interleukins IL-1α, 1β, 3, 7, 8, and 11, granulocyte-macrophage colony stimulating factor (GM-CSF), stem cell factor (SCF), transforming growth factor-β (TGF-β), tumor necrosis factor-α (TNF-α), interferon-α (INF-α), and basic fibroblast growth factor (bFGF). Additionaly, RNA corresponding to the receptors for IL-6, GM-CSF, SCF, INF-α,β, bFGF, and monocyte colony stimulating factor (M-CSF) were also expressed by the cells. The receptor for TNF-α was detected immunologically. Analysis at the protein level demonstrated that significant amounts of INF-α, TNF-α, GM-CSF, SCF, IL-1α, and a soluble form of the IL-6 receptor were produced by the cells. Addition of phorbol esters to CHRF-288-11 cells enhances their megakaryocytic phenotype; such treatment also results in increased secretion of INF-α, TNF-α, and GM-CSF. These results suggest that potential autocrine loops are established during the differentiation of CHRF-288-11 cells, which may alter the capability of the cell to differentiate. These findings are similar to those recently obtained for marrow-derived megakaryocytes (Jiang et al.) suggesting that CHRF-288-11 cells provide a useful model system for the study of cytokine release during megakaryocyte differentiation.     

Cytokine-induced selective increase of high-molecular-weight bFGF isoforms and their subcellular kinetics in cultured rat hippocampal astrocytes

Cytokines such as interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α) and epidermal growth factor (EGF) are probable factors responsible for up-regulation of basic fibroblast growth factor (bFGF) expression in reactive astrocytes following brain damage, however the effect of these cytokines on the expression of each bFGF-isoform has not been elucidated. Western biot analysis revealed the expression of 18, 22 and 24-kD bFGF isoforms in cultured rat hippocampal astrocytes, and the expression of high molecular weight (HMW)-isoforms (22 and 24-kD isoforms) but not of 18-kD isoform was selectively increased by cytokines. Immunofluorescent analysis demonstrated that bFGF content in the cytoplasm of astrocytes is initially increased by cytokines followed by nuclear targeting and localization in agreement with the previous evidence that HMW-isoforms possess a nuclear targeting signal. The present results suggest the important role of HMW-bFGF isoforms in the response of nervous tissue to injury.     

Establishment of a human fibroblast cell line producing tumor necrosis factor α (KMST-6/TNF) and growth inhibitory effects of its conditioned medium on malignant cells in culture

Summary To develop a new gene therapy model for cancer, a clonal cell line (KMST-6/TNF) which produces human tumor necrosis factor α (hTNF-α) has been developed by introducing hTNF-α cDNA into a human immortal fibroblast cell line (KMST-6). The conditioned medium (CM) of KMST-6/TNF cells inhibited the growth of various malignant human cell lines, but not that of normal human fibroblasts. Although the growth inhibitory effects of KMST-6/TNF CM were neutralized to a considerable degree by anti-TNF-α antibody, its inhibitory effects were more marked than the purified human natural TNF-α itself in the same units, suggesting that KMST-6/TNF CM contains some growth inhibitory substances other than TNF-α. However, interferons α, β, and γ were undetectable in the KMST-6/TNF CM.     

Differential responsiveness of late passage C-6 glial cells and advanced passages of astrocytes derived from aged mouse cerebral hemispheres to cytokines and growth factor: Glutamine synthetase activity

In this study, we were interested to compare the responsiveness to growth factors, NGF, b-FGF and EGF and cytokines, IL1β, and TNF-α, in late passages (74–79) C6 glial cells committed astrocytes and astrocytes of advanced passages (26–28) in cultures derived from aged mouse cerebral hemispheres (MACH). Cultures were grown in either DMEM or chemically defined medium (CDM/TIPS) in order to test the effects of growth factors or cytokines. The activity of glutamine synthetase (GS), a marker for astrocytes, was used as a test parameter. We found that treatment with growth factors increased GS activity in both glial cell culture systems with the exception of EGF in C-6 glial cells. Treatment with cytokines markedly decreased GS activity in the late passage C6 glial cells whereas only TNF-α had a similar effect on MACH astrocytes. In view of the generally opposite effects of growth factors and cytokines on GS activity, we-speculate that these molecules which are also endogenously present in glial cells may play a role in the maintenance of cellular homeostasis.     

Transforming growth factor-β1 modulates the effect of 1α,25-dihydroxyvitamin D3 on leukemic cells

Summary The human leukemic cells HL-60, U937, KG-1 and THP-1 incubated with transforming growth factor-β1 (TGF-β1) were studied by examining cell surface antigens and macrophage-specific activities. The addition of 0.5 ng/ml (20 pM) of TGF-β1 with 1α,25-dihydroxyvitamin D₃ [1α, 25(OH)₂D₃] induced more Leu-M3 (CD14)-positive cells (approximately 80%) than 5×10⁻⁸ M 1α,25(OH)₂D₃ alone did (30 to 50%), although original HL-60 cells did not express any Leu-M3 antigen at all. Tumor necrosis factor-α (TNF-α) with TGF-β1 and 1α,25(OH)₂D₃ was found to potentiate the expression of these surface antigens. Furthermore, the phagocytic activity was also induced strongly. The expression of CR3 (CD11b) antigen was also increased, and all Leu-M3-positive cells were found CR3-positive when HL-60, U937, and THP-1 cells were treated with these stimulants. In contrast, CR3 but not Leu-M3 was induced in KG-1 cells after the same treatment. This may indicate that the responsiveness of leukemic cells to TGF-β1 and 1α,25(OH)₂D₃ might vary depending on a differentiation stage of the target cells. Furthermore, K562 cells originated from a more undifferentiated precursor, were not able to respond to these two inducers. These results suggested that some of TGF-β superfamily proteins might represent potent modulators in hematopoiesis, especially in the development of monocytes-macrophages or their precursors.     

GM-CSF-induced granulation tissue formation: relationships between macrophage and myofibroblast accumulation

We have studied the formation of granulation tissue around osmotic minipumps delivering granulocyte macrophage-colony stimulating factor (GM-CSF) chronologically in the rat using electron microscopy and immunohistochemistry at the light and electron microscopic levels, with specific antibodies against α-smooth muscle (SM) actin and rat macrophages. At 2 and 3 days after pump implantation, GM-CSF application produced an extensive inflammatory reaction characterized by edema and the accumulation of polymorphonuclear cells and macrophages. Gradually, polymorphonuclear cells decreased in number and macrophages became arranged in large clusters. The expression of α-SM actin in fibroblastic cells of the granulation tissue started from the 4th day after pump implantation and progressed up to the 7th day. Double immunofluorescence staining showed macrophage clusters in relation to α-SM actinrich fibroblastic cells. Electron microscopic examination confirmed that the fibroblasts containing α-SM actinpositive stress fibers were found initially in close proximity to clustered macrophages. The delivery of plateletderived growth factor (PDGF) and tumor necrosis factor-α (TNF-α) by the osmotic minipump induced an accumulation of macrophages, but in a much smaller number compared with those seen after GM-CSF application; these macrophages were never assembled in clusters and, furthermore, TNF-α and PDGF did not stimulate α-SM actin expression in fibroblastic cells. Our results suggest that after GM-CSF administration, the cluster-like accumulation of macrophages plays an important role in stimulating α-SM actin expression in myofibroblasts. Our results may be relevant to the understanding of the processes leading to granulation tissue formation in this and other experimental models.     

The Role of TNF-<Emphasis Type="Italic">α</Emphasis> and its Receptors in the Production of <Emphasis Type="Italic">β</Emphasis>-1,4-galactosyltransferase I mRNA by Rat Primary Type-2 Astrocytes

β-1,4-galactosyltransferase I (β-1,4-GalT I) plays an important role in the synthesis of the backbone structure of adhesion molecules involved in leukocyte–endothelial cell interaction. The expression of β-1,4-GalT I mRNA increased in primary human endothelial cells after exposure to tumor necrosis factor-α (TNF-α). In the central nervous system (CNS), astrocytes play a pivotal role in immunity as immunocompetent cells by secreting cytokines and inflammatory mediators, there are two types of astrocytes. Type-1 astrocytes can secrete TNF-α when stimulated with Lipopolysaccharide (LPS), while the responses of type-2 astrocytes during inflammation are unknown. So we examined the expression change of β-1,4-GalT I mRNA in type-2 astrocytes after exposure to TNF-α and LPS. Real-time PCR showed that TNF-α or LPS affected β-1,4-GalT I mRNA expression in a time- and dose-dependent manner. RT-PCR analysis revealed that TNFR1 and TNFR2 were present in normal untreated type-2 astrocytes, and that TNF-α, TNFR1 and TNFR2 increased in type-2 astrocytes after exposure to TNF-α or LPS. Immunocytochemistry showed that TNFR1 was expressed in the cytoplasm, nucleus and processes of normal untreated type-2 astrocytes, and distributed mainly in the cytoplasm and processes after exposure to LPS. TNFR2 was mainly expressed in the nucleus of normal untreated type-2 astrocytes, and distributed mainly in the processes of type-2 astrocytes after exposure to LPS. Both anti-TNFR1 and anti-TNFR2 antibodies suppressed β-1,4-GalT I mRNA expression induced by TNF-α or LPS. From these results, we conclude that TNF-α signaling via both TNFR1 and TNFR2 translocated from nucleus to cytoplasm or processes is sufficient to induce β-1,4-GalT I mRNA. In addition, we observed that not only exogenous TNF-α but also TNF-α produced by type-2 astrocytes affected β-1,4-GalT I mRNA production in type-2 astrocytes. These results suggest that an autocrine loop involving TNF-α contributes to the production of β-1,4-GalT I mRNA in response to inflammation. Chunlin Xia is the co-first author.     

Tripeptide tyroserleutide enhances the antitumor effects of macrophages and stimulates macrophage secretion of IL-1β, TNF-α, and NO in vitro

Tyroserleutide (YSL) is a type of active, low molecular weight polypeptide, comprised of three amino acids, which has antitumor effects. YSL has various advantages over the other bioactive peptides such as its low molecular weight, simple construction, nonimmunogenicity, specificity, few side effects, and ease of synthesis. However, the biological activities contributing to it’s antitumor effects are not yet known. We studied the effects of YSL on the in vitro cytotoxic activity of BALB/c mice peritoneal macrophages (PEMφ) against the target tumor cell lines BEL-7402 and B16-F10. We also measured the concentrations of interleukin (IL)-1β, tumor necrosis factor (TNF)-α, and nitric oxide (NO) produced by YSL-activated Mφ, and we determined the concentrations of IL-1β and NO secreted by YSL-activated murine macrophage RAW264.7 cells. YSL activated Mφ in vitro, inhibited BEL-7402 proliferation, enhanced PEMφ antitumor effects, and stimulated IL-1β, TNF-α, and NO production by RAW264.7 cells. These data suggest that YSL activates the monocyte–macrophage system, which enhances Mφ antitumor effects against BEL-7402 and B16-F10 cells and stimulates the secretion by Mφ of cytotoxic effectors such as IL-1β, TNF-α, and NO.     

Interleukin-1, tumor necrosis factor-alpha,and transforming growth factor-beta 1 and integrative meniscal repair: influences on meniscal cell proliferation and migration

Introduction  

Interleukin-1 (IL-1) and tumor necrosis factor-α (TNF-α) are up-regulated in injured and osteoarthritic knee joints. IL-1 and TNF-α inhibit integrative meniscal repair; however, the mechanisms by which this inhibition occurs are not fully understood. Transforming growth factor-β1 (TGF-β1) increases meniscal cell proliferation and accumulation, and enhances integrative meniscal repair. An improved understanding of the mechanisms modulating meniscal cell proliferation and migration will help to improve approaches for enhancing intrinsic or tissue-engineered repair of the meniscus. The goal of this study was to examine the hypothesis that IL-1 and TNF-α suppress, while TGF-β1 enhances, cellular proliferation and migration in cell and tissue models of meniscal repair.     

Activation of EGF receptor family members suppresses the cytotoxic effects of tumor necrosis factor-α

Tumor necrosis factor (TNF)-α has a broad range of biological activities, which depend heavily on cell type and physiological condition. In a panel of human tumor cell lines we analyzed expression of the receptor tyrosine kinases EGFR, ErbB2 and ErbB3, and the response to TNF-α. Among the cell lines tested those resistant to TNF-α were found to express high levels of either EGFR, or ErbB2 and ErbB3. In TNF-sensitive breast and cervical carcinoma cells activation of EGFR or ErbB2 by the exogenous growth factors EGF and heregulin β1 resulted in a significant increase in the number of cells surviving TNF-α treatment. In contrast, inhibition of EGFR activation in TNF-resistant breast carcinoma cells by the novel antagonistic anti-EGFR antibody 14E1 sensitized the cells to the cytotoxic effects of TNF-α. A bacterially expressed fusion protein consisting of a 14E1 single-chain (sc) Fv antibody fragment linked to human TNF-α retained TNF-α activity. This scFv(14E1)-TNF-α molecule localized specifically to EGFR on the surface of tumor cells and activated the NF-κB pathway in co-cultured T cells, as demonstrated by electrophoretic mobility shift assays. Received: 6 May 1998 / Accepted: 16 July 1998     

Immunostimulatory effect by aqueous extract of <Emphasis Type="Italic">Hizikia fusiforme</Emphasis> in RAW 264.7 macrophage and whole spleen cells

Hizikia fusiforme is a commonly used food that possesses potent anti-bacterial, anti-fungal, and anti-inflammatory activities. The immunostimulatory activities of aqueous extract of Hizikia fusiforme (HFAE) in RAW 264.7 macrophages and whole spleen cells were investigated. HFAE activated RAW 264.7 macrophages to produce cytokines such as nitric oxide (NO), tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6) in a dose-dependent manner. In addition, HFAE induced the mRNA expression of TNF-α, IL-1β, and IL-6 in RAW 264.7 macrophages. Moreover, HFAE stimulated proliferation of whole spleen cells and reference mitogen. Taken together, the results demonstrate that HFAE potently activates the immune function by regulating NO, TNF-α, IL-1β, and IL-6 in RAW 264.7 macrophage and promoting spleen cell proliferation.     

Dehydroepiandrosterone and melatonin prevent Bacillus anthracis lethal toxin-induced TNF production in macrophages

The lethal toxin of Bacillus anthracis, which is composed of two separate proteinaceous exotoxins, namely protective antigen and lethal factor, is central to the pathogenesis of anthrax. Low levels of this toxin are known to induce release of cytokines such as tumor necrosis factor α (TNF-α). In the present study we investigated the effect of dehydroepiandrosterone (DHEA), melatonin (MLT), or DHEA + MLT on production of lethal toxin-induced TNF-α in mouse peritoneal macrophages. We found that treatment with DHEA significantly inhibited the TNF-α production caused by anthrax lethal toxin. Exposure of MLT to anthrax lethal toxin-treated macrophages also decreased the release of TNF-α to the extracellular medium as compared to the control. However, combined use of DHEA and MLT also inhibited TNF-α release, but not more than single therapies. These results suggest that DHEA and MLT may have a therapeutic role in reducing the increased cytokine production induced by anthrax lethal toxin. This revised version was published online in July 2006 with corrections to the Cover Date.     

The duality of the inflammatory response to traumatic brain injury

One and a half to two million people sustain a traumatic brain injury (TBI) in the US each year, of which approx 70,000–90,000 will suffer from long-term disability with dramatic impacts on their own and their families’ lives and enormous socio-economic costs. Brain damage following traumatic injury is a result of direct (immediate mechanical disruption of brain tissue, or primary injury) and indirect (secondary or delayed) mechanisms. These secondary mechanisms involve the initiation of an acute inflammatory response, including breakdown of the blood-brain barrier (BBB), edema formation and swelling, infiltration of peripheral blood cells and activation of resident immunocompetent cells, as well as the intrathecal release of numerous immune mediators such as interleukins and chemotactic factors. An overview over the inflammatory response to trauma as observed in clinical and in experimental TBI is presented in this review. The possibly harmful/beneficial sequelae of post-traumatic inflammation in the central nervous system (CNS) are discussed using three model mediators of inflammation in the brain, tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and transforming growth factor-β (TGF-β). While the former two may act as important mediators for the initiation and the support of post-traumatic inflammation, thus causing additional cell death and neurologic dysfunction, they may also pave the way for reparative processes. TGF-β, on the other hand, is a potent anti-inflammatory agent, which may also have some deleterious long-term effects in the injured brain. The implications of this duality of the post-traumatic inflammatory response for the treatment of brain-injured patients using anti-inflammatory strategies are discussed.     

Macrophage inflammatory protein (MIP)1α and MIP1β differentially regulate release of inflammatory cytokines and generation of tumoricidal monocytes in malignancy

The C–C chemokines, macrophage inflammatory protein (MIP)1α and MIP1β are potent chemoattractants for the monocytes, which form an important component of the stroma of tumor tissue and may regulate tumor growth and associated inflammation. We examined the role of MIP1α and MIP1β in inducing the release of inflammatory cytokines and the generation of tumoricidal monocytes from the peripheral blood monocytes (PBM) of healthy women and patients with carcinoma of breast (CaBr). Interleukin-1 (IL-1) and tumor necrosis factor (TNF) α release by the PBM was markedly stimulated by MIP1α in CaBr patients, but only marginally so in healthy women. In contrast, MIP1β stimulated the release of these cytokines by the PBM of healthy women, but failed to do so in CaBr patients. MIP1α, but not MIP1β, synergized with LPS in inducing the release of IL-1 from the PBM of both healthy women and CaBr patients. Both MIP1α and MIP1β augmented respiratory bursts in PBM and generated tumoricidal PBM that killed T24 cells, MIP1α being more effective in CaBr patients and MIP1β in healthy women. IFN-γ co-stimulated and IL-4 suppressed MIP1α and β-induced cytotoxicity in PBM. The synergy of IFN-γ was more marked with MIP1α than with MIP1β. The differential effects of MIP1α and MIP1β on the PBM of healthy women and CaBr patients co-related with the levels of expression of CCR1 and CCR5 in these monocytes. The expression of CCR5 was higher than that of CCR1 in the PBM of healthy women and the PBM of the CaBr patients showed overexpression of CCR1 and downregulation of CCR5.     

The effect of TGF-β1 and Smad7 gene transfer on the phenotypic changes of rat alveolar epithelial cells

The aim of this study was to investigate whether transforming growth factor-β1 (TGF-β1) could induce alveolar epithelial-mesenchymal transition (EMT) in vitro, and whether Smad7 gene transfer could block this transition. We also aimed to elucidate the possible mechanisms of these processes. The Smad7 gene was transfected to the rat type II alveolar epithelial cell line (RLE-6TN). Expression of the EMT-associated markers was assayed by Western Blot and Real-time PCR. Morphological alterations were examined via phase-contrast microscope and fluorescence microscope, while ultrastructural changes were examined via electron microscope. TGF-β1 treatment induced a fibrotic phenotype of RLE-6TN with increased expression of fibronectin (FN), α-smooth muscle actin (α-SMA) and vimentin, and decreased expression of E-cadherin (E-cad) and cytokeratin19 (CK19). After transfecting the RLE-6TN with the Smad7 gene, the expression of the mesenchymal markers was downregulated while that of the epithelial markers was upregulated. TGF-β1 treatment for 48 h resulted in the separation of RLE-6TN from one another and a change into elongated, myofibroblast-like cells. After the RLE-6TN had been transfected with the Smad7 gene, TGF-β1 treatment had no effect on the morphology of the RLE-6TN. TGF-β1 treatment for 48 h resulted in an abundant expression of α-SMA in the RLE-6TN. If the RLE-6TN were transfected with the Smad7 gene, TGF-β1 treatment for 48 h could only induce a low level of α-SMA expression. Furthermore, TGF-β1 treatment for 12 h resulted in the degeneration and swelling of the osmiophilic multilamellar bodies, which were the markers of type II alveolar epithelial cells. TGF-β1 can induce alveolar epithelialmesenchymal transition in vitro, which is dependent on the Smads signaling pathway to a certain extent. Overexpression of the Smad7 gene can partially block this process     

Cell contact interactions in rheumatology, The Kennedy Institute for Rheumatology, London, UK, 1-2 June 2000

The intricate interactions that regulate relationships between endogenous tissue cells and infiltrating immune cells in the rheumatic joint, particularly in rheumatoid arthritis (RA), were the subject of the meeting. A better understanding of these interactions might help to define intervention points that could be used to develop specific therapies. The presentations and discussions highlighted the fact that, once chronic inflammation is established, several pro-inflammatory loops involving tumour necrosis factor (TNF)-α and interleukin (IL)-1β can be defined. Direct cellular contact with stimulated T lymphocytes induces TNF-α and IL-1β in monocytes which in turn induce functions in fibroblast-like synoviocytes. The latter include the production of stromal cell-derived factor-1α (SDF-1α) which enhances the expression of CD40L in T cells, which stimulates SDF-1α production in synoviocytes, which in turn protects T and B cells from apoptosis and enhances cell recruitment thus favoring inflammatory processes. IL-1β and TNF-α also induce IL-15 in fibroblast-like synoviocytes, which induces the production of IL-17 which in turn potentiates IL-1β and TNF-α production in monocyte-macrophages. This underlines the importance of TNF-α and IL-1β in RA pathogenesis, and helps explain the efficiency of agents blocking the activity of these cytokines in RA. Factors able to block the induction of cytokine production (such as apolipoprotein A-I [apo A-I] and interferon [IFN]-β) might interfere more distally in the inflammatory process. Furthermore, stimulated T lymphocytes produce osteoclast differentiation factor (ODF), which triggers erosive functions of osteoclasts. Therefore, factors capable of affecting the level of T lymphocyte activation, such as IFN-β, IL-15 antagonist, or SDF-1α antagonist, might be of interest in RA therapy.     

Regulation of BCRP (ABCG2) and P-Glycoprotein (ABCB1) by Cytokines in a Model of the Human Blood–Brain Barrier

Brain capillary endothelial cells form the blood–brain barrier (BBB), a highly selective permeability membrane between the blood and the brain. Besides tight junctions that prevent small hydrophilic compounds from passive diffusion into the brain tissue, the endothelial cells express different families of drug efflux transport proteins that limit the amount of substances penetrating the brain. Two prominent efflux transporters are the breast cancer resistance protein and P-glycoprotein (P-gp). During inflammatory reactions, which can be associated with an altered BBB, pro-inflammatory cytokines are present in the systemic circulation. We, therefore, investigated the effect of the pro-inflammatory cytokines interleukin-1β (IL-1β), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) on the expression and activity of BCRP and P-gp in the human hCMEC/D3 cell line. BCRP mRNA levels were significantly reduced by IL-1β, IL-6 and TNF-α. The strongest BCRP suppression at the protein level was observed after IL-1β treatment. IL-1β, IL-6 and TNF-α also significantly reduced the BCRP activity as assessed by mitoxantrone uptake experiments. P-gp mRNA levels were slightly reduced by IL-6, but significantly increased after TNF-α treatment. TNF-α also increased protein expression of P-gp but the uptake of the P-gp substrate rhodamine 123 was not affected by any of the cytokines. This in vitro study indicates that expression levels and activity of BCRP, and P-gp at the BBB may be altered by acute inflammation, possibly affecting the penetration of their substrates into the brain.