Stimulate Thymocyte Proliferation Research Articles

Ghrelin augments murine T-cell proliferation by activation of the phosphatidylinositol-3-kinase, extracellular signal-regulated kinase and protein kinase C signaling pathways

Thymic atrophy occurs during normal aging, and is accelerated by exposure to chronic stressors that elevate glucocorticoid levels and impair the naïve T cell output. The orexigenic hormone ghrelin was recently shown to attenuate age-associated thymic atrophy. Here, we report that ghrelin enhances the proliferation of murine CD4+ primary T cells and a CD4+ T-cell line. Ghrelin induced activation of the ERK1/2 and Akt signaling pathways, via upstream activation of phosphatidylinositol-3-kinase and protein kinase C, to enhance T-cell proliferation. Moreover, ghrelin induced expression of the cell cycle proteins cyclin D1, cyclin E, cyclin-dependent kinase 2 (CDK2) and retinoblastoma phosphorylation. Finally, ghrelin activated the above-mentioned signaling pathways and stimulated thymocyte proliferation in young and older mice in vivo.

Jak3 activation in human lymphocyte precursor cells.

Although expression of the Jak3 tyrosine kinase in T lymphocytes has been thought to be restricted to mature, activated cells, mutations of Jak3 can lead to the development of a human severe combined immunodeficiency (SCID) characterized by an absence of peripheral T lymphocytes. We therefore examined in detail the expression of Jak3 throughout human T cell differentiation and show that Jak3 is in fact present throughout the entire developmental process, with high levels expressed in thymocytes. Jak3 is highly expressed in double negative (CD4- CD8-) cells, one of the earliest stages of thymocyte differentiation, and can be activated via the IL-7 receptor. IL-7 is known to stimulate thymocyte proliferation and initiate re-arrangement of the T cell receptor (TCR) beta gene, suggesting that the failure of mutated Jak3 proteins to transduce this signal may be responsible for failures in T cell development. While Jak3 SCID patients possess mature peripheral B cells, we demonstrate that the Jak3 tyrosine kinase is also expressed in human pre-B cells and can be activated by the pre-B cell growth factor IL-7.

DIVERGENT CELL-SPECIFIC EFFECTS OF ACTIVIN-A ON THYMOCYTE PROLIFERATION STIMULATED BY PHYTOHEMAGGLUTININ, AND INTERLEUKIN 1β OR INTERLEUKIN 6 IN VITRO

Activin-A is a member of the transforming growth factor-β (TGF-β) cytokine family. Based on studies in several cell systems, activin-A has been postulated to be a specific inhibitor of the actions of the inflammatory cytokine, interleukin 6. In cultures of adult rat thymocytes, activin-A inhibited sub-optimal phytohemagglutinin-induced and interleukin 1β-stimulated proliferation, as measured by [3H]-thymidine incorporation in vitro. In contrast with TGF-β1, which exerted similar inhibitory effects on thymocyte proliferation, activin-A activity was reduced by increasing the concentration of phytohemagglutinin or addition of the reducing agent, β-mercaptoethanol. Both activin-A and TGF-β1 inhibited the in vitro production of interleukin 6 by thymocytes in the presence of phytohemagglutinin and interleukin 1β. In the presence of exogenous interleukin 6, however, both activin-A and TGF-β1 stimulated thymocyte proliferation. These data suggest that activin-A inhibits thymocyte growth and differentiation, at least in part, by inhibiting endogenous production of interleukin 6, but stimulates thymocyte growth when exogenous interleukin 6 is present in vitro. These data indicate that activin interacts with other cytokines to exert complex regulation of T cell development, and is not an inhibitor of interleukin 6 action in all cell systems.

The effect of melittin on proliferation and death of thymocytes

The effect of melittin, an activator of phospholipase A 2, on proliferation and death of rat thymocytes in a broad concentration range was studied. Cell proliferation was estimated by the accumulation of colchicin metaphases, necrotic death was determined from lysis and staining of cells with trypan blue, and apoptosis was assessed from the type of DNA fragmentation, the amount of fragmented DNA, and the percentage of cells with subdiploid DNA. It was shown that low melittin concentrations (below 5 μg/ml) stimulate thymocyte proliferation. At high melittin concentrations, thymocytes die by the primary necrosis type. Throughout the concentration range studied, melittin does not produce apoptosis in thymocytes. Conversely, high melittin concentrations even inhibit thymocyte apoptosis in the control and after irradiation. An inhibitor of RNA synthesis actinomycin D does not affect thymocyte death in the presence of melittin. It is concluded that the activation of phospholipase A 2 can induce necrosis but not apoptosis and thus is not a necessary step in the signaling cascade that initiates apoptosis in thymocytes.

Replacement of pre-T cell receptor signaling functions by the CD4 coreceptor.

An important checkpoint in early thymocyte development ensures that only thymocytes with an in-frame T cell receptor for antigen beta (TCR-beta) gene rearrangement will continue to mature. Proper assembly of the TCR-beta chain into the pre-TCR complex delivers signals through the src-family protein tyrosine kinase p56lck that stimulate thymocyte proliferation and differentiation to the CD4+CD8+ stage. However, the biochemical mechanisms governing p56lck activation remain poorly understood. In more mature thymocytes, p56lck is associated with the cytoplasmic domain of the TCR coreceptors CD4 and CD8, and cross-linking of CD4 leads to p56lck activation. To study the effect of synchronously inducing p56lck activation in immature CD4-CD8- thymocytes, we generated mice expressing a CD4 transgene in Rag2-/- thymocytes. Remarkably, without further experimental manipulation, the CD4 transgene drives maturation of Rag2-/- thymocytes in vivo. We show that this process is dependent upon the ability of the CD4 transgene to bind Lck and on the expression of MHC class II molecules. Together these results indicate that binding of MHC class II molecules to CD4 can deliver a biologically relevant, Lck-dependent activation signal to thymocytes in the absence of the TCR-alpha or -beta chain.

Immunoregulatory effects of a synthetic monosaccharide.

Investigative attempts to identify novel therapy for inflammatory connective tissue diseases continue to evolve. Amiprilose hydrochloride (amiprilose HCl) is a synthetic carbohydrate shown to have anti-inflammatory effects in animal models of inflammatory arthritis and in a multicenter clinical trial. Interleukin-1 (IL-1) is an important mediator of immune regulation, inflammation and joint destruction in arthritis. In the present study, the effects of amiprilose HCl on IL-1 activity, production and receptor distribution were investigated. Drug effects on IL-2 production and receptor distribution on lymphocytes were also explored. Potential regulation of IL-1 activity was determined by monitoring the effects of amiprilose HCl on IL-1 stimulated proliferation of murine thymocytes and human synovial cells. Inhibitory effects on IL-1 beta and IL-2 production by stimulated human peripheral blood monocytes were measured by ELISA and lymphocyte IL-1 beta and IL-2 receptor distribution were analyzed by flow cytometry. The results from in vitro studies demonstrated that low concentrations of amiprilose HCl (1-100 micrograms/ml) stimulated thymocyte proliferation and enhanced the proliferative response of IL-1 stimulated human synovial fibroblasts. IL-1 beta production in cultures of human peripheral blood monocytes was significantly decreased after exposure of the cultures to varying doses of amiprilose HCl as determined by ELISA. Exposure of mitogen activated human peripheral blood lymphocytes to amiprilose HCl resulted in decreased IL-2 production at high concentrations of drug as compared to control. However, at doses of amiprilose HCl previously found to stimulate thymocyte proliferation (1-10 micrograms/ml), increased levels of culture supernatant IL-2 were observed. No amiprilose HCl mediated changes in lymphocyte IL-1 beta or IL-2 receptor expression were observed. The regulatory effects of amiprilose HCl on cytokines support the potential of this drug as a therapeutic agent for the treatment of inflammatory arthritis.

Relationship between dehydroepiandrosterone and calcitonin gene-related peptide in the mouse thymus.

Dehydroepiandrosterone (DHEA) and calcitonin gene-related peptide (CGRP) are naturally occurring substances that are reported to have both opposing and complementary effects on immune functions. In the current study, we sought to determine how they might work together to influence the mitogen-stimulated proliferation of thymocytes. In concanavalin A (ConA)-induced thymocyte proliferation assays, CGRP and DHEA each inhibited proliferation. When the CGRP antagonist CGRP-(8-37) was added to Con A-stimulated thymocytes, the proliferative response was significantly greater than the ConA response alone across a range of ConA doses. Moreover, CGRP-(8-37) blocked the inhibitory effect of DHEA. Individually, CGRP-(8-37), CGRP, DHEA, or their combination did not stimulate thymocyte proliferation in the absence of ConA. CGRP affects the proliferation of CD4+ T cells and thus may be a regional endogenous inhibitor of the proliferation of virgin mature T cells while they remain in the thymus. Furthermore, DHEA may act via endogenous CGRP on the thymus CD4+ T cell population.

IL-10, a novel growth cofactor for mature and immature T cells.

We identified a new cytokine, B cell-derived T cell growth factor (B-TCGF), that is produced by a murine B cell lymphoma and induces proliferation of mature and immature thymocytes in the presence of IL-2 and IL-4. Both adult and day 15 fetal thymocytes (CD4-8-, CD4+8-, CD4-8+) proliferate strongly in the presence of IL-2, IL-4, and B-TCGF. B-TCGF alone does not stimulate thymocyte proliferation. B-TCGF appears to be identical to a novel cytokine whose cDNA was recently isolated at our institution, cytokine synthesis-inhibitory factor (CSIF; IL-10). rIL-10 has B-TCGF activity, and mAb specific for IL-10 inhibit the B-TCGF activity present in CH12 supernatants. Further studies have shown that day 15 fetal thymocytes cultured in the presence of IL-10, IL-2, and IL-4 remain CD4- and CD8- but exhibit increased CD3 expression. Adult CD4- CD8- thymocytes cultured under the same conditions proliferate whether they are CD3+ or CD3-. The CD3- population becomes enriched in CD3+ cells after 4 days of culture. IL-10 is secreted by day 15 fetal thymocytes, adult thymocytes, and adult splenocytes when stimulated via their TCR. IL-10 is strongly homologous to the EBV gene BCRFI, and BCRFI has CSIF activity. In contrast to IL-10, BCRFI does not exhibit detectable thymocyte-stimulating activity, suggesting the existence of at least two functional epitopes on the IL-10 molecule.

Interleukin-4 (IL-4) inhibits secretion of IL-1 beta, tumor necrosis factor alpha, and IL-6 by human monocytes

Monocytes activated by lipopolysaccharide (LPS) and interferon gamma (IFN gamma) rapidly secrete a number of monokines with different functional properties. Interleukin–4 (IL–4), a T-cell derived cytokine, has been shown to reduce the production of monokines with cytostatic activity for tumor cells, chemotactic activity for monocytes, and factors that stimulate thymocyte proliferation. This latter activity is mediated by a number of monokines like IL–1, tumor necrosis factor alpha (TNF alpha), and IL–6. To elucidate which cytokines produced by monocytes are controlled by IL–4, we tested the effect of IL–4 on the secretion of IL–1 alpha, IL–1 beta, TNF alpha, and IL–6 induced by LPS or IFN gamma. IL–4 was found to inhibit the secretion of IL–1 beta and TNF alpha by activated monocytes almost 100%. The secretion of IL–6 was found to be reduced 70% to 85% in the presence of IL–4, whereas there was no effect on the secretion of IL–1 alpha (IL–1 alpha is mainly cell- associated). Time-course experiments demonstrate that IL–4 reduces the secretion of monokines for a prolonged period of time (greater than 40 hours). The reduced secretion of IL–1 beta and TNF alpha was specifically induced by IL–4 because anti-IL–4 antiserum completely restored normal monokine production. These data suggest that IL–4 plays a role in the regulation of immune responses by reducing the production of functionally important monokines.

Interleukin-4 (IL-4) Inhibits Secretion of IL-1β, Tumor Necrosis Factor a, and IL-6 by Human Monocytes

Monocytes activated by lipopolysaccharide (LPS) and interferon gamma (IFN gamma) rapidly secrete a number of monokines with different functional properties. Interleukin-4 (IL-4), a T-cell derived cytokine, has been shown to reduce the production of monokines with cytostatic activity for tumor cells, chemotactic activity for monocytes, and factors that stimulate thymocyte proliferation. This latter activity is mediated by a number of monokines like IL-1, tumor necrosis factor alpha (TNF alpha), and IL-6. To elucidate which cytokines produced by monocytes are controlled by IL-4, we tested the effect of IL-4 on the secretion of IL-1 alpha, IL-1 beta, TNF alpha, and IL-6 induced by LPS or IFN gamma. IL-4 was found to inhibit the secretion of IL-1 beta and TNF alpha by activated monocytes almost 100%. The secretion of IL-6 was found to be reduced 70% to 85% in the presence of IL-4, whereas there was no effect on the secretion of IL-1 alpha (IL-1 alpha is mainly cell-associated). Time-course experiments demonstrate that IL-4 reduces the secretion of monokines for a prolonged period of time (greater than 40 hours). The reduced secretion of IL-1 beta and TNF alpha was specifically induced by IL-4 because anti-IL-4 antiserum completely restored normal monokine production. These data suggest that IL-4 plays a role in the regulation of immune responses by reducing the production of functionally important monokines.

An Interleukin 1 Inhibitor Affects both Cell-Associated Interleukin 1-Induced T Cell Proliferation and PGE 2/Collagenase Production by Human Dermal Fibroblasts and Synovial Cells

Lipopolysaccharide (LPS) induces cell-associated interleukin 1 (IL 1) production in the human promonocytic cell line U937. Demonstration of cell-associated IL 1 activity was based on the ability of LPS-treated U937 cells, subsequently fixed with paraformaldehyde, to stimulate thymocyte proliferation in the presence of phytohemagglutinin. Like soluble IL 1 (sIL 1), cell-associated IL 1 is capable of inducing PGE 2 and/or collagenase production by dermal fibroblasts and human synovial cells in a dose-dependent manner. It is thus a mediator of the inflammatory response owing to a direct intercellular contact located at the membrane level, where bound molecules may trigger inflammation at a local site of action. We reported that the natural (~ 23 kDa) IL 1 inhibitor (IL I INH) from the urine of febrile patients inhibited all the sIL-1-induced biologic activities under investigation and that it acted by binding to the IL 1 receptor, thus blocking the interaction of the monokine with the receptor. Data demonstrate that the IL 1 INH also blocks cell-associated IL 1-induced T cell proliferation and PGE 2 production by both dermal fibroblasts and synovial cells as well as collagenase production by the latter cell type. Thus, as for the sIL 1, a feedback mechanism exists for cellassociated IL 1-induced bioactivities.

Interleukin 7 and interleukin 4 stimulate human thymocyte growth through distinct mechanisms

One of the major functions of cytokines is their ability to regulate cell growth and differentiation. The complexity of this process has been highlighted by recent studies on murine thymocytes; it has been shown that a number of cytokines interact to regulate thymocyte growth. We have investigated the effects of interleukin 4 (IL-4) and interleukin 7 (IL-7) on human thymocyte proliferation. Although maximal proliferation was dependent upon the presence of the mitogen phytohaemagglutinin (PHA), IL-7 alone stimulated thymocyte growth. In order to determine if this proliferation was due to the induction of IL-2, this pathway was inhibited by the addition of blocking antibody to the IL-2 receptor. Proliferation induced with IL-7 plus PHA, but not that induced by IL-7 alone, could be blocked by this treatment. In contrast, IL-4 stimulated thymocyte proliferation only in the presence of PHA; this proliferation was not inhibited by antibodies to the IL-2 receptor. Our findings show that both IL-7 and IL-4 can act as growth factors for human thymocytes, and that these cytokines stimulate proliferation through distinct mechanisms.

Schistosomal egg granuloma-derived fibroblast-stimulating factor is apparently distinct from interleukin-1

We have previously reported that egg granulomas isolated from livers of Schistosoma mansoni-infected euthymic mice in vitro elaborate a factor(s) that stimulates a variety of fibroblast responses including fibroblast proliferation and enhanced synthesis of extracellular matrix proteins. We have postulated that these factors play a role in the pathogenesis of hepatic fibrosis in schistosomiasis mansoni. Serum-free supernatants from egg granuloma cultures also stimulate thymocyte proliferation in an assay that defects interleukin-1 (IL-1). Thymocytes and fibroblasts are stimulated to proliferate by the same fractions of egg granuloma culture supernatant separated by gel filtration, isoelectric focusing, and ion-exchange chromatography. This suggested that granuloma-derived IL-1 is responsible for the observed fibroblast stimulation. Here we report that the ability of granuloma culture supernatants to stimulate the IL-1-sensitive D10.G4.1 cells but not fibroblasts is removed by treatment with immobilized anti-IL-1 antibody. We also observed that dialyzed culture supernatants from egg granulomas obtained from infected congenitally athymic (nude) mice also stimulate fibroblast proliferation. Treatment with anti-IL-1 antibody did not abrogate this response. In contrast to our experience with egg granulomas isolated from euthymic mice, IL-1- and fibroblast-stimulating activity could be separated by gel filtration and isoelectric focusing. We conclude that the fibroblast growth-stimulating activities elaborated by egg granulomas from S. mansoni-infected euthymic and athymic mice may be different but both appear to be distinct from IL-1.

Stimulation of glycosaminoglycan synthesis in cultured human dermal fibroblasts by interleukin 1. Induction of hyaluronic acid synthesis by natural and recombinant interleukin 1s and synthetic interleukin 1 beta peptide 163-171.

Hyaluronic acid (HA) is believed to play a critical role in wound healing and in morphogenesis. Factors controlling the production of HA by fibroblasts in normal and pathological states are not completely understood. In this report we have observed that natural human interleukin (IL-1)1 beta and human recombinant (hrIL)-1 alpha and beta are potent stimulators of HA production by fibroblasts in vitro. Hyaluronic acid is the major species of glycosaminoglycan (GAG) stimulated by IL-1 in fibroblasts. PGE2 does not appear to be involved directly in this IL-1 effect on fibroblasts, but stimulation of HA production by IL-1 is dependent on protein synthesis. The synthetic human IL-1 beta peptide 163-171 (Val-Gln-Gly-Glu-Glu-Ser-Asn-Asp-Lys), which has been previously shown to stimulate thymocyte proliferation but not fibroblast PGE2 production, is also able to stimulate fibroblast HA production. The synthesis and secretion of IL-1 by mononuclear phagocytes at sites of inflammation and immune reactions in vivo could potentially serve as a signal for fibroblasts to synthesize HA, which in turn could serve to facilitate and modulate reparative and immune processes by virtue of its ability to alter cell-cell, cell matrix, and cell-membrane receptor interactions.

A synergistic interaction of IL-6 and IL-1 mediates the thymocyte-stimulating activity produced by recombinant IL-1-stimulated fibroblasts.

We characterized the ability of normal human lung fibroblasts to elaborate thymocyte-stimulating activity, spontaneously, and in response to rIL-1. Supernatants from unstimulated fibroblasts did not contain thymocyte-stimulating activity, whereas supernatants from fibroblasts incubated with rIL-1 alpha or rIL-1 beta contained more thymocyte-stimulating activity than could be accounted for by passively transferred rIL-1 alone. This heightened thymocyte-stimulating activity was mediated by fibroblast-derived IL-6 inasmuch as it was neutralized by anti-serum against human rIL-6, and rIL-1-stimulated fibroblasts to accumulate messenger RNA for IL-6 and produce soluble IL-6 protein. However, IL-6 alone could not account for the intensity of this effect because rIL-6 only weakly stimulated thymocyte proliferation. In addition, antisera against the rIL-1 moiety that was used to prepare the supernatant had different effects on supernatants that contained and did not contain active IL-6. In the presence of IL-6 these antisera caused a greater decrease in thymocyte-stimulating activity than could be accounted for by passively transferred rIL-1 alone. When the IL-6 was neutralized the remaining thymocyte-stimulating activity could be quantitatively accounted for and neutralized by antisera against the rIL-1 that was passively transferred. Furthermore, rIL-6 and rIL-1 (alpha or beta) synergized in stimulating thymocyte proliferation. Thus, rIL-1 stimulates fibroblasts to produce a thymocyte-stimulating activity that is largely mediated by a synergistic interaction of fibroblast-derived IL-6 and IL-1. These findings suggest that fibroblast production of IL-6 may mediate or amplify some of the tissue effects of IL-1. In addition they suggest that biologic effects previously attributed to IL-1 may be due to IL-6 alone or the concerted action of IL-1 and IL-6.

Effect of aggregated immunoglobulins on mononuclear cell protein production in rheumatoid arthritis

The synthesis and secretion of proteins, especially interleukin 1 (IL-1), by mononuclear cells from patients with rheumatoid arthritis (RA) in response to heat-aggregated IgG (HAGG) has been investigated. Mononuclear cells were labeled with 35S-methionine during 1 hr of HAGG stimulation, and the intracellular and extracellular protein content was measured and compared by fluorography. HAGG dramatically accelerated protein release into the medium. This was observed in both normal and RA mononuclear cells but was much more marked with the latter. The phenomenon was correlated with the release of a IL-1 activity measurable in the thymocyte proliferation assay with a more pronounced labeling in the extracellular medium of a 14-kDa band not detectable in the intracellular pattern. Partially purified proteins between 14–20 kDa recovered by transblotting also stimulated thymocyte proliferation. These results therefore suggest a particular sensitivity of RA mononuclear cells to HAGG, resulting in a very rapid de novo synthesis and an accelerated secretion rate of IL-1.

Synergistic stimulation of fibroblast prostaglandin production by recombinant interleukin 1 and tumor necrosis factor.

Mononuclear cell production of cytokines that stimulate fibroblast prostaglandin (PG) elaboration is an important mechanism by which mononuclear cells regulate fibroblast function. However, the soluble factors mediating these PG-stimulatory effects are incompletely understood. We characterized the effects on PG production by confluent normal lung fibroblasts of recombinant interleukin 1 alpha (IL 1 alpha), interleukin 1 beta (IL 1 beta) and tumor necrosis factor (TNF), alone and in combination. All three cytokines stimulated fibroblast PG production with both IL 1 peptides being significantly more potent than TNF. In addition, TNF interacted in a synergistic fashion with both IL 1 peptides to augment fibroblast PGE elaboration further. The stimulatory effects of the cytokines were almost entirely caused by an increase in PGE2 production and were reversed when the cytokine(s) were removed. These changes in PG production could not be explained by alterations in cell number and were completely negated by specific anticytokine antibodies. Recombinant gamma interferon, although synergizing with TNF in regulating other cellular functions, did not interact with TNF to augment fibroblast PGE elaboration. In addition, the synergistic interaction of IL 1 and TNF did not extend to all biologic effects of IL 1 since TNF did not augment the ability of IL 1 to stimulate thymocyte proliferation.

Mesenchymal target cell specificity of egg granuloma-derived fibroblast growth factor in schistosomiasis.

We previously demonstrated that egg granulomas isolated from the liver of mice infected with Schistosoma mansoni secrete factors that stimulate fibroblast proliferation in vitro. Because this growth factor also stimulated thymocyte proliferation, we further investigated the mesenchymal target cell specificity of this factor. We now report that granulomas also secrete material that promotes growth of aortic smooth muscle and endothelial cells. We found that the activities that stimulate fibroblast and smooth muscle cell growth share similar physicochemical properties (Mr, 30-40 kilodaltons [kDa]; pI, approximately 6.5), a result suggesting that they are the same or related molecules. In contrast, fractions (Mr, less than 10 kDa; pI, 6.7-7.5) that maximally stimulated endothelial cells had minimal or no effects on the other two cell types. We postulate that soluble granuloma products might also stimulate in vivo proliferation of vascular smooth muscle and endothelial cells and contribute to the pathology of the intrahepatic vessels in schistosomiasis.

Ganglioside GM1 antibodies and B-cholera toxin bind specifically to embryonic chick dorsal root ganglion neurons but do not modulate neurite regeneration.

Polyclonal antibodies to ganglioside GM1 have been prepared and characterised by direct and competitive enzyme-linked immunoassay. An immunoglobulin fraction was prepared from a rabbit antisera showing high specificity and antibody titre for GM1 relative to the other major brain gangliosides. The anti-GM1 immunoglobulin fraction and B-cholera toxin specifically labelled neurons in primary cultures of embryonic chick dorsal root ganglia and there was a good correlation between the relative increase in binding of anti-GM1 immunoglobulin and B-cholera toxin following neuraminidase treatment of a variety of cell types. At antibody concentrations that show saturable binding to endogenous ganglioside in the neuronal membrane, the anti-GM1 immunoglobulin fraction did not interfere with the nerve growth factor (NGF)-mediated fibre outgrowth and neuronal survival as indexed by measurement of neurofilament protein levels. Similarly, at levels in excess of those shown to stimulate thymocyte proliferation, B-cholera toxin was also without effect. These data are not consistent with GM1 in the neuronal membrane functioning as a receptor molecule for NGF and/or other differentiation factors present in the tissue culture media.

Induction of membrane-associated interleukin 1 by tumor necrosis factor in human fibroblasts.

Highly purified recombinant human tumor necrosis factor (TNF) was found to induce interleukin 1 (IL 1) production in diploid human FS-4 fibroblasts. Demonstration of IL 1 activity was based on the ability of TNF-treated FS-4 cells, subsequently fixed with formaldehyde, to stimulate thymocyte proliferation in the presence of phytohemagglutinin. Incubation of FS-4 cells with the optimal dose of TNF (10 ng/ml) resulted in a marked increase in [3H] thymidine uptake by thymocytes co-cultured with formaldehyde-fixed FS-4 cells. Induction of this apparently membrane-associated IL 1 (MA-IL 1) activity was demonstrable at 6 hr and reached a plateau after 48 hr of incubation with TNF. FS-4 cells did not secrete soluble IL 1 in response to TNF. Dexamethasone suppressed the synthesis of TNF-induced MA-IL 1. A monoclonal antibody specific for TNF neutralized MA-IL 1 induction, indicating that the induction is due to TNF, and not to a contaminant in the TNF preparation. The ability of TNF to induce IL 1 synthesis in FS-4 fibroblasts at the transcriptional level was confirmed by S1 nuclease protection assay. Cytoplasmic RNA from uninduced FS-4 cells contained no demonstrable RNA hybridizing with a human IL 1-alpha cDNA probe and low levels of RNA hybridizing with an IL 1-beta cDNA. Induction with TNF resulted in the appearance of IL 1-alpha mRNA and a very significant increase in IL 1-beta mRNA, indicating that TNF induces the synthesis of both IL 1-alpha and IL 1-beta in FS-4 cells.