Cytokine regulation of interleukin 6 production by human endothelial cells

The influence of recombinant (r) human tumor necrosis factor alpha (rTNF-alpha), r human interleukin 1 beta (rIL-1 beta), and r human interferon gamma (rIFN-gamma) on the production of interleukin 6 (IL-6) by human endothelial cells (HEC) was investigated. The addition of 1-100 U/ml of either rTNF-alpha or rIL-1 beta to cultures of HEC monolayers caused a dose-related increase in IL-6 production as detected after 24 hr of incubation. In contrast to rIL-1 beta and rTNF-alpha, the use of up to 1000 U/ml of rIFN-gamma caused only a moderate increase in IL-6 production. However, significantly greater quantities of IL-6 were produced by HEC monolayers subjected to 1000 U/ml of rIFN-gamma in combination with 1-100 U/ml of rTNF-alpha. Furthermore, the addition of graded concentrations of human transforming growth factor beta (TGF-beta) to cultures resulted in a dose-related inhibition of rIL-1 beta- and rTNF-alpha-induced IL-6 production by HEC. The results demonstrate that rIL-1 beta and rTNF-alpha share the ability to stimulate HEC for production of IL-6 and indicate that TGF-beta may act as an immunosuppressive agent, at least partially, through its ability to inhibit the action of TNF-alpha and IL-1 on endothelial cells.     

Purification and characterization of the immunostimulatory properties of recombinant human interleukin-1 beta

In this study we have used a new method for human recombinant IL-1 beta (rIL-1 beta) purification and investigated its immunostimulatory biological activity. The IL-1 beta gene was cloned using a novel mRNA preparation from activated human blood monocytes. The purification protocol consists of extraction and two chromatographic steps using the new Soloza cation exchange resin. The purified protein was characterized electrophoretically, by amino acid analysis and reverse phase chromatography. The protein migrated on SDS-PAGE with a molecular weight of 18.200 but demonstrated the minor presence of aggregates (dimers and trimers). Specific activity of purified rIL-1 beta in comitogenic assay on mouse thymocytes was 10(8) U/mg protein. rIL-1 beta increased in a dose dependent manner proliferation of Con A-stimulated murine thymocytes, splenocytes, PHA-stimulated human peripheral blood lymphocytes and transformed B-cell lines. Comitogenic activity depended on the degree of lymphocyte preactivation and was similar to that of natural human IL-1 beta. rIL-1 beta enhanced IL-2 production by murine spleen cells and EL-4 cell line and IL-2 receptor expression by human peripheral blood mononuclear cells. It induced PGE2 release from human blood monocytes but had no effect on human neutrophil chemotaxis, phagocytosis and respiratory burst.     

Tumor necrosis factor-alpha enhances inhibitory effects of interleukin-1 beta on Leydig cell steroidogenesis

Human recombinant tumor necrosis factor-alpha (rTNF alpha) alone (up to 1000 units/ml) did not alter either basal or human chorionic gonadotropin (hCG)-induced testosterone formation in primary culture of rat Leydig cells. However, concomitant addition of rTNF alpha with human recombinant interleukin-1 beta (rIL-1 beta) enhanced the inhibitory effects of rIL-1 beta. The rIL-1 beta dose response curve was shifted to the left (IC50 changed from 1 ng/ml to 0.3 ng/ml). Even though rTNF alpha had no effect on testosterone formation, hCG-stimulated cyclic AMP formation was inhibited by rTNF alpha in a dose dependent manner. In the presence of both rTNF alpha and rIL-1 beta, hCG-induced cyclic AMP formation and binding of [125I]-hCG to Leydig cells were further inhibited. Testicular macrophages represent about 20% of the interstitial cells. TNF alpha and IL-1 may be produced locally by interstitial macrophages and have paracrine effects on Leydig cell function.     

Both interleukin-1 alpha and interleukin-1 beta are involved as accessory signals in primary antigen (tetanus toxoid) induced human T-cell activation

The function of interleukin-1 alpha and interleukin-1 beta (IL-1 alpha, IL-1 beta) in tetanus toxoid (TT) induced T-cell proliferation in cultures of peripheral blood mononuclear cells (PBL) obtained from healthy donors was assessed by using neutralizing antisera to IL-1 alpha and IL-1 beta. The neutralizing capacity and the specificity of the IL-1 antisera were tested by the use of the thymoma EL-4 NOB-1 cell line. Antisera to IL-1 beta effectively neutralized the proliferative capacity of human recombinant IL-1 beta but not of human recombinant IL-1 alpha and vice versa. Addition of either anti-IL-1 beta or anti-IL-1 alpha antiserum to the culture medium hardly affected TT induced T-cell proliferation. However, the proliferative T-cell response was consistently attenuated when a combination of IL-1 alpha and IL-1 beta antiserum was used. The antisera were never capable of completely abolishing the T-cell response to TT. We conclude that (a) IL-1 alpha and IL-1 beta are both necessary accessory signals for T-cell proliferation to antigen in vitro; (b) in T-cell proliferation IL-1 alpha and IL-1 beta are interchangeable; and (c) T-cell proliferation to antigen is only partially dependent on IL-1 as signal.     

Interleukin-1 receptors on human thyroid cells and on the rat thyroid cell line FRTL-5.

Cellular binding of interleukin-1 (IL-1) was tested on monolayers of human thyrocytes in secondary culture, on long-term cultures of human thyrocytes, and on the rat thyroid cell line FRTL-5. The human thyrocytes in secondary culture showed specific binding of human 125I-rIL-1 alpha. Scatchard plots of data obtained at 4 degrees C indicated the presence of a single population of receptors with a Kd of 30 to 170 pM and 2,000 to 6,000 receptors per cell. Incubation at room temperature resulted in internalization of the receptor-ligand complex. Parallel experiments were performed with the IL-1 receptor-positive murine T-cell lines EL-4 and NOB-1. The IL-1 receptors on these cells had Kd values one fifth to one tenth those on human thyroid cells in secondary culture. Both rIL-1 alpha and rIL-1 beta inhibited 125I-rIL-1 alpha binding to human thyrocytes and the murine T cells. In contrast to the cells in secondary culture, there was no specific binding of 125I-rIL-1 alpha to long-term cultivated human thyroid cells or to the FRTL-5 cells. We concluded that recently described differences in the response to IL-1 of different thyroid cell culture systems are most likely caused by differences in expression of IL-1 receptors.     

Up-regulation of hepatocyte growth factor gene expression by interleukin-1 in human skin fibroblasts.

Hepatocyte growth factor (HGF) functions as a hepatotrophic and renotrophic factor for regeneration of the liver and kidney. When 1 ng/ml of interleukin-1 alpha (IL-1 alpha) or interleukin-1 beta (IL-1 beta) was added to cultures of human skin fibroblasts, the production of HGF was 5-6 fold higher than levels in the controls. HGF mRNA level in the cells was increased to 4-fold higher levels at 6 h after exposure to IL-1 alpha. Tumor necrosis factor-alpha and interferon-gamma but no other cytokine tested had slightly stimulatory effects on HGF production. The tumor promoter, tetradecanoylphorbol 13-acetate (TPA) markedly enhanced the stimulatory effect of IL-1 alpha and IL-1 beta on the production of HGF. The stimulatory effect of both IL-1 alpha and IL-1 beta and the synergistical stimulation with TPA were completely abrogated by 10 ng/ml TGF-beta 1 or 1 microM dexamethasone. These results suggest that IL-1 alpha and IL-1 beta are positive regulators for expression of the HGF gene and are likely have a role in regeneration of tissues following the occurrence of inflammatory diseases.     

Regulation of acute phase reaction by transforming growth factor beta in cultured murine hepatocytes.

Transforming growth factor-beta (TGF beta 1), a multipotent immunoregulatory peptide produced by human platelets, has been shown to stimulate the synthesis of fibrinogen, contrapsin, complement component C3, and alpha-1-proteinase inhibitor by murine hepatocytes cultured for 2 days in DMEM containing 1 microM insulin and dexamethasone and 0.2% BSA. In the range of 10 pg to 10 ng/ml TGF-beta 1 did not elicit any change in albumin secretion. Two main inflammatory cytokines: interleukin-6 (IL-6) and interleukin-1 (IL-1), known to stimulate two different subsets of murine acute phase plasma proteins, failed to increase contrapsin and alpha-1-proteinase inhibitor production. Epidermal growth factor (EGF) in the concentration 1 ng to 10 ng/ml effectively counteracted the stimulatory effect of TGF-beta 1 on acute phase protein production. TGF-beta 1-induced fibrinogen protein levels were associated with increased beta-fibrinogen mRNA content. TGF-beta 1 appears to be an additional physiological factor responsible for the direct stimulation of normal mouse hepatocytes to acute phase response.     

Modulation of IL-1 inflammatory and immunomodulatory properties by IL-6.

Among the major cytokines present in inflammatory lesions interleukin-1 (IL-1), tumor necrosis factor alpha (TNF alpha) and interleukin-6 (IL-6) share many biological activities. Since IL-1 alpha, IL-1 beta and TNF alpha have been previously demonstrated to play an important role in connective tissue destruction by stimulating the production of prostaglandin E2 (PGE2) and collagenase, these functions were investigated in the presence or absence of natural human IL-6 (nhIL-6) or recombinant human IL-6 (rhIL-6). IL-6 was found 1 degree to stimulate immunoglobulin A production by the CESS B cell line up to 19 fold without being affected by the presence of IL-1 beta and 2 degrees to stimulate murine thymocytes proliferation up to 2-4 fold, with an increase up to 60-fold in costimulation with either IL-1 alpha or beta. IL-6 alone, even at very high concentrations (up to 200 U/ml and 50 ng/ml), did not induce PGE2 production by fibroblasts and synovial cells. However, IL-1 alpha or beta induced PGE2 production by human dermal fibroblasts and by human synovial cells was inhibited (in 5/8 experiments) up to 62% by addition of IL-6. On the contrary in 2/4 experiments TNF alpha-induced PGE2 production was increased (approximately 2 fold) by the addition of IL-6. IL-1 and TNF alpha-induced collagenase production in synovial cells remained unchanged in the presence of IL-6.(ABSTRACT TRUNCATED AT 250 WORDS)     

Decline in the production of interleukin-3 with age in mice

Previously, we and others have found that the ability to produce interleukin-1 (IL-1) and interleukin-2 (IL-2) declines with age in mice. The purpose of this study was to determine the influence of age on the capacity of mice to produce interleukin-3 (IL-3). Splenic cells (5 X 10(6)/ml) from young (3-4 months) and old (24-32 months) C57BL/6 mice were first assessed for their IL-3-producing capacities in response to varying doses of concanavalin A (Con A; 2-20 micrograms/ml) in a time-dependent manner. The results showed that the production of IL-3 by both young and old C57BL/6 mice was maximal on Days 3 and 4 in response to 20 micrograms/ml of Con A, and that of IL-2 was minimal (activity was less than 0.1 unit) on Day 4. Consequently, Day 4, was selected to assess the effect of age on IL-3 production by splenic cells. The results showed a twofold reduction in IL-3 production with age (P less than 0.05). Young-old splenic cell mixture experiments at ratios of 1:0, 3:1, 1:1, 1:3, and 0:1 indicated that the decrease in IL-3 production with age was not due to an increase in suppressor cell activity. Experiments based on mixtures of nylon wool-enriched splenic T-cell and adherent cells and on anti-MAC-1 plus complement-treated spleen cells indicated that (a) adherent cells are not required for T-cell production of IL-3, unlike IL-2 production, and (b) the decrease in IL-3 production with age is due solely to alteration in IL-3-producing T cells. Finally, a strong correlation was demonstrated between the production of IL-2 and IL-3 by spleen cells of individual young and old mice (r = 0.92, P less than 0.01). That production of both IL-2 and IL-3 is affected in a similar manner by age would suggest that a single class of helper T cells may be responsible for production of both lymphokines.     

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)     

Transforming growth factor-beta s are equipotent growth inhibitors of interleukin-1-induced thymocyte proliferation

The effects of two forms of transforming growth factor-beta, TGF-beta 1 and TGF-beta 2, upon the proliferative response of murine thymocytes were investigated in this study. TGF-beta 1 and TGF-beta 2 were found to be equipotent growth inhibitors of interleukin-1 (IL-1)- and phytohemagglutinin (PHA)-stimulated thymocytes when added at the initiation of the cultures. These factors suppressed the proliferative response in a dose-dependent fashion between 0.4 and 100 pM. The proliferative response was maximally inhibited (90% inhibition) at 100 pM. The half-maximal inhibitory dose (ID50) was 6 and 4 pM for TGF-beta 1 and TGF-beta 2, respectively. These factors were less effective or ineffective at suppressing the proliferation of thymocytes which had been prestimulated for 24 to 48 hr by IL-1 and PHA. Neither factor inhibited interleukin-2 (IL-2)-dependent thymocyte proliferation or the proliferation of an IL-2-dependent cytotoxic T cell line (CTL-L), suggesting that the anti-proliferative actions of these factors was by inhibition of cellular events triggered by IL-1. Furthermore, anti-TGF-beta 1 antibodies did neutralize the biological actions of TGF-beta 1 and these antibodies did block the binding of 125I-labeled TGF-beta 1 to cell surface receptors showing that the inhibitory action is mediated through specific receptors for TGF-beta 1 on thymocytes. These antibodies, however, did not neutralize the anti-proliferative action of TGF-beta 2. Although TGF-beta 1 and TGF-beta 2 exhibit very similar biological activities, these molecules are antigenically different and, therefore, have different tertiary structures.     

T4 cell activation by immobilized phytohemagglutinin: differential capacity to induce IL-2 responsiveness and IL-2 production

Soluble mitogens, such as PHA induce accessory cell (AC)-dependent T cell proliferation. One function of the AC is to create a stimulatory matrix. Therefore, experiments were carried out to determine whether PHA immobilized onto microtiter plates could stimulate T cells in the absence of AC. Peripheral blood T4 cells were cultured under limiting dilution conditions with either soluble or immobilized PHA with or without rIL-1 beta, rIL-2, r-TNF-alpha, an anti-CD28 mAb (9.3), or irradiated EBV-transformed B cells as AC. The frequency of proliferating T4 cells was assessed by examining wells microscopically, and the frequency of T4 cells producing IL-2 was assessed by examining the ability of supernatants to support CTLL-2 proliferation. The percentage of T4 cells growing and producing IL-2 was determined by a maximum likelihood procedure. Immobilized, but not soluble, PHA induced a mean of 20.0 +/- 2.6% of T4 cells to grow in the complete absence of AC in medium supplemented with rIL-2. Whereas rIL-1 beta, rTNF-alpha, and 9.3 were unable to support T4 cell growth in the absence of rIL-2, each enhanced the percentage of T4 cells responding to immobilized PHA in the presence of rIL-2. In contrast, both soluble and immobilized PHA were unable to induce T4 cell IL-2 production in the absence of AC, even when cultures were supplemented with rIL-1 beta or 9.3. In the presence of AC, a small percentage of T4 cells (5.4 to 11.7%) was stimulated to produce detectable amounts of IL-2 by either immobilized or soluble PHA. Moreover, in the presence of AC, a very small population (approximately 1%) of PHA-stimulated T4 cells proliferated without supplemental rIL-2. The data indicate that a matrix of immobilized PHA is sufficient for some T4 cells to be activated to respond to IL-2, whereas others require additional signals provided by rIL-1 beta, rTNF alpha, 9.3, or AC. In contrast, neither soluble nor immobilized PHA is sufficient to induce T cell IL-2 production. This response requires signals provided by intact AC.     

Differential biological activities of human interleukin-1 alpha and interleukin-1 beta

We examined the biological effects induced by both human recombinant interleukin-1 alpha (IL-1 alpha) and beta (IL-1 beta) in five different cell types of human, rat and mouse origin. IL-1 alpha and beta preparations were standardized in terms of biological activity in the EL-4/CTLL bioassay and, in parallel, employed to stimulate PGE2 secretion in human fibroblasts, mesangial cells (MC), C57B1/6 mouse MC, DBA/2 mouse macrophages and Sprague Dawley rat MC. In addition, the co-mitogenic effects of IL-1 alpha and beta were determined in freshly prepared Sprague Dawley rat thymocytes. No significant differences in IL-1 alpha and beta concentration dependent PGE2 production were detectable in the different cell types (MC, fibroblasts and macrophages) of human or mouse origin. Incubation of Sprague Dawley rat MC with both IL-1 alpha and beta resulted in a concentration dependent production of PGE2. However, in contrast to mouse or human MC the potency of IL-1 beta to induce PGE2 in Sprague Dawley rat MC was 26-fold higher compared to IL-1 alpha. In addition, the potency of IL-1 beta to enhance co-stimulated proliferation of Sprague Dawley thymocytes was 200-fold higher than that of equal biological activities of IL-1 alpha. When we tested the additive effects on Sprague Dawley cells, increasing IL-1 beta concentrations added to a fixed IL-1 alpha concentration resulted in a cumulative rise in both, PGE2 secretion by MC and thymocyte proliferation.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effect of interleukin-1, interleukin-6 and its interrelationship on the synthesis of serum amyloid A and C-reactive protein in primary cultures of adult human hepatocytes

During the acute phase response, synthesis of C-reactive protein and serum amyloid A is increased. To investigate whether the enhanced synthesis of these proteins are due to stimulatory effect of inflammatory mediators such as interleukin-1 (IL-1) and interleukin-6 (IL-6) produced by macrophages and monocytes, primary cultures of adult human hepatocytes were exposed to recombinant (r)IL-1, rIL-6 or rIL-1 and monospecific anti rIL-6 antibodies in the presence of 1 microM dexamethasone. The findings indicate that rIL-1 and rIL-6 both stimulate the liver synthesis of C-reactive protein and serum amyloid A, however monospecific anti rIL-6 antibodies reduce the stimulatory effect of rIL-1 on the synthesis of these proteins. These findings suggest that IL-6 plays a key role in the stimulation of synthesis of serum amyloid A and C-reactive protein by the human liver cells.     

Interleukin-2-induced production of interferon-gamma by resting human T cells and large granular lymphocytes: requirement for accessory cell factors, including interleukin-1

Between 5 and 20% of normal human lymphocytes were found to synthesize interferon-gamma (IFN-gamma) in primary cultures with recombinant interleukin-2 (rIL-2). After 22 hr, IFN-gamma-producing cells included CD5+ T lymphocytes, CD16+ large granular lymphocytes (LGL), and a population of CD5-, CD16- blast cells. Only a small proportion (0-7%) of IFN-gamma-synthesizing cells expressed HLA-DR. The production of IFN-gamma by all rIL-2-responding lymphocyte subsets was shown to require the presence of DR+ accessory cells, probably including nonadherent, esterase-negative monocytes and/or dendritic cells. Accessory cell function in lymphocyte preparations depleted of DR+ cells, or in purified (greater than or equal to 95%) suspensions of LGL, was fully replaced either by addition of 2% autologous, adherent monocytes or by monocyte culture supernatant. The activity of monocyte supernatant was greatly reduced by treatment with antiserum specific for human interleukin-1 beta (IL-1 beta), although a combination of rIL-1 beta and rIL-2 failed to stimulate IFN-gamma production in DR- lymphocytes. These results indicate that rIL-2-induced IFN-gamma synthesis in both T cells and LGL requires the synergistic activity of IL-1, and possibly of one or more other monokines, as yet unidentified.     

Purification and characterization of human recombinant interleukin-1 beta

A human interleukin-1 (IL-1) beta cDNA was cloned, and the region coding for the mature protein was expressed in Escherichia coli. The 17-kDa biologically active product was purified in 40% yield to apparent homogeneity, without chaotropes, from the soluble fraction of sonicated cell lysates. The recombinant IL-1 beta was characterized by amino acid analysis, NH2- and COOH-terminal sequence analysis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, spectroscopy, and biological assay. Specific biological activity was 4.6 X 10(8) units/mg in a co-mitogenic IL-2 induction assay using cultured EL-4 T-lymphocytes. The molar extinction coefficient was determined to be 10,300 cm-1 M-1 at 280 nm. NH2-terminal sequence analysis revealed that 70% of the product begins with the Ala corresponding to the NH2 terminus of the natural protein, while 30% begins with the following Pro. No initiator Met was observed. Both of the sulfhydryl groups are reactive to Ellman's reagent and to iodoacetamide under nonreducing conditions, indicating that the Cys residues do not form disulfide bonds. S-Carboxamidomethyl-Cys-rIL-1 beta retained biological activity in the IL-2 induction assay. Circular dichroism suggested an extensive beta sheet structure for rIL-1 beta.     

Regulation of mitogen-stimulated human T-cell proliferation, interleukin-2 production, and interleukin-2 receptor expression by protein kinase C inhibitor, H-7

Recently published reports suggest that the activation of protein kinase C (PKC) plays an important role in the activation pathway of many cell types. In this study, we examined the role of PKC in human T-cell proliferation, IL-2 production, and IL-2R expression, when cultured with the mitogen PHA, the PKC inhibitor H-7, and H-7 control HA1004. H-7 inhibited the PHA-stimulated [3H]thymidine uptake, IL-2 production, and IL-2R expression in a dose-related manner. Further, we found H-7 inhibited T-cell proliferation, IL-2 production, IL-2 mRNA from PHA plus PMA-stimulated cultures. We also found that H-7 inhibited the early-stage activation of PHA-stimulated cells. The presence of exogenous purified human IL-2 or rIL-4 partly reversed the immunosuppression caused by H-7. In contrast, HA1004 had no effect on cell proliferation, IL-2 production, or IL-2R expression. Our results demonstrate that PKC activation is one major pathway through which T-cells become activated.     

Augmentation of antitumor effect by combined administration with interleukin-2 and sizofiran,a single glucan,on murine EL-4 lymphoma

The antitumor effect of the combined administration with recombinant human interleukin-2 (rIL-2) and sizofiran (SPG), a single glucan of Shizophyllum commune Fries, was studied in vivo in C57BL/6 mice intraperitoneally inoculated with EL-4 lymphoma. The effect was evaluated by a) comparing the survival time of the mice, b) analysis of the intraperitoneal cell population in Giemsa-stained specimens, c) surface marker analysis of peritoneal exudative cells with flow cytometry, d) cytotoxic assay of cells against EL-4 and Yac-1 lymphoma, and e) elimination of some cell populations by monoclonal antibodies, to identify the antitumor-effector cells showing cytotoxic activity. The survival of mice given both rIL-2 and SPG was significantly longer than the control mice or those given SPG alone or rIL-2 alone. It was demonstrated that the administration of SPG and/or rIL-2 to the EL-4 lymphoma-bearing mice activated immune-response cells in the peritoneal cavity such as T lymphocytes, NK cells, or macrophages, which might be effective in reducing lymphoma cells. The combination of rIL-2 and SPG administration appears to activate the antitumor- immune response at the tumor site more effectively than when either agent was administered alone.