Effect of combinations of cytokines and hormones on synthesis of serum amyloid A and C-reactive protein in Hep 3B cells.

We have previously shown that induction of synthesis of the two major human acute phase proteins, serum amyloid A (SAA) and C-reactive protein (CRP), can be accomplished in the human hepatoma cell line Hep 3B, in the presence of dexamethasone, either by conditioned medium from LPS-stimulated monocytes or by the combination of IL-6 and IL-1. Neither of these cytokines alone caused significant induction of either SAA or CRP. In the present study we extended our earlier observations by evaluating the role of dexamethasone, the effect of different concentrations of IL-6 and IL-1 alpha in combination, and the possible role of TNF-alpha in regulating synthesis of SAA and CRP. Dexamethasone alone had no effect on induction of SAA or CRP. Incubation of Hep 3B cells with conditioned medium from LPS-stimulated monocytes, in the absence of dexamethasone, led to modest induction of SAA or CRP, but addition of dexamethasone potentiated this response in a dose-dependent manner. Similar results were obtained for the effect of dexamethasone on the induction of SAA by IL-6 plus IL-1 alpha. Checkerboard titration of IL-6 and IL-1 alpha revealed that increases in concentration of either cytokine led to dose-related increases in synthesis of both SAA and CRP as long as a minimal amount of the other cytokine was present. TNF-alpha alone had no significant effect on synthesis of either SAA or CRP, but the combination of IL-6 plus TNF-alpha led to substantial induction of SAA. This combination was less effective than the combination of IL-6 plus IL-1 alpha. No detectable effect of IL-6 plus TNF-alpha was observed on CRP synthesis. Both combinations of cytokines, IL-6 plus IL-1 alpha, and IL-6 plus TNF-alpha, caused increased SAA mRNA accumulation that roughly paralleled increase in synthesis. These data indicate that IL-6, IL-1 alpha, TNF-alpha, and dexamethasone in various combinations are all capable of influencing synthesis of SAA in Hep 3B cells, whereas only IL-6, IL-1 alpha, and dexamethasone can influence CRP synthesis.     

Interaction among hepatocyte-stimulating factors, interleukin 1, and glucocorticoids for regulation of acute phase plasma proteins in human hepatoma (HepG2) cells

Human hepatoma (HepG2) cells respond to unfractionated conditioned media of human squamous carcinoma (COLO-16) cells and lipopolysaccharide-stimulated human peripheral blood monocytes by increasing the synthesis of alpha 1-acid glycoprotein, haptoglobin, complement C3, alpha 1-antichymotrypsin, alpha 1-antitrypsin, and fibrinogen, while decreasing the synthesis of albumin. The regulation of the acute phase proteins is mediated by hepatocyte-stimulating factors (HSF) and interleukin 1 (IL-1) present in the conditioned medium. Purified HSF-I from COLO-16 cells stimulates preferentially alpha 1-acid glycoprotein synthesis, whereas COLO-HSF-II stimulates preferentially the synthesis of haptoglobin, fibrinogen, and alpha 1-antitrypsin. HSF from monocytes, which has been identified as interferon-beta 2 (B cell stimulating factor-2), displayed the same activity as COLO-HSF-II. Dexamethasone alone had no effect on acute phase plasma protein synthesis but enhanced the response to various HSF severalfold. IL-1 had a relatively low stimulatory activity on the synthesis of alpha 1-acid glycoprotein, haptoglobin, and alpha 1-antichymotrypsin but strongly reduced the basal expression of fibrinogen. The only synergistic action between IL-1 and HSF (or interferon-beta 2) was noted for the synthesis of alpha 1-acid glycoprotein. Tumor necrosis factor active on other hepatic cells failed to modulate significantly the expression of any plasma proteins in HepG2 cells. These studies showed that for an optimal HepG2-cell response a combination of HSF (or interferon-beta 2), IL-1, and dexamethasone is needed. This finding might indicate the identity of some of those hormones involved in regulation of the hepatic acute phase response in vivo.     

Modification of proinflammatory cytokine production by the antirheumatic agents tenidap and naproxen. A possible correlate with clinical acute phase response.

The cytokines IL-6, IL-1, and TNF play a key role in the pathogenesis of rheumatoid arthritis (RA) and initiate hepatic serum amyloid A (SAA) expression after injury. To provide a possible mechanistic explanation for the previous observation that plasma SAA concentrations decreased during treatment of RA patients with tenidap, but increased during treatment with naproxen, the present study compared the effects of tenidap and naproxen on the two stages of SAA expression: cytokine production by human PBMC and cytokine-stimulated SAA synthesis by human Hep3B hepatoma cells. Tenidap inhibited production of IL-6 greater than TNF greater than IL-1; the effect of naproxen on production of all three cytokines was lesser and least on IL-6. Indeed, an increase in IL-6 production was observed after exposure to naproxen. PBMC beta-2-microglobulin production and total protein synthesis were unaffected at concentrations and times at which effects on cytokine production were observed. Cell density was a significant factor in the extent to which cytokines were stimulated by LPS. Approximately physiologic cell densities, 0.5 to 1 x 10(6) cells/ml, were optimal for stimulation of IL-1-beta and IL-6 production by LPS; however, greater amounts of TNF were produced at lower cell densities. Because neither tenidap nor naproxen inhibited SAA synthesis by cytokine-stimulated Hep3B cells and because they differ most significantly in their effect on IL-6 production, the results support a role for IL-6 in the continued stimulation of SAA production during RA.     

Tumor necrosis factor (TNF) inhibits interleukin (IL)-1 and/or IL-6 stimulated synthesis of C-reactive protein (CRP) and serum amyloid A (SAA) in primary cultures of human hepatocytes

Interleukin (IL)-1, IL-6 and tumor necrosis factor (TNF) are considered as important mediators for the modulation of liver synthesis of acute phase proteins. However, studies of the direct effect of individual or a combination of these cytokines on the synthesis of acute phase proteins in human hepatocytes are still very limited. In this study, we have examined the synthesis of C-reactive protein (CRP) and serum amyloid A (SAA) in primary cultures of human hepatocytes exposed to recombinant(r)IL-1 alpha (100 U/ml), rIL-6 (2000 U/ml), rTNF alpha (30 U/ml) and to various combinations of these cytokines in the presence of 1 microM dexamethasone. Monoclonal antibodies to rTNF alpha and monospecific anti-rIL-6 sheep antiserum were also used to investigate the possible endogenous production of TNF or IL-6. The findings indicate: (1) IL-1 and IL-6 are stimulatory cytokines for the liver synthesis of CRP and SAA. Anti IL-6 abolishes the stimulatory effect of IL-1. These findings support the previous observation and indicate that IL-1 exerts its action on the enhanced synthesis of CRP and SAA at least in part via IL-6 production in the liver cell. (2) TNF is an inhibitory cytokine for the liver synthesis of CRP. It inhibits also the stimulatory effect of IL-1 and IL-6 on the synthesis of CRP and SAA. (3) Since anti-TNF enhances the stimulatory effect of IL-6 on the synthesis of CRP and SAA, it seems likely that TNF is also produced by the human hepatocytes. However, further studies for more direct evidence of the liver cell production of TNF, such as the detection of TNF messenger RNA are required.     

Human keratinocytes and monocytes release factors which regulate the synthesis of major acute phase plasma proteins in hepatic cells from man, rat, and mouse

Human keratinocytes and activated monocytes produces factors which can stimulate the proliferation of thymocytes. The same activity has also been implicated in regulating the expression of plasma proteins in liver cells during the acute phase reaction. To assess whether factors produced by such cells can directly influence liver cells to change the production of acute phase plasma proteins, we studied in tissue culture the response pattern of hepatic cells from three species: human hepatoma cells ( HepG2 cells), and primary cultures of rat and mouse hepatocytes. Conditioned media from the squamous carcinoma COLO-16 cells, normal epidermal cells, and activated peripheral monocytes were able to stimulate the synthesis of specific acute phase plasma proteins: alpha 1-antichymotrypsin in HepG -2 cells, alpha 1-antichymotrypsin, alpha 1-acid glycoprotein, alpha 1-acute phase protein, and alpha 2-macroglobulin in rat hepatocytes, and alpha 1-acid glycoprotein, haptoglobin, and hemopexin in mouse hepatocytes. Only in rat cells, dexamethasone was found to have further enhancing effect. The increased production of plasma proteins could be explained by an elevated level of functional mRNA. Comparing thymocyte-stimulating activities with the effects on plasma protein production, we found some difference both between the conditioned media of epidermal cells and monocytes, and between the responses of the three hepatic cell systems. Furthermore, gel chromatography of conditioned media resulted in partial separation of activities regulating liver cells and thymocytes. Since there is no strict correlation between thymocyte- and hepatocyte-stimulating activities, the presence of different sets of specific factors is assumed.     

Human hepatocyte-stimulating factor-III and interleukin-6 are structurally and immunologically distinct but regulate the production of the same acute phase plasma proteins

Human squamous carcinoma (COLO-16) cells synthesize and secrete hepatocyte-stimulating factor-III (HSF-III), a glycoprotein with Mr = 39,000, which stimulates the synthesis of several acute phase plasma proteins in human hepatoma (HepG2) cells. The qualitative response of HepG2 cells to HSF-III is essentially the same as that elicited by human recombinant interleukin-6 (IL-6). Although similar in hepatocyte-stimulating activity, HSF-III and IL-6 are distinct molecules which differ not only in size and charge but also in immunologic properties: no cross-recognition of HSF-III and IL-6 occurs using neutralizing antibodies against IL-6 and HSF-III, respectively. In addition, Northern blot hybridization of IL-6 cDNA to mRNA from COLO-16 cells revealed no detectable IL-6 message. HSF-III does not compete for binding to the IL-6 receptors suggesting that HepG2 cells carry receptors specific for each hormone. Both receptor types may trigger similar intracellular processes explaining the identical regulation of acute phase protein expression.     

The protozoan parasite, Theileria annulata, induces a distinct acute phase protein response in cattle that is associated with pathology

Acute phase proteins (APP) are synthesised in the liver in response to the systemic presence of high levels of pro-inflammatory cytokines. Bacteria are considered to be strong inducers of APP whereas viruses are weak or non-inducers of APP. Very few reports have been published on APP induction by parasites. Here, we report that the tick-borne protozoan parasite of cattle, Theileria annulata, induced an atypical acute phase response in cattle. Following experimental infection, serum amyloid A (SAA) appeared first, followed by a rise in alpha(1) acid glycoprotein (alpha(1)AGP) in all animals, whereas haptoglobin, which is a major APP in cattle, only appeared in some of the animals, and generally at a low level. All three APP only became elevated around or after the appearance of schizonts in draining lymph nodes and after the first observed temperature rise. Increased alpha(1)AGP levels coincided with the appearance of piroplasms. The production of SAA and alpha(1)AGP correlated strongly with each other, and also with some clinical measures of disease severity including the time to fever, development of leucopaenia, parasitaemia and mortality. These results are consistent with the hypothesis that T. annulata causes severe pathology in susceptible cattle by inducing high levels of pro-inflammatory cytokines.     

Induction of inflammatory cytokine release from cultured human monocytes by C-reactive protein.

The human acute phase protein, C-reactive protein (CRP), is capable of specifically binding to and modulating the function of mononuclear phagocytes. To investigate whether CRP can also affect the capacity of these cells to produce inflammatory cytokines, enzyme immunoassays and Western blot techniques were used to quantitate interleukin 1 beta (IL-1 beta), interleukin 6 (IL-6) and tumor necrosis factor alpha (TNF-alpha) produced by freshly-isolated normal human monocytes. CRP induced the rapid release of each cytokine, with significantly elevated levels in culture supernatants at 4 hours and maximal levels of TNF-alpha at 8 hours, and of IL-1 beta and IL-6 at 16 hours of culture. The effects of CRP were dose-dependent; greater than 10-fold increases of each cytokine were observed following culture with greater than or equal to 50 micrograms/ml CRP, concentrations which are often found in the presence of moderate to severe inflammation or tissue injury. The induction of cytokine release by CRP was unaffected by inclusion of 25 micrograms/ml polymyxin-B in culture media, but was completely abrogated by prior boiling of the CRP, a procedure which had no effect on induction of monocyte cytokine release by lipopolysaccharide. The dose-dependent induction of inflammatory cytokines by CRP provides further support for the hypothesis that interaction with mononuclear phagocytes constitutes an important biological role for this acute phase protein.     

Induction of interleukin-1 in human monocytes by the superantigen staphylococcal enterotoxin A requires the participation of T cells

Nanogram quantities of the bacterial superantigen Staphylococcal Enterotoxin A (SEA) induced significant amounts of extracellular IL-1 alpha and IL-1 beta in human peripheral blood mononuclear cells. Induction of maximal IL-1 alpha and IL-1 beta levels by lipopolysaccharide (LPS) required microgram quantities. LPS induced detectable extracellular IL-1 content within 3-6 hr and maximal levels were detected already after 12 hr. Induction of IL-1 production by SEA showed a delayed release with peak values after 24-48 hr. IL-1 beta was the major species of IL-1 seen in both SEA- and LPS-stimulated culture supernatants. SEA was in general a relatively stronger inducer of extracellular IL-1 alpha than LPS. SEA-induced extracellular IL-1 production in human monocytes was entirely dependent on the presence of T cells, whereas addition of T cells to LPS-stimulated purified human monocytes only marginally enhanced the extracellular IL-1 production. The capacity to induce extracellular IL-1 production in monocytes in response to SEA was high in the CD4+ 45RO+ memory T cell subset, whereas CD4+ 45RA+ naive T cells and CD8+ T cells had lower IL-1-inducing capacity. The T cell help for IL-1 production could not be replaced by a panel of T cell-derived recombinant lymphokines added to SEA-stimulated monocytes, including IFN-gamma and TNF, indicating the participation of cell membrane-bound ligands or hitherto unidentified soluble mediators.     

The acute-phase response of cultured rat hepatocytes. System characterization and the effect of human cytokines.

Hepatocytes were isolated from adult livers and cultured for periods of up to 5 days as monolayers at an initial density of 10(6) cells/10cm2 in Williams E medium containing insulin, dexamethasone and 5% foetal-calf serum. The daily production of 11 plasma proteins was measured by electroimmunoassay and compared with the concentrations of the same proteins in the plasma of normal rats and of those with experimental inflammation. Hepatocytes from normal rats synthesized proteins in relative amounts which were similar to the relative proportions of the same proteins in the plasma of turpentine-injected animals. The pattern changed only slowly during 5 days in culture, but it did so profoundly either when the medium was devoid of dexamethasone or when human cytokines (from endotoxin-stimulated monocytes or unstimulated human squamous-carcinoma cell line COLO-16) were added. The cytokines consistently increased the synthesis of alpha 2-macroglobulin and fibrinogen and depressed that of albumin; variable increases in the synthesis of alpha 1-acute-phase globulin, alpha 1-acid glycoprotein, haptoglobin and alpha 1-proteinase inhibitor, and variable decreases in transferrin synthesis, were seen, whereas the synthesis of antithrombin III, alpha 1-macroglobulin and prothrombin remained virtually unaffected. The cytokine effects on protein synthesis required the presence of dexamethasone. The hepatocyte-stimulating activity derived from monocytes chromatographed on Sephadex G-100 corresponding to 30 000 Da, as opposed to the lymphocyte-activating factor, which was eluted as a molecule of approx. 15 000 Da. This suggests that both activities probably reside with distinct molecular species in the preparations of human cytokines.     

IL-6 plays a critical role in the synergistic induction of human serum amyloid A (SAA) gene when stimulated with proinflammatory cytokines as analyzed with an SAA isoform real-time quantitative RT-PCR assay system

Serum amyloid A (SAA) is known to be a precursor of amyloid A (AA) protein in AA (secondary) amyloidosis and SAA1 to be mainly involved in AA amyloidosis. We established an SAA isoform real-time quantitative RT-PCR assay and found that beta-2 microglobulin is more stable as an internal control than GAPDH and beta-actin for our system. Either IL-6 and IL-1beta or IL-6 and TNFalpha, but not IL-1beta and TNFalpha, induced the synergistic induction of SAA1 and SAA2 genes. Anti-IL-6 receptor monoclonal antibody completely inhibited the synergistic induction of SAA1 and SAA2 during triple stimulation with IL-6, IL-1beta, and TNFalpha, but, IL-1 receptor antagonist or anti-TNFalpha monoclonal antibody was only partially inhibited in HepG2, Hep3B, and PLC/PRF/5 cells. Although the SAA1 promoter has no STAT3 consensus sequence, the JAK2 inhibitor-AG490 reduced SAA1 gene expression to 30%, suggesting the involvement of STAT3. We were able to demonstrate that IL-6 plays a critical role in the synergistic induction of human SAA gene when stimulated with proinflammatory cytokines.     

Cytokine regulation of IL-1 beta gene expression in the human polymorphonuclear leukocyte

Although recently polymorphonuclear leukocytes (PMN) have been identified as producers of IL-1 beta in response to LPS and granulocyte/monocyte colony stimulating factor, little is known regarding the ability of other cytokines to induce the production of IL-1 beta in the PMN. Inasmuch as IL-1 and TNF have been shown to be important priming agents, as well as agents that induce migration of PMN, we investigated their effect on IL-1 beta gene expression in human peripheral blood PMN. In the present study, we demonstrate that human peripheral blood PMN produce IL-1 beta in response to IL-1 alpha, IL-1 beta, and TNF-alpha. Control (unstimulated) human PMN had virtually undetectable levels of IL-1 beta mRNA. Either IL-1 beta or TNF, induced PMN to transiently express IL-1 beta mRNA with peak expression at 1 h, returning to untreated levels by 2 h. A dose response indicated that as little as 0.05 ng/ml of IL-1 beta or TNF resulted in IL-1 beta induction, with maximal effects at 1 ng/ml of IL-1 beta and 5 ng/ml of TNF. IL-1 alpha or IL-1 beta exhibited similar dose responses in IL-1 beta mRNA induction. Inasmuch as cytokines have been shown to have synergistic effects in cell function studies, we induced PMN with a combination of maximally effective doses of TNF plus IL-1 beta. They demonstrated a cooperative effect on IL-1 beta gene expression, in that mRNA levels were sustained for three hours. IL-1 beta Ag expression, as measured by ELISA, paralleled IL-1 beta mRNA expression with cell associated peak levels at 2 to 4 h. IL-1 beta Ag levels in PMN lysates and supernatants correlated with IL-1 beta mRNA levels, i.e., TNF + IL-1 greater than TNF greater than IL-1. Thus, these studies represent the first demonstration of IL-1 and TNF induction of IL-1 beta gene expression in the PMN. Furthermore, the time course of induction is unique to the PMN, with peak induction of mRNA at 1 h, which is consistent with the short lived nature of these cells in inflammatory lesions.     

Effects of cytokine combinations on acute phase protein production in two human hepatoma cell lines.

We evaluated the effects of binary combinations of four cytokines on production of the positive acute phase proteins alpha-1 antichymotrypsin, haptoglobin and fibrinogen, and the negative acute phase proteins albumin and alpha-fetoprotein (AFP) in two human hepatoma cell lines. The effects of the cytokine combinations on the five proteins varied; each protein exhibited a unique and specific pattern of response to the cytokine combinations. In Hep G2 cells, antichymotrypsin was induced by all four cytokines, IL-6, IL-1, TNF-alpha, and transforming growth factor beta 1 alone, and their effects in binary combinations could be attributed to additive or minimally synergistic interactions. Fibrinogen was induced only by IL-6 and this induction was inhibited by IL-1 alpha, TNF-alpha or transforming growth factor beta 1. Haptoglobin was also induced only by IL-6, but TNF-alpha was the only cytokine that inhibited this induction at all concentrations of IL-6. Each of the four cytokines alone down regulated production of AFP and albumin. However, binary combinations of the four cytokines were simply additive, for the most part, in inhibiting AFP production, whereas the inhibitory effects of combinations of cytokines on albumin production differed significantly from simple additive effects. These observations, taken together with studies of effects of cytokine combinations on other acute phase proteins, indicate that the various acute phase proteins respond differently to different combinations of cytokines and that the potential exists for highly specific regulation of synthesis of individual plasma proteins by cytokine interactions. These findings imply that the acute phase response in vivo represents the integrated sum of multiple, separately regulated changes in gene expression.     

Interleukin-1 and tumour necrosis factor production by human monocytoid cells: study on a single cell level.

Human IL-1 beta and TNF alpha production by normal and transformed monocytoid cells was studied using biological assays, cytokine specific ELISA and by immunocytochemical methods on a single cell level. Quiescent human blood monocytes and cultured in vitro transformed human monocytoid cell lines U-937, THP-1 and HL-60 did not contain IL-1 beta and TNF alpha in their cytoplasm. IL-1 beta synthesis and secretion was induced by LPS stimulation in nearly 90% monocytes, 15-20% U-937, 3-5% THP-1 and in no HL-60 cells. Normal human blood monocytes had a more rapid kinetics of IL-1 beta synthesis. IL-1 beta positive cells stained with antibodies to human IL-1 beta appeared at 1-2 hours after LPS application, while in monocytic cell lines only after 4-6 hours. Using immunoperoxidase staining of U-937 cells pulse labelled with 3H-thymidine, it was shown that proliferating cells did not synthetize IL-1 beta. Instead of IL-1 beta, TNF alpha could be induced by LPS in U-937 cells only after preliminary differentiation with PMA. Recombinant IL-1 beta induced a very low level of TNF alpha production in PMA-treated cells. Similarly recombinant TNF alpha alone induced IL-1 beta synthesis only in a few U-937 cells.     

A novel secretory pathway for interleukin-1 beta, a protein lacking a signal sequence

Interleukin 1 (IL-1) is a major soluble mediator of inflammation. Two human IL-1 genes, alpha and beta, have been isolated, which encode polypeptides with only 20-30% amino acid sequence homology. Unlike most secreted proteins, the two cytokines do not have a signal sequence, an unexpected finding in view of their biological role. Here we show that IL-1 beta is actively secreted by activated human monocytes via a pathway of secretion different from the classical endoplasmic reticulum--Golgi route. Drugs which block the intracellular transport of IL-6, of tumour necrosis factor alpha and of other secretory proteins do not inhibit secretion of IL-1 beta. Secretion of IL-1 beta is blocked by methylamine, low temperature or serum free medium, and is increased by raising the culture temperature to 42 degrees C or by the presence of calcium ionophores, brefeldin A, monensin, dinitrophenol or carbonyl cyanide chlorophenylhydrazone. IL-1 beta is contained in part within intracellular vesicles which protect it from protease digestion. In U937 cells large amounts of IL-1 beta are made but none is secreted. In these cells IL-1 beta is not found in the vesicular fraction, and all the protein is accessible to protease digestion. This suggests that intracellular vesicles that contain IL-1 beta are part of the protein secretory pathway. We conclude that IL-1 beta is released by activated monocytes via a novel mechanism of secretion which may involve translocation of intracellular membranes and is increased by stress conditions.