Murine eosinophils and IL-1: alpha IL-1 mRNA detection by in situ hybridization. Production and release of IL-1 from peritoneal eosinophils

The presence of IL-1 mRNA in eosinophils from mice infected with larvae of the parasite Mesocestoides corti was investigated by in situ hybridization technique. S35 labeled cDNA probe for alpha IL-1, hybridized with mRNA in murine eosinophils and macrophages. After 6 h of LPS stimulation eosinophils were able to express mRNA in their cytoplasm. This expression was highly increased by the addition of indomethacine. The IL-1 mRNA expression in murine macrophages was higher than in eosinophils in LPS-stimulated cells. This difference was statistically significant, p less than 0.001. To test if eosinophils may produce and release IL-1 in the culture medium, we isolated these cells in a Percoll gradient. Cell preparations with a purity exceeding 94% were cultured with various stimuli and their supernatants were tested for IL-1 activity. Eosinophils produced 169.65 +/- 73 U/ml when stimulated with LPS (n = 14). A dose-dependent response was obtained when the eosinophils were in the presence of the calcium ionophore A23187. Controls were performed to rule out the contribution of the contaminating population on the thymocyte proliferating activity. They were also used to detect other possible causes of interference in the assay, such as leukotrienes or TNF. IL-1 in supernatants was also detected using a conversion assay such as EL-4 thymoma cells. IL-1 activity was first detected in culture supernatants 18 h later, maximal production being in the first 24 h. In accordance with our hybridization results, an increase in IL-1 activity was obtained when eosinophils were stimulated with LPS and treated with indomethacine. The factor had a molecular mass between 16 to 20 kDa that corresponded to the described for murine IL-1. Inasmuch as IL-1 is an important mediator of inflammatory reactions this IL may enhance the proinflammatory action of eosinophils.     

Effects of kainic acid on messenger RNA levels of IL-1 beta, IL-6, TNF alpha and LIF in the rat brain.

We examined the kainic acid-induced changes of mRNA levels of several cytokines such as IL-1 beta, IL-6, TNF alpha and LIF in the rat brain regions using semiquantitative RT-PCR method. IL-1 beta mRNA was markedly increased in the cerebral cortex (CC), thalamus (THL) and hypothalamus (HT) 2 h after the injection of kainic acid in a convulsive dose (12 mg/kg i.p.), and tended to decrease 4 h after the injection. IL-6 mRNA was weakly induced in the hippocampus (HPP) 2 h after the injection of kainic acid and was markedly increased in the CC, HPP, THL, and HT at 4 h. The level of TNF alpha mRNA was highly elevated in the CC, HPP, striatum (STR), THL and HT at 2 and 4 h after the injection. LIF mRNA apparently expressed in the CC and HPP of control rats and was increased in the CC, HPP and HT by the treatment with kainic acid. These results indicate that mRNAs of several cytokines are increased in various brain regions with different time-courses by kainic acid.     

IL-1, IL-6, and PDGF mRNA expression in alveolar cells following stimulation with a tobacco-derived antigen.

To test the hypothesis that inflammatory cytokine production might be an early event in the development of the disease associated with smoking, we used alveolar cells from healthy nonsmokers stimulated with TGP as a model system. TGP, a phenol-rich glycoprotein which is present in tobacco leaves and cigarette smoke condensate, activates the immune system. It stimulates polyclonal B cell differentiation, induces primarily an IgE response, and activates human leukocytes to produce IL-1. Using in situ nucleic acid hybridization we show that the steady-state levels of IL-1 alpha, IL-1 beta, IL-6, platelet-derived growth factor (PDGF)-A, and PDGF-B mRNAs are consistently elevated in the alveolar cells of all donors following TGP stimulation. The kinetics of mRNA expression suggest that IL-1 alpha and IL-1 beta mRNAs are independently regulated in alveolar cells, while the regulation of PDGF-A and PDGF-B mRNA seems to be similar. The activated cells also synthesize elevated levels of IL-1 and IL-6. These findings lend support to the suggestion that some clinical consequences of smoking might be initiated and enhanced by the production of inflammatory cytokines. Moreover, IL-6 could also activate a polyclonal B cell response, which could lead to the synthesis of autoantibodies and thus cause immune-mediated tissue injury.     

Induction of HLA class I mRNA by cytokines in human fibroblasts: comparison of TNF, IL-1 and IFN-beta.

Expression of HLA class I antigens is known to be regulated by various cytokines at both the mRNA and protein levels. We have examined the induction of HLA-B7 by tumor necrosis factor alpha (TNF), interleukin 1 alpha (IL-1) and interferon beta (IFN-beta) in normal human diploid FS-4 fibroblasts. Optimal induction of HLA-B7 by TNF at 24 h was shown to require a continuous presence of TNF. Since TNF also induces IFN-beta in these cells and the latter cytokine itself has the capacity to upregulate HLA class I expression, we investigated the role of autocrine IFN-beta in the induction of HLA-B7 by TNF. Experiments with neutralizing polyclonal antibodies to recombinant IFN-beta showed that the induction of HLA-B7 mRNA by TNF was partially dependent on autocrine IFN-beta. However, TNF and IFN-beta induced HLA-B7 mRNA with similar kinetics and treatment with saturating concentrations of both TNF and IFN-beta resulted in an additive or possibly synergistic response. The latter findings support the idea that induction of HLA class I by TNF is not mediated solely by autocrine IFN-beta produced in response to TNF. In addition, experiments with the protein synthesis inhibitor cycloheximide suggested that the induction of mRNAs for both the heavy and light (beta 2-microglobulin) chains of the HLA class I antigen by TNF did not require de novo protein synthesis. IL-1 was also shown to increase steady-state mRNA levels of HLA-B7 with kinetics similar to those of TNF and IFN-beta in FS-4 cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Tumor necrosis factor and IL-1 in New Zealand Black/White mice. Enhanced gene expression and acceleration of renal injury

TNF and IL-1 are potent immunologic and inflammatory cytokines. We have previously reported increased levels of mRNA for TNF alpha and IL-1 beta in MRL-lpr mice with lupus nephritis. To determine whether the increased levels of TNF and IL-1 mRNA are a more general feature of mice with lupus nephritis we studied cytokine gene expression in female NZB x NZW F1 (NZB/W) mice by Northern blot analysis. Enhanced steady state levels of mRNA for TNF alpha and IL-1 beta, but not IL-1 alpha, were detected in the renal cortices of animals with lupus nephritis. To determine whether administration of TNF or IL-1 would accelerate renal injury and mortality, we injected murine rTNF alpha or rIL-1 alpha i.p. into female NZB/W or C3H/FeJ mice at two doses, 2.0 micrograms or 0.2 micrograms, three times weekly for 2 or 4 mo beginning at 2 or 4 mo of age. Administration of the lower dose of each cytokine accelerated renal disease and mortality rate when treatment was initiated at 4 mo of age. At the higher dose, neither cytokine promoted disease. Treatment administered from 2-4 mo of age did not accelerate renal disease. This observation suggests that in order to cause renal injury, these cytokines must interact with other pathologic features present in these animals after 4 mo of age. These findings support the hypothesis that TNF and IL-1 can contribute to nephritis in murine models of lupus. Taken together with previously published data, we propose that TNF and IL-1 have differential dose effects on renal disease. The dose of TNF and IL-1 and the stage of disease activity dictate the pathogenic action of these cytokines.     

Regulation of the expression of IL-6 in human monocytes

IL-6 is a cellular regulatory molecule with various cell-dependent functions. We have studied the control of IL-6 expression in human monocytes because they play a key role in the production of this molecule. The effects of adherence and different cytokines including CSF-1, IFN-gamma, IL-1 alpha, and granulocyte-macrophage-CSF were tested on IL-6 expression. IL-6 mRNA was usually not detected in the starting population of PBMC. Adherence induced IL-6 gene expression in monocytes in less than 2 h and subsequently IL-6 secretion. Priming of monocytes by adherence was more efficient for IL-6 overinduction by CSF-1. In contrast, high level induction of IL-6 by IFN-gamma in unfractionated PBMC did not require adherence and in situ hybridization revealed that IL-6 mRNA was present in monocytes but not in lymphocytes. A similar phenomenon was observed for IL-1 alpha and granulocyte-macrophage-CSF. Two cell lines, HL-60 and U937, in which monocytic differentiation occurs after induction by PMA, were subsequently investigated. IL-6 was not constitutively detectable in these two cell lines, whereas PMA treatment induced IL-6 expression. This effect was rapid (30 min) and transitory in HL-60, whereas IL-6 mRNA was still detected after 72 h of induction in U937. Addition of human rIL-6 on U937 and HL-60 cells inhibited their proliferation and enhanced expression of HLA class I Ag.     

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)     

Interleukin-1, interleukin-6, and tumor necrosis factor alpha are produced in the mouse uterus during the estrous cycle and are induced by estrogen and progesterone.

The ovarian steroids, estrogen and progesterone, regulate cellular and molecular changes which occur in the uterus during the estrous cycle. Cycles of protein synthesis, cell proliferation and differentiation, and cell death are the direct results of changes in hormone concentration. To explore the possibility that cytokines, which stimulate proliferation and differentiation of numerous types of cells, might be associated with those cyclic changes, the production of IL-1, IL-6, and TNF alpha was examined in the mouse uterus. Cytokine mRNA expression, bioactivity, and immunoreactivity were quantitated during the estrous cycle, following ovariectomy and exposure of ovariectomized mice to estrogen and progesterone. IL-1, IL-6, and TNF alpha mRNA was detected, and mRNA levels for each of the cytokines varied with the stage of the cycle. Cytokine bioactivity was expressed throughout the cycle, but levels of each cytokine were highest during proestrus and/or estrus. Immunoreactivity paralleled bioactivity. Uterus from ovariectomized mice contained little or no cytokine activity, and systemic administration of estrogen or progesterone resulted in the induction of IL-1 alpha and IL-1 beta mRNA expression. Significant amounts of IL-6 and TNF alpha mRNA appeared only following the exposure of ovariectomized mice to estrogen plus progesterone. Cytokine bioactivity and immunoreactivity also appeared following the administration of estrogen and/or progesterone. The highest activity levels for each cytokine were observed following the injection of estrogen plus progesterone. Cyclic expression of IL-1, IL-6, and TNF alpha in the uterus and their apparent regulation by estrogen and progesterone raise the possibility that cytokines and factors which are induced by cytokines are part of the regulatory process which is induced by ovarian hormones in the uterus of reproductive age females.     

Interleukin-18 (IL-18) mRNA expression and localization of IL-18 mRNA-expressing cells in the mouse uterus

Interleukin-18 (IL-18) belongs to the interleukin-1 family and was identified as an interferon-gamma inducing factor. We investigated IL-18 mRNA-expressing cells in the mouse uterus. By RNase protection assay, IL-18 mRNA and alpha subunit of IL-18 receptor mRNA were detected in the uterus. In the uterus, IL-18 mRNA levels increased during sexual maturation. In situ hybridization analysis demonstrated IL-18 mRNA-expressing cells in the mouse uterus of different ages. At 21 days of age, IL-18 mRNA-expressing cells were detected in the luminal epithelial cells and stromal cells although the IL-18 mRNA signal was weak. At 42 days of age, IL-18 mRNA signal was mainly detected in the stromal cells located near the myometrium, and in some of the luminal and glandular epithelial cells. In the uterus of 63-day-old adult mice, a strong hybridization signal for IL-18 mRNA was detected at estrus, but was weak at diestrus. IL-18 mRNA was mainly detected in the glandular epithelial cells and stromal cells. The effect of estradiol-17beta (E(2)) on IL-18 mRNA-expressing cells in the uterus was examined in ovariectomized mice. In oil-treated mice IL-18 mRNA signal was localized in luminal epithelial cells and stromal cells, while in E(2)-treated mice IL-18 mRNA signal was localized in stromal cells alone. These results suggest that the mouse uterus has an IL-18 system, and IL-18 exerts a physiological role within the uterus in a paracrine manner, and that IL-18 gene expression is regulated by estrogen.     

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.