Interleukin-8 production by the human cervix

OBJECTIVES: Our purpose was (1) to determine whether the human cervix is capable of producing interleukin-8 in vitro and to examine the possibility of stimulating an increase in any such output and (2) to examine the concomitant production of prostaglandins. STUDY DESIGN: Cervical tissue was obtained from 48 women, 29 pregnant women undergoing surgical termination of pregnancy (20 of whom were treated with the prostaglandin analog Cervagem), 14 nonpregnant, premenopausal women, and three postmenopausal women. Explants were cultured and the medium was assayed for interleukin-8 and prostaglandin E2. Analysis of variance and Newman-Keuls statistical tests were used. RESULTS: Significant quantities of interleukin-8 were produced by the tissue, and the data indicate that cervical explants from pregnant and nonpregnant women behave in a similar way when challenged by phorbol myristate acetate but that the postmenopausal cervix loses its capacity for interleukin-8 production. CONCLUSIONS: Human cervix is capable of producing large amounts of interleukin-8 in vitro, and it may be influenced by the steroid hormones. Thus interleukin-8 could be an excellent candidate for a prime role in neutrophil-mediated cervical ripening.     

Interleukin-6 downregulates factor XII production by human hepatoma cell line (HepG2)

Involvement of the contact system of coagulation in the pathogenesis of various inflammatory diseases is suggested by reduced plasma levels of factor XII (Hageman factor) and prekallikrein generally considered to result from activation of the contact system. However, in many of these diseases patients develop an acute-phase response and, therefore, an alternative explanation for the decreased levels of factor XII could be the downregulation of factor XII gene expression in the liver as described for negative acute-phase proteins. We report here that interleukin-6 (IL-6), the principal cytokine mediating the synthesis of most acute-phase proteins in the liver, downregulates the production of factor XII by the human hepatoma cell line HepG2 by up to 75%. The decrease in protein secretion correlated with an equivalent decrease of factor XII mRNA likely indicating a pretranslational control of factor XII gene expression by IL-6. Downregulation of factor XII production by IL-6 in vitro parallelled that of transthyretin, a known negative acute-phase protein. Moreover, we show that, in patients developing an acute-phase response after immunotherapy with IL-2, plasma levels of factor XII correlate (r = .76, P < .0001) with those of transthyretin. Taken together, these results suggest that factor XII behaves as a negative acute-phase protein.     

Responses of pre-implantation mouse embryos to thalidomide exposure in utero

On Day 1 of pregnancy, a thalidomide suspension (.05 ml) in saline at concentrations of 5 and 10 mcg/ml was infused into the uterine horns via the cervical route of 2 1/2-3 month old Swiss albino mice. Another group was infused on Day 0 at the doses mentioned. Parallel experiments were conducted after intrauterine infusions of saline. Following infusion, the mice were sacrificed on Days 2, 3, and 4 of gestation. Zygote and 4- and 8-cell stages of embryogenesis were most sensitive to the teratogen in terms of the induction of morphological anomalies and cell death. Drug effect was also observed in the postmorula embryos as abnormalities of varying degrees. There was an overall increase in the incidence of mitotic cells.     

Hyperglycemia induces apoptosis in pre-implantation embryos through cell death effector pathways

Although perinatal mortality rates have improved for pregnant diabetic women because of insulin therapy and tight metabolic control, infants of diabetics still experience significantly higher rates of congenital malformations and spontaneous miscarriages compared with those of non-diabetic women. Our results here indicate that hyperglycemic conditions, either in vivo or in vitro, modulate the expression of an apoptosis regulatory gene as early as the pre-implantation blastocyst stage in the mouse. Apoptosis in the mammalian pre-implantation blastocyst is a normal process, thought to protect the early embryo by eliminating abnormal cells. Here we demonstrate that expression of Bax, a Bcl-2-like protein, is increased at the blastocyst stage in the presence of high concentrations of glucose, and that these changes correlate morphologically with increased DNA fragmentation. Expression of Bax and caspase are necessary for this in vitro glucose-induced apoptotic event, and ceramide is involved in mediating this embryotoxic effect of glucose. We also show that these apoptotic cellular changes can be prevented in vivo by treating hyperglycemic mice with insulin before and immediately after conception. These findings emphasize the importance of tight glycemic control in diabetic women at the earliest stages after conception.     

Culture of pre-implantation Chinese hamster (Cricetulus griseus) embryos in vitro

Pre-implantation embryos from Chinese hamsters (Cricetulus griseus) were cultivated under completely defined conditions. The embryos were placed in drops of chemically defined medium under liquid paraffin and cultured in an atmosphere of 10% CO2 in air. By this method, development will proceed in vitro from the two-cell stage up to the blastula within 72 h. It is possible to stop the cultivation at different stages of development, to fix the embryos and analyse the chromosomes. The method described in detail seems to be appropriate for examination of the induction of genetic defects during the first days of embryogenesis.     

Interleukin-10 production by human carcinoma cell lines and its relationship to interleukin-6 expression

Recent data indicate a major role for IL-10 in suppressing immune and inflammatory reactions. To date, expression of human IL-10 has been attributed primarily to helper T lymphocytes, activated monocytes, and neoplastic B cells, and was often found to be associated with IL-6 expression. In this study we sought to determine whether non-hematopoietic human tumor cell lines produce IL-10 and, if so, what is the relationship between IL-10 and IL-6. Using ELISA, we determined IL-10 and IL-6 levels in culture supernatants of 48 cell lines established from carcinomas of the kidney, colon, breast and pancreas, malignant melanomas and neuroblastomas. IL-6 protein was secreted by 28 of the tumor cell lines; IL-10 was measurable in 15 cell lines. IL-6 secretion was maximal and most frequent in renal-cancer cell lines, while IL-10 production was found to be highest and most common among cell lines derived from colon carcinomas. IL-10 in conditioned medium of one of the colon carcinoma cell lines (CCL222) was bio-active, as demonstrated in the mouse MC/9 mast-cell-line assay and in human mixed-lymphocyte reactions. In both assays, IL-10 bio-activity was neutralized by an anti-IL-10 monoclonal antibody. Expression of IL-6 and IL-10 was confirmed by RNA analysis using message amplification by PCR and sequencing of amplified cDNA. LPS, IL-1 alpha, and TNF-alpha strongly enhanced the release of IL-6 by RCC cells, but only marginally affected IL-10 production in colon-carcinoma cells. IL-10 secretion by colon-carcinoma cells was moderately stimulated by IFN-gamma and IL-4. Dexamethasone suppressed the release of IL-6, but had no inhibitory effect on IL-10 secretion. Our results demonstrate that tumor cell lines established from certain types of human carcinomas are capable of expressing and releasing IL-6 and/or IL-10, suggesting a role of these cytokines in solid-tumor development and anti-tumor immunity.     

Interleukin-17 and interferon-gamma synergize in the enhancement of proinflammatory cytokine production by human keratinocytes

OBJECTIVES: To investigate the correlation of epidermal growth factor receptor (EGFR) expression and its ligands EGF and transforming growth factor-alpha (TGF-alpha) with disease outcome in a cohort of patients with superficial bladder cancer. METHODS: Tumor samples of 21 patients with transitional cell carcinoma of the bladder were analyzed by immunohistochemistry for expression of EGFR, EGF, and TGF-alpha. Disease-related events were recorded during a routine clinical follow-up and analyzed for possible correlation with the expression status of the above-mentioned proteins. RESULTS: All Stage pT1 transitional cell carcinomas expressed EGFR, and 10 of 21 (48%) tumors showed focal areas of strong EGF and/or TGF-alpha expression. Of these, 80% with EGF positivity (8 of 10) had recurrences, whereas only 9% of patients without EGF staining (1 of 11) did so. The same pattern was observed with TGF-alpha. A strong association was confirmed between EGF/TGF-alpha positivity and tumor recurrence (P <0.005). We also found that EGF and TGF-alpha were expressed in stroma and/or around the vessels of tumor tissue in 48% and 38% of the tumors, respectively. No association was found between the recurrence rate/vascular invasion and the stromal/vascular wall expression of the growth factors. CONCLUSIONS: Expression of EGF and TGF-alpha is correlated with tumor recurrence. Also, there is the ability of vessel walls to express EGF and TGF-alpha in superficial bladder cancer. Further clarification of the impact of this expression on angioinvasion of tumor cells may be helpful in understanding the nature of local invasion and metastasis.     

Interleukin-18 enhances lipopolysaccharide-induced interferon-gamma production in human whole blood cultures

Interleukin-18 (IL-18) is a newly described cytokine, formerly called interferon-gamma (IFN-gamma)-inducing factor. In a simple 24-h human whole blood culture, IFN-gamma was produced by the combination of lipopolysaccharide (LPS) plus IL-18. To liberate cytokines in the leukocyte and red cell compartments, the detergent Triton X-100 was added to the entire blood culture. The combination of low concentrations of LPS plus IL-18 induced a 3- to 5-fold greater production of IFN-gamma than did either stimulant alone. Tumor necrosis factor-alpha (TNF-alpha), IL-6, and IL-8 were also produced. The presence of IL-10 completely suppressed the production of IFN-gamma and reduced that of TNF-alpha, IL-6, and IL-8. Thus, IFN-gamma, TNF-alpha, IL-8, and IL-6 are produced in a single whole blood culture, making correlations in the synthesis of a T helper type 1 cytokine and proinflammatory cytokines with disease activity possible in a single culture.     

Interleukin-6 production in human fibroblasts derived from periodontal tissues is differentially regulated by cytokines and a glucocorticoid

Interleukin-6 (IL-6) is thought to be a major mediator of the host's defense against infection, and it regulates immune responses in inflamed tissue. In this study, we investigated the regulation of IL-6 production in human gingival fibroblasts (HGF) and human periodontal ligament fibroblasts (HPLF). Pro-inflammatory cytokines including interleukin (IL)-1 alpha, IL-1 beta and tumor necrosis factor (TNF)-alpha stimulated IL-6 production in HGF and HPLF in a time- and dose-dependent manner. This IL-1 alpha, IL-1 beta, or TNF-alpha-induced IL-6 production was enhanced, but the cAMP accumulation they induced was inhibited by the addition of indomethacin. This result suggests that endogenous prostaglandin E2 (PGE2) partially inhibits IL-1 or TNF-alpha-induced IL-6 production and that the enhancement of IL-6 production by IL-1 or TNF-alpha may not be caused through endogenous PGE2-induced cAMP-dependent pathway. Dexamethasone (DEX), a glucocorticoid which is a inhibitor of nuclear factor kappa B (NF-kappa B activation, markedly inhibited IL-1 (alpha or beta) or TNF-alpha-induced IL-6 production; so this production may be partially mediated through NF-kappa B. IL-1 (alpha or beta) and TNF-alpha enhanced IL-6 production synergistically. IL-6 production in HGF or HPLF stimulated with IL-1 beta was augmented by the addition of interferon (IFN)-gamma, but was slightly suppressed by the addition of IL-4. Endogenous IL-6 enhanced IL-1 (alpha or beta)-induced IL-6 production in the presence of IL-6 soluble receptor (IL-6sR). Accordingly, in inflamed periodontal tissues, gingival fibroblasts and periodontal ligament fibroblasts stimulated with pro-inflammatory cytokines such as IL-1 or TNF-alpha, may produce IL-6, and this production can be differentially modulated by endogenous PGE2, IL-6sR, T cell-derived cytokines such as IFN-gamma or IL-4, and glucocorticoids.     

Interleukin-15 augments superoxide production and microbicidal activity of human monocytes against Candida albicans

Overexpression of EGFR and c-erbB2 frequently occurs in human breast cancers, correlating with poor prognosis. Here we show that overexpression of EGFR and c-erbB2 in cell lines increases cell migration, an important step in metastasis formation. The effect of EGFR on migration is dependent on the addition of EGF to the cells. In contrast, c-erbB2 seems to act independently of its ligand in these assays. Overexpression of this receptor is sufficient to induce cell migration. In addition, we investigated the involvement of a number of signal transduction pathways known to be activated by the EGFR. We found that inactivation of MAPKK results in a decreased migration, while inactivation of PI3K increases migration.     

Nuclear-cytoplasmic interaction and development of goat embryos reconstructed by nuclear transplantation: production of goats by serially cloning embryos

We found previously that alcohol-preferring (P) rats have fewer serotonin (5-HT) neurons and fibers in key brain regions than alcohol-nonpreferring (NP) rats. Because 5-HT uptake blockers increase synaptic 5-HT content and 5-HT1A receptor antagonists increase 5-HT release by disinhibiting 5-HT autoinnervation, in the present study, our intent was to determine whether increased synaptic 5-HT content and/or 5-HT release in P rats would effectively reduce alcohol consumption. In experiment 1, the 5-HT antagonist WAY 100635 (WAY) was tested on adult female P rats maintained on 24-hr free-choice access to ethanol (10% v/v) and water. Twice daily doses of WAY (0.05, 0.1, 0.5, and 1.0 mg/kg, subcutaneously) were administered to each rat in a counterbalanced order. Baseline ethanol intake, derived from the mean ethanol intakes of the three previous non-drug days, was approximately 8 g/kg/day. Results indicated that 0.05, 0.1, and 0.5 mg/kg doses of WAY reduced 24-hr ethanol drinking by 25-30% (p < 0.01) without affecting 24-hr water intake or body weight. In the second experiment, the effects of WAY (0.5 mg/kg), fluoxetine (1.0 mg/kg), or a combination of both were tested in another group of female P rats. WAY and fluoxetine, each alone, reduced ethanol drinking by around 20% and, when combined, decreased ethanol intake by 50%, whereas the body weight and the total fluid intake were not significantly affected. Taken together, these results indicate that both fluoxetine and WAY preferentially reduce ethanol drinking in the P line of rats and, when administered together, reduce ethanol intake in an additive manner. It is proposed that coadministration of these two compounds with distinct mechanisms of action may be a new strategy for reducing alcohol intake.     

Interleukin-2-dependent augmentation of the anti-TNP antibody production by sodium butyrate in cultured murine splenic B cells

Previously we found that sodium butyrate (NaBu) markedly enhanced production of the antibody specific for a T-cell-dependent antigen, sheep red blood cells (SRBC) in murine splenocytes (Kishiro, Y., Ueda, K., Fujiwara, M. and Yamamoto, I., Jpn J. Phamacol., 1994 66, 369-376. To gain a better understanding of the target cells for NaBu's action on antibody responses, we have utilized the T-cell-independent antigen, trinitrophenyl-lypopolysaccharide (TNP-LPS) as a stimulant and have examined an effect of NaBu on the anti-TNP antibody production in vitro. NaBu markedly increased the anti-TNP plaque-forming cell (PFC) responses in murine whole splenocytes, but not in murine splenic B cells. Addition of T-cells or the concanavalin A supernatant (CAS) from murine splenocytes to the B cell cultures completely restored the enhancing effect of NaBu. This effect of CAS was totally blocked by an anti-interleukin (IL)-2 antibody and partially by an anti-IL-1 beta or anti-IL-4 antibody. The full enhancing effect of NaBu was also detected when IL-2 was added to the B cell cultures, while IL-2 alone had no stimulatory effect on the control PFC response. IL-1 beta alone significantly stimulated the antibody production and adding NaBu to this IL-1 beta-supplemented culture caused a further increase. Neither IL-4 alone nor NaBu plus IL-4 had any effect on the PFC response. NaBu did not affect the expression of the IL-2 receptor alpha- and beta-chains in B cells stimulated with TNP-LPS. These results suggest that NaBu is an agent that promotes B cell differentiation in vitro in an IL-2-dependent manner.     

Interleukin-8 expression by human neutrophils activated by Helicobacter pylori soluble proteins

A retrospective analysis was done in 60 consecutive patients who underwent anterior cervical fusion using vertebral grafts obtained from the fusion site at the Nagoya University and its affiliated hospitals by a single surgeon (MT). Follow-up results and technical advantages are reported. The average follow-up period was 33 months (range 6 to 55 months). Sufficient decompression of the anterior cervical pathology was performed successfully via a wider operative field. The symptoms and neurological score improved significantly without any new deficits in all patients except in one with a three-level fusion who needed re-operation for further decompression. No major graft complications such as graft extrusion or pseudoarthrosis occurred. Graft fracture was noted in five cases. However, good bony fusion was observed in all these cases without any further treatment. Normal cervical lordosis was preserved in most cases except in four, who lost lordotic alignment but did not show kyphosis. Major advantages of this method are a wider operative field, excellent graft fusion rate, and no need for an additional incision to obtain autogenous bone graft. These benefits seem have to contributed to satisfactory surgical results in this series.     

Interleukin 10 production by human melanoma

Interleukin 10 (IL-10) has the physiological role of down-regulating cell-mediated immunity. We have recently reported that mRNA for IL-10 was present in most metastatic melanoma tissues. The purpose of this investigation was to determine whether melanoma metastases produce IL-10 protein. Single-cell suspensions were prepared by enzymatic dissociation of 28 lymph node metastases and 7 s.c. metastases and cryopreserved. Of these 35 samples, 30 produced IL-10 after a 24-h incubation (median, 125.1 pg/ml). IL-10 production was slightly diminished after 25 Gy irradiation but almost completely abrogated after modification with the hapten dinitrophenyl. After 7 or 14 days in tissue culture, melanoma cells continued to produce IL-10 but only at about 10% of the levels of freshly dissociated tissues. Moreover, of eight melanoma cell lines established from these cultures, only one produced IL-10 protein. To determine whether IL-10 was produced by melanoma cells or tumor-associated leukocytes, single-cell suspensions were fractionated with anti-CD45 antibody-conjugated magnetic beads. In four of five samples, IL-10 production was increased by depletion of leukocytes, suggesting that the primary source was the melanoma cells themselves. This was confirmed by immunohistochemical staining of cytospin preparations and frozen tissue sections. Finally, 10 of 55 patients with clinically evident metastases showed elevations of circulating IL-10; three patients who had been melanoma-free developed high serum IL-10 levels, concurrent with the appearance of distant metastases. These data indicate that production of IL-10 is characteristic of metastatic melanomas and raise the possibility that this cytokine allows tumors to avoid or to modulate immunological attack.     

Interleukin-1 beta modulates prostaglandin and progesterone production by primate luteal cells in vitro

Increasing evidence suggests that cytokine products of the immune system may play a regulatory role in corpus luteum regulation in several species. The role of cytokines in primate luteal function, however, remains unclear. In the present study we examined the effects of interleukin-1 beta (IL-1 beta), tumor necrosis factor alpha (TNF alpha), and interferon-gamma (IFN-gamma) on progesterone and prostaglandin (PGE2, PGF2 alpha) production by primate luteal cells in vitro. Specifically, corpora lutea were removed from normally cycling cynomolgus monkeys (n = 30 corpora lutea) during either the early (Days 3-5 after the estimated LH surge), mid (Days 8-10), or late (Days 12-14) luteal phase of the menstrual cycle. The corpora lutea were dispersed into individual cells using collagenase, DNase, and hyaluronidase. Approximately 50,000 viable luteal cells per tube were incubated in Ham's F-10 medium with increasing concentrations of IL-1 beta (0.1-10 ng/ml), TNF alpha (1-100 ng/ml), or IFN-gamma (10-1000 U/ml) in the presence and absence of hCG for 8 h at 37 degrees C. TNF alpha and IFN-gamma had no effect on progesterone PGE2, or PGF2 alpha production during any phase of the cycle at the doses tested. In contrast, IL-1 beta significantly stimulated PGF2 alpha production in a dose-dependent manner during the mid and late luteal phases (p < 0.05). Human CG alone had no effect on PGE2 or PGF2 alpha production by dispersed luteal cells in vitro but inhibited IL-1 beta-stimulated PGF2 alpha production. As expected, hCG stimulated progesterone production by primate luteal cells in vitro. Interestingly, IL-1 beta inhibited this hCG stimulation of progesterone production. In summary, these date suggest that IL-1 beta is a potentially important modulator of prostaglandin production by the primate corpus luteum. In view of this, cytokine-mediated changes in prostaglandin production by the primate corpus luteum may participate in the physiological regulation of luteal function.     

Ectopic production of human placental lactogen by human breast tumors

Serum samples from 72 patients with established carcinoma of the breast were investigated for ectopic presence of hPL. Further, the relationship of ectopically-secreted hPL and hCG-beta in breast cancer was investigated. Ten of 72 patients examined had detectable hPL and 12 had detectable hCG-beta, at 1-2 ng/ml serum sensitivity of the assay. The presence of hPL in serum of breast cancer patients was found to be independent of that of hCG-Beta. Sera of 13 patients with cystic mastitis, five with fibroadenoma, two with acute inflammation of breast, 20 normal women (non-pregnant) and 20 normal men did not show any detectable serum hPL or hCG-beta at the above mentioned sensitivity of the assay. Since these hormones were not detectable in normal men, normal non-pregnant women, and in patients having other pathological conditions of breast, the possible use of them as markers in cancer is expected     

Interleukin-2 stimulates a late increase in phosphatidic acid production in the absence of phospholipase D activation

The use of optical trapping to create extremely compliant mechanical probes has ushered in a new field of biological inquiry, the mechanical and kinetic study of proteins at the single molecule level. This review focuses on three examples of such study and includes methods of extracting parameters of interest from the raw data such experiments generate.     

Interleukin-12 and interferon-gamma production in childhood idiopathic nephrotic syndrome

Cellular immune disturbances, and T lymphocyte function in particular, have been previously implicated in idiopathic nephrotic syndrome (INS) of childhood. There are different patterns of cytokine expression in various forms of glomerulonephritis, which suggests that local production of these peptides plays an important role in the pathogenesis and progression of glomerulonephritis. To investigate T-cell and monocyte/macrophage cytokine production in INS, interleukin-12 (IL-12) and interferon-gamma (IFN-gamma) production by peripheral blood mononuclear cells (PBMC) of 11 children with steroid-sensitive nephrotic syndrome (SSNS), 9 with focal segmental glomerulosclerosis (FSGS), and 17 healthy controls was determined. Children with SSNS were studied in relapse, during corticosteroid treatment, and in stable remission, off corticosteroid treatment. IL-12 was not detected in serum, urine, and in supernatants of unstimulated PBMC. IL-12 production by concanavalin A (Con A)-stimulated PBMC of children with SSNS and FSGS was not different from controls. IFN-gamma production by Con A-stimulated PBMC was decreased in children with relapsing SSNS, both in relapse and and during corticosteroid treatment. However, in stable remission it was similar to controls. Markedly decreased IFN-gamma production (P<0.001) was observed by pokeweed mitogen-stimulated PBMC of relapsing SSNS patients and moderately decreased production by PBMC of FSGS patients. This study has established a decreased production of IFN-gamma by PBMC of relapsing SSNS and FSGS patients, but does not allow differentiation between these two different conditions. IL-12 did not have a pathogenic role in either SSNS or FSGS.