Rh Interleukin Research Articles

Effect of inhibition proliferation in human lung adenocarcinoma A549 cells by cytokine‐induced killer cells

BackgroundAdenocarcinoma, the most common form of lung cancer, is one of main human malignant tumors. In this paper, we focus on the effect of antitumor activity of cytokine-induced killer (CIK) cells on human lung adenocarcinoma cell line A549.MethodsCIK cells were obtained by inducing peripheral blood mononuclear cells with recombinant human (rh) interferon-gamma, monoclonal anti-CD3 antibody, rh interleukin (IL)-1alpha, and rhIL-2, which were added into the culture. A549 cell viability of CIK cells was determined using MTS assay. Flow cytometry (FCM) experiments were performed to detect cell cycle changes. The expression of P27 in A549 cells treated by CIK cells was evaluated by Western blot.ResultThe percentage of CD3+CD16+CD56+ T cells in a representative peripheral blood mononucleated cell sample was 33.7 ± 1.3%. CIK cells, in dose and time dependent manners, inhibited the proliferation of A549. FCM demonstrated that A549 cells were accumulated in G2/M and G0/G1 phases when treated with CIK cells. FCM was used to analyze whether A549 cells treated with CIK cells induced apotosis or necrosis at 10:1 or 20:1. Compared to the control group, P27 was prominently upregulated in the CIK treated group.ConclusionWe propose that the pharmacological mechanisms of A549 cells inhibited by CIK cells can be estimated to possibly elicit different biological significance, which, in part, can be ascribed to a different mass transport rate in vitro.

Effect of inflammation-associated cytokines of biliary tract cancer on chemoresistance and therapeutic implications.

247 Background: Biliary tract cancer (BTC) is well-known to be commonly suffered from inflammations; cholangitis and/or liver abscess. The author previously showed interleukin-6 (IL-6) worked on anti-apoptotic process in BTC. Herein, we focused on inflammation-associated cytokines including not only IL-6 but also transforming growth factor-beta 1 (TGF-β1), and investigated the working processes, their relationship to chemoresistance, and therapeutic implications. Methods: We employed resected specimens and BTC cell lines. We evaluated IL-6/TGF-β1 expression, invasion, endothelial-mesenchymal transition (EMT), and chemoresistance to gemcitabine, with or without silencing of PI3K/Akt, MAPK, STAT, and Smad pathways. Results: Resected specimens expressed IL6 and TGF-β1 staining at the invasion front by immunohistochemical staining. In BTC cell lines, spontaneous expression of IL-6/TGF-β1 was related to malignant potencies such as EMT and chemoresistance. Rh IL-6/TGF-β1 induced invasion, EMT, and chemoresistance. Smad4 among cell signaling pathways functioned in a dominant manner; inhibition of Smad4 pathway reduced invasion, and reversed EMT and chemoresistance, in both rh IL-6/TGF-β1-treated cells, and we confirmed these results using SMAD4 siRNA. And, our established gemcitabine-resistant cells expressed high level of IL-6/TGF-β1, and promoted EMT. Inhibition of Smad4 also reversed chemoresistance in gemcitabine-resistant cells. We confirmed N-cadherin and Smad4 expression at the invasion front by immunohistochemistry. Conclusions: IL-6 and TGF-β1 resulted in chemoresistance, and inhibition of Smad4 would reverse chemoresistance in BTC.

Role of crosstalk between interleukin-6 and transforming growth factor-beta 1 in epithelial–mesenchymal transition and chemoresistance in biliary tract cancer

AimsThe mechanisms of progression in biliary tract cancer (BTC) with inflammation, including epithelial–mesenchymal transition (EMT), are not well understood. We focused on two inflammation-associated cytokines, interleukin-6 (IL-6) and transforming growth factor-beta 1 (TGF-β1), and investigated their expression and activity, as well as their relationship to key features of malignancy, in tumour samples from patients with BTC and in cultured BTC cells. MethodsWe employed five BTC cell lines (MzChA-1, gemcitabine-resistant MzChA-1, HuCCT-1, KMCH and CCLP-1) to evaluate IL-6/TGF-β1 expression, tumour cell invasion, EMT and chemoresistance to gemcitabine in the presence or absence of recombinant human (rh) IL-6 and TGF-β1. Possible pathways were evaluated with specific pathway inhibitors and small interfering RNA (siRNA). We also used 20 resected specimens from patients with BTC to verify the results in vitro. ResultsIL-6 and TGF-β1 expression was associated with features of malignancy such as EMT and chemoresistance in the four BTC cell lines. Addition of rh IL-6 and TGF-β1 increased endogenous IL-6 and TGF-β1 expression through crosstalk and induced cell invasion, EMT and chemoresistance. Smad4 functioned in this process in a dominant manner, and inhibition by SMAD4 siRNA reduced IL-6 and TGF-β1 expression, blocked invasion, and reversed EMT and chemoresistance in cells exposed to rh IL-6 and TGF-β1 and in gemcitabine-resistant cells. Immunohistochemistry in resected specimens revealed IL6, TGF-β1, N-cadherin and Smad4 staining at the invasion front. ConclusionCrosstalk between IL-6 and TGF-β1 is associated with malignant features, including EMT, and Smad4 works in a dominant manner to promote these features.

The HDAC inhibitor Givinostat modulates the hematopoietic transcription factors NFE2 and C-MYB in JAK2V617F myeloproliferative neoplasm cells

We investigated the mechanism of action of the histone deacetylase inhibitor Givinostat (GVS) in Janus kinase 2 (JAK2)(V617F) myeloproliferative neoplasm (MPN) cells. GVS inhibited colony formation and proliferation and induced apoptosis at doses two- to threefold lower in a panel of JAK2(V617F) MPN compared to JAK2 wild-type myeloid leukemia cell lines. By global gene expression analysis, we observed that at 6 hours, GVS modulated 293 common genes in the JAK2(V617F) cell lines HEL and UKE1, of which 19 are implicated in cell cycle regulation and 33 in hematopoiesis. In particular, the hematopoietic transcription factors NFE2 and C-MYB were downmodulated by the drug specifically in JAK2(V617F) cells at both the RNA and protein level. GVS also inhibited JAK2-signal transducer and activator of transcription 5-extracellular signal-regulated kinase 1/2 phosphorylation, but modulation of NFE2 and C-MYB was JAK2-independent, as shown using the JAK2 inhibitor TG101209. GVS had a direct effect on the NFE2 promoters, as demonstrated by specific enrichment of associated histone H3 acetylated at lysine 9. Modulation by GVS of NFE2 was also observed in freshly isolated CD34(+) cells from MPN patients, and was accompanied by inhibition of their proliferation and differentiation toward the erythroid lineage. We conclude that GVS acts on MPN cells through dual JAK2-signal transducer and activator of transcription 5-extracellular signal-regulated kinase 1/2 inhibition and downmodulation of NFE2 and C-MYB transcription.

IL-6, but not TNF-α, increases plasma YKL-40 in human subjects

Plasma levels of YKL-40 are elevated in patients with systemic infection, inflammatory disorders and cancer. Both monocytes/macrophages, neutrophils, and cancer cells have the capacity to produce YKL-40, but the regulation during the inflammatory response is unknown. To study the possible role of interleukin-6 (IL-6) and tumor necrosis factor (TNF)-α in the regulation of YKL-40 plasma levels, we included healthy men, who received either recombinant human (rh)IL-6 ( n = 6), rhTNF-α ( n = 8) or vehicle ( n = 7) for 3 h. The plasma levels of IL-6 and TNF-α reached ∼150 and ∼18 pg/ml, respectively, during the infusions. Following the IL-6 infusion, the plasma level of YKL-40 increased from ∼30 to ∼57 ng/ml ( p < 0.05) at 24 h, and returned to normal values after 48 h. The plasma level of YKL-40 did not change during TNF-α infusion or infusion of vehicle. These data demonstrate that IL-6, but not TNF-α, has a key-role in the regulation of plasma YKL-40 levels during inflammation.

Phenotype of Dendritic Cells Generated from Peripheral Blood Monocytes of Patients with Ovarian Cancer

Objectives The aim of our study was to generate dendritic cells (DCs) from peripheral blood monocytes (PBMC) of patients suffering from ovarian cancer. Materials and Methods Immature DCs were generated from PBMC cultured in RPMI 1640 medium with 2% human serum albumin (HSA), supplemented with recombinant human (rh) granulocyte macrophage colony stimulating factor (GM-CSF) and rh interleukin (IL)-4. After 5 days of culture, DC maturation was induced by the addition of an ovarian cancer cell line (CAOV3) lysate and after 6 days of rh tumor necrosis factor (TNF)-α for a further 2 days. The phenotype of the generated cells was assessed by flow cytometry for the expressions of human leukocyte antigen (HLA)-DR, costimulatory molecules (e.g., CD86, CD80), CD83, CD1a, and CD14. PBMC cultured in 2% HSA without rhIL-4, rhGM-CSF, rh-TNF-α, or tumor cell lysate formed the control group. Results The 2.41% (interquartile range, 1.51%–3.52%) of CD45 +/CD14 + cells in cultures with rhIL-4, rhGM-CSF, rhTNF-α and tumor cell lysate was significantly lower than in the control group (31.10%; interquartile range, 11.11%–64.06%). Cultures with rhIL-4, rhGM-CSF, rhTNF-α, and tumor cell lysate showed a higher percentage (19.96%; interquartile range, 9.30%–24.42%) of fully mature CD83 +/CD1a −/HLA-DR + DCs compared with control culture (6.02%; interquartile range, 3.01%–7.37%). There was no significant difference in the expression of the immature DC marker (CD1a) between the cultures. The expression of co-stimulatory markers (CD80, CD86, HLA-DR) was greater (24.29%; interquartile range, 18.68%–33.95%) on DCs from cultures with rhIL-4, rhGM-CSF, TNF-α, and tumor cell lysate versus controls (4.93%; interquartile range, 2.67%–9.09%). Conclusion Our data demonstrated that immature and mature DCs can be generated from adherent human PBMC from ovarian cancer patients cultured with rhIL-4, rhGM-CSF, rhTNF-α, and tumor cell lysates.

The Effects of IL-6 Infusion on the GH???&gt;IGF-I System in Humans

1567 Recent investigations have revealed that the early response to exercise leads to a growth-hormone resistant state rather than the expected anabolic activation of the growth hormone—> insulin like growth factor-I (GH—>IGF-I) axis. This phenomenon is believed to be a result of exercise induced stimulation of proinflammatory cytokines [such as interleukin-6 (IL-6)] known to directly inhibit anabolic activity of the GH—>IGF-1 axis. PURPOSE: To test the hypothesis that an IV infusion of IL-6 will induce changes in the GH—> IGF-I axis mediators similar to those observed in acute exercise in humans. METHODS: Twelve young healthy volunteers were randomly assigned to receive a low dose of recombinant human (rh)IL-6 (n = 6) or saline (n = 6) for 3 h. IL-6, GH, IGF-I (total and free), IGFBP-1&3, GHBP and Insulin were measured at baseline, 30 minutes, 1, 2 and 3 hours during the infusion and 1, 2 and 5 hours after the infusion was stopped. Significance level was set at p<0.05. RESULTS: Plasma IL-6 levels during rhIL-6 infusion were 140 pg/ml, corresponding to the levels obtained during very strenuous exercise. No significant effects on body temperature, mean arterial pressure, or heart rate were noted. During the rhIL-6 infusion an increase in GH levels was observed at 1 hour. In addition, a decrease in IGF-I was noted at 3 hours. Two hours post-rhIL-6 infusion an increase in IGFBP-1 was noted, irrespective to changes in insulin. No significant changes were observed for GHBP, IGFBP-3. CONCLUSIONS: These findings supports the hypothesis that physiological concentrations of IL-6, equivalent to those seen following strenuous exercise play a major role in exercise-induced changes in the GH—>IGF-I axis.

Concentration-dependent activity of mometasone furoate and dexamethasone on blood eosinophils isolated from atopic children: modulation of Mac-1 expression and chemotaxis

Treatment of asthma with corticosteroids results in downregulation of eosinophilic airway inflammation. We evaluated in vitro the activity of an “inhaled” corticosteroid, mometasone furoate (MF), and of a “systemic” corticosteroid, dexamethasone (DEX), on eosinophil functions, i.e. adhesion molecule expression and cell chemotaxis. Partially purified blood eosinophils were obtained from 18 asthmatic subjects sensitized to house dust mites. The expression of the macrophage antigen (Mac)-1 (CD11b/CD18) was measured by specific monoclonal antibody (mAb) staining and flow cytometry analysis at baseline or after stimulation with N-formyl-methionyl-leucyl-phenylalanine (fMLP) or with recombinant human (rh) granulocyte macrophage-colony stimulating factor (GM-CSF) plus a mAb anti-human (ah) IgE low affinity receptor [FcɛRII or CD23]. Cell chemotaxis toward the complement fragment 5a (C5a) or rh interleukin (IL)-5 was evaluated in Boyden microchambers by light microscopy. Eosinophils showed a significant increase in Mac-1 expression after activation with fMLP or with rh GM-CSF plus ah CD23 mAbs ( p<0.05, each comparison) and a remarkable chemotactic response to both C5a or rh IL-5 ( p<0.001, each comparison). To test the inhibitory activity of MF and DEX on eosinophil functions, the cells were preincubated for 3 h with four concentrations (0.1, 1, 10 and 100 nM) of each of the two drugs, before being activated by fMLP or by rh GM-CSF plus ah CD23 mAbs or tested with C5a or with rh IL-5. Independently of the stimulus used, both Mac-1 expression and eosinophil migration were effectively downregulated by preincubation with MF or DEX at 1, 10 and 100 nM ( p<0.05). The inhibitory activity on cell chemotaxis in response to both C5a or with rh IL-5 was higher for MF than DEX, but only at the highest concentration tested ( p<0.05, each comparison). These data demonstrate that concentrations of MF similar to those obtained in vivo are highly effective in inhibiting eosinophil functions involved in airway inflammation.

Carbohydrate Recognition of Interleukin-2 in Cell Proliferation

Publisher Summary This chapter focuses on carbohydrate-binding specificity of interleukin-2 (IL-2) in relation to T-cell proliferation. IL-2 has been widely studied as a mediator of cellular signaling in the immune system. IL-2 promotes the proliferation of IL-2-dependent T cells, and functions as an immunomodulator of activated B cells, macrophages, and natural-killer cells. IL-2 expresses its physiological functions through interaction with its receptor complex, which consists of three receptor subunits:α, β, and γ (IL-2Rα, IL2Rβ, and IL-2Rγ). The chapter discusses the preparation of IL-2, cell proliferation assay, bioassay of rhIL-2, preparation of oligosaccharides, and solid-phase-binding assay. Inhibitory effects of various high mannose-type glycans on IL-2 binding to ribonuclease B are investigated using the plate method. To clarify whether the carbohydrate recognition ability is indispensable for induction of IL-2-dependent cell proliferation, inhibitory effects of high mannose-type glycans on CTLL-2 cell proliferation are investigated. Inhibition of [35S]rh IL-2 binding to plates coated with ribonuclease B by oligomannosyl derivatives is also tabulated in the chapter.

Prostaglandin E2 Regulates the Level and Stability of Cyclooxygenase-2 mRNA through Activation of p38 Mitogen-activated Protein Kinase in Interleukin-1β-treated Human Synovial Fibroblasts

The p38 MAPK mediates transcriptional and post-transcriptional control of cyclooxygenase-2 (COX-2) mRNA following interleukin-1(IL-1)/lipopolysaccharide cellular activation. We explored a positive feedback, prostaglandin E(2) (PGE(2))-dependent stabilization of COX-2 mRNA mediated by the p38 MAPK cascade in IL-1 beta-stimulated human synovial fibroblasts. We observed a rapid (5 min), massive (>30-fold), and sustained (>48 h) increase in COX-2 mRNA, protein, and PGE(2) release following a recombinant human (rh) IL-1 beta signal that was inhibited by NS-398, a COX-2 inhibitor, and SB202190, a selective, cell-permeable p38 MAPK inhibitor. PGE(2) completely reversed NS-398-mediated inhibition but not SB202190-dependent inhibition. The eicosanoid didn't potentiate IL-1 beta-induced COX-2 expression nor did it activate COX-2 gene expression in quiescent cells. Transfection experiments with a human COX-2 promoter construct revealed a minor element of p38 MAPK-dependent transcriptional control after IL-1 beta stimulation. p38 MAPK synergized with the cAMP/cAMP-dependent protein kinase cascade to transactivate the COX-2 promoter. When human synovial fibroblasts were activated with rhIL-1 beta for 3-4 h (steady state) followed by washout, the elevated levels of COX-2 mRNA declined rapidly (<2 h) to control levels. If PGE(2), unlike EP2/3 agonists butaprost and sulprostone, was added to fresh medium, COX-2 mRNA levels remained elevated for up to 16 h. SB202190 or anti-PGE(2) monoclonal antibody compromised the stabilization of COX-2 mRNA by PGE(2). Deletion analysis using transfected chimeric luciferase-COX-2 mRNA 3'-untranslated region reporter constructs revealed that IL-1 beta increased reporter gene mRNA stability and translation via AU-containing distal regions of the untranslated region. This response was mediated entirely by a PGE(2)/p38 MAPK-dependent process. We conclude that the magnitude and duration of the induction of COX-2 mRNA, protein, and PGE(2) release by rhIL-1 beta is primarily the result of PGE(2)-dependent stabilization of COX-2 mRNA and stimulation of translation, a process involving a positive feedback loop mediated by the EP4 receptor and the downstream kinases p38 MAPK and, perhaps, cAMP-dependent protein kinase.

IL-6 attenuates apoptosis, while neither IL-6 nor IL-10 affect the numbers or protease phenotype of fetal liver-derived human mast cells.

The combination of recombinant human stem cell factor (rhSCF), rh interleukin (IL)-6 and rhIL-10 was reported to be optimal for mast cell development from cord blood progenitors and to induce chymase expression in all such mast cells earlier in their development than tryptase. The effects of rhIL-6 and rhIL-10 in various combinations on the rhSCF-dependent development of human mast cells from fetal liver progenitors were examined in serum-free media. Dispersed fetal liver cells were cultured in serum-free AIM-V medium with rhSCF alone, or with combinations of rhIL-6 and rhIL-10. Tryptase and chymase expression, surface Kit expression, metachromasia with toluidine blue and apoptosis were measured. Neither rhIL-6 nor rhIL-10 nor the two interleukins together, when included from day 0 of culture, affected the number or protease phenotype of mast cells at 1 or 3 weeks. Expression of tryptase paralleled the appearance of metachromasia and surface Kit, both of which preceded chymase expression, regardless whether a rabbit polyclonal or mouse monoclonal anti-chymase antibody preparation was used. On the other hand, rhIL-6 markedly attenuated baseline levels of apoptosis in the presence of rhSCF as well as apoptosis occurring after withdrawal of rhSCF, whereas rhIL-10 had no effect. RhIL-6 protected fetal liver-derived mast cells from apoptosis, particularly after withdrawal of rhSCF, but neither rhIL-6 nor rhIL-10 nor the combination of these interleukins affected the numbers or protease phenotype of these mast cells.

Lung epithelial H292 cells induce differentiation of immature human HMC-1 mast cells by interleukin-6 and stem cell factor.

Immature mast cells migrate into tissues where they differentiate into mature mast cells under the influence of local factors. In the airways of asthmatics increased numbers of chronically activated mast cells are located nearby the airway epithelium. The aim of this study was to evaluate whether and, if so, which products released by epithelial cells may affect mast cell proliferation and differentiation. We performed in vitro studies using the human lung mucoepidermoid carcinoma-derived H292 cell line and the immature human mast cell line, HMC-1. Proliferation was assessed by 3H-thymidine incorporation. Differentiation of HMC-1 cells was inferred from tryptase production. Exposure of HMC-1 cells to medium conditioned for 48 h by H292 cells resulted in a reduction of proliferation with 65 +/- 4.9% (mean +/- SEM, n = 9) at day 5. Culturing HMC-1 cells for 8 days in the presence of H292-conditioned medium resulted in morphological changes indicative of differentiation, and in a 3.0 +/- 0.4-fold increase of tryptase production (P = 0.0039, n = 9). Conditioned medium from H292 cells that were stimulated by LPS also inhibited HMC-1 proliferation. Inhibitory antibodies against two mediators from H292 cells, interleukin-6 (IL-6) and stem cell factor (SCF), abolished the increase in HMC-1 tryptase production induced by H292-conditioned medium. Recombinant human (rh) IL-6, but not rhSCF, reduced HMC-1 proliferation with 44% and 13% at day 3 and 5, respectively. Surprisingly, rhIL-6 did not increase HMC-1 tryptase production significantly whereas incubation with rhSCF did (1.5 +/- 0.1-fold, P = 0.002, n = 10) although the increase was less than observed for conditioned medium. Epithelial-derived IL-6 and SCF are implicated in differentiation of HMC-1 cells but additional factors are not excluded. As activated primary bronchial epithelial cells also express IL-6 and SCF, it should be considered that these cells are involved in mast cell differentiation within the airways, particularly in diseases where epithelial cells are activated, such as asthma.

Demonstration That Human Mast Cells Arise From a Progenitor Cell Population That Is CD34+, c-kit+, and Expresses Aminopeptidase N (CD13)

Human mast cells are known to arise from a CD34(+)/c-kit(+) progenitor cell population that also gives rise to neutrophils, eosinophils, basophils, and monocytes. To further characterize cells within the CD34(+)/c-kit(+) population that yield mast cells, this progenitor was additionally sorted for CD13, a myeloid marker known to appear early on rodent mast cells and cultured human mast cells, but not expressed or expressed at low levels on human tissue mast cells; and cultured in recombinant human (rh) stem cell factor (rhSCF), rh interleukin-3 (rhIL-3; first week only), and rhIL-6. Initial sorts revealed that although the majority of cells in culture arose from the CD34(+)/c-kit(+)/CD13(-) cell population, mast cells arose from a CD34(+)/c-kit(+)/CD13(+) progenitor cell that also gave rise to a population of monocytes. Sequential sorting confirmed that CD34(+)/c-kit(+)/CD13(+) cells in CD34(+)/c-kit(+)/CD13(-) sorts gave rise to the few mast cells observed in CD13(-) sorted cells. CD34(+)/c-kit(+)/CD13(+) cells plated as single cells in the presence of various cytokine combinations gave rise to pure mast cell, monocyte, or mixed mast cell/monocyte progeny. Addition of either rh granulocyte-macrophage colony-stimulating factor (rhGM-CSF) or rhIL-5 to the CD34(+)/c-kit(+)/CD13(+) progenitor cell population cultured in rhSCF, rhIL-3, and rhIL-6 did increase the number of total cells cultured and in the case of rhIL-5, did increase total mast cell numbers. Neither rhGM-CSF or rhIL-5 led to additional cell populations, ie, even with the addition of rhGM-CSF or rhIL-5, only mast cells and monocytes grew from CD34(+)/c-kit(+)/CD13(+) cells. Thus, human mast cells and a population of monocytes arise from precursor cells that express CD34, c-kit, and CD13; and within which, are mast cell, monocyte, and mast/monocyte (bipotential) precursors.

Effect of prostaglandin E2 on recombinant human bone morphogenetic protein-2-stimulated osteoblastic differentiation in human periodontal ligament cells.

Recombinant human (rh) bone morphogenetic protein-2 (BMP-2) stimulates osteoblastic differentiation in cells isolated from human periodontal ligament (HPLC), and this action of rhBMP-2 may be modulated by prostaglandins (PGs), which are local regulatory factors in the bone metabolism. In the present study, we investigated the effect of prostaglandin E2 (PGE2) on rhBMP-2-stimulated osteoblastic differentiation in cultured HPLC. rhBMP-2 (500 ng/ml)-stimulated alkaline phosphatase (ALPase) activity was enhanced by simultaneous treatment with low concentrations (10(-10)-10(-8) M) of PGE2, whereas a high concentration (10(-6) M) of PGE2 suppressed it. rhBMP-2 did not induce cyclo-oxygenase-2 (COX-2) mRNA expression or subsequent PGE2 production, whereas it remarkably suppressed rhIL-1 beta-induced COX-2 mRNA expression and PGE2 production. The rhBMP-2 action on osteoblastic differentiation in HPLC was also enhanced by co-treatment with 0.25 to 25 ng/ml of rh interleukin-1 beta (IL-1 beta). The ALPase activity stimulated by simultaneous treatment with rhBMP-2 and rhIL-1 beta was partially inhibited by addition of 10(-6) M of indomethacin, which completely inhibited rhIL-1 beta-induced PGE2 production. These results reveal that PGE2 at different concentrations exerts a biphasic effect on BMP-2-stimulated osteoblastic differentiation in HPLC, BMP-2 inhibits IL-1 beta-induced PGE2 production through suppressing COX-2 expression, and the BMP-2-stimulated osteoblastic differentiation may be enhanced by the endogenous PGE2 induced by BMP-2 and IL-1 beta. These suggest that BMP-2 action on osteoblastic differentiation in HPLC may be modulated by PGE2 in autocrine and paracrine fashions.

Intracellular interleukin-1 alpha production in human gingival fibroblasts is differentially regulated by various cytokines.

Interleukin-1 (IL-1) may play a critical role in immune and inflammatory responses in inflamed gingiva, and it is synthesized by a wide variety of host cells. In this study, we examined the regulatory effects of various cytokines on bioactive membrane IL-1 and intracellular IL-1 alpha production in cultured human gingival fibroblasts (HGF). Recombinant human (rh) IL-1 beta stimulated membrane IL-1 activity, which was mainly attributed to IL-1 alpha. rhIL-1 beta and rh tumor necrosis factor (TNF)-alpha stimulated HGF to produce intracellular IL-1 alpha, whereas rh interleukin-6 (IL-6), rh interleukin-4 (IL-4), and rh interferon (IFN)-gamma did not do so. Intracellular IL-1 alpha production induced by rhIL-1 beta or rhTNF-alpha may be partially related to protein kinase C (PKC) activation, because rhIL-1 beta or rhTNF-alpha-induced intracellular IL-1 alpha production was stimulated by pre-treatment with 12-o-tetradecanoylphorbol 13-acetate (TPA), a PKC activator, but was suppressed by the pre-treatment with 1-(5-isoquinoline-sulfonyl) -2-methylpiperazine dihydrochloride (H-7), which is a PKC inhibitor. rhIL-4 inhibited rhIL-1 beta- or rhTNF-alpha-induced intracellular IL-1 alpha production, but rhIL-6 had no effect on this production. Pre-treatment with rh IFN-gamma remarkably enhanced intracellular IL-1 alpha production induced by subsequent treatment with rhIL-1 beta or rhTNF-alpha. Simultaneous treatment with rhIFN-gamma and rhIL-1 beta inhibited rhIL-1 beta-induced intracellular IL-1 alpha production, but co-treatment with rhIFN-gamma and rhTNF-alpha enhanced rhTNF-alpha-induced intracellular IL-1 alpha production. These results suggest that in inflamed gingiva, pro-inflammatory cytokines such as IL-1 beta and TNF-alpha may induce bioactive intracellular IL-1 alpha production in human gingival fibroblasts and that this production can be differentially modulated by T-cell-derived cytokines such as IFN-gamma or IL4.

S-Phase Induction by Interleukin-6 Followed by Chemotherapy in Patients with Chronic Lymphocytic Leukemia and Non-Hodgkin'S Lymphoma

Interleukin-6 (IL-6) has in vitro demonstrated growth regulatory effects on tumor cells from patients with chronic lymphocytic leukemia (CLL) and lymphoma. The proliferation rate of these cells is usually very low and this is thought to be one of the reasons for the lack of a curative potential of cytostatic chemotherapy in CLL and low grade NHL. Recombinant human (rh) IL-6 might increase the in vivo proliferation rate leading to a higher sensitivity for chemotherapy. We tested this hypothesis by administering rhIL-6 to 9 CLL patients and 3 NHL patients in doses of 2.5μg/kg, 5μg/kg and 10μg/kg s.c. daily for 5 days followed by CHOP chemotherapy on the last day of rhIL-6 injection. Six patients had two treatment cycles. The proportion of cells in S-phase was determined by the bromodeoxyuridine labeling index (LI). Three patients achieved a partial remission, one patient had progressive disease and the remaining patients demonstrated no change. Two patients, who received 10 μg/kg/day rhIL-6, demonstrated a significant increase in LI, one of these was first observed in the second treatment cycle. A significant decrease was seen in two patients receiving 2.5μgkg and 5μg/kg respectively. Immunophenotypic assessment demonstrated that rhIL-6 increased the expression of CD20 in all CLL patients with a reversal after cessation of rhIL-6. We conclude that rhIL-6, in the dosage and schedule used in this study, did not increase the proportion of the cells in S-phase and that the growth stimulatory effects of rhIL-6 in CLL in vivo probably are insignificant. However, the role of rhIL-6 in CLL as inducer of increased CD20 expression prior to anti-CD20 antibody treatment remains to be determined.

Variability in the sensitivity of nine enzyme-linked immunosorbant assays (ELISAs) in the measurement of human interleukin 6

ELISAs employing several combinations of polyclonal and monoclonal anti-human IL-6 antibodies for the detection of interleukin 6 (IL-6) in cell culture supernatants were developed and compared. They were found to have differing sensitivities in the measurement of standard preparations of recombinant human (rh) IL-6 (WHO reference standard) as well as conventional preparations of IL-6 produced by stimulated human peripheral blood mononuclear cells. Thus, an ELISA that was optimal for detecting rhIL-6 standard was suboptimal for detecting IL-6 in cell culture supernatants. The presence of soluble IL-6 receptor (sIL-6R) or the signal-transducing gp130 molecule or serum did not alter the levels of IL-6 detected by these ELISAs. Therefore, the observed variability in sensitivity of the assays may be due to differences in epitope specificity of the various monoclonal antibodies used and the heterogeneity of IL-6 secreted into culture supernatants.

Interactions of Erythropoietin, Granulocyte Colony-Stimulating Factor, Stem Cell Factor, and Interleukin-11 on Murine Hematopoiesis During Simultaneous Administration

AbstractWe investigated how in vivo effects of single hematopoietic cytokines change if given in combination for a prolonged time. Mice were treated with every combination of recombinant human (rh) erythropoietin (EPO), rh granulocyte colony-stimulating factor (G-CSF), recombinant rat (rr) stem cell factor (SCF), and rh interleukin (IL)-11 by continuous infusion over 7 days (full factorial design with three dose levels for each cytokine). Burst-forming unit–erythroid (BFU-E), colony-forming unit–erythroid (CFU-E), and colony-forming unit–granulocyte-macrophage (CFU-GM) were determined in bone marrow and spleen, reticulocytes, hematocrit, granulocytes, and thrombocytes in the peripheral blood. An analysis of variance (ANOVA) and multiple comparison of means was used to evaluate the data. For several cell types, cytokine effects superimposed in an additive way if combined. However, in a large number of circumstances, nonadditive pairwise interactions were found. They differed in type and magnitude involving high-dose saturation, high-dose antagonistic effects, and even effect reversals (qualitative interactions). Hence, in general, it was not possible to foresee the combination effects on the basis of existing knowledge of single effects. On the other hand, the cytokine network was robust and no system hazards were observed under multiple cytokine combinations. The results illustrate that the cytokine network has nonlinear dynamic properties in vivo with dose-response characteristics of one cytokine being continuously modified by other cytokines.

Interactions of Erythropoietin, Granulocyte Colony-Stimulating Factor, Stem Cell Factor, and Interleukin-11 on Murine Hematopoiesis During Simultaneous Administration

We investigated how in vivo effects of single hematopoietic cytokines change if given in combination for a prolonged time. Mice were treated with every combination of recombinant human (rh) erythropoietin (EPO), rh granulocyte colony-stimulating factor (G-CSF), recombinant rat (rr) stem cell factor (SCF), and rh interleukin (IL)-11 by continuous infusion over 7 days (full factorial design with three dose levels for each cytokine). Burst-forming unit–erythroid (BFU-E), colony-forming unit–erythroid (CFU-E), and colony-forming unit–granulocyte-macrophage (CFU-GM) were determined in bone marrow and spleen, reticulocytes, hematocrit, granulocytes, and thrombocytes in the peripheral blood. An analysis of variance (ANOVA) and multiple comparison of means was used to evaluate the data. For several cell types, cytokine effects superimposed in an additive way if combined. However, in a large number of circumstances, nonadditive pairwise interactions were found. They differed in type and magnitude involving high-dose saturation, high-dose antagonistic effects, and even effect reversals (qualitative interactions). Hence, in general, it was not possible to foresee the combination effects on the basis of existing knowledge of single effects. On the other hand, the cytokine network was robust and no system hazards were observed under multiple cytokine combinations. The results illustrate that the cytokine network has nonlinear dynamic properties in vivo with dose-response characteristics of one cytokine being continuously modified by other cytokines.

Hematopoietic Growth Factors for the Treatment of Myelodysplastic Syndromes

Administration of recombinant human granulocyte colony-stimulating factor (rhG-CSF), rh granulocyte-macrophage colony-stimulating factor (rhGM-CSF) or rh interleukin-3 (rhIL-3) effectively stimulate and expand marrow myelopoiesis resulting in a dose-dependent increment of peripheral blood neutrophils in most patients with myelodysplasias (MDS). Clinical outcome with fewer infections have been reported in a few studies using rhG-CSF or rhGM-CSF, including a large randomized, controlled trial with rhGM-CSF. Clinical effective stimulation of megakaryopoiesis and erythropoiesis are however infrequent. Recently, rh erythropoietin (rhEpo) has been used to overcome the ineffective erythropoiesis in MDS to reduce transfusions needed. However, the efficiency has been low in most studies with marked differences in response rates. The most impressive clinical results were obtained in patients with milder forms of MDS combined with low prestudy endogenous S-Epo levels. The possible synergistic effect of combining rhEpo with rhG-CSF or rhGM-CSF has been studied with erythropoietic response rates of about 40%. The safety of the cytokine administration seems acceptable with no significant stimulation of leukemic myelopoiesis and subsequent progression into overt acute myeloid leukemia. In conclusion, combinations of hematopoietic growth factors may be of clinical benefit in some patients with MDS. However, due to the cost and inpredictable clinical outcome there is a need for extended laboratory research to understand the functional defects of MDS stem cells and progenitors.