Interleukin-1 release by alveolar macrophages in asthmatic patients and healthy subjects

A thymocyte proliferative response assay was used to compare spontaneous and lipopolysaccharide (LPS)-induced interleukin-1 (IL-1) release by alveolar macrophage (AM) in asthmatic patients and normal subjects. Twelve asthmatic patients and seven nonsmoking healthy subjects underwent a bronchoalveolar lavage (BAL). All asthmatic patients had a reversible airway obstruction and 7/12 were allergic. BAL AM were separated by adherence on tissue culture plates in medium RPMI-1640 supplemented with antibiotics and fetal calf serum, and were incubated with or without 10 micrograms/ml LPS for 20 h. Free-cell supernatants were tested by C3H/HeJ mice thymocyte proliferative assay. Unstimulated AM supernatant IL-1 activity was significantly higher in asthmatic patients (mean +/- SEM: 47.8 +/- 11.9 units/10(6) AM) in comparison with healthy subjects (4.8 +/- 2.3 units/10(6) AM; p less than 0.05, Mann-Whitney U test) but did not significantly differ between allergic (42.2 +/- 15.5 units/10(6) AM) and intrinsic asthmatic patients (55.8 +/- 20.7 units/10(6) AM). For healthy subjects, IL-1 activity was significantly higher in LPS-stimulated AM supernatants (85 +/- 20 units/10(6) AM, p less than 0.05; Mann-Whitney U test) in comparison with unstimulated ones; for asthmatic patients, unstimulated and LPS-stimulated AM supernatant IL-1 activity did not significantly differ. This finding is in accordance with previous work suggesting that AM from asthmatic patients have a weak suppressive activity upon lymphocyte proliferation and emphasize the enhanced AM releasability in asthma.     

Modulation of anti-Candida activity of human alveolar macrophages by interferon-gamma or interleukin-1-alpha

The fungicidal and bactericidal activities of human alveolar macrophages (AM) and peripheral blood monocytes (PBM) from 18 healthy volunteers were evaluated. The results showed that AM were able to phagocytize and kill Candida albicans, Pseudomonas aeruginosa, and Staphylococcus aureus. However, killing of the bacteria was already complete in 2 h, whereas killing of Candida required 4 to 6 h despite an early phagocytosis of yeast cells. The fungicidal activity of freshly collected AM and PBM was also tested after effector cell exposure to interferon-gamma (IFN-gamma), interleukin-1-alpha (IL-1 alpha), endotoxin lipopolysaccharide (LPS), or interleukin 2 (IL-2). It was found that treatment with IFN-gamma, IL-1 alpha, or LPS significantly augmented macrophage and PBM candidacidal activity, whereas the addition of IL-2 was ineffective. We also evaluated killing of C. albicans by AM cultured in vitro for different times. While phagocytosis was apparently unaffected, the candidacidal activity progressively decreased over the in vitro culture period, an effect that was largely reversed by cell exposure to IFN-gamma, IL-1 alpha, or LPS. In an experimental model in which mice infected with an agerminative C. albicans strain (PCA-2) resisted lethal microbial challenge, freshly harvested AM showed increased cytotoxic activity to Aspergillus fumigatus in vitro as well as enhanced IL-1 production. In conclusion, present data confirm the crucial role of AM in the surveillance of bacterial and fungal infections and indicate that treatment of these cells with IFN-gamma or IL-1 alpha is able to enhance their antimicrobial capability.     

Imbalance between interleukin-6 and adrenomedullin mRNA levels in peripheral blood mononuclear cells of patients with lupus nephritis.

In this study, we measured the mRNA levels of adrenomedullin (AM), C-type natriuretic peptide, vascular endothelial growth factor, interleukin-1beta (IL-1beta) and interleukin-6 (IL-6) in peripheral blood mononuclear cells (PBMC) of 34 patients with lupus nephritis (LN) (15 active and 19 inactive) and 30 healthy volunteers. mRNA levels were measured using a real-time quantitative PCR METHOD: Compared with healthy volunteers, IL-6 mRNA levels were elevated in LN patients (P < 0.005), while AM mRNA levels were decreased (P < 0.05). Also, IL-6 mRNA levels were higher and AM mRNA levels lower in active LN patients compared with inactive LN patients. In addition, IL-6 mRNA levels positively correlated and AM mRNA levels negatively correlated with SLE disease activity index and laboratory findings, such as blood urea nitrogen, serum creatinine, 50% haemolytic unit of complement and urinary excretion of protein over 24 h. Furthermore, IL-6 mRNA levels were negatively correlated with AM mRNA levels within the same LN patients. With regard to pathological findings, our results showed that IL-6 mRNA levels were higher, and AM mRNA levels significantly lower in patients with a high activity index compared to those with a low activity index. Following treatment with prednisolone, IL-6 mRNA levels in active LN patients decreased and AM mRNA levels increased to levels comparable to those in inactive LN and healthy volunteers. In vitro studies further demonstrated that elevated IL-6 mRNA levels in active LN patient PBMC were suppressed by the addition of adrenomedullin. Our results suggest that an imbalance between IL-6 and AM levels may play an important role in the progression of SLE, and that the mRNA levels of these genes in PBMC may be used as a disease activity index for SLE.     

Interleukin-1 stimulates rapid release of cytokine-induced neutrophil chemoattractant (CINC) in rat lungs

We found that intratracheal insufflation of interleukin-1 (IL-1) in rats rapidly increased lung lavage cytokine-induced neutrophil chemoattractant (CINC) concentrations, lung tissue myeloperoxidase (MPO) activity, and lung lavage neutrophil counts, and that CINC elevation preceded the migration of neutrophils into the lung. Further, we found that bolus CINC insufflation increased CINC concentrations in plasma, and we found that alveolar macrophages (AM) in lung tissue selections or AM recovered by lavage from rats given IL-1 intratracheally stained positively for CINC by immunohistochemistry. In addition, incubating rat AM with increasing doses of IL-1 in vitro progressively increased CINC concentrations in the culture medium. Our results suggest that the potent neutrophil chemoattractant CINC is rapidly produced and released by rat AM following challenge with IL-1 in vivo or in vitro, and support the hypothesis that CINC is an important mediator in the development of pulmonary inflammation in the rat.Parker B. Francis Fellow in Pulmonary Research.     

Enhancement of murine alveolar macrophage functions by orally administered beta-glucan.

The effect of orally administered SSG, a beta-1,3-glucan obtained from the culture filtrate of the fungus Sclerotinia sclerotiorum IFO 9395, on alveolar macrophage (AM) functions of CDF1 mice was examined. SSG administered orally (20, 40, 80 or 160 mg/kg) for 10 consecutive days enhanced the lysosomal enzyme activity of AM. The greatest enhancing effect was observed at 80 mg/kg of SSG. Multiple oral administrations of SSG (10 consecutive days) were needed to induce significant enhancing effects. Phagocytic activity and interleukin-1 (IL-1) production of AM were also augmented by oral administration of SSG, and the kinetics of the activated state differed depending on the kind of activity. However, H2O2 production of AM was not affected by SSG. Orally administered SSG also (40 or 80 mg/kg, 10 consecutive days) increased the number of AM and the greatest increment was observed 14 days after the first administration. On the other hand, the supernatant of Peyer's patch (PP) cells from mice administered SSG (80 mg/kg) orally stimulated the lysosomal enzyme activity of AM in vitro, and enhanced colony stimulating activity (CSA) was detected from this supernatant. These results demonstrate that SSG given by the oral route can activate murine AM both qualitatively and quantitatively, and it would mediated, at least in part, by the activation of PP cells in the intestine.     

Production of interleukin-2 by YAC-1 cells stimulated with interleukin-1 and its augmentation of the natural killer activity

The production of interleukin-2 (IL-2) by YAC-1 cells stimulated with interleukin-1 (IL-1) was examined in the in vitro culture system. The IL-2 activity was detectable in the culture supernatant of YAC-1 cells stimulated with either a mouse IL-1 preparation or human purified IL-1. This activity could be detected 1 h after stimulation with IL-1. The addition of monoclonal antibody reactive with mouse IL-2 receptor completely blocked the IL-2 activity in the culture supernatant of IL-1-stimulated YAC-1 cells. Further, the culture supernatant of IL-1-stimulated YAC-1 cells augmented the NK activity in mouse spleen cells. The role of the IL-2 activity in the culture supernatant of IL-1-stimulated YAC-1 cells on augmentation of the NK activity is discussed.     

Relationship between ineffective antigen presentation by murine alveolar macrophages and their immunosuppressive function.

Murine alveolar macrophages (AM) have been shown to be inefficient at providing accessory function for initiation of the in vitro plaque-forming cell (PFC) response. In the present study AM, which were obtained either from untreated mice (resident AM) or mice injected i.v. with BCG (activated AM) potently suppressed the PFC response of spleen cells from animals previously primed with sheep erythrocytes (SRBC). Addition of AM at a concentration of 10% with respect to spleen cells resulted in greater than 90% suppression of the PFC response. In order to determine if inefficient antigen presentation was associated with AM-mediated suppression, the role of IL-1 and Ia antigen was studied. Addition of exogenous recombinant IL-1 (rIL-1) stimulated the PFC response in control cultures, but had no effect on AM-mediated suppression. Resident AM could be activated with lipopolysaccharide or antigen to produce significant levels of IL-1. Membrane-bound IL-1, thought to be important in the presentation of particulate antigens, was detected on glutaraldehyde-fixed resident AM and was significantly elevated in BCG-activated macrophages. The frequency of cell surface Ia antigen expression was low in resident AM (4%), but could be increased (35%) after in vivo activation with BCG. Recombinant interferon-gamma (IFN-gamma), known to enhance expression of Ia antigen and production of IL-1, had no effect on AM-mediated suppression when used either to pretreat AM, when present during the entire period of culture, or when injected into mice before culture initiation. Treatment with IFN-gamma, however, resulted in a slight increase in the expression of Ia antigen. These results indicate that the immunosuppressive activity of AM is neither related to a defect in IL-1 production or expression nor to a deficiency in Ia antigen expression and therefore can not be explained by the inefficient antigen-presenting function of alveolar macrophages.     

Increases in lung prolyl hydroxylase and superoxide dismutase activities during bleomycin-induced lung fibrosis in hamsters

Intratracheal administration of bleomycin causes pulmonary fibrosis in hamsters. Using this model the activities of lung prolyl hydroxylase and superoxide dismutase and the accumulation of neutral salt soluble and insoluble collagens have been determined. One unit of bleomycin was injected intratracheally to hamsters, whereas control animals received an equivalent volume of sterile saline by the same route. Total lung prolyl hydroxylase activity was significantly elevated at all times following bleomycin treatment. The activity was increased as early as 2 days, peaked to a maximum value of 400% of the control at 14 days, followed by a sharp decline to 235% and 180% of the control activity at 21 and 28 days after bleomycin treatment, respectively. Except for the earliest time (2 days), lung prolyl hydroxylase specific activity was also significantly elevated at all times after bleomycin treatment. A significant increase in both total and specific activities of lung superoxide dismutase was also observed at all times after bleomycin treatment. Total activity peaked to a maximum value of 315% of the control activity at 14 days and the specific activity to a maximum value of 190% of the control at 21 days after bleomycin treatment. Thereafter, both activities declined, but were still significantly elevated over the control at 28 days after the treatment. Lung proline pool size was significantly increased at all times and attained a maximum value of 372% of the control at 14 days after bleomycin treatment. Increases in the lung prolyl hydroxylase and superoxide dismutase activities and in the proline pool size preceded the significant increases in neutral salt soluble and insoluble collagens which occurred at 7 days after bleomycin treatment and continued to be significantly elevated for the remaining period of the study.     

The effects of various drugs and immune complexes on the function of alveolar macrophages from patients with collagen-vascular diseases with interstitial pneumonia

Alveolar macrophages (AM) taken from sixteen patients with collagen-vascular diseases with interstitial pneumonia (CVD-IP+) were cultured with prednisolone (PSL), prostaglandin E2 (PGE2), colchicine (Colch), D-penicillamine or aggregated IgG (agg-IgG). The culture supernatants were tested on the fibroblast proliferation function and the amounts of fibronectin (Fn) and interleukin 1 (IL-1). The fibroblast proliferation was suppressed significantly by the supernatants of AM cultured with PGE2. The Fn production by AM was significantly suppressed by PGE2 and Colch. The IL-1 production by AM was not suppressed by these drugs, but was enhanced by Colch. In five patients with CVD-IP+, the fibroblast proliferation of AM supernatant was enhanced by agg-IgG. In these five patients, Fn and IL-1 production had a tendency to be increased by agg-IgG. The ratio of immune complexes (IC) in broncho alveolar lavage fluids to those in sera was significantly higher in patients with CVD-IP+ than in patients with CVD without IP suggesting a local production of IC in the lung in the patients with CVD-IP+.     

Stimulation of early eosinophil progenitors by a heat stable alveolar macrophage product from ovalbumin-sensitized and non-sensitized guinea pigs

Background Alveolar macrophages (AM) may participate in brochopulmonary hyperreactivity by secreting cytokines that recruit mature eosinophils, or induce eosinophil production from recruited circulating progenitors. Objective To define whether AM products can contribute to lung eosinophil production in immunized guinea pigs (GP), by analysing the effect of AM culture supernatatits (AM-SN) on in vitro eosinophilopoiesis. Methods Liquid and semi-solid bone marrow (BM) cultures were seeded witb SN from 95% pure AM exposed to LPS. Results AM-SN increased very significantly the long-term viability, cell proliferation and eosinophil production in liquid culture and supported formation of eosinophil-bearing mixed colonies, by acting on progenitors depleted of mature eosinophils. The effect on eosinophil production was not duplicated by natural or recombinant sources of GM-CSF (which nevertheless supported GM colony formation by GP BM), not by rhIL-8 (which was active on GP cells) and was not due to residual LPS. FPLC separation of active AM SN yielded a peak of apparent m.w. 43 kDa, active on both liquid and semi-solid cultures. The active moiety was heat- and trypsin-resistant. Neutralizing monoclonal antibodies to hGM-CSF, mGM-CSF, hIL-3 and mIL-3 failed to deplete the activity in AM-SN. Ovalbumin immunization induced its production by AM even without LPS challenge. Conclusions The lack of T lymphocytes among factor-producing AM, the properties of the active material, the inability of GM-CSF to reproduce these effects, and the failure of MoAbs to GM-CSF and to IL-3 to neutralize the activity indicate it is not due to the major eosinopoietic factors GM-CSF, IL-3 or IL-5.     

Effect of protein kinase C inhibitor (H-7) and calmodulin antagonist (W-7) on pertussis toxin-induced IL-1 production by human adherent monocytes. Comparison with lipopolysaccharide as a stimulator of IL-1 production.

Human adherent monocytes stimulated with 1 microgram/ml pertussis toxin (PT) produced interleukin-1 (IL-1), as measured by thymocyte co-stimulation assay and enzyme-linked immunosorbent assay (ELISA), specific for IL-1 alpha and IL-1 beta. To clarify the role of protein kinase C (PKC) and calmodulin in IL-1 production, we investigated the effects of a PKC inhibitor, H-7, and a calmodulin antagonist, W-7 on PT- and lipopolysaccharide (LPS)-induced IL-1 production by monocytes. Addition of 10 microM and 20 microM H-7 to the culture medium markedly suppressed both PT- and LPS-induced IL-1 production. PT-induced IL-1 production was significantly suppressed by 5 microM and 10 microM W-7. However, LPS-induced IL-1 production was not suppressed by W-7 at the concentrations tested. When monocytes were labelled with Quin 2/AM, IL-1 production by monocytes stimulated with PT and LPS was markedly suppressed. These results indicate that different pathways are involved in the IL-1 production by PT and LPS; both calmodulin- and PKC-dependent processes are necessary for the IL-1 production induced by PT, whereas LPS-induced IL-1 production is dependent on the PKC. Inhibition of IL-1 production by interfering with intracellular Ca2+ trafficking in Quin 2/AM-loaded monocytes may be associated with the inhibition of PKC and calmodulin activity.     

Secretion of monocyte chemotactic activity by alveolar macrophages.

The purpose of this study was to determine if alveolar macrophages (AMs) are a source of monocyte chemoattractants and the role bleomycin interaction with AMs may play in the recruitment of monocytes to the lung in a rodent model of bleomycin-induced pulmonary fibrosis. AMs isolated from rats with bleomycin-induced fibrosis secreted significantly greater amounts of monocyte chemoattractants than those isolated from normal rats. When AMs from normal rats were stimulated with bleomycin in vitro, monocyte chemotactic activity was secreted into the medium. Chemotactic activity secretion by AM stimulated with 0.01 to 0.1 micrograms/ml bleomycin was significantly higher than that of cells incubated in medium alone. This activity was truly chemotactic for monocytes, but caused only minimal migration of normal AMs. Bleomycin itself at concentrations of 1 pg/ml to 10 micrograms/ml had no monocyte chemoattractant activity. Characterization of the chemotactic activity in conditioned media (CM) from bleomycin-stimulated AM demonstrated that the major portion of the activity bound to gelatin, was heterogeneous, with estimated molecular weights of 20 to 60 kd, and was inactivated by specific antifibronectin antibody. These findings suggest that fibronectin fragments are primarily responsible for the monocyte chemotactic activity secreted by AMs. Through increased secretion of such chemotactic substances, AMs could play a key role in the recruitment of peripheral blood monocytes into the lung in inflammatory lung disease and fibrosis.     

实验性肺纤维化大鼠肺泡巨噬细胞脂质过氧化先于其细胞因子的释放

目的:动态观察博莱霉素(BLM)致大鼠肺纤维化过程中肺泡巨噬细胞(AM)脂质过氧化损伤和抗氧化损伤指标的变化,探讨与其释放肿瘤坏死因子(TNF-α)和血小板源生长因子(PDGF)的关系。方法:分离AM测定其中丙二醛(MDA)含量和谷胱甘肽过氧化物酶(GSH-Px)活性;行体外AM纯培养,测定其培养上清中上述两种细胞因子含量。结果:(1)AM中MDA第1d即明显升高,第3d至高峰,急性期过后则逐渐降至正常;而GSH-Px则早期处于极低状态,14d后逐渐恢复近于正常。(2)AM释放上述两种细胞因子于第3d开始明显增高,并呈动态变化。(3)AM脂质过氧化损伤先于其释放TNF-α和PDGF。结论:推测AM脂质过氧化损伤所导致的抗氧化物质缺乏可能对其激活发挥重要作用。     

Effects of three cysteine pro-drugs on bleomycin-induced lung fibrosis in hamsters.

The cysteine pro-drug Z2196 ((2RS, 4R)-2-methylthiazolidine carboxylic acid) and two drugs with methyl esters attached to Z2196 (Z2197 and Z2199) were evaluated for antifibrotic effects in the hamster bleomycin model of lung fibrosis. Each drug or phosphate-buffered saline (PBS) was given daily (300 mg/kg intraperitoneally) for 2 days before intratracheal instillation of bleomycin (7.5 units/kg) or saline for an additional 13 days. Lung collagen measured as hydroxyproline was significantly increased to 138% of the control groups in the PBS + Bleomycin treated group, but the Z2196 + Bleomycin group was increased to 108% and was not statistically different from controls. Protein content of bronchoalveolar lavage supernatant in PBS + Bleomycin treated hamsters was significantly increased to 326% of controls. The protein content of bronchoalveolar lavage supernatant for all cysteine pro-drug + Bleomycin treated hamsters was increased to 160% of PBS + Bleomycin treated hamsters. All the Bleomycin treated hamsters had significantly more cells and more neutrophils recovered in bronchoalveolar lavage than controls. The PBS + Bleomycin treated hamsters had significantly more lymphocytes in bronchoalveolar lavage than all the other treatment groups. The Z2196 + Bleomycin and Z2197 + Bleomycin hamsters had significantly less monocytes in BALF than PBS + Bleomycin hamsters. The lung total sulfhydryl and nonprotein sulfhydryl in PBS + Bleomycin treated hamsters were increased to 210% and 253% of controls, respectively, whereas in Z2196 + Bleomycin hamsters they were increased to 152% and 153%, respectively. Histopathology of PBS + Bleomycin hamsters showed a diffuse mixed mononuclear alveolitis, multifocal fibrosis and peribronchiolar fibrosis, whereas Z2196 + Bleomycin hamsters showed notably less alveolitis and fibrosis.(ABSTRACT TRUNCATED AT 250 WORDS)     

IL4-10 fusion protein: a novel immunoregulatory drug combining activities of interleukin 4 and interleukin 10

The objective of this study was to test the capacity of a newly developed fusion protein of interleukin 4 (IL-4) and IL-10 [IL4-10 fusion protein (FP)] to shift multiple pro-inflammatory pathways towards immune regulation, and to inhibit pro-inflammatory activity in arthritis models. The effects of IL4-10 FP in comparison with IL-4, IL-10 and IL-4 plus IL-10 on pro- and anti-inflammatory mediators, T cells and immunoglobulin (Ig) receptors in favour of immunoregulatory activity were studied. In addition, the capacity of IL4-10 FP to inhibit pro-inflammatory activity in ex-vivo and in-vivo arthritis models was investigated. IL4-10 FP robustly inhibited pro-inflammatory cytokine [IL-1β, tumour necrosis factor (TNF)-α, IL-6 and IL-8] production in whole blood cultures, mediated by both the IL-10 and the IL-4 moiety. IL4-10 fusion protein induced IL-1 receptor antagonist (IL-1RA) production and preserved soluble TNF receptor (sTNFR) levels, strongly increasing IL-1RA/IL-1β and sTNFR/TNF-α ratios. In addition, IL4-10 FP strongly inhibited T helper (Th) type 1 and 17 cytokine secretion, while maintaining FoxP3 expression and up-regulating Th2 activity. In addition, while largely leaving expression of activating Fc gamma receptor (FcγR)I, III and Fc epsilon receptor (FcεR) unaffected, it significantly shifted the FcγRIIa/FcγRIIb ratio in favour of the inhibitory FcγRIIb. Moreover, IL4–10 FP robustly inhibited secretion of pro-inflammatory cytokines by rheumatoid arthritis synovial tissue and suppressed experimental arthritis in mice, without inducing B cell hyperactivity. IL4-10 fusion protein is a novel drug, signalling cells to induce immunoregulatory activity that overcomes limitations of IL-4 and IL-10 stand-alone therapy, and therefore has therapeutic potential for inflammatory diseases such as rheumatoid arthritis.     

Pharmacological inhibition of Mannheimia haemolytica lipopolysaccharide and leukotoxin-induced cytokine expression in bovine alveolar macrophages

The inflammatory cytokines tumor necrosis factor-alpha (TNFalpha), interleukin-1 beta (IL-1beta), and interleukin-8 (IL-8) are believed to contribute to the pathogenesis of lung injury in bovine pneumonic mannheimiosis (BPM) caused by Mannheimia (Pasteurella) haemolytica. Inflammatory cytokines may, therefore, represent therapeutic targets to be modulated for the purpose of treating or preventing this important disease of cattle. The purpose of this study was to evaluate the ability of six pharmacological agents to suppress the expression of TNFalpha, IL-1beta, and IL-8 genes and proteins in bovine alveolar macrophages (AM) exposed to M. haemolytica lipopolysaccharide (LPS) and leukotoxin (LktA) in vitro. The compounds tested included dexamethasone (DEX), tetrahydropapaveroline (THP), pentoxifylline (PTX), rolipram (ROL), SB203580 (SB), and thalidomide (THL). Cytokine expression was induced by the addition of purified M. haemolytica LPS and LktA to AM cell cultures following pretreatment with inhibitor compounds. Secretion of TNFalpha, IL-1beta, and IL-8 proteins into the cell culture supernatant was measured using enzyme-linked immunosorbent assays, and steady-state accumulation of cytokine-specific mRNA was measured by northern blot analysis. Dose-dependent inhibition of cytokine secretion occurred in response to pretreatment of AM with DEX (TNFalpha, IL-1beta, IL-8), THP (TNFalpha, IL-1beta, IL-8), PTX (TNFalpha, IL-1beta, IL-8), ROL (TNFalpha, IL-1beta), and SB (TNFalpha, IL-8). Significant dose-dependent inhibition of cytokine mRNA expression occurred in response to pretreatment with DEX (TNFalpha, IL-1beta, IL-8), THP (TNFalpha, IL-1beta, IL-8), and PTX (TNFalpha). DEX was the most effective inhibitor by far; pretreatment with this compound yielded greater than 95% inhibition of cytokine gene and protein expression over a broad range of concentrations. These findings demonstrate that DEX, THP, PTX, ROL, and SB are capable of suppressing inflammatory cytokine secretion by bovine AM in vitro. If pulmonary cytokine secretion may be similarly inhibited in vivo, anti-cytokine therapy may represent a novel strategy for the management of BPM.     

PTEN对缺氧复氧心肌H9c2细胞凋亡、氧化损伤及免疫炎症因子水平的影响

目的:研究第10号染色体缺失的磷酸酶及张力蛋白同源物(PTEN)对缺氧复氧条件下心肌H9c2细胞凋亡、氧化损伤及免疫炎症因子水平的影响。方法:用H9c2细胞构建缺氧复氧模型。细胞转染PTEN小干扰RNA(siRNA)和阴性对照siRNA,缺氧复氧处理后,RT-PCR和Western blot分别测定细胞中PTEN的mRNA和蛋白水平。MTT法测定细胞活力,流式细胞术测定细胞凋亡,用黄嘌呤氧化法测定细胞中超氧化物歧化酶(SOD)活性,用硫代巴比妥酸法测定丙二醛(MDA)含量,用二硝基苯肼法测定培养液上清中乳酸脱氢酶(LDH)活性,ELISA测定培养液上清中肿瘤坏死因子α(TNF-α)、白细胞介素1β(IL-1β)和白细胞介素6(IL-6)水平,Western blot法测定细胞中cleaved caspase-3、Bax和Fas L的蛋白水平。结果:缺氧复氧处理后H9c2细胞中PTEN的mRNA和蛋白水平均明显升高(P 0. 05)。转染PTEN siRNA后的细胞中PTEN的mRNA和蛋白水平明显下降(P 0. 05)。缺氧复氧处理后H9c2细胞活力下降,凋亡率升高,细胞中cleaved caspase-3、Bax和Fas L的蛋白水平升高,MDA水平也升高,SOD活性下降,培养液上清中LDH、TNF-α、IL-1β和IL-6的水平升高(P 0. 05),而下调PTEN可以部分拮抗缺氧复氧对H9c2细胞活力、凋亡率、MDA水平、SOD水平及培养液上清中LDH、TNF-α、IL-1β和IL-6水平的影响。结论:下调PTEN可以减轻缺氧复氧诱导的心肌H9c2细胞氧化损伤,减少H9c2细胞凋亡,降低TNF-α、IL-1β和IL-6的水平。     

Pharmacokinetics and cytokine production in heroin and morphine-treated mice

The parallelism between serum levels of heroin and morphine (M) metabolites and the production of interleukin-1beta (IL-1beta), interleukin-2 (IL-2), interleukin-10 (IL-10), tumor necrosis factor alpha (TNF-alpha), transforming growth factor-beta1 (TGF-beta1), and interferon-gamma (IFN-gamma) from murine splenocyte cultures following s.c. injection with 20 mg/kg heroin or M in C57/BL mice is described. The pharmacokinetic profiles of M and inactive morphine-3-glucuronide (M3G) in morphine-treated mice nearly overlapped those in heroin-treated mice, with the only difference being the presence of 6-monoacetylmorphine (AM) in profiles of the latter group. Heroin and M significantly increased production of IL-1beta, IL-2, TNF-alpha and IFN-gamma at 3, 20 and 40 min from treatment, peaking at 20 min, though the effect was very brief. At 24 h production was greatly inhibited, and this depressive effect lasted longer than the stimulatory effect. At 48 h only a partial recovery was observed. Heroin and M also had a highly stimulatory effect on the release of anti-inflammatory cytokines such as TGF-beta1 and IL-10, though this effect was observed after 120 min, peaking at 24 h and then somewhat decreasing at 48 h. This study demonstrates that the more rapid and pronounced immune response to heroin treatment was due to the presence of AM. Both heroin and M produced a biphasic effect on cytokine production: the central opioid or non-opioid receptors are involved in exogenous opiod-induced stimulatory effects, whereas peripheral opioid or non-opioid receptors are involved in depressive effects. Deficient or excess expression of these key mediators may predispose the host to aberrant defence mechanisms.     

Regulation of fibronectin synthesis by interleukin-1 and interleukin-6 in rat hepatocytes.

The authors have observed previously that recombinant human interleukin-1 (rhIL-1) administered into rats increased plasma fibronectin (Fn) level concomitant with the increase of Fn in the liver. Because IL-1 induces interleukin-6 (IL-6) in certain cell types, the IL-1 effect might be mediated by IL-6. To evaluate this possibility, the effect of recombinant human interleukin-6 (rhIL-6), rhIL-1 alpha, and rhIL-1 beta on Fn synthesis in cultured rat hepatocytes was studied. It was shown that rhIL-6 increased Fn synthesis in hepatocytes, in contrast, rhIL-1 alpha, rhIL-I beta and TNF did not have any effect on Fn synthesis. When we studied the interaction of IL-1 and IL-6, IL-1 did not exhibit any synergistic effect with IL-6. Conditioned medium (CM) from rhIL-1 stimulated peripheral blood monocytes (PBM) increased the Fn synthesis, and its activity was neutralized significantly by anti-rhIL-6 antibodies. The CM from rhIL-1-stimulated PBM was analyzed by enzyme linked immunosorbent assay and revealed the increase of IL-6. Furthermore, it was found that intraperitoneal administration of rhIL-1 induced IL-6 into blood. The administration of rhIL-6 into rats increased circulating Fn levels. These results strongly suggest that the in vivo effect of IL-1 on Fn synthesis is mediated by IL-6.