Soluble HLA-G molecules induce apoptosis in natural killer cells

Membrane-bound human leukocyte antigen-G (HLA-G) molecules are primarily expressed by cytotrophoblasts of the fetus. They are thought to protect the fetus from immunologic attack by the maternal immune system and have recently been associated with transplantation graft acceptance. In addition, soluble HLA-G molecules (sHLA-G) have been shown to play a role in the success of pregnancies, but are upregulated in certain cancers. However, the exact mechanism for this regulation has remained elusive. The aim of this study was to examine the mechanism by which sHLA-G interact with natural killer (NK) cells in vitro. sHLA-G effectively blocked NK lysis of target cells via fracticide killing of NK cells by apoptosis. These studies support the protective role of sHLA-G in immunologic reactions by interacting with NK cells, thus providing a regulatory function.     

晚期糖基化终末产物诱导人卵巢颗粒细胞株COV434凋亡并上调促炎因子HMGB1自分泌

目的:探讨晚期糖基化终末产物(advanced glycation end products,AGEs)是否诱导人卵巢颗粒细胞株COV434凋亡,并探究其可能的机制。方法:采用不同浓度AGEs牛血清白蛋白与人卵巢颗粒细胞株COV434共同孵育,流式细胞术观察细胞凋亡率,Western blot观察caspase-3和cleaved caspase-3的蛋白水平,ELISA检测细胞培养液上清中高迁移率族蛋白B1(high mobility group box 1 protein,HMGB1)的含量。结果:与对照组比较,100 mg/L AGEs组和200 mg/L AGEs组早、晚期凋亡率显著增加,各组caspase-3蛋白水平的差异无统计学显著性,但与对照组比较,100 mg/L AGEs组和200 mg/L AGEs组cleaved caspase-3的蛋白水平显著增加(P0.05)。此外,与对照组比较,100 mg/L AGEs组和200 mg/L AGEs组细胞培养液上清中HMGB1促炎介质的水平显著上升(P0.05)。结论:晚期糖基化终末产物诱导人卵巢颗粒细胞株COV434凋亡可能与促炎反应有关。     

Inhibitory effect of statins on inflammatory cytokine production from human bronchial epithelial cells

Statins are 3-hydroxy-3-methylglutaryl-co-enzyme A reductase inhibitors of cholesterol biosynthesis, and have been reported to exert pleiotropic effects on cellular signalling and cellular functions involved in inflammation. Recent reports have demonstrated that previous statin therapy reduced the risk of pneumonia or increased survival in patients with community-acquired pneumonia. However, the precise mechanisms responsible for these effects are unclear. In the present study, we examined the effects of statins on cytokine production from lipopolysaccharide (LPS)-stimulated human bronchial epithelial cells (BEAS-2B). Interleukin (IL)-6 and IL-8 mRNA expression and protein secretion in LPS-stimulated cells were inhibited significantly by the lipophilic statin pitavastatin and the hydrophilic statin pravastatin. As these inhibitory effects of statin were negated by adding mevalonate, the anti-inflammatory effects of statins appear to be exerted via the mevalonic cascade. In addition, the activation levels of Ras homologue gene family A (RhoA) in BEAS-2B cells cultured with pitavastatin were significantly lower than those without the statin. These results suggest that statins have anti-inflammatory effects by reducing cytokine production through inhibition of the mevalonic cascade followed by RhoA activation in the lung.     

Studies on the interactions of Haemophilus parasuis with porcine epithelial tracheal cells: Limited role of LOS in apoptosis and pro-inflammatory cytokine release

Haemophilus parasuis colonizes the upper respiratory tract of swine and causes Glässer's disease. We recently demonstrated that H. parasuis can adhere to newborn pig tracheal (NPTr) cells. However, the molecular mechanisms involved in upper respiratory tract colonization by H. parasuis are unknown. The aim of this work was to investigate the role of H. parasuis lipooligosaccharide (LOS) in bacterial adhesion to NPTr cells, the ability of the bacteria and its LOS to induce NPTr cells apoptosis, and their stimulating effect on cytokine release. Our results showed that LOS is partially involved in adhesion to NPTr cells. H. parasuis induced NPTr cells apoptosis in a caspase-3 dependent fashion, but LOS did not seem to be involved in such a process. H. parasuis and, to a lesser extent, its LOS stimulated IL-8 and IL-6 release by NPTr cells. In addition, H. parasuis serotype 4 field isolates induced higher levels of these mediators than did serotype 5 isolates. These results suggest that bacterial adhesion, induction of apoptosis and cytokine release are important events for H. parasuis colonization, but LOS appears to have a limited role in these processes.     

Fluticasone and budesonide inhibit cytokine release in human lung epithelial cells and alveolar macrophages

Glucocorticoids are potent anti-inflammatory agents capable of influencing cytokine release in a number of cell types. The aim of the present study was to investigate whether glucocorticoids, frequently used in the treatment of asthma, interfere with cytokine secretion by lung epithelial cells and alveolar macrophages in vitro. Inhalation of swine dust induces airway inflammation with influx of inflammatory cells and release of proinflammatory cytokines in the lungs. Therefore, human lung epithelial cells (A549) and human alveolar macrophages were stimulated with swine dust or lipopolysaccharide (LPS), and the inhibitory effect of budesonide and fluticasone propionate on cytokine release was studied in a dose-response (10(-13)-10(-8) M) manner. The time course for the steroid effect was also investigated. Both steroids caused a dose-dependent, almost total, inhibition of swine dust-induced IL-6 and IL-8 release from epithelial cells and LPS-induced IL-6 and TNF-alpha from alveolar macrophages. The steroids only partially inhibited IL-8 release from alveolar macrophages. Budesonide was approximately 10 times less potent than fluticasone propionate. Preincubation with the steroids did not inhibit cytokine release more than simultaneous incubation with stimulus and steroid. In conclusion, budesonide and fluticasone propionate, in concentrations that probably occur in the airway lining fluid during inhalational therapy, inhibited cytokine release from human lung epithelial cells (IL-6, IL-8) and alveolar macrophages (TNF-alpha, IL-6, IL-8). In vitro, the onset of this effect was rapid.     

Haemophilus influenzae and Streptococcus pneumoniae induce different intracerebral mRNA cytokine patterns during the course of experimental bacterial meningitis

Using in situ hybridization with radiolabelled oligonucleotide probes, we studied the mRNA expression of IL-1β, IL-4, IL-6, IL-10, IL-12, tumour necrosis factor-alpha (TNF-α), TNF-β, interferon-gamma (IFN-γ), and transforming growth factor-beta (TGF-β) in the brain during the lethal course of experimental meningitis in a rat model inoculated intracisternally with Haemophilus influenzae type b (Hib) or Streptococcus pneumoniae and in uninfected control rats inoculated with the same volume of PBS. The production of IL-1β, IL-4, IL-6 and IFN-γ was also evaluated by immunohistochemistry. In the brain of Hib-inoculated rats, there was marked mRNA expression of IL-1β, IL-6, TNF-α, IL-12 and IFN-γ. IL-1β, IL-6 and TNF-α were up-regulated throughout the observation period at 2, 8 and 18 h post-inoculation (p.i.), with similar patterns of induction. The Th1 cytokines IFN-γ and TNF-β were up-regulated within 8 h p.i. IL-10 and TGF-β were down-regulated at 18 h p.i., while IL-4 was not detected. In contrast, the brain of S. pneumoniae-inoculated rats showed lower levels of IL-1β, IL-6 and TNF-α, but higher levels of TNF-β and detectable mRNA expression of IL-4 when compared with Hib-inoculated rats. IL-12, IFN-γ, IL-10 and TGF-β exhibited similar patterns of induction in the brains of Hib- and S. pneumoniae-inoculated rats. At 18 h p.i., immunohistochemistry showed similar patterns of IL-1β, IL-4, IL-6 and IFN-γ as mRNA expression in the brains of Hib- and S. pneumoniae-inoculated rats. The differences of cytokine profiles induced by the two bacterial strains may imply that different immunomodulating approaches should be considered, depending on etiology.     

Natural killer, natural killer T, helper and cytotoxic T cells in the decidua from sporadic miscarriage

PROBLEM: The aim of this cohort study was to assess natural killer (NK) cell and natural killer T (NKT) cell populations and cytokine expressions of helper T (Th) and cytotoxic T (Tc) cells in the decidua of sporadic miscarriage (MS) and induced abortion (IA). METHODS: The deciduae were obtained from consecutive 40 women whose pregnancies ended in the first trimester MS, and the fetal chromosome karyotypes were analyzed. The cell populations were measured by flow cytometry. RESULTS: No significant differences in NK cell or NKT cell percentages were found among MS with normal chromosome karyotype (MSNK, n = 14), MS with abnormal karyotype (MSAK, n = 26) and IA (n = 14). Interferon (IFN)-gamma(+) cell percentage and interleukin (IL)-4(+)/tumor necrosis factor (TNF)-alpha(+) ratio of Th cells in MS groups were increased, while IL-4(+) cell percentage, IL-4(+)/IFN-gamma(+) and IL-4(+)/TNF-alpha(+) ratios of Tc cells in MS groups were decreased as compared with those in IA. No significant difference in these parameters between MSNK and MSAK was found. CONCLUSIONS: These results suggested that Tc1 predominance existed in the decidua of MS. Th1 predominance was not found in MSNK.     

CpG-ODN诱导白血病HL60细胞分化和凋亡的研究

目的:研究含CpG基序的寡核苷酸对白血病HL60细胞的作用。方法:设计合成含CpG基序的寡核苷酸(CpG-ODN)、不含CpG基序的寡核苷酸(nonCpG-ODN)和含甲基化CpG基序的寡核苷酸(ZpG-ODN)分别作用于白血病HL60细胞,MTT法测定HL60细胞抑制效应,硝基四氮唑蓝还原实验和细胞表面CD14分子表达状况分析HL60细胞诱导分化结果,流式细胞仪和透射电镜观察HL60细胞的凋亡现象,免疫组化检测HL60细胞后caspase3、Bcl-2和Bax的表达状况。结果:CpG-ODN对白血病HL60细胞有诱导分化和诱导凋亡作用,nonCpG-ODN和ZpG-ODN无此作用。结论:CpG-ODN可直接诱导白血病HL60细胞分化和凋亡,此为白血病免疫治疗提供了新的途径。     

Isolates of the Enterobacter cloacae complex induce apoptosis of human intestinal epithelial cells

Strains of the Enterobacter cloacae complex are becoming increasingly important human pathogen. The aim of the study was to identify, by sequencing the hsp60 gene, the species of clinical isolates phenotypically identified as E. cloacae and to examine them for virulence-associated properties: the ability of adhesion, invasion to HEp-2 cells and the induced apoptosis of infected epithelial cells. The majority of the strains were identified as Enterobacter hormaechei with E. hormaechei subsp. steigerwaltii being the most frequent subspecies. Other strains belonged to E. hormaechei subsp. oharae, E. cloacae cluster III, and E. cloacae cluster IV. The strains were examined for virulence-associated properties: the ability to adhesion and invasion to HEp-2 cells and the apoptosis induction of infected epithelial cells. All strains revealed adherence ability and most of them (71%) were invasive to epithelial cells. Analyses of cellular morphology and DNA fragmentation in the HEp-2 cells exhibited typical features of cells undergoing apoptosis. We observed morphological changes, including condensation of nuclear chromatin, formation of apoptotic bodies and blebbing of cell membrane. The lowest apoptotic index did not exceed 6%, whereas the highest reached 49% at 24 h and 98% at 48 h after infection. Forty strains (73%) induced fragmentation of nuclear DNA and characteristic intranucleosomal pattern with the size of about 180–200 bp in DNA extracted from infected cells at 48 h after infection. The results indicated that the bacteria of the E. cloacae complex may adhere to and penetrate into epithelial cells and induce apoptosis, which could be an important mechanism contributing to the development diseases.     

Short-chain fatty acids induce pro-inflammatory cytokine production alone and in combination with toll-like receptor ligands

Citation Mirmonsef P, Zariffard MR, Gilbert D, Makinde H, Landay, AL, Spear GT. Short‐chain fatty acids induce pro‐inflammatory cytokine production alone and in combination with Toll‐like receptor ligands. Am J Reprod Immunol 2012; 67: 391–400 Problem Short‐chain fatty acids (SCFAs), produced at relatively high levels by anaerobic bacteria in bacterial vaginosis (BV), are believed to be anti‐inflammatory. BV, a common alteration in the genital microbiota associated with increased susceptibility to HIV infection, is characterized by increased levels of both pro‐inflammatory cytokines and SCFAs. We investigated how SCFAs alone or together with Toll‐like receptor (TLR) ligands affected pro‐inflammatory cytokine secretion. Method of study Cytokines were measured by ELISA. Flow was used for phenotyping and reactive oxygen species (ROS) measurement. Results Short‐chain fatty acids, at 20 mm , induced interleukin (IL)‐8, IL‐6, and IL‐1β release, while lower levels (0.02–2 mm ) did not induce cytokine secretion. Levels >20 mm were toxic to cells. Interestingly, lower levels of SCFAs significantly enhanced TLR2 ligand‐ and TLR7 ligand‐induced production of IL‐8 and TNFα in a time‐ and dose‐dependent manner, but had little effect on lipopolysaccharide‐induced cytokine release. SCFAs mediated their effects on pro‐inflammatory cytokine production at least in part by inducing the generation of ROS. Conclusion Our data suggest that SCFAs, especially when combined with specific TLR ligands, contribute to a pro‐inflammatory milieu in the lower genital tract and help further our understanding of how BV affects susceptibility to microbial infections.     

Soluble HLA-A,-B,-C and -G molecules induce apoptosis in T and NK CD8+ cells and inhibit cytotoxic T cell activity through CD8 ligation

There is convincing evidence that soluble HLA-A,-B,-C (sHLA-A,-B,-C) and soluble HLA-G (sHLA-G) antigens can induce apoptosis in CD8(+) activated T cells although there is scanty and conflicting information about the mechanism(s) by which sHLA-A,-B,-C antigens and sHLA-G antigens induce apoptosis. In this study we have compared the apoptosis-inducing ability of sHLA-A,-B,-C antigens with that of sHLA-G1 antigens in CD8(+) T lymphocytes and CD8(+) NK cells. Furthermore we have compared the inhibitory effect of sHLA-A,-B,-C antigens and of sHLA-G1 antigens on the activity of EBV-specific CD8(+) cytotoxic T lymphocytes (CTL). sHLA molecules were purified from serum and from the supernatant of HLA class I-negative cells transfected with one gene encoding either classical or non-classical HLA class I antigens. Both classical and non-classical sHLA class I molecules trigger apoptosis in CD8(+) T lymphocytes and in CD8(+) NK cells, which lack the T cell receptor, and their apoptotic potency is comparable. The binding of sHLA-A,-B,-C and sHLA-G1 molecules to CD8 leads to Fas ligand (FasL) up-regulation, soluble FasL (sFasL) secretion and CD8(+) cell apoptosis by Fas/sFasL interaction. Moreover, classical and non-classical sHLA class I molecules inhibit the cytotoxic activity of EBV-specific CD8(+) CTL. As the amount ofsHLA-G molecules detectable in normal serum is significantly lower than that of sHLA-A,-B,-C molecules, the immunomodulatory effects of sHLA class I molecules purified from serum are likely to be mainly attributable to classical HLA class I antigens. As far as the potential in vivo relevance of these findings is concerned, we suggest that classical sHLA class I molecules may play a major immunoregulatory role in clinical situations characterized by activation of the immune system and elevated sHLA-A,-B,-C serum levels. In contrast, non-classical HLA class I molecules may exert immunomodulatory effects in particular conditions characterized by elevated sHLA-G levels such as pregnancy and some neoplastic diseases.     

4-羟基壬烯醛诱导支气管上皮细胞凋亡

目的探讨4-羟基壬烯醛(4-HNE)对支气管上皮细胞(16-HBE)凋亡的诱导作用及其机制。方法将支气管上皮细胞(16-HBE)分为空白对照组、10μmol/L、30μmol/L、50μmol/L4-HNE作用4h组,观察4-HNE作用4h后的细胞凋亡情况。Western印迹方法检测磷酸化(p-)SAPK-JNK和caspase-3蛋白表达的情况。结果细胞经30μmol/L、50μmol/L4-HNE作用后,姬姆萨染色可见明显细胞凋亡改变。对照组、10μmol/L、30μmol/L、50μmol/L4-HNE组的AnnexinV-PI染色凋亡细胞数分别为1.94±1.03,2.03±1.04,43.36±1.3,65.92±3.45。30μmol/L和50μmol/L4-HNE组与对照组及10μmol/L4-HNE组比较,差异有统计学意义(P<0.01)。各组p-SAPK-JNK蛋白表达差异无统计学意义(P>0.05)。30μmol/L、50μmol/L4-HNE刺激组caspase-3的表达较对照组和10μmol/L4-HNE组差异有统计学意义(P<0.01)。结论30、50μmol/L4-HNE可通过caspase-3的激活引起支气管上皮细胞的凋亡。     

Inhibition of apoptosis in Bacteroides fragilis enterotoxin-stimulated intestinal epithelial cells through the induction of c-IAP-2

Enterotoxigenic Bacteroides fragilis produces an approximately 20-kDa heat-labile enterotoxin (BFT) that plays an essential role in generating mucosal inflammation. Although it is well known that proinflammatory signals are expressed in BFT-stimulated intestinal epithelial cells, cell death processes have not been elucidated. BFT induced apoptosis in HT-29 cells, but the apoptosis was first apparent 36 h after stimulation. During the early period of BFT stimulation, expression of cellular inhibitor of apoptosis protein-2 (c-IAP2) increased, and inhibition of c-IAP2 augmented the apoptotic cell death. Inhibition of BFT-induced COX-2 expression decreased prostaglandin E(2) (PGE(2)) production, which led not only to a decrease of c-IAP2 activity but also to an enhancement of DNA fragmentation in the early period of BFT stimulation. Furthermore, apoptosis inhibition through PGE(2) and c-IAP2 was mainly regulated by a p38 mitogen-activated protein kinase (MAPK). These results suggest that the inhibition of apoptosis may be mediated by a sequential pathway, including MAPK, COX-2, PGE(2) and c-IAP2, in the early period of stimulation. The delay in the onset of epithelial cell apoptosis after enterotoxigenic B. fragilis infection may be important to the host since it can provides sufficient time for epithelial cells to generate signals for the activation of mucosal inflammation and it may increase the chances of bacterial colonization.     

Inhibition of mediator and cytokine release from dispersed nasal polyp cells by terfenadine

The mechanism of action of H₁-blockers requires elucidation because they may possess properties unrelated to the blockage of histamine at its receptor level. A study was performed with enzymatically dispersed cells obtained from nasal polyps to examine the effect of terfenadine (0.1–10 μmol) on the release of leukotrienes (LT) (LTC₄/D₄ and LTB₄) after stimulation by anti-IgE, and on the spontaneous release of cytokines (granulocyte/macrophage-colony stimulating factor [GM-CSF] and tumor necrosis factor-alpha [TNF-α]) released from cells cultured for 6 h. Terfenadine inhibited significantly, and in a dose-dependent manner, the release of LTC₄/D₄, LTB₄, TNF-α, and GM-CSF. IC₅₀ values were determined for LTC₄/D₄ (8 μmol), LTB₄ (9.9 μmol), TNF-α (6.1 μmol), and GM-CSF (4 μmol). Terfenadine was found to possess new antiallergic properties with a novel in vitro model which mimics more closely inflammatory cells of allergic rhinitis or asthma.     

卵巢上皮性肿瘤神经内分泌细胞增殖与凋亡研究

目的： 观察卵巢上皮性肿瘤组织中神经内分泌细胞（NECs）的形态特点及增殖与凋亡状况，以探讨其生物学及临床意义。 方法： 手术切除的卵巢上皮性肿瘤标本79例，其中良性20例，交界性18例，恶性41例，正常卵巢组织22例作对照，采用嗜铬素A（CgA）免疫组化染色显示NECs，并进行CgA/Ki67及TUNEL/CgA双重染色，观察NECs的增殖与凋亡状况。 结果： 卵巢上皮性肿瘤组织NECs的阳性率、分布范围及染色强度均高于正常卵巢组织。卵巢上皮性肿瘤中NECs形态多样，具有神经元样突起，延伸到毗邻细胞或基底膜，NECs之间亦有突起相互接触。双重免疫组化染色显示NECs均呈TUNEL染色阴性，部分细胞Ki67染色阳性。 结论： 卵巢上皮性肿瘤组织中NECs与肿瘤细胞一样可增殖，但不发生凋亡，其分泌产物可促进周围非NECs的增殖，抑制其凋亡。     

Natural killer cells and T cells induce different types of skin reactions during recall responses to haptens

The role of T cells in contact hypersensitivity (CHS) to haptens has been well described. However, recent reports demonstrated that CHS-like reactions to experimental haptens could be induced in mice deficient in T cells and B cells, as a result of adaptive-like features of NK cells. Here, we compared hapten-specific inflammatory reactions induced by memory T cells or NK cells. Classical CHS protocols were applied to WT or T- and B-cell deficient mice. Adoptive transfers of hapten-specific T cells and NK cells were also performed. Liver NK cells from hapten-primed mice induced specific recall responses to haptens upon transfer in CD3ε-deficient mice, thus confirming the existence of "memory" NK cells in the liver. We investigated the nature of the inflammation generated in these transfer conditions and found that hapten-induced skin inflammation mediated by CD8(+) T cells or "memory" NK cells are different. Indeed, ear swelling induced by memory NK cells was transient and not associated with cellular infiltrate and inflammation markers, characteristic for T-cell-mediated responses. Thus, NK cells and T cells mediate distinct forms of skin inflammation. NK cell-mediated pathogenesis does not rely on cellular infiltrate and could be involved in atypical forms of adverse drug reactions.     

IL-4 influences the differentiation and the susceptibility to activation-induced cell death of human naive CD8+ T cells

It is now well established that the cytokine environment influences the activation, differentiation, proliferation and death of T lymphocytes during the primary response to antigen. Using an in vitro model, we investigated the influence of IL-4, added at the onset of TCR stimulation, on phenotypic and functional markers of naive CD8+ T cell activation including the up-regulation of activation markers, proliferation as well as the susceptibility to activation-induced cell death (AICD). We report that IL-4, unlike IL-2 added at the onset of repeated TCR stimulation of naive CD8+ T cells prevents AICD, in part due to its ability to maintain the level of the survival-related protein Bcl-2. Moreover, TCR-triggered activation of naive CD8+ T cells in the presence of IL-4 leads to the development of a CD8+ T cell subset that proliferates normally, but which fails to exhibit characteristic activation parameters such as the up-regulation of CD25 and Granzyme B. Taken together, these results demonstrate that exposure to IL-4 during primary activation influences CD8+ T cell differentiation by inducing the development of a sub-population of AICD-resistant, proliferation-competent cells that do not show some of the typical features of CD8+ T cell activation.     

The effect of interleukin (IL)‐21 and CD4+CD25++ T cells on cytokine production of CD4+ responder T cells in patients with myasthenia gravis

Impairment of the suppressive function of regulatory T (T_reg) cells has been reported in myasthenia gravis (MG). In this study, cytokine‐related mechanisms that may lead to the defect of T_reg were investigated in patients with anti‐acetylcholine receptor antibody‐positive MG (AChR + MG). Proliferation and cytokine production of responder T (T_resp) cells in response to polyclonal activation were measured in a suppression assay. The effect of interleukin (IL)‐21 on suppression was evaluated in vitro in co‐culture. IL‐21 increased the proliferation of T_resp cells in T_resp/T_reg co‐cultures. T_resp cells from patients with MG secreted significantly lower levels of IL‐2. In patients with MG, IL‐2 levels did not change with the addition of T_reg to cultures, whereas it decreased significantly in controls. In T_resp/T_reg co‐cultures, IL‐4, IL‐6 and IL‐10 production increased in the presence of T_reg in patients. Interferon (IFN)‐γ was decreased, whereas IL‐17A was increased in both patient and control groups. IL‐21 inhibited the secretion of IL‐4 in MG and healthy controls (HC), and IL‐17A in HC only. The results demonstrated that IL‐21 enhances the proliferation of T_resp cells in the presence of T_reg. An effect of IL‐21 mainly on T_resp cells through IL‐2 is implicated.