S-O_2-1菌苗诱生干扰素的研究 Ⅱ小鼠脾脏细胞体外诱生

本文表明S-O_2-1菌苗具有较强的诱导小鼠脾细胞产生干扰素的能力。将事先用菌苗免疫的C57BL/6小鼠脾细胞体外培养时,即使不加菌苗也能产生较高滴度的干扰素活性,该活性在免疫后4天制备的脾细胞培养中开始出现,免疫后7天则达高峰(7.47±0.31 log_2U/ml),以后逐渐下降。若培养时添加菌苗,干扰素高峰出现稍晚些,于免疫后10天达高峰,但滴度明显上升(9.10±0.26 log_2U/ml)。另外,C57BL/6正常小鼠的脾细胞(5×10~6细胞/ml)添加菌苗(0.1亿菌/ml)体外培养12小时后可开始测出干扰活性,培养48小时后活性达高峰(8.23±0.30log_2U/ml)。该干扰素具有小鼠Ⅱ-型干扰素相同的特性。我们在实验中还发现,将S-O_2-1菌苗与刀豆素A(Con A)合用诱生的干扰素滴度明显高于两者单独使用时诱生滴度的累加值。     

S-O_2-1细菌核糖体制剂对小鼠淋巴细胞的作用

<正> 我们实验室曾报道了 S-O_2-1细菌核糖体制剂是低毒、高效的免疫增强剂,能激活小鼠的单核巨噬细胞系统,增强小鼠腹腔粘附细胞对S_(180)肿瘤细胞的体外细胞毒作用,还能促进小鼠NK细胞活性,并能在体外诱生Ⅰ型干扰素和抑瘤因子。但关于该核糖体对淋巴细胞的作用还不清楚,本文观察S-O_2-1菌核糖体制剂对淋巴细胞的作用,从中探讨其免疫调节与抑瘤作用机理。     

婴儿白细胞体外诱生干扰素水平的研究

本文观察脐血、婴儿(8～10月龄)及成人静脉血白细胞经不同诱生剂作用后,产生干扰素的水平。发现人出生时诱生α干扰素的水平随诱生剂不同而有所差异,NDV诱导脐血产生的α干扰素效价与成人血相当;但SPA菌体诱导的脐血α干扰素(4.36±2.45log_2u/50μl)明显低于成人者(9.73±1.93log_2u/50μl)。PHA或ConA诱导脐血产生的γ干扰素量都明显低于成人水平。于8～10月龄时SPA菌体诱生α干扰素水平已无异于成人,而PHA或ConA作用下γ干扰素的效价仍逊于成人。这种干扰素,特别是γ干扰素,产生不足可能是新生儿易患严重胞内病原体感染的一个重要的免疫因素。     

S-O_2-1菌苗对小鼠淋巴细胞的作用

动物实验和初步临床应用表明有抑制肿瘤生长作用的S-O_2-1菌苗,在体外能直接刺激小鼠脾细胞淋巴细胞的增殖,并协同增强脾细胞的Con A增殖反应、S-O_2-1菌苗诱导和增强小鼠腹腔粘附细胞的抑制活性,后者表现在直接抑制脾细胞和肠系膜淋巴结细胞对Con A和S-O_2-1菌苗的增殖反应。腹腔转移菌苗活化的腹腔粘附细胞能抑制受体小鼠对SRBC的PFC应答。提示:S-O_2-1菌苗诱导增强巨噬细胞的抑制活性,以致间接抑制淋巴细胞的免疫应答。     

S-O_2-1菌苗对S_(180)肿瘤细胞DNA合成的抑制作用

用小鼠 S_(180)肿瘤细胞作为靶细胞,以~3H-TdR 的掺入抑制百分率作为抗肿瘤活性的指标,在体外培养条件下,S-O_2-1 菌苗能刺激正常小鼠及带瘤小鼠的免疫活性细胞,释放对 S_(180)肿瘤细胞 DNA 合成有明显抑制作用的可溶性物质。实验提示,该类免疫因子的产生能增强免疫细胞对肿瘤细胞的细胞毒效应。同时发现 S-O_2-1菌苗本身对 S_(180)肿瘤细胞还具有直接杀伤作用。     

S-O_2-1菌苗对小鼠免疫细胞的作用

本文探讨 S-O_2-1菌苗对小鼠淋巴细胞和单核吞噬系统的影响。正常小鼠接种菌苗后,脾脏T细胞百分率剧烈下降、胸腺显著缩小。脾脏细胞的抗 SRBC抗体应答和 GVH反应性明显降低。经 ALS处理的免疫低下小鼠接种菌苗后,抗 SRBC应答和GVH反应性的抑制更加明显。这些结果表明该菌苗具有抑制T细胞功能活性。另一方面,接种菌苗的小鼠,无论对无机碳粒还是对鸡红细胞,都显著加快了廓清速率,其腹腔细胞形成 EA花环能力也成倍上升,提示菌苗对单核吞噬系统有强烈刺激作用。以上资料证明,S-O_2-1菌苗的免疫刺激效应不在T细胞方面,而是针对单核吞噬系统。     

在人扁桃体细胞中诱生γ干扰素

本文初步研究了用人扁桃体细胞诱生γ干扰素及其影响因素。试验结果显示:细胞浓度在5×10~7/ml时产生的γ干扰素效价最高;PHA、PWM、ConA等T细胞促分裂剂诱生γ干扰素的适宜浓度范围分别为50～400μg/ml,2.5～40μg/ml,5～80μg/ml;诱生时间均以72小时为宜;2-ME对人γ干扰素的产生没有影响。本文还将人扁桃体细胞和外周血白细胞对4种γ干扰素诱生利(PHA、PWM、ConA、SEA)和3种α干扰素诱生剂(NDV、SPA菌体、CP)的反应进行了比较。     

小鼠腹腔巨噬细胞诱生一氧化氮的实验研究

应用细胞因子,细菌内毒素和甲醛固定的细菌刺激小鼠腹腔巨噬细胞观察其诱生一氧化氮(NO)的能力,结果发现LPS,IFN-γ+IL-2能够诱生NO,IFN-γ和高浓度的M-CSF与LPS具有协同作用。细菌和BCG也具有诱生NO的能力。还观察了巨噬细胞诱生NO的动力学变化。     

类淋巴细胞干扰素的研究——Ⅰ.干扰素诱生条件

在静止培养条件下,对 Namalwa 细胞诱生干扰素的条件进行了研究。先对单项因素进行试验,包括诱生病毒,细胞密度,促进剂处理,制备液种类和制备温度;然后进行多因素正交优选法试验,寻找最适诱生条件。结果表明,Namalwa 细胞密度为4～6x10~6/ml,NDV-F 和仙台病毒诱生干扰素能力良好,如用前者则细胞宜先用1mM 丁酸钠处理48小时,再用 NDV-F 诱生,用仙台病毒诱生则细胞不须先作处理,诱生后加入不含血清的1640液作为制备液,在36℃培养18℃20小时后,离心收取上清,即为粗制干扰素,效价平均10,000～20,000国际单位/ml,最高可达60,000国际单位/ml。     

S-O_2-1菌苗的免疫效应和抗肿瘤活性

本文报告我们自己分离的革兰氏阴性杆菌(S-O_2-1)菌苗对小鼠的免疫效应和抗肿瘤活性的研究。实验证明该菌苗主要作用于单核巨噬细胞系统,还能激发免疫淋巴细胞释放淋巴因子,对移植艾氏腹水癌的小鼠有较高的保护力,并能抑制癌细胞在小鼠腹腔内增殖,表明该菌苗有一定程度的佐剂性能。     

LPS binding protein is important in the airway response to inhaled endotoxin

BACKGROUND: Inhaled endotoxin is a risk factor for asthma exacerbation, and endotoxin inhalation by itself recapitulates many of the classical features of asthma in mice, including reversible airflow obstruction and inflammation, airways hyperresponsiveness, and airway remodeling. OBJECTIVE: Our objective was to determine the importance of LPS binding protein (LBP) in the response to inhaled LPS. METHODS: We challenged LBP-deficient mice (C57BL/6(LBP-/-)) and C57BL/6 mice with inhaled endotoxin for 4 hours, 5 days, or 4 weeks, followed by 3 days of recovery. RESULTS: LBP in the lung was significantly increased in LPS-exposed C57BL/6 mice from all 3 groups. Only LPS-exposed C57BL/6 mice had significantly enhanced airway responsiveness to inhaled methacholine. Total lavage cells in LPS-exposed C57BL/6(LBP-/-) mice were significantly reduced compared with those seen in LPS-exposed C57BL/6 mice; however, the percentage of PMNs was similarly increased in both the C57BL/6 and C57BL/6(LBP-/-) mice. TNF-alpha, IL-1 beta, and IL-6 protein concentrations in whole-lung lavage fluid from C57BL/6(LBP-/-) mice were also significantly reduced when compared with those seen in C57BL/6 mice. In C57BL/6(LBP-/-) mice submucosal cell proliferation was significantly reduced in the 1-week group when compared with that seen in similarly exposed C57BL/6 mice. The 4-week exposed C57BL/6 mice had significantly thickened airway submucosa and significantly increased lavaged TGF-beta(1) protein compared with that seen in C57BL/6(LBP-/-) mice. CONCLUSIONS: These findings indicate that LBP is one of the critical molecules regulating the acute and chronic airway response to inhaled LPS.     

The IL-1 type 1 receptor is required for the development of LPS-induced airways disease

BACKGROUND: The contribution of IL-1beta signaling through the IL-1 type 1 receptor (IL-1R1) to the development of persistent LPS-induced airway disease has not been investigated. OBJECTIVE: To determine the importance of signaling through the IL-1 type 1 receptor in the development of LPS-induced airway disease. METHODS: We exposed IL-1R1-deficient (C57BL/6(IL-1RI-/-)) mice to an aerosol of LPS or filtered air for 1 day, 1 week, or 4 weeks. RESULTS: After 4 weeks of LPS inhalation, C57BL/6(IL-1RI-/-) mice failed to develop significant submucosal thickening, whereas C57BL/6 mice had significantly thickened submucosa in small, medium, and large airways compared with those of unexposed control mice. Cell proliferation in the airways of both the 1-week and 4-week LPS-exposed C57BL/6(IL-1RI-/-) mice was significantly reduced compared with LPS-exposed C57BL/6 mice. mRNA for type III alpha-3 procollagen was significantly elevated over baseline in C57BL/6 yet remained unchanged compared with baseline in C57BL/6(IL-1RI-/-) mice after 1 week or 4 weeks of LPS inhalation. mRNA for tissue inhibitor of metalloprotease 1 in C57BL/6 mice in the 1-week and 4-week groups was significantly elevated over both control mice and C57BL/6(IL-1RI-/-) mice. CONCLUSION: These data support the hypothesis that signaling through the IL-1 receptor modulates extracellular matrix homeostasis in response to inhaled LPS. CLINICAL IMPLICATIONS: Attenuating IL-1R1-mediated signaling might be an effective therapy against the development of airway remodeling in chronic inflammatory diseases.     

Antibody-mediated protection against Cryptococcus neoformans pulmonary infection is dependent on B cells

The pathogenesis of pulmonary Cryptococcus neoformans infection and the efficacy of passive immunoglobulin G1 (IgG1) administration were investigated in B-cell-deficient and C57BL/6J mice. C57BL/6J mice lived longer than B-cell-deficient mice after both intratracheal and intravenous infections. Administration of IgG1 prior to infection prolonged the survival of C57BL/6J mice but had no effect on the survival or numbers of CFU in the lungs of B-cell-deficient mice. C. neoformans infection in B-cell-deficient mice resulted in significantly higher levels of gamma interferon (IFN-gamma), monocyte chemoattractant protein-1 (MCP-1), and macrophage inflammatory protein-1alpha (MIP-1alpha) than in C57BL/6J mice. IgG1 administration reduced IFN-gamma and MCP-1 levels in C57BL/6J mice but not in B-cell-deficient mice. In addition, compared to its effect in C57BL/6J mice, C. neoformans infection in FcRgammaIII-deficient, athymic, and SCID mice significantly increased IFN-gamma and MCP-1 levels. IgG1 administration was associated with reduced IFN-gamma levels in C57BL/6J mice but not in FcRgammaIII-deficient, athymic, and SCID mice. These observations suggest that IgG1-mediated protection in this system is a consequence of alterations in the inflammatory response that translate into less damage to the host without directly reducing the fungal burden. For hosts with impaired immunities, the ineffectiveness of passive antibody (Ab) may reflect an inability to down-regulate inflammation and avoid self-damage. The results indicate an important role for B cells in host defense against C. neoformans infection and demonstrate a surprising dependence of Ab-mediated protection on B cells in this system.     

Spleen cell cytokine secretion in Mycobacterium bovis BCG-infected mice.

Three susceptible mouse strains, i.e., BALB/c (H-2d), C57BL/6 (H-2b), and major histocompatibility complex-congenic BALB.B10 (H-2b), were infected intravenously with 4 x 10(6) CFU of live Mycobacterium bovis BCG and analyzed 4 weeks later for in vitro spleen cell cytokine secretion in response to purified protein derivative (PPD), BCG culture filtrate (CF), BCG cellular extract, total BCG, the purified extracellular 30-32-kDa antigen (the fibronectin-binding antigen 85), or the intracellular 65-kDa heat shock protein. C57BL/6 and BALB.B10 mice produced 5- to 10-fold more gamma interferon and interleukin-2 (IL-2) when stimulated with CF, PPD, and antigen 85 than BALB/c mice did. When stimulated with BCG extract and whole BCG, gamma interferon and IL-2 levels were generally lower and comparable in the three strains. IL-4 was detected in spleen cell culture supernatants from infected BALB/c mice but not from C57BL/6 or BALB.B10 mice. IL-5 could not be detected. C57BL/6 and BALB.B10 spleen cells also produced more tumor necrosis factor alpha and IL-6 after stimulation with PPD and CF than BALB/c cells did. Finally, BCG vaccination generated efficient protective immunity in C57BL/6 and BALB.B10 mice but not in BALB/c mice. These data suggest that secreted mycobacterial CF antigens selectively induce a strong TH1 response in BCG-infected C57BL/6 and BALB.B10 mice, whereas in BALB/c mice this response is partly counterbalanced by TH2 cells.     

Lymphocyte population kinetics during the development of the immune system. B cell persistence and life-span can be determined by the host environment

We have investigated the kinetic behaviour of LPS reactive Bcells during the development of the immune system. By studyingthe persistence of LPS reactive spleen cells transferred fromadult C57BL/6 donor mice into histocompatible C57BL/10 Sc.CrLPS non-responder mice we have confirmed that B cell populationsobtained from adult donor mice decay rapidly after transferinto adult recipients. In contrast, the same cell populationsafter transfer into neonatal reclpients are able to divide andmaintain their numbers for {small tilde}2-5 weeks in the host'sspleen. In fact, comparison of hosts at different ages showthat with the increasing age of the host the fate of donor Bcells evolves to mimic the behaviour observed upon transferinto adult recipients. Kinetic studies of LPS reactive spleencells obtained from newborn (2 weeks old) C57BL/6 donors aftertransfer into adult C57BL/10 Sc.Cr mice have shown that youngdonor cells were able to keep at constant numbers in the adultenvironment for the first {small tilde}10–15 days aftertransfer and to decay afterwards in the host's spleen at thesame rate as observed upon transfer of spleen cells from adultdonors into adult hosts. These studies provide the first evidencefor the different kinetic behaviour of lymphoid B cell populationsin developing and mature immune systems, confirm that cell persistencecorrelates with cell activation and division, and show thatB cell life-span is also dependent on the host environment.     

Listeria monocytogenes infection in caspase-11-deficient mice

Caspase-11 (Cas11) is a cysteine protease involved in programmed cell death and cytokine maturation. Through activation of Cas1 (interleukin-1beta [IL-1beta]-converting enzyme), Cas11 is directly involved in the maturation of IL-1beta and IL-18. Apoptosis is mediated through Cas3. Given the role of apoptosis and cytokine signaling during the innate immune response in intracellular infection, we examined Cas11-deficient (Cas11(-/-)) mice during infection with Listeria monocytogenes. Cas11(-/-) and wild-type C57BL/6 mice were equally susceptible to intravenous infection with L. monocytogenes, resulting in similar bacterial burdens in tissue and similar survival rates. By contrast, enhanced susceptibility was observed in control mice on a mixed genetic 129/C57BL/DBA2 background. Cas11(-/-) and wild-type mice infected with Listeria had similar hepatic microabscess formation in terms of histologic appearance, size, and number. Apoptosis of L. monocytogenes-infected hepatocytes in vivo and in vitro in primary culture was not altered by the absence of Cas11. Serum IL-18 and IL-1beta levels were similar in Cas11(-/-) mice and controls. Endotoxin (lipopolysaccharide [LPS])-challenged Cas11(-/-) mice were deficient in the production of gamma interferon. IL-1beta responses in Cas11(-/-) were normal with intravenous administration of LPS but decreased with intraperitoneal administration. Our findings suggest that Cas11 deficiency does not impair the immune response to infection with L. monocytogenes. Apoptosis and maturation of IL-18 and IL-1beta were normal despite Cas11 deficiency. LPS-induced proinflammatory pathways are altered by the absence of Cas11. While Cas11-mediated Cas1 and Cas3 activation is crucial for cytokine maturation and apoptosis during inflammation, alternative pathways allow normal inflammatory and apoptotic responses during infection with L. monocytogenes.     

Intranasal infection of beige mice with Mycobacterium avium complex: role of neutrophils and natural killer cells.

Beige mice show increased susceptibility to intranasal infection with organisms of the Mycobacterium avium complex (MAC) compared with their immunocompetent congenics, C57BL/6 mice. This increased susceptibility was clear 2 weeks postinfection, before the activation of the specific immune response. T lymphocytes from 4-week infected beige mice, cultured in vitro, produced amounts of gamma interferon similar to those found in cells from C57BL/6 mice. Macrophage activation, as judged by NO production and lysis of the macrophage target P815, occurred in the lungs of beige mice. Despite the inability of bone marrow-derived NK cells from beige mice to lyse NK-susceptible YAC-1 cells, their gamma interferon production was normal. Monoclonal antibody to NK1.1 was used to deplete C57BL/10 mice of lytic activity against YAC-1 cells without exacerbating infection between 2 and 6 weeks of observation, making it unlikely that any deficiency in NK cells was the cause of susceptibility in beige mice. There was a striking influx of neutrophils in the lungs of beige mice compared with C57BL/6. More than half of the MAC organisms appeared associated with the neutrophils of beige mice, while in C57BL/6 mice, most MAC organisms were associated with cells of macrophage/monocyte morphology. Injection of monoclonal antibody specific for neutrophils failed to eliminate those cells from the lungs of beige mice. However, in C57BL/6 mice, neutrophil numbers were reduced by 95% without exacerbating the infection. We conclude that, although neutrophils are not essential to the relative resistance of C57BL/6 mice, the known deficiencies in both neutrophils and macrophages account for the susceptibility of beige mice.     

Interleukin 12 is produced in vivo during endotoxemia and stimulates synthesis of gamma interferon.

Gamma interferon (IFN-gamma) is produced in response to circulating lipopolysaccharide (LPS) and contributes to the lethality of endotoxic shock. To address the cellular source of IFN-gamma production in vivo, T cells and B cells were magnetically purified from C57BL/6 mouse spleens 5 h following endotoxin injection. IFN-gamma RNA was abundant in splenic CD4+ and CD8+ T cells and in a T- and B-cell-depleted population of splenocytes containing 34% NK1.1+ natural killer (NK) cells. Because interleukin 12 (IL-12) is a known inducer of IFN-gamma synthesis by cultured T cells and NK cells, we examined whether IL-12 might be involved in IFN-gamma release during endotoxemia. mRNA encoding the p40 subunit of IL-12 increased markedly in the spleens of C57BL/6 mice at 2 h after LPS injection, whereas p35 IL-12 mRNA was constitutively expressed at all times. Bioactive IL-12 (p70 heterodimer) was detected in mouse serum at 2 to 4 h after LPS injection. Similar results were obtained using a p40 subunit-specific enzyme-linked immunosorbent assay. Endotoxin-insensitive C3H/HeJ mice generated threefold less IL-12 p70 and IFN-gamma at these times than endotoxin-sensitive C3H/HeOuJ mice. Pretreatment of mice with polyclonal anti-mouse IL-12 antibody reduced IFN-gamma levels present at 6 h post-LPS nearly sixfold in three separate experiments. These studies support a role for IL-12 as a proximal stimulator of IFN-gamma release during endotoxemia.     

Influence of age on the production and regulation of interleukin-1 in mice.

The decrease in T-cell proliferation with age is due, in part, to the decline in the production of IL-2. Since IL-1 is needed to trigger IL-2 production, we determined the IL-1 producing capacity of peritoneal macrophages of young (2-4 months) and old (24-26 months) BALB/c and C57BL/6 mice. Mice were stimulated with LPS, and their peritoneal macrophages were obtained 3 days later, purified, and assessed for IL-1 production by coculturing them with splenic T cells at a ratio of 1:5 in the presence of LPS. Supernatants were obtained 4 days later when the PGE2 and IL-2 activities were minimal and IL-1 activity maximal. IL-1 activity was assessed for their ability to augment the proliferative activity of indicator thymocytes in their response to PHA stimulation. The results revealed that (i) IL-1 production by cells of old BALB/c and C57BL/6 mice is reduced to about 40% and 30% that of young mice, respectively; (ii) indomethacin enhances IL-1 production by cells of both young and old mice to the same extent; and (iii) reduction in the IL-1 producing capacity by cells of old mice results from altered activities of both the IL-1 producing peritoneal macrophages and the augmenting T cells.