The effect of yoghurt on the cytotoxic and phagocytic activity of macrophages in tumour‐bearing mice

In a previous paper, it was demonstrated that feeding yoghurt was able to inhibit the growth of an intestinal tumour induced chemically with 1,2‐dimethylhydrazine (DMH). This effect was due to the increase in IgA‐producing cells and a diminution of the inflammatory immune response. In this paper the phagocytic and cytotoxic capacity of macrophages both involved and not involved in the target organ are studied. The study was aimed at determining whether in the intestinal tumour inhibition demonstrated previously the systemic immune response was also increased. The cytotoxic capacity and ß‐glucuronidase enzyme levels of the peritoneal macrophages were analyzed together with the cytolytic effect of the serum on tumour cells and the phagocytic activity of the macrophages infiltrating the intestinal mucosa. Groups of mice were split into three experimental groups. One group was treated with DMH. The others were treated with DMH, and their diets were supplemented with yoghurt for 7 or 10 consecutive days, during 24 weeks. It was demonstrated that feeding yoghurt for 7 or 10 days increased cytotoxic and ß‐glucuronidase levels in peritoneal macrophages, and also the cytolytic capacity of serum, reaching values significantly higher than those in the DMH control. Enhancement of the phagocytic activity of the macrophages associated with the large intestine was also observed. This increase in the macrophage activity involved in the systemic and mucosal immune responses could also be responsible for the tumour inhibition observed in the group of mice fed with yoghurt. The presence in the serum of lytic factors (cytokines) which were released by immune cells activated by feeding yoghurt may also have had a role in tumour inhibition.     

Control of dual-specificity phosphatase-1 expression in activated macrophages by IL-10

Ligation of Toll-like receptors (TLR) on macrophages induces cytokines and mediators important for the control of pathogens. Macrophage activation has to be tightly controlled to prevent hyper-inflammation. Accordingly, the hallmarks of TLR-triggered signaling, nuclear translocation of NF-kappaB and phosphorylation of mitogen-activated protein kinases (MAPK), are transient events. We have mined microarray datasets for changes in the expression of phosphatases in resting and TLR-activated macrophages. Several members of the dual-specificity phosphatases (DUSP) were induced upon triggering TLR4 with LPS. Up-regulation of DUSP1 mRNA was transient after stimulation with LPS alone, but addition of the immunosuppressive cytokine IL-10 resulted in robust, continued DUSP1 expression. IL-10 also synergized with the anti-inflammatory glucocorticoid dexamethasone in the induction of DUSP1 mRNA expression in activated macrophages, as well as in the inhibition of IL-6 and IL-12 production. Increased expression of DUSP1 in IL-10-treated activated macrophages was correlated with a faster down-regulation of p38 MAPK activation. Thus, these data suggest an operational link between IL-10 and inhibition of p38 MAPK via sustained expression of DUSP1.     

IL─10与IL─1ra对大鼠腹腔Mφ诱生细胞因子的影响

通过建立大鼠佐剂性关节炎（ＡＡ）动物模型，研究了ＩＬ－１０和ＩＬ－１ｒａ对正常及ＡＡ大鼠腹腔Ｍφ分泌细胞因子的调节作用，并利用分子杂交技术在基因水平上对其作用机制进行了初步探讨。结果表明：ＡＡ大鼠腹腔Ｍφ呈高度活化状态，产生大量炎症细胞因子ＩＬ－１、ＩＬ－６、ＩＬ－８和ＴＮＦ等，ＩＬ－１０和ＩＬ－１ｒａ对正常及ＡＡ大鼠腹腔Ｍφ细胞因子的产生均呈显著抑制作用，并呈剂量依赖关系。ＩＬ－１０和ＩＬ－１ｒａ具有协同抑制作用。进一步研究发现ＩＬ－１０和ＩＬ－１ｒａ能抑制ＬＰＳ诱导的腹腔ＭφＩＬ－８ｍＲＮＡ表达，表明其抑制作用发生在基因水平上。提示ＩＬ－１０和ＩＬ－１ｒａ是有效的免疫抑制剂，从而为其临床抗炎应用提供了重要的理论和实验依据     

紫杉醇对骨髓细胞体外诱导分化的巨噬细胞影响

目的:研究紫杉醇对小鼠骨髓分化巨噬细胞的直接影响。方法:常规方法无菌制备BALB/c小鼠骨髓细胞,用含M-CSF的RPMI1640培养液培养7d,同时加入不同浓度紫杉醇,通过流式细胞术对骨髓单核细胞分化的巨噬细胞的表型分子、吞噬功能进行测定,采用迟发型过敏反应(DTH)方法检测巨噬细胞免疫原性。结果:紫杉醇明显降低骨髓单核细胞分化成巨噬细胞的数量;F4/80 巨噬细胞表面分子CD80、CD14表达升高,而I-Ad表达降低;紫杉醇提高分化的巨噬细胞吞噬鸡红细胞的能力;但使其免疫原性降低。结论:紫杉醇能使骨髓单核细胞分化的巨噬细胞细胞吞噬能力提高,但其免疫原性下降,提示紫杉醇可能具有调节巨噬细胞免疫功能的作用。     

Differential signaling of cmvIL‐10 through common variants of the IL‐10 receptor 1

Human IL‐10 (hIL‐10) signaling is mediated by receptors consisting of two subunits, IL‐10 receptor 1 (IL‐10R1) and IL‐10 receptor 2. Two common variants of the IL‐10R1 (Ser 138 Gly (single‐nucleotide polymorphism 3, SNP3) and Gly 330 Arg (SNP4)) are associated with diverse disease phenotypes. Viral homologs to hIL‐10, such as cmvIL‐10, utilize the same IL‐10 receptor complex as part of viral immune evasion strategies. For the present study we hypothesized that IL‐10R1 variants alter the ability of viral IL‐10 to utilize the IL‐10R1 signaling pathway. HeLa cell clones expressing different IL‐10R1 haplotypes (WT or any variant) were incubated with hIL‐10 or cmvIL‐10. In cells expressing IL‐10R1‐WT, cmvIL‐10 (both non‐glycosylated‐ and HeLa‐expressed) resulted in equal or slightly stronger STAT3 phosphorylation compared with hIL‐10. In clones expressing IL‐10R1‐SNP3, IL‐10R1‐SNP4 or IL‐10R1‐SNP3+4, the cmvIL‐10 showed significantly less STAT3 phosphorylation, especially when HeLa‐expressed cytokines were used. Time course experiments demonstrated a slower kinetic of cmvIL‐10 STAT3 activation through the variant IL‐10R1. Similarly, IL‐10R1 variants decreased the cmvIL‐10‐induced SOCS3 and signaling lymphocytic activation molecule mRNA expression. These data suggest that the IL‐10R1 variants differentially reduce the signaling activity of cmvIL‐10 and thereby may affect CMV's ability to escape from the host's immune surveillance.     

PD‐1 modulates steady‐state and infection‐induced IL‐10 production in vivo

Programmed death‐1 (PD‐1) plays an important role in mediating immune tolerance through mechanisms that remain unclear. Herein, we investigated whether PD‐1 prevents excessive host tissue damage during infection with the protozoan parasite, Toxoplasma gondii. Surprisingly, our results demonstrate that PD‐1‐deficient mice have increased susceptibility to T. gondii, with increased parasite cyst counts along with reduced type‐1 cytokine responses (IL‐12 and IFN‐γ). PD‐1^?/? DCs showed no cell intrinsic defect in IL‐12 production in vitro. Instead, PD‐1 neutralization via genetic or pharmacological approaches resulted in a striking increase in IL‐10 release, which impaired type‐1‐inflammation during infection. Our results indicate that the absence of PD‐1 increases IL‐10 production even in the absence of infection. Although the possibility that such increased IL‐10 protects against autoimmune damage is speculative, our results show that IL‐10 suppresses the development of protective Th1 immune response after T. gondii infection.     

Lck regulates IL-10 expression in memory-like Th1 cells

The Src family kinase Lck is thought to facilitate Th2 differentiation; however, its role in Th1 cells has not been well explored. Using mice that lack Lck in mature T cells, we find that lck^−/− Th1 skewed cells have normal expression of T‐bet and produce IFN‐γ at WT levels. However, there is a 3‐fold increase in IL‐10 producing cells in the mutant cultures. These cells do not have elevated levels of IL‐4, GATA3, IL‐17 or Foxp3, indicating that they are not Th2, Th17, or Foxp3⁺ T regulatory cells (Treg). Nor do these cells behave in a similar manner as the type 1 Treg. Most of the IL‐10 in the lck^−/− Th1 cultures is derived from the memory/activated subset, as the cytokine profile from Th1 cultures established from purified CD62L⁺ (naïve) cells are similar to WT cells. Furthermore, this IL‐10 expression appears to be dependent on IL‐12 and correlates with elevated c‐Maf. These data highlight a previously unappreciated role for Lck in regulating IL‐10 in Th1 cells.     

Dimeric galectin-1 induces IL-10 production in T-lymphocytes: an important tool in the regulation of the immune response

Galectin-1, a beta-galactoside binding protein that can occur as both a monomer and a homodimer, binds to leucocyte membrane antigens such as CD7, CD43, and CD45, and has immune-regulatory functions in several animal models of autoimmune disease. However, its mechanism of action is only partially understood. In this study, a marked increase in IL-10 mRNA and protein levels was demonstrated in non-activated and activated CD4(+) and CD8(+) T-cells, following treatment with a high concentration (dimeric form), but not a low concentration (monomeric form), of recombinant galectin-1 protein. IL-10 is known to suppress TH1 type immune responses and upregulation of IL-10 may thus contribute to the immune-regulatory function of galectin-1. Galectin-1 was strongly expressed on the endothelial cells of human kidney allografts, suggesting a role in the regulation of immune responses in transplantation. Administration of high concentrations of galectin-1 may be a useful tool in the treatment of T-cell-mediated diseases.     

Modulation of the expression of M150 on macrophages by Th1/Th2 cytokines and co-stimulatory molecules CD40, B7-1, B7-2 and ICAM-1

M150 is an 150-kDa protein associated with the surface of macrophages and is responsible chiefly for the activation of Th1 cells. It is a unique subset of the lysosome-associated membrane protein-1 glycoprotein and its co-stimulatory activity depends on its post-translational modification, which has a distinct glycosylation pattern restricted to macrophages. In the present study, we have observed that M150 is expressed constitutively on peritoneal but not splenic macrophages isolated from mice of different genetic backgrounds: Balb/c, C57BL/6 and C3He. However, M150 was expressed not only on peritoneal but also on splenic macrophages of non-obese diabetic (NOD) mice. Expression on splenic macrophages was induced by culture with lipopolysaccharide (LPS). Expression could also be significantly up-regulated by interferon (IFN)-gamma and granulocyte-macrophage colony stimulating factor (GM-CSF) but was inhibited by interleukin (IL)-10; IL-4 exhibited no effect. Further, cross-linking of B7-2, CD40, ICAM-1 but not B7-1 enhanced the level of M150 significantly. IFN-gamma and GM-CSF acted synergistically with CD40. The significance of these findings is that cytokines IFN-gamma, GM-CSF and IL-10 and the co-stimulatory molecules B7-2, CD40 and ICAM-1 can regulate the expression of M150 on macrophages.     

Activation of murine invariant NKT cells promotes susceptibility to candidiasis by IL‐10 induced modulation of phagocyte antifungal activity

Invariant NKT (iNKT) cells play an important role in a variety of antimicrobial immune responses due to their ability to produce high levels of immune‐modulating cytokines. Here, we investigated the role of iNKT cells in host defense against candidiasis using Jα18‐deficient mice (Jα18^?/?), which lack iNKT cells. Jα18^?/? mice were more resistant to the development of lethal candidiasis than wild‐type (WT) mice. In contrast, treatment of WT mice with the iNKT cell activating ligand α‐galactosylceramide markedly enhanced their mortality after infection with Candida albicans. Serum IL‐10 levels were significantly elevated in WT mice in response to infection with C. albicans. Futhermore, IL‐10 production increased after in vitro coculture of peritoneal macrophages with iNKT cells and C. albicans. The numbers of peritoneal macrophages, the production of IL‐1β and IL‐18, and caspase‐1 activity were also significantly elevated in Jα18^?/? mice after infection with C. albicans. The adoptive transfer of iNKT cells or exogenous administration of IL‐10 into Jα18^?/? reversed susceptibility to candidiasis to the level of WT mice. These results suggest that activation of iNKT cells increases the initial severity of C. albicans infection, most likely mediated by IL‐10 induced modulation of macrophage antifungal activity.     

IL‐10 produced by activated human B cells regulates CD4+ T‐cell activation in vitro

IL‐10‐producing regulatory B cells have been identified in mice and shown to downregulate inflammation, making them potentially important for maintenance of tolerance. In this study, we isolated B cells from human blood and spleen, and showed that after a short period of ex vivo stimulation a number of these cells produced IL‐10. The IL‐10‐producing B cells did not fall within a single clearly defined subpopulation, but were enriched in both the memory (CD27⁺) and the transitional (CD38^high) B‐cell compartments. Combined CpG‐B+anti‐Ig stimulation was the most potent IL‐10 stimulus tested. B cells stimulated in this way inhibited CD4⁺CD25^? T‐cell proliferation in vitro by a partially IL‐10‐dependent mechanism. These findings imply that manipulating IL‐10 production by human B cells could be a useful therapeutic strategy for modulating immune responses in humans.     

Roles of tumour necrosis factor-alpha (TNF-alpha), transforming growth factor-beta (TGF-beta), and IL-10 in the modulation of intercellular adhesion molecule-1 (ICAM-1) expression by macrophages during mycobacterial infection

Profiles of ICAM-1 expression on cultured murine peritoneal macrophages infected with Mycobacterium avium complex (MAC) were examined, with special reference to modulating roles of TNF-alpha, TGF-beta, and IL-10. When macrophages were infected with MAC, ICAM-1 expression, measured by microscopic counting of ICAM-1+ macrophages stained with anti-ICAM-1 antibody, ELISA, and flow cytometric analysis, was rapidly increased, peaking at day 3 (early-phase up-regulation) due to endogenous TNF-alpha, and thereafter gradually declined to the normal level within 1 week or more (late-phase down-regulation). The late-phase ICAM-1 down-regulation was also seen in macrophages phagocytosing heat-killed MAC and those stimulated with lipopolysaccharide but not in macrophages phagocytosing latex beads. ICAM-1 mRNA expression was augmented markedly at day 1 after MAC infection and thereafter decreased. While TNF-alpha and IL-10 production by MAC-infected macrophages was observed during the first 3 days, TGF-beta production was initiated from day 3 and continued until day 14. Exogenously added TGF-beta strongly inhibited the early-phase increase in ICAM-1 expression by infected macrophages, and the blockade of endogenous TGF-beta with anti-TGF-beta antibody markedly inhibited late-phase ICAM-1 down-regulation. Moderate blocking effect was also observed for anti-IL-10 antibody. On the other hand, late-phase ICAM-1 down-regulation was not prevented by the addition of exogenous TNF-alpha. Therefore, TGF-beta and IL-10, especially the former, appear to play active roles in the late-phase down-regulation of ICAM-1 in MAC-infected macrophages during long-term cultivation.     

HIV-1 does not alter in vitro and in vivo IL-10 production by human monocytes and macrophages

The present study analyses the ability of HIV-1 to modulate IL-10 production in cells of monocyte-macrophage lineage cultured in the presence of macrophage colony-stimulating factor (M-CSF). Both monocytes and macrophages spontaneously produced low amount of IL-10. Lipopolysaccharide (LPS) induced a strong IL-10 response in fresh monocytes and in M-CSF-treated macrophages. In contrast, macrophages cultured in the absence of M-CSF exhibited a marked decrease in their susceptibility to LPS stimulation. M-CSF increased the IL-10 response of macrophages to LPS by enhancing both the expression of membrane-bound CD14, the protein that serves as LPS receptor, and the sensibility of CD14-expressing cells to LPS stimulation. Neither spontaneous nor LPS-induced expression of IL-10 was modulated in monocytes and macrophages by infection with eight monocytotropic strains, as demonstrated by ELISA and cytofluorimetric analysis. In contrast, all the HIV-1 strains primed macrophages for an increased IL-6 response to LPS stimulation. To determine whether IL-10 production was associated with in vivo infection, monocytes from AIDS individuals were analysed for IL-10 production. We found that neither spontaneous nor LPS-induced IL-10 production were different between healthy controls and HIV-infected patients. Taken together, these data strongly suggest that HIV-1 infection of monocytes-macrophages does not play a significant role in the regulation of IL-10 in infected patients. This study also emphasizes the role of M-CSF activation in the regulation of the cytokine response in macrophages.     

IgG1 antimycobacterial antibodies can reverse the inhibitory effect of pentoxifylline on tumour necrosis factor alpha (TNF-alpha) secreted by mycobacterial antigen-stimulated adherent cells

Chronic inflammation associated with cachexia, weight loss, fever and arthralgia is the hallmark of advanced mycobacterial diseases. These symptoms are attributed to the chronic stimulation of tumour necrosis factor (TNF)-alpha. Mycobacterial components directly stimulate adherent cells to secrete TNF-alpha. We have shown recently that IgG1 antimycobacterial antibodies play a role in augmenting TNF-alpha in purified protein derivative (PPD)-stimulated adherent cells from non-BCG-vaccinated donors. We now show that IgG1 antibodies can also augment TNF-alpha expression in stimulated adherent cells obtained from BCG-vaccinated donors and this augmentation is not linked to interleukin (IL)-10 secretion. In addition IgG1 antimycobacterial antibodies can reverse the effect of TNF-alpha blockers such as pentoxifylline and thalidomide. These studies therefore have clinical implications for anti-inflammatory drug treatments which are used increasingly to alleviate symptoms associated with chronic inflammation.     

人参皂甙Rb1对小鼠腹腔巨噬细胞体外吞噬及细胞因子和NO分泌的影响

目的:研究人参皂甙Rb1(Ginsenoside Rb1)对小鼠腹腔巨噬细胞体外吞噬及细胞因子和一氧化氮(NO)分泌的影响。方法:分离制备小鼠腹腔巨噬细胞悬液;MTT法检测不同终浓度的人参皂甙Rb1对巨噬细胞的毒性;流式细胞术检测Rb1对巨噬细胞吞噬直径1μm微球的影响;用Griess试剂盒检测Rb1对巨噬细胞NO量的影响;使用流式液相蛋白定量检测技术Cytometric Bead Array(CBA)检测Rb1对LPS刺激巨噬细胞分泌细胞因子的影响。结果:终浓度为5、10、20μmol/L的Rb1对巨噬细胞的吞噬功能具有促进作用,明显抑制LPS诱导的吞噬作用(P<0.05);Rb1能抑制巨噬细胞NO的产生(P<0.01);Rb1能够调节LPS诱导的细胞因子的产生。结论:Rb1对巨噬细胞可能具有双向调节作用,通过抑制LPS信号的转导,调节巨噬细胞因子的分泌,抑制其活化,使NO减少;对于未活化的巨噬细胞,可能通过上调其吞噬功能,增强固有免疫系统来抵御病原体侵入。     

Variability of the inhibition by total immunoglobulin of in vitro autoantibody-mediated erythrophagocytosis by mouse macrophages

Several autoimmune diseases, mainly autoantibody-mediated, are attenuated by infusion of total IgG (IVIg). The efficacy varies widely from one patient to another. Using an experimental model of in vitro phagocytosis of autoantibody-coated erythrocytes by mouse macrophages, we analysed the possible causes for such a variability. Our results indicated that the efficacy of the phagocytosis inhibition depends upon different factors, such as the isotype and the extent of polymerization of the immunoglobulin used for the treatment as well as the genetic background of the mice and the state of macrophage activation that can be influenced by concomitant viral infection. The development of an in vitro assay for the phagocytic activity of macrophages might improve the selection of patients susceptible to benefit from IVIg treatment.