LPS induces rapid IL-10 release by M-CSF-conditioned tolerogenic dendritic cell precursors

Dendritic cells (DC) obtained by culturing myeloid precursors in GM-CSF undergo maturation and induce an efficient T cell response when stimulated with microbial products. DC precursors themselves also recognize microbial products, and it remains unclear how these stimulated DC precursors modulate the immune response. We show here that M-CSF-conditioned human DC precursors responded to LPS, Mycobacteria bovis, and inflammatory cytokines by a rapid and robust production of IL-10, largely superior to that observed with immature DC or monocytes. The endogenous IL-10 restrained the DC precursors from converting into professional APC, as blocking the IL-10 receptor in the presence of LPS resulted in the formation of efficient T cell stimulators. LPS stimulation concomitant with DC differentiation gave rise to immature DC, which were tolerant to a secondary LPS exposure. Furthermore, the LPS-activated DC precursors reduced bystander DC maturation and anti-CD3/CD28-triggered T cell activation. These data suggest that when exposed to inflammatory or microbial signals, M-CSF-conditioned DC precursors can participate in the modulation of inflammation and immune response by rapid release of IL-10.     

Adult bone marrow contains precursors for CD5+ B cells

To determine directly whether B cell precursors of adult origin are capable of generating CD5⁺ B cells, we reconstituted neonatal C3H.SCID mice with adult C57BL/6 bone marrow and analyzed splenic B cells 10 months later. Surface staining and flow cytometry revealed that the B cells were of donor origin and that 30% were CD5⁺. This confirms that in vivo generated CD5⁺ B cells can be adult derived. After anti-IgM (but not lipopolysaccharide) stimulation in vitro, virtually all of the B cells from the bone marrow-reconstituted mice expressed surface CD5. Sequence analysis of expressed V_HDJ_H genes from the CD5⁺ B cells present after anti-IgM stimulation revealed a high frequency of N nucleotide addition in CDR3 regions. The presence of N nucleotides indicates that these sequences were derived from CD5⁺ B cells of adult origin rather than from long-lived fetal precursor B cells present in either the adult bone marrow at the time of transfer or adult spleen. These experiments demonstrate conclusively that adult bone marrow contains precursors for CD5⁺ B cells and that unlike fetal liver-derived precursors these express terminal deoxynucleotidyl transferase.     

Circulating IL-8 and IL-10 in euthyroid sick syndromes following bone marrow transplantation

It is generally agreed that euthyroid sick syndromes (ESS) are associated with an increased production of cytokines. However, there has been scarce data on the relationship thyroid hormone changes and cytokines among the patients undergoing bone marrow transplantation (BMT). Because interleukin-8 (IL-8) has been identified as a potent proinflammatory and interleukin-10 (IL-10) as an antiinflammatory cytokine, we studied the relation between thyroid hormone parameters and these cytokines following BMT. We studied 80 patients undergoing allogeneic BMT. Serum T3 decreased to nadir at post-BMT 3 weeks. Serum T4 was the lowest at the post-BMT 3 months. Serum TSH sharply decreased to nadir at 1 week and gradually recovered. Serum free T4 significantly increased during 3 weeks and then returned to basal level. Mean levels of serum IL-8 significantly increased at 1 week after BMT. Mean levels of serum IL-10 significantly increased until 4 weeks after BMT. No significant correlation was found between serum thyroid hormone parameters and cytokines (IL-8, IL-10) after adjusting steroid doses during the entire study period. In conclusion, ESS developed frequently following allogeneic BMT and cytokine levels were increased in post-BMT patients. However, no significant correlation was found between serum thyroid hormone parameters and these cytokines.     

Induction of IL-10 producing CD4+ T cells with regulatory activities by stimulation with IL-10 gene-modified bone marrow derived dendritic cells

Dendritic cells (DCs) can induce both tolergenic as well as effective immune responses in the lung. Pulmonary DCs producing interleukin (IL)-10 mediated tolerance induced by respiratory exposure to antigen. IL-10 is an important immunosuppressive cytokine, which inhibits maturation and function of DC. To assess whether IL-10 producing DCs can exert the tolergenic effect through the differentiation of regulatory T cells, bone marrow derived DCs were genetically modified by IL-10 expressing adenovirus. IL-10 gene modified DCs (Ad-IL-10-DC) displayed a characteristic phenotype of immature DCs. Here we showed that in vitro repetitive stimulation of naïve DO11·10 CD4⁺ T cells with Ad-IL-10-DCs resulted in a development of IL-10 producing T-cell regulatory cells. These T cells could not proliferate well but also lost their ability to produce interferon-γ upon restimulation with irradiated splenocytes and ovalbumin peptide. Furthermore, in co-culture experiments these T cells inhibited the antigen-driven proliferation of naïve CD4+ T cells in a dose-dependent manner. Our findings demonstrated that IL-10 producing DCs had the potential to induce the differentiation of Tr1-like cells and suggested their therapeutic use.     

Distinct mouse bone marrow macrophage precursors identified by differential expression of ER-MP12 and ER-MP20 antigens

The characterization of early branch points in the differentiation of leukocytes requires identification of precursor cells in the bone marrow. Recently, we produced two monoclonal antibodies, ER-MP12 and ER-MP20, which in two-color flow-cytometric analysis divide the murine bone marrow into six defined subsets. Here we show, using fluorescence-activated cell sorting followed by macrophage colony-stimulating factor-stimulated culture in soft agar, that precursors of the mononuclear phagocyte system reside only within the ER-MP12^hi20^?, ER-MP12⁺20⁺ and ER-MP12^?20^hi bone marrow subsets. Together, these subsets comprise 15% of nucleated bone marrow cells. Furthermore, we provide evidence that the macrophage precursors present in these subsets represent successive stages in a maturation sequence where the most immature ER-MP12^hi20^? cells develop via the ER-MP12⁺20⁺ stage into ER-MP12^?20^hi monocytes.     

ICAM-1 costimulation induces IL-2 but inhibits IL-10 production in superantigen-activated human CD4+ T cells.

We have previously reported that costimulatory pathways including B7-CD28 and lymphocyte function-associated antigen-3 (LFA-3)-CD2 shape distinct activation profiles in human CD4+ T cells. We now show that superantigen (SAg), in combination with intracellular adhesion molecule-1 (ICAM-1) costimulation drives a proliferative response accompanied by high levels of interleukin-2 (IL-2) and moderate levels of interferon-gamma (IFN-gamma) and tumour necrosis factor (TNF). This response profile differs from that observed in B7 or LFA-3 costimulated T cells because our previous results showed that B7-CD28 costimulation was accompanied by high levels of IL-2, IFN-gamma and TNF, whereas LFA-3 was a potent inducer of IFN-gamma and TNF, but had little influence on IL-2 production. The ICAM-1-induced IL-2 production could efficiently be abrogated with monoclonal antibody (mAb) against ICAM-1 or LFA-1, showing that the activation is dependent of a functional ICAM-1-LFA-1 pathway. SAg-induced IL-2, IFN-gamma and TNF were detected in both CD4+ and CD8+ T cells, whereas production of IL-10 was restricted to CD4+ T cells. A major finding in the present study was that ICAM-1 costimulation strongly inhibits IL-10 production in CD4+ T cells. Our data demonstrate that ICAM-1 costimulation is sufficient to induce large amounts of IL-2. The presence of ICAM-1 results in suppression of IL-10 production in T helper (Th) cells, which may favour the development of Th1 and not Th2 T cells.     

Mycobacterium tuberculosis heat-shock protein 70 impairs maturation of dendritic cells from bone marrow precursors, induces interleukin-10 production and inhibits T-cell proliferation in vitro

In different inflammatory disease models, heat-shock proteins (hsp) and hsp-derived peptides have been demonstrated to possess anti-inflammatory properties. While some studies have shown that hsp can directly interact with antigen-presenting cells, others report that bacterial hsp can induce specific T cells with regulatory phenotypes. Effective characterization of the immunomodulatory effects of hsp 70, however, has historically been confounded by lipopolysaccharide (LPS) contamination. In this study, we compared the effects of LPS-free Mycobacterial tuberculosis hsp 70 (TBhsp70) and its possible contaminants on dendritic cells (DC). We demonstrate herein that LPS-free TBhsp70 inhibits murine DC maturation in vitro, while LPS-contaminated TBhsp70 induces DC maturation. Mock recombinant preparations have no effect. In contrast to LPS, TBhsp70 does not induce tumour necrosis factor-alpha production by DC, but interleukin-10. In vivo, only LPS-contaminated TBhsp70 induces up-regulation of CD86 in splenic mature DC. Finally, TBhsp70 inhibited phytohaemagglutinin-induced T-cell proliferation. Our results support the hypothesis that TBhsp70 does not have inflammatory potential, but rather has immunosuppressive properties.     

Alternative activation of macrophage by IL-10.

Macrophage activation by proinflammatory stimuli is suppressed by IL-10. We tested the hypothesis that IL-10 induces an alternative state of macrophage activation rather than solely mediating suppression. We therefore searched for genes the expression of which might be up-rather than downregulated in response to IL-10. Total RNA was obtained from mouse macrophages J774 A.1 before or after stimulation with IL-10 (20 ng/ml). Poly(A)+RNA was isolated in both cases in order to obtain driver and tester mRNA. Subtraction suppression hybridization was performed using the PCR-select cDNA subtraction procedure. After evaluation of the subtraction efficiency the subtracted cDNA library was cloned into pCRII.1. A total of 1,300 clones were obtained. Southern blot hybridization analysis was performed as the first screening step of this total number of clones. 140 (10.7%) were identified as upregulated in response to IL-10. Sequence analyses so far showed perfect or near perfect matches with already known genes for the majority of clones. Our results clearly indicate that IL-10 stimulates the expression of a large number of genes in macrophages. We conclude that IL-10 induces in macrophages a noninflammatory state of reactivity which may serve to contain proinflammatory conditions.     

Differential effects of IL-10 on prostaglandin H synthase-2 expression and prostaglandin E2 biosynthesis between spleen and bone marrow macrophages

Different populations of mononuclear phagocytes (MO) show considerable diversity of cellular function including prostaglandin E2 (PGE2) biosynthesis. Certain bacterial components enhance PGE2 biosynthesis differentially in selected populations of MO. Interleukin (IL)-10 is proposed to inhibit modulation of PGE2 biosynthesis by down-regulating prostaglandin G/H synthase-2 (PGHS-2) expression. To assess whether IL-10 regulates PGE2 biosynthesis and PGHS-2 expression, splenic and bone marrow MO were isolated from IL-10-deficient (IL-10(-/-)), C57Bl/6 [wild-type (WT) control], and Balb/c (comparison control) mice and were treated with lipopolysaccharide (LPS) and/or interferon-gamma (IFN-gamma) as a model of bacterial inflammation. LPS-induced PGHS-2 expression was similar for splenic MO isolated from the three strains of mice. However, PGE2 released by LPS-treated splenic MO was significantly higher in IL-10(-/-) and Balb/c than in WT cells. In the presence of LPS and IFN-gamma, PGHS-2 expression and PGE2 release by IL-10(-/-) and Balb/c splenic MO were enhanced compared with stimulation with LPS alone or IFN-gamma alone. However, there was no significant increase in PGE2 release from WT splenic MO treated with LPS plus IFN-gamma despite increased PGHS-2 expression. In sharp contrast, PGHS-2 expression and PGE2 release by bone marrow MO were greatly enhanced in IL-10(-/-) cells compared with control cells. Our results indicate that IL-10 regulation of MO PGE2 biosynthesis and PGHS-2 expression is compartment-dependent and that PGE2 production is not linked directly to PGHS-2 levels. Furthermore, our findings emphasize strain-specific differences between C57Bl/6 and Balb/c mice, and Balb/c appears more similar to the IL-10(-/-) than to the C57Bl/6 with respect to prostanoid production.     

IL-10 regulation of macrophage VEGF production is dependent on macrophage polarisation and hypoxia

Vascular endothelial growth factor A (VEGF) is critical for vascular remodelling during tissue repair subsequent to inflammation or injury, but under pathological conditions, VEGF induces tissue damaging angiogenesis. Macrophages generate VEGF that supports angiogenesis, when they adapt to their environment and respond with a co-ordinated set of signals to promote or resolve inflammation. Depending on the stimulus, the phenotype of macrophage activation is broadly classified into M1 (NOS2⁺) and M2 (arginase-1⁺). In recent studies, IL-10, an anti-inflammatory cytokine that suppresses the M1 phenotype, has been shown to dampen the angiogenic switch and subsequent neovascularisation. However, as we show here, these effects are context dependent. In this study, we have demonstrated that IL-10 inhibits M1 bone marrow-derived macrophages (BMDMs) VEGF, stimulated by LPS/CGS21680 (adenosine A2A receptor agonist), but does not prevent VEGF production from M2 macrophages stimulated with prostaglandin E2 (PGE2). Furthermore, we show that hypoxic-conditioned BMDM generated VEGF was maintained in the presence of IL-10, but was suppressed when concomitantly stimulated with IFN-γ. Finally, LPS/PGE2 generated an arginase-1⁺ M2 macrophage that in addition to generating VEGF produced significant quantities of IL-10. Under these conditions, neither in IL-10 deficient macrophages nor following IL-10 neutralization was VEGF production affected. Our results indicate IL-10 suppressed M1 but not M2 derived VEGF, and that activation signals determined the influence of IL-10 on VEGF production. Consequently, therapies to suppress macrophage activation that as a result generate IL-10, or utilising IL-10 as a potential anti-angiogenic therapy, may result in a paradoxical support of neovascularisation and thus on-going tissue damage or aberrant repair.     

Intracellular IL-4, IL-10, and IFN-gamma levels of leukemic cells and bone marrow T cells in acute leukemia

The quantitative levels of intracellular cytokines IL-4, IL-10, and IFN-gamma (ie, the number of bound PE-conjugated antibody molecules/cell) of leukemic cells and bone marrow T cells (bmT cells) of acute leukemia patients were analyzed by flow cytometry. One hundred, thirty-one (95 AML, 25 ALL, 11 ABL) patients were studied. The leukemic cell IL-4 level was highest in the monocytic AML group (1735 +/- 1056) and lowest in the dysplastic AML group (960 +/- 545). The IFN-gamma level was highest in the acute promyelocytic leukemia (APL) group (495 +/- 159), and lowest in the ALL group (252 +/- 119). The IL-10 level was not significantly different among the diagnosis groups. In bmT cells, the IL-10 level was highest in the dysplastic AML group (972 +/- 1049) and lowest in the APL group (397 +/- 352). The leukemic cell cytokine levels were lowest and bmT cell cytokine levels were highest in the dysplastic AML group. There were no significant correlations of these cytokine levels with 2-yr survival rate, complete remission (CR) rate, or relapse rate. The cytokine levels of bmT cells at the time of CR became normal and were not different among the diagnosis groups. In summary, leukemic cell and bmT cell cytoplasmic expression profiles of IL-4, IL-10, and IFN-gamma are characteristic for each diagnostic group of acute leukemia patients and the profiles of bmT cells are normal at the time of CR.     

Expression of bone marrow macrophage receptors in cytostatic-induced myelodepression

Expression of macrophage sialoadhesin and erythroblast receptors involved into the hemopoietic islet formation is studied in cyclophosphamide-treated CBA mice. The number of hemopoietic islets and the content of immature granulocytic and erythroid cells were determined. Cyclophosphane reduces the expression of sialoadhesin and erythroblast receptors. It can be hypothesized that the disturbances in granulocyte differentiation result from impaired sialoadhesin-mediated interaction between hemopoietic cells and macrophages. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 126, No. 7, pp. 25–27, July, 1998     

FK506 induces biphasic Ca2+ release from microsomal vesicles of rat pancreatic acinar cells

The effect of the immunosuppressant drug FK506 on microsomal Ca2+ release was investigated in rat pancreatic acinar cells. When FK506 (0.1-200 microM) was added to the microsomal vesicles at a steady state of ATP-dependent 45Ca2+ uptake, FK506 caused a dose-dependent and a biphasic release of 45Ca2+. Almost 10% of total 45Ca2+ uptake was released at FK506 concentrations up to 10 microM (Km=0.47 microM), and 60% of total 45Ca2+ uptake was released at FK506 concentrations over 10 microM (Km=55 microM). Preincubation of the vesicles with cyclic ADP-ribose (cADPR, 0.5 microM) increased the FK506 (< or =10 microM)-induced 45Ca2+ release (Ozawa T, Biochim Biophys Acta 1693: 159-166, 2004). Preincubation with heparin (200 microg/ml) resulted in significant inhibition of the FK506 (30 microM)-induced 45Ca2+ release. Subsequent addition of inositol 1,4,5-trisphosphate (IP3, 5 microM) after FK506 (100 microM)-induced 45Ca2+ release did not cause any release of 45Ca2+. These results indicate that two types of FK506-induced Ca2+ release mechanism operate in the endoplasmic reticulum of rat pancreatic acinar cells: a high-affinity mechanism of Ca2+ release, which involves activation of the ryanodine receptor, and a low-affinity mechanism of Ca2+ release, which involves activation of the IP3 receptor.     

HTA-HSP70BCG对小鼠骨髓树突状细胞的冲击作用

目的 :用榄香烯复合瘤苗抗原 卡介苗热休克蛋白复合物 (HTA HSP70 BCG)冲击正常小鼠骨髓树突状细胞 ,检测其抗原递呈功能。方法 :用HTA HSP70 BCG刺激 ,并与GM CSM和IL 4联合培养的骨髓树突状细胞 ,用MTT法测增殖活性 ,用透射电镜检查形态学改变 ,用流式细胞仪检测FITC labeleddextran的摄取。结果 :与单用HTA或HSP70 BCG刺激的树突状细胞相比 ,HTA HSP70 BCG刺激的树突状细胞增殖指数明显增大 (2 10 7± 0 0 13) ,对脾不粘附细胞的激活作用明显增强 (增殖指数为 1 92 7±0 0 73) ,FITC labeleddextran摄取率明显增高 (达 5 8 6 1% ) ,形态上更趋于成熟。结论 :HTA HSP70 BCG 对树突状细胞具有较强的冲激作用 ,增强其抗原递呈功能。     

转IL—2基因的骨髓基质细胞对骨髓移植后免疫功能重建的促进作用

目的探讨了用逆转录病毒载体转入小鼠IL-2基因的基质细胞系QXMSC1对异基因骨髓移植后免疫功能重建的促进作用.方法将小鼠IL-2cDNA片段连接到逆转录病毒载体PLXSN上,构建重组逆转录病毒载体PL2SN(含小鼠IL-2cDNA).用磷酸钙共沉淀法将PL2SN转入单嗜性包装细胞系CRE,获得G418抵抗细胞株后,以上清感染双嗜性包装细胞系CRIP,G418筛选后获得高滴度的包装细胞系CRIPIL-2.感染NIH3T3细胞测定CRIPIL-2培养上清病毒滴度为3.4×105cfu/ml.感染骨髓基质细胞系QXMSC1(H-2d),G418筛选,有限稀释法得10个单克隆细胞株,选择表达IL-2最高的细胞株为实验用细胞QXMSC1IL-2,用于以后的实验.供体小鼠BALB/c(H-2d)骨髓以抗T细胞单抗anti-Thy1.2加补体去除骨髓中T细胞,受体小鼠C57BL/6(H-2b)经γ射线致死剂量照射后,输入供体骨髓1×107/只,同时输入基质细胞QXMSC1IL-25×105/只.在第30天、60天检测BMT小鼠脾细胞对LPS、ConA的反应,脾细胞产生IL-2的能力,BMT小鼠产生抗体生成细胞(PFC)的能力及产生DTH的能力.流式细胞仪检测了QXMSC1IL-2对BMT小鼠T细胞亚群的影响.结果电泳及酶切鉴定证实构建的PL2SN质粒.QXMSC1IL-2细胞系IL-2的分泌量为857U(1×106*24h).异基因骨髓移植,共输入QXMSC1IL-2能明显增加BMT小鼠脾细胞对LPS、ConA的反应.脾细胞产生IL-2的能力增强.PFC数目增加,DTH反应增强.T细胞亚群中CD4+/CD8+的比例有所恢复.结论转入IL-2的骨髓基质细胞系可促进骨髓移植后免疫功能重建.     

Respiratory syncytial virus induces prostaglandin E2, IL-10 and IL-11 generation in antigen presenting cells

Bronchiolitis caused by respiratory syncytial virus (RSV) infection is a major cause of hospitalization in children under 1 years of age. The disease characteristically does not induce protective immunity. However, a mononuclear peribronchiolar and perivascular infiltrate during RSV infection is suggestive of an immune-mediated pathogenesis. Macrophages and dendritic cells (DCs) play an essential role in the initiation and maintenance of immune response to pathogens. To analyse interactions of RSV and immune cells, human cord blood derived macrophages and dendritic cells were infected with RSV. Both cells were found to be infected with RSV resulting in the activation of macrophages and maturation of dendritic cells as reflected by enhanced expression of several surface antigens. In the next set of experiments, generation of mediators was compared between cells infected with RSV, parainfluenza (PIV3) and influenza virus as well as ultracentrifuged virus free supernatant. Whereas the supernatant did not induce release of mediators, all three live virus infections induced IL-6 production from macrophages and DC. Influenza virus infection induced predominantly IL-12 p75 generation in DC. In contrast, RSV induced strong IL-11 and prostaglandin E2 release from both macrophages and DCs. In addition, RSV but not influenza and parainfluenza virus induced a strong IL-10 generation particularly from macrophages. Since IL-10, IL-11 and PGE2 are known to act immunosuppressive rather than proinflammatory, these mediators might be responsible for the delayed protective RSV specific immune response.