金针菇子实体多糖FVPB2对小鼠T淋巴细胞和巨噬细胞的免疫调节作用

从金针菇子实体中分离纯化得到均一多糖FVPB2,其分子量为15kDa,是由葡萄糖、半乳糖、岩藻糖和甘露糖组成的吡喃型杂多糖,利用C57BL/6小鼠脾淋巴细胞和骨髓巨噬细胞研究FVPB2对免疫功能的影响,体外免疫实验表明,FVPB2能促进T淋巴细胞激活并分泌肿瘤坏死因子和干扰素γ细胞因子,同时还能够促进巨噬细胞产生一氧化氮,分泌白介素-1β、白介素-6和肿瘤坏死因子-α细胞因子。本研究以首次从金针菇子实体中获得均一多糖FVPB2为研究对象,观察其对T细胞和巨噬细胞的免疫调节活性,研究结果表明其具有良好的免疫调节活性和潜在的生物学功能。     

CD58/LFA-3 and IL-12 provided by activated monocytes are critical in the in vitro expansion of CD56+ T cells

A small proportion of human CD3⁺ T lymphocytes are known to co-express CD56, an antigen usually restricted in its expression to natural killer (NK) cells. Whereas the in vivo function of CD3⁺ CD56⁺ T cells remains unknown, we and others have previously shown that both in vitro and in vivo, these cells can mediate a significantly greater degree of MHC-unrestricted cytotoxicity against a variety of human tumor cells when compared to either CD3⁺ CD56⁻ T cells or lymphokine activated killer (LAK) cells. While the mechanisms regulating the in vivo expansion of CD56⁺ T cells are not known, here we demonstrate the importance of CD2-mediated IL-12-dependent signals in the in vitro expansion of CD56⁺ T cells. Specifically, we show that activated monocytes provide a contact dependent factor (CD58/LFA-3) and a soluble factor (IL-12), both critical for the in vitro expansion of CD56⁺ T cells. The biological and therapeutic implications of these findings are discussed. Received: 4 May 2000 / Accepted: 25 August 2000     

Strategies for improving antitumor activity utilizing IL-2: Preclinical models and analysis of antitumor activity of lymphocytes from patients receiving IL-2

Conclusions Considerable enthusiasm remains for the successful utilization of the immune system for the immunotherapy of human cancers. Immunotherapeutic maneuvers have been able to mediate impressive antitumor responses for some patients with advanced and refractory malignancies. Unfortunately, the number of patients who benefit from current immunotherapies is low, while the toxicity for many of the patients receiving these treatments is high. It is becoming quite clear that the development of successful immunotherapeutic strategies will involve a carefully chosen combination of immunotherapeutic modalities or of immunotherapy combined with either surgery, radiation therapy, or chemotherapy. The use of an IL-2 based regimen which is clinically tolerable and can provide significant immune activation continues to remain central to many of these treatment approaches. Preclinicalin vitro and animal model systems can evaluate promising treatment strategies, including combination approaches. As an effective immunotherapeutic approach will likely require use of a combination of biologically active agents, the scheduling of these therapies may have profound importance both for optimal antitumor responses as well as clinical tolerance.     

Gp130 activation by soluble interleukin-6 receptor/interleukin-6 enhances osteoblastic differentiation of human bone marrow-derived mesenchymal stem cells

Interleukin-6 (IL-6) promotes osteodifferentiation in bone-located progenitors; however, it is not known whether this cytokine affects the differentiation of bone marrow-located osteoprogenitors. To address this issue, we prepared human bone marrow-derived mesenchymal stem cells (MSCs), which were characterized by a cell surface phenotype and multipotential nature. It was observed that in the presence of IL-6, MSCs were not differentiated into the osteogenic lineage, as evidenced by a failure to induce alkaline phosphatase activity, an earlier marker of osteodifferentiation. The lack of effect of IL-6 correlates with the observation that MSCs do not express a membrane-bound or soluble IL-6 receptor (sIL-6R). The incompetence of IL-6 was not reversed by the addition of sIL-6R alone or the sIL-6R/IL-6 complex, as it occurs in other IL-6R-negative cells. However, after MSC osteocommittment by dexamethasone, sIL-6R or the sIL-6R/IL-6 complex enhanced alkaline phosphatase activity. The effect of sIL-6R or sIL-6R/IL-6 proved to be dependent on gp130 availability, which is expressed by MSCs, and involves stat-3 phosphorylation. These data suggest that IL-6R deficiency may represent for bone marrow-located mesenchymal progenitors a sort of protective mechanism to escape the osteogenic effect of IL-6, which is produced by the MSC itself as well as by other marrow stromal cells.     

The roles of IL-12 and IL-23 in CD8 T cell-mediated immunity against Listeria monocytogenes: Insights from a DC vaccination model

Listeria monocytogenes infection induces a strong inflammatory response characterized by the production of IL-12 and IFN-γ and protective immunity against this pathogen is dependent on CD8⁺ T cells (CTL). Recent studies have suggested that these inflammatory cytokines affect the rate of memory CD8⁺ T cell generation as well as the number of short-lived effector cells generated. The role of the closely related cytokine, IL-23, in this response has not been examined. We hypothesized that IL-12 and IL-23 produced by dendritic cells collectively enhance the generation and function of memory cells. To test this hypothesis, we employed a DC vaccination approach. Mice lacking IL-12 and IL-23 were vaccinated with wild-type (WT), IL-12^−/−, or IL-12/23^−/− DC and protection to Lm was monitored. Mice vaccinated with WT and IL-12^−/− DC were resistant to lethal challenge with Lm. Surprisingly, mice vaccinated with IL-12/23^−/− DC exhibited significantly reduced protection when challenged. Protection correlated with the relative size of the memory pools generated. In summary, these data indicate that IL-23 can partially compensate for the lack of IL-12 in the generation protective immunity against Lm.     

T cells in murine lupus: propagation and regulation of disease

MRL/Mp-lpr/lpr mice develop a spontaneous lupus syndrome, including hypergammaglobulinemia, autoantibodies, glomerulonephritis, and lymphadenopathy. To investigate the role of lymphocyte subsets in the pathogenesis of disease, lupus-prone MRL mice deficient in T cells, T cells, or both were generated. Mice deficient in T cells developed a partially penetrant lupus syndrome, characterized by lymphadenopathy, elevated levels of class-switched immunoglobulins, an increased incidence of antinuclear antibodies, and immune deposits in kidneys which progressed to renal insufficiency over time. In comparison to wild type animals, T cell-deficient animals developed an accelerated and exacerbated disease phenotype, characterized by accelerated hypergammaglobulinemia and enhanced autoantibody production and mortality. Repertoire analysis of these latter animals identified polyclonal expansion (V) of CD4+B220-cells. Mice lacking both and T cells failed to generate class-switched autoantibodies and immune complex renal disease. First, these findings demonstrate that murine lupus in the setting of Fas-deficiency does not absolutely require the presence of T cells, and they also suggest that a significant basis for MRL/lpr disease, including renal disease, involves T cell-independent, T cell dependent, polyreactive B cell autoimmunity, upon which T cell-dependent mechanisms aggravate specific autoimmune responses. Second, these data indicate that T cells partake in the regulation of systemic autoimmunity, presumably via their effects on CD4+B220-T cells that provide B cell help. Finally, these results demonstrate that MRL/lpr B cells, despite their intrinsic abnormalities, cannot per se cause tissue injury without T cell help.Abbreviations snRNPs small nuclear ribonucleoprotein particles     

Caspase-1 activity is required for neuronal differentiation of PC12 cells: cross-talk between the caspase and calpain systems

Previously, we have found that caspase-1 activity is increased during myoblast differentiation to myotubes. Here we show that caspase-1 activity is required for PC12 differentiation to neuronal-like cells. Caspase-1 is shown to be activated (by immunoblotting and by assessing activity in cell extracts) in the PC12 cells following the initial stage of differentiation. The inhibition of caspase-1 arrests PC12 cells at an intermediate stage of differentiation and prevents neurite outgrowth in these cells; the inhibition is reversed upon the removal of the inhibitor. Calpastatin (calpain endogenous specific inhibitor, and a known caspase substrate) is diminished at the later stages of PC12 cell differentiation, and diminution is prevented by caspase-1 inhibition. The degradation of fodrin (a known caspase and calpain substrate) is found in the advanced stage of differentiation. Caspase-1 has been implicated in the activation of proinflammatory cytokines, and in cell apoptosis. The involvement of caspase-1 in two distinct differentiation processes (myoblast fusion and neuronal differentiation of PC12 cells) indicates a function for this caspase in differentiation processes, and suggests some common mechanisms underlying caspase roles in such processes.     

Astrocytes as antigen-presenting cells: expression of IL-12/IL-23

Interleukin-12 (IL-12, p70) a heterodimeric cytokine of p40 and p35 subunits, important for Th1-type immune responses, has been attributed a prominent role in multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE). Recently, the related heterodimeric cytokine, IL-23, composed of the same p40 subunit as IL-12 and a unique p19 subunit, was shown to be involved in Th1 responses and EAE. We investigated whether astrocytes and microglia, CNS cells with antigen-presenting cell (APC) function can present antigen to myelin basic protein (MBP)-reactive T cells, and whether this presentation is blocked with antibodies against IL-12/IL-23p40. Interferon (IFN)-gamma-treated APC induced proliferation of MBP-reactive T cells. Anti-IL-12/IL-23p40 antibodies blocked this proliferation. These results support and extend our previous observation that astrocytes and microglia produce IL-12/IL-23p40. Moreover, we show that stimulated astrocytes and microglia produce biologically active IL-12p70. Because IL-12 and IL-23 share p40, we wanted to determine whether astrocytes also express IL-12p35 and IL-23p19, as microglia were already shown to express them. Astrocytes expressed IL-12p35 mRNA constitutively, and IL-23 p19 after stimulation. Thus, astrocytes, under inflammatory conditions, express all subunits of IL-12/IL-23. Their ability to present antigen to encephalitogenic T cells can be blocked by neutralizing anti-IL-12/IL-23p40 antibodies.     

Chloroquine induces activation of nuclear factor-kappaB and subsequent expression of pro-inflammatory cytokines by human astroglial cells

Chloroquine, an antimalarial lysosomotropic base, is known for its anti-inflammatory effects and therefore used for treatment of autoimmune diseases. Given its anti-inflammatory effects, it has been under clinical trials to modify neurodegenerative processes. In this study, we examined whether chloroquine has an anti-inflammatory effect in the CNS by determining the in vitro effects of chloroquine on LPS-induced expression of cytokines by glial cells. We observed that (i) chloroquine augmented LPS-induced expression of pro-inflammatory cytokines such as lymphotoxin (LT)-beta, tumor necrosis factor (TNF)-alpha, interleukin (IL)-1alpha, IL-1beta and IL-6 in human astroglial cells, while the same treatment suppressed LPS-induced expression of cytokines in monocytic and microglial cells; (ii) chloroquine alone induced expression of pro-inflammatory cytokines in a dose- and time-dependent manner in astroglial cells; (iii) other lysosomotropic agents such as ammonium chloride and bafilomycin A1 had minimal effects on cytokine expression; and (iv) chloroquine induced the activation of nuclear factor-kappa B in astroglial cells, which is a required component of chloroquine induction of cytokines. These results suggest that chloroquine may evoke either anti- or pro-inflammatory responses in the CNS depending on the cellular context.     

Specific inhibitor of MEK-mediated cross-talk between ERK and p38 MAPK during differentiation of human osteosarcoma cells

Osteosarcoma is the most common primary malignant bone tumor, accounting for approximately 20% of all primary sarcomas in bone. Although treatment modalities have been improved over the past decades, it is still a tumor with a high mortality rate in children and young adults. Based on histological considerations, osteosarcoma arises from impaired differentiation of these immature cells into more mature types and that correction of this impairment may reduce malignancy and increase the efficiency of chemotherapy. The purpose of this study was to determine the effect of specific inhibitors of MAPK extracellular signaling-regulated kinase (ERK) kinase (MEK) and p38 on the differentiation of human osteosarcoma cell line SaOS-2 cells. We found that PD98059, a specific inhibitor of MEK, inhibited the serum-stimulated proliferation of SaOS-2 cells; whereas SB203580, a specific inhibitor of p38 MAPK, had little effect on it. SB203580 suppressed ALPase activity, gene expression of type I collagen, and expression of ALP and BMP-2 mRNAs; whereas PD98059 upregulated them dose dependently. In addition, immunoblot and immunostaining analysis revealed that phosphorylation of ERK was increased by treatment with SB203580; whereas PD98059 increased the phosphorylation of p38, which implies a seesaw-like balance between ERK and p38 phosphorylation. We suggest that osteosarcoma cell differentiation is regulated by the balance between the activities of the ERK and p38 pathways and that the MEK/ERK pathway negatively regulates osteosarcoma cell differentiation, whereas the p38 pathway does so positively. MEK inhibitor may thus be a good candidate for altering the expression of the osteosarcoma malignant phenotype.     

Cytokine production by dendritic cells genetically engineered to express IL-4: induction of Th2 responses and differential regulation of IL-12 and IL-23 synthesis

Dendritic cells (DCs) retrovirally transduced with IL-4 have recently been shown to inhibit murine collagen-induced arthritis and associated Th1 immune responses in vivo, but the mechanisms that underly these effects are not yet understood. In this report we demonstrate that IL-4-transduced DCs loaded with antigen led to lower T cell production of IFN-gamma, increased production of IL-4, and an attenuated, delayed type hypersensitivity response. We hypothesized that the ability of such DCs to regulate the Th1 immune response in vivo depends in part on their capacity to produce IL-12 and IL-23. Quantitative mRNA analysis revealed that IL-4-transduced DCs stimulated with CD40 ligand expressed higher levels of IL-12p35 mRNA, but lower levels of mRNA for IL-23p19 and the common subunit p40 found in both IL-12 and IL-23, compared with control DCs. These results, which indicate that expression of the IL-12 and IL-23 subunits is differentially regulated in IL-4-transduced DCs, were confirmed by ELISA of the IL-12 and IL-23 heterodimers. Thus, therapeutic suppression of Th1 -mediated autoimmunity (as recently shown in murine collagen-induced arthritis) and induction of Th2 responses in vivo by IL-4-transduced DCs occurs despite their potential to produce increased levels of IL-12, but could reflect, in part, decreased production of IL-23.     

ALK activation induces Shc and FRS2 recruitment: Signaling and phenotypic outcomes in PC12 cells differentiation

Activation of the neuronal receptor tyrosine kinase ALK (anaplastic lymphoma kinase) promoted the neuron-like differentiation of PC12 cells through specific activation of the ERK MAP-kinase pathway. However, the nature of primary signaling events initiated is still poorly documented. Here, we established that Shc and FRS2 adaptors were recruited and phosphorylated following antibody-based ALK activation. We further demonstrated that Shc was recruited to the consensus phosphotyrosine site NPTpY(1507) and FRS2 was likely recruited to a novel non-orthodox phosphotyrosine site within ALK. Finally, we characterized a functional role for Shc and likely FRS2 in ALK-dependant MAP-kinase activation and neuronal differentiation of PC12 cells. These findings hence open attractive perspectives concerning specific characteristics of ALK in the control of the mechanisms driving neuronal differentiation.