Establishment of lymphotoxin beta receptor signaling-dependent cell lines with follicular dendritic cell phenotypes from mouse lymph nodes

Follicular dendritic cells (FDCs) have been shown to play a crucial role in the positive selection of high-affinity B cells that are generated by somatic hypermutation in germinal center (GC). Because of technical difficulties in preparing and maintaining pure FDCs, a role for FDCs in this complicated process has not been fully elucidated. In this study, we established a cell line designated as pFL that retained major FDC phenotypes from a three-dimensional culture of mouse lymph node cells. pFL cells proliferated slowly in response to an agonistic anti-lymphotoxin beta receptor mAb and TNF-alpha. A more rapidly growing clone, named FL-Y, with similar requirements for growth was isolated from a long-term culture of pFL. Analysis of surface markers in these two cell lines by immunostaining, flow cytometry, and DNA microarray revealed the expression of genes, including those of CD21, FcgammaRIIB, lymphotoxin beta receptor, ICAM-1, VCAM-1, IL-6, and C4, which have been shown to be characteristic of FDCs. In addition, B cell-activating factor was expressed in these two cell lines. At the pFL or FL-Y:B cell ratio of 1:100, the cell lines markedly sustained B cell survival and Ab production during 2 wk of culture, while most B cells collapsed within 1 wk in the absence of the FDC-like cells. Interestingly, expression of typical GC markers, Fas and GL-7, was notably augmented in B cells that were cocultured with Th cells on these two cell lines. Thus, pFL and FL-Y cells may be useful for providing insight into the functional role for FDCs in GC.     

一种拮抗HIV-1并靶向DC的融合基因的设计及表达鉴定

艾滋病病毒 (Human immunodeficiency virus,HIV) 通过与靶细胞膜的融合感染宿主细胞,研究表明阻断HIV与受体靶分子的结合可以阻止HIV进入宿主细胞,抑制HIV病毒的感染。设计合成了一个包含CD4和CCR5与HIV-1结合的主要功能结构区,及Flt3-L和Mip-3α分子的融合基因,构建了2个融合基因的真核表达载体pABK-CKR5-CD4/Flt3L-Mip3α (pABK-HIV-MF) 和pABK-CKR5-CD4 (pABK-HIV-MT),在人胚肾293细胞中进行了表达。RT-PCR、细胞免疫荧光技术、ELISA和Western blotting检测结果表明融合基因在真核细胞中获得了正确的表达,这为进一步研究其对于HIV-1的拮抗并靶向树突状细胞 (DC) 清除研究奠定了基础。     

Assessment of the genetic susceptibility of sheep to scrapie by protein misfolding cyclic amplification and comparison with experimental scrapie transmission studies

The susceptibility of sheep to scrapie is influenced mainly by the prion protein polymorphisms A136V, R154H, and Q171R/H. Here we analyzed the ability of protein misfolding cyclic amplification (PMCA) to model the genetic susceptibility of sheep to scrapie. For this purpose, we studied the efficiency of brain homogenates from sheep with different PrP genotypes to support PrP^Sc amplification by PMCA using an ARQ/ARQ scrapie inoculum. The results were then compared with those obtained in vivo using the same sheep breed, genotypes, and scrapie inoculum. Genotypes associated with susceptibility (ARQ/ARQ, ARQ/AHQ, and AHQ/ARH) were able to sustain PrP^Sc amplification in PMCA reactions, while genotypes associated with resistance to scrapie (ARQ/ARR and ARR/ARR) were unable to support the in vitro conversion. The incubation times of the experimental infection were then compared with the in vitro amplification factors. Linear regression analysis showed that the efficiency of in vitro PrP^Sc amplification of the different genotypes was indeed inversely proportional to their incubation times. Finally, the rare ARQK₁₇₆/ARQK₁₇₆ genotype, for which no in vivo data are available, was studied by PMCA. No amplification was obtained, suggesting ARQK₁₇₆/ARQK₁₇₆ as an additional genotype associated with resistance, at least to the isolate tested. Our results indicate a direct correlation between the ability of different PrP genotypes to undergo PrP^C-to-PrP^Sc conversion by PMCA and their in vivo susceptibility and point to PMCA as an alternative to transmission studies and a potential tool to test the susceptibility of numerous sheep PrP genotypes to a variety of prion sources.     

Follicular dendritic cell of the knock-in mouse provides a new bioassay for human prions

Infectious prion diseases initiate infection within lymphoid organs where prion infectivity accumulates during the early stages of peripheral infection. In a mouse-adapted prion infection, an abnormal isoform (PrP(Sc)) of prion protein (PrP) accumulates in follicular dendritic cells within lymphoid organs. Human prions, however, did not cause an accumulation of PrP(Sc) in the wild type mice. Here, we report that knock-in mouse expressing humanized chimeric PrP demonstrated PrP(Sc) accumulations in follicular dendritic cells following human prion infections, including variant Creutzfeldt-Jakob disease. The accumulated PrP(Sc) consisted of recombinant PrP, but not of the inoculated human PrP. These accumulations were detectable in the spleens of all mice examined 30 days post-inoculation. Infectivity of the spleen was also evident. Conversion of humanized PrP in the spleen provides a rapid and sensitive bioassay method to uncover the infectivity of human prions. This model should facilitate the prevention of infectious prion diseases.     

Enhanced inhibition of tissue factor by the extended form of an endothelial cell glycoprotein (an extrinsic pathway inhibitor)

We have identified, in the supernatant medium of cultured endothelial cells, an additional inhibitor of tissue factor that is eluted at higher salt concentrations during heparin-Sepharose chromatography and is a much more potent inhibitor of the activation of the coagulation cascade than the species we studied earlier (Colburn and Buonassisi: In Vitro Cellular and Developmental Biology 24: 1133-1136, 1988). The inhibitor we described earlier has been shown to be functionally and immunologically identical to a rabbit plasma extrinsic pathway inhibitor, EPI (Warn-Cramer et al.: Thrombosis Research 61:515-527, 1991). The new species (molecular mass, 47 kDa) is susceptible to proteolytic cleavage which results in a sharp reduction of its ability to inhibit tissue factor and an increase in electrophoretic mobility compatible with a molecular mass of 45 kDa, the same as that of the inhibitor reported earlier. Based on these data, we suggest that the new inhibitor yields, through proteolytic cleavage of an amino acid sequence of about 25 residues, the N-glycan-sulfated compound previously described.     

Follicular dendritic cell sarcoma of the cervical lymph node diagnosed on fine needle aspiration cytology

Follicular dendritic cell sarcomas (FDCS) are rare tumours of lymph nodes and extranodal tissues which are grouped with the histiocytic and dendritic cell neoplasms. The diagnosis is usually made after thorough clinical and pathological examination with immunohistochemical analysis. Difficulties persist in diagnosing FDCS on cytological preparations. We report herein a case of a 57-year-old female who presented with a right neck mass of 5 months duration. Computed Tomography (CT) imaging of the neck reported a necrotic right level IIb lymph node and asymmetric fullness of the right palatine tonsil. Fine needle aspiration (FNA) biopsy revealed numerous spindle, oval and stellate neoplastic cells, arranged singly and in syncytia with moderate nuclear pleomorphism, vesicular chromatin pattern, and prominent nucleoli, sprinkled with small lymphocytes. The tumour cells were strongly diffusely positive for CD21, CD23, and D2-40 immunostaining on cell bock sections, but were negative for CD1a and CD34, supporting the diagnosis of FDCS. Follow-up surgical pathology on the resection showed histopathological features and an immunohistochemical profile consistent with FDCS.     

Tumor-derived TGF-beta reduces the efficacy of dendritic cell/tumor fusion vaccine

Dendritic cell (DC)-based antitumor vaccine is a novel cancer immunotherapy that is promising for reducing cancer-related mortality. However, results from early clinical trials were suboptimal. A possible explanation is that many tumors secrete immunosuppressive factors such as TGF-beta, which may hamper host immune response to DC vaccine. In this study, we demonstrated that TGF-beta produced by tumors significantly reduced the potency of DC/tumor fusion vaccines. TGF-beta-secreting (CT26-TGF-beta) stable mouse colon cancer cell lines were generated using a retroviral vector expressing TGF-beta. A non-TGF-beta-secreting (CT26-neo) cell line was generated using an empty retroviral vector. The efficacies of DC/tumor fusion vaccines were assessed in vitro and in vivo. DC/CT26-TGF-beta fusion cells failed to induce a strong T cell proliferative response in vitro, mainly due to the effect of TGF-beta on T cell responsiveness rather than DC stimulatory capability. Animals vaccinated with DC/CT26-TGF-beta fusion vaccine had lower tumor-specific CTL activity and had significantly lower survival after tumor challenge as compared with animals immunized with DC/CT26-neo hybrids (45 vs 77%, p < 0.05). Ex vivo exposure of DCs to TGF-beta did not appear to lessen the efficacy of DC vaccine. These data suggest that tumor-derived TGF-beta reduces the efficacy of DC/tumor fusion vaccine via an in vivo mechanism. Neutralization of TGF-beta produced by the fusion cells may enhance the effectiveness of DC-based immunotherapy.     

Reversal of virus-induced systemic shock and respiratory failure by blockade of the lymphotoxin pathway

At present, little is known about the pathogenesis of acute virus-induced shock and pulmonary failure. A chief impediment in understanding the underlying disease mechanisms and developing treatment strategies has been the lack of a suitable animal model. This study describes a mouse model of virus-induced systemic shock and respiratory distress, and shows that blockade of the lymphotoxin beta receptor pathway reverses the disease.     

Cutting Edge: CpG DNA inhibits dendritic cell apoptosis by up-regulating cellular inhibitor of apoptosis proteins through the phosphatidylinositide-3'-OH kinase pathway.

CpG DNA has been recognized as a powerful stimulant of dendritic cells (DCs). In this study, we demonstrate that CpG DNA inhibits spontaneous apoptosis of DCs. CpG DNA up-regulated cellular inhibitor of apoptosis proteins (cIAPs) as well as Bcl-2 and Bcl-x(L), but down-regulated active caspase-3. Although CpG DNA activated p38 mitogen-activated protein kinase, extracellular signal-related kinase, and phosphatidylinositide-3'-OH kinase (PI3K), only the blocking of PI3K inhibited the CpG DNA-induced DC survival. Moreover, while the expression of Bcl-2 and Bcl-x(L) depends on both PI3K and p38 mitogen-activated protein kinase, the up-regulation of cIAPs and the down-regulation of active caspase-3 by CpG DNA require PI3K activation, suggesting PI3K-dependent up-regulation of cIAPs in the antiapoptotic activity of CpG DNA in DCs. This study indicates that CpG DNA provides a survival signal to DCs, which might be one of mechanisms by which bacterial DNA stimulates and maintains the innate immune responses.     

Microsecond unfolding kinetics of sheep prion protein reveals an intermediate that correlates with susceptibility to classical scrapie

The microsecond folding and unfolding kinetics of ovine prion proteins (ovPrP) were measured under various solution conditions. A fragment comprising residues 94–233 of the full-length ovPrP was studied for four variants with differing susceptibilities to classical scrapie in sheep. The observed biexponential unfolding kinetics of ovPrP provides evidence for an intermediate species. However, in contrast to previous results for human PrP, there is no evidence for an intermediate under refolding conditions. Global analysis of the kinetic data, based on a sequential three-state mechanism, quantitatively accounts for all folding and unfolding data as a function of denaturant concentration. The simulations predict that an intermediate accumulates under both folding and unfolding conditions, but is observable only in unfolding experiments because the intermediate is optically indistinguishable from the native state. The relative population of intermediates in two ovPrP variants, both transiently and under destabilizing equilibrium conditions, correlates with their propensities for classical scrapie. The variant susceptible to classical scrapie has a larger population of the intermediate state than the resistant variant. Thus, the susceptible variant should be favored to undergo the PrP^C to PrP^Sc conversion and oligomerization.     

SP600125, an inhibitor of Jnk pathway, reduces viability of relatively resistant cancer cells to doxorubicin

We identified four breast cancer cell lines and one stomach cancer cell line resistant to the cytotoxic effects of doxorubicin (DOX) and examined their sensitivity to other chemotherapeutic agents. SP600125, an inhibitor of the Jnk pathway, reduced the cellular viability of all five DOX-resistant cancer cell lines. Jnk1 siRNA also reduced the viability of the one DOX-resistant cell line in which it was tested. Similar results were produced in an in vivo mouse model, in which the volume of tumors derived from the DOX-resistant cell line was reduced more effectively by treatment with SP600125 than by treatment with DOX, whereas those from a DOX-sensitive cell line were reduced only by DOX treatment. Overall, these results may contribute to the development of chemotherapeutic treatments for patients with DOX-resistant tumors.     

Lung cancer-derived galectin-1 mediates dendritic cell anergy through inhibitor of DNA binding 3/IL-10 signaling pathway

Lung cancer, one of the leading causes of death worldwide, is often associated with a state of immune suppression, but the molecular and functional basis remains enigmatic. Evidence is provided in this paper supporting the role of lung cancer-derived soluble lectin, galectin-1, as a culprit in dendritic cell (DC) anergy. We have shown that galectin-1 is highly expressed in lung cancer cell lines, together with the serum and surgical samples from lung cancer patients. Functionally, lung cancer-derived galectin-1 has been shown to alter the phenotypes of monocyte-derived DCs (MdDCs) and impair alloreactive T cell response, concomitant with the increase of CD4(+)CD25(+)FOXP3(+) regulatory T cells. The regulatory effect of galectin-1 is mediated, in part, through its ability to induce, in an Id3 (inhibitor of DNA binding 3)-dependent manner, the expression of IL-10 in monocytes and MdDCs. This effect is inhibited by the addition of lactose, which normalizes the phenotypic and functional alterations seen in MdDCs. Of note, significant upregulation of IL-10 was seen in tumor-infiltrating CD11c(+) DCs in human lung cancer samples. This was also noted in mice transplanted with lung cancer cells, but not in those receiving tumor cells with galectin-1 knockdown. Furthermore, a significant reduction was noted in lung cancer incidence and in the levels of IL-10-expressing, tumor-infiltrating DCs, in mice receiving galectin-1-silenced tumor cells. These results thus suggest that the galectin-1/IL-10 functional axis may be crucial in lung cancer-mediated immune suppression, and that galectin-1 may serve as a target in the development of lung cancer immunotherapy.     

Purkinje cell dendritic arbors in chick embryos following chronic treatment with an N-methyl-D-aspartate receptor antagonist

The normal development of Purkinje cell dendrites is dependent on afferent innervation. To investigate the role of neuronal activity in Purkinje cell dendritic development, chick embryos were chronically treated with a potent, selective, and systemically active competitive N-methyl-D -aspartate (NMDA) receptor antagonist, NPC 12626. The NMDA receptor was chosen as a target for pharmacological blockade because of the importance of the NMDA receptor in synaptic plasticity and stabilization in development. Chick embryos were given daily injections of NPC 12626 (25 to 100 mg/kg) from embryonic day 14 (E14) to E17. The initial injections of NPC 12626 dramatically blocked embryo movements, but activity levels partially recovered following subsequent injections. Embryo movements were reduced by 24% at the end of the experiment. Embryos were killed on E18, and their brains processed for Golgi-Cox staining. The morphology of Golgi-stained Purkinje cells in drug-treated embryos was similar to control embryos. Morphometric analysis showed, however, that chronic treatment with NPC 12626 resulted in a 19% reduction in Purkinje cell dendritic tree area and a 13% reduction in the number of dendritic branch points. The overall width and height of the drug-treated dendritic trees were not significantly different from controls, suggesting that NPC 12626 reduced Purkinje cell dendritic area by interfering with branch formation. The volume of the granule cell layer and the heights of the molecular and external granule cell layers was not reduced, suggesting that NPC 12626 treatment did not simply delay development. These results suggest that activation of the NMDA receptor may mediate the afferent-target interactions in the cerebellum that regulate the elaboration of Purkinje cell dendrites. © 1995 John Wiley & Sons, Inc.     

Arrest of the cell cycle reduces susceptibility of target cells to perforin-mediated lysis

Cytotoxic T lymphocytes secrete a pore-forming cytolysin, perforin, that damages membranes of target cells. They also ligate Fas receptors on target cells and provoke apoptotic death. A20 (B lymphoma) and P815 (mastocytoma) cell lines were examined for their susceptibility to perforin-mediated lysis and to Fas-induced apoptosis after blockade of the cell cycle at the G₁/S interface. Cells were arrested at the G₁/S interface by inhibition of DNA synthesis with thymidine or aphidicolin. Subsequently, the treated cells were incubated either with CTL cytotoxic granules or the Fas-specific monoclonal antibody Jo-2. We show that arrest of the cell cycle at the G₁/S interface markedly reduced the susceptibility of target cells to perforin-mediated lysis. In contrast, growth arrest with thymidine or aphidicolin increased susceptibility of A20 and P815 cells to Fas-mediated apoptosis. Susceptibility to lysis by intact CTLs was not affected significantly by blockade of target cells with aphidicolin or thymidine. When cells surviving exposure to perforin-containing granules were isolated on Ficoll density gradients and cell-cycle profiles were examined by flow cytometry, the ratio of G₁ to G₂cells increased among the survivors exposed to granules in contrast to controls incubated with buffer alone. The data suggest that cells in G₁ phase of the cell cycle are less susceptible to the perforin pathway than cells in G₂and S phases but are more susceptible to the Fas pathway. J. Cell. Biochem. 69:425–435, 1998. © 1998 Wiley-Liss, Inc.     

Chronic theatment with an endothelin-converting enzyme inhibitor reduces the development of hypoxia-induced pulmonary hypertension in rat

The aim of this study was to investigate how blocking functional endothelin-converting enzyme activity may offer a new approach to inhibition of changes in pulmonary vessels reactivity due to development of hypoxia-induced pulmonary arterial hypertension in rats. This data shows that treatment with endothelin-converting enzyme blocker PP36 significantly reduced pathological changes due to hypoxia-induced pulmonary hypertension. One of the reasons may be the increased production and role of nitric oxide in pulmonary artery tone.     

Studies on the mechanism of action of guinea pig lymphotoxin. I. Membrane active substances prevent target cell lysis by lymphotoxin.

The effect of various pharamacologic agents on lysis of L cells by guinea pig lymphotoxin (LT) was studied. Among the many drugs tested, only those that affect plasma membrane functions were found to interfere with the cytolytic action of LT. Dimethyl sulfoxide or lidocaine potentiated L cell resistance against lysis. Stronger protection was provided by ouabain. Addition of ouabain to cells previously injured by LT was also effective in reducing cell death. Attempts to detect metabolic disturbances in cells before LT cytolysis were unsuccessful. The biosynthetic rate of DNA, RNA, or protein, and the total cellular content of ATP were not significantly changed in LT-treated cells. The results suggest that LT disturbs some plasma membrane functions of the target cell, perhaps ion transport systems, and consequently induces ionic imbalances between the intra-and extracellular milieu which eventually cause cell death.     

Increased expression of HSP70 by colon cancer cells is not always associated with access to the dendritic cell cross-presentation pathway

Dendritic cells (DCs) are highly specialized antigen-presenting cells endowed with the unique ability to not only present exogenous antigens upon exposure to MHC II, but also to cross-present these upon exposure to MHC I. This property was exploited to generate the tumor-specific CD8 cytotoxic lymphocyte (CTL) response in DCs-based cancer vaccine protocols. In this context, the source of tumor antigens remains a critical challenge. A crude tumor in the context of danger signals is believed to represent an efficient source of tumor antigens (TAs) for DCs loading. In our previous work, increased DCs cross-presentation of antigens from necrotic gastric carcinoma cells paralleled up-regulation of the heat shock protein hsp70. We studied the expression of hsp70 on primary colon carcinoma cells and its relevance in the cross-priming of anti-tumor CTL by tumor-loaded DCs. Hsp70 was expressed on all three of the tumors studied, but was never detected in the peritumoral normal mucosa (NM). The uptake of the tumor induced a trend towards down-modulation of the monocyte-specific marker CD14, but had no effect on the chemokine receptors CCR4 and CCR7. The IFN-γ enzyme-linked immunospot assay (ELIspot) showed cross-priming of CTL by tumor-loaded but not NM-loaded DCs in four of the six cases studied. The CTL response generated in DC+tumor cultures was directed towards the tumor, but not towards NM, and it was characterized by refractoriness to polyclonal (Ca ionophores, PKC activators) stimuli. Of the three CTL-generating tumors, only one expressed hsp70. This data indicates a tumor-specific expression of hsp70, but does not support its relevance in the DC cross-presentation of TAs.     

Enhancement of tumor cell susceptibility to lymphokine-activated killer cells by treatment with the streptococcal preparation OK432

We investigated whether tumor cell lysis by LAK cells was augmented by treatment with OK432in vitro. NK and LAK activity against K562 cells was not enhanced by their treatment with OK432. In contrast, the susceptibility of OK432-treated Daudi and KATO-III cells to lysis by LAK cells was enhanced. Succinate dehydrogenase activity and RNA synthesis were impaired in Daudi and KATO-III cells by treatment with OK432, and moreover the expression of HLA Class I antigen and ₂-microglobulin was inhibited in OK432-treated KATO-III cells. Thus, it is suggested that the enhancement of the susceptibility of OK432-treated tumor cells with regard to succinate dehydrogenase activity, RNA synthesis, and HLA Class I antigen expression.