Divergent activities of protein kinases in IL-6-induced differentiation of a human B cell line

Lymphokines including IL-2, IL-4, and IL-6 are involved in the induction of Ig production by activated B cells. We have investigated the role of protein kinases in IL-6-induced IgM secretion by SKW6.4 cells, an IL-6 responsive B cell line. IL-6-stimulated IgM production was inhibited by elevated intracellular cAMP induced either by the addition of dibutyryl cAMP or cholera toxin. The inhibitory effect of elevated intracellular cAMP was blocked by n-(2-(Methylamino)ethyl)-5-isoquinolinesulfonic dihydrochloride (H8), an inhibitor of protein kinase A. H8 did not affect IgM secretion induced by IL-6. In contrast, the addition of 1-(5-isoquinolinesulfonyl)-2-methylpiperizine dihydrochloride (H7), an inhibitor of protein kinase C activity, markedly inhibited IL-6-stimulated IgM production by SKW6.4 cells. H7 and elevated intracellular cAMP inhibited IgM mRNA expression and subsequent IgM synthesis by SKW6.4 cells. SKW6.4 proliferation, as determined by [3H]thymidine incorporation, was not markedly affected by IL-6, dibutyryl cAMP, cholera toxin, H7 or H8. PMA, an activator of protein kinase C, directly stimulated significant IgM secretion by SKW6.4 cells. When added to PMA-stimulated SKW6.4 cells, IL-6 stimulated additional IgM production. This observation suggested that IL-6 could stimulate differentiation without activating protein kinase C. This was confirmed by demonstrating that IL-6 did not stimulate production of diacylglycerol, did not induce the translocation of protein kinase C from the cytosolic compartment to the plasma membrane and could induce SKW6.4 cells to produce IgM after depletion of their cellular protein kinase C by PMA. Taken together these results suggests that IL-6-stimulated IgM production requires utilization of an H7-inhibitable protein kinase that can be inhibited by a protein kinase A-dependent pathway. Despite the fact that PMA can stimulate IgM production in SKW6.4 cells, IL-6 appears to use a protein kinase pathway other than protein kinase C to induce IgM production.     

Fibronectin-associated Fas ligand rapidly induces opposing and time-dependent effects on the activation and apoptosis of T cells

Recently, it has been shown that Fas ligand (FasL) interacts with the extracellular matrix (ECM) protein fibronectin (FN), and that the bound FasL retains its cytotoxic efficacy. Herein, we examined the ramifications of FasL-ECM protein interactions throughout a specific time period, in the absence or presence of additional activating molecules, assuming that these complexed interactions occur during inflammation. We found that exposure of purified human T cells to FN-associated recombinant FasL for as brief as 5-10 min at 0.1-100 ng/ml induced their adhesion in beta(1) integrin- and FasR-dependent manners while activating the intracellular protein kinase, Pyk-2. The FN-associated FasL stops the CXCL12 (stromal cell-derived factor 1alpha)-induced chemotaxis of T cells by inhibiting the chemokine-induced extracellular signal-regulated kinase signaling and cytoskeletal rearrangement. This short term exposure of T cells to the FN-bound FasL (1 ng/ml), which was followed by T cell activation via the CD3 complex, resulted in 1) increased secretion of IFN-gamma (measured after 24 h), and 2) enhanced T cell apoptosis (measured after 72 h). Thus, in the context of inflamed ECM and depending on the time after FasL activation, its concentration, and the nature of other contextual mediators, FasL initially retains effector T cells at sites of inflammation and, later, induces T cell apoptosis and return to homeostasis.     

Negative Regulation of Human Astrocytes by Interferon (IFN) α in Relation to Growth Inhibition and Impaired Glucose Utilization

The present study assessed the direct effects of IFNs on human astrocytes. Human astrocytes were exposed to human recombinant IFNs, and the proliferation of cells was measured. Type I IFN receptor mRNA and protein expression, the phosphoprotein levels of signaling molecules including JNK, ERK1/2, IκB, p38MAPK, Stat3, and the expression of cytokines were determined respectively. In addition, cellular glucose consumption was measured as well as Glut-1 protein and activation of GSK-3β/mTOR signal were determined. The expression of Type I IFN receptor was detected in cultured human astrocytes. 2?IU/ml IFNα2a and IFNα2b significantly decreased the proliferation of human astrocytes respectively, compared to control. IFNβ had no significant effect on the proliferation of the cells. The phosphorylation of JNK stimulated by all IFNs detected was more pronounced and sustained than ERK1/2 and IκB. No effects were observed on the activation of p38MAPK and Stat3. Moreover, Treatment with IFNα, especially with IFNα2b, decreased glucose consumption and stimulated phosphorylation of GSK-3β and mTOR, but decreased the expression of Glut-1. In contrast, IFNβ had no significant effect on either glucose consumption or activation of GSK-3β/mTOR signals. INFα2b significantly decreased the levels of IL-8 whereas the levels of GM-CSF were increased. The present study demonstrates direct inhibitory effects of IFNα on cell proliferation, cell signaling and glucose utilization in human astrocytes.     

Phorbol 12-myristate 13-acetate inhibits death receptor-mediated apoptosis in Jurkat cells by disrupting recruitment of Fas-associated polypeptide with death domain.

Regulation of death receptor-mediated apoptosis is incompletely understood. Previous studies have demonstrated that phorbol 12-myristate 13-acetate (PMA), a protein kinase C activator, inhibits Fas (CD95)-mediated apoptosis in Jurkat (type II) cells but not SKW6.4 (type I) cells. In this study, we demonstrated that PMA also protects Jurkat cells from apoptosis induced by tumor necrosis factor-alpha and the tumor necrosis factor-alpha-related apoptosis-inducing ligand (TRAIL). Interestingly, PMA failed to protect Jurkat cells from apoptosis induced by other agents, including etoposide, camptothecin, and gamma-irradiation. Analysis of the initial events induced by agonistic anti-Fas antibodies revealed that PMA inhibited Fas binding to Fas-associated polypeptide with death domain (FADD) in Jurkat cells but not in SKW6.4 cells. Although the protein kinase inhibitor bisindoylmaleimide VIII increased apoptosis induced by agonistic anti-Fas antibody, tumor necrosis factor-alpha, and TRAIL, these effects were not observed with the protein kinase C inhibitor H7 and were not associated with increased FADD recruitment to Fas. These results indicate that PMA inhibits death signaling induced by a number of discrete receptors and suggest that the effects are mediated at the level of receptor-mediated adaptor molecule recruitment.     

Phosphatidylserine exposure in Fas type I cells is mitochondria-dependent

Previous studies have demonstrated that Fas-triggered activation of effector caspases and subsequent nuclear apoptosis either is mitochondria-independent (type I cells) or relies on mitochondrial amplification of the initial stimulus (type II cells). We show herein that Bcl-2 overexpression in a prototypic type I cell line (SKW6.4) promotes mitochondrial generation of ATP and blocks Fas-triggered plasma membrane externalization of phosphatidylserine (PS). Moreover, overexpression of Bcl-2 attenuates macrophage engulfment of Fas-triggered cells. Fas-mediated DNA fragmentation, on the other hand, remains unaffected in SKW6.4-bcl-2 cells. These studies thus demonstrate that PS externalization and clearance of cell corpses are mitochondria-dependent events, and show that these events can be dissociated from other features of the apoptotic program, in Fas type I cells.     

Fas inhibition attenuates lipopolysaccharide-induced apoptosis and cytokine release of rat type II alveolar epithelial cells

The aim of this study is to investigate whether silencing of Fas could have an influence on type II alveolar epithelial cell (AEC) apoptosis and inflammatory cytokine production, which prevents alveolar healing after acute lung injury (ALI). Rat primary type II AECs were isolated by elastase cell dispersion and IgG panning. The cells were transfected with Fas-specific small interfering RNA (siRNA) followed by treatment with lipopolysaccharide (LPS), Fas ligand (FasL) or both. The effects of siRNA-mediated silencing of Fas on LPS-induced apoptosis and cytokine release were then assessed. Notably, LPS, either alone or together with FasL, significantly stimulated type II AEC apoptosis and the release of tumor necrosis factor-alpha (TNF-α) and monocyte chemoattractant protein 1 (MCP-1) (P < 0.05 versus the control without treatment). Moreover, the effects exerted by both LPS and FasL were considerably counteracted by pretreatment with Fas-siRNA (P < 0.05 versus treatment with LPS and FasL). In conclusion, inhibition of Fas can diminish LPS-induced apoptosis and inflammatory cytokine production in type II AECs, and Fas specific siRNAs may have therapeutic potentials for intervention of ALI/ARDS.     

Forced involution of the functionally differentiated mammary gland by overexpression of the pro-apoptotic protein bax

The mammary gland is a developmentally dynamic, hormone-responsive organ that undergoes proliferation and differentiation within the secretory epithelial compartment during pregnancy. The epithelia are maintained by pro-survival signals (e.g., Stat5, Akt1) during lactation, but undergo apoptosis during involution through inactivation of cell survival pathways and upregulation of pro-apoptotic proteins. To assess if the survival signals in the functionally differentiated mammary epithelial cells can override a pro-apoptotic signal, we generated transgenic mice that express Bax under the whey acidic protein (WAP) promoter. WAP-Bax females exhibited a lactation defect and were unable to nourish their offspring. Mammary glands demonstrated: (1) a reduction in epithelial content, (2) hallmark signs of mitochondria-mediated cell death, (3) an increase in apoptotic cells by TUNEL assay, and (4) precocious Stat3 activation. This suggests that upregulation of a single pro-apoptotic factor of the Bcl-2 family is sufficient to initiate apoptosis of functionally differentiated mammary epithelial cells in vivo.     

The adaptor protein Grb2 regulates cell surface Fas ligand in Schwann cells

Fas Ligand (FasL, CD178) is a cytokine that may be secreted or expressed as a transmembrane ligand at the cell surface, and induces apoptosis by binding to the “death receptor” Fas (CD95). Here, we show that Grb2, an SH3 domain-containing adaptor protein, binds to the proline-rich domain of FasL and regulates its cell surface expression. We found that knocking down Grb2 expression decreased the amount of FasL at the cell surface and increased the abundance of intracellular vesicles containing FasL. Furthermore, we showed that Grb2 acts as an adaptor for FasL to interact with adaptin β, a molecule known to regulate trafficking. Our data reveal that Grb2 facilitates the association of FasL with adaptinβ, and promotes sorting of FasL to the cell surface. As FasL is a potent regulator of cell death, dynamic regulation of its cell surface localization is critical for controlling local tissue remodeling and inflammation.     

Overexpression and structure--function analysis of a bioengineered IL-2/IL-6 chimeric lymphokine.

A synthetic chimeric IL-2/IL-6 gene was synthesized to engineer a bifunctional lymphokine which was overproduced in Escherichia coli. Following denaturation of the inclusion bodies in 6 M guanidine and refolding and reoxidation in the presence of a redox system, the fusion protein (rIL-2/IL-6) was purified to homogeneity and shown to react with both monospecific anti-IL-2 and anti-IL-6 antisera. A collagen-like spacer was introduced between the two cytokine moieties to generate IL-2 and IL-6 molecules upon collagenase digestion. After cleavage, the two subunits, purified in a single-step procedure, were found to be correctly reoxidized and functionally as active as their native counterparts. Circular dichroism studies of rIL-2/IL-6 revealed that both cytokine subunits refolded independently and exhibited the alpha-helical structures characteristic of the corresponding wild-type lymphokines. The chimera displayed full IL-2 activity in the CTLL-2 cell proliferation assay. It also retained the IL-6 property to enhance IgM synthesis in SKW6.4 cells, induce the proliferation of B-cell hybridomas and stimulate the production of fibrinogen in hepatocytes. Because IL-2 amplifies the cellular immune response and IL-6 up-regulates the humoral response, this bifunctional lymphokine represents a potentially useful therapeutic adduct and may serve as an immunomodulator to enhance the host's response to vaccination.     

Cell surface sialylation plays a role in modulating sensitivity towards APO-1-mediated apoptotic cell death

APO-1/Fas(CD95), a member of the tumour necrosis factor (TNF)/nerve growth factor (NGF) receptor superfamily transduces apoptotic signals into apoptosis sensitive cells. In metabolic labelling experiments using the highly APO-1 positive cell lines HUT78 (adultT cell leukemia) and SKW6.4 (Blymphoblastoid cell line) APO-1 was characterised as a long living protein with a complex glycosylation pattern involving terminal sialic acid groups which account for 8-kDa of its apparent molecular weight on SDS-PAGE. APO-1 expression and the degree of sialylation were determined in additionalT and B cell lines. On the group I Burkitt's lymphoma cell line BL60 transfected with human APO-1 (K50) low sialylated species were detected only on the cell surface, suggesting that sialylation might be functionally important. Removal of terminal sialic acid groups by treatment of B and T cell lines with Vibrio cholerae neuraminidase (VCN) augmented sensitivity towards anti-APO-1 and human APO-1 ligand induced apoptosis. Similarly, VCN-treated U937 cells were rendered more sensitive to TNFalpha-induced cell death. Thus, sialylation may be one mechanism to regulate sensitivity towards ligand-mediated cell death in this receptor family.     

人FasL基因转染成熟树突状细胞对T淋巴细胞增殖和凋亡的影响

探讨转染人FasL基因的成熟树突状细胞(DC)对异体T淋巴细胞增殖和凋亡的影响,为实现临床器官移植免疫耐受提供初步实验依据.从健康成年人外周静脉血中获得成熟树突状细胞.将人FasL基因成功转染成熟树突状细胞,检测其表面分子的表达和自身凋亡情况,并对其抗原递呈功能进行分析.从异体健康成人外周血中获取T淋巴细胞,将转染成功的树突状细胞与T淋巴细胞混合培养,检测其对T淋巴细胞增殖和凋亡的影响.结果表明:人FasL基因转染没有明显影响成熟树突状细胞表面分子CD40、CD80、CD86和HLA-DR的表达;没有诱导树突状细胞自身发生凋亡;没有影响DC的抗原递呈功能.转染FasL基因后的树突状细胞使异体T淋巴细胞刺激指数明显下降,凋亡增加.因此认为,人FasL基因转染对成熟树突状细胞的表面分子表达、自身凋亡、抗原递呈等生物学性状无影响;转染FasL基因的树突状细胞使异体T淋巴细胞的增殖能力减弱,并能明显诱导T淋巴细胞凋亡.     

Administration of an antigen at a high dose generates regulatory CD4+ T cells expressing CD95 ligand and secreting IL-4 in the liver

Ags administered orally at a high dose are absorbed in immunogenic forms and perfuse the liver, which raises a question regarding the relevance of hepatic lymphocyte activation to the systemic hyporesponsiveness against the ingested Ag. Oral administration of 100 mg of OVA to the mice led to massive cell death of OVA-specific (KJ1-26+)CD4+ T cells by Fas-Fas ligand (FasL)-mediated apoptosis in the liver, which was associated with the emergence of hepatic KJ1-26+CD4+ T cells expressing FasL. Hepatic CD4+ T cells in OVA-fed mice secreted large amounts of IL-4, IL-10, and TGF-beta(1) upon restimulation in vitro and inhibited T cell proliferation. Adoptive transfer of these hepatic CD4+ T cells to naive mice and subsequent antigenic challenge led to suppression of T cell proliferation as well as IgG Ab responses to OVA; this effect was mostly abrogated by a blocking Ab to FasL. i.p. administration of an Ag at a high dose also generated hepatic CD4+FasL+ T cells with similar cytokine profile as T cells activated by oral administration of Ags at a high dose. Finally, we did not see an increase in FasL+ cells in the hepatic CD4+Vbeta8+ T cell subset of MRL/lpr/lpr mice given staphylococcal enterotoxin B, indicating the requirement for Fas-mediated signals. These hepatic CD4+FasL+ regulatory cells may explain the tolerogenic property of the liver and play roles in systemic hyporesponsiveness induced by an Ag administered at a high dose.