Hypoxia affects dendritic cell survival: role of the hypoxia-inducible factor-1α and lipopolysaccharide.

Dendritic cells (DC) are the most potent antigen-presenting cells and during their life cycle they are exposed to different oxygen tensions. Similarly to inflamed and tumor tissues, lymphoid organs are characterized by a hypoxic microenvironment; thus, the modality by which hypoxia may affect DC is important for regulating both the quality and the intensity of the immune response. Here, we show that human monocyte-derived DC, exposed to hypoxia, expressed high levels of the hypoxia-inducible factor (HIF)-1α, associated with upregulation of BNIP3 and BAX expression. This was paralleled with downregulation of the anti-apoptotic molecule Bcl-2, enhanced caspase-3 activity and poly (ADP-ribose) polymerase cleavage, along with cell death. Transfection of HIF-1α siRNA protected DC from the effects of hypoxia. Of interest, when hypoxic DC were maturated with lipopolysaccharide (LPS), we did not observe an increased cell death, while HIF-1α accumulation and BNIP3 expression were still significantly upregulated. In contrast with immature DC, mature DC expressed higher levels of Bcl-2, and, more importantly, of phosphorylated Akt. Transfection of HIF-1α siRNA to mature DC resulted in a significant upregulation of Akt phosphorylation as well. Moreover, inhibition of PI3K/Akt pathway resulted in an increased cell death of hypoxic mature DC. We may conclude that a prolonged exposure to hypoxia induces a cell death program which could be prevented by HIF-1α inhibition and/or LPS maturation. Our results may contribute to further understand the physiology of DC and the molecular mechanisms involved in the survival of DC, with important implications in the regulation of the immune response.

The adaptor protein p66Shc is a positive regulator in the angiogenic response induced by hypoxic T cells.

Immune cells play an important role in the onset of angiogenesis. Here, we report that VEGF represents the major proangiogenic factor expressed by T cells exposed to hypoxia, a common feature of inflammation and tumor microenvironment. The supernatants of hypoxic T cells were highly angiogenic when delivered on the chick embryo CAM. The angiogenic response was abrogated by a neutralizing anti-VEGF antibody and mimicked by rVEGF. Interestingly, VEGF induction by hypoxia was up-regulated in Jurkat T cells overexpressing the adaptor protein p66Shc but not the inactive S36 p66Shc mutant, and it was abolished in p66Shc-/- mouse splenocytes. Accordingly, the angiogenic response induced by the supernatants from hypoxic p66Shc-/- splenocytes was reduced dramatically when compared with the wild-type controls. In conclusion, hypoxic T cells may contribute to the onset of angiogenesis through a novel VEGF-mediated mechanism, where p66Shc acts as a positive regulator.

Inhibition of Bcr-Abl phosphorylation and induction of apoptosis by pyrazolo[3,4-d]pyrimidines in human leukemia cells.

A series of pyrazolo[3,4-d]pyrimidines, previously found to be Src inhibitors, was tested for their ability to inhibit proliferation of three Bcr-Abl-positive human leukemia cell lines (K-562, KU-812, and MEG-01), on the basis of the experimental evidence that various Src inhibitors are also active against Bcr-Abl kinase (the so called dual Src/Abl inhibitors). They reduce Bcr-Abl tyrosine phosphorylation and promote apoptosis of the Bcr-Abl-expressing cells. A cell-free enzymatic assay on isolated c-Abl confirmed that such compounds directly inhibit Abl activity. Finally, molecular modeling simulations were also performed to hypothesize the binding mode of the compounds into the Abl binding site.

p66Shc is involved in promoting HIF-1alpha accumulation and cell death in hypoxic T cells.

Hypoxia results in adaptationally appropriate alterations of gene expression through the activation of hypoxia-inducible factor (HIF)-1 to overcome any shortage of oxygen. Peripheral blood mononuclear cells may be exposed to low oxygen tensions for different times as they migrate between blood and various tissues. We and others have previously shown that T-cell adaptation to hypoxia is characterized by a modulation of cytokine expression and an inhibition of T-cell activation. We have recently demonstrated that the adaptor protein p66Shc negatively regulates T-cell activation and survival. We here show that hypoxia enhances HIF-1alpha accumulation and vascular endothelial growth factor production in T cells. Hypoxic T cells expressed high levels of p21(WAF1/CIP1), of the pro-apoptotic molecules BNIP3, a classic HIF target gene, and BAX, as well as low levels of the anti-apoptotic molecule BCLxl, associated with an induction of cell death. We found out that hypoxic T cells expressed p66Shc. Furthermore, using T-cell transfectants expressing p66Shc, as well as T cells derived from mice p66Shc-/-, we defined a role of p66Shc in T-cell responses to hypoxia. Of interest, hypoxic p66Shc-positive transfectants expressed higher level of HIF-1alpha than negative controls. Thus, p66Shc may play an important role in downstream hypoxic signaling, involving HIF-1alpha protein accumulation and cell death in T lymphocytes.

Thrombin inhibits IFN-gamma production in human peripheral blood mononuclear cells by promoting a Th2 profile.

Thrombin, the key enzyme of the coagulation cascade, is involved in inflammation. It was proposed recently that thrombin activity may play an important role in allergic inflammation. Interferon-gamma (IFN-gamma) is a potent Th1-related cytokine secreted by activated T cells and is usually downregulated in allergic inflammation. We recently demonstrated that thrombin enhances interleukin-10 (IL-10) in peripheral blood mononuclear cells (PBMC). Thus, we hypothesized that thrombin may promote a Th2 profile. We here report that human alpha- thrombin downregulates IFN-gamma expression at both protein and mRNA levels in activated PBMCs. The use of proteolytically inactive thrombin and of the specific thrombin receptor agonist peptide, SFLLRN, shows that this downregulation is thrombin specific and requires thrombin proteolytic activity. The addition of an anti- IL-10 monoclonal antibody (mAb) to thrombin-treated PBMCs abolishes IFN-gamma downregulation, suggesting that thrombin exerts its effect through IL-10, a Th2-related cytokine. Furthermore, IFN-gamma reduction was accompanied by increased IL-4 release, as well as by an increase in the proinflammatory cytokine IL-1. In conclusion, the observation that thrombin affects the production of IFN-gamma (Th1 profile) and IL-4 (Th2 profile) provides further evidence for the role played by thrombin in modulating Th1/Th2 cytokine balance, which could be particularly relevant in allergic inflammation.

Cutting edge: IL-1beta mediates the proangiogenic activity of osteopontin-activated human monocytes.

Inflammation plays an important role in the onset of angiogenesis. In the present study, we show that osteopontin (OPN), a proinflammatory mediator involved in tissue repair, induces IL-1beta up-regulation in human monocytes. This was accompanied by the enhanced production of TNF-alpha, IL-8, and IL-6, a decreased release of IL-10, and increased p38 phosphorylation. The supernatants of OPN-treated monocytes were highly angiogenic when delivered on the chick embryo chorioallantoic membrane. The angiogenic response was completely abrogated by a neutralizing anti-IL-1 Ab, thus indicating that this cytokine represents the major proangiogenic factor expressed by OPN-activated monocytes. Accordingly, rIL-1beta mimicked the proangiogenic activity of OPN-treated monocyte supernatants, and IL-1R (type I) was found to be expressed in the chorioallantoic membrane. In conclusion, OPN-activated monocytes may contribute to the onset of angiogenesis through a mechanism mediated by IL-1beta.

Pyrazolo[3,4-d]pyrimidines as potent antiproliferative and proapoptotic agents toward A431 and 8701-BC cells in culture via inhibition of c-Src phosphorylation.

We report here the synthesis of new pyrazolo[3,4-d]pyrimidine derivatives along with their biological properties as inhibitors of isolated Src and cell line proliferation (A431 and 8701-BC cells). Such compounds block the growth of cancer cells by interfering with the phosphorylation of Src, and they act as proapoptotic agents through the inhibition of the anti apoptotic gene BCL2. Several of them were found to be more active than the reference compound (1-(tert-butyl)-3-(4-chlorophenyl)-4-aminopyrazolo[3,4-d]pyrimidine, PP2) in inhibiting cell proliferation and in inducing apoptosis, and as active as PP2 in the inhibition of the phosphorylation of isolated Src. Moreover, molecular modeling simulations have been performed to hypothesize the way, at the molecular level, by which the inhibitors were able to act as antiproliferative agents.

Thrombin-mediated IL-10 up-regulation involves protease-activated receptor (PAR)-1 expression in human mononuclear leukocytes.

Thrombin, the key enzyme of the coagulation cascade, exerts cellular effects through activation of the protease-activated receptors (PARs). Interleukin (IL)-10, besides its anti-inflammatory properties, is considered a major denominator of the immunosuppressive effect during human endotoxemia. We have recently shown that thrombin inhibits IL-12 production in human mononuclear cells and that such inhibition is accompanied by IL-10 up-regulation. To our knowledge, there are no data available to show that thrombin mediates IL-10 production by its interactions with PAR-1. We here report that human alpha-thrombin enhances IL-10 expression in human peripheral blood mononuclear cells and in established monocytic cell lines and that this up-regulation requires PAR-1 expression. The use of proteolytically inactive thrombin reveals that such enhancement requires thrombin proteolytic activity. Addition of PAR-1 agonist peptides, such as SFLLRN, results in a significant increase of IL-10 production. PAR-1 expression is required for thrombin-induced IL-10 production, as shown by experiments performed with antisense or sense PAR-1 oligonucleotides. Treatment with thrombin or SFLLRN of monocytic cell lines, such as U937 and Mono Mac-6, results in an increased IL-10 production. This suggests that the observed IL-10 up-regulation may be the result of a direct interaction with monocytes. The observation that thrombin-mediated up-regulation of IL-10 may require the expression of the PAR-1 receptor identifies a new, functional link between inflammation and coagulation. Our results may also contribute to better design therapeutic strategies to treat several disorders, characterized by the presence of inflammatory as well as coagulant responses.

Early response to bleomycin is characterized by different cytokine and cytokine receptor profiles in lungs.

The sensitivity to the fibrosis-inducing effect of bleomycin varies considerably from species to species, the reasons for which are unknown. The variability of the response in different strains of mice is well documented. Recent evidence indicates that the upregulated expression of cytokines and cytokine receptors may be involved. We evaluated the expression pattern of some cytokines and their receptors in C57Bl/6J bleomycin-sensitive and Balb/C bleomycin-resistant mice. Animals from both strains received, under ether anesthesia, either saline (50 microl) or bleomycin (0.1 U/50 microl) intratracheally. At various times after the treatment, the lungs were analyzed for cytokines and cytokine receptors by histochemistry and their mRNA by RNase protection assay. A significantly increased expression of TNF-alpha and IL-1beta was observed in both strains. However, an upregulated lung expression for TNF-alpha and IL-1 receptors was observed in C57Bl/6J-sensitive animals only. This profile is evident from 63 h onward. In addition to TNF-alpha, bleomycin administration also resulted in the upregulated expression of TGF-beta in the lungs of both strains at 8 h and in an enhanced expression of TGF-beta receptors I and II in C57Bl/6J mice only. The upregulation of TGF-beta receptor expression was preceded in this strain by an increased expression of IL-4, IL-13, and IL-13 receptor-alpha (at 8 h after bleomycin) and followed by an upregulation of gp130 and IL-6. The difference we observed in the cytokine receptor profile may offer an additional explanation for the different fibrogenic response of the two mouse strains to bleomycin.

Pyrazolo[3,4-d]pyrimidines endowed with antiproliferative activity on ductal infiltrating carcinoma cells.

Novel 1,4,6-trisubstituted pyrazolo[3,4-d]pyrimidines are reported with preliminary in vitro activity data indicating that several of them are potent inhibitors (better than the reference compound) of Src phosphorylation of the breast cancer cells 8701-BC, known to overexpress Src. The ability of such compounds to significantly reduce 8701-BC cell proliferation suggests that this scaffold could be a promising lead for the development of antitumoral agents able to block Src phosphorylation of breast cancer cells.

Human alpha-thrombin stimulates proliferation of interferon-gamma differentiated, growth-arrested U937 cells, overcoming differentiation-related changes in expression of p21CIP1/WAF1 and cyclin D1.

In addition to its central role in blood coagulation and hemostasis, human alpha-thrombin is a growth factor for a variety of cell types. We recently demonstrated that interferon-gamma (IFNgamma)-differentiated U937 cells show increased expression of the proteolytically activated receptor for thrombin (PAR-1) relative to undifferentiated U937. In the present study we show that cell proliferation is inhibited in IFNgamma-differentiated cells relative to undifferentiated U937. Addition of thrombin to the differentiated cells, however, overcomes the inhibition and restores the cells to a highly proliferative state. Ribonuclease protection assays indicate that the IFNgamma-induced growth arrest is associated with an increased expression of the cyclin-dependent kinase inhibitor p21(CIP1/WAF1) and downregulation of cyclin D(1). Treatment of cells with thrombin downregulates p21(CIP1/WAF1) expression in these cells and upregulates cyclin D(1) mRNA expression, thus overcoming the differentiation-related effects in a coordinated manner. Treating differentiated cells with the PAR-1 activation peptide, SFLLRN, stimulates proliferation and has effects similar to those of thrombin on expression of p21(CIP1/WAF1). Thus, it appears that these thrombin stimulated proliferative effects are mediated through activation of PAR-1. These results may help explain how thrombin can overcome growth arrest in normal tissue to initiate tissue repair and why thrombin and thrombin-like enzymes may contribute to unrestricted proliferation observed in certain malignancies.

Thrombin enhancement of interleukin-1 expression in mononuclear cells: involvement of proteinase-activated receptor-1.

In addition to its central role in blood coagulation and hemostasis, human alpha-thrombin is considered a pro-inflammatory molecule. We have previously demonstrated that differentiated monocytes express the proteolytically activated receptor for thrombin (PAR-1) and that thrombin enhances the release of interleukin (IL)-6 in human monocytes. In the present study we show that thrombin upregulates the production of both IL-1alpha and IL-1beta in phytohemagglutin (PHA)-activated human peripheral blood mononuclear cells (PBMC). Treating PHA-activated PBMC with the PAR-1 activation peptide, SFLLRN, mimics the effects of thrombin on IL-1alpha and IL-1beta production. Thus, it appears that these pro-inflammatory effects induced by thrombin may be mediated through activation of PAR-1. ELISA and RNase protection assays indicate that thrombin and SFLLRN peptide upregulates IL-1 expression at both protein and mRNA levels. Thrombin directly affects monocyte IL-1 expression, since treatment of differentiated U937 cells with thrombin and SFLLRN enhances IL-1 production. These results may help explain how thrombin can enhance IL-1 expression in normal tissue to initiate tissue repair and why thrombin and thrombin-like enzymes may contribute to inflammatory responses observed in several pathophysiological conditions.