Wan-Wan Lin

A cytokine-mediated link between innate immunity, inflammation, and cancer.

It has been established that cancer can be promoted and/or exacerbated by inflammation and infections. Indeed, chronic inflammation orchestrates a tumor-supporting microenvironment that is an indispensable participant in the neoplastic process. The mechanisms that link infection, innate immunity, inflammation, and cancer are being unraveled at a fast pace. Important components in this linkage are the cytokines produced by activated innate immune cells that stimulate tumor growth and progression. In addition, soluble mediators produced by cancer cells recruit and activate inflammatory cells, which further stimulate tumor progression. However, inflammatory cells also produce cytokines that can limit tumor growth. Here we provide an overview of the current understanding of the role of inflammation-induced cytokines in tumor initiation, promotion, and progression.

Carcinoma-produced factors activate myeloid cells through TLR2 to stimulate metastasis

Metastatic progression depends on genetic alterations intrinsic to cancer cells as well as the inflammatory microenvironment of advanced tumours. To understand how cancer cells affect the inflammatory microenvironment, we conducted a biochemical screen for macrophage-activating factors secreted by metastatic carcinomas. Here we show that, among the cell lines screened, Lewis lung carcinoma (LLC) were the most potent macrophage activators leading to production of interleukin-6 (IL-6) and tumour-necrosis factor-α (TNF-α) through activation of the Toll-like receptor (TLR) family members TLR2 and TLR6. Both TNF-α and TLR2 were found to be required for LLC metastasis. Biochemical purification of LLC-conditioned medium (LCM) led to identification of the extracellular matrix proteoglycan versican, which is upregulated in many human tumours including lung cancer, as a macrophage activator that acts through TLR2 and its co-receptors TLR6 and CD14. By activating TLR2:TLR6 complexes and inducing TNF-α secretion by myeloid cells, versican strongly enhances LLC metastatic growth. These results explain how advanced cancer cells usurp components of the host innate immune system, including bone-marrow-derived myeloid progenitors, to generate an inflammatory microenvironment hospitable for metastatic growth.

Involvement of p38 mitogen-activated protein kinase in lipopolysaccharide-induced iNOS and COX-2 expression in J774 macrophages

Both the nitrite and prostaglandin E2 (PGE2) release caused by lipopolysaccharide (LPS) in J774 macrophages are inhibited by SB 203580, a specific p38 mitogen‐activated protein kinase (MAPK) inhibitor, in a concentration‐dependent manner. The 50% inhibitory concentration (IC50) for nitrite and PGE2 responses was 1 μm and 0·5 μm, respectively. Inhibition was marked following simultaneous treatment with SB 203580 and LPS, and was much reduced when SB 203580 was added 6 hr after LPS treatment. In parallel, LPS induction of inducible NO synthase (iNOS) and cyclo‐oxygenase‐2 (COX‐2) proteins and their steady‐state levels of mRNA were reduced by SB 203580. LPS activation of nuclear factor‐kappa B (NF‐κB), activator protein‐1 (AP‐1) and p38 MAPK was also inhibited by SB 203580. These results suggest a crucial role of p38 MAPK in regulation of the transcriptional level of endotoxin LPS‐induced iNOS and COX‐2 protein expression.

Effects of cannabinoids on LPS‐stimulated inflammatory mediator release from macrophages: Involvement of eicosanoids

Δ9‐Tetrahydrocannabinol (Δ9‐THC) is the major psychoactive component of marijuana and elicits pharmacological actions via cannabinoid receptors. Anandamide (AEA) and 2‐arachidonoyl‐glycerol (2‐AG) are endogenous ligands for cannabinoid receptors, which because of their structural similarities to arachidonic acid (AA), AEA, and 2‐AG could serve as substrates for lipoxygenases and cyclooxygenases (COXs) that metabolize polyunsaturated fatty acids to potent bioactive molecules. In this study, we have compared the effects of Δ9‐THC, AEA, 2‐AG, and another cannabinoid agonist, indomethacin morpholinylamide (IMMA), on lipopolysaccharide (LPS)‐induced NO, IL‐6, and PGE2 release from J774 macrophages. Δ9‐THC, IMMA, and AEA diminish LPS‐induced NO and IL‐6 production in a concentration‐dependent manner. 2‐AG inhibits the production of IL‐6 but slightly increases iNOS‐dependent NO production. Δ9‐THC and IMMA also inhibit LPS‐induced PGE2 production and COX‐2 induction, while AEA and 2‐AG have no effects. These discrepant results of 2‐AG on iNOS and COX‐2 induction might be due to its bioactive metabolites, AA and PGE2, whose incubation cause the potentiation of both iNOS and COX‐2 induction. On the contrary, the AEA metabolite, PGE2‐ethanolamide, influences neither the LPS‐induced NO nor IL‐6 production. Taken together, direct cannabinoid receptor activation leads to anti‐inflammatory action via inhibition of macrophage function. The endogenous cannabinoid, 2‐AG, also serves as a substrate for COX‐catalyzing PGE2 production, which in turn modulates the action of CB2. J. Cell. Biochem. 81: 715–723, 2001.

Silymarin protects dopaminergic neurons against lipopolysaccharide‐induced neurotoxicity by inhibiting microglia activation

An inflammatory response in the central nervous system mediated by activation of microglia is a key event in the early stages of the development of neurodegenerative diseases. Silymarin is a polyphenolic flavanoid derived from milk thistle that has anti‐inflammatory, cytoprotective and anticarcinogenic effects. In this study, we first investigated the neuroprotective effect of silymarin against lipopolysaccharide (LPS)‐induced neurotoxicity in mesencephalic mixed neuron–glia cultures. The results showed that silymarin significantly inhibited the LPS‐induced activation of microglia and the production of inflammatory mediators, such as tumour necrosis factor‐α and nitric oxide (NO), and reduced the damage to dopaminergic neurons. Therefore, the inhibitory mechanisms of silymarin on microglia activation were studied further. The production of inducible nitric oxide synthase (iNOS) was studied in LPS‐stimulated BV‐2 cells as a model of microglia activation. Silymarin significantly reduced the LPS‐induced nitrite, iNOS mRNA and protein levels in a dose‐dependent manner. Moreover, LPS could induce the activation of p38 mitogen‐activated protein kinase (MAPK) and c‐jun N‐terminal kinase but not extracellular signal‐regulated kinase. The LPS‐induced production of NO was inhibited by the selective p38 MAPK inhibitor SB203580. These results indicated that the p38 MAPK signalling pathway was involved in the LPS‐induced NO production. However, the activation of p38 MAPK was not inhibited by silymarin. Nevertheless, silymarin could effectively reduce LPS‐induced superoxide generation and nuclear factor kappaB (NF‐κB) activation. It suggests that the inhibitory effect of silymarin on microglia activation is mediated through the inhibition of NF‐κB activation.

High Glucose Induces Human Endothelial Cell Apoptosis Through a Phosphoinositide 3-Kinase–Regulated Cyclooxygenase-2 Pathway

Objectives—Diabetes mellitus causes endothelial dysfunction. The precise molecular mechanisms by which hyperglycemia causes apoptosis in endothelial cells are not yet well understood. The aim of this study was to explore the role of cyclooxygenase-2 (COX-2) and the possible involvement of phosphoinositide 3-kinase (PI3K) signaling in high glucose (HG)–induced apoptosis in human umbilical vein endothelial cells (HUVECs). Methods and Results—For detection of apoptosis, the morphological Hoechst staining and Annexin V/propidium iodide staining were used. Glucose upregulated COX-2 protein expression, which was associated with the induction of prostaglandin (PG) E2 (PGE2), caspase-3 activity, and apoptosis. Unexpectedly, we found that PI3K inhibitors could suppress COX-2 expression, PGE2 production, caspase-3 activity, and the subsequent apoptosis under HG condition. Glucose-induced activation of PI3K resulted in the downstream effector Akt phosphorylation. PI3K inhibitors effectively attenuated the intracellular reactive oxygen species (ROS) generation and nuclear factor &kgr;B (NF-&kgr;B) activation. Blocking the PI3K and Akt activities with the dominant-negative vectors greatly diminished the HG-triggered NF-&kgr;B activation and COX-2 expression and apoptosis. Conclusions—These results suggest that HG, via PI3K/Akt signaling, induces NF-&kgr;B–related upregulation of COX-2, which in turn triggers the caspase-3 activity that facilitates HUVEC apoptosis. Also, HG may cause ROS generation in HUVECs through a PI3K/Akt–dependent pathway.

PKC‐ and ERK‐dependent activation of IκB kinase by lipopolysaccharide in macrophages: enhancement by P2Y receptor‐mediated CaMK activation

Although accumulating studies have identified IκB kinase (IKK) to be essential for controlling NF‐κB activity in response to several cytokines, the upstream kinases that control IKK activity are still not completely known. We have previously reported that G protein‐coupled P2Y6 receptor activation by UTP potentiates lipopolysaccharide (LPS)‐induced IκB phosphorylation and degradation, and NF‐κB activation in J774 macrophages. In this study, we investigated the upstream kinases for IKK activation by UTP and LPS. In murine J774 macrophages, LPS‐induced NF‐κB activation was inhibited by the presence of PDTC, D609, Ro 31‐8220, PD 098059 and SB 203580. Accompanying NF‐κB activation, LPS induced IκB degradation and IKK activation were reduced by PDTC, D609, Ro 31‐8220 and PD 098059, but not by SB 203580. Although UTP itself slightly induced IKK activation, this response was synergistic with LPS. BAPTA/AM and KN‐93 (a calcium/calmodulin‐dependent protein kinase (CaMK) inhibitor) attenuated UTP‐ but not LPS‐stimulated IKK activity. Synergistic IKK activation between LPS and thapsigargin was further demonstrated in peritoneal macrophages. LPS and UTP co‐stimulation additively increased p65 NF‐κB phosphorylation. In vitro kinase assays revealed that LPS and UTP induced extracellular signal‐regulated protein kinase (ERK) and p38 mitogen‐activated protein kinase activation were respectively inhibited by PD098059 and SB 203580. Taken together, we demonstration that Gq protein‐coupled P2Y6 receptor activation can potentiate LPS‐stimulated IKK activity. While PKC and ERK participate in IKK activation by LPS and UTP, the phosphatidylinositide‐phospholipase C‐dependent activation of CaMK plays a major role in UTP potentiation of the LPS response.

Signal transduction for inhibition of inducible nitric oxide synthase and cyclooxygenase-2 induction by capsaicin and related analogs in macrophages

Although capsaicin analogs might be a potential strategy to manipulate inflammation, the mechanism is still unclear. In this study, the effects and action mechanisms of vanilloid analogs on iNOS and COX‐2 expression were investigated in RAW264.7 macrophages. Capsaicin and resiniferatoxin (RTX) can inhibit LPS‐ and IFN‐γ‐mediated NO production, and iNOS protein and mRNA expression with similar IC50 values of around 10 μM. Capsaicin also transcriptionally inhibited LPS‐ and PMA‐induced COX‐2 expression and PGE2 production. However, this effect exhibited a higher potency (IC50: 0.2 μM), and RTX failed to elicit such responses at 10 μM. Interestingly, we found that capsazepine, a competitive TRPV1 antagonist, did not prevent the inhibition elicited by capsaicin or RTX. Nevertheless, it mimicked vanilloids in inhibiting iNOS/NO and COX‐2/PGE2 induction with an IC50 value of 3 μM. RT–PCR and immunoblotting analysis excluded the expression of TRPV1 in RAW264.7 macrophages. The DNA binding assay demonstrated the abilities of vanilloids to inhibit LPS‐elicited NF‐κB and AP‐1 activation and IFN‐γ‐elicited STAT1 activation. The reporter assay of AP‐1 activity also supported this action. The kinase assay indicated that ERK, JNK, and IKK activation by LPS were inhibited by vanilloids. In conclusion, vanilloids can modulate the expression of inflammatory iNOS and COX‐2 genes in macrophages through interference with upstream signalling events of LPS and IFN‐γ. These findings provide new insights into the potential benefits of the active ingredient in hot chilli peppers in inflammatory conditions.

Soluble decoy receptor 3 induces angiogenesis by neutralization of TL1A, a cytokine belonging to tumor necrosis factor superfamily and exhibiting angiostatic action

TL1A is a member of the tumor necrosis factor superfamily and plays an important role in regulating endothelial cell apoptosis. A previous study shows TL1A is able to interact with death receptor 3 and decoy receptor 3 (DcR3). Here, we demonstrate that DcR3 is able to induce angiogenesis in human umbilical vein endothelial cells (HUVECs). DcR3 promotes HUVEC proliferation and migration and up-regulates matrix metalloproteinase-2 mRNA expression and enzyme activity. Furthermore, DcR3 enhances EC differentiation into cord vascular-like structures in vitro, as well as neovascularization in vivo. The effects of DcR3 on HUVECs are also mimicked by anti-TL1A and antideath receptor 3 antibodies. In contrast, human aortic endothelial cells, which do not express TL1A, are not responsive to DcR3 treatment, including cell proliferation, migration, and angiogenic differentiation. These data demonstrate DcR3 might not only help tumor cells to escape immune surveillance but also induce angiogenesis by blocking TL1A action in endothelial cells. The pathological role of DcR3 in promoting cancer progress raises the possibility to target DcR3 for antiangiogenic therapy in the future.

CLEC5A is critical for dengue virus-induced inflammasome activation in human macrophages

Persistent high fever is one of the most typical clinical symptoms in dengue virus (DV)-infected patients. However, the source of endogenous pyrogen (eg, IL-1β) and the signaling cascade leading to the activation of inflammasome and caspase-1, which are essential for IL-1β and IL-18 secretion, during dengue infection have not been elucidated yet. Macrophages can be polarized into distinct phenotypes under the influence of GM-CSF or M-CSF, denoted as GM-Mϕ and M-Mϕ, respectively. We found that DV induced high levels of IL-1β and IL-18 from GM-Mϕ (inflammatory macrophage) and caused cell death (pyroptosis), whereas M-Mϕ (resting macrophage) did not produce IL-1β and IL-18 on DV infection even with lipopolysaccharide priming. This observation demonstrates the distinct responses of GM-Mϕ and M-Mϕ to DV infection. Moreover, up-regulation of pro-IL-1β, pro-IL-18, and NLRP3 associated with caspase-1 activation was observed in DV-infected GM-Mϕ, whereas blockade of CLEC5A/MDL-1, a C-type lectin critical for dengue hemorrhagic fever and Japanese encephalitis virus infection, inhibits NLRP3 inflammasome activation and pyrotopsis in GM-Mϕ. Thus, DV can activate NLRP3 inflammasome via CLEC5A, and GM-Mϕ plays a more important role than M-Mϕ in the pathogenesis of DV infection.