Hematopoietic prostaglandin D synthase-derived prostaglandin D2 ameliorates adjuvant-induced joint inflammation in mice.

Although prostaglandins (PGs) are known to be involved in the progression of arthritis, the role of PGD2 remains unclear. In this study, we evaluated the role of PGD2 in joint inflammation using genetically modified mice. Injection of complete Freund's adjuvant (CFA) increased the production of PGD2 and induced paw swelling and cartilage erosion in wild-type (WT) mice. These phenomena were accompanied with an increase in the mRNA levels of TNF-α, IL-6, IL-1ß, and matrix-degrading metalloproteinase-9. Knockdown of hematopoietic PGD synthase (H-PGDS) abolished the PGD2 production and exacerbated all of the arthritic manifestations in the inflamed paw. Immunostaining revealed that infiltrating macrophages strongly expressed H-PGDS in the CFA-injected paw. Morphologic studies revealed vascular hyperpermeability and angiogenesis in the inflamed WT paw. H-PGDS deficiency was accelerated, whereas daily administration of a PGD2 receptor D prostanoid (DP) agonist attenuated the CFA-induced hyperpermeability and angiogenesis. We further confirmed that DP deficiency exacerbated, whereas the administration of the DP agonist improved, the CFA-induced arthritic manifestations. The findings demonstrate that H-PGDS-derived PGD2 ameliorates joint inflammation by attenuating vascular permeability and subsequent angiogenesis and indicates the therapeutic potential of a DP agonist for arthritis.-Tsubosaka, Y., Maehara, T., Imai, D., Nakamura, T., Kobayashi, K., Nagata, N., Fujii, W., Murata, T. Hematopoietic prostaglandin D synthase-derived prostaglandin D2 ameliorates adjuvant-induced joint inflammation in mice.

A deficiency in the prostaglandin D2 receptor CRTH2 exacerbates adjuvant-induced joint inflammation.

Although the cyclooxygenase metabolites PGs are known to be involved in the progression of arthritis, the role of PGD2 remains unclear. In this study, we evaluated the contribution of signaling mediated through a PGD2 receptor, chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2), in the progression of adjuvant-induced joint inflammation. Injection of CFA into the ankle joint stimulated PGD2 production and induced paw swelling in both CRTH2-naive (WT) and CRTH2(-/-) mice. CRTH2(-/-) mice presented more severe arthritic manifestations than did WT mice. Through bone marrow transplantation experiments between WT and CRTH2(-/-) mice, we showed that CRTH2 deficiency in bone marrow-derived immune cells is involved in disease progression. Morphological studies showed that CRTH2 deficiency accelerated the infiltration of macrophages into the inflamed paw. Consistent with this finding, we observed that treatment with the macrophage inactivator GdCl3 or the macrophage-depleting agent liposomal clodronate improved arthritis symptoms in CRTH2(-/-) mice. Adoptive transfer of CRTH2(-/-) macrophages exacerbated joint inflammation in WT mice. In addition, CRTH2 deficiency accelerated, whereas CRTH2 agonism inhibited, the expression of a macrophage-activating cytokine (GM-CSF) and a chemokine receptor (CXCR2) in CFA-treated peritoneal macrophages. Together, these observations demonstrate that PGD2-CRTH2 signaling plays a protective role in joint inflammation by attenuating the infiltration of macrophages.

Opposing immunomodulatory roles of prostaglandin D2 during the progression of skin inflammation.

The effects of PGD2 are extremely context dependent. It can have pro- or anti-inflammatory effects in clinically important pathological conditions. A greater mechanistic insight into the determinants of PGD2 activity during inflammation is thus required. In this study, we investigated the role of PGD2 in croton oil-induced dermatitis using transgenic (TG) mice overexpressing hematopoietic PGD synthase. Administration of croton oil caused tissue swelling and vascular leakage in the mouse ear. Compared with wild-type animals, TG mice produced more PGD2 and showed decreased inflammation in the early phase, but more severe manifestations during the late phase. Data obtained from bone marrow transplantation between wild-type and TG mice indicated that PGD2 produced by tissue resident cells in the TG mice attenuated early-phase inflammation, whereas PGD2 produced from hematopoietic lineage cells exacerbated late-phase inflammation. There are two distinct PGD2 receptors: D-prostanoid receptor (DP) and chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2). In TG mice, treatment with a DP antagonist exacerbated inflammation in the early phase, whereas treatment with a CRTH2 antagonist attenuated inflammation during the late phase. In vitro experiments showed that DP agonism enhanced vascular endothelial barrier formation, whereas CRTH2 agonism stimulated neutrophil migration. Collectively, these results show that when hematopoietic PGD synthase is overexpressed, tissue resident cell-derived PGD2 suppresses skin inflammation via DP in the early phase, but hematopoietic lineage cell-derived PGD2 stimulates CRTH2 and promotes inflammation during the late phase. DP-mediated vascular barrier enhancement or CRTH2-mediated neutrophil activation may be responsible for these effects. Thus, PGD2 represents opposite roles in inflammation, depending on the disease phase in vivo.

Anti-inflammatory role of PGD2 in acute lung inflammation and therapeutic application of its signal enhancement.

We investigated the role of prostaglandin D2 (PGD2) signaling in acute lung injury (ALI), focusing on its producer-effector interaction in vivo. Administration of endotoxin increased edema and neutrophil infiltration in the WT mouse lung. Gene disruption of hematopoietic PGD synthase (H-PGDS) aggravated all of the symptoms. Experiments involving bone marrow transplantation between WT and H-PGDS-deficient mice showed that PGD2 derived from alveolar nonhematopoietic lineage cells (i.e., endothelial cells and epithelial cells) promotes vascular barrier function during the early phase (day 1), whereas neutrophil-derived PGD2 attenuates its own infiltration and cytokine expression during the later phase (day 3) of ALI. Treatment with either an agonist to the PGD2 receptor, DP, or a degradation product of PGD2, 15-deoxy-Δ(12,14)-PGJ2, exerted a therapeutic action against ALI. Data obtained from bone marrow transplantation between WT and DP-deficient mice suggest that the DP signal in alveolar endothelial cells is crucial for the anti-inflammatory reactions of PGD2. In vitro, DP agonism directly enhanced endothelial barrier formation, and 15-deoxy-Δ(12,14)-PGJ2 attenuated both neutrophil migration and cytokine expression. These observations indicate that the PGD2 signaling between alveolar endothelial/epithelial cells and infiltrating neutrophils provides anti-inflammatory effects in ALI, and suggest the therapeutic potential of these signaling enhancements.

VEGF-induced blood flow increase causes vascular hyper-permeability in vivo.

VEGF is known to cause vascular leak, its detailed mechanisms in vivo remain unclear. Here, we investigated the mechanisms underlying VEGF-induced vascular hyper-permeability focusing on two major regulators of vascular permeability: blood flow and endothelial barrier function. Administration of VEGF caused vascular hyper-permeability and tissue swelling in mouse ears, which were abolished by VEGF receptor-2 blockade. Intravital imaging showed that VEGF dilated ear arteries but not veins, and laser Doppler velocimetry showed that VEGF quickly increased tissue blood flow along with arterial dilation. Whole-mount immunostaining showed that VEGF phosphorylated endothelial nitric oxide synthase (eNOS) at residue Ser1177 and disrupted the alignment of vascular endothelial-cadherin (VE-cadherin) around the endothelial cell borders in mouse ear skin, indicating endothelial nitric oxide (NO) production and barrier disruption. Administration of the nitric oxide synthesis inhibitor, L-NAME, as well as the vasoconstrictor phenylephrine, abolished all VEGF-induced responses, including blood flow increase, dye leakage, and tissue swelling. However, these two treatments did not alter the intracellular localization of VE-cadherin-induced by VEGF. These observations underscore the importance of vascular dilation and, subsequent increase in blood flow, as well as, endothelial barrier disruption in the mechanisms of VEGF-induced vascular hyper-permeability.

Prostaglandin D2-DP signaling promotes endothelial barrier function via the cAMP/PKA/Tiam1/Rac1 pathway.

OBJECTIVE: Prostaglandin D(2) (PGD(2)) is one of the prostanoids produced during inflammation. Although PGD(2) is known to decrease endothelial permeability through D prostanoid (DP) receptor stimulation, the detailed mechanism is unknown. METHODS AND RESULTS: Treatment with PGD(2) (0.1-3 µmol/L) or the DP receptor agonist, BW245C (0.1-3 µmol/L), dose-dependently increased transendothelial electrical resistance and decreased the FITC-dextran permeability of human umbilical vein endothelial cells. Both indicated decreased endothelial permeability. These phenomena were accompanied by Tiam1/Rac1-dependent cytoskeletal rearrangement. BW245C (0.3 µmol/L) increased the intracellular cAMP level and subsequent protein kinase A (PKA) activity. Pretreatment with PKA inhibitory peptide, but not gene depletion of exchange protein directly activated by cAMP 1 (Epac1), attenuated BW245C-induced Rac1 activation and transendothelial electric resistance increase. In vivo, application of 2.5% croton oil or histamine (100 µg) caused vascular leakage indexed by dye extravasation. Pretreatment with BW245C (1 mg/kg) attenuated the dye extravasation. Gene deficiency of DP abolished, or inhibition of PKA significantly reduced, the DP-mediated barrier enhancement. CONCLUSIONS: PGD(2)-DP signaling reduces vascular permeability both in vivo and in vitro. This phenomenon is mediated by cAMP/PKA/Tiam1-dependent Epac1-independent Rac1 activation and subsequent enhancement of adherens junction in endothelial cell.

Bile acid receptor TGR5 agonism induces NO production and reduces monocyte adhesion in vascular endothelial cells.

OBJECTIVE: TGR5 is a G-protein-coupled receptor for bile acids. So far, little is known about the function of TGR5 in vascular endothelial cells. APPROACH AND RESULTS: In bovine aortic endothelial cells, treatment with a bile acid having a high affinity to TGR5, taurolithocholic acid (TLCA), significantly increased NO production. This effect was abolished by small interfering RNA-mediated depletion of TGR5. TLCA-induced NO production was also observed in human umbilical vein endothelial cells measured via intracellular cGMP accumulation. TLCA increased endothelial NO synthase(ser1177) phosphorylation in human umbilical vein endothelial cells. This response was accompanied by increased Akt(ser473) phosphorylation and intracellular Ca(2+). Inhibition of these signals significantly decreased TLCA-induced NO production. We next examined whether TGR5-mediated NO production affects inflammatory responses of endothelial cells. In human umbilical vein endothelial cells, TLCA significantly reduced tumor necrosis factor-α-induced adhesion of monocytes, vascular cell adhesion molecule-1 expression, and activation of nuclear factor-κB. TLCA also inhibited lipopolysaccharide-induced monocyte adhesion to mesenteric venules in vivo. These inhibitory effects of TLCA were abrogated by NO synthase inhibition. CONCLUSIONS: TGR5 agonism induces NO production via Akt activation and intracellular Ca(2+) increase in vascular endothelial cells, and this function inhibits monocyte adhesion in response to inflammatory stimuli.

Halichlorine reduces monocyte adhesion to endothelium through the suppression of nuclear factor-kappaB activation.

Halichlorine is a marine alkaloid isolated from the marine sponge Halichondria okadai KADOTA, and its pathophysiological effect on vascular cells remains unknown. Here, we examined the anti-atherosclerosis activity of halichlorine on endothelial cells by assessing the expression of adhesion molecules. In bovine aortic endothelial cells (BAECs), pretreatment with halichlorine (10 microM, 2 h) inhibited lipopolysaccharide (LPS) (3 microg/ml, 3 h)-induced mRNA expressions of vascular cell adhesion molecule (VCAM-1), intercellular adhesion molecule (ICAM-1), and E-selectin. Consistently, pretreatment with halichlorine (10 microM, 2 h) reduced LPS (3 microg/ml)-induced monocyte (U937) adhesion to endothelial monolayer. To investigate the mechanism underlying this phenomenon, we examined the effect of halichlorine on nuclear factor-kappaB (NF-kappaB) activity in endothelial cells. Treatment with LPS (3 microg/ml) for 1 h increased the ratio of cells showing NF-kappaB p65 translocation from cytosol to nucleus. Pretreatment with halichlorine (10 microM, 2 h) significantly inhibited the LPS-induced NF-kappaB p65 translocation. Finally, we examined the cytotoxicity of halichlorine on endothelial cells and found that halichlorine (10 microM, 24 - 48 h) did not influence BAECs proliferation and viability. Herein, we provided the first evidence that halichlorine inhibits LPS-induced NF-kappaB activation, which results in the suppression of VCAM-1, ICAM-1, and E-selectin gene expression and monocyte-adhesion to endothelial cells.

T Cell Activation Depends on Extracellular Alanine.

T cell stimulation is metabolically demanding. To exit quiescence, T cells rely on environmental nutrients, including glucose and the amino acids glutamine, leucine, serine, and arginine. The expression of transporters for these nutrients is tightly regulated and required for T cell activation. In contrast to these amino acids, which are essential or require multi-step biosynthesis, alanine can be made from pyruvate by a single transamination. Here, we show that extracellular alanine is nevertheless required for efficient exit from quiescence during naive T cell activation and memory T cell restimulation. Alanine deprivation leads to metabolic and functional impairments. Mechanistically, this vulnerability reflects the low expression of alanine aminotransferase, the enzyme required for interconverting pyruvate and alanine, whereas activated T cells instead induce alanine transporters. Stable isotope tracing reveals that alanine is not catabolized but instead supports protein synthesis. Thus, T cells depend on exogenous alanine for protein synthesis and normal activation.

Histamine Induces Vascular Hyperpermeability by Increasing Blood Flow and Endothelial Barrier Disruption In Vivo.

Histamine is a mediator of allergic inflammation released mainly from mast cells. Although histamine strongly increases vascular permeability, its precise mechanism under in vivo situation remains unknown. We here attempted to reveal how histamine induces vascular hyperpermeability focusing on the key regulators of vascular permeability, blood flow and endothelial barrier. Degranulation of mast cells by antigen-stimulation or histamine treatment induced vascular hyperpermeability and tissue swelling in mouse ears. These were abolished by histamine H1 receptor antagonism. Intravital imaging showed that histamine dilated vasculature, increased blood flow, while it induced hyperpermeability in venula. Whole-mount staining showed that histamine disrupted endothelial barrier formation of venula indicated by changes in vascular endothelial cadherin (VE-cadherin) localization at endothelial cell junction. Inhibition of nitric oxide synthesis (NOS) by L-NAME or vasoconstriction by phenylephrine strongly inhibited the histamine-induced blood flow increase and hyperpermeability without changing the VE-cadherin localization. In vitro, measurements of trans-endothelial electrical resistance of human dermal microvascular endothelial cells (HDMECs) showed that histamine disrupted endothelial barrier. Inhibition of protein kinase C (PKC) or Rho-associated protein kinase (ROCK), NOS attenuated the histamine-induced barrier disruption. These observations suggested that histamine increases vascular permeability mainly by nitric oxide (NO)-dependent vascular dilation and subsequent blood flow increase and maybe partially by PKC/ROCK/NO-dependent endothelial barrier disruption.

γ-Oryzanol reduces adhesion molecule expression in vascular endothelial cells via suppression of nuclear factor-κB activation.

γ-Oryzanol (γ-ORZ) is a mixture of phytosteryl ferulates purified from rice bran oil. In this study, we examined whether γ-ORZ represents a suppressive effect on the lipopolysaccharide (LPS)-induced adhesion molecule expression on vascular endothelium. Treatment with LPS elevated the mRNA expression of vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), and E-selectin in bovine aortic endothelial cells (BAECs). Pretreatment with γ-ORZ dose-dependently decreased the LPS-mediated expression of these genes. Western blotting also revealed that pretreatment with γ-ORZ dose-dependently inhibited LPS-induced VCAM-1 expression in human umbilical vein endothelial cells. Consistently, pretreatment with γ-ORZ dose-dependently reduced LPS-induced U937 monocyte adhesion to BAECs. In immunofluorescence, LPS caused nuclear factor-κB (NF-κB) nuclear translocation in 40% of BAECs, which indicates NF-κB activation. Pretreatment with γ-ORZ, as well as its components (cycloartenyl ferulate, ferulic acid, or cycloartenol), dose-dependently inhibited LPS-mediated NF-κB activation. Collectively, our results suggested that γ-ORZ reduced LPS-mediated adhesion molecule expression through NF-κB inhibition in vascular endothelium.

Halichlorine is a novel L-type Ca(2+) channel inhibitor isolated from the marine sponge Halichondria okadai Kadota.

Halichlorine, isolated from a marine sponge Halichondria okadai Kadota, has a unique structure and its physiological activity is virtually unknown. In the present study, we investigated the direct effect of halichlorine on vascular contractility. In endothelium-denuded rat aorta, while the treatment of halichlorine (0.01-10microM) did not induce vascular contraction, halichlorine (0.01-10microM) dose-dependently inhibited both the steady-state precontractions induced by high K(+) (65.4mM) and phenylephrine (1microM). The vasodilator effect of halichlorine (10microM) on high K(+) (65.4mM)-induced contraction was more potent than that on phenylephrine (1microM)-induced contraction (65.4mM high K(+): 72.7+/-3.4%; 1microM phenylephrine: 34.7+/-2.3%). To investigate the mechanism underlying the suppressive effect of halichlorine on vascular contractility, we examined the effect of halichlorine on intracellular Ca(2+) concentration in vascular smooth muscle with a fluorescent Ca(2+) indicator, fura-2. Treatment of halichlorine (10microM) significantly inhibited the sustained [Ca(2+)](i) elevation induced by high K(+) (65.4mM) (45.3+/-5.5%). Furthermore, current measurements by whole-cell mode patch-clamp recording in rat aortic smooth muscle cells (A7r5 cells) demonstrated that halichlorine (10microM) decreased the current density of the L-type Ca(2+) channel (peak Ca(2+)-channel current densities: -2.09+/-0.27pA/pF for control; -0.58+/-0.07pA/pF for halichlorine). These results suggest that halichlorine inhibits L-type Ca(2+) channels in vascular smooth muscle cells, which inhibit intracellular Ca(2+) influx, and then reduce vascular contractions.