Immunocytochemical localization of substance P neurokinin-1 receptors in rat gingival tissue

The distributions of substance P (SP) and the neurokinin-1 receptor (NK1-R), the receptor preferentially activated by SP, were examined in rat gingiva by immunocytochemical methods with light and electron microscopy. SP-immunoreactive nerve fibers were located preferentially in the junctional epithelium (JE) but few in the other oral and oral sulcular epithelia. NK1-R immunoreactivity was found in the endothelial cells (capillaries and postcapillary venules underlying the JE). NK1-R-labeled and -unlabeled unmyelinated nerve fibers were located close to the blood vessels and partially or completely covered by a Schwann cell sheath. In the JE, labeled naked axons without Schwann cell sheaths were observed. Neutrophils and macrophages in the connective tissue underlying the JE and in the JE were also labeled with NK1-R. Furthermore, NK1-R was found in the JE cells. Basically, immunoreaction products for NK1-R were found throughout various cells (endothelial cells, neutrophils, and JE cells) at invaginations of the plasma membrane and in vesicular and granular structures that are probably endosomes and are found close to both the plasma membrane and the nucleus. This is a first report, demonstrating the presence of NK1-R in the gingival tissue in the normal nonstimulated condition. Furthermore, it is thought that SP may modulate the permeability of blood vessels beneath the JE, the production of antimicrobial agents in neutrophils, and the proliferation and endocytotic ability of JE cells through NK1-R.     

IL-17 promotes bone erosion in murine collagen-induced arthritis through loss of the receptor activator of NF-kappa B ligand/osteoprotegerin balance

IL-17 is a T cell-derived proinflammatory cytokine in experimental arthritis and is a stimulator of osteoclastogenesis in vitro. In this study, we report the effects of IL-17 overexpression (AdIL-17) in the knee joint of type II collagen-immunized mice on bone erosion and synovial receptor activator of NF-kappa B ligand (RANKL)/receptor activator of NF-kappa B/osteoprotegerin (OPG) expression. Local IL-17 promoted osteoclastic bone destruction, which was accompanied with marked tartrate-resistant acid phosphatase activity at sites of bone erosion in cortical, subchondral, and trabecular bone. Accelerated expression of RANKL and its receptor, receptor activator of NF-kappa B, was found in the synovial infiltrate and at sites of focal bone erosion, using specific immunohistochemistry. Interestingly, AdIL-17 not only enhanced RANKL expression but also strongly up-regulated the RANKL/OPG ratio in the synovium. Comparison of arthritic mice from the AdIL-17 collagen-induced arthritis group with full-blown collagen-arthritic mice having similar clinical scores for joint inflammation revealed lower RANKL/OPG ratio and tartrate-resistant acid phosphatase activity in the latter group. Interestingly, systemic OPG treatment prevented joint damage induced by local AdIL-17 gene transfer in type II collagen-immunized mice. These findings suggest T cell IL-17 to be an important inducer of RANKL expression leading to loss of the RANKL/OPG balance, stimulating osteoclastogenesis and bone erosion in arthritis.     

Hydrogen sulfide acts as a mediator of inflammation in acute pancreatitis: in vitro studies using isolated mouse pancreatic acinar cells

Hydrogen sulphide (H(2)S) is synthesized from L-cysteine via the action of cystathionine-gamma-lyase (CSE) and cystathionine-beta-synthase (CBS). We have earlier shown that H(2)S acts as a mediator of inflammation. However the mechanism remains unclear. In this study, we investigated the presence of H(2)S and the expression of H(2)S synthesizing enzymes, CSE and CBS, in isolated mouse pancreatic acini. Pancreatic acinar cells from mice were incubated with or without caerulein (10(-7) M for 30 and 60 min). Caerulein increased the levels of H(2)S and CSE mRNA expression while CBS mRNA expression was decreased. In addition, cells pre-treated with DL-propargylglycine (PAG, 3 mM), a CSE inhibitor, reduced the formation of H(2)S in caerulein treated cells, suggesting that CSE may be the main enzyme involved in H(2)S formation in mouse acinar cells. Furthermore, substance P (SP) concentration in the acini and expression of SP gene (preprotachykinin-A, PPT-A) and neurokinin-1 receptor (NK-1R), the primary receptor for SP, are increased in secretagogue caerulein-treated acinar cells. Inhibition of endogenous production of H(2)S by PAG significantly suppressed SP concentration, PPT-A expression and NK1-R expression in the acini. To determine whether H(2)S itself provoked inflammation in acinar cells, the cells were treated with H(2)S donor drug, sodium hydrosulphide (NaHS), (10, 50 and 100 muM), that resulted in a significant increase in SP concentration and expression of PPT-A and NK1-R in acinar cells. These results suggest that the pro-inflammatory effect of H(2)S may be mediated by SP-NK-1R related pathway in mouse pancreatic acinar cells.     

Delineation of the endocytic pathway of substance P and its seven-transmembrane domain NK1 receptor.

Many of the actions of the neuropeptide substance P (SP) that are mediated by the neurokinin 1 receptor (NK1-R) desensitize and resensitize, which may be associated with NK1-R endocytosis and recycling. We delineated this endocytic pathway in transfected cells by confocal microscopy using cyanine 3-SP and NK1-R antibodies. SP and the NK1-R were internalized into the same clathrin immunoreactive vesicles, and then sorted into different compartments. The NK1-R was colocalized with a marker of early endosomes, but not with markers of late endosomes or lysosomes. We quantified the NK1-R at the cell surface by incubating cells with an antibody to an extracellular epitope. After exposure to SP, there was a loss and subsequent recovery of surface NK1-R. The loss was prevented by hypertonic sucrose and potassium depletion, inhibitors of clathrin-mediated endocytosis. Recovery was independent of new protein synthesis because it was unaffected by cycloheximide. Recovery required endosomal acidification because it was prevented by an H(+)-ATPase inhibitor. The fate of internalized 125I-SP was examined by chromatography. SP was intact at the cell surface and in early endosomes, but slowly degraded in perinuclear vesicles. We conclude that SP induces clathrin-dependent internalization of the NK1-R. The SP/NK1-R complex dissociates in acidified endosomes. SP is degraded, whereas the NK1-R recycles to the cell surface.     

NK-1 receptor desensitization and neutral endopeptidase terminate SP-induced pancreatic plasma extravasation

Substance P (SP) induces plasma extravasation and neutrophil infiltration by activating the neurokinin-1 receptor (NK1-R). We characterized the mechanisms regulating this response in the rat pancreas. Anesthetized rats were continuously infused with SP, and plasma extravasation was quantified using Evans blue (EB) dye. Continuous infusion of SP (8 nmol. kg(-1). h(-1)) resulted in a threshold increase in EB at 15 min, a peak effect at 30 min (150% increase), and a return to baseline by 60 min. The NK1-R antagonist CP-96,345 blocked SP-induced plasma extravasation. After 60 min, the NK1-R was desensitized to agonist challenge. Resensitization was first detected at 20 min and increased until full recovery was seen at 30 min. Inhibition of the cell-surface protease neutral endopeptidase (NEP) by phosphoramidon potentiated the effect of exogenous SP; therefore endogenous NEP attenuates SP-induced plasma extravasation. Thus the continuous infusion of SP stimulates plasma extravasation in the rat pancreas via activation of the NK1-R, and these effects are terminated by both desensitization of the NK1-R and the cell-surface protease NEP.     

Stromal cell-derived factor-1-CXC chemokine receptor 4 interactions play a central role in CD4+ T cell accumulation in rheumatoid arthritis synovium

Rheumatoid arthritis (RA) is characterized by the accumulation of CD4(+) memory T cells in the inflamed synovium. To address the mechanism, we analyzed chemokine receptor expression and found that the frequency of CXC chemokine receptor (CXCR)4 expressing synovial tissue CD4(+) memory T cells was significantly elevated. CXCR4 expression could be enhanced by IL-15, whereas stromal cell-derived factor (SDF)-1, the ligand of CXCR4, was expressed in the RA synovium and could be increased by CD40 stimulation. SDF-1 stimulated migration of rheumatoid synovial T cells and also inhibited activation-induced apoptosis of T cells. These results indicate that SDF-1-CXCR4 interactions play important roles in CD4(+) memory T cell accumulation in the RA synovium, and emphasize the role of stromal cells in regulating rheumatoid inflammation.     

RANK ligand, RANK, and OPG expression in type II collagen-induced arthritis mouse

Rheumatoid arthritis (RA) is a systemic disorder characterized by synovial inflammation and subsequent destruction and deformity of synovial joints. The articular lesions start with synovitis, focal erosion of unmineralized cartilage, and then culminate in the destruction of subarticular bone by pannus tissue. Periarticular osteopenia and systemic osteoporosis follow as late complications of RA. Osteoclasts, specialized cells that resorb bone, play a central role in developing these osteolytic lesions. To elucidate the mechanism of osteoclastogenesis and bone destruction in autoimmune arthritis, we investigated the expression of RANK ligand (RANKL), RANK, and osteoprotegerin (OPG) mRNA in a mouse type II collagen-induced arthritis (CIA) model by in situ hybridization. The results indicated that most of the TRAP-positive mono- and multinucleated cells in the inflamed and proliferating synovium and in the pannus were RANK-positive authentic osteoclasts and their precursors. In the inflamed synovium and pannus of the mouse CIA model, synovial fibroblastic cells around these RANK-positive cells were strongly positive for RANKL. Moreover, RANKL-positive osteoblasts on the endosteal bone surface, at a distance from the affected synovial joints, increased significantly in the mouse CIA model prior to periarticular osteopenia and systemic osteoporosis. These data indicated that the RANKL-RANK system plays an important role for osteoclastogenesis in both local and systemic osteolytic lesions in autoimmune arthritis, and can therefore be a good target for therapeutic intervention.     

Role of C-reactive protein in osteoclastogenesis in rheumatoid arthritis

IntroductionC-reactive protein (CRP) is one of the biomarkers for the diagnosis and assessment of disease activity in rheumatoid arthritis (RA). CRP is not only the by-product of inflammatory response, but also plays proinflammatory and prothrombotic roles. The aim of this study was to determine the role of CRP on bone destruction in RA.MethodsCRP levels in RA synovial fluid (SF) and serum were measured using the immunoturbidimetric method. The expression of CRP in RA synovium was assessed using immunohistochemical staining. CD14+ monocytes from peripheral blood were cultured with CRP, and receptor activator of nuclear factor-κB ligand (RANKL) expression and osteoclast differentiation were evaluated using real-time PCR, counting tartrate resistant acid phosphatase (TRAP)-positive multinucleated cells and assessing bone resorbing function. CRP-induced osteoclast differentiation was also examined after inhibition of Fcγ receptors.ResultsThere was a significant correlation between CRP levels in serum and SF in RA patients. The SF CRP level was correlated with interleukin (IL)-6 levels, but not with RANKL levels. Immunohistochemical staining revealed that compared with the osteoarthritis synovium, CRP was more abundantly expressed in the lining and sublining areas of the RA synovium. CRP stimulated RANKL production in monocytes and it induced osteoclast differentiation from monocytes and bone resorption in the absence of RANKL.ConclusionsCRP could play an important role in the bony destructive process in RA through the induction of RANKL expression and direct differentiation of osteoclast precursors into mature osteoclasts. In the treatment of RA, lowering CRP levels is a significant parameter not only for improving disease activity but also for preventing bone destruction.     

Vascular endothelial growth factor signaling in VE-cadherin expression and tube-like formation by rheumatoid arthritic synovial fibroblast-like cells

An increase in the vasculature is one of most representative changes in the synovial tissue of joints in rheumatoid arthritis (RA) and is closely associated with disease progression. Although the vasculatures are believed to be a result of VE-cadherin-dependent angiogenesis and a possible therapeutic target of the disease, synovial fibroblastic cells express VE-cadherin and form tube-like structures, suggesting that vasculatures in RA synovium may not simply result from angiogenesis. This paper analyzes a mechanism of VE-cadherin expression by rheumatoid arthritic synovial fibroblast-like cells (RSFLs) and their involvement in the tube-like formation. A representative angiogenic factor, vascular endothelial growth factor (VEGF), and its binding to a predominant receptor (VEGFR2) activated VE-cadherin expression and the signaling pathways of ERK/MAPK and PI3K/AKT/mTOR. Treatment of RSFLs with signaling pathway inhibitors, VEGFR2 siRNA and a VEGF-antagonizing mimicking peptide inhibited VE-cadherin expression dose-dependently. VEGF-stimulated tube-like formation by RSFLs on Matrigel was hindered by the mimicking peptide and inhibitor treatment. This data demonstrates that RSFLs activated by VEGF binding of VEGFR2 express VE-cadherin and formed tube-like structure under the control of ERK/MAPK and PI3K/AKT/mTOR pathways suggesting that the inhibition suppresses vascular development in RA synovium.     

Characterization of synovial cell clones isolated from rheumatoid arthritis patients: possible involvement of TNF-alpha in reduction of osteoprotegerin in synovium

To elucidate the role of the synovium in bone destruction by osteoclasts in rheumatoid arthritis (RA), primary synovial cells isolated from RA patients were cultured and characterized. The cultured primary cells did not produce RANKL (TRANCE/ODF/OPGL/TNFSF11/CD254), an inducer of osteoclast differentiation, but constitutively produced its inhibitor, osteoprotegerin (OPG). Addition of TNF-alpha to the primary cultures of synovial cells reduced the cell viability and strongly suppressed OPG production. We then established nine synovial cell clones, including SYM-1, responsible for OPG production from primary synovial cell cultures. TNF-alpha induced apoptosis of SYM-1 cells within 24h and decreased OPG levels, while infliximab, a chimerical form of the anti-TNF-alpha antibody drug, suppressed the apoptosis and restored OPG levels. These results suggest the existence of fibroblastic cells producing OPG in the synovium, while TNF-alpha suppresses OPG production by inducing apoptosis in those cells. Further, infliximab is considered to inhibit bone destruction through restoration of OPG levels in RA.     

Basic fibroblast growth factor accelerates matrix degradation via a neuro-endocrine pathway in human adult articular chondrocytes

Pain-related neuropeptides released from synovial fibroblasts, such as substance P, have been implicated in joint destruction. Substance P-induced inflammatory processes are mediated via signaling through a G-protein-coupled receptor, that is, neurokinin-1 tachykinin receptor (NK(1)-R). We determined the pathophysiological link between substance P and its receptor in human adult articular cartilage homeostasis. We further examined if catabolic growth factors such as basic fibroblast growth factor (bFGF or FGF-2) or IL-1beta accelerate matrix degradation via a neural pathway upregulation of substance P and NK(1)-R. We show here that substance P stimulates the production of cartilage-degrading enzymes, such as matrix metalloproteinase-13 (MMP-13), and suppresses proteoglycan deposition in human adult articular chondrocytes via NK(1)-R. Furthermore, we have demonstrated that substance P negates proteoglycan stimulation promoted by bone morphogenetic protein-7, suggesting the dual role of substance P as both a pro-catabolic and anti-anabolic mediator of cartilage homeostasis. We report that bFGF-mediated stimulation of substance P and its receptor NK(1)-R is, in part, through an IL-1beta-dependent pathway.     

Protein kinase C-mediated desensitization of the neurokinin 1 receptor

An understanding of the mechanisms that regulate signaling bythe substance P (SP) or neurokinin 1 receptor (NK1-R) is of interestbecause of their role in inflammation and pain. By using activators andinhibitors of protein kinase C (PKC) and NK1-R mutations of potentialPKC phosphorylation sites, we determined the role of PKC indesensitization of responses to SP. Activation of PKC abolishedSP-induced Ca²⁺ mobilization in cells that expresswild-type NK1-R. This did not occur in cells expressing aCOOH-terminally truncated NK1-R (NK1-R324), which may correspond toa naturally occurring variant, or a point mutant lacking eightpotential PKC phosphorylation sites within the COOH tail (NK1-RSer-338, Thr-339, Ser-352, Ser-387, Ser-388, Ser-390, Ser-392,Ser-394/Ala, NK1-RKC4). Compared with wild-type NK1-R, thet_1/2 of SP-induced Ca²⁺mobilization was seven- and twofold greater in cells expressing NK1-R324 and NK1-RKC4, respectively. In cells expressing wild-type NK1-R, inhibition of PKC caused a 35% increase in thet_1/2 of SP-induced Ca²⁺mobilization. Neither inhibition of PKC nor receptor mutation affecteddesensitization of Ca²⁺ mobilization to repeated challengewith SP or SP-induced endocytosis of the NK1-R. Thus PKC regulatesSP-induced Ca²⁺ mobilization by full-length NK1-R and doesnot regulate a naturally occurring truncated variant. PKC doesnot mediate desensitization to repeated stimulation or endocytosis ofthe NK1-R.

     

Desensitization of the Neurokinin-1 Receptor (NK1-R) in Neurons: Effects of Substance P on the Distribution of NK1-R, Gαq/11, G-Protein Receptor Kinase-2/3, and β-Arrestin-1/2

Observations in reconstituted systems and transfected cells indicate that G-protein receptor kinases (GRKs) and β-arrestins mediate desensitization and endocytosis of G-protein–coupled receptors. Little is known about receptor regulation in neurons. Therefore, we examined the effects of the neurotransmitter substance P (SP) on desensitization of the neurokinin-1 receptor (NK1-R) and on the subcellular distribution of NK1-R, G_αq/11, GRK-2 and -3, and β-arrestin-1 and -2 in cultured myenteric neurons. NK1-R was coexpressed with immunoreactive G_αq/11, GRK-2 and -3, and β-arrestin-1 and -2 in a subpopulation of neurons. SP caused 1) rapid NK1-R–mediated increase in [Ca²⁺]_i, which was transient and desensitized to repeated stimulation; 2) internalization of the NK1-R into early endosomes containing SP; and 3) rapid and transient redistribution of β-arrestin-1 and -2 from the cytosol to the plasma membrane, followed by a striking redistribution of β-arrestin-1 and -2 to endosomes containing the NK1-R and SP. In SP-treated neurons G_αq/11 remained at the plasma membrane, and GRK-2 and -3 remained in centrally located and superficial vesicles. Thus, SP induces desensitization and endocytosis of the NK1-R in neurons that may be mediated by GRK-2 and -3 and β-arrestin-1 and -2. This regulation will determine whether NK1-R–expressing neurons participate in functionally important reflexes.     

Hyaluronan Inhibits Tlr-4-Dependent RANKL Expression in Human Rheumatoid Arthritis Synovial Fibroblasts

The Toll-like receptor (TLR) signaling pathway is activated in synovial fibroblast cells in patients with rheumatoid arthritis (RA). The receptor activator of nuclear factor-κB (RANK) and its ligand, RANKL, are key molecules involved in the differentiation of osteoclasts and joint destruction in RA. Hyaluronan (HA) is a major extracellular component and an important immune regulator. In this study, we show that lipopolysaccharide (LPS) stimulation significantly increases RANKL expression via a TLR-4 signaling pathway. We also demonstrate that HA suppresses LPS-induced RANKL expression, which is dependent on CD44, but not intercellular adhesion molecule-1 (ICAM-1). Our study provides evidence for HA-mediated suppression of TLR-4-dependent RANKL expression. This could present an alternative target for the treatment of destructed joint bones and cartilages in RA.     

Osteoarthritic tissues modulate functional properties of sensory neurons associated with symptomatic OA pain

Osteoarthritis (OA) is an age-related degenerative disease of cartilaginous tissues that is accompanied by hyperalgesia. Molecular cause and effect relationships between OA and pain remain to be elucidated. In this study, we have developed an experimental ex vivo organ co-culture system with dorsal root ganglia (DRGs) and knee synovial tissues from OA patients or unaffected human subjects. Our results suggest that tissues may generate symptomatic pain by altering the functional properties of sensory neurons. Specifically, we find that the expression levels of genes associated with neuronal pathways (e.g., SP, NK1, NK2, NPYR1, NPYR2, α2δ1) or inflammation (COX2/PTGS2 and IL6/interferon β2) are clearly elevated in DRG explants cultured in the presence of OA derived synovial tissues. These findings are consistent with a model in which cytokines and pain molecules produced by knee synovium sensitize nociceptive neurons in tissues peripheral to joint cartilage.     

生物肽SP对NK细胞NKG2D/NKG2A受体表达的影响

选择NK92-MI细胞为研究体系,研究SP对NK细胞的杀伤活性及功能性受体NKG2D/NKG2A表达的影响,以探讨SP对NK细胞功能的调节作用机制。采用MTT法测定NK92-MI细胞对K562细胞的杀伤活性;采用Real-Time PCR和流式细胞术检测NK92-MI细胞活化性受体NKG2D和抑制性受体NKG2A的基因表达和膜表达。10-14～10-8 mol/L的SP在体外可明显增强NK92-MI细胞的杀伤活性。该浓度范围的SP均可上调NKG2D/NKG2A的mRNA水平;10-14～10-8 mol/L的SP均上调NKG2D/NKG2A的膜表达,较低浓度（10-14 mol/L）的SP仅使NKG2D表达上调,而NKG2A表达无明显变化;SP刺激NKG2D膜表达增加的程度高于NKG2A。生物肽SP调节NK细胞功能性受体NKG2D/NKG2A的表达,可能是SP增强NK细胞杀伤活性的一种原因。     

The Effect of Vascular Endothelial Growth Factor on Osteoclastogenesis in Rheumatoid Arthritis

Vascular endothelial growth factor (VEGF) has angiogenic, inflammatory, and bone-destructive roles in rheumatoid arthritis (RA). We aimed to determine the unique role of VEGF in osteoclastogenesis in RA. VEGF-induced receptor activator of nuclear factor ҡB ligand (RANKL) expression was determined in RA synovial fibroblasts by real-time PCR, luciferase assays, and ELISA. Osteoclastogenesis in peripheral blood monocytes cultured with VEGF was assessed by determining the numbers of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells. Synovial fluid RANKL was correlated with VEGF concentration in the RA patients. VEGF stimulated the expression of RANKL in RA synovial fibroblasts. The RANKL promoter activity was upregulated by VEGF in the synovial fibroblasts transfected with RANKL-reporter plasmids. The VEGF-induced RANKL expression was decreased by the inhibition of both VEGF receptors (VEGFR) 1 and 2, Src, protein kinase C (PKC) and p38 MAPK. VEGF induced osteoclast differentiation from monocytes in the absence of RANKL and this was decreased by the inhibition of VEGFR1 and 2, Src, PKC and p38 MAPK. On coculturing with VEGF-prestimulated RA synovial fibroblasts, the monocytes differentiated into osteoclasts, and the osteoclastogenesis decreased by inhibition of Src and PKC pathways. VEGF plays dual roles on osteoclastogenesis in RA: direct induction of osteoclastogenesis from the precursors and stimulation of RANKL production in synovial fibroblasts, which is mediated by Src and PKC pathways. The axis of VEGF and RANKL could be a potential therapeutic target for RA-associated bone destruction.