Ramasamy Tamizhselvi

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

Hydrogen sulphide (H2S) is synthesized from L‐cysteine via the action of cystathionine‐γ‐lyase (CSE) and cystathionine‐β‐synthase (CBS). We have earlier shown that H2S acts as a mediator of inflammation. However the mechanism remains unclear. In this study, we investigated the presence of H2S and the expression of H2S 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 H2S 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 H2S in caerulein treated cells, suggesting that CSE may be the main enzyme involved in H2S 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 H2S by PAG significantly suppressed SP concentration, PPT‐A expression and NK1‐R expression in the acini. To determine whether H2S itself provoked inflammation in acinar cells, the cells were treated with H2S donor drug, sodium hydrosulphide (NaHS), (10, 50 and 100 μM), 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 H2S may be mediated by SP‐NK‐1R related pathway in mouse pancreatic acinar cells.

Substance P enhances NF-κB transactivation and chemokine response in murine macrophages via ERK1/2 and p38 MAPK signaling pathways

The neuropeptide substance P (SP), as a major mediator of neuroimmunomodulatory activity, modulates diverse functions of immune cells, including macrophages. In the current study, we focused on the yet uncertain role of SP in enhancing the inducible/inflammatory chemokine response of macrophages and the signaling mechanism involved. We studied the effect on the murine monocyte/macrophage cell line RAW 264.7 as well as isolated primary macrophages. Our data show that SP, at nanomolar concentrations, elicited selective chemokine production from murine macrophages. Among the chemokines examined, macrophage inflammatory protein-2 and monocyte chemoattractant protein-1 are two major chemokines that were synthesized by macrophages in response to SP. Furthermore, SP treatment strongly induced the classic pathway of IkappaB-dependent NF-kappaB activation and enhanced DNA binding as well as transactivation activity of the transcription factor. SP-evoked transcriptional induction of chemokines was specific, since it was blocked by treatment with selective neurokinin-1 receptor antagonists. Moreover, SP stimulation of macrophages activated the ERK1/2 and p38 MAPK but not JNKs. Blockade of these two MAPK pathways with specific inhibitors abolished SP-elicited nuclear translocation of phosphorylated NF-kappaB p65 and NF-kappaB-driven chemokine production, suggesting that the two MAPKs lie in the signaling pathways leading to the chemokine response. Collectively, our data demonstrate that SP enhances selective inflammatory chemokine production by murine macrophages via ERK/p38 MAPK-mediated NF-kappaB activation.

Inhibition of hydrogen sulfide synthesis attenuates chemokine production and protects mice against acute pancreatitis and associated lung injury.

Objectives: The present study investigated whether chemokines are involved in hydrogen sulfide (H2S)-associated pathogenesis of acute pancreatitis and associated lung injury. Methods: We have examined the effect of DL-propargylglycine, a cystathionine &ggr;-lyase inhibitor, on the synthesis of CC chemokine monocyte chemotactic protein 1, Regulated upon Activation, Normal T-cell Expressed, and Secreted, and macrophage inflammatory protein-1&agr; (MIP-1&agr;), and CXC chemokine MIP-2 in an in vitro and in vivo model of cerulein-induced acute pancreatitis and associated lung injury. In addition, the pancreatic acinar cells were treated with H2S donor drug, sodium hydrosulfide. The expression ofthese chemokines in the pancreatic acini, pancreas, and lungs wasdetermined by quantitative real-time reverse transcriptase polymerase chain reaction, enzyme-linked immunosorbent assay, and immunohistochemistry. Results: After treatment with DL-propargylglycine, reverse transcriptase polymerase chain reaction, and enzyme-linked immunosorbent assay demonstrated down-regulation of cerulein-induced increase in monocyte chemotactic protein 1, MIP-1&agr;, and MIP-2 expression but had no apparent effect on Regulated upon Activation, Normal T-cell Expressed, and Secreted expression. Conclusions: These results suggest that the proinflammatory effect of H2S may be mediated by chemokines.

Pro-inflammatory effects of hydrogen sulphide on substance P in caerulein-induced acute pancreatitis

Hydrogen sulphide (H2S), a novel gasotransmitter, has been recognized to play an important role in inflammation. Cystathionine‐γ‐lyase (CSE) is a major H2S synthesizing enzyme in the cardiovascular system and DL‐propargylglycine (PAG) is an irreversible inhibitor of CSE. Substance P (SP), a product of preprotachykinin‐A (PPT‐A) gene, is a well‐known pro‐inflammatory mediator which acts principally through the neurokinin‐1 receptor (NK‐1R). We have shown an association between H2S and SP in pulmonary inflammation as well as a pro‐inflammatory role of H2S and SP in acute pancreatitis. The present study was aimed to investigate the interplay between pro‐inflammatory effects of H2S and SP in a murine model of caerulein‐induced acute pancreatitis. Acute pancreatitis was induced in mice by 10 hourly intraperitoneal injections of caerulein (50 (g/kg). PAG (100 mg/kg, i.p.) was administered either 1 hr before (prophylactic) or 1 hr after (therapeutic) the first caerulein injection. PAG, given prophylactically as well as therapeutically, significantly reduced plasma H2S levels and pancreatic H2S synthesizing activities as well as SP concentrations in plasma, pancreas and lung compared with caerulein‐induced acute pancreatitis. Furthermore, prophylactic as well as therapeutic administration of PAG significantly reduced PPT‐A mRNA expression and NK‐1R mRNA expression in both pancreas and lung when compared with caerulein‐induced acute pancreatitis. These results suggest that the pro‐inflammatory effects of H2S may be mediated by SP‐NK‐1R pathway in acute pancreatitis.

Hydrogen sulfide induces ICAM-1 expression and neutrophil adhesion to caerulein-treated pancreatic acinar cells through NF-κB and Src-family kinases pathway

We have earlier shown that mouse pancreatic acinar cells produce hydrogen sulfide (H(2)S), which plays a key role in the pathogenesis of acute pancreatitis (AP). H(2)S-dependent induction of inflammation is mediated by the activation of transcription factor NF-kappaB. We now provide evidence that activation of Src family kinases (SFKs) is crucial in signaling H(2)S-induced intracellular adhesion molecule (ICAM)-1 expression via NF-kappaB. Stimulation of acini with H(2)S resulted in a time-dependent activation of SFKs. In order to better understand this effect of H(2)S, acinar cells were stimulated with caerulein after addition of H(2)S donor, NaHS. Inhibition of SFKs impaired H(2)S-induced NF-kappaB activity and ICAM-1 expression in caerulein treated acinar cells. We also observed that H(2)S-induced up-regulation of ICAM-1 enhanced the adhesion of neutrophils onto acinar cells. Analysis of NF-kappaB pathway revealed that the effect of SFKs inhibition correlated with IkappaBalpha degradation and NF-kappaB DNA binding function. Interestingly, H(2)S-induced association of SFKs with translocation of NF-kappaB, and inhibition of SFKs prevented this response, indicating that this interaction may depend on activation of SFKs. These data suggest that H(2)S, by activating the phosphorylation of SFKs, may promote the transcriptional activity of NF-kappaB and eventually lead to an upregulation of ICAM-1 expression.

Role of protein kinase C and phosphoinositide 3-kinase-Akt in substance P-induced proinflammatory pathways in mouse macrophages

Neuropeptide modulation of immune cell function is an important mechanism of neuroimmune intersystem crosstalk. Substance P (SP) is one such key neuropeptide involved. In this study, we investigated the yet unexplored cellular mechanisms of SP‐mediated inflammatory responses in macrophages using a mouse macrophage‐like cell line RAW 264.7 and isolated peritoneal macrophages. We found that the conventional PKCα and novel PKCδ and ε were selectively activated by SP via its primary neurokinin‐1 receptor (NK‐1R) on the cells. Activation of these PKC isoforms mediated the activation of downstream extracellular signal‐regulated kinase‐1/2 (ERK1/2) and the transcription factor NF‐κB, which drove the transcription of inducible chemokines in macrophages. Additionally, phosphoinositide 3‐kinase (PI3K)‐Akt was also activated by SP/NK‐1R in macrophages. Inhibition of PI3K‐Akt pathway attenuated ERK1/2 and NF‐κB activation, suggesting it also played a part in SP‐induced cellular inflammatory response. Kinetic analysis indicated that PKC isoforms induced early ERK1/2 activation, while PI3K‐Akt contributed to the pathway at later time points. It was further demonstrated that PKC and PI3K‐Akt were activated independent of each other. Collectively, our results suggest that SP/NK‐1R activates two convergent proinflammatory signaling pathways, PKCs and PI3K‐Akt, resulting in ERK1/2 and NF‐κB activation and chemokine production in mouse macrophages.—Sun, J., Ramnath, R. D., Tamizhselvi, R., Bhatia, M. Role of protein kinase C and phosphoinositide 3‐kinase‐Akt in substance P‐induced proinflammatory pathways in mouse macrophages. FASEB J. 23, 997–1010 (2009)

Effect of Hydrogen Sulfide on the Phosphatidylinositol 3-Kinase-Protein Kinase B Pathway and on Caerulein-Induced Cytokine Production in Isolated Mouse Pancreatic Acinar Cells

We have shown earlier that mouse pancreatic acinar cells produce hydrogen sulfide (H2S) and play a role in the pathogenesis of acute pancreatitis. It is noteworthy that recent evidence indicates that H2S has anti-inflammatory effects. To date, the mechanism by which H2S directly reduces inflammation has not been elucidated. In the present study, we hypothesized that H2S inhibits the production of proinflammatory cytokines by activation of the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) pathway. Pancreatic acinar cells were treated with the H2S donor, sodium hydrogen sulfide (NaHS) (5, 10, and 30 μM). To better understand the effect of H2S in inflammation, pancreatic acinar cells were stimulated with caerulein after the addition of NaHS (5, 10, and 30 μM). We observed that H2Sat the 5 μM concentration down-regulates the activation of NF-κB and degradation of IκBα. However, H2S(5 μM) activates PI3K as reflected by AKT phosphorylation. We found that H2S-mediated activation of PI3K in caerulein-treated acinar cells correlated with the down-regulation of extracellular signal-regulated kinase 1/2 phosphorylation, whereas phosphorylation of p38 and c-Jun NH2-terminal kinase and mitogen-activated protein kinases was unchanged. The PI3K inhibitor LY294002 [2-(4-morpholinyl)-8-phenyl-1(4H)-benzopyran-4-one hydrochloride] abolished the H2S-mediated activation of AKT and increases tumor necrosis factor α and interleukin 1β levels in caerulein-treated acinar cells. These findings indicate that the phosphatidylinositol 3-kinase plays a negative role in NaHS-treated pancreatic acinar cells and suggest a role for H2S in the PI3K/AKT pathway in acute pancreatitis.

Extracellular Signal-Regulated Kinase 1/2 and c-Jun NH2-Terminal Kinase, through Nuclear Factor-κB and Activator Protein-1, Contribute to Caerulein-Induced Expression of Substance P and Neurokinin-1 Receptors in Pancreatic Acinar Cells

The neuropeptide substance P (SP) has emerged to be an important proinflammatory mediator in acute pancreatitis (AP). The presence of substance P and its receptor, neurokinin-1 receptor (NK1R) has been shown in the pancreas and the pancreatic acinar cells. In this study, we investigated the unexplored mechanisms that mediate SP and NK1R expression using an in vitro AP model. Pancreatic acinar cells were obtained from pancreas of male Swiss mice. Isolated cells were treated with caerulein to mimic secretagogue pancreatitis. A concentration-dependent study that subjected the cells to 60 min of stimulation by caerulein showed that SP and the transcript from its gene preprotachykinin-A (PPT-A), and NK1R were up-regulated at a supraphysiological concentration of 10−7 M. A concentration-dependent study on intracellular kinases, extracellular signal-regulated kinase (ERK1/2), and c-Jun N-terminal kinase (JNK) and also transcription factors nuclear factor-κB (NF-κB) and activator protein-1 (AP-1) showed that they were activated when the caerulein concentration was 10−7 M. Inhibition of JNK reversed the up-regulation of PPT-A, SP, and NK1R. However, inhibition of ERK1/2 reversed the up-regulation of NK1R but not of PPT-A and SP. Furthermore, we found that specific ERK1/2 and JNK inhibitors reduce NF-κB and AP-1 activity. Taken together, our results suggest that supraphysiological concentrations of caerulein up-regulate the expression of SP and NK1R in pancreatic acinar cells, and the signaling molecules that are involved in this up-regulation include ERK1/2, JNK, NF-κB, and AP-1.

Preprotachykinin-A gene deletion regulates hydrogen sulfide-induced toll-like receptor 4 signaling pathway in cerulein-treated pancreatic acinar cells.

Objective: This study aimed to determine the effect of hydrogen sulfide (H2S) on Toll-like receptor 4 (TLR4)-mediated innate immune signaling in acute pancreatitis (AP) via substance P. Methods: Male Swiss mice were treated with hourly intraperitoneal injections of cerulein (50 &mgr;g/kg) for 10 hours. dl-propargylglycine ([PAG] 100 mg/kg, intraperitoneally), an inhibitor of H2S formation, was administered 1 hour after the induction of AP. Pancreatic acinar cells from male preprotachykinin-A gene-knockout mice (PPTA-/-) and their wild-type counterparts were incubated with or without cerulein (10-7 M for 60 minutes). To better understand the effect of H2S in inflammation, acinar cells were stimulated with cerulein after addition of H2S donor, sodium hydrosulfide. In addition, cerulein-treated pancreatic acinar cells were pretreated with PAG (30 &mgr;M) for 1 hour. Results: The H2S inhibitor PAG eliminated TLR4, interleukin 1 receptor-associated kinase 4, tumor necrosis factor receptor-associated factor 6, and nuclear factor-&kgr;B (NF-&kgr;B) levels in in vitro and in vivo models of cerulein-induced AP. PPTA gene deletion reduced TLR4, myeloid differentiation factor 88, interleukin 1 receptor-associated kinase 4, tumor necrosis factor receptor-associated factor 6, and NF-&kgr;B in cerulein-treated pancreatic acinar cells, whereas administration of sodium hydrosulfide resulted in a further rise in TLR4 and NF-&kgr;B levels in cerulein-treated pancreatic acinar cells. Conclusion: The present findings show for the first time that in AP, H2S may up-regulate the TLR4 pathway and NF-&kgr;B via substance P.

Neurokinin A engages neurokinin-1 receptor to induce NF-κB-dependent gene expression in murine macrophages: implications of ERK1/2 and PI 3-kinase/Akt pathways

Neurokinin A (NKA) belongs to the tachykinin neuropeptide family. Its biological functions are primarily mediated by the neurokinin (NK)-2 receptor. NKA has been implicated in several inflammatory conditions. However, there are limited data about the mechanism of its pathogenetic action. Here, we investigated proinflammatory effects of NKA on peripheral immune cells using the mouse macrophage/monocyte cell line RAW 264.7 and primary peritoneal macrophages. The signaling mechanistic pathways involved were also studied. In mouse macrophages with no detectable NK-2 receptors, NKA induces the upregulation of NK-1 but not NK-2 receptor expression. Furthermore, NKA engages this NK-1 receptor, resulting in inflammatory-like responses involving activation of the transcription factor nuclear factor (NF)-kappaB and induction of NF-kappaB-responsive proinflammatory chemokine expression. NKA activates NF-kappaB as evidenced by induced phosphorylation (leading to degradation) of its inhibitory protein IkappaBalpha, increased cellular levels of the transactivation-active phospho(Ser(276))-p65 and its nuclear translocation, as well as enhanced DNA-binding activity of NF-kappaB. These responses are specifically inhibited by selective NK-1 receptor antagonists but not NK-2 receptor antagonists, thereby excluding the role of NK-2 receptor. Further investigation on the upstream signaling mechanisms suggests that two NF-kappaB-activating pathways (extracellular signal-regulated kinase 1/2 and phosphatidylinositol 3-kinase/protein kinase B) are activated by NKA. Specific inhibitors of the two pathways block NF-kappaB-dependent chemokine expression. The inhibitory effects are mediated through regulation of nuclear translocation, DNA-binding activity, and/or transactivation activity of NF-kappaB. Together, we provide novel evidence that NKA engages NK-1 receptors on mouse macrophages to elicit NF-kappaB-dependent cellular responses. The findings reveal cellular mechanisms that may underlie NKA-mediated inflammatory and immunological conditions.