Raina Devi Ramnath

Hydrogen sulfide is a novel mediator of lipopolysaccharide-induced inflammation in the mouse

Hydrogen sulfide (H2S) is synthesized in the body from l‐cysteine by several enzymes including cystathionine‐γ‐lyase (CSE). To date, there is little information about the potential role of H2S in inflammation. We have now investigated the part played by H2S in endotoxin‐induced inflammation in the mouse. E. coli lipopolysaccharide (LPS) administration produced a dose (10 and 20 mg/kg ip)‐ and time (6 and 24 h)‐dependent increase in plasma H2S concentration. LPS (10 mg/kg ip, 6 h) increased plasma H2S concentration from 34.1 ± 0.7 µM to 40.9 ± 0.6 µM (n=6, P<0.05) while H2S formation from added l‐cysteine was increased in both liver and kidney. CSE gene expression was also increased in both liver (94.2±2.7%, n=6, P<0.05) and kidney (77.5±3.2%, n=6, P<0.05). LPS injection also elevated lung (148.2±2.6%, n=6, P<0.05) and kidney (78.8±8.2%, n=6, P<0.05) myeloperoxidase (MPO, a marker of tissue neutrophil infiltration) activity alongside histological evidence of lung, liver, and kidney tissue inflammatory damage. Plasma nitrate/nitrite (NOx) concentration was additionally elevated in a time‐ and dose‐dependent manner in LPS‐injected animals. To examine directly the possible proinflammatory effect of H2S, mice were administered sodium hydrosulfide (H2S donor drug, 14 µmol/kg ip) that resulted in marked histological signs of lung inflammation, increased lung and liver MPO activity, and raised plasma TNF‐α concentration (4.6±1.4 ng/ml, n=6). In contrast, dl‐propargylglycine (CSE inhibitor, 50 mg/kg ip), exhibited marked anti‐inflammatory activity as evidenced by reduced lung and liver MPO activity, and ameliorated lung and liver tissue damage. In separate experiments, we also detected significantly higher (150.5±43.7 µM c.f. 43.8±5.1 µM, n=5, P<0.05) plasma H2S levels in humans with septic shock. These findings suggest that H2S exhibits proinflammatory activity in endotoxic shock and suggest a new approach to the development of novel drugs for this condition.

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.

Role of MCP-1 in endotoxemia and sepsis☆

Sepsis is a complex clinical syndrome resulting from a harmful host inflammatory response to infection. Similarly, lipopolysaccharide (LPS) induced endotoxemia is marked by the activation of inflammatory responses, which can lead to shock, multiple organ damage and even death. Inflammatory mediator, chemokines are known to play an important role in the pathogenesis of sepsis and endotoxemia. Monocyte chemoattractant protein (MCP)-1, a prototype of CC chemokines, is a potent chemoattractant and a regulatory mediator involved in a variety of inflammatory diseases. The objective of this study is to investigate the role of MCP-1, by using bindarit, a blocker of MCP-1 synthesis, in murine models of sepsis and endotoxemia. Treatment with bindarit both prophylactically and therapeutically significantly (P<0.05) reduced MCP-1 levels in the lungs and liver in both sepsis and endotoxemia. In addition, prophylactic and therapeutic treatment with bindarit significantly (P<0.05) protected mice against sepsis and endotoxemia, as evidenced by the attenuation in lung and liver myeloperoxidase (MPO) activity, an indicator of neutrophil recruitment. The protective effect of bindarit was further confirmed by histological examination of lung and liver sections. Treatment with bindarit reduced lung and liver injury as indicated by decreased thickening of alveolar and neutrophil infiltration in CLP-induced sepsis and LPS-induced endotoxemia. Considering these results, we propose that anti-MCP-1 strategies may be of potential therapeutic value in the treatment of sepsis and endotoxemia.

Effect of mitogen-activated protein kinases on chemokine synthesis induced by substance P in mouse pancreatic acinar cells.

Substance P, acting via its neurokinin 1 receptor (NK1 R), plays an important role in mediating a variety of inflammatory processes. Its interaction with chemokines is known to play a crucial role in the pathogenesis of acute pancreatitis. In pancreatic acinar cells, substance P stimulates the release of NFκB‐driven chemokines. However, the signal transduction pathways by which substance P‐NK1 R interaction induces chemokine production are still unclear. To that end, we went on to examine the participation of mitogen‐activated protein kinases (MAPKs) in substance P‐induced synthesis of pro‐inflammatory chemokines, monocyte chemoanractant protein‐1 (MCP‐I), macrophage inflammatory protein‐lα (MIP‐lα) and macrophage inflammatory protein‐2 (MIP‐2), in pancreatic acini. In this study, we observed a time‐dependent activation of ERK1/2, c‐Jun N‐terminal kinase (JNK), NFκB and activator protein‐1 (AP‐1) when pancreatic acini were stimulated with substance P. Moreover, substance P‐induced ERK 1/2, JNK, NFκB and AP‐1 activation as well as chemokine synthesis were blocked by pre‐treatment with either extracellular signal‐regulated protein kinase kinase 1 (MEK1) inhibitor or JNK inhibitor. In addition, substance P‐induced activation of ERK 112, JNK, NFκB and AP‐1‐driven chemokine production were attenuated by CP96345, a selective NK1 R antagonist, in pancreatic acinar cells. Taken together, these results suggest that substance P‐NK1 R induced chemokine production depends on the activation of MAPKs‐mediated NFκB and AP‐1 signalling pathways in mouse pancreatic acini.

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)

Involvement of SRC family kinases in substance P-induced chemokine production in mouse pancreatic acinar cells and its significance in acute pancreatitis.

Substance P is known to play a key role in the pathogenesis of acute pancreatitis. Src family kinases (SFKs) are known to be involved in cytokine signaling. However, the involvement of SFKs in substance P-induced chemokine production and its role in acute pancreatitis have not been investigated yet. To that end, we have used primary preparations of mouse pancreatic acinar cells as our model to show that substance P/neurokinin 1 receptor (NK1R) induced activation of SFKs. SFKs mediated the activation of mitogen-activated protein kinases [extracellular signal-regulated kinase (ERK), c-Jun NH2-terminal kinase (JNK)], transcription factors [signal transducer and activator of transcription (STAT) 3, nuclear factor (NF) κB, activator protein-1 (AP-1)], and production of chemokines in pancreatic acinar cells. We further tested the significance of the SFK signaling pathway in acute pancreatitis. Our results show, for the first time, that treatment of mice with the potent and selective SFK inhibitor PP2 [4-amino-5-(4-chlorophenyl)-7-(t-butyl) pyrazolo [3,4-d] pyrimidine], but not its negative inhibitor PP3 (4-amino-7-phenylpyrazol [3,4-d] pyrimidine), reduced the severity of pancreatitis. This was proven by significant attenuation of hyperamylasemia, pancreatic myeloperoxidase activity, chemokines, and water content. Histological evidence of diminished pancreatic injury also confirmed the protective effect of the inhibition of SFKs. Moreover, treatment with the substance P receptor antagonist CP96345 [(2S,3S)-cis-2-(diphenylmethyl)-N-((2-methoxyphenyl)-methyl)-1-azabicyclo(2.2.2.)-octan-3-amine] attenuated acute pancreatitis-induced activation of SFKs, ERK, JNK, STAT3, NFκB, and AP-1. The proposed signaling pathway through which substance P mediates acute pancreatitis is through substance P/NK1R-SFKs-(ERK, JNK)-(STAT3, NFκB, AP-1) chemokines. In light of our study, we propose that drugs targeting the substance P-mediated signaling pathways could prove beneficial in improving treatment efficacy in acute pancreatitis.

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.

Role of calcium in substance P‐induced chemokine synthesis in mouse pancreatic acinar cells

Substance P (SP) and chemokines play critical roles in acute pancreatitis. SP elevates cytosolic calcium in pancreatic acinar cells and elevated cytosolic calcium is thought to be an early event in the pathogenesis of acute pancreatitis. SP induces production of chemokines MCP‐1, MIP‐1α and MIP‐2 in pancreatic acinar cells, however the exact mechanism by which SP stimulates the production of these pro‐inflammatory mediators remain undetermined. The aim of the present study is to investigate the role of calcium in SP‐induced chemokine production in pancreatic acinar cells and to establish the signal transduction mechanisms involved.

Inflammatory mediators in sepsis: Cytokines, chemokines, adhesion molecules and gases

Sepsis is a systemic inflammatory response syndrome in response to severe infection. An overwhelming systemic response brought about by the release of various inflammatory mediators can lead to shock, multiple organ damage and death. Cytokines play an important role in the pathogenesis of sepsis and are regulated by a complex network of pro- and anti-inflammatory mediators. Various chemokines sequester neutrophils into the target organ, further augmenting inflammation. Chemokine receptor antagonism represents a major therapeutic approach against sepsis. Adhesion molecules mediate the migration of leukocytes towards the site of inflammation and their activation. Gaseous mediators such as nitric oxide and hydrogen sulfide are also involved in the pathogenesis of inflammation. Our review summarizes the current understanding of the roles of various inflammatory mediators in sepsis.