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.     

Monosodium urate crystals synergize with IFN-gamma to generate macrophage nitric oxide: involvement of extracellular signal-regulated kinase 1/2 and NF-kappa B

Elevated NO production has been detected in patients suffering from various arthropathies; however, its role and regulation during gouty arthritis remain largely unexplored. Monosodium urate (MSU) crystals, the causative agent of gout, have been shown to induce NO generation in vivo and inducible NO synthase (iNOS) expression in human monocytes. The present study was designed to evaluate the ability of MSU crystals to modulate macrophage (M phi) iNOS expression and NO synthesis and to investigate the molecular mechanisms underlying these cellular responses. We found that MSU crystals did not induce NO production in murine J774 M phi. However, a synergistic effect on the level of iNOS expression and NO generation was observed in cells exposed to MSU crystals in combination with IFN-gamma. Characterization of the second messengers involved revealed the requirement of IFN-gamma-mediated Janus kinase 2/STAT1 alpha activation even though MSU crystals did not modulate this signaling cascade by themselves. MSU crystals exerted their up-regulating effect by increasing extracellular signal-regulated kinase (ERK) 1/2 phosphorylation and NF-kappa B nuclear translocation in response to IFN-gamma. The use of specific inhibitors against either NF-kappa B or the ERK1/2 pathway significantly reduced MSU + IFN-gamma-inducible NF-kappa B activity, iNOS expression, and NO production. Altogether, these data indicate that MSU crystals exert a potent synergistic effect on the IFN-gamma-inducible M phi NO generation via ERK1/2- and NF-kappa B-dependent pathways. Understanding the molecular mechanisms through which MSU crystals amplify M phi responses to proinflammatory cytokines such as IFN-gamma will contribute to better define their role in NO regulation during gout, in particular, and inflammation, in general.     

Interleukin-1beta stimulates macrophage inflammatory protein-1alpha and -1beta expression in human neuronal cells (NT2-N)

Chemokines are important mediators in immune responses and inflammatory processes of neuroimmunologic and infectious diseases. Although chemokines are expressed predominantly by cells of the immune system, neurons also express chemokines and chemokine receptors. We report herein that human neuronal cells (NT2-N) produce macrophage inflammatory protein-1alpha and -1beta (MIP-1alpha and MIP-1beta), which could be enhanced by interleukin (IL)-1beta at both mRNA and protein levels. The addition of supernatants from human peripheral blood monocyte-derived macrophage (MDM) cultures induced MIP-1beta mRNA expression in NT2-N cells. Anti-IL-1beta antibody removed most, but not all, of the MDM culture supernatant-induced MIP-1beta mRNA expression in NT2-N cells, suggesting that IL-1beta in the MDM culture supernatants is a major factor in the induction of MIP-1beta expression. Investigation of the mechanism(s) responsible for IL-1beta-induced MIP-1alpha and -1beta expression demonstrated that IL-1beta activated nuclear factor kappa B (NF-kappaB) promoter-directed luciferase activity in NT2-N cells. Caffeic acid phenethyl ester, a potent and specific inhibitor of activation of NF-kappaB, not only blocked IL-1beta-induced activation of the NF-kappaB promoter but also decreased IL-1beta-induced MIP-1alpha and -1beta expression in NT2-N cells. These data suggest that NF-kappaB is at least partially involved in the IL-1beta-mediated action on MIP-1alpha and -1beta in NT2-N cells. IL-1beta-mediated up-regulation of beta-chemokine expression may have important implications in the immunopathogenesis of inflammatory diseases in the CNS.     

Differential expression and regulation of macrophage inflammatory protein (MIP)-1alpha and MIP-2 genes by alveolar and peritoneal macrophages in LPS-hyporesponsive C3H/HeJ mice

A point mutation in Toll-like receptor 4 (Tlr4) gene in C3H/HeJ mice underlies a defect in LPS-induced cytokine production by peritoneal macrophages (PMphi;). Whether the C-C and the C-X-C chemokines are induced differently by LPS between alveolar macrophages (AMphi;) and PMphi; in this mice remains unclear. Thus, we examined the expression and regulation of macrophage inflammatory protein-1alpha (MIP-1alpha) and macrophage inflammatory protein-2 (MIP-2) in C3H/HeJ macrophages. These results showed that the accumulation of MIP-1alpha and MIP-2 mRNA increased dose dependently in response to LPS. PMphi; responded to LPS to produce significantly higher levels of both chemokine mRNA and protein than AMphi;. In addition, both macrophages produced much more MIP-2 than MIP-1alpha by the same doses of LPS stimulation. Moreover, the chemokine production by C3H/HeN macrophages was significantly higher than that of the C3H/HeJ macrophages. IFN-gamma suppressed the LPS-induced MIP-1alpha release but enhanced the LPS-induced MIP-2 secretion in both macrophages. These results show that the chemokine production was induced and regulated differentially in AMphi; and PMphi;.     

Inhibition of endotoxin-induced macrophage chemokine production by vasoactive intestinal peptide and pituitary adenylate cyclase-activating polypeptide in vitro and in vivo.

Inflammatory chemokines recruit various populations of immune cells that initiate and maintain the inflammatory response against foreign Ags. Although such a response is necessary for the elimination of the Ag, the inflammation has to be eventually resolved in a healthy organism. Neuropeptides such as vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP), released after antigenic stimulation, contribute to the termination of an inflammatory response primarily by inhibiting the production of proinflammatory cytokines. Here we investigated the effects of VIP and PACAP on chemokine production. We report that VIP and PACAP inhibit the expression of the macrophage-derived CXC chemokines macrophage inflammatory protein-2 and KC (IL-8), and of the CC chemokines MIP-1alpha, MIP-1beta, monocyte chemoattractant protein 1, and RANTES in vivo and in vitro. The inhibition of chemokine gene expression correlates with an inhibitory effect of VIP/PACAP on NF-kappaB binding and transactivating activity. The VIP/PACAP inhibition of both chemokine production and of NF-kappaB binding and transactivating activity is mediated through the specific VIP receptor VPAC1, and involves both cAMP-dependent and -independent intracellular pathways. In an in vivo model of acute peritonitis, the inhibition of chemokine production by VIP/PACAP leads to a significant reduction in the recruitment of polymorphonuclear cells, macrophages, and lymphocytes into the peritoneal cavity. These findings support the proposed role of VIP and PACAP as key endogenous anti-inflammatory agents and describe a novel mechanism, i.e., the inhibition of the production of macrophage-derived chemokines.     

Regulation of macrophage chemokine expression by lipopolysaccharide in vitro and in vivo.

The host response to Gram-negative LPS is characterized by an influx of inflammatory cells into host tissues, which is mediated, in part, by localized production of chemokines. The expression and function of chemokines in vivo appears to be highly selective, though the molecular mechanisms responsible are not well understood. All CXC (IFN-gamma-inducible protein (IP-10), macrophage inflammatory protein (MIP)-2, and KC) and CC (JE/monocyte chemoattractant protein (MCP)-1, MCP-5, MIP-1alpha, MIP-1beta, and RANTES) chemokine genes evaluated were sensitive to stimulation by LPS in vitro and in vivo. While IL-10 suppressed the expression of all LPS-induced chemokine genes evaluated in vitro, treatment with IFN-gamma selectively induced IP-10 and MCP-5 mRNAs, but inhibited LPS-induced MIP-2, KC, JE/MCP-1, MIP-1alpha, and MIP-1beta mRNA and/or protein. Like the response to IFN-gamma, LPS-mediated induction of IP-10 and MCP-5 was Stat1 dependent. Interestingly, only the IFN-gamma-mediated suppression of LPS-induced KC gene expression was IFN regulatory factor-2 dependent. Treatment of mice with LPS in vivo also induced high levels of chemokine mRNA in the liver and lung, with a concomitant increase in circulating protein. Hepatic expression of MIP-1alpha, MIP-1beta, RANTES, and MCP-5 mRNAs were dramatically reduced in Kupffer cell-depleted mice, while IP-10, KC, MIP-2, and MCP-1 were unaffected or enhanced. These findings indicate that selective regulation of chemokine expression in vivo may result from differential response of macrophages to pro- and antiinflammatory stimuli and to cell type-specific patterns of stimulus sensitivity. Moreover, the data suggest that individual chemokine genes are differentially regulated in response to LPS, suggesting unique roles during the sepsis cascade.     

Implications of proteasome inhibition: an enhanced macrophage phenotype

The objective of this study was to elucidate the role of the cellular proteasome on endotoxin-mediated activation of the macrophage. To study this role, THP-1 cells were stimulated with lipopolysaccharide (LPS) with selective cells being pretreated with the proteasome inhibitor, lactacystin or MG-132. LPS stimulation led to the phosphorylation and degradation of IRAK, followed by activation of JNK/SAPK, ERK 1/2, and p38. Subsequently, LPS induced the degradation of IkappaB, and the nuclear activation of NF-kappaB and AP-1. Activation of these pathways was associated with the production of IL-6, IL-8, IL-10, and TNF-alpha. Proteasome inhibition with either lactacystin or MG-132 attenuated LPS-induced IRAK degradation, and enhanced activation of JNK/SAPK, ERK 1/2, and p38. Proteasome inhibition, also, led to increased LPS-induced AP-1 activation, and attenuated LPS-induced IkappaB degradation resulting in abolished NF-kappaB activation. Proteasome inhibition led to significant modulation of LPS-induced cytokine production; increased IL-10, no change in IL-6, and decreased IL-8, and TNF-alpha. Thus, this study demonstrates that cellular proteasome is critical to regulation of LPS-induced signaling within the macrophage, and inhibition of the proteasome results in a conversion to an anti-inflammatory phenotype.     

Substance P treatment stimulates chemokine synthesis in pancreatic acinar cells via the activation of NF-kappaB

Acinar cell injury early in acute pancreatitis leads to a local inflammatory reaction and to the subsequent systemic inflammatory response, which may result in multiple organ dysfunction and death. Inflammatory mediators, including chemokines and substance P (SP), are known to play a crucial role in the pathogenesis of acute pancreatitis. It has been shown that pancreatic acinar cells produce the chemokine monocyte chemoattractant protein-1 (MCP-1) in response to caerulein hyperstimulation, demonstrating that acinar-derived MCP-1 is an early mediator of inflammation in acute pancreatitis. Similarly, SP levels in the pancreas and pancreatic acinar cell expression of neurokinin-1 receptor, the primary receptor for SP, are both increased during secretagogue-induced experimental pancreatitis. This study aims to examine the functional consequences of exposing mouse pancreatic acinar cells to SP and to determine whether it leads to proinflammatory signaling, such as production of chemokines. Exposure of mouse pancreatic acini to SP significantly increased synthesis of MCP-1, macrophage inflammatory protein-1alpha (MIP-1alpha), as well as MIP-2. Furthermore, SP also increased NF-kappaB activation. The stimulatory effect of SP was specific to chemokine synthesis through the NF-kappaB pathway, since the increase in chemokine production was completely attenuated when pancreatic acini were pretreated with the selective NF-kappaB inhibitor NF-kappaB essential modulator-binding domain peptide. This study shows that SP-induced chemokine synthesis in mouse pancreatic acinar cells is NF-kappaB dependent.