Blockade of neurokinin-1 receptor attenuates CC and CXC chemokine production in experimental acute pancreatitis and associated lung injury

Accumulating evidence suggests the neuropeptide substance P (SP) and its receptor neurokinin-1 receptor (NK-1R) play a pivotal role in the pathogenesis of acute pancreatitis (AP). However, the mechanisms remain unclear. The present study investigated whether chemokines as proinflammatory molecules are involved in SP-NK-1R-related pathogenesis of this condition. We observed temporally and spatially selective chemokine responses in secretagogue caerulein-induced AP in mice. CC chemokines monocyte chemotactic protein (MCP)-1 and macrophage inflammatory protein-1alpha (MIP-1alpha) and CXC chemokine MIP-2 were elevated after AP induction. Time-dependent, tissue-specific analysis of their mRNA and protein expression suggested that they are early mediators in the condition and mediate local as well as systemic inflammatory responses. In contrast, another CC chemokine regulated on activation, T cells expressed and secreted (RANTES) was only involved in local pancreatic inflammation at a later stage of the disease. Either prophylactic or therapeutic treatment with a potent selective NK-1R antagonist CP-96,345 significantly suppressed caerulein-induced increase in MCP-1, MIP-1alpha, and MIP-2 expression but had no apparent effect on RANTES expression. The suppression effect of CP-96,345 on MCP-1, MIP-1alpha, and MIP-2 expression was concordantly demonstrated by immunohistochemistry, which, additionally, suggested that chemokine immunoreactivity was localized to acinar cells and the infiltrating leukocytes in the pancreas and alveolar macrophages, epithelial cells, and endothelial cells in the lungs. Our data suggest that SP, probably by acting via NK-1R on various chemokine-secreting cells in the pancreas and lungs, stimulates the release of chemokines that aggravate local AP and the development of its systemic sequelae.     

The effect of MCP-1 depletion on chemokine and chemokine-related gene expression: evidence for a complex network in acute inflammation

The expression of chemokines has been suggested to involve an interdependent network, with the absence of a single chemokine affecting the expression of multiple other chemokines. Monocyte chemoattractant protein (MCP-1), a member of C-C chemokine superfamily, plays a critical role in the recruitment and activation of leukocytes during acute inflammation. To examine the effect of the loss of MCP-1 on expression of the chemokine network, we compared the mRNA expression profiles of MCP-1(-/-) and wild type mice during the acute inflammatory phase of excisional wounds. Utilizing a mouse cDNA array containing 514 chemokine and chemokine related genes, the loss of MCP-1 was observed to cause a significant upregulation of nine genes (Decorin, Persephin, IL-1beta, MIP-2, MSP, IL1ra, CCR5, CCR3, IL-11) and significant downregulation of two genes (CCR4 and CD3Z) in acute wounds. The array data was confirmed by semi-quantitative RT-PCR. The effect of MCP-1 deletion on chemokine expression was further examined in isolated macrophages. Compared to wild type, LPS-stimulated peritoneal macrophages from MCP-1(-/-) mice showed a significant increase in the expression of RANTES, MIP-1beta, MIP-1alpha and MIP-2 mRNA. The data suggest that loss of a single chemokine perturbs the chemokine network not only in the setting of acute inflammation but even in an isolated inflammatory cell, the macrophage.     

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.     

Inhibition of endotoxin-induced macrophage chemokine production by VIP and PACAP in vitro and in vivo

Inflammatory chemokines recruit immune cells which initiate and maintain the inflammatory response. Although such a response is necessary for the elimination of the antigen, the inflammation has to be eventually resolved. Peptides such as vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP), released following 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 MIP-2 and KC (IL-8), and of the CC chemokines MIP-1a, MIP-1b, MCP-1 and RANTES in vivo and in vitro. The decrease of chemokine gene expression correlates with an inhibitory effect of VIP/PACAP on NFkB binding. 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 PMNs, 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 chemokine expression by IL-10 in lung inflammation

We have been interested in understanding the mechanisms regulating the inflammatory process underlying acute lung injury. The current studies have employed a model of acute lung inflammation in mice triggered by bacterial lipopolysaccharide. The development of this injury was associated with increased expression of the chemokines, MIP-1alpha and MIP-2, that coordinate recruitment of neutrophils to the lung. IL-10 is a potent, endogenous anti-inflammatory molecule that has been shown to decrease lung inflammation partly on the basis of TNF-alpha and IL-1beta inhibition. In these studies we tested the hypothesis that endogenous IL-10 modulates chemokine expression using the IL-10 knock-out mouse, and then explored the molecular mechanisms by which IL-10 might do so. The results demonstrate that significant elevations in both chemokines were observed in the absence of IL-10 and that these findings were associated with significant increases in lung neutrophil accumulation. In vitro studies defined two, gene-specific, mechanisms by which IL-10 regulated chemokine expression: mRNA destabilization and NF-kappaB inhibition. These results suggested that IL-10 is an important, endogenous regulator of chemokine expression in acute lung inflammation.     

Differential activation of innate immune responses by adenovirus and adeno-associated virus vectors

Adenovirus vectors induce acute inflammation of infected tissues due to activation of the innate immune system and expression of numerous chemokines and cytokines in transduced target cells. In contrast, adeno-associated virus (AAV) vectors are not associated with significant inflammation experimentally or clinically. We tested the ability of AAV vectors to induce the expression of chemokines in vitro and to activate the innate immune system in vivo. In human HeLa cells and murine renal epithelium-derived cells (REC cells) the adenovirus vector AdlacZ induced the expression of multiple inflammatory chemokines including RANTES, interferon-inducible protein 10 (IP-10), interleukin-8 (IL-8), MIP-1beta, and MIP-2 in a dose-dependent manner. The use of AAVlacZ did not induce the expression of these chemokines above baseline levels despite 40-fold-greater titers than AdlacZ and greater amounts of intracellular AAVlacZ genomes according to Southern and slot blot analysis. This finding confirmed that the lack of AAVlacZ induction of chemokine was not due to reduced transduction. In DBA/2 mice, the intravenous administration of 2.5 x 10(11) particles of AAVlacZ resulted in the rapid induction of liver tumor necrosis factor alpha (TNF-alpha), RANTES, IP-10, MIP-1beta, MCP-1, and MIP-2 mRNAs. However, 6 h following injection, chemokine mRNA levels returned to baseline. As expected, administration of 10-fold less AdlacZ caused an induction of liver TNF-alpha and chemokine mRNAs that persisted for more than 24 h posttransduction. Whereas intravenous administration of 2.5 x 10(11) particles of AAVlacZ triggered a transient infiltration of neutrophils and CD11b(+) cells into liver, this response stood in contrast to widespread inflammation and toxicity induced by AdlacZ. Kupffer cell depletion abolished AAVlacZ but not AdlacZ-induced chemokine expression and neutrophil infiltration. In summary, these results show that AAV vectors activate the innate immune system to a lesser extent than do adenovirus vectors and offer a possible explanation for the reduced inflammatory properties of AAV compared to adenovirus vectors.     

Fibrinogen stimulates macrophage chemokine secretion through toll-like receptor 4

Extravascular fibrin deposition is an early and persistent hallmark of inflammatory responses. Fibrin is generated from plasma-derived fibrinogen, which escapes the vasculature in response to endothelial cell retraction at sites of inflammation. Our ongoing efforts to define the physiologic functions of extravasated fibrin(ogen) have led to the discovery, reported here, that fibrinogen stimulates macrophage chemokine secretion. Differential mRNA expression analysis and RNase protection assays revealed that macrophage inflammatory protein-1alpha (MIP-1alpha), MIP-1beta, MIP-2, and monocyte chemoattractant protein-1 are fibrinogen inducible in the RAW264.7 mouse macrophage-like cell line, and ELISA confirmed that both RAW264.7 cells and primary murine thioglycolate-elicited peritoneal macrophages up-regulate the secretion of monocyte chemoattractant protein-1 >100-fold upon exposure to fibrinogen. Human U937 and THP-1 precursor-1 (THP-1) monocytic cell lines also secreted chemokines in response to fibrinogen, upon activation with IFN-gamma and differentiation with vitamin D(3), respectively. LPS contamination could not account for our observations, as fibrinogen-induced chemokine secretion was sensitive to heat denaturation and was unaffected by the pharmacologic LPS antagonist polymyxin B. Nevertheless, fibrinogen- and LPS-induced chemokine secretion both apparently required expression of functional Toll-like receptor 4, as each was diminished in macrophages derived from C3H/HeJ mice. Thus, innate responses to fibrinogen and bacterial endotoxin may converge at the evolutionarily conserved Toll-like recognition molecules. Our data suggest that extravascular fibrin(ogen) induces macrophage chemokine expression, thereby promoting immune surveillance at sites of inflammation.     

Inducible expression of inflammatory chemokines in respiratory syncytial virus-infected mice: role of MIP-1alpha in lung pathology

Lower respiratory tract disease caused by respiratory syncytial virus (RSV) is characterized by profound airway mucosa inflammation, both in infants with naturally acquired infection and in experimentally inoculated animal models. Chemokines are central regulatory molecules in inflammatory, immune, and infectious processes of the lung. In this study, we demonstrate that intranasal infection of BALB/c mice with RSV A results in inducible expression of lung chemokines belonging to the CXC (MIP-2 and IP-10), CC (RANTES, eotaxin, MIP-1beta, MIP-1alpha, MCP-1, TCA-3) and C (lymphotactin) families. Chemokine mRNA expression occurred as early as 24 h following inoculation and persisted for at least 5 days in mice inoculated with the highest dose of virus (10(7) PFU). In general, levels of chemokine mRNA and protein were dependent on the dose of RSV inoculum and paralleled the intensity of lung cellular inflammation. Immunohisthochemical studies indicated that RSV-induced expression of MIP-1alpha, one of the most abundantly expressed chemokines, was primarily localized in epithelial cells of the alveoli and bronchioles, as well as in adjoining capillary endothelium. Genetically altered mice with a selective deletion of the MIP-1alpha gene (-/- mice) demonstrated a significant reduction in lung inflammation following RSV infection, compared to control littermates (+/+ mice). Despite the paucity of infiltrating cells, the peak RSV titer in the lung of -/- mice was not significantly different from that observed in +/+ mice. These results provide the first direct evidence that RSV infection may induce lung inflammation via the early production of inflammatory chemokines.     

Cutaneous injection of human subjects with macrophage inflammatory protein-1 alpha induces significant recruitment of neutrophils and monocytes

Macrophage inflammatory protein (MIP-1 alpha), a member of the CC chemokine subfamily, has been shown to attract T cells and monocytes in vitro and to be expressed at sites of inflammation. Although the in vitro activities of MIP-1 alpha have been well documented, the in vivo biological activities of MIP-1 alpha in humans have not been studied. To address this, we challenged human subjects by intradermal injection with up to 1000 pmol of MIP-1 alpha and performed biopsies 2, 10, and 24 h later. Although no acute cutaneous or systemic reactions were noted, endothelial cell activation, as indicated by the expression of E-selectin, was observed. In agreement with its in vitro activity, monocyte, lymphocyte, and, to a lesser degree, eosinophil infiltration was observed, peaking at 10-24 h. Surprisingly, in contrast to its reported lack of in vitro neutrophil-stimulating activity, a rapid infiltration of neutrophils was observed in vivo. This neutrophil infiltration occurred as early as 2 h, preceding the appearance of other cells, and peaked at 10 h. Interestingly, we found that neutrophils in whole blood, but not after isolation, expressed CCR1 on their cell surface. This CCR1 was thought to be functional as assessed by neutrophil CD11b up-regulation following whole-blood MIP-1 alpha stimulation. These studies substantiate the biological effects of MIP-1 alpha on monocytes and lymphocytes and uncover the previously unrecognized activity of MIP-1 alpha to induce neutrophil infiltration and endothelial cell activation, underscoring the need to evaluate chemokines in vivo in humans.     

Induction of the chemokine beta peptides, MIP-1 alpha and MIP-1 beta, by lipopolysaccharide is differentially regulated by immunomodulatory cytokines gamma-IFN, IL-10, IL-4, and TGF-beta.

The macrophage occupies a central role in the host response to invasion, exerting its control over the developing inflammatory response largely through the elaboration of an assortment of endogenous mediators including many cytokines. The beta chemokine peptides, macrophage inflammatory protein [MIP]-1 alpha and MIP-1 beta, are two such effectors markedly up-regulated in macrophages following exposure to bacterial lipopolysaccharide (LPS). These highly homologous peptides, like the other members of the beta chemokine family, exhibit diverse but partially overlapping biological activity profiles, suggesting that the cellular participants and intensity of an inflammatory response may in part be regulated by selective expression of these chemokines. Studies reported here demonstrate that, in contrast to the "balanced" MIP-1 alpha/MIP-1 beta chemokine responses of LPS-stimulated macrophage cultures in vitro, circulating levels of MIP-1 beta are significantly higher than those of MIP-1 alpha following LPS administration in vivo. Further studies have revealed that several immunomodulatory cytokines known to be up-regulated in vivo as a consequence of exposure to an invasive stimulus (gamma-IFN, IL-10, IL-4, and transforming growth factor [TGF]-beta) down-regulated the LPS-induced release of MIP-1 alpha by macrophages in vitro, but spared the MIP-1 beta response. This altered pattern of secretion may explain, at least in part, the high circulating levels of MIP-1 beta relative to MIP-1 alpha observed in vivo in response to LPS challenge.     

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;.     

Induction of β-family chemokines mRNA in human embryonic astrocytes by inflammatory cytokines

The cellular infiltration found during CNS inflammation consists of monocytes and activated T cells, suggesting the presence of cell-specific chemotactic signals during inflammatory responses. Astrocyte chemokine expression might contribute to site-specific leukocyte infiltration within the CNS. To investigate the factors that regulate astrocyte chemokine expression, we examined the ability of human fetal astrocytes to induce β-family chemokine mRNA. Astrocyte-derived monocyte chemoattractant protein-1 (MCP-1), RANTES, macrophage inflammatory protein-1α (MIP-1α), and MIP-1β mRNA were easily induced by lipopolysaccharide and/or the proinflammatory cytokines (IFNγ and/or TNF-α), respectively. Addition of both IFNγ and TNF-α together did not lead to an additive effect but resulted in the inhibition of MCP-1 and MIP-1β mRNA expression, indicating that interaction between chemokines and cytokines may play a key role in regulating the local immune response of resident and infiltrating cells at the site of lesion. Interestingly, ultraviolet light-inactivated measles virus, but not cytomegalovirus, strongly induced expression of MCP-1, RANTES, MIP-1α, and MIP-1β mRNA in human embryonic astrocytes, especially MCP-1 and MIP-1β. An association occurs between the β-family chemokine expression in astrocytes and inflammatory factors/virus, suggesting a possible role for β-family chemokines in the pathogenesis of CNS inflammatory disease.     

CpG oligodeoxynucleotides induce murine macrophages to up-regulate chemokine mRNA expression

Intramuscular injection of synthetic oligodeoxynucleotides (ODN) expressing unmethylated CpG motifs trigger the rapid development of a local inflammatory response. In vitro studies demonstrate that macrophages exposed to CpG ODN up-regulate expression of mRNA encoding the chemokines MIP-1alpha, MIP-1beta, MIP-2, RANTES, JE/MCP-1, and IP-10. Within 6 h of in vivo administration, CpG ODN induce a significant increase in chemokine mRNA levels at the site of injection and draining lymph nodes. These chemokines may contribute to the migration and stimulation of inflammatory cells that contribute to the development of CpG ODN-induced immune responses.     

Chemokine receptor CCR5 deficiency exacerbates cerulein-induced acute pancreatitis in mice

Acute pancreatitis (AP) is an inflammatory disease involving the production of different cytokines and chemokines and is characterized by leukocyte infiltration. Because the chemokine receptor CCR5 and its ligands [the CC chemokines CCL3/MIP-1alpha, CCL4/MIP-1beta, and CCL5/regulated upon activation, normal T cell expressed and secreted (RANTES)] regulate leukocyte chemotaxis and activation, we investigated the expression of CCR5 ligands and the role of CCR5 and its ligands in experimental AP in mice. AP was induced by hourly intraperitoneal injections of cerulein in CCR5-deficient (CCR5(-/-)) or wild-type (WT) mice. Induction of AP by cerulein resulted in an early increase of pancreatic CCL2, CCL3, and CCL4 mRNA expression, whereas CCL5 mRNA expression occurred later. CCR5(-/-) mice developed a more severe pancreatic injury than WT mice during cerulein-induced AP, as assessed by a more pronounced increase in serum amylase and lipase levels and by more severe pancreatic edema, inflammatory infiltrates (mainly neutrophils), and necrosis. CCR5(-/-) mice also exhibited increased production of CCL2/MCP-1, CCL3/MIP-1alpha, and CCL4/MIP-1beta during the course of cerulein-induced AP. In vivo simultaneous neutralization of CC chemokines with monoclonal antibodies in CCR5(-/-) mice reduced the severity of cerulein-induced AP, indicating a role of CC chemokines in exacerbating the course of AP in the absence of CCR5. Moreover, simultaneous neutralization of CCR5 ligands in WT mice also reduced the severity of cerulein-induced AP. In conclusion, lack of the chemokine receptor CCR5 exacerbates experimental cerulein-induced AP and leads to increased levels of CC chemokines and a more pronounced pancreatic inflammatory infiltrate, suggesting that CCR5 expression can modulate severity of AP.     

Role of CCR5 in the pathogenesis of IL-13-induced inflammation and remodeling

IL-13 is a major effector at sites of Th2 inflammation and tissue remodeling. In these locations, it frequently coexists with the CCR5 chemokine receptor and its ligands MIP-1alpha/CCL3 and MIP-1beta/CCL4. We hypothesized that CCR5 induction and activation play important roles in the pathogenesis of IL-13-induced tissue responses. To test this hypothesis, we evaluated the effects of IL-13 on the expression of CCR5 in the murine lung. We also compared the effects of lung-targeted transgenic IL-13 in mice treated with anti-CCR5 or an Ab control and mice with wild-type or null CCR5 loci. These studies demonstrate that IL-13 is a potent stimulator of epithelial cell CCR5 expression. They also demonstrate that CCR5 neutralization or a deficiency of CCR5 significantly decreases IL-13-induced inflammation, alveolar remodeling, structural and inflammatory cell apoptosis, and respiratory failure and death. Lastly, these studies provide mechanistic insights by demonstrating that CCR5 is required for optimal IL-13 stimulation of select chemokines (MIP-1alpha/CCL3, MIP-1beta/CCL4, MCP-1/CCL-2), matrix metalloproteinase-9 and cell death regulators (Fas, TNF, TNFR1, TNFR2, Bid), optimal IL-13 inhibition of alpha1-antitrypsin, and IL-13-induction of and activation of caspases-3, -8, and-9. Collectively, these studies demonstrate that CCR5 plays a critical role in the pathogenesis of IL-13-induced inflammation and tissue remodeling.