PAC1 Receptor

Emerging Target for Septic Shock Therapy

CARMEN MARTINEZ,a ALICIA ARRANZ,b YASMINA JUARRANZ,b

CATALINA ABAD,b MARIA GARCIA-GOMEZ,b

FLORENCIA ROSIGNOLI,b JAVIER LECETA,b AND ROSA P. GOMARIZb

aDepartment of Cell Biology, Faculty of Medicine, Complutense University,28040 Madrid, SpainbDepartment of Cell Biology, Faculty of Biology, Complutense University,28040 Madrid, Spain

ABSTRACT: Septic shock is a systemic response to severe bacterial in-fections, generally caused by Gram-negative bacterial endotoxins, withmultiple manifestations such as hypotension, tissue injury, disseminatedintravascular coagulation, and multi-organ failure. All these effects, areinduced by the generation of pro-inflammatory and vasodilator media-tors, cell adhesion molecules, coagulation factors, and acute-phase pro-teins. Vasoactive intestinal polypeptide (VIP) and pituitary adenylatecyclase-activating polypeptide (PACAP) are two immunopeptides withanti-inflammatory properties exerted through type 1 and 2 VIP recep-tors (VPAC1 and VPAC2, respectively), and PACAP receptor (PAC1). Thepresent results recapitulate the protective role of PAC1 in an experimentalmodel of lethal endotoxemia using a knockout for the PAC1 receptor. Ourresults demonstrate that VIP and PACAP decrease lipopolysaccharide(LPS)-induced interleukin-6 (IL-6) production, neutrophil infiltrationand intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesionmolecule-1 (VCAM-1), and fibrinogen expression through PAC1 recep-tor, providing an advantage to design more specific drugs complementingstandard intensive care therapy in septic shock.

KEYWORDS: PAC1; endotoxemia; neutrophil infiltration; fibrinogen;IL-6

INTRODUCTION

Septic shock is a systemic response to severe bacterial infections, generallycaused by Gram-negative bacterial endotoxins that trigger a cascade of biolog-ical events. During septic shock, the activation of coagulation and the release

Address for correspondence: Carmen Martınez, Ph.D., Department of Cell Biology, Faculty ofMedicine, Complutense University, Ciudad Universitaria, 28040 Madrid, Spain. Voice: 34-913944971;fax: 34-913944981.

e-mail: cmmora@bio.ucm.es

Ann. N.Y. Acad. Sci. 1070: 405–410 (2006). C© 2006 New York Academy of Sciences.doi: 10.1196/annals.1317.053

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of a large number of mediators of inflammation contribute to the pathogen-esis of disseminated intravascular coagulation and multi-organ failure.1 Therecruitment of leukocytes into sites of inflammation by interactions with thevascular endothelium leads to the release of a wide range of pro-inflammatorycytokines such as interleukin-6 (IL-6) and tumor necrosis factor-� (TNF-�),which are pivotal mediators of septic shock. These cytokines initiate a widerange of systemic responses including a dramatic increase of acute-phase (AP)proteins such as serum amyloid A (SAA), which is synthesized in the liver.Coagulation factors as fibrinogen are also very rapidly released in response toendotoxins and inflammatory cytokines triggering blood clot formation. Allthese molecules associated with nitric oxide (NO), which leads to the hypoten-sion that defines septic shock, are important markers of inflammation andbelong to the growing list of potential therapeutic targets in the pathogenesisof sepsis. Though significant advances have been made in our understandingof the septic cascade, most of the early trials have failed to improve survival.Interference with adhesion molecules, inflammatory agents, and coagulationabnormalities as a whole might offer hopeful, therapeutic possibilities. Thepresent article summarizes the protective role of the member of VIP/PACAPreceptor family PAC1, mediating the blockade of several important markersinto the complexities of sepsis.

VIP AND PACAP IN SEPTIC SHOCK

Vasoactive intestinal polypeptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP) are two immunopeptides that exert their im-munological effects through binding to the family 2 of G protein–coupledreceptors, VIP receptors type 1 and 2 (VPAC1 and VPAC2, respectively),and PACAP receptor (PAC1).2 In recent years, VIP and PACAP effects in-hibiting endotoxin-induced production of proinflammatory factors such asTNF-�, IL-6, IL-12, NO, and interferon-� (IFN-� ), and stimulating anti-inflammatory cytokines, such as IL-103,4 have been described. Applicationof the anti-inflammatory properties of VIP and PACAP in a murine model,5

has demonstrated that both immunopeptides protect from endotoxemia throughinhibition of TNF-� and IL-6. Although our studies using specific VIP agonistssuggested that VPAC1 was the main mediator,6 PAC1 has recently emerged asan anti-inflammatory receptor improving survival in a murine model of lethalendotoxemia, as shown by using mice defective in PAC1 expression (PAC1−/−)compared with their wild-type counterparts.7

HOW DOES PAC1 RECEPTOR PROTECT FROM ENDOTOXICSHOCK?

The first findings on the role of PAC1 in endotoxic shock pointed out itsinvolvement mediating the inhibition of VIP/PACAP on IL-6 production after

MARTINEZ et al.: PAC1 RECEPTOR PROTECTS FROM ENDOTOXIC SHOCK 407

a lethal dose (1 mg) of lipopolysaccharide7 (LPS). However, it did not par-ticipate in the inhibition of TNF-� in accordance with previous studies usingVPAC1 and VPAC2 agonists.6 The best correlation of plasma cytokine levelswith mortality from septic shock has been made with IL-6,8 which appearsto orchestrate a variety of inflammatory responses amplifying leukocyte re-cruitment through an increase of the local production of chemokines, and theexpression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell ad-hesion molecule-1 (VCAM-1), as well as inducing the hepatic AP response.9,10

Thus, IL-6/IFN-� activate nuclear factors for the regulation of ICAM-1 geneexpression,11 and an IL-6-response element has been described in the promo-tor of � -chain of human fibrinogen.12 Therefore, we speculated that PAC1could modulate neutrophil recruitment, adhesion molecule expression, as wellas AP proteins. Firstly, we evaluated the effect of VIP/PACAP (5 nmol) onmyeloperoxidase activity (MPO), as a quantitative measure of neutrophil in-filtration in liver and intestinal extracts. Both peptides inhibited MPO activityin wild-type but not in PAC1−/−. This fact was confirmed by histopathologi-cal analysis. Transmigration of leukocytes involves endothelial cell adhesion,including ICAM-1 and vascular VCAM-1 which increase after LPS admin-istration. VIP and PACAP reduced soluble ICAM-1 (ICAM) and the adhe-sion molecule transcripts in wild-type, whereas no differences were observedin PAC1−/− mice. Next, we showed the effects of VIP and PACAP on the

FIGURE 1. Endotoxic shock induces different pathological outcomes that VIP andPACAP abrogate through activation of VIP/PACAP receptors, notably of PAC1 receptor.

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MARTINEZ et al.: PAC1 RECEPTOR PROTECTS FROM ENDOTOXIC SHOCK 409

hepatic synthesis of the coagulation-related molecule �-fibrinogen duringLPS-induced endotoxemia. Although treatment with VIP/PACAP resulted ina downregulation of �-fibrinogen in wild-type mice, it failed to reduce LPS-induced �-fibrinogen mRNA expression in PAC1−/− mice. Therefore, the ef-fect of VIP and PACAP on neutrophil recruitment and ICAM-1, VCAM-1, and�-fibrinogen expression could be at least partly explained by their inhibitoryeffect on IL-6 production mediated through PAC1.13 These results were cor-roborated using the PAC1 antagonist PACAP (6–38). In contrast, VIP andPACAP inhibited the production of the AP protein SAA and the reactive oxy-gen NO in a PAC1-independent way4,14 (FIG. 1). The analysis of intracellularsignal pathways involved in the inhibitory effects of VIP and PACAP suggestsan important involvement of the inositol/phosphate/PLC cascade.13 TABLE 1summarizes information of advances in the identification of VIP/PACAP ef-fects mediated through PAC1 in the experimental endotoxic shock model.

In conclusion, this study provides some important insights regarding theinvolvement of PAC1 into the complexities of sepsis and may suggest newapproaches for the therapy of septic shock.

ACKNOWLEDGMENTS

This work was supported by grants BFI 2002-03489 from Ministerio deCiencia y Tecnologıa (Spain) and a predoctoral fellowship from Ministerio deCiencia y Tecnologıa (to A.A).

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