点击显示 收起
Experimental Physiopathology Laboratory, Universidade do Extremo Sul Catarinense, Criciúma
Departments of Biochemistry and Pharmacology, Institute for Basic Health Sciences, Graduate Program in Medical Sciences
Department of Internal Medicine, Academic Hospital, Federal University of Rio Grande do Sul
Center for Memory Research, Biomedical Research Institute, Pontifical Catholic University of Rio Grande do Sul, Porto Alegre
Department of Internal Medicine, Academic Hospital, So Paulo University, So Paulo, Brazil
ABSTRACT
Rationale: Several new therapeutic strategies have been described for the treatment of sepsis, but to date none are related to alterations in the bombesin/gastrin-releasing peptide (GRP) receptor pathways.
Objectives: To determine the effects of a selective GRP receptor antagonist, RC-3095, on cytokine release from macrophages and its in vivo effects in the cecal ligation and puncture (CLP) model of sepsis and in acute lung injury induced by intratracheal instillation of LPS.
Methods: We determined the effects of RC-3095 in the CLP model of sepsis and in acute lung injury induced by intratracheal instillation of LPS. In addition, we determined the effects of RC-3095 on tumor necrosis factor- (TNF-), interleukin (IL)-1, IL-10, and nitric oxide release from activated macrophages.
Measurements and Main Results: The GRP antagonist attenuated LPS- or CLP-induced TNF-, IL-1, and nitric oxide release in cultured macrophages and decreased the mRNA levels of inducible nitric oxide synthase. The administration of RC-3095 (0.3 mg/kg) 6 h after sepsis induction improved survival in the CLP model, and diminished lung damage after intratracheal instillation of LPS. These effects were associated with attenuation on the circulating TNF- and IL-1 levels and decreased myeloperoxidase activity in several organs.
Conclusions: We report that a selective GRP receptor antagonist attenuates the release of proinflammatory cytokines in vitro and in vivo and improves survival in "established" sepsis. These are consistent with the involvement of a new inflammatory pathway relevant to the development of sepsis.
Key Words: cytokine gastrin-releasing peptide lipopolysaccharide macrophage RC-3095
Septic shock has become one of the most frequent causes of morbidity and mortality in intensive care units (1). Treatment of sepsis consists of supporting blood pressure, organ blood flow, and ventilation, along with an emphasis on antibiotics and eradicating the source(s) of infection. Despite significant advances in the understanding of pathogenesis of sepsis and its management, only a few therapeutic strategies have been introduced that could reduce mortality from septic shock (2).
Although commonly initiated by an infection, the pathogenesis of sepsis is characterized by an overwhelming systemic inflammatory response that can lead to lethal multiple organ failure (2). To date, antiinflammatory strategies have produced modest clinical effects in critically ill patients (3). Several new therapeutic strategies have been described in the literature for the treatment of sepsis and its consequences (4–8), but none of these is related to alterations in the bombesin/gastrin-releasing peptide (GRP) receptor pathways. GRP receptor pathways were previously shown to participate in the control of central nervous system and gastrointestinal system functions (9–11), cancer growth (12), and immune cell regulation (13–15), thus being implicated in the pathogenesis of inflammatory conditions (16, 17).
Because activated macrophages have been shown to secrete GRP (13) and macrophages seem to be central in the development of sepsis and septic shock (18, 19), we here study the effects of a selective GRP receptor antagonist, (D-Tpi6,Leu13[CH2NH]-Leu14) bombesin(6–14) (RC-3095; Tpi is 2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole-3-carboxylic acid), on cytokine release from macrophages. In addition, we determine the in vivo effects of RC-3095 in the cecal ligation and puncture model of sepsis and in acute lung injury (ALI) induced by intratracheal instillation of LPS.
METHODS
All experimental procedures involving animals were performed in accordance with the National Institutes of Health (Bethesda, MD) Guide for the Care and Use of Laboratory Animals with the approval of the local ethics committee.
Materials
GRP receptor antagonist RC-3095, originally synthesized in the Schally laboratory by solid-phase methods (20), was made by Zentaris (Frankfurt am Main, Germany).
Cecal Ligation Puncture Model
Male Wistar rats, 2 to 3 mo old, subjected to cecal ligation and puncture (CLP) as previously described and standardized in our laboratory (5), were used in this study (for details, see the online supplement). For the purpose of biochemical measurements and histopathologic analyses (see below), 24 rats were made septic by CLP. The animals were divided into four groups: (1) sham operated; (2) CLP; (3) CLP plus "basic support" (saline administered subcutaneously at 50 ml/kg 6 and 12 h after CLP plus ceftriaxone administered subcutaneously at 30 mg/kg and clindamycin administered subcutaneously at 25 mg/kg every 6 h for 3 d, starting 6 h after CLP); and (4) same as group 3 but with RC-3095 administered subcutaneously at 0.3 mg/kg, once a day for 2 d, starting 6 h after CLP. Blood was drawn from the caudal vein 12 and 24 h after CLP to determine organ damage plasma markers and plasma cytokines (see below). Twenty-four hours after treatment administration the rats were killed by decapitation followed by the harvesting of lung, liver, kidney, heart, and ileum samples, which were immediately stored at –70°C until assayed for myeloperoxidase activity, or were fixed for histopathologic analyses.
In a separate cohort of animals mortality was evaluated. Animals exposed to CLP were randomly assigned to receive or not receive RC-3095 (subcutaneously at 0.03, 0.3, and 3 mg/kg) once per day for 2 d starting at 6 h with "basic support." All animals were then returned to their cages with free access to food and water and were monitored for 10 d.
As an index of neutrophil infiltration, we measured myeloperoxidase activity in tissue homogenates as previously described (21) (for details, see the online supplement). For histopathologic analyses after fixation, excised tissues were embedded in paraffin and then routinely stained with hematoxylin and eosin. An experienced pathologist performed blinded histopathologic analyses.
ALI Model
Adult male Wistar rats weighing approximately 250 to 300 g were used in this study. Rats were anesthetized by an intraperitoneal injection of ketamine (80 mg/kg) and ALI was induced by intratracheal instillation of LPS (Escherichia coli 055:B5; Sigma, St. Louis, MO) at a dose of 100 μg/100 g body weight.
Twelve hours after LPS instillation, the rats were killed and bronchoalveolar lavage (BAL) was performed. BAL fluid (BALF) was centrifuged and the resultant cell-free supernatant was analyzed for various biochemical parameters (see below). The cell pellet was used to determine the total cell count and differential (see below). In a separate cohort of animals, ALI was induced as described above and lung tissue was analyzed. In brief, 12 h after LPS instillation, the rats were killed and samples from the lung were isolated and fixed in 4% formalin solution for histopathologic analyses.
The animals were divided into three groups: group 1, instillation of isotonic saline; group 2, ALI treated with saline; group 3, ALI treated with RC-3095 (0.3 mg/kg, administered subcutaneously 3 h after ALI; n = 36). To estimate the degree of alveolar cell injury and alveolar–capillary membrane compromise, BALF total cell count and differential, BALF protein, and lactate dehydrogenase (LDH) content were determined. BALF cells, stained with Giemsa or trypan blue exclusion dye, were evaluated with a Neubauer chamber. BALF total protein content was determined by Lowry assay. BALF total LDH content was determined with commercially available kits (LabTrade do Brasil, So Paulo, Brazil).
Peritoneal Macrophage Experiments
Peritoneal macrophages were prepared from freshly isolated peritoneal exudates of Wistar rats (normal rats or rats 4 h after CLP; for details, see the online supplement). Macrophages isolated from normal rats were exposed to LPS (100 ng/ml for 4 h) and then assigned to receive RC-3095 (1 or 10 μg/ml) for 2 h or not to receive RC-3095. After this period the medium was recovered for determination of tumor necrosis factor- (TNF-), interleukin (IL)-1, and IL-10 by ELISA with commercial available kits (R&D Systems, Minneapolis, MN). Macrophages isolated from CLP-exposed animals were cultured for 4 h and treated with or without RC-3095 for 2 h. After this period the medium was recovered for determination of TNF-, IL-1, and IL-10.
RAW 264.7 Experiments
To determine oxidative burst and nitrite content, and to perform reverse transcription-polymerase chain reaction (RT-PCR) analyses of inducible nitric oxide synthase (iNOS) mRNA (see below), RAW 264.7 cultures (for details, see the online supplement) were exposed to LPS (100 ng/ml)–supplemented medium (RPMI 1640) or RPMI 1640 alone and, 4 h later, RC-3095 (1 or 10 μg/ml) was added for 2 h. Several times after this period cells and/or medium were collected for analyses.
In the reverse transcriptase–polymerase chain reaction (RT-PCR) experiments, RAW 264.7 cell RNA was extracted and RT-PCR was performed (for details, see the online supplement). iNOS mRNA levels were expressed as the ratio of signal intensity for the target genes in relation to that for the coamplified glyceraldehyde-3-phosphate dehydrogenase.
Nitrite concentration was measured in the medium as an index of macrophage release of nitric oxide, as previously described (22) (for details, see the online supplement). Nitrite values were expressed as a percentage relative to control (cultured macrophages without LPS addition).
Macrophage oxidative burst was assessed in RAW 264.7 cells by the dichlorofluorescein technique as previously described (23).
Statistical Analyses
Results are expressed as means and p < 0.05 was considered significant. Differences between multiple groups were determined by one-way analysis of variance followed by a Newman-Keuls test. Differences between two groups were determined by t test. The survival curves of the different treatment groups were compared by log-rank test. All statistical analyses were performed with SPSS 12.0 for Windows (SPSS, Chicago, IL).
RESULTS
The GRP antagonist attenuated LPS- or sepsis-induced TNF- and IL-1 release in cultured peritoneal macrophages (Figure 1A). This was more pronounced at 10 μg/ml (Figure 1A), but was evident at 1 μg/ml only for TNF- (data not shown). In addition, RC-3095 treatment decreased iNOS mRNA levels (Figure 1B) in LPS-stimulated RAW 264.7 cells 30 min (Figure 1B), but not 2 h, after treatment (data not shown), suggesting that RC-3095 effects were reversible. As demonstrated for TNF- release, these effects were clearer at 10 μg/ml, but were evident at 1 μg/ml (data not shown). This was supported by the observed decrease in nitrite accumulation in the culture medium 6 h after LPS administration (Figure 1C). The decrease in proinflammatory cytokines and nitric oxide release from macrophages could attenuate the inflammatory response and diminish damage in inflammatory conditions such as sepsis. In addition, RC-3095 treatment did not interfere with the release of the antiinflammatory cytokine IL-10 (Figure 1A) or with the macrophage oxidative burst in RAW 264.7 macrophages (data not shown).
To determine the effects of RC-3095 in vivo we performed experiments in the CLP model of rodent sepsis and in ALI induced by LPS. The administration of RC-3095 (0.3 mg/kg) 6 h after sepsis induction improved survival in the CLP model (Figure 2). Higher doses of RC-3095 did not enhance its effects, and lower doses presented a less significant impact on mortality (Figure 2). The administration of RC-3095 1 h before or 1 h after CLP without basic support significantly improved survival by approximately 50% (data not shown). The effects on mortality were associated with attenuation of circulating TNF- and IL-1 levels, but not IL-10 levels (Table 1), 12 and 24 h after sepsis induction. Attenuation of the inflammatory response is also evidenced by decreased myeloperoxidase activity in the lung, liver, and ileum (Figure 3) 24 h after treatment. RC-3095 administration attenuated the damage in pancreas, liver, and kidney as assessed by plasma markers of organ injury (Table 1). The GRP antagonist diminished ileal inflammatory infiltration (Figures 4A and 4B), alveolar edema and inflammatory infiltration (Figures 4C and 4D), and renal tubular necrosis (Figures 4E and 4F) when compared with basic support. Quantitative blood cultures were similar in control CLP animals or CLP animals treated only with RC-3095, suggesting a lack of direct antibacterial effects associated with this compound.
GRP antagonist also attenuated the alveolar inflammatory infiltration and alveolar exudation induced by intratracheal LPS (Figures 5A and 5B). Myeloperoxidase activity was reduced from 31 ± 4 mU/mg protein in the saline group to 18 ± 2 mU/mg protein in the RC group (p < 0.05, t test). These findings were supported by the BALF content of inflammatory cells, LDH activity, protein, TNF-, and IL-1 content (Table 2). RC-3095 administration reduced BALF total inflammatory cell content, protein exudation, and TNF- and IL-1 content (Table 2). In addition, RC-3095 treatment diminished BALF LDH content (as an index of alveolar cell injury; Table 2).
DISCUSSION
Here, we report on the beneficial effects of the selective bombesin/GRP receptor antagonist, RC-3095, in a well-established model for experimental sepsis and acute lung injury. RC-3095 modulates the release of proinflammatory cytokines (TNF- and IL-1) by activated macrophages, leading to a diminution of inflammatory infiltration and organ dysfunction, thus improving mortality in a clinically relevant model of sepsis.
Although it seems that the overwhelming inflammatory response is central to the pathogenesis of septic shock, currently used antiinflammatory strategies have a limited clinical effect in patients with sepsis or acute respiratory distress syndrome (3). This could be secondary to several factors, including the heterogeneous inflammatory response associated with these conditions (2) and the misleading design of preclinical studies (24, 25). Thus, we tested the effects of RC-3095 in the CLP model of sepsis with antibiotics and fluid resuscitation, administering the treatments late after sepsis induction to more closely resemble clinical practice.
Receptors for GRP have been detected in various cells of the gastrointestinal system; the pancreas; smooth muscles of the digestive tract, bladder, and uterus; the neuronal elements of the myenteric plexus; and in the nervous system (26, 27). Lymphocytes, neutrophils, eosinophils, macrophages, mast cells, and endothelial cells express receptors for GRP (28). The expression of GRP receptors depends on cell differentiation, and GRP can stimulate or inhibit responses, depending on the maturity and state of activation of the target cell (13, 28, 29). Previous work had shown that GRP exerts a significant effect on different aspects of immune system function in vitro and in vivo (13–17); but it was unknown whether this pharmacologic strategy could be used in vivo to improve survival in sepsis. In our study, we demonstrate that RC-3095 inhibits macrophage release of TNF- and IL-1, and this could block several processes associated with sepsis progression. These effects on cytokine release attenuate neutrophil infiltration, organ damage in sepsis, and ALI. Interestingly, RC-3095 did not modulate the release of the antiinflammatory IL-10, suggesting that the intracellular pathway modulated by bombesin/GRP is selective to proinflammatory cytokines. Some of the actions of bombesin/GRP seem to involve the activation of protein kinase C (14). The effects of bombesin, a homolog of GRP, on cyclooxygenase-2 expression in intestinal cell lines requires an increase in Ca+2; activation of extracellular signal-regulated kinase-1 and -2 and p38MAPK; and increased activation and expression of the transcription factors Elk-1, ATF-2, c-Fos, and c-Jun (30).
Little is known about the physiologic effects of GRP on immune function and their possible use in a pharmacologic approach to inflammatory conditions. With the use of a specific antagonist of the GRP receptor we have demonstrated that GRP receptor inhibition could decrease TNF- and IL-1 release from activated macrophages, and that this could represent a possible pharmacologic target by which to control systemic inflammation. In short, RC-3095 was able to attenuate damage and to improve survival in a relevant animal model of sepsis and ALI. This is the first report of a protective effect of a bombesin/GRP antagonist in these conditions and of the possible role of GRP in the development of sepsis.
Acknowledgments
The authors thank Dr. Joao Rocha for histopathologic analyses and Dr. Elidio Angioleto for microbiological analyses.
FOOTNOTES
Supported by the South American Office for Anticancer Drug Development and by Zentaris GmbH.
This article has an online supplement, which is accessible from this issue's table of contents at www.atsjournals.org
Originally Published in Press as DOI: 10.1164/rccm.200507-1118OC on September 28, 2005
Conflict of Interest Statement: F.D.-P. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. L.P.D.L. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. C.R. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. M.R.M. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. A.R. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. D.P.G. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. A.Z.-F. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. L.F.d.S. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. M.A. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. D.F.B. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. E.A.B. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. M.C. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. L.R.M.B. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. F.G.S. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. J.C.F.M. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. R.R. is an inventor in a provisional patent application for the therapeutic use of gastrin-releasing peptide receptor antagonists in the treatment of inflammatory conditions. G.S. is an inventor in a provisional patent application for the therapeutic use of gastrin-releasing peptide receptor antagonists in the treatment of inflammatory conditions.
REFERENCES
Sands KE, Bates DW, Lanken PN, Graman PS, Hibberd PL, Kahn KL, Parsonnet J, Panzer R, Orav EJ, Snydman DR. Epidemiology of sepsis syndrome in 8 academic medical centers. JAMA 1997;278:234–240.
Hotchkiss RS, Karl IE. The pathophysiology and treatment of sepsis. N Engl J Med 2003;348:138–150.
Eichacker PQ, Parent C, Kalil A, Esposito C, Cui X, Banks SM, Gerstenberger EP, Fitz Y, Danner RL, Natanson C. Risk and the efficacy of antiinflammatory agents: retrospective and confirmatory studies of sepsis. Am J Respir Crit Care Med 2002;166:1197–1205.
Giacometti A, Cirioni O, Ghiselli R, Mocchegiani F, D'Amato G, Circo R, Orlando F, Skerlavaj B, Silvestri C, Saba V, et al. Cathelicidin peptide sheep myeloid antimicrobial peptide-29 prevents endotoxin-induced mortality in rat models of septic shock. Am J Respir Crit Care Med 2004;169:187–194.
Ritter C, Andrades ME, Reinke A, Menna-Barreto S, Moreira JC, Dal-Pizzol F. Treatment with N-acetylcysteine plus deferoxamine protects rats against oxidative stress and improves survival in sepsis. Crit Care Med 2004;32:342–349.
Tzeng HP, Ho FM, Chao KF, Kuo ML, Lin-Shiau SY, Liu SH. -Lapachone reduces endotoxin-induced macrophage activation and lung edema and mortality. Am J Respir Crit Care Med 2003;168:85–91.
Wang H, Liao H, Ochani M, Justiniani M, Lin X, Yang L, Al-Abed Y, Wang H, Metz C, Miller EJ, et al. Cholinergic agonists inhibit HMGB1 release and improve survival in experimental sepsis. Nat Med 2004;10:1216–1221.
Yang H, Ochani M, Li J, Qiang X, Tanovic M, Harris HE, Susarla SM, Ulloa L, Wang H, DiRaimo R, et al. Reversing established sepsis with antagonists of endogenous high-mobility group box 1. Proc Natl Acad Sci USA 2004;101:296–301.
Yamada K, Santo-Yamada Y, Wada E, Wada K. Role of bombesin (BN)-like peptides/receptors in emotional behavior by comparison of three strains of BN-like peptide receptor knockout mice. Mol Psychiatry 2002;7:113–117.
Mellar CA, Henriques JAP, Schwartsmann G, Roesler R. The bombesin/gastrin releasing peptide receptor antagonist RC-3095 blocks apomorphine but not MK-801-induced stereotypy in mice. Peptides 2004;25: 585–588.
Roesler R, Henriques JA, Schwartsmann G. Neuropeptides and anxiety disorders: bombesin receptors as novel therapeutic targets. Trends Pharmacol Sci 2004;25:241–242.
Schwartsmann G. Dexamethasone and gastrin-releasing peptide receptors in human lung cells. Lung Cancer 2004;46:129.
Genton L, Kudsk KA. Interactions between the enteric nervous system and the immune system: role of neuropeptides and nutrition. Am J Surg 2003;186:253–258.
Medina S, Del Rio M, Victor VM, Hernanz A, De la Fuente M. Changes with ageing in the modulation of murine lymphocyte chemotaxis by CCK-8S, GRP and NPY. Mech Ageing Dev 1998;102:249–261.
Medina S, Rio MD, De la Cuadra B, Guayerbas N, De la Fuente M. Age-related changes in the modulatory action of gastrin-releasing peptide, neuropeptide Y and sulfated cholecystokinin octapeptide in the proliferation of murine lymphocytes. Neuropeptides 1999;33:173–179.
Subramaniam M, Sugiyama K, Coy DH, Kong Y, Miller YE, Weller PF, Wada K, Wada E, Sunday ME. Bombesin-like peptides and mast cell responses: relevance to bronchopulmonary dysplasia Am J Respir Crit Care Med 2003;168:601–611.
Grimsholm O, Rantapaa-Dahlqvist S, Forsgren S. Levels of gastrin- releasing peptide and substance P in synovial fluid and serum correlate with levels of cytokines in rheumatoid arthritis. Arthritis Res Ther 2005;7:R416–R426.
Dal-Pizzol F. Alternative activated macrophage: a new key for systemic inflammatory response syndrome and sepsis treatment Crit Care Med 2004;32:1971–1972.
Shasby DM, McCray P. Sepsis and innate immunity. Am J Respir Crit Care Med 2004;169:144–145.
Radulovic S, Cai R-Z, Serfozo P, Groot K, Redding TW, Pinski J, Schally AV. Biological effects and receptor binding affinities of new pseudononapeptide bombesin/GRP receptor antagonists with N-terminal D-Trp or D-Tpi. Int J Pept Protein Res 1991;38:593–600.
Liaudet L, Pacher P, Mabley JG, Virag L, Soriano FG, Hasko G, Szabo C. Activation of poly(ADP-ribose) polymerase-1 is a central mechanism of lipopolysaccharide-induced acute lung inflammation. Am J Respir Crit Care Med 2002;165:372–377.
Marletta MA, Yoon PS, Iyengar R, Leaf CD, Wishnok JS. Macrophage oxidation of L-arginine to nitrite and nitrate: nitric oxide is an intermediate. Biochemistry 1988;27:8706–8711.
Wan CP, Myung E, Lau BH. An automated micro-fluorometric assay for monitoring oxidative burst activity of phagocytes. J Immunol Methods 1993;159:131–138.
Ritter C, Andrades M, Reinke A, Moreira JC, Dal-Pizzol F. Drug intervention trials in sepsis. Lancet 2004;364:498.
Polderman KH, Girbes AR. Drug intervention trials in sepsis: divergent results. Lancet 2004;363:1721–1723.
Ohki-Hamazaki H, Iwabuchi M, Maekawa F. Development and function of bombesin-like peptides and their receptors. Int J Dev Biol 2005;49: 293–300.
Thomas RP, Hellmich MR, Townsend CM, Evers BM. Role of gastrointestinal hormones in the proliferation of normal and neoplastic tissues. Endocr Rev 2003;24:571–599.
Furness JB, Kunze WA, Clerc N. The intestine as a sensory organ: neural, endocrine, and immune responses. Am J Physiol 1999;277:922–928.
Rozengurt E. G protein-coupled receptors in gastrointestinal physiology. V. Gastrointestinal peptide signaling through tyrosine phosphorylation of focal adhesion proteins. Am J Physiol 1998;275:177–182.
Guo YS, Hellmich MR, Wen XD, Townsend CM Jr. Activator protein-1 transcription factor mediates bombesin-stimulated cyclooxygenase-2 expression in intestinal epithelial cells. J Biol Chem 2001;276:22941–22947.