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-α (TNF-α), interleukin (IL)-1β, IL-10, and nitric oxide release from activated macrophages.
Measurements and Main Results: The GRP antagonist attenuated LPS- or CLP-induced TNF-α, IL-1β, 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-α and IL-1β 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.
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,Leu13ψ[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.
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.
GRP receptor antagonist RC-3095, originally synthesized in the Schally laboratory by solid-phase methods (20), was made by Zentaris (Frankfurt am Main, Germany).
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.
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, São Paulo, Brazil).
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-α (TNF-α), interleukin (IL)-1β, 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-α, IL-1β, and IL-10.
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).
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).
The GRP antagonist attenuated LPS- or sepsis-induced TNF-α and IL-1β 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-α (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-α 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-α and IL-1β 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.
Time after CLP
|12 h||24 h||12 h||24 h||12 h||24 h|
|Urea, mg/dl||48 ± 1.3||54 ± 2.1||47 ± 1.7||26 ± 0.6*||27 ± 0.9*||27 ± 0.5*|
|Creatinine, mg/dl||0.3 ± 0.01||0.4 ± 0.03||0.4 ± 0.03||0.3 ± 0.01||0.2 ± 0.01||0.2 ± 0.02|
|AST, IU/L||418 ± 23||497 ± 31||425 ± 26||393 ± 32||276 ± 19*||301 ± 21*|
|ALT, IU/L||87 ± 7||102 ± 12||102 ± 10||87 ± 9||59 ± 7*||52 ± 10|
|Amylase, IU/L||3,136 ± 100||2,970 ± 99||2,956 ± 129||2,863 ± 131||2,010 ± 102*||1,900 ± 121*|
|TNF-α, pg/ml||3,400 ± 198||2,600 ± 175||2,100 ± 99*||1,200 ± 102*||1,130 ± 92†||1,021 ± 102*|
|IL-1β, pg/ml||430 ± 41||338 ± 37||367 ± 44||201 ± 32*||209 ± 24*||178 ± 21*|
|IL-10, pg/ml||220 ± 21||233 ± 18||215 ± 18||227 ± 11||234 ± 22||244 ± 25|
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-α, and IL-1β content (Table 2). RC-3095 administration reduced BALF total inflammatory cell content, protein exudation, and TNF-α and IL-1β content (Table 2). In addition, RC-3095 treatment diminished BALF LDH content (as an index of alveolar cell injury; Table 2).
Total Cell Count (× 105)
Total Protein (mg/ml)
LDH Activity (IU/L)
|Control||51 ± 3.4||40 ± 3.2||1.2 ± 0.1||UD||UD|
|LPS||500 ± 21*||78 ± 6.5*||16.2 ± 2.3*||41 ± 6||31 ± 3|
|LPS + RC||97 ± 4.6†||37 ± 2.1||8.3 ± 1.2†||8 ± 0.9†||15 ± 2†|
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-α and IL-1β) 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-α and IL-1β, 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-α and IL-1β 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.
The authors thank Dr. Joao Rocha for histopathologic analyses and Dr. Elidio Angioleto for microbiological analyses.
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