Pituitary Adenylate Cyclase-Activating Polypeptide Prevents Mortality Caused by Septic Peritonitis in Mice

Sepsis is initiated and perpetuated by overproduction of inflammatory cytokines and chemokines, resulting in organ injury and death in ∼30–50% of patients (1). Despite recent advances in our understanding of the molecular mechanism of sepsis, most of its complications remain refractory to treatment (2). Chemokine production by tissue macrophages (Mfs) promotes the recruitment of inflammatory cells, whereas increased expression of adhesion molecules facilitates leukocyte–endothelial cell interactions (3). Subsequent overproduction of inflammatory mediators injures organs including the lung, liver and kidney. Among these, lung injury and dysfunction represent the first step in the development of multiple organ failure, and acute respiratory distress syndrome is progressive and associated with mortality (4, 5).

Cecal ligation and puncture (CLP) is a frequently used experimental animal model of sepsis (6). This model closely resembles bowel perforation with tissue necrosis, leading to polymicrobial infection similar to acute peritonitis. The CLP model results in an early hyperdynamic phase followed by a hypodynamic phase, as in human septic peritonitis (7). Yang et al. reported that, in a murine CLP model, treatment with anti–high-mobility group box chromosomal protein 1 (HMGB1) Abs and antibiotics after CLP surgery significantly reduced mortality (8). Alternatively, it was reported from our laboratory that inhibition of Kupffer cells, the resident hepatic Mfs, increased serum HMGB1 levels and exacerbated lung injury and mortality in rats after CLP (9). Thus, the hepatic Mfs play a key role in septic peritonitis.

Pituitary adenylate cyclase-activating polypeptide (PACAP), first isolated from the ovine hypothalamus, belongs to the secretin glucagon vasoactive intestinal peptide (VIP) superfamily and exists in two amidated forms, PACAP27 and PACAP38 (10). PACAP plays a pivotal role in mediating vasodilation and bronchodilation (11) via three specific receptors: PACAP receptor 1 (PAC1R) and two VIP/PACAP receptors (12). PACAP has neuroprotective effects at very low concentrations, with intracerebroventricular infusion of PACAP in the rat preventing neuronal loss in the hippocampus and decreasing the extent of infarction after global and focal ischemia (13). It was also reported that both VIP and PACAP inhibit TNF-α production by LPS-activated RAW 246.7 cells in a dose- and time-dependent manner (14). TNF-α, a proinflammatory cytokine produced by activated Mfs, plays an essential role in normal and pathological inflammatory reactions. Because TNF-α plays a central role in inflammatory diseases, including endotoxin shock, multiple sclerosis, cerebral malaria, and various autoimmune conditions, the downregulatory effect of PACAP may have a significant therapeutic potential against inflammation. In addition, PACAP enhanced IL-10 production by activated Mfs, indicating an anti-inflammatory activity (14). IL-10 acts in vivo in conjunction with the inhibition of proinflammatory cytokines TNF-α to reduce the magnitude of the immune response. Separately, PACAP treatment increases plasma corticosterone and adrenocorticotropic hormone (ACTH) levels (15). Based on these results, PACAP may prevent acute lung injury and mortality in septic peritonitis by inhibiting proinflammatory cytokines and enhancing anti-inflammatory cytokines as direct effects, and activation of the hypothalamus-sympathetic adrenal axis. The aim of this study was to investigate the specific effects of PACAP in septic peritonitis using an animal model.

C57BL/6J mice (purchased from Japan SLC, Shizuoka, Japan) were used in all experiments. The experimental protocol followed our Institutional and the National Research Council criteria for the care and use of laboratory animals in research. All animals used for this study were housed in sterilized cages in a facility with a 12 h night/day cycle. Temperature and relative humidity were maintained at 23 ± 2°C and 50 ± 10%, respectively. The University of Yamanashi staff maintains these animal facilities, and veterinarians are always available to ensure animal health. All animals were given humane care in compliance with governmental regulations and institutional guidelines, and studies were performed according to protocols approved by the appropriate institutional review board.

Septic peritonitis was induced by CLP as described previously (6, 16). Briefly, the mice were anesthetized with inhaled diethyl ether, and then a 15-mm midline lower abdominal incision was made to expose the cecum, which was then ligated below the ileocecal junction, maintaining intestinal continuity. The cecum was punctured twice with a 21-gauge needle, and a small amount of cecal contents was expressed through the puncture wound. The incision was then closed, and 2 ml of sterile saline was administered s.c. for fluid resuscitation. After surgery, the mice were placed on a heating pad until they recovered from the anesthesia. The mice were given food and water ad libitum throughout the study. Survival was monitored for 7 d (n = 10 in each group).

Animals were treated with PACAP (50 μg/animal, i.v.) or saline vehicle at designated times before or after CLP.

Blood samples were collected via the inferior vena cava at designated times after CLP (n = 8). The samples were centrifuged at 1200 × g for 10 min at 4°C, and the serum was stored at −80°C until assay. Determination of serum HMGB1, TNF-α, and IL-10 levels was performed using ELISA kits (Central Institute, Shino-Test, Kanagawa, Japan).

Tissue samples were collected at designated times after CLP or sham operation and stored at −80°C for further analysis. These samples were fixed in formalin, embedded in paraffin, and serially sectioned. Some sections were stained with H&E to assess inflammation and necrosis. The pathology was evaluated in a blind manner by one of the authors and by an expert in rodent pathology.

Nine hours after CLP, mice were anesthetized and laparotomized. The lungs were placed in a tared plastic petri dish for weighing and then dried in a vacuum lyophilizer at −50°C and atmospheric pressure of 10 mmHg for 72 h (Refrigeration for Science, Island Park, NY). This process removed virtually all gravimetrically detectable water. The dry lung weight was determined, and wet/dry weight ratios were calculated to assess pulmonary edema (16).

Blood was collected via the portal vein 9 h after CLP in pyrogen-free heparinized syringes and centrifuged at 1200 rpm for 10 min. Plasma was stored at −20°C in pyrogen-free glass tubes until assay using a Limulus Amebocyte Lysate test kit (Kinetic-QCL; BioWhittaker, Walkersville, MD) (17).

Peripheral whole blood and peritoneal lavage collection were performed using aseptic technique (18). Serial dilutions were plated onto tryptic soy agar prepoured petri dishes supplemented with 5% sheep’s blood (Hardy Diagnostics) and incubated aerobically overnight at 37°C. Plates with 20–200 colonies were counted, and CFUs were normalized per gram of tissue collected.

The hepatic Mfs (Kupffer cells) were isolated by collagenase digestion and differential centrifugation using Nycodenz (Nycomed Pharma, Oslo, Norway) (19), as described in our previous work, with modifications as described elsewhere (9).

The alveolar Mfs were collected, and lavage of the lungs was repeated 20 times until only occasional cells were present in the lavage fluid. Thereafter, the lung tissue was sliced into small pieces. The interstitial lung Mfs were isolated according to the method of Holt et al. (20), as modified by Sjöstrand et al. (21). For isolation of the peritoneal Mfs, the contents of the peritoneum were lavaged by injection of 50 ml of PBS. Peripheral blood was also collected from the aorta, and peripheral mononuclear cells were isolated by differential centrifugation using NycoPrep 1.077A (Nycomed Pharma) (16). Isolated cells were seeded onto culture dishes and incubated for 1 h in DMEM (Life Technologies Laboratories Life Technologies, Grand Island, NY) supplemented with 10% FCS and antibiotics. After incubation, adhesive cells were collected using a scraper and washed three times with PBS for further experiments.

Nuclear protein extracts from the liver and the lung were prepared on ice as described by Dignam et al. (22) with minor modifications (23, 24). Protein concentration was determined using the Bradford protein concentration assay kit (Bio-Rad Laboratories, Hercules, CA) (25). A gel mobility shift assay was used to assess the amount of active protein involved in protein–DNA interactions (26). The specificity of protein binding in nuclear extracts was confirmed in competition and supershift experiments. Labeled and unlabeled oligonucleotides contained the consensus sequence for NF-κB (27). Data were quantitated by scanning autoradiograms with GelScan XL (Amersham Pharmacia Biotech, Uppsala, Sweden).

Determination of cytokine production by isolated Mfs was performed using ELISA kits for TNF-α (R&D Systems, Minneapolis, MN), HMGB1 (Central Institute, Shino-Test), and IL-10 (R&D Systems).

Measurements of plasma corticosterone and ACTH were performed using ELISA kits (corticosterone; Abcam, Cambridge, MA and ACTH; OriGene Technologies, Rockville, MD).

Data are expressed as the mean ± SEM. ANOVA with Bonferroni post hoc test or Fisher exact test (for survival experiments) was used to determine significance when appropriate. A p value < 0.05 was considered significant.

In the sham operation groups, all animals survived after surgery, and treatment with PACAP did not affect mortality. Mortality was 100% at 7 d after CLP in animals given a saline vehicle (Fig. 1). In contrast, mortality was 30% in animals treated with PACAP just after CLP (PACAP 0 h). There was a significant difference between the two groups. Furthermore, in animals treated with PACAP at 1 or 2 h after CLP, the mortality was also significantly reduced compared with the vehicle group (mortality, PACAP after 1 h; 50% and PACAP after 2 h; 70%).

Mortality in animals treated with PACAP 1 h after CLP was not reduced compared with that in animals treated with PACAP just after CLP. This was most likely due to the effect of PACAP as a vasoactive intestinal peptide on the intestine. Indeed, after PACAP treatment, intestinal secretion increased, leading to leakage of stool from the perforated holes after CLP. This may have exacerbated peritonitis and mortality.

In the sham operation groups, no organ injury was observed (data not shown). Severe interstitial lung edema and hemorrhage were observed in the control group (Fig. 2). In contrast, lung injury was significantly reduced by the PACAP treatment.

Tissue injury in other organs, including the liver, the kidney, and the small intestine, was also analyzed. As a result, injury in the liver and the kidney was minimal. In addition, mild mucosal injury (arrows) was detected in the small intestine in animals without PACAP treatment after CLP (Fig. 2). This injury was also prevented by PACAP treatment.

Reflecting the acute lung injury (Fig. 2), the increased wet/dry weight ratio in the control group was significantly reduced in the PACAP-treated group compared with the vehicle group (Fig. 3A).

Plasma endotoxin levels were minimal in all the sham operation groups (data not shown). In contrast, endotoxin levels were significantly higher in both the vehicle and PACAP groups 9 h after CLP (Fig. 3B). There were no significant differences between the two groups.

There were no significant differences in bacterial load both in the peripheral blood and the peritoneal lavage between PACAP-treated and nontreated groups (Fig. 3C).

Serum TNF-α levels were minimal in the sham operation group (data not shown). In contrast, the levels peaked at 9 h and gradually decreased until 24 h after CLP in the control group (Fig. 4A). In the PACAP group, they also peaked at 9 h after CLP; however, they were significantly lower compared with the control group after CLP.

Serum HMGB1 levels were minimal in the sham operation group (data not shown). In contrast, serum HMGB1 levels increased gradually up to 24 h after CLP in the control group (Fig. 4B). In the PACAP group, levels also increased gradually up to 24 h after CLP; however, they were significantly lower compared with the control group at 12, 18 and 24 h after CLP.

Serum IL-10 levels were minimal in the sham operation group (data not shown). In contrast, the levels peaked at 12 h and gradually decreased until 24 h after CLP in the control group (Fig. 4C). In the PACAP group, they also peaked at 12 h after CLP and were significantly greater compared with the control group after CLP.

In the sham operation group, the plasma corticosterone and ACTH levels were significantly greater in animals treated with PACAP compared with vehicle in animals that underwent sham operation (Fig. 5). Although these levels increased in both groups after CLP, the values were significantly greater in the PACAP group than in the vehicle group.

Production of inflammatory and anti-inflammatory cytokines by isolated tissue Mfs were measured in vitro (Fig. 6). Production of inflammatory cytokines HMGB1 and TNF-α was significantly inhibited by PACAP treatment in the isolated Kupffer cells, compared with those in the other tissue Mfs (Fig. 6A, 6B).

Production of anti-inflammatory cytokine IL-10 was significantly increased by PACAP treatment in the isolated Kupffer cells and interstitial lung Mfs, compared with those in the other tissue Mfs (Fig. 6C).

The active form of the pleiotropic transcription factor NF-κB was minimal in hepatic Mfs from normal rats (data not shown). However, NF-κB activity increased in tissue Mfs isolated from CLP animals (Fig. 7A, 7B), and the activity was greatest in the hepatic Mfs. PACAP significantly inhibited NF-κB activity in the hepatic Mfs but not in Mfs from the other tissues (Fig. 7B).

To confirm that protein binding in nuclear extracts to the labeled oligonucleotide probe was specific for the active form of NF-κB, gel shift assays were carried out either in the presence of excess unlabeled ds-oligonucleotide with a consensus sequence for NF-κB binding or with Abs specific for the NF-κB p50 and p60 subunit (Fig. 7C). No binding was detected with no nuclear extract added (lane 7). Nuclear extracts from hepatic Mfs (same as in lane 3 in Fig. 7A) were used for competition experiments (200-fold excess of the unlabeled oligonucleotide, lane 8) and supershift experiments (p50 or p65 antiserum, lanes 9 and 10, respectively).

HMGB1 is actively released into the serum by activated monocytes/Mfs and passively diffuses from necrotic cells. Therefore, the CLP model has two possible sources of HMGB1: monocyte/Mfs activated by endotoxin or inflammatory cytokines such as TNF-α and the necrotic cecum (28). In the current study, serum HMGB1 concentrations increased at time points earlier than 3 h after CLP (Fig. 1). Furthermore, there were no significant differences in pathological changes of the cecum between the groups studied. Serum HMGB1 concentrations were reported to increase until 4 h after CLP in a modified model that preserves blood flow (8). These results may indicate that the expression of HMGB1 in the early phase after CLP is predominantly from inflammatory cells including Mfs activated by endotoxin. Thus, HMGB1 plays a pivotal role in septic peritonitis.

It was reported that PACAP inhibits TNF-α production by LPS-activated RAW 246.7 cells in a dose- and time-dependent manner (14). Moreover, in this study, PACAP also inhibited production of TNF-α by isolated hepatic Mfs (Fig. 6). Importantly, in the current study, treatment of PACAP in vitro significantly inhibited activation of NF-κB in the isolated tissue Mfs, particularly in the hepatic Mfs (Fig. 7) (29). Thus, PACAP inhibits initiation of the inflammatory cytokine cascade, including proinflammatory cytokine TNF-α induced by NF-κB activation. In the current study, although there were no significant differences in plasma endotoxin levels between animals with and without PACAP treatment (Fig. 3A), serum TNF-α levels were inhibited in the PACAP group compared with the control group (Fig. 4). Thus, inhibition of TNF-α production by Kupffer cells via NF-κB activation due to PACAP treatment most likely leads to inhibition of serum TNF-α levels, reducing the acute lung injury and mortality after CLP (Figs. 1, 2, 3B).

PACAP reportedly enhanced the production of IL-10 by activated Mfs, indicating an anti-inflammatory activity (30). Consistent with this, in the current study, PACAP increased the production of IL-10 by the Kupffer cells and the interstitial lung Mfs, as well as the serum IL-10 levels (Figs. 4C, 6C). IL-10 acts in conjunction with the inhibition of proinflammatory cytokine IL-6 in vivo. In this study, serum IL-6 levels were also blunted in the animals treated with PACAP (data not shown). Reflecting these results, the mortality and acute lung injury after CLP were prevented by PACAP treatment (Figs. 1, 2).

It was recently reported that peripherally administered orexin penetrates the blood–brain barrier (BBB) under endotoxin shock, suppressing cytokine production and improving survival, indicating a direct action of orexin in the CNS (31). In vivo, PACAP can exert neuroprotective effects after i.v. injection, because it is transported in sufficient quantities by a saturable system across the BBB (32). In this study, PACAP increased both ACTH and corticosterone levels and prevented mortality after CLP (Figs. 1, 5). Because PACAP transport across the BBB varies among regions of the brain (33), PACAP could potentially penetrate into the CNS because of BBB dysregulation under the condition of systemic inflammation including sepsis.

Reversible adrenal insufficiency has been frequently diagnosed in critically ill patients with sepsis who have either low basal cortisol levels or low cortisol responses to ACTH stimulation (34). It was reported that PACAP increased ACTH after CLP and corticosterone levels after endotoxin administration (34, 35). In this study, peripheral i.v. injection of PACAP also significantly increased the plasma corticosterone and ACTH levels after CLP (Fig. 5). Thus, PACAP could induce corticosterone release from the brain, leading to reduced inflammatory cytokine levels in the peripheral blood (Fig. 4A, 4B). Taken together, PACAP may also have a direct action in the CNS, and further investigation is needed to clarify this issue.

The authors have no financial conflicts of interest.