Abstract
Severe acute pancreatitis (AP) is a disease associated with high mortality and characterized by overwhelming systemic inflammation. Older people have a prolonged hospital stay and worst prognosis, when affected by this disease. Our group hypothesized, thus, that the systemic inflammatory response in the elderly would promote more organ damage when compared to the young. We sought to investigate the effect of systemic inflammation on the gene expression of cytokines, chemokines, and growth factors in the hearts of older and younger rats in an animal model of AP. AP was induced in all rats by injection of 0.5 mL of 2.5% taurocholate. There were two healthy age-matched control groups. An array of 79 cytokines, chemokines, and growth factors was measured in samples of cardiac tissue taken from the AP rats after 10 h, and from control rats. Older healthy rats had significantly higher levels of interleukin-10 (IL-10) and CCL1 gene expression than younger ones (P < 0.05), but all other measurements were similar among the study groups. This study indicates the systemic inflammation may show unique features for different organs in the body, but older animals with systemic inflammation are similar to the young regarding the cardiac inflammatory response.
Introduction
Acute pancreatitis (AP) is a disease characterized by local and systemic inflammation, and its severe form is associated with high morbidity and mortality rates. 1 Advanced AP is characterized by infected necrosis and can lead to the involvement of distant organs and the development of multiple organ dysfunction.2,3 AP has various etiological factors, the most common of which are gallstones and excess alcohol consumption, which together represent 75% of cases. 4 Regardless of the initial mechanism, the inflammatory process is triggered by injury to the pancreatic acinar cells, leading to recruitment of immune cells and production of molecules such as cytokines and chemokines, which play an important role in the pathogenesis of this disease. 5 Pancreatic inflammation induces profound disturbances in homeostasis, leading to tissue injury in other organs such as the intestines. 6 On the other hand, organs such as the brain 7 have structural and cellular mechanisms of protection against inflammation, and neurologic manifestations of this disease are rare. What about the heart?
It has been described that patients with heart failure exhibit cardiac injury, leading to the production of a variety of pro-inflammatory cytokines, activation of T cells, formation of autoantibodies, and activation of the complement system. 8 This suggests that a robust innate immune response can also be activated in the heart, so we hypothesized that the heart could display cardioprotective mechanisms during systemic inflammation. We have previously demonstrated that the intensity of systemic inflammation associated with AP is similar in young and old rats but that the duration of systemic inflammation is much longer in older animals. It was found that older rats had more severe intestinal injury and, as a consequence, there was greater bacterial translocation, leading to prolonged systemic infection, multiple organ failure, and higher mortality. 6 Advanced age is considered to be an independent prognostic factor for a poorer prognosis in AP but the mechanisms involved are not fully understood. 9
Sustained inflammatory response in distant organs like the heart may be implicated in this age-related vulnerability. CC chemokines are the largest subfamily of chemokines, which are important components of the innate immune system.
Chemokines trigger leukocyte trafficking and are implicated in cardiovascular disease pathophysiology. The aim of this study was to investigate whether the cardiac inflammatory response and chemokines expression could be more exacerbated in the elderly, during severe pancreatic injury.
Materials and methods
Animal model of AP
The experiments were performed at the Emergency Medicine Department (LIM-51—Faculdade de Medicina da Universidade de São Paulo) and at the Heart Institute (InCor—Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo). The Protocol was approved by the Ethics Committee on the use of animals (Protocol # 141/14).
Male Wistar rats, either young (8 weeks), from 200 to 350 g body weight, or aged 18 months, from 600 g to 850 g, were divided into the following groups: young animals with induced pancreatitis (n = 4), young animal controls (n = 6), elderly animals with induced pancreatitis (n = 5), and elderly animal controls (n = 5). AP was induced by the retrograde injection of 0.5 mL of 2.5% sodium taurocholate (Sigma-Aldrich, St Louis, MO, USA) directly into the pancreatic duct at a constant infusion rate using a pump. Since we found in a pilot study that rat pancreas weight did not increase with age, we used the same amount of sodium taurocholate in young and elderly rats.
After 10 h, rats were euthanized, hearts were perfused with saline, and samples of cardiac tissue were collected and stored at −80°C for later analysis.
Analysis of gene expression
Total RNA was isolated from cardiac tissue according to the standard protocol for extraction using Trizol (Invitrogen, Carlsbad, USA). Quantification of RNA extracted from cardiac tissue was carried out spectrophotometrically at 260 nm in NanoVue™ Plus equipment (GE Healthcare, Piscataway, NJ, USA), and the A260/A280 ratio was determined. The polymerase chain reaction (PCR) kit Array (RT2 First Strand Kit-Rat Cytokines and Chemokines) was used to analyze the cytokines, chemokines, and growth factors that are listed in Table 1, following the protocols of complementary DNA (cDNA) synthesis and real-time PCR according to the manufacturer’s recommendations. Subsequently, samples were subjected to PCR Step 1 (Applied Biosystems Carlsbad, CA, USA). Data were analyzed using the RT2 Profiler PCR Array Data Analysis Web-based Portal, using the Rplp1 gene as housekeeping gene.
Cytokine, chemokine, and growth factor genes analyzed in this study.
Statistical analysis
The characteristics of AP rats and controls were compared using the Kruskal–Wallis or Mann–Whitney U test, as appropriate. A P-value <0.05 was considered statistically significant. The analysis was performed using GraphPad InStat 3 software (GraphPad InStat Inc., San Diego, CA, USA).
Results
In healthy rats, aged animals exhibited significantly higher levels of interleukin-10 (IL-10) (Figure 1(c)) and CCL1 (Figure 2(c)) compared with young controls. All other mediators investigated showed no differences between aged and young healthy or AP animals. Figures 1 and 2 show the most important findings.
Gene expression of (a) TNFα, (b) IL-6, and (c) IL-10, in the heart tissue of young and old rats subjected to an animal model of acute pancreatitis compared to healthy controls.
Gene expression of (a) CXCL1, (b) CXCL3, (c) CCL1, (d) CCL7, (e) CCL11, and (f) CCL19 in the heart tissue of young and old rats subjected to an animal model of acute pancreatitis compared with healthy controls.
Discussion
Recent studies have indicated that older adults have elevated levels of pro-inflammatory cytokines and acute phase reactants in the steady state.10–12 In this study, we found a similar inflammatory response in the heart of young and aged rats submitted to severe AP injury.
Several studies have shown that AP in elderly patients leads to greater morbidity and mortality. Experimental studies and clinical trials, however, do not show marked changes in the systemic inflammatory response in the elderly with major clinical diseases, such as AP or septic shock. 13 It is possible that specific changes at the local level in key organs are responsible for the greater morbidity and mortality in the elderly with AP. Experimental work in our laboratory showed an increase in pro-inflammatory cytokines and a reduction in anti-inflammatory cytokines in the terminal ileum of elderly rats with pancreatitis compared with young rats with pancreatitis, although there were no detectable differences in serum levels. 6 It is possible that the heart could be affected, not only by circulating cytokines associated with AP 14 but also by local production of cytokines, as was recently demonstrated by Meyer et al. 15 In that study, the authors observed a significant elevation of interleukin-6 (IL-6) gene expression in the myocardial tissue of Wistar rats, 2 h after experimental AP was induced. Cardiac structural changes were also found, including vacuolar degeneration, pyknosis, and core tissue loss, and were associated with local production of cytokines in the myocardium. 15
In this study, we found increased levels of IL-10 and CCL1 in the heart tissue of healthy aged rats compared with young healthy rats. Interestingly, Damas et al. 16 found increased levels of chemokines CC and CXC and their receptors in patients with heart failure, suggesting a pathogenic role for chemokines in chronic heart failure. In our study, however, the gene expression levels of all the other chemokines investigated were similar among the study groups.
Chemokines belong to a family of inflammatory cytokines that control the chemotaxis of leukocytes into inflamed tissue. They form a sophisticated communication system to chemo-attract all immune cells. Chemokines are classified independent of their function, but based on their amino acid composition, specifically on the presence of a conserved tetra-cysteine motif. 17 There are two major subclasses: CXC and CC chemokines. We now know that this complex system of approximately 50 chemokines and 20 G protein-coupled, seven-transmembrane signaling receptors is also important in adaptive immunity.18,19 The chemokines also participate in other biological events, such as cell proliferation, organogenesis, and cardiogenesis. 20 Some studies have shown involvement of chemokines in the pathogenesis of various heart diseases, such as atherosclerosis, 21 myocarditis, 22 and ischemic reperfusion injury, 23 suggesting a possible pathogenic role of these mediators in the progression to heart failure. 24 Chemokines have been considered important mediators of inflammation and host defense. 20 Thus, the chemokine system may represent a previously unrecognized pathogenic factor in the development of heart disease, and may be a family of mediators with detrimental effects on the myocardium. 20 Despite our negative results, we believe other studies are important to evaluate this topic.
Systemic inflammation has been erroneously regarded as a uniform process characterized by a cytokine storm that elicits a state of overall inflammation in the body. This study indicates that systemic inflammation is compartmentalized and has unique features, depending on the organ investigated. In this rat model of AP, however, the heart remained similarly protected in aged and young animals.
Footnotes
Declaration of conflicting interests
The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Funding
The author(s) received no financial support for the research, authorship, and/or publication of this article.