- Citado por SciELO
versión impresa ISSN 1130-0108
Rev. esp. enferm. dig. vol.96 no.8 ago. 2004
Pulmonary injuries and cytokine levels after the intraperitoneal administration
of pancreatic homogenates in rats
G. Mozo, M. L. del Olmo, A. Caro-Patón, E. Reyes1, L. Manzano1, A. Belmonte, A. Almaraz and M. Álvarez-Mon1
Department of Medicine. University of Valladolid. Spain. 1Department of Immunology. University of Alcalá de Henares. Madrid. Spain
Introduction: our objective was to investigate the effects of the administration of pancreatic homogenates, with or without enzymatic activation, to healthy animals regarding cytokine serum levels and the development of pulmonary distress.
Material and methods: 106 male Wistar rats, divided into three groups, were studied: group A, intraperitoneal administration of homogenates activated with enterokinase; group B, homogenates without enterokinase; and group C, control group with administration of physiological saline solution. Each group was divided into 4 subgroups according to the time of sacrifice: 0, 2, 6 and 24 hours. We studied the pulmonary and pancreatic histology, serum parameters of renal and hepatic function, and serum levels of IL-1ß, IL-6 and TNFa.
Results: there was no mortality in any group. Pancreatic disorders in A and B groups were noted at 24 hours. These two groups had statistically significant higher transaminase serum levels than those of the control group, as well as statistically significant higher creatinine levels in group A. IL-1ß showed a statistically significant higher level at 6 h in both groups, A and B, but was higher in group A, which also exhibited significant pulmonary histologic damage with respect to controls at 6 h.
Conclusions: the higher IL-1ß level in group A may result from production by peritoneal macrophages under the influence of homogenate enzymatic activation. This may be the reason for lung damage.
Key words: Pancreatic ascites. Pancreatic homogenates. Interleukin-1ß. TNFa. Lung.
Mozo G, del Olmo ML, Caro-Patón A, Reyes E, Manzano L, Belmonte A, Almaraz A, Álvarez-Mon M. Pulmonary injuries and cytokine levels after the intraperitoneal administration of pancreatic homogenates in rats. Rev Esp Enferm Dig 2004; 96: 527-538.
Correspondencia: María Lourdes del Olmo Mar´tinez. C/ Federico Landrove, 14, 3º. 47014 Valladolid. e-mail: email@example.com
During the course of severe acute pancreatitis (AP) the inflammatory process, which is initially localized and limited to the pancreas, may spread with the induction of a systemic inflammatory response that can progress and even cause multiple organ failure (MOF) (1-3). In these cases, there is an accumulation of leukocytes, mainly polymorphonuclear (PMN) cells, in various organs such as the kidney, liver and lung, in parallel to the severity of AP (4-6). The PMN adhesion process to vascular endothelial cells -and the subsequent extravasation and parenchymal infiltration- is mediated by proinflammatory cytokines such as TNFa, IL-1ß, IL-6 and IL-8. These are initially released from acinus cells and damaged peripancreatic tissues, and later produced in other organs (3,7-12).
Many works have thoroughly studied the contribution of pancreatic ascites to the pathophysiology, development and degeneration of AP and its systemic involvement (13-23). It has been demonstrated that during AP there is an increase in peritoneal permeability (24,25) that contributes to pancreatic enzyme transfer via the peritoneum to the thoracic duct (26). Moreover, in pancreatic ascites there are proinflammatory cytokines whose origin seems to be activated peritoneal macrophages (15,19,20,27), which contribute to the spreading of inflammation. Nevertheless, these cytokines do not seem to be the only inflammatory factors in ascites, since the latter will still induce systemic effects even in their absence (15,17-19,28-33). Peritoneal lavage has been proposed (34-36) as a measure to eliminate proinflammatory factors present in pancreatic ascites that may contribute to patient deterioration (1) by producing damaging effects on hemodynamics and organs such as the kidney, liver and lung (28,31,37-42). In the lung they can induce an adult respiratory distress syndrome (ARDS), as observed in experimental models when injecting this exudate into the pulmonary artery (33) or following an intravenous (17) or intraperitoneal method (i.p.) (24).
Experimental studies have employed i.p. administration of pancreatic homogenenates as a method to simulate pancreatic ascites (43). It seems that a homogenate, once injected in a healthy rat, acquires fibrinolytic properties similar to those present in pancreatic ascites, which it did not previously possess. This appears to indicate that homogenates interact with the peritoneum (43).
The aim of this work was to study the effects produced by the administration of pancreatic homogenates with or without previous enterokinase activation on serum cytokine levels as well as on the development of alterations of pulmonary morphology in healthy animals.
MATERIAL AND METHODS
A total of 106 male Wistar rats were studied. The process was developed according to the rules established by the Ethics Committee of Valladolid Medicine School, as well as observing the rules stipulated in Decree 223/1988 of March 14 and those of Order October 13, 1989. The animals remained in cages of 5 individuals with alternating cycles of 12 light/dark hours, and free access to standard food and water until 12 hours before the beginning of experiments, when they were deprived of food but allowed free access to water.
The rats were distributed in three groups:
Group A. A total of 28 animals that received a pancreatic homogenate previously activated with enterokinase (EK).
Group B. A total of 28 animals that received a pancreatic homogenate without previous activation with EK.
Group C. A total of 50 control animals that received physiological saline solution in the same doses and times as those in groups A and B.
At the same time, each group was divided into subgroups according to the different sacrifice times: 0, 2, 6, and 24 hours after the administration of the homogenate.
Preparation and administration of the pancreatic homogenized solution
Eight female Wistar rats were used. They were fasted for 12 hours, but allowed free access to water, prior to sacrifice. Following anesthesia by an IP injection of 25 mg of sodium Pentothal (B. Braun Medical, Barcelona), a laparotomy and pancreas extraction using an aseptic technique were performed. The specimen obtained was immediately suspended in the proportion of 250 mg of tissue to 1.5 ml of sterile Tris buffer solution, pH 7.8, with Triton X 100. It was homogenized cold and centrifuged at a temperature of 4 ºC. Each 20 ml of supernatant were incubated for 30 minutes at 37 ºC with 2.22 ml of EK (Sigma, Londons) (5 mg/ml in a Tris buffer, pH 7.8), which was intended for animals in group A. In group B, the homogenate was incubated without EK.
Trypsin activity in homogenates was measured by using the Erlanger-Kokowsky-Cohen method (44), resulting in 120 IU/g of protein in the EK-activated homogenates and zero activity in samples incubated without EK. We previously observed that the i.p. administration of Triton X 100 (1/1000) did not induce any damage in the rat.
The prepared homogenates were i.p. injected at a dose of 0.5 ml in each animal, and this moment was considered zero time.
Animals were killed according to the established protocol by means of exsanguination by cardiac puncture following anesthesia with 25 mg of sodium pentothal injected i.p. Following a limited thoracotomy and right lung extraction, a wide medium laparotomy for pancreas extraction was also performed.
Biochemical and cytokine determinations
The blood obtained was kept on ice for 90 minutes until clot formation. After centrifugation the serum was taken and kept frozen until use at -20 ºC for biochemical determinations, and at -80 ºC for cytokine determinations.
Amylase, lipase, urea, creatinine, AST, ALT and glucose were determined in serum using a Hitachi 917 automatic analyzer. Hematocrit was determined by capillary centrifugation of heparinized blood.
Similarly, serum TNFa, IL-1ß and IL-6 levels were determined using specific ELISA kits for rats and for each cytokine. A kit with a sensitivity of 20-1000 pg/ml (Diaclone, France) was used for TNFa, one with a sensitivity of 10-1500 pg/ml (Endogen, USA) for IL-6, and one with a sensitivity of 3-2000 pg/ml (Biosource International, USA) for IL-1ß. The reading was done with a Delta Soft II spectrophotometer, version 4.1F (Biometallics. Inc.), with wavelengths of 450 nm using a LCII Macintosh computer.
The fresh right lung and pancreas of each animal were weighed and placed in a 10% formaldehyde solution. They were stained with hematoxylin/eosin. Both organs were studied with an optical microscope (Zeiss II, Inc., New York) using 200, 400 and 1000 magnification values. The parameters assessed in each organ were adjusted according to a scale from 0 to 3, and are listed in table I.
A comprehensive pancreatic and pulmonary histologic evaluation was established by calculating the mean of all histologic parameters for each organ following a method established by other authors (45,46).
The fresh organ weight/total corporal weight ratio for each animal -expressed as a percentage- was used as an estimation of tissular edema (lung or pancreas).
The results are expressed as mean ± standard error of mean (SEM). The SPSS 6.1.2. software program was used. We used Mann Whitney' non-parametric U test for comparison of means, and established p < 0.05 as the statistically significant value.
There was no mortality in any group of the study.
Enzymatic levels in both homogenates
Lipase and amylase levels in homogenates without EK activation were higher than those in activated homogenates (lipase 122.1 KU/ml versus 97.2 KU/ml, amylase 298.7 U/ml against 258.3 U/ml).
Transaminase levels were also higher in homogenates without EK activation than in activated homogenates (AST 6430 mU/ml against 3450 mU/ml, ALT 2135 mU/ml against 312 mU/ml).
Total protein results showed a greater concentration in homogenates without EK activation (1.81 g/dl vs 1.13 g/dl in homogenates with EK activation).
There were no macroscopic pancreatic alterations in groups A and B with relation to controls, nor were there any differences in the pancreatic weight/corporal weight ratio.
Significant higher levels of serum amylase at 2 and 6 h were evident only in group B when compared to the control group. Serum lipase showed slightly higher values in both groups (A and B) at 2 and 6 h, with statistically significant differences with respect to the control group (Table II). At 6 h, group B had significantly higher levels of serum amylase than group A. The same occurred at 2 and 6 h with serum lipase (Table II).
The histological study of the pancreas showed the presence of inflammatory acinar infiltration and pancreatic edema at 24 h in group A (with EK), a significant difference with respect to the control group. In the group with EK-free homogenates (B) we noticed inflammatory infiltration of acini and slight apoptosis at 24 h, with the presence of acinar edema at 2, 6 and 24 h, all of which was significant with respect to the control group and at 2 and 24 h with respect to group A (Table III).
Serum transaminases and renal function parameters
At 2 and 6 h both experimental groups showed AST levels significantly higher than the control group, but ALT levels were higher only in group B with respect to groups C and A (Table II).
There were no differences in urea between groups. Only in group A did creatinine stand at significantly higher levels than in group C at 6 h. No differences were observed either in glucose level or hematocrit (Table II).
Serum levels of IL-1ß, TNFa and lL-6 (Table II)
The determination of serum IL-1ß showed significantly higher levels at 6 h in both groups A and B compared to the control group, the highest value occurring in group A (58.8 pg/ml) in contrast to group B (22.4 pg/ml). The same occurred at 0 hrs in group B (Table II).
IL-6 results showed no detectable values in any group.
TNFa results showed no statistically significant differences when comparing between groups A and B, and these with the control group at the various time points of the experiment.
Pulmonary histologic alterations
In group A we observed a greater alteration of lung architecture at 6 h compared to the control group, together with a more important presence of alveolo-capillary pulmonary edema during the same time period with statistically significant differences. The remaining parameters showed no significant findings. In group B no alteration was noted versus the control group in any of the parameters analyzed (Table IV, Fig. 1).
The evaluation of the right lung weight/body weight ratio showed no difference between groups.
According to our study, no significant pancreatic alteration occurred in experimental groups A and B, as was expected. The very mild presence of an inflammatory infiltrate and interstitial edema in both groups at 24 h may be associated with a possible late attack on the pancreas, probably by an immunologic mechanism (the administration of pancreatic homogenates coming from another animal may generate an immune response against the viscera itself). A more remote possibility, however, would be that the IL-1ß increase produced in these groups at 6 h could have triggered late selective chemotaxis on the pancreas. In any case, these alterations are so mild that they seem to have have no influence both on pulmonary involvement and cytokine production, since such phenomena develop in earlier stages.
Given the absence of pancreatic injury, serum lipase and amylase increases may be due to the peritoneal absorption of enzymes contained in the administered homogenates, which also occurs in pancreatic ascites (47,48). Differences in serum lipase and amylase between groups A and B -higher in B- correspond to the higher lipase and amylase activity observed in homogenates not activated by EK. It seems reasonable to think that the lower enzyme levels in EK-activated homogenates may be a result of their digestion by the trypsin present therein.
Higher ALT levels at 2 and 6 h in group B may also be interpreted in this way. However, several studies have demonstrated the presence of liver cell apoptosis in healthy animals after the i.p. administration of pancreatic homogenates (42). Similarly, the pancreatic homogenates used in our study may have had that same effect.
The absence of detectable serum IL-6 levels and of significant differences in TNFa may be explained by the fact that the injuries induced were not of sufficient intensity, as has been evidenced in other experimental works and in humans (39,49,50).
IL-1ß levels remained elevated in both experimental groups, showing significant differences compared with the control group after 6 hours, when this cytokine reached peak levels. As homogenates come from a healthy rat without pancreatic inflammation, it appears that this is not the origin of this cytokine. Although we have not studied the peritoneum from a histologic point of view, there have been previous works using models equivalent to ours which ruled out the presence of inflammatory signs in the peritoneum (31), suggesting that IL-1ß elevation in groups A and B is not due to the existence of peritoneal inflammation. It seems that neither the pancreas nor the lung are likely to be the origin of this cytokine, since no injury existed prior to the significant increase of IL-1ß. Therefore, we suggest that the most likely origin of this cytokine is peritoneal macrophages. A number of studies have demonstrated that pro-inflammatory cytokines and regulatory proteins for the transcription of pro-inflammatory genes, such as NFκB, are produced by peritoneal and splenic macrophages upon stimulation by pancreatic homogenates lacking endotoxine and cytokines (15,18-20,28-33).
If we take into account that the pancreatic enzymes released to the circulation do not seem to influence the production of cytokines by macrophages (19,20), there must be a factor of pancreatic origin contained in our homogenates that is not dependent on the prior existence of pancreatic injury interacting with the peritoneal macrophages and stimulating them to produce IL-1ß.
The fact that in group A, IL-1ß levels at hour 6 were higher than in group B, and that only group A rats had significant pulmonary injuries, suggests that the unknown factor may be found in larger amounts in activated homogenates (a product of the transformation by activated enzymes of some pancreatic protein?). Another possibility would be that the presence of activated enzymes contributed to the effect of this factor on peritoneal macrophages, and although it appears that enzymes are not directly responsible for these phenomena, they are likely to help them (18,20,21,23).
As for the study of pulmonary morphology, our findings are consistent with an alteration of normal pulmonary structure and the presence of mild pulmonary edema, statistically significant in comparison with groups B and C also at 6 h. The absence of differences in lung weight may be due to the low grade of edema, with absence of macroscopic findings, together with a sample error, since removed right lungs had not exactly the same size in all animals, and a clear parallel relationship is not always present between lung size and body size.
It is difficult to establish the role that activated enzymes of homogenates may have played in generating these lung injuries. A recent work shows greater lung injury associated with AP when administering intravenous EK in order to activate pancreatic enzymes, which remained outside the acini. However, this procedure caused a further aggravation of pancreatic injury; therefore, increased lung damage must be more attributable to greater AP severity than to the activity of pancreatic enzymes on the lung (51). We know that activated enzymes do not induce pulmonary injury by themselves (18). Furthermore, it is clear that a narrow relationship exists between the presence of pro-inflammatory cytokines and the appearance of pulmonary injury, not only in AP but also in other diseases (52-56). With these facts in mind, we must associate pulmonary alterations present in group A with the higher IL-1ß levels detected. Nevertheless, IL-1ß was higher in group B at 2 h, although without significant differences versus group A. This suggests that lung damage is not induced by this cytokine alone. Therefore, it may be that some other factor generated within the EK-activated homogenate is involved in this lesion.
In summary, pulmonary injuries were observed only in animals treated with activated homogenates, where IL-1ß levels were significantly higher. This may imply that pancreatic enzymes are potential facilitators of a supposed interaction between activated homogenates and peritoneal macrophages. In case of true pancreatic ascites all these elements in conjunction with pancreatic inflammation would converge, thus undoubtedly generating higher levels of pro-inflammatory cytokines and systemic alterations.
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