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PROFILES OF PRO-INFLAMMATORY CYTOKINES IN THE SERUM OF RABBITS AFTER EXPERIMENTALLY INDUCED ACUTE PANCREATITIS
doi:10.1006/cyto.2001.0977, available online at http://www.idealibrary.com on
PROFILES OF PRO-INFLAMMATORY CYTOKINES IN THE SERUM OF RABBITS AFTER EXPERIMENTALLY INDUCED ACUTE PANCREATITIS Maher O. Osman,1 Borbala Gesser,2 Janne T. Mortensen,2 Kouji Matsushima,3 Steen L. Jensen,1 Christian G. Larsen2 In a recent study we have demonstrated that interleukin 8 (IL-8) and tumour necrosis factor alpha (TNF-) serum levels correlate positively with the severity of acute pancreatitis (AP), induced by bile acid injected into the pancreatic duct of rabbits. In this article we describe the effect of an IL-10 analogue IT9302 and a monoclonal anti-IL-8 (mon. IL-8) antibody on the content of several pro-inflammatory cytokines in the serum of rabbits, after induction of AP. We found that the serum content of inflammatory cytokines IL-8, IL-1, TNF- and monocyte chemoattractant protein 1 (MCP-1) are increased during AP. Injection of IT9302 or mon. IL-8 antibody, diminish the concentration of these cytokines in the serum, with the exception that mon. IL-8 antibody actually increased the circulating level of MCP-1. In addition, intravenous administration of IT9302 increased the serum levels of IRAP, an IL-1 receptor antagonistic cytokine. Furthermore, intravenous injection of mon. IL-8 antibody increased serum levels of IL-4. It can be concluded that both the human IL-10 analogue IT9302 and mon. IL-8 antibody are able to alter the pro-inflammatory cytokine levels in rabbits suffering from experimentally induced AP. 2002 Elsevier Science Ltd.
Pro-inflammatory cytokines such as interleukin (IL)-1, tumour necrosis factor alpha (TNF-), IL-6 and IL-8 are elevated during acute pancreatitis (AP) and are considered to be involved in the pathogenesis of pancreatitis-associated multiple organ dysfunction.1–5 Elevated serum levels of these cytokines have been demonstrated clinically as well as experimentally in different animal models of AP.6,7 In a recent study, we have demonstrated that IL-8 and TNF- serum levels correlated positively with the severity of experimentally induced AP (ei-AP) induced by the intraductal injection of bile acid into the pancreatic duct of rabbits. Elevated cytokine levels were From the Departments of 1Surgery and 2Dermatology, Aarhus University Hospital, Aarhus University, Aarhus, Denmark; 3 Department of Molecular Preventive Medicine, University of Tokyo, Japan Correspondance to: Borbala Gesser, Department of Dermatology, Marselisborg Hospital, DK-8000 Aarhus, Denmark. Tel: +45 8949 1906; Fax: +45 8949 1906; E-mail [email protected] Received 8 May 2000; received in revised form 28 June 2001; accepted for publication 7 September 2001 2002 Elsevier Science Ltd. 1043–4666/02/010053+07 $35.00/0 KEY WORDS: acute pancreatitis/rabbit/serum cytokines/Western blotting CYTOKINE, Vol. 17, No. 1 (January), 2002: pp 53–59
associated with marked up-regulation of the adhesion molecule CD11b/CD18 in pancreas and lung tissues, as well as with significant leukopenia in the peripheral blood.8 IL-8 is a potent neutrophil chemotactic and activation cytokine and has been thought to play an important role in the recruitment of leukocytes responsible for the induction of the systemic inflammatory response complicating severe AP.5 Interleukin 8 is the major neutrophil chemotactic factor in the lung.9 IL-8 stimulates the binding activity of the leukocyte adhesion receptor CD11b/CD18 on human neutrophils.10 MCP-1 is a monocyte chemoattractant and activating factor and regulates adhesion molecule expression in human monocytes.11,12 The presence of MCP-1 has been shown in rat during chronic lung injury13 but has not been studied in acute pancreatitis. In this study we applied the Western blotting technique in order to study the profile of different cytokines, IL-8, IL-4, IL-1, TNF-, MCP-1, IL-1 receptor antagonistic protein (IRAP) and IL-2, in the serum of rabbits with ei-AP. The rabbit model of ei-AP has been chosen for several reasons. First, it has been shown that rabbit proteins are phylogenetically more closely related to primate (human) proteins than other rodents proteins.14–16 Second, specific rabbit 53
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antibodies against IL-8 are at hand. Thereby species differences between animal and antibody are avoided when investigating the involvement of IL-8 during the induction of AP.17 Finally, the rabbit has a large blood volume compared to the rat making sampling of relatively large blood volumes possible for analyses of different inflammatory cytokines simultaneously. The possible therapeutic potential of IL-10 in acute pancreatitis has previously been demonstrated.18–20 Thus mice pre-treated with murine IL-10 developed less acinar necrosis or even prevented necrosis in experimental models of pancreatitis.18,19 Also, interleukin 10 reduced the severity of acute pancreatitis in rats.20 The effect of IL-10 was attributed to a down-regulation of TNF- mRNA and TNF- protein. An IL-10 agonist IT9302 is a nonapeptide21 with homology to the C-terminal end of human IL-10. IT9302 has been shown to possess some activities that mimic those of human IL-10, and is a potential candidate to be tested in acute pancreatitis. In this study we describe that this analogue has the capacity to reduce the content of several pro-inflammatory cytokines in the circulation of rabbits with ei-AP.
RESULTS To evaluate the contents of cytokines in the serum of rabbit, we freeze dried fractions of 200 l serum and then redissolved the material in 200 l gel lysis buffer.22 The material was then separated by electrophoresis using 15 or 20% sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) gels and analysed by Western blotting.
Serum content of pro-inflammatory cytokines Group A: rabbits with ei-AP The IL-8 content in the serum reached a maximum between 3 and 6 h, with a peak concentration around 2 ng/ml. The IL-1 serum levels showed a maximum between 1 and 3 h, with a maximum level slightly above 2 ng/ml. The TNF- level reached its maximum between 6 and 9 h, significantly higher than 2 ng/ml, but lower than 50 ng/ml. With respect to MCP-1, it appeared that only a dimeric 15-kDa form was detectable in the serum, with a peak around 6 h. The concentration was between 2 and 50 ng/ml (Fig. 1). Group B: rabbits treated with IT9302 before ei-AP was inflicted IL-8 was not detectable even at 3 or 6 h, (Fig. 2). The levels of IL-1 were likewise significantly suppressed at 1 and 3 h compared with the control
Figure 1. Content of cytokines in serum of rabbits with experimentally induced acute pancreatitis. Serum samples were freeze dried and re-dissolved in equal volumes of gel lysis buffer and cytokines were identified by Western blotting. The data represent a time course study in one animal.
group A. Further, TNF- was not detectable in the serum in contrast to that of the control group. The MCP-1 immunostaining revealed the presence of the monomeric 8-kDa form at 1 and 3 h around 50 ng/ml. A baseline amount of the 15-kDa form (approximately 2 ng/ml) was detected at 6 and 9 h, but at the same time more than 2 ng/ml of the monomeric 8-kDa form was present (Fig. 2). Group C: rabbits treated with WS-4 (mon. IL-8) before induction of ei-AP Presence of IL-8 was not detectable at any timepoints and IL- 1 (not shown) as well as TNF- were below the detection level compared with the control group A. Interestingly, the MCP-1 immunostaining
Pro-inflammatory cytokines in rabbit serum after experimentally induced acute pancreatitis / 55
Figure 3. Content of cytokines in the serum of rabbits with ei-AP that received a single intravenous bolus injection (1 mg/kg dissolved in equal volumes of saline) of the monoclonal neutralizing IL-8 antibody WS-4, 30 min before induction of acute pancreatitis. The data represents a time course study in one animal.
Figure 2. Content of cytokines in the serum of rabbits with ei-AP and treated with two doses of IT9302.
induction of IL-4 in the WS.4 group C, at 6 and 9 h more than 2 ng/ml (Fig. 3).
The first dose (100 g/kg) was injected subcutaneously and the second dose (100 g/kg) diluted in 40 ml saline was injected intravenously, both administered half an hour before the injection of 5% bile acid. The data represent a time course study in one animal.
Negative control group (saline injected into the pancreatic duct)
showed an increase of the dimeric 15-kDa form throughout 1, 3, 6, 9 h reaching a level above 50 ng/ml (Fig. 3).
Serum content of anti-inflammatory cytokines In the positive control group A and the WS-4 treated group C, no presence of IRAP was detected. In group B, serum IRAP was increased from 1 h to 3, 6 and 9 h, to a level around 2 ng/ml (Fig. 2). Immuno staining did not reveal presence of IL-4 in the positive control group A, but showed small levels in the IT9302 group B at 6 h and revealed a clear
Others have demonstrated the induction of IL-2 simply by injection of saline into the pancreatic duct. As method control experiment we measured the level of IL-2.23 Thus in group A, IL-2 was present at 0 and 1 h and at a lower concentration than the saline group. In group B, IL-2 was not detectable (Fig. 4). Only the saline treated rabbit maintained the IL-2 at different times.
Serum content of myeloperoxidase Group A showed high levels of myeloperoxidase (MPO) identified as an 80-kDa band on the gel.24 MPO was detectable from 0 h, reached highest level at 1 h and remained high until 6 h. Group B, revealed a lower content of myeloperoxidase from 0 h and
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Figure 4. Content of IL-2 in the serum of rabbits treated with saline only, alternatively non-treated or IT9302-treated before induction of ei-AP.
Figure 5. An example of quantitation of the immunostaining on Hybond-ECL nitrocellulose membranes (chemiluminescence).
throughout 1, 3, 6 and 9 h compared with group A. In group C, myeloperoxidase was detected at time 0, but undetectable at time 1, 3, 6 and 9 h (Fig. 5).
DISCUSSION Pro-inflammatory cytokines are pivotal mediators for the pathophysiological mechanism leading to acute pancreatitis.6–8 Thus IT9302, an IL-10-analogue and a potent endogenous suppressor of pro-inflammatory cytokine activity when injected into rabbits with ei-AP, increases the survival of animals, preferentially by diminishing the severity of pancreatitis-associated multiple organ dysfunction.25,26 In the control group 60% of the rabbits died before the end of the experiment (after 6 to 9 h) whereas in the IT9302-treated group all animals survived.25 Similarly in the WS-4 treated group, 90% of the rabbits were alive at 12 h.26 In the present study, we demonstrated that induction of pro-inflammatory cytokines, such as IL-1 (1 h), IL-8 (3 h), TNF- (6 h) and MCP-1 (6 h) (Fig. 1) accompanies the development of ei-AP, reflected by their increased concentration in sera of rabbits with ei-AP. The cellular origins of these cytokines are principally believed to be from the pancreatic tissue, including damaged vascular tissue and infiltrating leukocytes.27,28 Not surprisingly, MPO is an early
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mediator, already detectable at baseline (0 h) (Fig. 6), during the severe inflammatory response and tissue damage, which is the result of ei-AP.8,25,26 IL-1 has been shown to be a significant cytokine for the development of AP.29 Also, our data suggest that IL-1 is orchestrating an array of proinflammatory cytokines such as the CXC chemokine IL-8 and the CC chemokine MCP-140 during the development of AP. This, and IL-8 in particular, further enhance leukocyte activation, principally by exerting chemotactic activity on neutrophils,5,9 thereby attracting them to the inflammatory focus and by inducing the release of enzymes from leukocytes such as MPO from neutrophils.30 The relevance of this mechanism appears to be supported by our observation that treating the rabbits with IT9302 or neutralizing monoclonal anti-IL-8 antibody, reduced the content of MPO in the serum. The effect of anti-IL-8 therapy on the outcome of the ei-AP as well as the reduction of TNF- and as expected, on IL-8 itself, generally supports that IL-8 is a pivotal mediator during ei-AP.39 The MCP-1 induction in the anti-IL-8 antibody treated animals could be explained by the fact that IgG complexes activate monocytes to secrete MCP-1.31 TNF- also appears to be important for the development of pancreatitis-associated syndromes such as adult respiratory distress syndrome (ARDS).32–34 We found that the level of TNF- is increased following the induction of ei-AP, and that this increase could be reduced by treating the rabbits with IT9302. It is therefore likely that part of the observed pronounced effect of IT9302 on the survival of rabbits with ei-AP is explained by a reduction of the TNF- load in the circulation.20 However, IT9302 also induced the production of IRAP-1, a natural IL-1 antagonist and does reduce the production of most pro-inflammatory cytokines.35 Further, the effect of IT9302 on chemokine activities could be parallel to that of natural IL-10, which is known to weaken the function of the chemokine receptors by reducing the receptor-mediated signal transduction.36 Together, this reveals a scenario where severe local tissue damage results in profound local production of pro-inflammatory cytokines, which may engage in a local enhancement of the inflammatory response, by inducing secondary mediators, by attracting leukocytes and by inducing the local release of tissue damaging leukocyte derived enzymes such as MPO. Further, the peripheral circulation, eased by the rich vascularization of the pancreas and its region, readily and partially absorbs this burst of potent peptide mediators. These circumstances possibly govern the condition of hypercytokinaemia leading to the induction of multiple organ dysfunction such as ARDS.
Pro-inflammatory cytokines in rabbit serum after experimentally induced acute pancreatitis / 57
It can be concluded that both the human IL-10 analogue IT9302 and the monoclonal anti-IL-8 antibody are able to alter cytokine levels, which correlate with a reduction in the inflammatory response associated with AP in rabbits. Consequently, these observations as well as earlier observations by us and others18–20,25,26,37,38 suggest that modulation of cytokine activity appears to be a means to prevent and or treat acute pancreatitis.
MATERIALS AND METHODS Animals We used adult male and female New Zealand white rabbits, weighing 3.9–5.2 kg. The experimental protocol was approved by the Danish Experimental Animal Committee.
Induction of acute pancreatitis The procedure of induction of acute pancreatitis has been described in detail elsewhere.8 Briefly, under general anaesthesia all rabbits underwent a 4-cm long midline laparotomy. The pancreatic duct was visualized entering the duodenum at a point approximately 20 cm from the pylorus. Acute pancreatitis was induced by the retrograde intraductal injection of 2.0 ml of 5.0% chenodeoxycholic bile acid (200 mg plus 4.0 ml ethanol, Sigma Chemicals Co., St. Louis, Missouri, USA, product no. C9377) through a polyethylene catheter inserted into the intramural aspect of the duct. After injection, the pancreatic duct was ligated and the abdomen was closed.8
Experimental design Twenty-eight rabbits were divided equally into three groups. Group A (controls, n=10) received an injection of 2.0 ml 5% chenodeoxyclolic bile acid, followed by duct ligation. They were then given a single intravenous saline bolus injection followed by infusion of 40 ml saline for 30 min until induction of acute pancreatitis. Group B (n=8) was treated with human IT9302, a nonapeptide with homology to the C-terminal portion of human IL-10,21 in two doses. The peptide/saline solution was freshly prepared for each day. The first dose (100 g/kg) was injected subcutaneously, and the second dose (100 g/ml) diluted in 40 ml saline (40 ml/30 min) intravenously, both then given half an hour before the injection of 5% bile acid, as described in the control group.25 Group C (n=10) received a single intravenous bolus injection (1 mg/kg dissolved in equal volumes of saline) of the monoclonal neutralizing anti-IL-8 antibody (WS-4) (Prof. Kouji Matsushima, Tokyo, Japan), 30 min before induction of acute pancreatitis, followed by infusion of 40 ml saline for 30 min until induction of acute pancreatitis.26 Nine rabbits were also used as a negative control group, without induction of AP, but only receiving saline. Animals were observed for a total period of 12 h, after which surviving rabbits were killed. Arterial blood samples were drawn 5 min before, and then 1, 3, 6, 9 and 12 h after induction of acute pancreatitis. Blood samples were immediately centrifuged at 3000 rpm for 10 min at 5C. The
Figure 6. The effect of IT9302 and WS-4 on the content of myeloperoxidase (MPO) in serum during the course of ei-AP. Immunoblotting was performed using different amounts of recombinant hTNF- and developed by using a polyclonal rabbit antihuman TNF- antibody. The detection limit by this technique is approximately 2ng/ ml.
supernatants were separated using sterile pipettes. All samples were coded and stored at 80C until analysed by Western blotting technique. At the end of the experiment (12 h) all animals underwent autopsy and the lung and the pancreas were investigated for the adhesion complexes CD11b/CD18.8,25,26 When animals died earlier than 12 h the autopsy was taken earlier.
Assays Serum samples of 200 l were freeze dried and dissolved in an equal volume of gel lysis buffer.22 We examined three rabbit serum samples (40 l) from each group at times 0, 1, 3, 6 and 9 h, for each cytokine. ECL-Western blotting of rabbit serum IL-8, IL-1, IL-4, IRAP and IL-2 was performed, using specific goat anti-human antibodies. MCP-1 was detected using a mouse anti-human MCP-1 antibody. All three animals tested showed similar immunostaining, but only data from one animal are shown in the figures. One-dimensional 15% or 20% SDS-PAGE gels were run and the serum samples were prepared in gel lysis buffer 1 h before and kept in 40C water until they were applied to the gels. The proteins were transferred to Hybond-ECL nitrocellulose membranes (Amersham RPN 2020 D, Little Chalfont, UK) and blocked with 5% bovine serum albumin (Sigma) in Tris-buffered saline, pH 7.8 containing 0.1% Tween-20. The blots were then incubated with 50 g of the specific cytokine antibody in 50 ml Tris buffer with 0.1% Tween-20, followed by 10 l of horse radish peroxidase labelled secondary antibody (cat. No. P447, P449 or P217, Dako, Copenhagen, Denmark) in 50 ml Tris buffer with 0.1% Tween-20. The secondary antibody labelling was followed by
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TABLE 1. Western blotting was performed to identify the cytokines. The serum samples and recombinant cytokines, which served as positive control, were run on 15% or 20% SDS-PAGE gels. The recombinant cytokines were reconstituted in PBS with 4% bovine serum albumin, and antibodies were applied as described in the Materials and Methods.
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anti-human TNF- antibody (code IP-310) is from Genzyme, Kings Hill, UK. Goat anti-human IL-1 receptor antagonist protein antibody (AB-280-NA) and mouse anti-human MCAF antibody (MAB 279) are from R&D Systems, UK. The secondary antibodies were, horseradish peroxidase labelled of either goat anti-mouse IgG (cat. No. P447), rabbit anti-goat IgG (cat. No. P449) or swine anti-rabbit IgG (cat. No. P217) from Dako, DK.
Acknowledgements This research was supported by a research grant from Nycomed Dak A/S, Copenhagen, and by SRG A/S, Svendborg.
REFERENCES
enhanced chemiluminescence activation (cat. no. RPN 2106, ECL, Amersham) and the immunostaining was detected by autoradiography. Films (Kodak X-OMAT-S, Tastrup, Denmark) were exposed for 90 s or 2 min.22 Positive controls were recombinant human cytokines and were also used for quantitation of cytokines in the serum samples. For quantitation of the IL-8 bands, we ran Western blotting of recombinant human or rabbit IL-8 standards in parallel wells of 15% or 20% SDS-PAGE gels (recombinant rabbit IL-8 was from K. Matsushima, Tokyo, Japan). The same principle was used for quantitation of IRAP, TNF-, IL-2, IL-4 and MCP-1 in a similar way (Fig. 5). For positive controls see Table 1.
Determination of the serum content of myeloperoxidase (MPO) Serum samples were further diluted in gel lysis buffer. After protein measurement, a sample of 50 g protein was applied to 10% SDS-PAGE gels. For positive control, a pro-myelocytic human cell line HL-60 (ECACC, Salisbury, UK) was used.24 Human myeloperoxidase is expressed as an 80-kDA protein. Determination of the serum content of myeloperoxidase was by rabbit anti-human MPO Ab (A 0398, Dako) and Western blotting.
Cytokine antibodies The following primary antibodies were used: monoclonal mouse anti-human IL-8 antibody (WS-4), goat antihuman IL-8 antibody (AB-208-NA), goat anti-human IL-4 antibody (AB-204-NA), goat anti-human IL-1 antibody (AB-201-NA) and goat anti-human IL-2 antibody (AB-202NA), are from R&D Systems, Abingdon, UK. Rabbit
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PROFILES OF PRO-INFLAMMATORY CYTOKINES IN THE SERUM OF RABBITS AFTER EXPERIMENTALLY INDUCED ACUTE PANCREATITIS Maher O. Osman,1 Borbala Gesser,2 Janne T. Mortensen,2 Kouji Matsushima,3 Steen L. Jensen,1 Christian G. Larsen2 In a recent study we have demonstrated that interleukin 8 (IL-8) and tumour necrosis factor alpha (TNF-) serum levels correlate positively with the severity of acute pancreatitis (AP), induced by bile acid injected into the pancreatic duct of rabbits. In this article we describe the effect of an IL-10 analogue IT9302 and a monoclonal anti-IL-8 (mon. IL-8) antibody on the content of several pro-inflammatory cytokines in the serum of rabbits, after induction of AP. We found that the serum content of inflammatory cytokines IL-8, IL-1, TNF- and monocyte chemoattractant protein 1 (MCP-1) are increased during AP. Injection of IT9302 or mon. IL-8 antibody, diminish the concentration of these cytokines in the serum, with the exception that mon. IL-8 antibody actually increased the circulating level of MCP-1. In addition, intravenous administration of IT9302 increased the serum levels of IRAP, an IL-1 receptor antagonistic cytokine. Furthermore, intravenous injection of mon. IL-8 antibody increased serum levels of IL-4. It can be concluded that both the human IL-10 analogue IT9302 and mon. IL-8 antibody are able to alter the pro-inflammatory cytokine levels in rabbits suffering from experimentally induced AP. 2002 Elsevier Science Ltd.
Pro-inflammatory cytokines such as interleukin (IL)-1, tumour necrosis factor alpha (TNF-), IL-6 and IL-8 are elevated during acute pancreatitis (AP) and are considered to be involved in the pathogenesis of pancreatitis-associated multiple organ dysfunction.1–5 Elevated serum levels of these cytokines have been demonstrated clinically as well as experimentally in different animal models of AP.6,7 In a recent study, we have demonstrated that IL-8 and TNF- serum levels correlated positively with the severity of experimentally induced AP (ei-AP) induced by the intraductal injection of bile acid into the pancreatic duct of rabbits. Elevated cytokine levels were From the Departments of 1Surgery and 2Dermatology, Aarhus University Hospital, Aarhus University, Aarhus, Denmark; 3 Department of Molecular Preventive Medicine, University of Tokyo, Japan Correspondance to: Borbala Gesser, Department of Dermatology, Marselisborg Hospital, DK-8000 Aarhus, Denmark. Tel: +45 8949 1906; Fax: +45 8949 1906; E-mail [email protected] Received 8 May 2000; received in revised form 28 June 2001; accepted for publication 7 September 2001 2002 Elsevier Science Ltd. 1043–4666/02/010053+07 $35.00/0 KEY WORDS: acute pancreatitis/rabbit/serum cytokines/Western blotting CYTOKINE, Vol. 17, No. 1 (January), 2002: pp 53–59
associated with marked up-regulation of the adhesion molecule CD11b/CD18 in pancreas and lung tissues, as well as with significant leukopenia in the peripheral blood.8 IL-8 is a potent neutrophil chemotactic and activation cytokine and has been thought to play an important role in the recruitment of leukocytes responsible for the induction of the systemic inflammatory response complicating severe AP.5 Interleukin 8 is the major neutrophil chemotactic factor in the lung.9 IL-8 stimulates the binding activity of the leukocyte adhesion receptor CD11b/CD18 on human neutrophils.10 MCP-1 is a monocyte chemoattractant and activating factor and regulates adhesion molecule expression in human monocytes.11,12 The presence of MCP-1 has been shown in rat during chronic lung injury13 but has not been studied in acute pancreatitis. In this study we applied the Western blotting technique in order to study the profile of different cytokines, IL-8, IL-4, IL-1, TNF-, MCP-1, IL-1 receptor antagonistic protein (IRAP) and IL-2, in the serum of rabbits with ei-AP. The rabbit model of ei-AP has been chosen for several reasons. First, it has been shown that rabbit proteins are phylogenetically more closely related to primate (human) proteins than other rodents proteins.14–16 Second, specific rabbit 53
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antibodies against IL-8 are at hand. Thereby species differences between animal and antibody are avoided when investigating the involvement of IL-8 during the induction of AP.17 Finally, the rabbit has a large blood volume compared to the rat making sampling of relatively large blood volumes possible for analyses of different inflammatory cytokines simultaneously. The possible therapeutic potential of IL-10 in acute pancreatitis has previously been demonstrated.18–20 Thus mice pre-treated with murine IL-10 developed less acinar necrosis or even prevented necrosis in experimental models of pancreatitis.18,19 Also, interleukin 10 reduced the severity of acute pancreatitis in rats.20 The effect of IL-10 was attributed to a down-regulation of TNF- mRNA and TNF- protein. An IL-10 agonist IT9302 is a nonapeptide21 with homology to the C-terminal end of human IL-10. IT9302 has been shown to possess some activities that mimic those of human IL-10, and is a potential candidate to be tested in acute pancreatitis. In this study we describe that this analogue has the capacity to reduce the content of several pro-inflammatory cytokines in the circulation of rabbits with ei-AP.
RESULTS To evaluate the contents of cytokines in the serum of rabbit, we freeze dried fractions of 200 l serum and then redissolved the material in 200 l gel lysis buffer.22 The material was then separated by electrophoresis using 15 or 20% sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) gels and analysed by Western blotting.
Serum content of pro-inflammatory cytokines Group A: rabbits with ei-AP The IL-8 content in the serum reached a maximum between 3 and 6 h, with a peak concentration around 2 ng/ml. The IL-1 serum levels showed a maximum between 1 and 3 h, with a maximum level slightly above 2 ng/ml. The TNF- level reached its maximum between 6 and 9 h, significantly higher than 2 ng/ml, but lower than 50 ng/ml. With respect to MCP-1, it appeared that only a dimeric 15-kDa form was detectable in the serum, with a peak around 6 h. The concentration was between 2 and 50 ng/ml (Fig. 1). Group B: rabbits treated with IT9302 before ei-AP was inflicted IL-8 was not detectable even at 3 or 6 h, (Fig. 2). The levels of IL-1 were likewise significantly suppressed at 1 and 3 h compared with the control
Figure 1. Content of cytokines in serum of rabbits with experimentally induced acute pancreatitis. Serum samples were freeze dried and re-dissolved in equal volumes of gel lysis buffer and cytokines were identified by Western blotting. The data represent a time course study in one animal.
group A. Further, TNF- was not detectable in the serum in contrast to that of the control group. The MCP-1 immunostaining revealed the presence of the monomeric 8-kDa form at 1 and 3 h around 50 ng/ml. A baseline amount of the 15-kDa form (approximately 2 ng/ml) was detected at 6 and 9 h, but at the same time more than 2 ng/ml of the monomeric 8-kDa form was present (Fig. 2). Group C: rabbits treated with WS-4 (mon. IL-8) before induction of ei-AP Presence of IL-8 was not detectable at any timepoints and IL- 1 (not shown) as well as TNF- were below the detection level compared with the control group A. Interestingly, the MCP-1 immunostaining
Pro-inflammatory cytokines in rabbit serum after experimentally induced acute pancreatitis / 55
Figure 3. Content of cytokines in the serum of rabbits with ei-AP that received a single intravenous bolus injection (1 mg/kg dissolved in equal volumes of saline) of the monoclonal neutralizing IL-8 antibody WS-4, 30 min before induction of acute pancreatitis. The data represents a time course study in one animal.
Figure 2. Content of cytokines in the serum of rabbits with ei-AP and treated with two doses of IT9302.
induction of IL-4 in the WS.4 group C, at 6 and 9 h more than 2 ng/ml (Fig. 3).
The first dose (100 g/kg) was injected subcutaneously and the second dose (100 g/kg) diluted in 40 ml saline was injected intravenously, both administered half an hour before the injection of 5% bile acid. The data represent a time course study in one animal.
Negative control group (saline injected into the pancreatic duct)
showed an increase of the dimeric 15-kDa form throughout 1, 3, 6, 9 h reaching a level above 50 ng/ml (Fig. 3).
Serum content of anti-inflammatory cytokines In the positive control group A and the WS-4 treated group C, no presence of IRAP was detected. In group B, serum IRAP was increased from 1 h to 3, 6 and 9 h, to a level around 2 ng/ml (Fig. 2). Immuno staining did not reveal presence of IL-4 in the positive control group A, but showed small levels in the IT9302 group B at 6 h and revealed a clear
Others have demonstrated the induction of IL-2 simply by injection of saline into the pancreatic duct. As method control experiment we measured the level of IL-2.23 Thus in group A, IL-2 was present at 0 and 1 h and at a lower concentration than the saline group. In group B, IL-2 was not detectable (Fig. 4). Only the saline treated rabbit maintained the IL-2 at different times.
Serum content of myeloperoxidase Group A showed high levels of myeloperoxidase (MPO) identified as an 80-kDa band on the gel.24 MPO was detectable from 0 h, reached highest level at 1 h and remained high until 6 h. Group B, revealed a lower content of myeloperoxidase from 0 h and
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Figure 4. Content of IL-2 in the serum of rabbits treated with saline only, alternatively non-treated or IT9302-treated before induction of ei-AP.
Figure 5. An example of quantitation of the immunostaining on Hybond-ECL nitrocellulose membranes (chemiluminescence).
throughout 1, 3, 6 and 9 h compared with group A. In group C, myeloperoxidase was detected at time 0, but undetectable at time 1, 3, 6 and 9 h (Fig. 5).
DISCUSSION Pro-inflammatory cytokines are pivotal mediators for the pathophysiological mechanism leading to acute pancreatitis.6–8 Thus IT9302, an IL-10-analogue and a potent endogenous suppressor of pro-inflammatory cytokine activity when injected into rabbits with ei-AP, increases the survival of animals, preferentially by diminishing the severity of pancreatitis-associated multiple organ dysfunction.25,26 In the control group 60% of the rabbits died before the end of the experiment (after 6 to 9 h) whereas in the IT9302-treated group all animals survived.25 Similarly in the WS-4 treated group, 90% of the rabbits were alive at 12 h.26 In the present study, we demonstrated that induction of pro-inflammatory cytokines, such as IL-1 (1 h), IL-8 (3 h), TNF- (6 h) and MCP-1 (6 h) (Fig. 1) accompanies the development of ei-AP, reflected by their increased concentration in sera of rabbits with ei-AP. The cellular origins of these cytokines are principally believed to be from the pancreatic tissue, including damaged vascular tissue and infiltrating leukocytes.27,28 Not surprisingly, MPO is an early
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mediator, already detectable at baseline (0 h) (Fig. 6), during the severe inflammatory response and tissue damage, which is the result of ei-AP.8,25,26 IL-1 has been shown to be a significant cytokine for the development of AP.29 Also, our data suggest that IL-1 is orchestrating an array of proinflammatory cytokines such as the CXC chemokine IL-8 and the CC chemokine MCP-140 during the development of AP. This, and IL-8 in particular, further enhance leukocyte activation, principally by exerting chemotactic activity on neutrophils,5,9 thereby attracting them to the inflammatory focus and by inducing the release of enzymes from leukocytes such as MPO from neutrophils.30 The relevance of this mechanism appears to be supported by our observation that treating the rabbits with IT9302 or neutralizing monoclonal anti-IL-8 antibody, reduced the content of MPO in the serum. The effect of anti-IL-8 therapy on the outcome of the ei-AP as well as the reduction of TNF- and as expected, on IL-8 itself, generally supports that IL-8 is a pivotal mediator during ei-AP.39 The MCP-1 induction in the anti-IL-8 antibody treated animals could be explained by the fact that IgG complexes activate monocytes to secrete MCP-1.31 TNF- also appears to be important for the development of pancreatitis-associated syndromes such as adult respiratory distress syndrome (ARDS).32–34 We found that the level of TNF- is increased following the induction of ei-AP, and that this increase could be reduced by treating the rabbits with IT9302. It is therefore likely that part of the observed pronounced effect of IT9302 on the survival of rabbits with ei-AP is explained by a reduction of the TNF- load in the circulation.20 However, IT9302 also induced the production of IRAP-1, a natural IL-1 antagonist and does reduce the production of most pro-inflammatory cytokines.35 Further, the effect of IT9302 on chemokine activities could be parallel to that of natural IL-10, which is known to weaken the function of the chemokine receptors by reducing the receptor-mediated signal transduction.36 Together, this reveals a scenario where severe local tissue damage results in profound local production of pro-inflammatory cytokines, which may engage in a local enhancement of the inflammatory response, by inducing secondary mediators, by attracting leukocytes and by inducing the local release of tissue damaging leukocyte derived enzymes such as MPO. Further, the peripheral circulation, eased by the rich vascularization of the pancreas and its region, readily and partially absorbs this burst of potent peptide mediators. These circumstances possibly govern the condition of hypercytokinaemia leading to the induction of multiple organ dysfunction such as ARDS.
Pro-inflammatory cytokines in rabbit serum after experimentally induced acute pancreatitis / 57
It can be concluded that both the human IL-10 analogue IT9302 and the monoclonal anti-IL-8 antibody are able to alter cytokine levels, which correlate with a reduction in the inflammatory response associated with AP in rabbits. Consequently, these observations as well as earlier observations by us and others18–20,25,26,37,38 suggest that modulation of cytokine activity appears to be a means to prevent and or treat acute pancreatitis.
MATERIALS AND METHODS Animals We used adult male and female New Zealand white rabbits, weighing 3.9–5.2 kg. The experimental protocol was approved by the Danish Experimental Animal Committee.
Induction of acute pancreatitis The procedure of induction of acute pancreatitis has been described in detail elsewhere.8 Briefly, under general anaesthesia all rabbits underwent a 4-cm long midline laparotomy. The pancreatic duct was visualized entering the duodenum at a point approximately 20 cm from the pylorus. Acute pancreatitis was induced by the retrograde intraductal injection of 2.0 ml of 5.0% chenodeoxycholic bile acid (200 mg plus 4.0 ml ethanol, Sigma Chemicals Co., St. Louis, Missouri, USA, product no. C9377) through a polyethylene catheter inserted into the intramural aspect of the duct. After injection, the pancreatic duct was ligated and the abdomen was closed.8
Experimental design Twenty-eight rabbits were divided equally into three groups. Group A (controls, n=10) received an injection of 2.0 ml 5% chenodeoxyclolic bile acid, followed by duct ligation. They were then given a single intravenous saline bolus injection followed by infusion of 40 ml saline for 30 min until induction of acute pancreatitis. Group B (n=8) was treated with human IT9302, a nonapeptide with homology to the C-terminal portion of human IL-10,21 in two doses. The peptide/saline solution was freshly prepared for each day. The first dose (100 g/kg) was injected subcutaneously, and the second dose (100 g/ml) diluted in 40 ml saline (40 ml/30 min) intravenously, both then given half an hour before the injection of 5% bile acid, as described in the control group.25 Group C (n=10) received a single intravenous bolus injection (1 mg/kg dissolved in equal volumes of saline) of the monoclonal neutralizing anti-IL-8 antibody (WS-4) (Prof. Kouji Matsushima, Tokyo, Japan), 30 min before induction of acute pancreatitis, followed by infusion of 40 ml saline for 30 min until induction of acute pancreatitis.26 Nine rabbits were also used as a negative control group, without induction of AP, but only receiving saline. Animals were observed for a total period of 12 h, after which surviving rabbits were killed. Arterial blood samples were drawn 5 min before, and then 1, 3, 6, 9 and 12 h after induction of acute pancreatitis. Blood samples were immediately centrifuged at 3000 rpm for 10 min at 5C. The
Figure 6. The effect of IT9302 and WS-4 on the content of myeloperoxidase (MPO) in serum during the course of ei-AP. Immunoblotting was performed using different amounts of recombinant hTNF- and developed by using a polyclonal rabbit antihuman TNF- antibody. The detection limit by this technique is approximately 2ng/ ml.
supernatants were separated using sterile pipettes. All samples were coded and stored at 80C until analysed by Western blotting technique. At the end of the experiment (12 h) all animals underwent autopsy and the lung and the pancreas were investigated for the adhesion complexes CD11b/CD18.8,25,26 When animals died earlier than 12 h the autopsy was taken earlier.
Assays Serum samples of 200 l were freeze dried and dissolved in an equal volume of gel lysis buffer.22 We examined three rabbit serum samples (40 l) from each group at times 0, 1, 3, 6 and 9 h, for each cytokine. ECL-Western blotting of rabbit serum IL-8, IL-1, IL-4, IRAP and IL-2 was performed, using specific goat anti-human antibodies. MCP-1 was detected using a mouse anti-human MCP-1 antibody. All three animals tested showed similar immunostaining, but only data from one animal are shown in the figures. One-dimensional 15% or 20% SDS-PAGE gels were run and the serum samples were prepared in gel lysis buffer 1 h before and kept in 40C water until they were applied to the gels. The proteins were transferred to Hybond-ECL nitrocellulose membranes (Amersham RPN 2020 D, Little Chalfont, UK) and blocked with 5% bovine serum albumin (Sigma) in Tris-buffered saline, pH 7.8 containing 0.1% Tween-20. The blots were then incubated with 50 g of the specific cytokine antibody in 50 ml Tris buffer with 0.1% Tween-20, followed by 10 l of horse radish peroxidase labelled secondary antibody (cat. No. P447, P449 or P217, Dako, Copenhagen, Denmark) in 50 ml Tris buffer with 0.1% Tween-20. The secondary antibody labelling was followed by
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TABLE 1. Western blotting was performed to identify the cytokines. The serum samples and recombinant cytokines, which served as positive control, were run on 15% or 20% SDS-PAGE gels. The recombinant cytokines were reconstituted in PBS with 4% bovine serum albumin, and antibodies were applied as described in the Materials and Methods.
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anti-human TNF- antibody (code IP-310) is from Genzyme, Kings Hill, UK. Goat anti-human IL-1 receptor antagonist protein antibody (AB-280-NA) and mouse anti-human MCAF antibody (MAB 279) are from R&D Systems, UK. The secondary antibodies were, horseradish peroxidase labelled of either goat anti-mouse IgG (cat. No. P447), rabbit anti-goat IgG (cat. No. P449) or swine anti-rabbit IgG (cat. No. P217) from Dako, DK.
Acknowledgements This research was supported by a research grant from Nycomed Dak A/S, Copenhagen, and by SRG A/S, Svendborg.
REFERENCES
enhanced chemiluminescence activation (cat. no. RPN 2106, ECL, Amersham) and the immunostaining was detected by autoradiography. Films (Kodak X-OMAT-S, Tastrup, Denmark) were exposed for 90 s or 2 min.22 Positive controls were recombinant human cytokines and were also used for quantitation of cytokines in the serum samples. For quantitation of the IL-8 bands, we ran Western blotting of recombinant human or rabbit IL-8 standards in parallel wells of 15% or 20% SDS-PAGE gels (recombinant rabbit IL-8 was from K. Matsushima, Tokyo, Japan). The same principle was used for quantitation of IRAP, TNF-, IL-2, IL-4 and MCP-1 in a similar way (Fig. 5). For positive controls see Table 1.
Determination of the serum content of myeloperoxidase (MPO) Serum samples were further diluted in gel lysis buffer. After protein measurement, a sample of 50 g protein was applied to 10% SDS-PAGE gels. For positive control, a pro-myelocytic human cell line HL-60 (ECACC, Salisbury, UK) was used.24 Human myeloperoxidase is expressed as an 80-kDA protein. Determination of the serum content of myeloperoxidase was by rabbit anti-human MPO Ab (A 0398, Dako) and Western blotting.
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