IT 9302, a synthetic interleukin-10 agonist, diminishes acute lung injury in rabbits with acute necrotizing pancreatitis

IT 9302, a synthetic interleukin-10 agonist, diminishes acute lung injury in rabbits with acute necrotizing pancreatitis Maher O. Osman, MD, Niels O. Jacobsen, MD, Jørgen U. Kristensen, MD, Bent Deleuran, MD, DMSci, Borbola Gesser, Christian G. Larsen, MD, DMSci, and Steen L. Jensen, MD, DMSci, Aarhus, Denmark

Background. Proinflammatory cytokines (eg, tumor necrosis factor [TNF]–α, interleukin [IL]-1, and IL8) are believed to play an important role in the pathogenesis of acute necrotizing pancreatitis (ANP) and its systemic complications. Recently, IL-10 has emerged as a major anti-inflammatory cytokine, inhibiting the secretion and activities of inflammatory cytokines. Further, a protective effect of IL-10 has recently been shown in experimental acute pancreatitis. The purpose of this study was to test the potential role of a newly developed IL-10 agonist, IT 9302, in a model of ANP in rabbits. Methods. ANP was induced in 18 rabbits by retrograde injection of 5% chenodeoxycholic acid in the pancreatic duct, followed by duct ligation. The rabbits were allocated to pretreatment with intravenous physiologic saline solution or IT 9302 (200 µg/kg) 30 minutes before the induction of ANP. Results. Injection of IT 9302 resulted in a significant reduction in the blood levels of TNF-α and IL-8 from 3 to 6 hours. IT 9302 also reduced the amount of ascitic fluid and significantly inhibited neutrophil infiltration and margination, as well as the number of CD11b- and CD18-positive cells in the lung tissues. By contrast, the local pancreatic necrosis, as well as the biochemical changes such as serum amylase, lipase, and calcium, was severe and similar in both groups. Survival was improved significantly after treatment with IT 9302. Conclusions. As expected, IT 9302 cannot change the degree of ANP induced by 5% bile acid but does reduce mortality rates and the development of acute lung injury, probably through the inhibition of circulating levels of TNF-α, IL-8, and the expression of the adhesion molecule complex CD11b/CD18. (Surgery 1998;124:584-92.) From the Departments of Surgery L, Pathology, Rheumatology, and Dermatology, Aarhus University Hospital, Aarhus University, Aarhus, Denmark

ACUTE NECROTIZING PANCREATITIS (ANP) is a common and potentially fatal disease, its pathogenesis is incompletely resolved, and its treatment is largely nonspecific. Acute lung injury is a frequent complication of the severe disease and has extensively been shown to contribute significantly to the high morbidity and mortality rates associated with the disease.1 Activated leukocytes and the proinflammatory cytokines (eg, tumor necrosis factor [TNF]–α, interleukin [IL]-1, IL-6, and IL-8) seem to play a Supported by a grant from Fabrikant Frands Køhler Nielsens og Hustrus Mindelegat and grants from the Danish Rheumatism Association and Mads Clausens Foundation (B.D.). Accepted for publication March 16, 1998. Reprint requests: Maher Omar Osman, MD, MS-Chi, c/o Professor Steen Lindkaer Jensen, Department of Surgery L, Aarhus University Hospital, Norrebrogade 44, DK-8000 Aarhus C, Denmark. Copyright © 1998 by Mosby, Inc. 0039-6060/98/$5.00 + 0

584 SURGERY

11/56/90361

predominant role in the progression of severe acute pancreatitis (AP) from a local inflammatory necrosis into a systemic inflammatory response.2,3 Several groups have shown a correlation between the severity of clinical and experimental AP and subsequent elevations in serum levels of TNF-α, IL1, and IL-8.4-6 Antagonism of TNF-α and IL-1 in experimental AP has resulted in an amelioration of the physiologic and biochemical changes of AP and increased survival.7-9 Recently, IL-10 has emerged as a potent antiinflammatory cytokine, inhibiting the production of other cytokines.10 In vivo, IL-10 protected mice against lethal shock induced by staphylococcal enterotoxin B.11 IL-10 has also reduced the amount of pancreatic damage in a murine model of cerulein-induced AP, presumably by inhibiting the production of TNF-α. 12 More recently, nonapeptide IT 9302, with homology to the C-terminal portion of human IL-10, has been found to possess some activities that mimic those of IL-10 in vitro.13 It is our hypothesis that IT

Surgery Volume 124, Number 3

Osman et al 585

Fig 1. A, Leukocyte count in control group (ANP 5%) and group pretreated with IT 9302, with significant difference after 3 hours. B, TNF-α serum levels in control group and group pretreated with IT 9302. Significant difference between two groups at 3 and 6 hours was demonstrated. C, Serum IL-8 levels in control group and group pretreated with IT 9302, with significant reduction in group treated with IT 9302 (P = .026 and .005, respectively).

9302 pretreatment of rabbits with severe AP induced by bile injection into the pancreatic duct would reduce the systemic manifestations of the disease. This study was designed to investigate the potential effects of IT 9302 in a rabbit model of severe ANP.14 MATERIAL AND METHODS Animals. Adult male and female New Zealand White rabbits, weighing 3.9 to 5.2 kg, were used. The animals were housed at the animal farm of Aarhus University at least 1 week before use and according to the requirements for animal care as stipulated by the Danish Experimental Animal Committee, who also approved the experimental protocol. Anesthetics and surgical procedures. Overnightfasted animals, but with free access to tap water, were anesthetized with a mixture of Dormicum (2 mg/kg) and Hypnorm (0.3 mL/kg), both injected

intramuscularly. The animals were kept anesthetized throughout the experimental period of 12 hours by repeated intramuscular injections of Hypnorm (0.15 mL/kg). The operative procedures have been described in detail elsewhere.14 Briefly, all rabbits underwent a 4-cm long midline laparotomy, and the pancreatic duct was visualized entering the duodenum at a point approximately 20 cm from the pylorus. The duct was cannulated, and AP was induced by retrograde intraductal injection of 2.0 mL 5% chenodeoxycholic bile acid (clear faint yellow solution at 200 mg plus 4.0 mL ethanol, product no. C9377; Sigma Chemical Co, St Louis, Mo) for 21⁄2 minutes (ie, in a pressure-, volume-, and time-controlled manner).14 After injection, the pancreatic duct was ligated and the abdomen was closed in 2 layers. Experimental design. Eighteen animals were allocated into 2 groups. Group A (controls) (n = 10) underwent retrograde intraductal injection

586 Osman et al

Surgery September 1998

Fig 2. Output of ascitic fluid, with significant reduction of output in treated animals (P < .001). Outputs of amylase and lipase in ascitic fluid have also been reduced significantly by IT 9302 (P = .016 and .028, respectively). Further, total protein (Prot.) output and free hemoglobin (Hgb.) output in ascitic fluid were also reduced significantly by IT 9302 pretreatment.

of 2.0 mL 5% bile acid, ductal ligation, and a subcutaneous bolus injection of 0.9% saline solution (4.0 mL) followed by infusion of 40 mL 0.9% saline solution for 30 minutes before induction of AP. Group B was treated with human IT 9302 (n = 8) in 2 doses; the first dose (100 µg/kg) was injected subcutaneously and the second dose (100 µg/kg, diluted in 40 mL saline solution [40 mL/30 min]) intravenously, both 1⁄2 hour before the injection of 5% bile acid as described in the control group. Arterial blood samples were drawn at –30 minutes, +5 minutes, and 3, 6, 9, and 12 hours after laparotomy for determination of amylase, lipase, leukocyte, glucose, calcium, and cytokine levels (IL-8 and TNF-α). The volume of each blood sample (6.0 mL) was replaced by 0.9% saline solution. Four milliliters of physiologic saline solution was infused per hour in all animals throughout the experimental period. All blood samples, except blood for leukocyte count, were immediately centrifuged at 3000 rpm for 10 minutes at 5°C. The supernatants were separated

with sterile pipettes. All samples were coded and stored at –80°C until analyzed. At the end of the observation period, surviving rabbits were killed and ascitic fluid was quantified and aliquots separated for determination of amylase, lipase, free hemoglobin, and free protein levels. Finally, all animals underwent autopsy and the pancreata, lungs, and kidneys were excised and divided into 2 parts, 1 part fixed in 10% formalin for histologic grading and the second part frozen immediately in liquid nitrogen for immunohistochemical analysis of the adhesion complex CD11b/CD18. Functional studies. Leukocyte count was measured in ethylenediaminetetraacetic acid–preserved blood samples, with a Coulter counter model T 890. Serum and ascitic fluid amylase and lipase levels were measured by photometry and the enzymatic reaction by kinetic turbidimetry, respectively. Results are expressed as units per liter. Serum glucose and calcium levels were measured by an enzymatic glucose oxygenation dehydrogenation (GOD) and photometry, respectively. Results are

Surgery Volume 124, Number 3

Osman et al 587

Fig 3. A, Alveolar lung tissue with edema grade 2. There is heavy infiltration of alveolar walls with inflammatory cells, most of which are identified as granulocytes at higher magnification. (Hematoxylin-eosin stain; original magnification ×190.) B, Alveolar lung tissue from animals in group B. There is practically no edema and fewer inflammatory cells. (Hematoxylin-eosin stain; original magnification ×190.)

expressed as millimolars. Ascitic fluid total protein and free hemoglobin levels were measured by the Biuret method and spectrophotometry, respectively. Results are expressed in grams per liter and millimolars, respectively. Serum TNF-α levels were determined with an enzyme-linked immunosorbent assay kit from Genzyme (code no. 80-3807-00), according to the manufacturer’s guidelines. IL-8 measurement in rabbit serum was carried out by a special IL-8 enzyme-linked immunosorbent assay kit (a gift from Professor Kouji Matsushima, Tokyo, Japan). The method has been published in detail elsewhere15 but with slight modification. Briefly, monoclonal anti-IL-8 (1.5 µg/mL) WS-4 antibody and peroxidase-conjugated anti-guinea pig antibody (PO 141; Dako Corp) were used as capture and detection antibodies, respectively. Histologic examination. Paraffin-embedded sections from the pancreas, right lung, and right kidney were stained with hematoxylin-eosin. Sections of lung and kidney were stained further for fibrin (Mallory) and lung sections also for elastic tissue (van Gieson). All sections were evaluated blindly. The specimens were scored for necrosis, inflammatory cellular infiltration, and the presence of vascular thrombosis according to the following criteria. Pancreas. The volume fraction of necrotic pancreatic parenchyma was determined by point counting on hematoxylin-eosin–stained sections

and expressed as necrotic tissue/total pancreatic parenchyma × 100. The inflammatory response to necrosis was evaluated semiquantitatively as follows: grade 0, absent; grade 1, slight focal inflammation; grade 2, moderate granulocytic margination of necrosis; and grade 3, widespread and pronounced granulocytic margination of necrosis. The amount of fat necrosis was also semiquantitated: grade 0, absent; grade 1, less than 1⁄3 of peripancreatic fat showing necrosis; grade 2, from 1⁄3 to 2⁄3 of fat with necrosis; and grade 3, more than 2⁄3 of peripancreatic fat to be necrotic. Finally, the presence of vascular thrombosis was also described. Lung. In hematoxylin-eosin–stained sections from each animal, the degree of edema was semiquantitated: grade 0, none; grade 1, slight edema of the alveolar walls; grade 2, moderate edematous thickening of alveolar walls with occasional alveoli containing coagulated edema fluid; and grade 3, extensive occurrence of alveolar and interstitial edema. The number of neutrophils per high-power field (×750) was counted in 10 random fields of alveolar lung tissue, avoiding fields containing bronchi or larger vessels. Further, the presence of margination of neutrophils in vascular lumina was noted. In some of the animals (control animals and animals with experimental AP14) focal interstitial mononuclear infiltrates were found in the lungs. These changes were ascribed to an asymptomatic infection with Bordetella bronchiseptica, which regularly can be cultured from

588 Osman et al

the airways of laboratory rabbits and are known to cause such infiltrates.16 The infiltrates were semiquantitated as following: 0, none; 1, few; 2, moderate; and 3, pronounced. In sections stained for fibrin, the presence or absence of capillary thrombosis or thrombosis of larger vessels was noted. Kidney. In sections stained for fibrin, the presence or absence of glomerular microthrombosis was determined. Immunohistochemical detection of CD11b/ CD18. Biopsy specimens from the lung and pancreas were snap frozen in liqiud nitrogen and kept at –80°C until processing. The method has been described in detail elsewhere.14 The immunohistochemical staining of the lung tissue was quantified by computer by random selection by a grid. Because of the severe destruction of the pancreas, counting for statistical purposes was not possible in this organ. Statistical analysis. Results are expressed as means and standard errors of the mean. Differences between the two groups were judged by the nonparametric Mann-Whitney rank-sum test or the unpaired Student t test. A P value < .05 was considered significant. RESULTS Survival. In group A, six rabbits (60%) died before the end of the experiment (after 6 to 9 hours), whereas in group B all animals survived the entire observation period, with a significant difference between the two groups (P < .05). Serum amylase, lipase, calcium, and glucose levels. In all animals a marked elevation of serum amylase and lipase levels was observed, starting early after the induction of AP and reaching a peak after 6 hours. Pretreatment with IT 9302 did not result in a significant reduction in the mean amylase and lipase levels (data not shown). The serum calcium level was decreased throughout the course of pancreatitis in all animals without any significant effect for IT 9302 pretreatment. The control animals manifested hyperglycemia after 6 hours followed by a decline, reaching a low level after 12 hours. In the IT 9302-pretreated group, hypoglycemia was evident during the entire experimental period, with a significant difference between both groups only after 6 hours (P = .031) (data not shown). Leukocyte count. Control animals were significantly leukopenic 3 hours after the induction of AP, whereas in the group pretreated with IT 9302 the leukocyte count was unchanged by that time (Fig 1, A), with a statistically significant difference compared with the control group (P = .004).

Surgery September 1998

Fig 4. Number of granulocytes per high-power field of 10 random lung sections shows statistical difference between group treated with IT 9302 and control group (ANP 5%) (P < .001).

Thereafter no significant difference between the groups was detected. Serum TNF-α and IL-8. In the untreated group there was marked elevation of inflammatory cytokines in the circulation, with TNF-α markedly rising at both 3 and 6 hours, describing a biphasic curve (Fig 1, B), whereas maximum IL-8 induction was after 6 hours (Fig 1, C). Both TNF-α and IL-8 levels in the circulation were significantly downregulated for 3 to 6 hours in the group pretreated with IT 9302 (P = .016 and .012 for TNF-α and .026 and .005 for IL-8 at 3 and 6 hours, respectively). Ascitic fluid. Pretreatment with IT 9302 resulted in a significant reduction in the output of the ascitic fluid per hour (Fig 2) (P < .001). A significant reduction in the concentration of amylase and lipase (Fig 2) in the ascitic fluid was also observed (P = .016 and .028, respectively). Further, a significant reduction in the ascitic fluid output of total protein (P = .05) and free hemoglobin (P = .044) (Fig 2) was also noted in the treated group compared with the control animals. Morphologic findings Pancreas. In both groups of animals there was the same degree of widespread parenchymal coagulative necrosis. In the control group the mean volume of necrotic tissue was 73.5% ± 2.9%. In the group pretreated with IT 9302 the mean value was 71.4% ± 6.6%. Likwise, there seemed to be no difference between the two groups regarding the inflammatory response in the pancreas. Rather, the individual differences found in the degree of inflammation seemed to be related to the time of survival after injection of bile acid. Thus the control animals that survived for only 6 or 9 hours showed practically no inflammation (either grade 0 or 1), whereas in all longer surviving animals of both groups grade 2 or 3 inflammatory response was seen (data not shown). The same was true for the degree of fat necrosis. Thrombosis of pancre-

Osman et al 589

Surgery Volume 124, Number 3

Fig 5. Distribution of CD11b-expressing leukocytes in rabbit lung tissue. In untreated group (a), high numbers of CD11b-positive cells were present in lung tissue, whereas in group B (b) amount of CD11b-expressing cells in lung was reduced significantly, as counted in randomized computer grids. (Streptavidine alkaline phosphatase technique; positive staining appears as red, hematoxylin counterstained.)

atic vessels was found among 6 of 10 animals in the control group and 6 of 8 animals in the animals pretreated with IT 9302. Lungs. In the lungs of the control animals (Fig 3, A), a moderate to severe edema was observed, as well as a pronounced infiltrate of neutrophils in the alveolar walls. As shown in Figs 3, B, and 4, these changes were reduced significantly in the group pretreated with IT 9302. The neutrophils were scattered diffusely throughout the lung tissue and not particularly related to the interstitial mononuclear infiltrates found in all animals (data not shown). In almost all animals of group A (9 of 10) and group B (6 of 8), a more or less pronounced leukocytic vascular margination and perivascular infiltrates of polymorphonuclear neutophils was noted. Foci of vascular thrombosis in large vessels and microthrombi were seen in the control group (6 of 10) and in only 3 of 8 animals in the treated group. Kidney. In the control group, 6 of the animals showed few glomerular microthrombi, whereas the rabbits pretreated with IT 9302 did not show signs of vascular thrombosis. In other respects, the kidney appeared normal. Immunohistochemical detection of CD11b/ CD18. Immunohistochemical presence of CD11b and CD18 was noted in all animals examined. In the lung, CD11b was detected in all animals of the control group (Fig 5, a). It gave rise to a cytoplasmic staining localized to cells that were mainly neutrophils, observed throughout the lung tissue. In the group pretreated with IT 9302, CD11bpositive cells were observed in the lung, but their number and staining intensity were greatly reduced (P < .001) compared with group A (Fig 5,

b). They were localized mainly around bronchioles and not in the lung tissue. CD18 was detected in all animals in both lung and pancreatic tissues. Although more cells expressed CD18 than CD11b, serial sections revealed that cells appeared to be CD11b/CD18 positive, suggesting that this complex is of importance for the inflammatory process in rabbits. The highest expression of CD18 was observed in the lung and pancreas of control animals (Fig 6, a). The lungs of the group pretreated with IT 9302 had greatly reduced numbers of CD18-positive cells compared with group A (P < .001) (Fig 6, b). The pancreas was too severely damaged to make counting for statistical purposes, but the CD11b- and CD18-positive cells were observed in all glands of control animals and some of the pretreated group. DISCUSSION This study demonstrated that pretreatment with IT 9302 in animals with ANP causes (1) a significant reduction of TNF-α and IL-8 concentrations in the systemic blood; (2) a significant improvement of the leukocyte count after 3 hours from the induction of AP; (3) a significant downregulation of the adhesion molecule complex CD11b/CD18 in the lung and pancreatic tissues; (4) a marked attenuation of the lung inflammatory reaction, evidenced by a reduction in neutrophil infiltration, the degree of edema, and vascular thrombosis in the lung interstitial tissue; (5) a significant reduction in the output of ascitic fluid and its contents of amylase, lipase, and total proteins; and (6) a significant improvement in survival of treated animals.

590 Osman et al

Surgery September 1998

Fig 6. Distribution of CD18-expressing leukocytes in rabbit lung. In untreated rabbits (a), high numbers of CD18-positive cells were present in lung tissue. Serial sections revealed cells to be either double positive or localized in vicinity of each other. In animals pretreated with IT 9302, amount of CD18-expressing cells in lung (b) was reduced significantly, as counted in randomized computer grids. (Streptavidine alkaline phosphatase technique; positive staining appears as red, hematoxylin counterstained.) Bar indicates 10 µm.

In this study only prophylactic treatment was carried out. There are several reasons for this. First, the model of bile acid infusion is not suitable for a delay treatment arm because of irreversibility of the local pancreatic changes and, second, delayed treatment has been shown to work with virtually all cytokine antagonists.7-9 Thus the novelty of our study can be shown without a delayed treatment group. Our findings suggest that pretreatment of animals with IT 9302 has an inhibitory effect on the systemic manifestations of AP, reducing the development of acute lung injury and thus improving animal survival. These systemic effects of IT 9302 could be explained by its anti-inflammatory activity, inhibiting the production of other cytokines, as well as the adhesion molecules, leading to inhibition of the neutrophil-induced tissue destruction. The in vivo anti-inflammatory effects exerted by IT 9302 in this model of AP are consistent with the in vitro effects recently reported by Gesser et al.13 IT 9302 has been shown to mimic and possess some IL-10 activities, which include inhibition of spontaneous IL-8 production, induction of IL-1 receptor antagonist protein production by human monocytes, down-regulation of TNF-α production by CD+8 T cells, induction of IL-4 production by CD+4 T cells, and suppression of the chemotactic response of human monocytes toward MCAF/ MCP-1. IT 9302 does not induce stimulation of endogenous IL-10 synthesis.13 Moreover, our findings of no difference in severity of pancreatitis, locally in the inflamed pancreas, are inconsistent with the earlier reports investigating the outcome of treating animals

with experimental pancreatitis by the administration of IL-10. 12,17,18 By contrast, in one study using the diet-induced model, 17 IL-10 significantly reduced serum amylase and pancreatic histologic score when administered either prophylactically or therapeutically. However, the systemic or local levels of inflammatory cytokines have not been measured in this study. 17 Furthermore, none of the previous studies has shown the effect of IL-10 treatment on the systemic complications of AP. Again, the effect of IL10 on the circulating IL-8 levels, as well as on the local expression of the adhesion molecule complex CD11b/CD18, has never been demonstrated in AP. It seems of huge pharmaceutical interest that the analog IT 9302, which consists of only 9 amino acids, exerts similar inhibitory effects on the circulating cytokine levels as the entire IL-10 molecule. The protective role for IL-10 or its agonist IT 9302 in experimental AP might be explained through its inhibitory effect on the macrophage-derived cytokines, including IL-1, TNF-α, and IL-8, 10 or by its induction of IL-1 receptor antagonist protein, 13 which has been shown to ameliorate significantly the manifestations of AP in several animal models.8,9 The proinflammatory cytokines are known to be important mediators to create the necessary chemotactic gradient for leukocyte infiltration and also stimulate adhesion molecule CD11b/CD18 expression, thereby mediating the development of an acute inflammatory response.19 The inflammatory cytokines IL-1, IL-6, and TNF-α are produced rapidly after the induction of AP, mimicking its severity. Intrapancreatic cytokine tissue levels sig-

Osman et al 591

Surgery Volume 124, Number 3

nificantly precede in time and exceed in amount levels reached in the serum.20 Moreover, IL-1 and TNF-α gene expression is induced locally during AP, resulting in large amounts of intrapancreatic IL-1 and TNF-α proteins.5,21 We have demonstrated the production of IL-8 by the pancreatic acinar and ductal cells.14 Local cytokine production is followed by distant up-regulation of these proinflammatory cytokines, as has been reported by Hughes et al22 and recently by Norman et al.23 Our findings showing that systemic complications of AP such as acute lung injury can be blocked independently of the severity of the local morphologic pancreatic changes further confirm the distant role of inflammatory cytokines, as has been described previously by Norman et al.24 Furthermore, the adhesion complex CD11b/ CD18, induced by inflammatory cytokines, has been demonstrated to play a crucial role in neutrophil-induced tissue damage by enhancing neutrophil migration and adhesiveness.25 Antibody blockade of the adhesion molecule CD18 in a bileinduced rat model of experimental AP reduced the neutrophil-induced acute lung injury and improved survival,26 suggesting an important role for the adhesion molecules. Taken together, our findings confirm the role of the proinflammatory cytokines, as well as the adhesion molecules, in the progression of severe AP and the development of its systemic complications.2,3 Our findings of systemic effects of IT 9302 without accompanying local effects on the morphologic changes of AP correspond with other similar previous studies. In a similar model of bileinduced ANP in rats, Grewal et al27 and Tanaka et al28 have demonstrated an amelioration of the biochemical changes of AP and improvement of animal survival after the administration of anti-TNF-α polyclonal antibody and IL-1 receptor antagonist, respectively, without significant effect on the local pancreatic damage. In conclusion, IT 9302 cannot prevent the AP induced by the injection of 5% bile acid, but it could prevent acute lung injury, resulting in a reduction of mortality rates from 60% to 0%. This is most likely a result of the anti-inflammatory properties of IT 9302. Further efforts, however, should be made to evaluate the therapeutic role of IT 9302 in other models of AP with milder severity. We thank Walter Gyldenlowe and his staff at the research farm, Aarhus University, for their support and never-failing interest.

REFERENCES 1. Jacobs ML, Daggett WM, Civetta JM, Vasu MA, Lawson DW, Warshaw AL, et al. Acute pancreatitis: analysis of factors influencing survival. Ann Surg 1977;185:43-51. 2. Rinderknecht H. Fatal pancreatitis: a consequence of excessive leukocyte stimulation? Int J Pancreatol 1988;3:105-12. 3. Gross V, Lesser H-G, Heinisch A, Scholmerich J. Inflammatory mediators and cytokines: new aspects of the pathophysiology and assessment of acute pancreatitis? Hepatogastroenterology 1993;42:522-30. 4. Grewal HP, Kotb M, Mohey el-Din AB, Ohman M, Salem A, Gaber L, et al. Induction of tumor necrosis factor in severe acute pancreatitis and its subsequent reduction after hepatic passage. Surgery 1994;115:213-21. 5. Fink GW, Norman JG. Intrapancreatic interleukin-1β gene expression by specific leukocyte populations during acute pancreatitis. J Surg Res 1996;63:369-73. 6. Gross V, Andreesen R, Lesser H-G, Ceska M, Liehl E, Lausen M, et al. Interleukin-8 and neutrophil activation in acute pancreatitis. Eur J Clin Invest 1992;22:200-3. 7. Hughes CB, Gaber LW, Mohey el-Din AB, Grewal HP, Kotb M, Mann L, et al. Inhibition of TNF-α improves survival in an experimental model of acute pancreatitis. Am Surg 1996;62:8-13. 8. Norman JG, Franz MG, Fink GS, Messina J, Fabri PJ, Gower WR, et al. Decreased mortality of severe acute pancreatitis after proximal cytokine blockade. Ann Surg 1995; 221:625-34. 9. Norman JG, Franz M, Messina J, Riker A, Fabri PJ, Rosemurgy AS, et al. Interleukin-1 receptor antagonist decreases severity of experimental acute pancreatitis. Surgery 1995;117:648-55. 10. Fiorentino DF, Zlotink A, Mosmann TR, Howard M, O’Garra A. IL-10 inhibits cytokine production by activated macrophages. J Immunol 1991;147:3815-22. 11. Bean A, Freiberg R, Andrade S, Menon S, Zlotnick A. Interleukin-10 protects mice against staphylococcal enterotoxin-B-induced lethal shock. Infect Immunol 1993; 61:4937-9. 12. Van Laethem JL, Marchant A, Delvaux A, Goldman M, Robberecht P, Velu T, et al. Interleukin-10 prevents necrosis in murine experimental acute pancreatitis. Gastroenterology 1995;108:1917-22. 13. Gesser B, Leffers H, Jinquan T, Vestergaard C, Kirstein N, Pedersen S, et al. Identification of a functional domain on human IL-10. Proc Natl Acad Sci USA 1997;94:14620-5. 14. Osman MO, Lausten SB, Jacobsen NO, Kristensen JU, Deleuran B, Larsen CG, et al. Graded experimental acute pancreatitis: monitoring of a renewed rabbit model focusing on the production of TNF-α, interleukin-8 and CD11b/CD18. Eur J Gastroenterol Hepatol In press. 15. Ikeda N, Mukaida N, Kaneko S, Fujioka N, Su S-B, Nariuchi H, et al. Prevention of endotoxin-induced acute lethality in Propionibacterium acnes–primed rabbits by an antibody to leukocyte integrin β2 with concomitant reduction of cytokine production. Infect Immunol 1995; 63:4812-7. 16. Uzal FA, Feinstein RE, Rehbinder C, Persson L. A study of lung lesions in asymptomatic rabbits naturally infected with B. bronchseptica. Scand J Lab Anim Sci 1989;16:3-13. 17. Kusske AM, Rongione AJ, Ashley SW, McFadden DW, Reber HA. Interleukin-10 prevents death in lethal necrotizing pancreatitis in mice. Surgery 1996;120:284-9. 18. Rongione AJ, Kusske AM, Kwan K, Ashley SW, Reber HA, McFadden DW. Interleukin-10 reduces severity of acute pancreatitis in the rat. Gastroenterology 1997;112:960-7.

592 Osman et al

19. Lowry SF. Cytokine mediators of immunity and inflammation. Arch Surg 1993;128:1235-41. 20. Norman J, Franz M, Riker A, Fabri PJ, Gower W. Rapid elevation of systemic cytokine production during acute pancreatitis and their origination within the pancreas. Surg Forum 1994;XLV:148-60. 21. Norman JG, Fink GW, Franz MG. Acute pancreatitis induces intrapancreatic tumor necrosis factor gene expression. Arch Surg 1995;130:966-70. 22. Hughes CB, Henry J, Kotb M, Lobaschevsky A, Sabek O, Gaber AO. Up-regulation of TNF-α mRNA in the rat spleen following induction of acute pancreatitis. J Surg Res 1995;59:687-93. 23. Norman JG, Yang J, Fink G, Carter G, Ku G, Denham W, et al. Severity and mortality of experimental pancreatitis are dependent upon interleukin-1 converting enzyme (ICE). J Interferon Cytokine Res 1997;17:113-8. 24. Norman JG, Fink GW, Denham W, Yang J, Carter G, Sexton C, et al. Tissue specific cytokine production during experi-

Surgery September 1998

25.

26.

27.

28.

mental acute pancreatitis: a probable mechanism for distant organ dysfunction. Dig Dis Sci 1997;42:1783-8. Jaeschke T, Farhood A, Smith W. Neutrophil-induced liver cell injury in endotoxic shock is a CD11b/CD18-dependent mechanism. Am J Physiol 1991;261:G1051-6. Inoue S, Nakao A, Kishimoto W, Murakami H, Itoh K, Harada A, et al. Anti-neutrophil antibody attenuates the severity of acute lung injury in rats with experimental acute pancreatitis. Arch Surg 1995;130:93-8. Grewal HP, Mohey el-Din AB, Gaber L, Kotb M, Gaber AO. Amelioration of the physiologic and biochemical changes of acute pancreatitis using anti-TNF-α polyclonal antibody. Am J Surg 1994;167:214-8. Tanaka N, Murata A, Uda K, Toda H, Kato T, Hayashida H, et al. Interleukin-1 receptor antagonist modifies the changes in vital organs induced by acute necrotizing pancreatitis in a rat experimental model. Crit Care Med 1995; 23:901-8.