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Immunomodulation with interleukin-10 and interleukin-4 compared with ketorolac tromethamine for prevention of postoperative adhesions in a murine model
FERTILITY AND STERILITYt VOL. 71, NO. 1, JANUARY 1999 Copyright © 1998 American Society for Reproductive Medicine Published by Elsevier Science Inc. Printed on acid-free paper in U.S.A.
Immunomodulation with interleukin-10 and interleukin-4 compared with ketorolac tromethamine for prevention of postoperative adhesions in a murine model Christine H. Holschneider, M.D., Farshid Nejad, B.S., and F. J. Montz, M.D., K.M. Gynecologic Oncology Service, Department of Obstetrics and Gynecology, University of California, Los Angeles (UCLA) School of Medicine, Los Angeles, California
Objective: To assess the effects of two macrophage down-regulating cytokines (interleukin [IL]-10 and IL-4) and the nonsteroidal anti-inflammatory drug ketorolac tromethamine on postoperative adhesion formation. Design: Randomized controlled trial. Setting: Research center vivarium. Animal(s): Six-week-old Swiss Webster mice. Intervention(s): One hundred eighty animals were randomized to serve as nonsurgical controls or to undergo a standardized adhesion-inducing procedure. Subsequently, animals were randomized to nine different treatment groups to receive no injections, phosphate-buffered saline (PBS) only, IL-10, IL-4, ketorolac, IL-10 plus IL-4, IL-10 plus ketorolac, IL-4 plus ketorolac, or all three agents. Main Outcome Measure: Adhesion scores on postoperative day 10. Result(s): Postoperative adhesion scores were significantly reduced in all groups of animals treated with IL-10 or ketorolac. Animals treated with IL-4 showed a nonsignificant trend toward reduction of adhesions. There were significantly more animals with adhesion scores of #3 in the IL-10 and ketorolac treatment groups than in the control groups receiving no treatment or PBS only. Conclusion(s): Although treatment with IL-10 and ketorolac did not completely prevent adhesion formation, treatment with these drugs did lead to a significant reduction in adhesion formation. Adhesions also tended to be thin and filmy rather than thick and vascular. Addition of IL-4 did not augment these effects. (Fertil Sterilt 1999;71:67–73. ©1998 by American Society for Reproductive Medicine.) Key Words: Postoperative adhesions, intraperitoneal adhesions, cytokines, NSAIDs, IL-10, IL-4, ketorolac, murine model, mouse model Received May 19, 1998; revised and accepted August 12, 1998. Presented in part at the 44th Annual Meeting of the Society for Gynecologic Investigation, San Diego, California, March 19 –22, 1997. Reprint requests and present address: F. J. Montz, M.D., K.M., Department of Obstetrics and Gynecology, Johns Hopkins Hospital, 600 North Wolfe, Houck 248, Baltimore, Maryland 21287 (FAX: 410-614-9607). 0015-0282/98/$19.00 PII S0015-0282(98)00393-8
Intraperitoneal adhesions are a major health care problem and source of morbidity, such as infertility, pelvic pain, and bowel obstruction, as well as increased intraoperative complications at subsequent surgeries. Adhesion-related morbidity substantially drains limited health care resources, accounting for 282,000 hospitalizations in 1988 alone and an associated $1.18 billion expenditure. These numbers do not include outpatient care and indirect costs due to loss of productivity and consumer spending (1). Over the last several decades, adhesion-related research has evolved around two main goals: to further understanding of the patho-
physiology and cellular and molecular mechanisms of adhesion formation and to develop agents that will prevent adhesion formation. The cellular events that occur after peritoneal injury and during adhesion formation have been thoroughly described. Peritoneal injury leads to extravasation of serum and cellular elements. By 12 hours after the injury, the wound is covered predominantly by polymorphonuclear cells entangled in fibrin strands, which are soon outnumbered by macrophages. When normal fibrinolysis occurs, islands of mesothelial cells proliferate throughout the wound and completely cover the defect within 67
4 –5 days (2). If normal fibrinolysis is inhibited, macrophages persist and fibroblasts proliferate. Within 5 days, the fibrin network between adherent structures is replaced by fibrous adhesions of bundles of collagen and fibroblasts (3). These cellular events appear to be orchestrated at least in part by cytokines that function as chemoattractants and immunostimulants. Interleukin (IL)-6 (4), transforming growth factor (TGF)-a (5), epidermal growth factor (5), TGF-b (6, 7), and IL-1a (8, 9) have been found to be adhesiogenic, whereas antibodies to IL-6 (4), tumor necrosis factor (TNF)-a (10), and IL-1 (10) reduce postoperative adhesion formation. A thorough elucidation of the role and characteristics of centrally active cytokines might make possible the development of adhesion-preventing agents by targeted immunomodulation. We propose that IL-10, also known as cytokine synthesis inhibiting factor, may be one of these centrally active mediators that inhibits the expression of cytokines such as IL-1, TNF-a, or IL-6 (11). In previous investigations, we (12, 13) demonstrated the ability of IL-10 to minimize postoperative adhesion formation. Another cytokine that may have antiadhesive effects is IL-4, with its inhibiting effects on monocytes and macrophages (14). In vitro findings demonstrate that IL-10 and IL-4 inhibit production of cytokines (e.g., adhesiogenic IL-6) in monocytes and macrophages by different mechanisms (15). In this study, we looked for possible synergistic inhibitory effects by IL-10 and IL-4 on postoperative adhesion formation. We further compared the IL-10 – and IL-4 –mediated immunomodulation with the adhesion-preventing effects of ketorolac tromethamine, a widely used nonsteroidal antiinflammatory drug (NSAID) that is known to reduce postoperative adhesion formation in animal models (16, 17). This randomized trial involved evaluation of the effects of IL-10, IL-4, and ketorolac individually and in combination on postoperative adhesion formation in a murine model.
MATERIALS AND METHODS Female 6-week-old Swiss Webster mice (Simonsen Laboratories Inc., Gilroy, CA) were used for our investigation, which was approved by the Animal Research Committee of the University of California–Los Angeles (UCLA). All procedures were performed in accordance with the standards described in the National Institutes of Health Guide for the Care and Use of Laboratory Animals, in compliance with the Federal Animal Welfare Act. The animals were housed at the UCLA Vivarium. Before and after surgery, the mice had access to water and food ad libidum. The animals were randomized either to serve as nonsurgical controls (n 5 90) or to undergo a standardized surgical procedure (n 5 90), as described earlier (12). Briefly, 68
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anesthesia was induced with xylazine (10 mg/kg IM) and ketamine (150 mg/kg IM). Anesthesia was maintained as necessary with halothane via cotton. A sharp midline incision was made extending from the symphysis 3 cm cephalad. The peritoneal cavity was explored for any evidence of preexisting adhesions, and findings were documented. Subsequently, a standardized peritoneal injury was induced by sharp abrasion of a 2-cm2 area of the left parietal peritoneum, leading to microhemorrhage. The incision was closed with a single layer of sutures, using 2-0 Dexon (Davis & Geck, Wayne, NJ). All animals were operated on by the same surgeon. Each animal underwent surgery before allocation to a study arm, to minimize investigator bias. Immediately after surgery, the mice were randomized to nine different treatment groups, with 10 animals per group. Treatments were given as follows: Injections of recombinant murine IL-10 (PeproTech Inc., Rocky Hill, NJ) or recombinant murine IL-4 (PeproTech Inc.) were given intraperitoneally (IP) 0, 24, 48, and 72 hours postoperatively. Both IL-10 and IL-4 were administered at doses of 1 mg/kg in 1 mL of phosphate-buffered saline (PBS), as determined by dose-response curves. Ketorolac was given at the maximum recommended human dose with a 1 mg/kg IM load immediately postoperatively, followed by 0.5 mg/kg IM injections every 6 hours for the first 72 hours after surgery. Similarly, the nonsurgical control mice were randomized to complementary treatment groups, with 10 animals per group. Both IL-10 and IL-4 were administered at doses of 1 mg/kg in 1 mL of PBS. With the use of a separate cohort of female Swiss Webster mice, dose-response curves had been established for both IL-10 and IL-4, covering ranges from 10 ng/kg to 100 mg/kg and from 10 ng/kg to 10 mg/kg, respectively, in 10-fold increments, with 10 animals per dosing group. The ranges of biologic activity as reported by the manufacturer for IL-10 (0.2–20 ng/mL) and IL-4 (0.1–10 ng/mL) were fully covered by the ranges of our doseresponse curves. As shown previously (13), IP administration of IL-10 led to a significant dose-dependent reduction in adhesion formation (Fig. 1A), with a maximum effect reached at 1 mg/kg. By contrast, administration of IL-4 alone did not lead to a significant change in postoperative adhesion formation (Fig. 1B). Therefore, we decided to administer IL-4 at a dose of 1 mg/kg, well within the range of reported biologic activity. Groups 1–9 did not undergo surgery. Group 1 served as the untreated control group. Group 2 received IP injections of 1 mL of PBS at 0, 24, 48, and 72 hours. Group 3 was treated with IL-10, group 4 received IL-4, and group 5 received ketorolac. Groups 6, 7, and 8 underwent treatment with IL-10 and IL-4, IL-10 and ketorolac, and IL-4 and ketorolac, respectively. Group 9 received all three agents (IL-10, IL-4, and ketorolac). Groups 10 –18 underwent the standardized surgical pro-
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FIGURE 1 (A) Effects of different doses of IL-10 on postoperative adhesion formation reported as geometric means of adhesion scores with 95% confidence intervals (CIs). Group 1: no injections; group 2: PBS only; group 3: 10 ng/kg; group 4: 100 ng/kg; group 5: 1 mg/kg; group 6: 10 mg/kg; and group 7: 100 mg/kg. (Reprinted from Holschneider et al. [13]). (B) Effects of different doses of IL-4 on postoperative adhesion formation reported as geometric means of adhesion scores with 95% CIs. Group 1: no injections; group 2: PBS only; group 3: 10 ng/kg; group 4: 100 ng/kg; group 5: 1 mg/kg; and group 6: 10 mg/kg.
cedure. Group 10 had no further treatment. Group 11 received PBS, and groups 12, 13, and 14 were treated with IL-10, IL-4, and ketorolac, respectively. Group 15 was given IL-10 and IL-4, group 16 received IL-10 and ketorolac, and group 17 received IL-4 and ketorolac. Group 18 received all three agents (IL-10, IL-4, and ketorolac). FERTILITY & STERILITYt
After 10 days, all animals were killed with CO2. An exploratory celiotomy was performed through a vertical midline incision in control animals or to the right of the old skin incision in previously operated animals. All adhesions present were assessed with the use of a standardized qualitative and quantitative scoring system (Table 1). The adhe69
dence intervals. Nominal variables were compared with the use of x2 tests.
TABLE 1 Adhesion scoring system.
RESULTS
Adhesion score Extent None Confined to traumatized area Confined to nontraumatized area In traumatized and nontraumatized areas Type Filmy Opaque, nonvascular Opaque, vascular Dense
0 1 2 3 1 3 extent 5 2 3 extent 5 3 3 extent 5 4 3 extent 5
Note: Modified from Hershlag et al. (8).
sions were scored by a single operator who was blinded to the treatment regimen. However, it was impossible to blind the investigator to whether the animal had undergone surgery because the scar was always clearly visible. Adhesion scores were transformed with natural logarithms to account for heterogeneous variance among groups. Statistical analysis for continuous variables was performed with the use of analysis of variance, followed by t-tests with Bonferroni corrections for multiple comparisons. Data are presented as geometric means with 95% confi-
None of the animals who underwent surgery had any adhesions at the time of initial celiotomy. The standardized surgical procedure of peritoneal abrasion causing microhemorrhage led to adhesion formation in all animals who underwent surgery (Fig. 2). However, postoperative adhesion scores were significantly reduced (P ,.005) in all groups of animals treated with IL-10, ketorolac, or a combination thereof (groups 12 and 14 –18), compared with the groups of mice who did not undergo postoperative treatment (group 10) or who received PBS only (group 11). When all postoperative IL-10 or ketorolac treatment groups (groups 12 and 14 –18) were compared, no significant difference was found in the postoperative adhesion scores (P 5 .57). Similarly, in terms of adhesion scores, treatment with ketorolac alone did not differ significantly from treatment with ketorolac plus IL-10 or IL-4 or both (P 5 .94). Animals treated with IL-4 showed only marginal reduction of adhesion formation (P 5 .053). Two animals in the nonsurgical control groups had minimal evidence of filmy peritoneal adhesions (Fig. 2), possibly preexisting but more likely secondary to multiple IP
FIGURE 2 Postoperative adhesion scores reported as geometric means with 95% confidence intervals (CIs) for nonsurgical control animals (groups 1–9) and animals who underwent surgery (groups 10 –18). For 3 days, animals received either no injections (groups 1 and 10), PBS only (groups 2 and 11), IL-10 (groups 3 and 12), IL-4 (groups 4 and 13), ketorolac (groups 5 and 14), IL-10 plus IL-4 (groups 6 and 15), IL-10 plus ketorolac (groups 7 and 16), IL-4 plus ketorolac (groups 8 and 17), or IL-10 plus IL-4 plus ketorolac (groups 9 and 18).
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els as well as data from studies using significantly different adhesion scoring systems. To overcome some of these difficulties of comparison, we performed a randomized study designed to include a readily available standard.
TABLE 2 Animals with only filmy or minimal opaque adhesions (adhesion score #3). Adhesion score #3 Treatment
n
%
10 11 12 13 14 15 16 17 18
3 2 9 5 8 4 8 7 8
30 20 90 50 89 50 89 78 80
(no treatment) (PBS) (IL-10) (IL-4) (ketorolac) (IL-10 1 IL-4) (IL-10 1 ketorolac) (IL-4 1 ketorolac) (IL-10 1 IL-4 1 ketorolac)
Note: All animals had at least filmy adhesions. PBS 5 phosphate-buffered saline; IL 5 interleukin.
injections. All animals tolerated the injections with IL-10, IL-4, and ketorolac well, without any evidence of obvious side effects. Adhesion formation still proceeded in all treatment groups; however, there were significantly more (P ,.05) animals with only filmy or minimal opaque adhesions, leading to adhesion scores of #3, in the IL-10 or ketorolac treatment groups than in the control groups receiving no treatment or PBS only (Table 2). Five of the animals who underwent surgery were excluded from analysis. One animal was found dead several hours after surgery. The cause of death is unknown. The animal had an apparently normal immediate recovery from anesthesia and received one dose of ketorolac postoperatively. Postmortem inspection of the peritoneal cavity did not reveal any evidence of intraperitoneal hemorrhage. The other four animals excluded from analysis had wound dehiscence in the first 24 hours postoperatively. In all four cases, sutures had broken probably because of the animals’ frequent biting and scratching of the sutures in the period immediately after surgery. It is unlikely that the treatment modalities used contributed to a weakening of the wounds because all cases of wound dehiscence occurred within the 1st postoperative day and in each case a suture was broken.
DISCUSSION Numerous challenges must be met to perform sufficiently valid and clinically meaningful adhesion prevention research. The first challenge is to develop an animal model with readily and reliably inducible adhesions. Our model of peritoneal injury in Swiss Webster mice fulfills these criteria, in that adhesion formation occurred in all mice who underwent surgery. A second difficulty is that of interpreting and comparing data obtained through use of various animal modFERTILITY & STERILITYt
For numerous reasons, the NSAID ketorolac appears to be an ideal standard. In several animal studies (16, 17), ketorolac has been shown to reduce postoperative adhesion formation, while not inducing a confounding local immune reaction, because it is given by IM injection. Ketorolac is widely used and is approved for human administration. For practical purposes, any new adhesion-preventing agent might not be considered for human use unless it was clearly superior to the NSAID control in a randomized trial. As we gain a better understanding of the molecular and biochemical events that occur during peritoneal healing and postoperative adhesion formations, investigations will be aimed at directed suppression of adhesiogenic cytokines or augmentation of cytokines that inhibit adhesion formation. Investigators currently believe that IL-1a (8, 9), IL-2 (18), IL-6 (4), platelet-derived growth factor (18), epidermal growth factor (5), TGF-a (5), and TGF-b (7) all act to potentiate adhesion formation, and that antibodies against IL-1 (10), IL-6 (4), and TNF-a (10) have been shown to reduce postoperative adhesions. In this study, we evaluated the effects of two cytokines, IL-10 and IL-4, both of which were postulated to inhibit postoperative adhesion formation. We confirmed a significant reduction in postoperative adhesions in IL-10 –treated animals. There are multiple mechanisms by which IL-10 can reduce adhesion formation. Interleukin-10, produced by type 2 helper T cells, inhibits profibrotic cytokine production by type 1 helper T cells (11, 19), peripheral blood mononuclear cells, and macrophages (11, 20, 21). One of these adhesiogenic cytokines suppressed by IL-10 is IL-2, which is known to stimulate the synthesis of two highly fibrogenic cytokines, TGF-b and platelet-derived growth factor-B (18). Similarly, IL-10 acts on monocytes and macrophages to suppress the synthesis of the potent adhesion-forming mediators IL-1 and IL-6 (11, 14). Further reduction in IL-1 activity may be effected by the IL-10 –induced up-regulation of the antiinflammatory IL-1 receptor antagonist (22). Numerous studies in both humans and animals have demonstrated that IL-4 inhibits the production of adhesiogenic cytokines such as TNF-a (23, 24) or IL-1 (25, 26) by monocytes and macrophages. Interleukin-4 further up-regulates the expression of the anti-inflammatory IL-1 receptor antagonist (25). Differential (15) and synergistic (27) effects between IL-10 and IL-4 have been described in particular for the reduction of IL-6 expression. We therefore hypothesized that IL-4 would have the effect of an inhibition in postoperative adhesions similar to that observed in IL-10 –treated animals. However, the trend toward reduction in adhesion formation was not statistically significant with IL-4 treatment. 71
This lack of statistical significance could be attributed to the limited statistical power of our study, as there were only 10 animals per treatment arm. Interleukin-4 did not augment adhesion-suppressing effects of IL-10 or ketorolac in any of the combination treatment groups. One potential explanation for the significant observed and unexpected difference in potency between IL-10 and IL-4 to reduce adhesions may stem from the different half-lives of these cytokines. Intravenously administered IL-10 has a half-life of approximately 3 hours (28), whereas IL-4 is rapidly cleared after IV administration, having a half-life of 19 minutes (29). In the majority of treated animals, administration of IL-10 and ketorolac resulted in postoperative adhesions being thin and filmy; in contrast, the adhesions in control animals who underwent surgery were generally thick, dense, and vascular. Although there is some evidence that NSAIDs up-regulate IL-10 production in murine peritoneal macrophages (30), it remains to be proved whether ketorolac and IL-10 prevent postoperative adhesion formation by the same mechanism. One may speculate that neither IL-10 nor ketorolac has significant impact on the events that initiate adhesion formation and that thin and filmy adhesions therefore result. However, IL-10 and ketorolac appear effectively to reduce neovascularization, as well as fibroblast migration, proliferation, and collagen production, thus preventing the formation of thick and vascular adhesions. In our study, animals were observed for 10 days postoperatively. This follow-up duration was chosen because peritoneal healing is virtually completed within 5– 8 days after injury (2, 31). Similarly, after 7 to 10 days, adhesions are generally characterized by the presence of fibroblasts, collagen formation, and small blood vessels (3). Studies on the kinetics of postoperative adhesion formation have found the peritoneum’s susceptibility to the initiation of adhesion formation to be limited to the first 36 hours postoperatively (32). Although these data support a 10-day follow-up after 3 days of treatment as being long enough for full evaluation of treatment effects, there remains a small possibility that IL-10 and ketorolac might merely delay, rather than prevent, postoperative adhesion formation. This possibility could be excluded only in a future study with several months of follow-up. In summary, we conclude that IL-10 and ketorolac tromethamine reduce postoperative adhesion formation significantly and to an equivalent degree. Although IL-10 and ketorolac do not completely prevent adhesions, they tend to cause postoperative adhesions to be thin and filmy rather than thick, dense, and vascular. Given concomitantly, IL-4 does not augment the IL-10 – or ketorolac-induced reduction in adhesions. Immunomodulation with IL-10 is not superior to treatment with the NSAID ketorolac with regard to prevention of postoperative adhesions. However, further elucidation of the mechanisms of action of IL-10 may contribute 72
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significantly to our understanding of the pathophysiology of postoperative adhesion formation. References 1. Ray NF, Larsen JW Jr, Stillman RJ, Jacobs RJ. Economic impact of hospitalizations for lower abdominal adhesiolysis in the United States in 1988. Surg Gynecol Obstet 1993;176:271– 6. 2. Raftery AT. Regeneration of parietal and visceral peritoneum: an electron microscopical study. J Anat 1973;115:375–92. 3. Milligan DW, Raftery AT. Observations on the pathogenesis of peritoneal adhesions: a light and electron microscopical study. Br J Surg 1974;61:274 – 80. 4. Saba AA, Kaidi AA, Godziachvili V, Dombi GW, Dawe EJ, Libcke JH, et al. Effects of interleukin-6 and its neutralizing antibodies on peritoneal adhesion formation and wound healing. Am Surg 1996; 62:569 –72. 5. Chegini N, Simms J, Williams RS, Masterson BJ. Identification of epidermal growth factor, transforming growth factor-a, and epidermal growth factor receptor in surgically induced pelvic adhesions in the rat and intraperitoneal adhesions in the human. Am J Obstet Gynecol 1994;171:321–7. 6. Williams RS, Rossi AM, Chegini N, Schultz G. Effect of transforming growth factor b on postoperative adhesion formation and intact peritoneum. J Surg Res 1992;52:65–70. 7. Chegini N, Gold LI, Williams RS, Masterson BJ. Localization of transforming growth factor beta isoforms TGF-b1, TGF-b2, and TGF-b3 in surgically induced pelvic adhesions in the rat. Obstet Gynecol 1994;83:449 –54. 8. Hershlag A, Otterness IG, Bliven ML, Diamond MP, Polan ML. The effect of interleukin-1 on adhesion formation in the rat. Am J Obstet Gynecol 1991;165:771– 4. 9. McBride WH, Mason K, Withers HR, Davis C. Effect of interleukin 1, inflammation, and surgery on the incidence of adhesion formation and death after abdominal irradiation in mice. Cancer Res 1989;49: 169 –73. 10. Kaidi AA, Nazzal M, Gurchumelidze T, Ali MA, Dawe EJ, Silva YJ. Preoperative administration of antibodies against tumor necrosis factoralpha (TNF-a) and interleukin-1 (IL-1) and their impact on peritoneal adhesion formation. Am Surg 1995;61:569 –72. 11. Fiorentino DF, Zlotnik A, Mosmann TR, Howard M, O’Garra A. IL-10 inhibits cytokine production by activated macrophages. J Immunol 1991;147:3815–22. 12. Montz FJ, Holschneider CH, Bozuk M, Gotlieb WH, Martinez-Maza O. Interleukin 10: ability to minimize postoperative intraperitoneal adhesion formation in a murine model. Fertil Steril 1994;61:1136 – 40. 13. Holschneider CH, Cristoforoni PM, Ghosh K, Punyasavatsut M, Abed E, Montz FJ. Endogenous versus exogenous IL-10 in postoperative adhesion formation in a murine model. J Surg Res 1997;70:138 – 43. 14. Te Velde AA, Huijbens RJF, Heije K, de Vries JE, Figdor CG. Interleukin-4 (IL-4) inhibits secretion of IL-1b, tumor necrosis factor a, and IL-6 by human monocytes. Blood 1990;76:1392–7. 15. Takeshita S, Gage JR, Kishimoto T, Vredevoe DL, Martinez-Maza O. Differential regulation of IL-6 gene transcription and expression by IL-4 and IL-10 in human monocytic cell lines. J Immunol 1996;156: 2591– 8. 16. Montz FJ, Monk BJ, Lacy SM, Fowler JM. Ketorolac tromethamine, a nonsteroidal anti-inflammatory drug: ability to inhibit post-radical pelvic surgery adhesions in a porcine model. Gynecol Oncol 1993;48: 76 –9. 17. DiZerega GS. Contemporary adhesion prevention. Fertil Steril 1994; 61:219 –35. 18. Kovacs EJ, Brock B, Silber IE, Neuman JE. Production of fibrogenic cytokines by IL-2 treated peripheral blood leukocytes: expression of TGF-b and PDGF-B chain genes. Obstet Gynecol 1993;82:29 –36. 19. Mosmann TR, Moore KW. The role of IL-10 in crossregulation of TH1 and TH2 responses. Immunol Today 1991;12:A49 –A53. 20. Zlotnik A, Moore KW. Interleukin-10. Cytokine 1991;3:366 –71. 21. Taga K, Tosato G. IL-10 inhibits human T-cell proliferation and IL-2 production. J Immunol 1992;148:1143– 8. 22. Howard M, O’Garra A. Biological properties of interleukin 10. Immunol Today 1992;13:198 –200. 23. Mijatovic T, Kruys V, Caput D, Defrance P, Huez G. Interleukin-4 and -13 inhibit tumor necrosis factor-alpha mRNA translational activation in lipopolysaccharide-induced mouse macrophages. J Biol Chem 1997; 272:14394 – 8. 24. Suk K, Erickson KL. Differential regulation of tumor necrosis factor-a mRNA degradation in macrophages by interleukin-4 and interferon-g. Immunology 1996;87:551– 8. 25. Hart PH, Ahern MJ, Smith MD, Finlay-Jones JJ. Comparison of the suppressive effects of interleukin-10 and interleukin-4 on synovial fluid
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29. Ghosh AK, Smith NK, Prendiville J, Thatcher N, Crowther D, Stern PL. A phase I study of recombinant human interleukin-4 administered by the intravenous and subcutaneous route in patients with advanced cancer: immunological studies. Eur Cytokine Netw 1993;4:205–11. 30. Shimizu T, Sano C, Sato K, Tomioka H. Effects of various steroidal and non-steroidal anti-inflammatory drugs on in-vitro IL-10 production of murine peritoneal macrophages infected with Mycobacterium avium complex. Kansenshogaku Zasshi 1997;71:910 –7. 31. Raftery TA. Regeneration of parietal and visceral peritoneum: a light microscopical study. Br J Surg 1973;60:293–9. 32. Harris ES, Morgan RF, Rodeheaver GT. Analysis of the kinetics of peritoneal adhesion formation in the rat and evaluation of potential antiadhesive agents. Surgery 1995;117:663–9.
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Immunomodulation with interleukin-10 and interleukin-4 compared with ketorolac tromethamine for prevention of postoperative adhesions in a murine model Christine H. Holschneider, M.D., Farshid Nejad, B.S., and F. J. Montz, M.D., K.M. Gynecologic Oncology Service, Department of Obstetrics and Gynecology, University of California, Los Angeles (UCLA) School of Medicine, Los Angeles, California
Objective: To assess the effects of two macrophage down-regulating cytokines (interleukin [IL]-10 and IL-4) and the nonsteroidal anti-inflammatory drug ketorolac tromethamine on postoperative adhesion formation. Design: Randomized controlled trial. Setting: Research center vivarium. Animal(s): Six-week-old Swiss Webster mice. Intervention(s): One hundred eighty animals were randomized to serve as nonsurgical controls or to undergo a standardized adhesion-inducing procedure. Subsequently, animals were randomized to nine different treatment groups to receive no injections, phosphate-buffered saline (PBS) only, IL-10, IL-4, ketorolac, IL-10 plus IL-4, IL-10 plus ketorolac, IL-4 plus ketorolac, or all three agents. Main Outcome Measure: Adhesion scores on postoperative day 10. Result(s): Postoperative adhesion scores were significantly reduced in all groups of animals treated with IL-10 or ketorolac. Animals treated with IL-4 showed a nonsignificant trend toward reduction of adhesions. There were significantly more animals with adhesion scores of #3 in the IL-10 and ketorolac treatment groups than in the control groups receiving no treatment or PBS only. Conclusion(s): Although treatment with IL-10 and ketorolac did not completely prevent adhesion formation, treatment with these drugs did lead to a significant reduction in adhesion formation. Adhesions also tended to be thin and filmy rather than thick and vascular. Addition of IL-4 did not augment these effects. (Fertil Sterilt 1999;71:67–73. ©1998 by American Society for Reproductive Medicine.) Key Words: Postoperative adhesions, intraperitoneal adhesions, cytokines, NSAIDs, IL-10, IL-4, ketorolac, murine model, mouse model Received May 19, 1998; revised and accepted August 12, 1998. Presented in part at the 44th Annual Meeting of the Society for Gynecologic Investigation, San Diego, California, March 19 –22, 1997. Reprint requests and present address: F. J. Montz, M.D., K.M., Department of Obstetrics and Gynecology, Johns Hopkins Hospital, 600 North Wolfe, Houck 248, Baltimore, Maryland 21287 (FAX: 410-614-9607). 0015-0282/98/$19.00 PII S0015-0282(98)00393-8
Intraperitoneal adhesions are a major health care problem and source of morbidity, such as infertility, pelvic pain, and bowel obstruction, as well as increased intraoperative complications at subsequent surgeries. Adhesion-related morbidity substantially drains limited health care resources, accounting for 282,000 hospitalizations in 1988 alone and an associated $1.18 billion expenditure. These numbers do not include outpatient care and indirect costs due to loss of productivity and consumer spending (1). Over the last several decades, adhesion-related research has evolved around two main goals: to further understanding of the patho-
physiology and cellular and molecular mechanisms of adhesion formation and to develop agents that will prevent adhesion formation. The cellular events that occur after peritoneal injury and during adhesion formation have been thoroughly described. Peritoneal injury leads to extravasation of serum and cellular elements. By 12 hours after the injury, the wound is covered predominantly by polymorphonuclear cells entangled in fibrin strands, which are soon outnumbered by macrophages. When normal fibrinolysis occurs, islands of mesothelial cells proliferate throughout the wound and completely cover the defect within 67
4 –5 days (2). If normal fibrinolysis is inhibited, macrophages persist and fibroblasts proliferate. Within 5 days, the fibrin network between adherent structures is replaced by fibrous adhesions of bundles of collagen and fibroblasts (3). These cellular events appear to be orchestrated at least in part by cytokines that function as chemoattractants and immunostimulants. Interleukin (IL)-6 (4), transforming growth factor (TGF)-a (5), epidermal growth factor (5), TGF-b (6, 7), and IL-1a (8, 9) have been found to be adhesiogenic, whereas antibodies to IL-6 (4), tumor necrosis factor (TNF)-a (10), and IL-1 (10) reduce postoperative adhesion formation. A thorough elucidation of the role and characteristics of centrally active cytokines might make possible the development of adhesion-preventing agents by targeted immunomodulation. We propose that IL-10, also known as cytokine synthesis inhibiting factor, may be one of these centrally active mediators that inhibits the expression of cytokines such as IL-1, TNF-a, or IL-6 (11). In previous investigations, we (12, 13) demonstrated the ability of IL-10 to minimize postoperative adhesion formation. Another cytokine that may have antiadhesive effects is IL-4, with its inhibiting effects on monocytes and macrophages (14). In vitro findings demonstrate that IL-10 and IL-4 inhibit production of cytokines (e.g., adhesiogenic IL-6) in monocytes and macrophages by different mechanisms (15). In this study, we looked for possible synergistic inhibitory effects by IL-10 and IL-4 on postoperative adhesion formation. We further compared the IL-10 – and IL-4 –mediated immunomodulation with the adhesion-preventing effects of ketorolac tromethamine, a widely used nonsteroidal antiinflammatory drug (NSAID) that is known to reduce postoperative adhesion formation in animal models (16, 17). This randomized trial involved evaluation of the effects of IL-10, IL-4, and ketorolac individually and in combination on postoperative adhesion formation in a murine model.
MATERIALS AND METHODS Female 6-week-old Swiss Webster mice (Simonsen Laboratories Inc., Gilroy, CA) were used for our investigation, which was approved by the Animal Research Committee of the University of California–Los Angeles (UCLA). All procedures were performed in accordance with the standards described in the National Institutes of Health Guide for the Care and Use of Laboratory Animals, in compliance with the Federal Animal Welfare Act. The animals were housed at the UCLA Vivarium. Before and after surgery, the mice had access to water and food ad libidum. The animals were randomized either to serve as nonsurgical controls (n 5 90) or to undergo a standardized surgical procedure (n 5 90), as described earlier (12). Briefly, 68
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anesthesia was induced with xylazine (10 mg/kg IM) and ketamine (150 mg/kg IM). Anesthesia was maintained as necessary with halothane via cotton. A sharp midline incision was made extending from the symphysis 3 cm cephalad. The peritoneal cavity was explored for any evidence of preexisting adhesions, and findings were documented. Subsequently, a standardized peritoneal injury was induced by sharp abrasion of a 2-cm2 area of the left parietal peritoneum, leading to microhemorrhage. The incision was closed with a single layer of sutures, using 2-0 Dexon (Davis & Geck, Wayne, NJ). All animals were operated on by the same surgeon. Each animal underwent surgery before allocation to a study arm, to minimize investigator bias. Immediately after surgery, the mice were randomized to nine different treatment groups, with 10 animals per group. Treatments were given as follows: Injections of recombinant murine IL-10 (PeproTech Inc., Rocky Hill, NJ) or recombinant murine IL-4 (PeproTech Inc.) were given intraperitoneally (IP) 0, 24, 48, and 72 hours postoperatively. Both IL-10 and IL-4 were administered at doses of 1 mg/kg in 1 mL of phosphate-buffered saline (PBS), as determined by dose-response curves. Ketorolac was given at the maximum recommended human dose with a 1 mg/kg IM load immediately postoperatively, followed by 0.5 mg/kg IM injections every 6 hours for the first 72 hours after surgery. Similarly, the nonsurgical control mice were randomized to complementary treatment groups, with 10 animals per group. Both IL-10 and IL-4 were administered at doses of 1 mg/kg in 1 mL of PBS. With the use of a separate cohort of female Swiss Webster mice, dose-response curves had been established for both IL-10 and IL-4, covering ranges from 10 ng/kg to 100 mg/kg and from 10 ng/kg to 10 mg/kg, respectively, in 10-fold increments, with 10 animals per dosing group. The ranges of biologic activity as reported by the manufacturer for IL-10 (0.2–20 ng/mL) and IL-4 (0.1–10 ng/mL) were fully covered by the ranges of our doseresponse curves. As shown previously (13), IP administration of IL-10 led to a significant dose-dependent reduction in adhesion formation (Fig. 1A), with a maximum effect reached at 1 mg/kg. By contrast, administration of IL-4 alone did not lead to a significant change in postoperative adhesion formation (Fig. 1B). Therefore, we decided to administer IL-4 at a dose of 1 mg/kg, well within the range of reported biologic activity. Groups 1–9 did not undergo surgery. Group 1 served as the untreated control group. Group 2 received IP injections of 1 mL of PBS at 0, 24, 48, and 72 hours. Group 3 was treated with IL-10, group 4 received IL-4, and group 5 received ketorolac. Groups 6, 7, and 8 underwent treatment with IL-10 and IL-4, IL-10 and ketorolac, and IL-4 and ketorolac, respectively. Group 9 received all three agents (IL-10, IL-4, and ketorolac). Groups 10 –18 underwent the standardized surgical pro-
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FIGURE 1 (A) Effects of different doses of IL-10 on postoperative adhesion formation reported as geometric means of adhesion scores with 95% confidence intervals (CIs). Group 1: no injections; group 2: PBS only; group 3: 10 ng/kg; group 4: 100 ng/kg; group 5: 1 mg/kg; group 6: 10 mg/kg; and group 7: 100 mg/kg. (Reprinted from Holschneider et al. [13]). (B) Effects of different doses of IL-4 on postoperative adhesion formation reported as geometric means of adhesion scores with 95% CIs. Group 1: no injections; group 2: PBS only; group 3: 10 ng/kg; group 4: 100 ng/kg; group 5: 1 mg/kg; and group 6: 10 mg/kg.
cedure. Group 10 had no further treatment. Group 11 received PBS, and groups 12, 13, and 14 were treated with IL-10, IL-4, and ketorolac, respectively. Group 15 was given IL-10 and IL-4, group 16 received IL-10 and ketorolac, and group 17 received IL-4 and ketorolac. Group 18 received all three agents (IL-10, IL-4, and ketorolac). FERTILITY & STERILITYt
After 10 days, all animals were killed with CO2. An exploratory celiotomy was performed through a vertical midline incision in control animals or to the right of the old skin incision in previously operated animals. All adhesions present were assessed with the use of a standardized qualitative and quantitative scoring system (Table 1). The adhe69
dence intervals. Nominal variables were compared with the use of x2 tests.
TABLE 1 Adhesion scoring system.
RESULTS
Adhesion score Extent None Confined to traumatized area Confined to nontraumatized area In traumatized and nontraumatized areas Type Filmy Opaque, nonvascular Opaque, vascular Dense
0 1 2 3 1 3 extent 5 2 3 extent 5 3 3 extent 5 4 3 extent 5
Note: Modified from Hershlag et al. (8).
sions were scored by a single operator who was blinded to the treatment regimen. However, it was impossible to blind the investigator to whether the animal had undergone surgery because the scar was always clearly visible. Adhesion scores were transformed with natural logarithms to account for heterogeneous variance among groups. Statistical analysis for continuous variables was performed with the use of analysis of variance, followed by t-tests with Bonferroni corrections for multiple comparisons. Data are presented as geometric means with 95% confi-
None of the animals who underwent surgery had any adhesions at the time of initial celiotomy. The standardized surgical procedure of peritoneal abrasion causing microhemorrhage led to adhesion formation in all animals who underwent surgery (Fig. 2). However, postoperative adhesion scores were significantly reduced (P ,.005) in all groups of animals treated with IL-10, ketorolac, or a combination thereof (groups 12 and 14 –18), compared with the groups of mice who did not undergo postoperative treatment (group 10) or who received PBS only (group 11). When all postoperative IL-10 or ketorolac treatment groups (groups 12 and 14 –18) were compared, no significant difference was found in the postoperative adhesion scores (P 5 .57). Similarly, in terms of adhesion scores, treatment with ketorolac alone did not differ significantly from treatment with ketorolac plus IL-10 or IL-4 or both (P 5 .94). Animals treated with IL-4 showed only marginal reduction of adhesion formation (P 5 .053). Two animals in the nonsurgical control groups had minimal evidence of filmy peritoneal adhesions (Fig. 2), possibly preexisting but more likely secondary to multiple IP
FIGURE 2 Postoperative adhesion scores reported as geometric means with 95% confidence intervals (CIs) for nonsurgical control animals (groups 1–9) and animals who underwent surgery (groups 10 –18). For 3 days, animals received either no injections (groups 1 and 10), PBS only (groups 2 and 11), IL-10 (groups 3 and 12), IL-4 (groups 4 and 13), ketorolac (groups 5 and 14), IL-10 plus IL-4 (groups 6 and 15), IL-10 plus ketorolac (groups 7 and 16), IL-4 plus ketorolac (groups 8 and 17), or IL-10 plus IL-4 plus ketorolac (groups 9 and 18).
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els as well as data from studies using significantly different adhesion scoring systems. To overcome some of these difficulties of comparison, we performed a randomized study designed to include a readily available standard.
TABLE 2 Animals with only filmy or minimal opaque adhesions (adhesion score #3). Adhesion score #3 Treatment
n
%
10 11 12 13 14 15 16 17 18
3 2 9 5 8 4 8 7 8
30 20 90 50 89 50 89 78 80
(no treatment) (PBS) (IL-10) (IL-4) (ketorolac) (IL-10 1 IL-4) (IL-10 1 ketorolac) (IL-4 1 ketorolac) (IL-10 1 IL-4 1 ketorolac)
Note: All animals had at least filmy adhesions. PBS 5 phosphate-buffered saline; IL 5 interleukin.
injections. All animals tolerated the injections with IL-10, IL-4, and ketorolac well, without any evidence of obvious side effects. Adhesion formation still proceeded in all treatment groups; however, there were significantly more (P ,.05) animals with only filmy or minimal opaque adhesions, leading to adhesion scores of #3, in the IL-10 or ketorolac treatment groups than in the control groups receiving no treatment or PBS only (Table 2). Five of the animals who underwent surgery were excluded from analysis. One animal was found dead several hours after surgery. The cause of death is unknown. The animal had an apparently normal immediate recovery from anesthesia and received one dose of ketorolac postoperatively. Postmortem inspection of the peritoneal cavity did not reveal any evidence of intraperitoneal hemorrhage. The other four animals excluded from analysis had wound dehiscence in the first 24 hours postoperatively. In all four cases, sutures had broken probably because of the animals’ frequent biting and scratching of the sutures in the period immediately after surgery. It is unlikely that the treatment modalities used contributed to a weakening of the wounds because all cases of wound dehiscence occurred within the 1st postoperative day and in each case a suture was broken.
DISCUSSION Numerous challenges must be met to perform sufficiently valid and clinically meaningful adhesion prevention research. The first challenge is to develop an animal model with readily and reliably inducible adhesions. Our model of peritoneal injury in Swiss Webster mice fulfills these criteria, in that adhesion formation occurred in all mice who underwent surgery. A second difficulty is that of interpreting and comparing data obtained through use of various animal modFERTILITY & STERILITYt
For numerous reasons, the NSAID ketorolac appears to be an ideal standard. In several animal studies (16, 17), ketorolac has been shown to reduce postoperative adhesion formation, while not inducing a confounding local immune reaction, because it is given by IM injection. Ketorolac is widely used and is approved for human administration. For practical purposes, any new adhesion-preventing agent might not be considered for human use unless it was clearly superior to the NSAID control in a randomized trial. As we gain a better understanding of the molecular and biochemical events that occur during peritoneal healing and postoperative adhesion formations, investigations will be aimed at directed suppression of adhesiogenic cytokines or augmentation of cytokines that inhibit adhesion formation. Investigators currently believe that IL-1a (8, 9), IL-2 (18), IL-6 (4), platelet-derived growth factor (18), epidermal growth factor (5), TGF-a (5), and TGF-b (7) all act to potentiate adhesion formation, and that antibodies against IL-1 (10), IL-6 (4), and TNF-a (10) have been shown to reduce postoperative adhesions. In this study, we evaluated the effects of two cytokines, IL-10 and IL-4, both of which were postulated to inhibit postoperative adhesion formation. We confirmed a significant reduction in postoperative adhesions in IL-10 –treated animals. There are multiple mechanisms by which IL-10 can reduce adhesion formation. Interleukin-10, produced by type 2 helper T cells, inhibits profibrotic cytokine production by type 1 helper T cells (11, 19), peripheral blood mononuclear cells, and macrophages (11, 20, 21). One of these adhesiogenic cytokines suppressed by IL-10 is IL-2, which is known to stimulate the synthesis of two highly fibrogenic cytokines, TGF-b and platelet-derived growth factor-B (18). Similarly, IL-10 acts on monocytes and macrophages to suppress the synthesis of the potent adhesion-forming mediators IL-1 and IL-6 (11, 14). Further reduction in IL-1 activity may be effected by the IL-10 –induced up-regulation of the antiinflammatory IL-1 receptor antagonist (22). Numerous studies in both humans and animals have demonstrated that IL-4 inhibits the production of adhesiogenic cytokines such as TNF-a (23, 24) or IL-1 (25, 26) by monocytes and macrophages. Interleukin-4 further up-regulates the expression of the anti-inflammatory IL-1 receptor antagonist (25). Differential (15) and synergistic (27) effects between IL-10 and IL-4 have been described in particular for the reduction of IL-6 expression. We therefore hypothesized that IL-4 would have the effect of an inhibition in postoperative adhesions similar to that observed in IL-10 –treated animals. However, the trend toward reduction in adhesion formation was not statistically significant with IL-4 treatment. 71
This lack of statistical significance could be attributed to the limited statistical power of our study, as there were only 10 animals per treatment arm. Interleukin-4 did not augment adhesion-suppressing effects of IL-10 or ketorolac in any of the combination treatment groups. One potential explanation for the significant observed and unexpected difference in potency between IL-10 and IL-4 to reduce adhesions may stem from the different half-lives of these cytokines. Intravenously administered IL-10 has a half-life of approximately 3 hours (28), whereas IL-4 is rapidly cleared after IV administration, having a half-life of 19 minutes (29). In the majority of treated animals, administration of IL-10 and ketorolac resulted in postoperative adhesions being thin and filmy; in contrast, the adhesions in control animals who underwent surgery were generally thick, dense, and vascular. Although there is some evidence that NSAIDs up-regulate IL-10 production in murine peritoneal macrophages (30), it remains to be proved whether ketorolac and IL-10 prevent postoperative adhesion formation by the same mechanism. One may speculate that neither IL-10 nor ketorolac has significant impact on the events that initiate adhesion formation and that thin and filmy adhesions therefore result. However, IL-10 and ketorolac appear effectively to reduce neovascularization, as well as fibroblast migration, proliferation, and collagen production, thus preventing the formation of thick and vascular adhesions. In our study, animals were observed for 10 days postoperatively. This follow-up duration was chosen because peritoneal healing is virtually completed within 5– 8 days after injury (2, 31). Similarly, after 7 to 10 days, adhesions are generally characterized by the presence of fibroblasts, collagen formation, and small blood vessels (3). Studies on the kinetics of postoperative adhesion formation have found the peritoneum’s susceptibility to the initiation of adhesion formation to be limited to the first 36 hours postoperatively (32). Although these data support a 10-day follow-up after 3 days of treatment as being long enough for full evaluation of treatment effects, there remains a small possibility that IL-10 and ketorolac might merely delay, rather than prevent, postoperative adhesion formation. This possibility could be excluded only in a future study with several months of follow-up. In summary, we conclude that IL-10 and ketorolac tromethamine reduce postoperative adhesion formation significantly and to an equivalent degree. Although IL-10 and ketorolac do not completely prevent adhesions, they tend to cause postoperative adhesions to be thin and filmy rather than thick, dense, and vascular. Given concomitantly, IL-4 does not augment the IL-10 – or ketorolac-induced reduction in adhesions. Immunomodulation with IL-10 is not superior to treatment with the NSAID ketorolac with regard to prevention of postoperative adhesions. However, further elucidation of the mechanisms of action of IL-10 may contribute 72
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