- Email: [email protected]
Interleukin 10: ability to minimize postoperative intraperitoneal adhesion formation in a murine model*
Reproductive animal research Vol. 61, No.6, June 1994
FERTILITY AND STERILITY Copyright
@
Printed on acid-free paper in U. S. A.
1994 The American Fertility Society
Interleukin 10: ability to minimize postoperative intraperitoneal adhesion formation in a murine model*
Fredrick J. Montz, M.D.t Christine H. Holschneider, M.D. Michael Bozuk, B.S.
Walter H. Gotlieb, M.D., Ph.D. Dto Martinez-Maza, Ph.D.
Gynecologic Oncology Service, Department of Obstetrics and Gynecology, University of California at Los Angeles (UCLA) Center for Health Sciences, Los Angeles, California
Objective: To determine the ability ofinterleukin 10 (IL-10) to suppress postoperative intraperitoneal adhesion formation. Design: Randomized, controlled trial. Setting: University animal research facility. Animals: Six -week -old Swiss Webster mice undergoing a standardized intraperitoneal operative procedure. Interventions: Animals were randomized to "surgery" or "no surgery" and then further randomized to receive intraperitoneal injections of 1 mL phosphate-buffered saline (PBS) or 1 Ilg/kg IL-lO in 1 mL PBS. Vehicle-only doses were given immediately after surgery and then every 24 hours for a total of four injections. Interleukin 10 injections were similarly given but with an added preoperative injection 30 minutes before surgery in one half of the animals. Main Outcome Measure: Adhesion formation. Results: Animals treated with vehicle or IL-10 but not undergoing surgical intervention had no intraperitoneal adhesions. Animals undergoing surgery who were treated with IL-lO, with or without a preoperative dose, had significantly lower postoperative adhesion scores than did control animals who postoperatively received PBS only. Conclusion: Interleukin-lO is effective at limiting postoperative intraperitoneal adhesion formation with minimal evident systemic side effects. Fertil Steril1994;61:1136-40 Key Words: Intraperitoneal adhesions, postoperative adhesion prevention, cytokines, interleukin 10, IL-10, CSIF, murine model
Intraperitoneal adhesions are a major source of patient morbidity and mortality as well as a substantial drain on limited health care resources. In 1988 alone there were approximately 282,000 hospitalizations in the United States during which lower abdominal adhesiolysis was performed, accounting for 948,727 days of inpatient care. These hospital-
Received July 2, 1993; revised and accepted
* Supported by the Bertha Warshaver Rubin Research Fund, Los Angeles, California. t Reprint requests: Fredrick J. Montz, M.D., Department of Obstetrics and Gynecology, UCLA, CRS 24-127, lO833 Le Conte Avenue, Los Angeles, California 90024 (FAX: 310-2063670). 1136
Montz et at.
Interleukin 10 and adhesion prevention
izations were responsible for an estimated $1.18 billion in expenditures, an estimate that does not include outpatient and indirect costs due to loss of productivity or decreased consumer spending (1). It is acknowledged widely that the vast majority of intraperitoneal adhesions that necessitate adhesiolysis represent sequelae from prior surgery (2). Though the serial cellular events that transpire after a peritoneal injury have been defined thoroughly (3), the exact role of cytokines in peritoneal repair and adhesion formation is yet to be elucidated completely. Preliminary data that state numerous cytokines such as IL-1 (4, 5) as well as IL-2, transforming growth factor {3 (TGF-{3), and platelet-derived growth factor B (PDGF-B) (6) are Fertility and Sterility
potentiators of postoperative adhesion formation exist. We proposed to evaluate the ability of a naturally occurring cytokine synthesis inhibiting factor (IL-10) (7,8) to suppress postsurgical adhesion formation in a well-standardized animal model. MATERIALS AND METHODS
After obtaining approval from the Animal Research Committee at the University of California at Los Angeles (UCLA), 60 six-week-old female Swiss Webster mice (Simonsen Laboratories, Inc., Gilroy, CA) were used for our investigations. The animals were housed at the UCLA Vivarium. All animal procedures were performed in accordance with the standards described in the National Institute of Health Guide for the Care and Use of Laboratory Animals, in compliance with the Federal Animal Welfare Act. Before surgery, the animals were allowed access to water and chow ad libidum. Anesthesia was induced with halothane inhalation. Animals were then injected with 30 mg/kg SC pentobarbital. Adequate anesthesia was maintained as necessary with halothane via a cotton ball. A sharp midline incision was made extending 2 cm from the symphysis to the upper abdomen. Intraperitoneal exploration was performed and any adhesions present were quantified. Subsequently, a standardized left lower abdominal wall injury was produced using sharp abrasion so as to induce microhemorrhage and peritoneal disruption over a 2 cm z area. The anterior abdominal wall was closed using a running 3-0 Maxon suture (American Cyanamid Company, Danbury, CT) for the peritoneum-fascia-muscle in an en bloc technique and the skin was closed using a running 3-0 silk suture. Animals were randomized into six separate groups. Group I underwent no surgery but received 1 mL of endotoxin-free phosphate-buffered saline (PBS) administered transabdominally into the peritoneal cavity using aseptic technique at time 0 and then 24, 48, and 72 hours thereafter. Group II also did not undergo surgery and received 1 JIg/kg recombinant IL-10 (Peprotec, Rocky Hill, NJ) in 1 mL IP PBS in the time schedule described above. This dose of IL-10 was chosen to lie well within the range of biologic activity of IL-lO, which is reported by the manufacturer to be between 0.2 and 20 ng/mL. Group III underwent surgery only and received no postoperative injections. Group IV underwent surgery and IL-10 treatment in the same manner as group II, with the first administration being immediately after closure of the anterior abdominal wall. Vol. 61, No.6, June 1994
Table 1
Adhesion Scoring* Adhesion score
Extent None Confined to traumatized area Confined to nontraumatized area Intraumatized and nontraumatized areas Type Filmy Opaque, nonvascular Opaque, vascular Dense
* Modified from
o 1 2
3 1 X extent = 2 X extent = 3 X extent = 4 X extent = sum =
Hershlag et al. (5).
Group V received 1 JIg/kg IL-lO in 1 mL PBS administered percutaneously into the peritoneal cavity 30 minutes before surgery and postoperatively received the same regimen as group IV. Group VI underwent surgery and then received intra-abdominal vehicle alone in a manner similar to group I, with the first administration given immediately after closure of the anterior abdominal wall. After recovery from anesthesia, animals were allowed immediate and unlimited access to water and chow. To minimize suffering of the animals during any subsequent intraperitoneal injections, short anesthesia with halothane was given immediately before injection. On postoperative day 7, the animals were killed using carbon dioxide euthanasia, explored, and adhesions quantified using a modification of the method of Hershlag and associates (5). This technique quantifies adhesions by extent and type to obtain a composite adhesion score for a given animal (Table 1). To minimize investigator bias, all animals that received postoperative injections of IL-10 or placebo were operated first and then randomized to different regimen groups. All adhesions were scored by the same investigator, who was blinded regarding the specific injection regimen. However, it was impossible to achieve blinding as to whether the animal was operated on because the incision was always visible. Statistical analysis was performed using the SAS statistical package (SAS Institute, Inc., Cary, NC). Adhesion scores were transformed with natural logarithms before statistical analysis to account for heterogeneous variance among groups. Following standard statistical practice, 0.5 was added to all values before transformation to account for zero values. This log transformation implies that group comparisons should be discussed as relative rather than absolute differences. As a result of the log Montz et al.
Interleukin 10 and adhesion prevention
1137
differ significantly in mean adhesion scores nor did groups III and VI. Based on statistical power calculations, eight animals per group are needed to detect a fourfold difference in adhesion scores. Thus, we are fairly certain that this study population had adequate numbers so as not to miss detecting differences between groups III and VI and IV and V on the order of magnitude of the apparent effect of IL-I0 itself.
transformation, group means are actually geometric means (inverse transformed values of the means or the log scale) and confidence intervals are reported rather than standard deviations because variation around the mean is not symmetrical. One-way analysis of variance with Tukey's pairwise comparison method was used to compare mean adhesion scores among groups. Alternatively, a nonparametric analysis using Wilcoxon's test or a pairwise t-test followed by Bonferroni adjustments were used. Regardless of the statistical tool used, the results obtained showed the same statistical significance.
DISCUSSION
It is a widely accepted fact that postoperative adhesions are a common cause of morbidity and potential mortality in patients undergoing intra-abdominal surgery in general (9, 10) and radical pelvic surgery in particular (11). Numerous adjuvants have been used throughout the history of surgery in an attempt to minimize the formation of such adhesions. The rationale for using the majority of these agents has been empirical, with little understanding of what transpires at a molecular level in normal and aberrant peritoneal healing. Recently, agents have been investigated that reflect our slowly expanding knowledge ofthe molecular and biochemical events of peritoneal healing (12,13). Further investigations must be undertaken to elucidate and modulate the basic mechanisms of mesothelial repair and thereby lead to rational drug design and therapy development. When reflecting on ways that the native peritoneal environment can be manipulated at a molecular level so as to limit adhesion formation, a logical novel tack would be to attempt to modify the cytokine response to a peritoneal injury. This could be aimed at suppression of cytokines, which have been demonstrated to promote adhesiogenesis, or at augmentation of cytokines inhibitory for adhesion formation. Although our understanding ofthe quantitative and temporal order of the cytokine response to a peritoneal injury
RESULTS
In no cases were intraperitoneal adhesions evident at the time ofthe initial laparotomy. No abnormalities of healing of the anterior abdominal wall incision such as wound disruption or infection were noted in any of the animal groups that had undergone surgery regardless of which study regimen they were assigned. One animal from each of the two groups that had undergone surgery and received IL-I0 died in the postoperative period. There were no adhesions observed at posteuthanasia autopsy in mice that had not undergone surgery but had been injected only with PBS with (group II; mean adhesion score = 0) or without IL10 (group I; mean adhesion score = 0). Remarkably, at time of exploration, postoperative adhesions were found universally in animals that were injected either with PBS only or received no postoperative intraperitoneal injections, while being noted to a lesser extent and degree in the majority ofthe IL-lO-treated animals (Table 2). Both groups receiving IL-I0 (IV, V) had significantly lower mean adhesion scores than both control groups (III, VI), (1.1 and 1.2 versus 4.3 and 4.2, respectively; P < 0.05). Groups IV and V did not
Table 2
Adhesion Scores* Group
No. of adhesions
III (surgery only):j: VI (surgery + postoperative PBS) IV (surgery + postoperative IL-10)§ V (surgery + IL-10 preoperative + postoperative)
10 10 9 9
* The overall t-test P value for the analysis of variance is = 0.002. t Groups III and VI have significantly greater mean adhesion scores than groups IV and V.
P
1138
Montz et al.
Interleukin 10 and adhesion prevention
Range (2 to (1 to (0 to (0 to
10) 10) 3) 6)
Geometric meant
95% confidence
interval 2.9 2.3 0.37 0.26
4.3 4.2 1.1
1.2
to to to to
6.3 7.5 2.3 3.3
:j: Groups III and VI do not differ significantly. § Groups IV and V do not differ significantly.
Fertility and Sterility
is infantile, it appears that IL-10' (4, 5), IL-2, PDGF, and TGF-~ (6) all act to potentiate adhesion formation. The extent of postoperative adhesion formation is probably a product of enhanced deposition of fibrin as well as cytokine-mediated stimulation of fibroblast proliferation and collagen production. It is our opinion that the most rational choice for an agent to be tested to suppress the adhesiogenic effect of the above described cytokines is IL-10. Interleukin 10 has been identified recently and its gene cloned based on its ability to inhibit cytokine synthesis (14). It is produced by the type 2 CD4+ helper T cell clones (Th-2) and inhibits the cytokine production of the type 1 (Th-1) subset in murine systems (15). Th-1 and Th-2 are the two subpopulations of T helper cells that control the nature of the immune response by secreting what are considered characteristic and mutually antagonistic sets of cytokines (16). Th-1 cells specifically produce IL-2 and interferon-,), (IFN-')') whereas Th-2 cells produce IL-4, IL-5, IL-6, and IL-10. Interleukin 10 appears to inhibit the production ofIL-2 and IFN -')' by the Th -1 cells (15) as well as the proliferation of human peripheral blood T cells and the synthesis of IFN -')' and other cytokines by stimulated peripheral blood monocytes (8, 17). It also has been demonstrated that IL-10 inhibits the production of IL-1, TNF-O', and IL-6 by lipopolysaccharide-activated macrophages (18). This anti -T cell-anticytokine-antimacrophage activity has been used in animal models in attempts to modulate the inflammatory response and associated pathological disease processes such as endotoxic shock (19) or parasitic infections (20, 21). In addition to these inhibitory activities, it has been noted that IL-10 can act to stimulate B cell growth and differentiation (22) as well as to serve as a costimulatory agent with other cytokines (23). It is exciting to speculate as to the potential mechanisms by which IL-10 limits the formation of postoperative peritoneal adhesions. It is probable that this phenomenon is a product of multiple events: Direct Effects on Macrophages
It has been well documented that the macrophage is a predominant cell in the repair of a peritoneal injury, implicating it in a central role at directing the events of peritoneal healing (3). This role probably is mediated via the production of cytokines, one of the most important of which known to be produced by macrophages is IL-10'. Interleukin Vol. 61, No.6, June 1994
10' has been noted to promote adhesion formation in a murine model (5). Interleukin 10 could, through the suppression of the production of IL-1 by macrophages, limit IL-1 activity and therefore adhesion formation would be curtailed. In addition, IL-10 may exert an antagonizing effect on IL-1 activity by inducing the expression of IL-1 receptor antagonists in macrophages (24). Direct Suppression of the Th-l Cells
As described above, one of the most remarkable functions of IL-10 is its ability to limit the capacity of the Th-1 cells to produce IL-2 and IFN-')'. Interleukin 2 has been demonstrated to potentiate adhesion formation via stimulation of TGF -~ and PDGF-B production (6). Both of these growth factors are highly fibrogenic cytokines. Thus, an IL10-mediated reduction of IL-2 production indirectly may reduce fibroblast stimulation and equate to a limiting of collagenous adhesion formation. Indirect Suppression of Macrophage Function
Interleukin 2 and IFN -')' both directly stimulate macrophage activity via paracrine regulatory mechanisms. Interleukin 10 could, through inhibition of IL-2 and IFN-')' production by the Th-1 cells, reduce macrophage activity via an indirect effect. Similarly, as described above, if macrophage activity is suppressed, it would be reasonable to predict a decrease in adhesion formation. One animal in each of our two study groups that had undergone surgery and received IL-10 died in the postoperative period. The cause of these fatal outcomes in not known yet appeared to be related to an inadvertent overdose of halothane anesthesia given before each intraperitoneal injection rather than IL-10. However, in vivo toxicity studies will be a most important next step in the evaluation of IL10 to assure its safety as an adhesion-preventing agent. The finding that IL-10 is able to limit postoperative intraperitoneal adhesion formation is a potent stimulus for further research. First, the necessity of defining the specific molecular biology response to peritoneal injury is emphasized. Further understanding of what transpires in the "normal" and "abnormal" mesothelial repair process will allow us to make rational decisions regarding potential modifications of the peritoneal response to injury and ways to prevent adhesions. Second, with the availability of recombinant technologies, should IL-10 prove to be a potent adhesionpreventing agent at doses that are well tolerated by Montz et al.
Interleukin 10 and adhesion prevention
1139
the patient, the potential to limit significantly the postsurgical adhesion formation and associated morbidity, mortality, and cost to the health care system would be immense. Acknowledgement. The authors thank Peter D. Christenson, Ph.D., Department of Biomathematics, University of California, Los Angeles, for the statistical analysis.
REFERENCES 1. Ray NF, Larsen JW Jr, Stillman RJ. Economic impact of hospitalizations for lower abdominal adhesiolysis in the United States in 1988. Surg Gynecol Obstet 1993;176:271-6. 2. Strangel JJ, Nisbet JD II, Settles H. Formation and prevention of postoperative abdominal adhesions. J Reprod Med 1984;29:143-56. 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. 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. 5. Hershlag A, Otterness IG, Bliven ML, Diamond MP, Polan ML. The effect of interleukin-1 on adhesion formation in the rat. Am J Obstet GynecoI1991;165:771-4. 6. Kovacs EJ, Brock B, Silber IE, Neuman JE. Production of fibrinogenic cytokines by Interleukin-2-treated peripheral blood leukocytes: expression of TGF -iJ and PDGF B chain genes. Obstet Gynecol 1993;82:29-36. 7. Fiorentino DF, Bond MW, Mossman TR. Two types of mouse T helper cell IV. TH2 clones secrete a factor that inhibits cytokine production by TH1 clones. J Exp Med 1989;170:2081-95. 8. Taga K, Tosato G. IL-10 inhibits human T cell proliferation and IL-2 production. J ImmunoI1992;148:1143-8. 9. Menzies D. Peritoneal adhesions: incidence, cause, and prevention. Surg Annu 1992;1:27-45. 10. Menzies D, Ellis H. Intestinal obstruction from adhesions: how big is the problem? Ann R Coli Surg EngI1990;72:60-4. 11. Montz FJ, Holschneider CH, Solh S, Schuricht LC, Monk BJ. Small bowel obstruction following radical hysterectomy: risk factors, incidence and operative findings. Gynecol Oncol. In press.
1140
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Interleukin 10 and adhesion prevention
12. Montz FJ, Fowler JM, Wolff AJ, Lacey SM, Mohler M. The ability of recombinant tissue plasminogen activator to inhibit post radical pelvic surgery adhesions in the dog model. Am J Obstet Gynecol 1991;165:1539-42. 13. Montz FJ, Monk BJ, Lacey SM, Fowler JM. Ketorolac tromethamine, a nonsteroidal anti-inflammatory drug: ability to inhibit post-radical pelvic surgery adhesions in a procine model. Gynecol Oncol 1993;48:76-9. 14. Moore KW, Vieira P, Fiorentino DF, Trounstine ML, Khan TA, Mosmann TR. Homology of cytokines synthesis inhibitory factor (lL-10) to the Epstein Barr virus gene BCRF!. Science 1990;248:1230-4. 15. Fiorentino DF, Zlotnik A, Vieira P, Mosmann TR, Howard M, Moore KW, et al. IL-lO acts on the antigen presenting cell to inhibit cytokine production TH1 cells. J Immunol 1991;146:3444-51. 16. Mosmann TR, Moore KW. The role of1L-10 incrossregulation of TH1 and TH2 responses. Immunol Today 1991;12:A49-A53. 17. Zlotnik A, Moore KW. Interleukin-10. Cytokines 1991; 3:366-71. 18. 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. 19. Gerard C, Bruyns C, Marchant A, Abramowicz D, Vandenabeele P, Delvaux A, et al. IL-10 reduces the release of TNF and prevents lethality in experimental endotoxemia. J Exp Med 1993;177:547-50. 20. Heinzel FP, Sadick MD, Mutha SS, Locksley RM. Production of interferon 'Y, interleukin-2, interleukin-4 and interleukin-10 by CD4+ and lymphocytes in vivo during healing and progressive murine leishmaniasis. Proc Natl Acad Sci USA 1991;88:7011-5. 21. Silva JS, Morrissey PJ, Grabstein KH, Mohler KM, Anderson D, Reed SG. Interleukin 10 and interferon 'Y regulation of experimental trypanosoma cruzi infection. J Exp Med 1992;175:169-74. 22. Rousset F, Garcia E, Defrance T, Peronne C, Vezzio N, Hsu DH, et al. Interleukin 10 is a potent growth and differentiation factor for activated human B-lymphocytes. Proc Natl Acad Sci USA 1992;89:1890-3. 23. Defrance T, Vanbervliet B, Briere F, Durand I, Rousset F, Banchereau J. Interleukin 10 and transforming growth factor iJ cooperate to induce anti CD40 activated naive human B-cells to secrete immunoglobulin A. J Exp Med 1992;175:671-82. 24. Howard M, O'Garra A. Biological properties of interleukin 10. Immunol Today 1992;13:198-200.
Fertility and Sterility
FERTILITY AND STERILITY Copyright
@
Printed on acid-free paper in U. S. A.
1994 The American Fertility Society
Interleukin 10: ability to minimize postoperative intraperitoneal adhesion formation in a murine model*
Fredrick J. Montz, M.D.t Christine H. Holschneider, M.D. Michael Bozuk, B.S.
Walter H. Gotlieb, M.D., Ph.D. Dto Martinez-Maza, Ph.D.
Gynecologic Oncology Service, Department of Obstetrics and Gynecology, University of California at Los Angeles (UCLA) Center for Health Sciences, Los Angeles, California
Objective: To determine the ability ofinterleukin 10 (IL-10) to suppress postoperative intraperitoneal adhesion formation. Design: Randomized, controlled trial. Setting: University animal research facility. Animals: Six -week -old Swiss Webster mice undergoing a standardized intraperitoneal operative procedure. Interventions: Animals were randomized to "surgery" or "no surgery" and then further randomized to receive intraperitoneal injections of 1 mL phosphate-buffered saline (PBS) or 1 Ilg/kg IL-lO in 1 mL PBS. Vehicle-only doses were given immediately after surgery and then every 24 hours for a total of four injections. Interleukin 10 injections were similarly given but with an added preoperative injection 30 minutes before surgery in one half of the animals. Main Outcome Measure: Adhesion formation. Results: Animals treated with vehicle or IL-10 but not undergoing surgical intervention had no intraperitoneal adhesions. Animals undergoing surgery who were treated with IL-lO, with or without a preoperative dose, had significantly lower postoperative adhesion scores than did control animals who postoperatively received PBS only. Conclusion: Interleukin-lO is effective at limiting postoperative intraperitoneal adhesion formation with minimal evident systemic side effects. Fertil Steril1994;61:1136-40 Key Words: Intraperitoneal adhesions, postoperative adhesion prevention, cytokines, interleukin 10, IL-10, CSIF, murine model
Intraperitoneal adhesions are a major source of patient morbidity and mortality as well as a substantial drain on limited health care resources. In 1988 alone there were approximately 282,000 hospitalizations in the United States during which lower abdominal adhesiolysis was performed, accounting for 948,727 days of inpatient care. These hospital-
Received July 2, 1993; revised and accepted
* Supported by the Bertha Warshaver Rubin Research Fund, Los Angeles, California. t Reprint requests: Fredrick J. Montz, M.D., Department of Obstetrics and Gynecology, UCLA, CRS 24-127, lO833 Le Conte Avenue, Los Angeles, California 90024 (FAX: 310-2063670). 1136
Montz et at.
Interleukin 10 and adhesion prevention
izations were responsible for an estimated $1.18 billion in expenditures, an estimate that does not include outpatient and indirect costs due to loss of productivity or decreased consumer spending (1). It is acknowledged widely that the vast majority of intraperitoneal adhesions that necessitate adhesiolysis represent sequelae from prior surgery (2). Though the serial cellular events that transpire after a peritoneal injury have been defined thoroughly (3), the exact role of cytokines in peritoneal repair and adhesion formation is yet to be elucidated completely. Preliminary data that state numerous cytokines such as IL-1 (4, 5) as well as IL-2, transforming growth factor {3 (TGF-{3), and platelet-derived growth factor B (PDGF-B) (6) are Fertility and Sterility
potentiators of postoperative adhesion formation exist. We proposed to evaluate the ability of a naturally occurring cytokine synthesis inhibiting factor (IL-10) (7,8) to suppress postsurgical adhesion formation in a well-standardized animal model. MATERIALS AND METHODS
After obtaining approval from the Animal Research Committee at the University of California at Los Angeles (UCLA), 60 six-week-old female Swiss Webster mice (Simonsen Laboratories, Inc., Gilroy, CA) were used for our investigations. The animals were housed at the UCLA Vivarium. All animal procedures were performed in accordance with the standards described in the National Institute of Health Guide for the Care and Use of Laboratory Animals, in compliance with the Federal Animal Welfare Act. Before surgery, the animals were allowed access to water and chow ad libidum. Anesthesia was induced with halothane inhalation. Animals were then injected with 30 mg/kg SC pentobarbital. Adequate anesthesia was maintained as necessary with halothane via a cotton ball. A sharp midline incision was made extending 2 cm from the symphysis to the upper abdomen. Intraperitoneal exploration was performed and any adhesions present were quantified. Subsequently, a standardized left lower abdominal wall injury was produced using sharp abrasion so as to induce microhemorrhage and peritoneal disruption over a 2 cm z area. The anterior abdominal wall was closed using a running 3-0 Maxon suture (American Cyanamid Company, Danbury, CT) for the peritoneum-fascia-muscle in an en bloc technique and the skin was closed using a running 3-0 silk suture. Animals were randomized into six separate groups. Group I underwent no surgery but received 1 mL of endotoxin-free phosphate-buffered saline (PBS) administered transabdominally into the peritoneal cavity using aseptic technique at time 0 and then 24, 48, and 72 hours thereafter. Group II also did not undergo surgery and received 1 JIg/kg recombinant IL-10 (Peprotec, Rocky Hill, NJ) in 1 mL IP PBS in the time schedule described above. This dose of IL-10 was chosen to lie well within the range of biologic activity of IL-lO, which is reported by the manufacturer to be between 0.2 and 20 ng/mL. Group III underwent surgery only and received no postoperative injections. Group IV underwent surgery and IL-10 treatment in the same manner as group II, with the first administration being immediately after closure of the anterior abdominal wall. Vol. 61, No.6, June 1994
Table 1
Adhesion Scoring* Adhesion score
Extent None Confined to traumatized area Confined to nontraumatized area Intraumatized and nontraumatized areas Type Filmy Opaque, nonvascular Opaque, vascular Dense
* Modified from
o 1 2
3 1 X extent = 2 X extent = 3 X extent = 4 X extent = sum =
Hershlag et al. (5).
Group V received 1 JIg/kg IL-lO in 1 mL PBS administered percutaneously into the peritoneal cavity 30 minutes before surgery and postoperatively received the same regimen as group IV. Group VI underwent surgery and then received intra-abdominal vehicle alone in a manner similar to group I, with the first administration given immediately after closure of the anterior abdominal wall. After recovery from anesthesia, animals were allowed immediate and unlimited access to water and chow. To minimize suffering of the animals during any subsequent intraperitoneal injections, short anesthesia with halothane was given immediately before injection. On postoperative day 7, the animals were killed using carbon dioxide euthanasia, explored, and adhesions quantified using a modification of the method of Hershlag and associates (5). This technique quantifies adhesions by extent and type to obtain a composite adhesion score for a given animal (Table 1). To minimize investigator bias, all animals that received postoperative injections of IL-10 or placebo were operated first and then randomized to different regimen groups. All adhesions were scored by the same investigator, who was blinded regarding the specific injection regimen. However, it was impossible to achieve blinding as to whether the animal was operated on because the incision was always visible. Statistical analysis was performed using the SAS statistical package (SAS Institute, Inc., Cary, NC). Adhesion scores were transformed with natural logarithms before statistical analysis to account for heterogeneous variance among groups. Following standard statistical practice, 0.5 was added to all values before transformation to account for zero values. This log transformation implies that group comparisons should be discussed as relative rather than absolute differences. As a result of the log Montz et al.
Interleukin 10 and adhesion prevention
1137
differ significantly in mean adhesion scores nor did groups III and VI. Based on statistical power calculations, eight animals per group are needed to detect a fourfold difference in adhesion scores. Thus, we are fairly certain that this study population had adequate numbers so as not to miss detecting differences between groups III and VI and IV and V on the order of magnitude of the apparent effect of IL-I0 itself.
transformation, group means are actually geometric means (inverse transformed values of the means or the log scale) and confidence intervals are reported rather than standard deviations because variation around the mean is not symmetrical. One-way analysis of variance with Tukey's pairwise comparison method was used to compare mean adhesion scores among groups. Alternatively, a nonparametric analysis using Wilcoxon's test or a pairwise t-test followed by Bonferroni adjustments were used. Regardless of the statistical tool used, the results obtained showed the same statistical significance.
DISCUSSION
It is a widely accepted fact that postoperative adhesions are a common cause of morbidity and potential mortality in patients undergoing intra-abdominal surgery in general (9, 10) and radical pelvic surgery in particular (11). Numerous adjuvants have been used throughout the history of surgery in an attempt to minimize the formation of such adhesions. The rationale for using the majority of these agents has been empirical, with little understanding of what transpires at a molecular level in normal and aberrant peritoneal healing. Recently, agents have been investigated that reflect our slowly expanding knowledge ofthe molecular and biochemical events of peritoneal healing (12,13). Further investigations must be undertaken to elucidate and modulate the basic mechanisms of mesothelial repair and thereby lead to rational drug design and therapy development. When reflecting on ways that the native peritoneal environment can be manipulated at a molecular level so as to limit adhesion formation, a logical novel tack would be to attempt to modify the cytokine response to a peritoneal injury. This could be aimed at suppression of cytokines, which have been demonstrated to promote adhesiogenesis, or at augmentation of cytokines inhibitory for adhesion formation. Although our understanding ofthe quantitative and temporal order of the cytokine response to a peritoneal injury
RESULTS
In no cases were intraperitoneal adhesions evident at the time ofthe initial laparotomy. No abnormalities of healing of the anterior abdominal wall incision such as wound disruption or infection were noted in any of the animal groups that had undergone surgery regardless of which study regimen they were assigned. One animal from each of the two groups that had undergone surgery and received IL-I0 died in the postoperative period. There were no adhesions observed at posteuthanasia autopsy in mice that had not undergone surgery but had been injected only with PBS with (group II; mean adhesion score = 0) or without IL10 (group I; mean adhesion score = 0). Remarkably, at time of exploration, postoperative adhesions were found universally in animals that were injected either with PBS only or received no postoperative intraperitoneal injections, while being noted to a lesser extent and degree in the majority ofthe IL-lO-treated animals (Table 2). Both groups receiving IL-I0 (IV, V) had significantly lower mean adhesion scores than both control groups (III, VI), (1.1 and 1.2 versus 4.3 and 4.2, respectively; P < 0.05). Groups IV and V did not
Table 2
Adhesion Scores* Group
No. of adhesions
III (surgery only):j: VI (surgery + postoperative PBS) IV (surgery + postoperative IL-10)§ V (surgery + IL-10 preoperative + postoperative)
10 10 9 9
* The overall t-test P value for the analysis of variance is = 0.002. t Groups III and VI have significantly greater mean adhesion scores than groups IV and V.
P
1138
Montz et al.
Interleukin 10 and adhesion prevention
Range (2 to (1 to (0 to (0 to
10) 10) 3) 6)
Geometric meant
95% confidence
interval 2.9 2.3 0.37 0.26
4.3 4.2 1.1
1.2
to to to to
6.3 7.5 2.3 3.3
:j: Groups III and VI do not differ significantly. § Groups IV and V do not differ significantly.
Fertility and Sterility
is infantile, it appears that IL-10' (4, 5), IL-2, PDGF, and TGF-~ (6) all act to potentiate adhesion formation. The extent of postoperative adhesion formation is probably a product of enhanced deposition of fibrin as well as cytokine-mediated stimulation of fibroblast proliferation and collagen production. It is our opinion that the most rational choice for an agent to be tested to suppress the adhesiogenic effect of the above described cytokines is IL-10. Interleukin 10 has been identified recently and its gene cloned based on its ability to inhibit cytokine synthesis (14). It is produced by the type 2 CD4+ helper T cell clones (Th-2) and inhibits the cytokine production of the type 1 (Th-1) subset in murine systems (15). Th-1 and Th-2 are the two subpopulations of T helper cells that control the nature of the immune response by secreting what are considered characteristic and mutually antagonistic sets of cytokines (16). Th-1 cells specifically produce IL-2 and interferon-,), (IFN-')') whereas Th-2 cells produce IL-4, IL-5, IL-6, and IL-10. Interleukin 10 appears to inhibit the production ofIL-2 and IFN -')' by the Th -1 cells (15) as well as the proliferation of human peripheral blood T cells and the synthesis of IFN -')' and other cytokines by stimulated peripheral blood monocytes (8, 17). It also has been demonstrated that IL-10 inhibits the production of IL-1, TNF-O', and IL-6 by lipopolysaccharide-activated macrophages (18). This anti -T cell-anticytokine-antimacrophage activity has been used in animal models in attempts to modulate the inflammatory response and associated pathological disease processes such as endotoxic shock (19) or parasitic infections (20, 21). In addition to these inhibitory activities, it has been noted that IL-10 can act to stimulate B cell growth and differentiation (22) as well as to serve as a costimulatory agent with other cytokines (23). It is exciting to speculate as to the potential mechanisms by which IL-10 limits the formation of postoperative peritoneal adhesions. It is probable that this phenomenon is a product of multiple events: Direct Effects on Macrophages
It has been well documented that the macrophage is a predominant cell in the repair of a peritoneal injury, implicating it in a central role at directing the events of peritoneal healing (3). This role probably is mediated via the production of cytokines, one of the most important of which known to be produced by macrophages is IL-10'. Interleukin Vol. 61, No.6, June 1994
10' has been noted to promote adhesion formation in a murine model (5). Interleukin 10 could, through the suppression of the production of IL-1 by macrophages, limit IL-1 activity and therefore adhesion formation would be curtailed. In addition, IL-10 may exert an antagonizing effect on IL-1 activity by inducing the expression of IL-1 receptor antagonists in macrophages (24). Direct Suppression of the Th-l Cells
As described above, one of the most remarkable functions of IL-10 is its ability to limit the capacity of the Th-1 cells to produce IL-2 and IFN-')'. Interleukin 2 has been demonstrated to potentiate adhesion formation via stimulation of TGF -~ and PDGF-B production (6). Both of these growth factors are highly fibrogenic cytokines. Thus, an IL10-mediated reduction of IL-2 production indirectly may reduce fibroblast stimulation and equate to a limiting of collagenous adhesion formation. Indirect Suppression of Macrophage Function
Interleukin 2 and IFN -')' both directly stimulate macrophage activity via paracrine regulatory mechanisms. Interleukin 10 could, through inhibition of IL-2 and IFN-')' production by the Th-1 cells, reduce macrophage activity via an indirect effect. Similarly, as described above, if macrophage activity is suppressed, it would be reasonable to predict a decrease in adhesion formation. One animal in each of our two study groups that had undergone surgery and received IL-10 died in the postoperative period. The cause of these fatal outcomes in not known yet appeared to be related to an inadvertent overdose of halothane anesthesia given before each intraperitoneal injection rather than IL-10. However, in vivo toxicity studies will be a most important next step in the evaluation of IL10 to assure its safety as an adhesion-preventing agent. The finding that IL-10 is able to limit postoperative intraperitoneal adhesion formation is a potent stimulus for further research. First, the necessity of defining the specific molecular biology response to peritoneal injury is emphasized. Further understanding of what transpires in the "normal" and "abnormal" mesothelial repair process will allow us to make rational decisions regarding potential modifications of the peritoneal response to injury and ways to prevent adhesions. Second, with the availability of recombinant technologies, should IL-10 prove to be a potent adhesionpreventing agent at doses that are well tolerated by Montz et al.
Interleukin 10 and adhesion prevention
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the patient, the potential to limit significantly the postsurgical adhesion formation and associated morbidity, mortality, and cost to the health care system would be immense. Acknowledgement. The authors thank Peter D. Christenson, Ph.D., Department of Biomathematics, University of California, Los Angeles, for the statistical analysis.
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12. Montz FJ, Fowler JM, Wolff AJ, Lacey SM, Mohler M. The ability of recombinant tissue plasminogen activator to inhibit post radical pelvic surgery adhesions in the dog model. Am J Obstet Gynecol 1991;165:1539-42. 13. Montz FJ, Monk BJ, Lacey SM, Fowler JM. Ketorolac tromethamine, a nonsteroidal anti-inflammatory drug: ability to inhibit post-radical pelvic surgery adhesions in a procine model. Gynecol Oncol 1993;48:76-9. 14. Moore KW, Vieira P, Fiorentino DF, Trounstine ML, Khan TA, Mosmann TR. Homology of cytokines synthesis inhibitory factor (lL-10) to the Epstein Barr virus gene BCRF!. Science 1990;248:1230-4. 15. Fiorentino DF, Zlotnik A, Vieira P, Mosmann TR, Howard M, Moore KW, et al. IL-lO acts on the antigen presenting cell to inhibit cytokine production TH1 cells. J Immunol 1991;146:3444-51. 16. Mosmann TR, Moore KW. The role of1L-10 incrossregulation of TH1 and TH2 responses. Immunol Today 1991;12:A49-A53. 17. Zlotnik A, Moore KW. Interleukin-10. Cytokines 1991; 3:366-71. 18. 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. 19. Gerard C, Bruyns C, Marchant A, Abramowicz D, Vandenabeele P, Delvaux A, et al. IL-10 reduces the release of TNF and prevents lethality in experimental endotoxemia. J Exp Med 1993;177:547-50. 20. Heinzel FP, Sadick MD, Mutha SS, Locksley RM. Production of interferon 'Y, interleukin-2, interleukin-4 and interleukin-10 by CD4+ and lymphocytes in vivo during healing and progressive murine leishmaniasis. Proc Natl Acad Sci USA 1991;88:7011-5. 21. Silva JS, Morrissey PJ, Grabstein KH, Mohler KM, Anderson D, Reed SG. Interleukin 10 and interferon 'Y regulation of experimental trypanosoma cruzi infection. J Exp Med 1992;175:169-74. 22. Rousset F, Garcia E, Defrance T, Peronne C, Vezzio N, Hsu DH, et al. Interleukin 10 is a potent growth and differentiation factor for activated human B-lymphocytes. Proc Natl Acad Sci USA 1992;89:1890-3. 23. Defrance T, Vanbervliet B, Briere F, Durand I, Rousset F, Banchereau J. Interleukin 10 and transforming growth factor iJ cooperate to induce anti CD40 activated naive human B-cells to secrete immunoglobulin A. J Exp Med 1992;175:671-82. 24. Howard M, O'Garra A. Biological properties of interleukin 10. Immunol Today 1992;13:198-200.
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