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Interaction of Interceed oxidized regenerated cellulose with macrophages: A potential mechanism by which Interceed may prevent adhesions
Interaction of Interceed oxidized regenerated cellulose with macrophages: A potential mechanism by which Interceed may prevent adhesions Sujatha Reddy, MD, Nalini Santanam, PhD, P. Pravin Reddy, MD, John A. Rock, MD, Aria A. Murphy, MD, and Sampath Parthasarathy, PhD Atlanta, Georgia OBJECTIVE: The objective of the study was to determine whether Interceed oxidized regenerated
cellulose (Johnson & Johnson Medical, Arlington, Tex.), because of its polyanionic nature, may compete for the macrophage scavenger receptor. STUDY DESIGN: RAW macrophages were incubated with Interceed oxidized regenerated cellulose and known scavenger receptor ligands. The production of interleukin-113 by mouse peritoneal macrophages was measured in the presence of Interceed cellulose. RESULTS" When macrophages were incubated with Interceed cellulose, increasing concentrations inhibited the uptake of fluorescent acety[ low-density lipoprotein. In the presence of Interceed cellulose there was a decrease in the production of interleukin-113 by mouse macrophages. CONCLUSION: These results suggest that the interaction of Interceed oxidized regenerated cellulose with macrophages with scavenger receptors may result in a decreased secretion of matrix components, inflammatory mediators, and cellular growth factors. Thus Interceed cellulose may function as a biologic barrier in preventing adhesions. (Am J Obstet Gynecol 1997;177:1315-21 .)
Key words: Scavenger receptor, solubilized Interceed oxidized regenerated cellulose, po!yanion, acetyl low-density lipoprotein, interleukin-1
Postoperative adhesion formation results from the fibroproliferative end products of the inflammatory process. Macrophages are crucial to this process. Approximately 3% of all laparotomies are performed for adhesion-induced intestinal obstructions. ~'2 A great deal of research has been directed toward the reduction and prevention of adhesions because of the morbidity and cost associated with adhesions. In 1988 the cost of abdominopelvic adhesiolysis was ~/1179.9 million.3 Recently, a n u m b e r of products, such as Interceed oxidized regenerated cellulose (Johnson & Johnson Medical, Arlington, Tex.), for preventing adhesions intraoperatively have become available.< 5 Interceed cellulose has been shown to decrease postoperative adhesions. < 7 The exact mechanism of action of Interceed cellulose is unknown, but it i s felt that it functions as a physical barrier separating opposing surfaces. 8 However, Interceed cellulose rapidly dissolves from its mesh form into a soft gelatinous mass in vivo. This occurs in about 2 days. Therefore it is difficult to attribute the efficacy solely to its From the Department of Obstetrics and @necology, EmoU University. Second Prize Presidential Resident~Fellow Paper; presented at the Twenty-third Annual Meeting of the ,Society of Gynecologic Surgeons, New Orle,ans, Louisiana, FebruaU 24-26, 199Z Reprint requests: Sampath Parthasarathy, PhD, Department of Obstetrics and Gynecology, Emo~3~Universiiy, Atlanta, GA 30322. Copyright © 1997 by Mosby-Year Book, Inc. 0002-9378/97 $5.00 + 0 6/6/85176
ability to act as a physical barrier, suggesting it may also have biologic activity that may account for its ability to prevent adhesions. Adhesion formation occurs as a result of the inflammation, granulation, and scar formation that follow trauma. A variety of stimuli are known to induce injury and adhesions. These include surgical trauma, infection, ischemia, and radiation. After injury and the inflammatory reaction, fibrin arrives at the site of injury through exudation. Fibrin acts as a g]ue between structures. If the fibrin is not removed through fibrinolytic action, the temporary adhesions become p e r m a n e n t fibrous adhesions. Through granulation, macrophages and fibroblasts invade the fibrin network, and collagen and other connective tissue elements are laid down. Macrophages present in the h u m a n peritoneal cavity are potent mediators of inflammation. They are capable of generating inflammatory cytokines and growth factors for fibroblasts. They also are intricately involved in the production and dissolution of extracellular matrix components such as collagen. In this study we tested the ability of Interceed cellulose to compete for the macrophage scavenger receptor. The attachment of macrophages to matrix components by way of its scavenger receptor(s) has been a topic of novel interest.9, 10 Macrophage scavenger receptors bind and internalize a n u m b e r of polyanionic molecules such as 1315
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acetylated low-density lipoproteins (LDL), fucoidin, polyinosinic acid, and oxidized LDL. 11' 12 These ligands and their components are potent activators of the monocyte-macrophage inflammatory process and induce the synthesis and secretion of cytokines such as interleukin-l]3 (IL-I[3)} 316
Material and methods Interceed oxidized regenerated cellulose was obtained from Johnson & Johnson Medical. DiI (3,3'-dioctadecylindocarbocyanine) was obtained from Molecular Probes, Plano, Texas. RPMI 1640 medium was purchased from Mediatech, Herndon, Virginia. 1-Carbon-14-1abeled oleic acid was purchased from New England Nuclear, Boston. Preparation of LDL and aeetylated LDL. Blood was collected from the healthy donor and LDL was prepared from the plasma by ultracentrifugation as described in previous publications}7 DiI-labeled acetylated LDL was prepared as described by Pitas et al} s Solubilization of Interceed cellulose. Approximately 100 mg of Interceed mesh was treated with ]00 Ixl of a m m o n i u m hydroxide in 2 ml of phosphate-buffered saline solution. The solution was vigorously mixed with mild warming. Interceed mesh readily goes into solution u n d e r these conditions. The control tube contained only a m m o n i u m hydroxide. Nitrogen gas was gently bubbled through the tubes to remove excess ammonia. The pH of the solution was neutral. Interceed and control solutions were filter sterilized by passage through a 0.45 Ixm filter. Fluorescence measurement. RAW macrophages were cultured on 24-well plates. The cells were washed and then incubated with increasing concentrations of solubilized Interceed cellulose (0 to 100 ~g per well) in the presence of fluorescent DiI-acetylated LDL (5 ~g) for 48 hours. At the end of incubation the cells were washed, and the fluorescence was visualized with use of the Nikon fluorescent microscope. The fluorescent material from the cells was then extracted with methanol and quantitated with use of a Shimadzu Spectro Fluorometer (excitation 590 nm, emission 570 nm). is 14C-labeled oleate incorporation. To determine the uptake and degradation of acetyl LDL by cells, the incorporation of 14C-labeled oleate into cellular cholesteryl esters was measured in the presence of solubilized Interceed cellulose. RAW macrophages were incubated with 25 p~g of ace@ LDL and 100 nmol of 14C-labeled oleate (9000 disintegrations/min/nmol) in the presence of 125 ~g of solubilized Interceed cellulose for 24 hours at 37 ° C. After the cells were washed, cellular lipids were exn-acted and separated by thin-layer chromatography. Spots corresponding to cholesteryl esters were visualized and the radioactivity was determined. 19 Interleukin-1 (IL-1) production. Mouse peritoneal macrophages were harvested 19 and plated in culture
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dishes at 2 × 105 cells per well in RPMI 1640 medium containing 10% fetal calf serum. After 24 hours cells were washed and incubated with 100 txg of solubilized Interceed cellulose in the presence or absence of either 10 nmol of 13-hydroperoxy linoleate (generated by the action of soybean lipoxygenase on linoleic acid) or 25 peg of oxidized LDL or 1 txg of lipopolysaccharide (known stimulants of IL-1 production). Cells were incubated for 24 hours and the amount of IL-113 released into the culture supernatant was quantified with use of a commercial kit for mouse IL-1[3 (Intertest-l[3X, available from Genzyme Diagnostics, Cambridge, Mass.). The commercial kit is a solid-phase enzyme-linked immunosorbent assay (ELISA) kit that uses the multiple-antibody sandwich principle. Briefly, 100 Ixl of the culture supernatants was added to each well in the 96-well microtiter plate precoated with monoclonal antimouse IL-113 and incubated for 1 hour. After the u n b o u n d material was washed, biotinylated polyclonal antimonoclonal IL-l[3, which binds to the captured monoclonal IL-l[3 was added. After incubation and washing, the polyclonal antibody was removed and the streptavidin conjugated to horseradish peroxidase was added to the wells and incubated. The plate was washed again and then incubated with the substrate solution, which initiated a peroxidase-catalyzed color change that was measured at an absorbance of 450 n m with use of an ELISA plate reader. The concentration of the unknown antigen is quantitated with use of the standard curve obtained from known standards provided by the manufacturer.
Results RAW macrophages and the cell-free control wells were incubated in 24-well dishes with 25 mm z pieces of Interceed cellulose for 24 hours. Microscopic examination showed that Interceed cellulose remained intact in cell-free control incubations (Fig. 1, A). In contrast, most of Interceed cellulose in incubations that contained RAW cells was present as partly digested materials or as completely dissolved solutions (Fig. 1, B). No evidence of toxicity could be seen u n d e r microscopy. In fact, during the incubation attachment of macrophages to Interceed fibers could be seen, which eventually dissolved to form a clear solution. This observation suggests that macrophages may digest Interceed fibers. However, in these studies it was not possible to determine whether Interceed cellulose was taken up by the cells or remained outside in the medium. Because Interceed cellulose appeared to function in its dissolved form, we solubilized it as described in the Material and methods section. We tested the ability of solubilized Interceed cellulose to affect the uptake and internalization of acetyl LDL, a ligand for the macrophage scavenger receptor} 9 When RAW macrophages were incubated with fluorescent DiI ace@ LDL, the cells took up the acetylated LDL as
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Fig. 1. RAW macrophages and no-cell controls were cultured in 24-well dishes in presence and absence of 5 mm 2 pieces of Interceed cellulose. After 24-hour incubation period, cells were observed with Nikon light microscope. A, Interceed cellulose, in no-ceil control well. Fibers remained intact after 24 hours. B, Interceed cellulose, when incubated with RAW macrophages, appeared either partly digested or completely dissolved. Macrophages can be seen aligning along Interceed fiber.
observed by increased fluorescence u n d e r the fluoresc e n t microscope. Cytoplasmic droplets of fluorescent material could be seen (Fig. 2, A). W h e n m a c r o p h a g e s were incubated with a c e @ LDL and increasing concentrations o f solubilized I n t e r c e e d cellulose, it was observed that there was a d o s e - d e p e n d e n t decrease in the fluorescence, suggesting that the I n t e r c e e d cellulose c o m p e t e d with the acetyl LDL for the scavenger receptor. Fig. 2, B, shows the almost c o m p l e t e absence of fluorescence w h e n cells were incubated with 2.5 txg of fluorescent acetyl LDL in the p r e s e n c e of 100 txg of solubilized I n t e r c e e d cellulose. Control incubations with an equal a m o u n t of a m m o n i u m hydroxide solution c o r r e s p o n d i n g to the I n t e r c e e d c o n c e n t r a t i o n did n o t decrease the fluorescence, suggesting that it was n o t the a m m o n i a but the
I n t e r c e e d cellulose that decreased the fluorescence. W h e n the fluorescent material was extracted with methanol and quantified, there was a d o s e - d e p e n d e n t decrease in cell-associated fluorescence in the presence of I n t e r c e e d cellulose (Table I). As a measure of scavenger r e c e p t o r activity, RAW macrophages were incubated with 25 b~g acetyl LDL in the presence or absence of soluble I n t e r c e e d cellulose (125 b~g) and 100 n m o l of 14C-labeled oleate. T h e radioactive oleate i n c o r p o r a t e d into the cholesterol esters was as shown in Table II. It was observed that I n t e r c e e d cellulose decreased the oleate i n c o r p o r a t i o n by 42% c o m p a r e d with the control. In the presence of I n t e r c e e d cellulose the thioglycol a t e q n d u c e d mouse peritoneal m a c r o p h a g e s p r o d u c e d less IL-113 c o m p a r e d with the control (Table III). Inter-
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A
B Fig. 2. RAW macrophages cultured in 24-well dishes were incubated with and without Interceed cellulose in presence of fluorescent Dil ace~l LDL (as described in Material and methods section) for 24 to 48 hours. A, Control cells had increased fluorescence as seen under fluorescent microscope. B, Cells incubated with Interceed cellulose had a dose-dependent decrease in fluorescence.
T a b l e I. A m o u n t of f l u o r e s c e n c e e x t r a c t e d f r o m DiI-labeled acetyl LDL RAW m a c r o p h a g e s
Cellsample Control TC-7 10 Ixg TC-7 20 Ixg TC-7 50 >g TC-7 100 ixg Ammonium hydroxide
l
bTuorescenceunits 162.4 lt2.0 55.0 44.0 30.0 187.0
Raw macrophages cultured in 24-well plates were incubated with 2.5 Ixg/ml DiI-acetyl LDL in presence or absence of increasing concentrations of Interceed cellulose (0 to 100 p,g). After 24 hours' incubation, amount of fluorescent-labeled acetyl LDL taken up by macrophages was quantitated after extraction with methanol and measured on a spectrofluorometer (Ex 520 nm and Em 570 nm). Table I is representative average of two individual experiments.
ceed cellulose s e e m e d to d e c r e a s e the IL-I[3 p r o d u c t i o n s t i m u l a t e d by lipopolysaccharide, as seen by lower a m o u n t s of IL-1[3 r e l e a s e d into the m e d i u m . This suggests t h a t I n t e r c e e d cellulose may b e able to r e d u c e t h e inflammatow response of the macrophages.
Comment I n t e r c e e d oxidized r e g e n e r a t e d cellulose is a n a d h e sion b a r r i e r a p p r o v e d for use to p r e v e n t p o s t o p e r a t i v e adhesions. Its m e c h a n i s m o f action is u n k n o w n , b u t it has b e e n t h o u g h t to f u n c t i o n as a physical barrier. It has b e e n o b s e r v e d in a n i m a l studies t h a t t h e r e is a c o m p l e t e dissolution o f I n t e r c e e d cellulose in t h e p e r i t o n e a l cavity after 1 to 2 days b u t it c o n t i n u e s to p r e v e n t a d h e sions.20.2~ This o b s e r v a t i o n suggests t h a t I n t e r c e e d cellulose has biologic activity. W o m e n with e n d o m e t r i o s i s are
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Table II. I n t e r c e e d cellulose inhibits i n c o r p o r a t i o n of I4C-labeled oleate into m a c r o p h a g e cholesterol esters in p r e s e n c e of acetyl LDL Incubation conditions Acetyl LDL (25 ~xg/ml) Acetyl LDL (25 ~xg/ml) plus Interceed cellulose (125 p~g/ml)
14C-labeled oleate incorporated in cholesterol ester (nmol) 7.2 4.2
RAW macrophages were cultured in 6-well plates and incubated with acetyl LDL (25 b~g/ml) with and without Interceed cellulose (125 Ixg/ml), in presence of 100 nmol of 14C-labeled oleate. Macrophages were incubated at 37 ° C for 24 hours. The 14C-labeled oIeate incorporated into cholesterol ester was quantitated as described in Material and Methods section. Table II is representative average of two individual experiments. felt to have an increased n u m b e r of m a c r o p h a g e s in the peritoneal cavity32'2~ These m a c r o p h a g e s may be responsible for the dissolution of I n t e r c e e d cellulose in vivo and may result in its increased biologic activity. As observed in our study, w h e n RAW m a c r o p h a g e s were i n c u b a t e d with increasing concentrations of I n t e r c e e d cellulose, the I n t e r c e e d cellulose completely dissolved ,vithin 48 hours. Macrophages play an i m p o r t a n t p r o i n f l a m m a t o r y role in various diseases. 24' 25 It is well known that macrophages play a vital role in the f o r m a t i o n of adhesions. 26 These cells take up various anionic c o m p o u n d s such as fucoidin, polydnosinic acid, oxidized LDL, etc., by its scavenger receptors. 11 ' 12 Studies have suggested that the uptake of these ligands by m a c r 0 p h a g e s may increase its p r o i n f l a m m a t o r y capacity. ~3-16 I n t e r c e e d cellulose is taken up by m a c r o p h a g e s by the scavenger receptors and thereby may p r e v e n t adhesions. To establish w h e t h e r I n t e r c e e d cellulose affects macrophage uptake of scavenger ligands, we also used fluorescent-labeled acetyl LDL. Fluorescent-labeled acetyl LDL is a ligand for scavenger receptors. As these ligands bind to receptors the cells take on a characteristic fluoresc e n c e u n d e r microscopy. The presence of I n t e r c e e d cellulose decreased the uptake of fluorescent-labeled LDL. As a measure of scavenger r e c e p t o r activity, radioactive oleate i n c o r p o r a t i o n into the cholesterol ester in m a c r o p h a g e s was studied. It was observed that I n t e r c e e d cellulose decreased the oleate incorporation. We n e x t studied the effect of I n t e r c e e d cellulose on cellular b i o c h e m i c a l production. Bacterial lipopolysaccharide is a known ligand for the scavenger receptor. IL-113 p r o d u c t i o n is known to be m e d i a t e d by the scave n g e r receptor. In the p r e s e n c e of lipopolysaccharide I n t e r c e e d cellulose s e e m e d to block the e x p e c t e d increase in IL-113 caused by lipopolysaccharide. In conclusion, we have shown that I n t e r c e e d cellulose affects the ability of known scavenger r e c e p t o r ligands to interact with macrophages. The primary interaction of
] 319
Table III. IL-I[3 p r o d u c t i o n by macrophages Sample Control Interceed cellulose (100 Ixg/ml) LPS (1 ixg/ml) LPS plus Interceed cellulose Ammonium hydroxide
IL-l[3 (pg) 134 116 155 118 137
± 11 _+ 8 2 37 -~ 8 + 21
Mouse peritoneal macrophages grown on 24-well plates were incubated with Interceed cellulose (10O Izg/ml) in presence or absence of 1 brg/ml lipopolysaccharide for 24 hours. At end of incubation, 100 Ixl of culture supernatant was used to measure IL-1[3 released with use of commercially available ELISA kit as explained in Material and Methods section. Table III is representative average of three individual experiments. LPS, Lipopolysaccharide.
I n t e r c e e d cellulose may be on activated macrophages and their scavenger receptors, which are i m p o r t a n t in p r o m o t i n g the growth of ectopic e n d o m e t r i u m and the f o r m a t i o n of adhesions. This study is the first to provide preliminary evidence that I n t e r c e e d oxidized regenerated cellulose may function as a biologic barrier in preventing adhesion formation.
REFERENCES
1. Menzies D, Ellis H. Intestinal obstruction from adhesions-how big is the problem? Ann R Coil Surg Ing 1990;72:60-3. 2. Ellis H. The cause and prevention of postoperative intraperitoneal adhesions. Surg Gynecol Obstet 1971;133:497511. 3. Ray NF, LarsenJW, Stillman RJ,Jacobs RJ. Economic impact of hospitalization for lower abdominal adhesiolysis in the United States in 1988. Surg Gynecol Obstet 1993;176:271-6. 4. Franklin RR. Reduction of ovarian adhesions by the use of Interceed: Ovarian Adhesion Study Group. Obstet Gynecol 1995;86:335-40. 5. Nordic Adhesion Prevention Study Group. The efficacy of Interceed(TC7)* for prevention of refmxnation of postoperative adhesions on ovaries, fallopian tubes, and fimbriae in microsurgical operations for fertility: a multicenter study. FertiI Steril 1995;63:709-14. 6. Mais V, Ajossa S, Marongiu D, Peiretti RF, Guerriero S, Melis GB. Reduction of adhesion reformation after laparoscopic endometriosis surgmT: a randomized trial with an oxidized regenerated cellulose absorbable barrier. Obstet Gynecol 1995;86:512-5. 7. Sekiba K. Use of Interceed (TC7) absorbable adhesion barrier to reduce postoperative adhesion reformation in infertility and endometriosis surgery: the Obstetrics and Gynecology Adhesion Prevention Committee. Obstet Gynecol 1992;79:518-22. 8. diZerega GS. Contemporary adhesion prevention. Fertil Steril 1994;61:219-35. 9. Fraser I, Hughes D, Gordon S. Divalent cation-independent macrophage adhesion inhibited by monoclonal antibody to murine scavenger receptor. Nature 1993;364:343-6. 10. Hughes DA, Fraser IP, Gordon S. Murine macrophage scavenger receptor: in vivo expression and function as receptor for macrophage adhesion in lymphoid and nonlymphoid organs. EurJ Immunol 1995;25:466-73. 11. Pearson AM, Rich A, Krieger M. Polynucleotide binding to macrophage scavenger receptors depends on the formation of base-quartet-stabilized four-stranded helices. J Biol Chem 1993;268:3546-54.
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12. Parthasarathy S. Modified lipoproteins in the pathogenesis of atherosclerosis. Austin (TX): RG Landes; 1994. 13. Palkama T, Majuri ML, Mattila P, Hurme M, Renkonen R. Regulation of endothelial adhesion molecules by" ligands binding to the scavenger receptor. Clin Exp Immunol 1993;92:353-60. 14. Palkama T. Induction of interleukin-1 production by ligands binding to the scavenger receptor in human monocytes and the THP-1 cell line. Imnmnology 1991;74:432-8. 15. Thomas CE,Jackson RL, Ohlweiler DF, Ku G. Multiple lipid oxidation products in low density lipoproteins induce interlenkin-1 beta release from human blood mononuclear cells. J Lipid Res 1994;35:417-27. 16. Ku G, Thomas CE, Akeson AL, Jackson RL. Induction of interleukin 1 beta expression from human peripheral blood monocyte derived macrophages by 9-hydroxyoctadecadienoic acid. J Biol Chem 1992;267:14183-8. 17. Santanam N, Parthasarathy S. Paradoxical actions of antioxidants in the oxidation of low density lipoprotein by peroxidases. J Ctin Invest 1995;95:2594-600. 18. Dejager S, Mietus Snyder M, Friera A, Pitas RE. Dominant negative mutations of the scavenger receptor: native receptor inactivation by expression of truncated variants. J Clin Invest 1993;99:894-902. 19. Parthasarathy S, Printz DJ, Boyd D, Joy L, Steinberg D. Macrophage oxidation of low density lipoprotein generates a modified form recognized by the scavenger receptor. Arteriosclerosis 1986;6:505-10. 20. Haney AF, Doty E. Comparison of the peritoneal cells elicited by oxidized regenerated cellulose (Interceed) and expanded polytetrafluoroethylene (Gore-Tex Surgical Membrane) in a nmrine model..~n J Obstet Gynecol 1992;166:1137-49: 91. Diamond MP, Linsky CB, Cunningham T, Kamp L, Pines E, DeCherney AH, et al. Synergistic effects of INTERCEED (TCT) and heparin in reducing adhesion formation in the rabbit uterine horn model. Fertil Steril 1991;55:389-94. 99 HalmeJ, Becker S, Wing R. Accentuated cyclic activation of peritoneal macrophages in patients with endometriosis. Am J Obstet Gynecol 1984;148:85-90. 23. Uda S. The role of the peritoneal macrophage on the infertility associated with endometriosis. Osaka City Med J 1989;38:11-26. 24. Nathan CF, Tsunawaki S. Secretion of toxic oxygen products by macrophages: regulatory cytokines and their effects on the oxidase. Ciba Found Syrup 1986;118:211-30. 25. Yamamoto N, Willett NP, Lindsay DD. Participation of serum proteins in the inflammation-primed activation of macrophages. Inflammation 1994;18:311-92. 26. Haney AF. Endometriosis, macrophages, and adhesions [review]. Prog Clin Biol Res 1993;381:19-44. Discussion Dl~. MICHAELP. DIAMOND, Detroit, Michigan. Prevention of adhesions are a major problem each of us faces in every surgical procedure we perform because of the potential consequences of pain, infertility, small bowel obstruction, and difficult reoperative procedures. Nonetheless, the frequency with which adhesions develop has often been underappreciated, in part because a second operative procedure is used to identify them, yet there is often no clinical reason to justify such a procedure. We have previously reported that among women undergoing laparotomy for infertility adhesions were identified in 91 of 106 or 86% of subjects at second-look laparoscopy in spite of application of microsurgical principles and use of the carbon dioxide laser. More recently, we have also demonstrated that after laparoscopic adhesiolyses adhesions developed in 66 of
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68, or 97%, of women. Thus laparoscopic surgery is not a panacea for prevention of adhesions. Unfortunately, these observations are not unique to infertility, patients and the procedures they undergo. A study of m e n and women undergoing colectomy identified that 94% of control subjects had adhesions to the anterior abdominal wall incision line. Thus clearly there is a place for adjuvants to try to reduce postoperative adhesion development and tile clinical consequences. Currently there are two products approved by the Food and Drug Administration for the reduction of postoperative adhesions, Interceed and Seprafilm, each which are regulated as devices on the basis of the concept that they separate potentially opposing surfaces during the brief time period of days during which reperitonealization occurs. Dr. Reddy and colleagues are to be congratulated for this challenging hypothesis, that Interceed cellulose exerts its efficacy solely by this barrier mechanism. It has been known that this material, which is composed of oxidized regenerated cellulose, gelates after placement. Interceed is degraded into oligomers of varying length that get smaller in time as has been shown by highpressure liquid chromatography. This process occurs by both enzymatic and macrophage-dependent processes. In studies in which lead-labeled Interceed has been ingested, a peritoneal macrophage scanning electron micrographs have shown Interceed breakdown products within the cytoplasm in vacuoles and associated with cellular membranes. This article has provided evidence that the breakdown products may be biologically active. In this study solubilized Interceed inhibited uptake of acetylated LDL and fucoidin. Because each of these molecules normally serves as potent activators of the inflammatory process, including the synthesis and secretion of growth factors and cytokines, their inhibition may lessen inflammatory adhesion development. The clinical significance of this observation is that, if Interceed's efficacy is due to the breakdown products rather than to action as a barrier, then benefit may be realized at other sites throughout the abdominopehdc cavity distant from where it has been placed. Furthe> more, variation in efficacy may be the result of expression of function at scavenger receptors. In closing, I would like to ask three questions. (1) What limitations should be considered in view of having performed these studies in RAW macrophages, a celt line derived from rodents, as opposed to individually ha> vested macrophages from humans and have you assessed this issue? (2) Have you had the opportunity to assess the effect of blocking the scavenger receptor with Interceed on inflammatory process markers such as growlh factor or cytokine secretion? I too have thought that Interceed's action may in part be as a biologically active molecule rather than solely as a barrier; however, I had envisioned other possible actions. This raises the question of which biologic activity is associated with its efficacy. I wonder whether you have had the opportunity to statistically
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correlate whether animals in which efficacy is shown exhibit blockage of the scavenger receptors. In other words, do you have thoughts on how you might try to differentiate Interceed's efficacy as a barrier as opposed to biologically active membrane affecting scavenger receptor or other mechanisms? Dr. Reddy (Closing). I am looking forward to comparing cultured RAW macrophages to freshly collected cells from donors. I anticipate that the freshly gathered macrophages will have a more intense response than will the cultured cells. The cultured macrophages probably manifest a blunted response. As far as animal studies, we hope
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to prove that Interceed functions in its soluble form in mice. We would do this by producing tissue injury and then inserting soluble and intact Interceed and then comparing the n u m b e r and type of adhesions at second look. We also plan to determine how Interceed affects IL-I~ production. We are considering experiments with tritiated proline suture to determine the effect of Interceed on collagen formation. By showing that Interceed affects the production of these biochemical substances we would have further evidence that Interceed functions as a biologic barrier.
cellulose (Johnson & Johnson Medical, Arlington, Tex.), because of its polyanionic nature, may compete for the macrophage scavenger receptor. STUDY DESIGN: RAW macrophages were incubated with Interceed oxidized regenerated cellulose and known scavenger receptor ligands. The production of interleukin-113 by mouse peritoneal macrophages was measured in the presence of Interceed cellulose. RESULTS" When macrophages were incubated with Interceed cellulose, increasing concentrations inhibited the uptake of fluorescent acety[ low-density lipoprotein. In the presence of Interceed cellulose there was a decrease in the production of interleukin-113 by mouse macrophages. CONCLUSION: These results suggest that the interaction of Interceed oxidized regenerated cellulose with macrophages with scavenger receptors may result in a decreased secretion of matrix components, inflammatory mediators, and cellular growth factors. Thus Interceed cellulose may function as a biologic barrier in preventing adhesions. (Am J Obstet Gynecol 1997;177:1315-21 .)
Key words: Scavenger receptor, solubilized Interceed oxidized regenerated cellulose, po!yanion, acetyl low-density lipoprotein, interleukin-1
Postoperative adhesion formation results from the fibroproliferative end products of the inflammatory process. Macrophages are crucial to this process. Approximately 3% of all laparotomies are performed for adhesion-induced intestinal obstructions. ~'2 A great deal of research has been directed toward the reduction and prevention of adhesions because of the morbidity and cost associated with adhesions. In 1988 the cost of abdominopelvic adhesiolysis was ~/1179.9 million.3 Recently, a n u m b e r of products, such as Interceed oxidized regenerated cellulose (Johnson & Johnson Medical, Arlington, Tex.), for preventing adhesions intraoperatively have become available.< 5 Interceed cellulose has been shown to decrease postoperative adhesions. < 7 The exact mechanism of action of Interceed cellulose is unknown, but it i s felt that it functions as a physical barrier separating opposing surfaces. 8 However, Interceed cellulose rapidly dissolves from its mesh form into a soft gelatinous mass in vivo. This occurs in about 2 days. Therefore it is difficult to attribute the efficacy solely to its From the Department of Obstetrics and @necology, EmoU University. Second Prize Presidential Resident~Fellow Paper; presented at the Twenty-third Annual Meeting of the ,Society of Gynecologic Surgeons, New Orle,ans, Louisiana, FebruaU 24-26, 199Z Reprint requests: Sampath Parthasarathy, PhD, Department of Obstetrics and Gynecology, Emo~3~Universiiy, Atlanta, GA 30322. Copyright © 1997 by Mosby-Year Book, Inc. 0002-9378/97 $5.00 + 0 6/6/85176
ability to act as a physical barrier, suggesting it may also have biologic activity that may account for its ability to prevent adhesions. Adhesion formation occurs as a result of the inflammation, granulation, and scar formation that follow trauma. A variety of stimuli are known to induce injury and adhesions. These include surgical trauma, infection, ischemia, and radiation. After injury and the inflammatory reaction, fibrin arrives at the site of injury through exudation. Fibrin acts as a g]ue between structures. If the fibrin is not removed through fibrinolytic action, the temporary adhesions become p e r m a n e n t fibrous adhesions. Through granulation, macrophages and fibroblasts invade the fibrin network, and collagen and other connective tissue elements are laid down. Macrophages present in the h u m a n peritoneal cavity are potent mediators of inflammation. They are capable of generating inflammatory cytokines and growth factors for fibroblasts. They also are intricately involved in the production and dissolution of extracellular matrix components such as collagen. In this study we tested the ability of Interceed cellulose to compete for the macrophage scavenger receptor. The attachment of macrophages to matrix components by way of its scavenger receptor(s) has been a topic of novel interest.9, 10 Macrophage scavenger receptors bind and internalize a n u m b e r of polyanionic molecules such as 1315
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acetylated low-density lipoproteins (LDL), fucoidin, polyinosinic acid, and oxidized LDL. 11' 12 These ligands and their components are potent activators of the monocyte-macrophage inflammatory process and induce the synthesis and secretion of cytokines such as interleukin-l]3 (IL-I[3)} 316
Material and methods Interceed oxidized regenerated cellulose was obtained from Johnson & Johnson Medical. DiI (3,3'-dioctadecylindocarbocyanine) was obtained from Molecular Probes, Plano, Texas. RPMI 1640 medium was purchased from Mediatech, Herndon, Virginia. 1-Carbon-14-1abeled oleic acid was purchased from New England Nuclear, Boston. Preparation of LDL and aeetylated LDL. Blood was collected from the healthy donor and LDL was prepared from the plasma by ultracentrifugation as described in previous publications}7 DiI-labeled acetylated LDL was prepared as described by Pitas et al} s Solubilization of Interceed cellulose. Approximately 100 mg of Interceed mesh was treated with ]00 Ixl of a m m o n i u m hydroxide in 2 ml of phosphate-buffered saline solution. The solution was vigorously mixed with mild warming. Interceed mesh readily goes into solution u n d e r these conditions. The control tube contained only a m m o n i u m hydroxide. Nitrogen gas was gently bubbled through the tubes to remove excess ammonia. The pH of the solution was neutral. Interceed and control solutions were filter sterilized by passage through a 0.45 Ixm filter. Fluorescence measurement. RAW macrophages were cultured on 24-well plates. The cells were washed and then incubated with increasing concentrations of solubilized Interceed cellulose (0 to 100 ~g per well) in the presence of fluorescent DiI-acetylated LDL (5 ~g) for 48 hours. At the end of incubation the cells were washed, and the fluorescence was visualized with use of the Nikon fluorescent microscope. The fluorescent material from the cells was then extracted with methanol and quantitated with use of a Shimadzu Spectro Fluorometer (excitation 590 nm, emission 570 nm). is 14C-labeled oleate incorporation. To determine the uptake and degradation of acetyl LDL by cells, the incorporation of 14C-labeled oleate into cellular cholesteryl esters was measured in the presence of solubilized Interceed cellulose. RAW macrophages were incubated with 25 p~g of ace@ LDL and 100 nmol of 14C-labeled oleate (9000 disintegrations/min/nmol) in the presence of 125 ~g of solubilized Interceed cellulose for 24 hours at 37 ° C. After the cells were washed, cellular lipids were exn-acted and separated by thin-layer chromatography. Spots corresponding to cholesteryl esters were visualized and the radioactivity was determined. 19 Interleukin-1 (IL-1) production. Mouse peritoneal macrophages were harvested 19 and plated in culture
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dishes at 2 × 105 cells per well in RPMI 1640 medium containing 10% fetal calf serum. After 24 hours cells were washed and incubated with 100 txg of solubilized Interceed cellulose in the presence or absence of either 10 nmol of 13-hydroperoxy linoleate (generated by the action of soybean lipoxygenase on linoleic acid) or 25 peg of oxidized LDL or 1 txg of lipopolysaccharide (known stimulants of IL-1 production). Cells were incubated for 24 hours and the amount of IL-113 released into the culture supernatant was quantified with use of a commercial kit for mouse IL-1[3 (Intertest-l[3X, available from Genzyme Diagnostics, Cambridge, Mass.). The commercial kit is a solid-phase enzyme-linked immunosorbent assay (ELISA) kit that uses the multiple-antibody sandwich principle. Briefly, 100 Ixl of the culture supernatants was added to each well in the 96-well microtiter plate precoated with monoclonal antimouse IL-113 and incubated for 1 hour. After the u n b o u n d material was washed, biotinylated polyclonal antimonoclonal IL-l[3, which binds to the captured monoclonal IL-l[3 was added. After incubation and washing, the polyclonal antibody was removed and the streptavidin conjugated to horseradish peroxidase was added to the wells and incubated. The plate was washed again and then incubated with the substrate solution, which initiated a peroxidase-catalyzed color change that was measured at an absorbance of 450 n m with use of an ELISA plate reader. The concentration of the unknown antigen is quantitated with use of the standard curve obtained from known standards provided by the manufacturer.
Results RAW macrophages and the cell-free control wells were incubated in 24-well dishes with 25 mm z pieces of Interceed cellulose for 24 hours. Microscopic examination showed that Interceed cellulose remained intact in cell-free control incubations (Fig. 1, A). In contrast, most of Interceed cellulose in incubations that contained RAW cells was present as partly digested materials or as completely dissolved solutions (Fig. 1, B). No evidence of toxicity could be seen u n d e r microscopy. In fact, during the incubation attachment of macrophages to Interceed fibers could be seen, which eventually dissolved to form a clear solution. This observation suggests that macrophages may digest Interceed fibers. However, in these studies it was not possible to determine whether Interceed cellulose was taken up by the cells or remained outside in the medium. Because Interceed cellulose appeared to function in its dissolved form, we solubilized it as described in the Material and methods section. We tested the ability of solubilized Interceed cellulose to affect the uptake and internalization of acetyl LDL, a ligand for the macrophage scavenger receptor} 9 When RAW macrophages were incubated with fluorescent DiI ace@ LDL, the cells took up the acetylated LDL as
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Fig. 1. RAW macrophages and no-cell controls were cultured in 24-well dishes in presence and absence of 5 mm 2 pieces of Interceed cellulose. After 24-hour incubation period, cells were observed with Nikon light microscope. A, Interceed cellulose, in no-ceil control well. Fibers remained intact after 24 hours. B, Interceed cellulose, when incubated with RAW macrophages, appeared either partly digested or completely dissolved. Macrophages can be seen aligning along Interceed fiber.
observed by increased fluorescence u n d e r the fluoresc e n t microscope. Cytoplasmic droplets of fluorescent material could be seen (Fig. 2, A). W h e n m a c r o p h a g e s were incubated with a c e @ LDL and increasing concentrations o f solubilized I n t e r c e e d cellulose, it was observed that there was a d o s e - d e p e n d e n t decrease in the fluorescence, suggesting that the I n t e r c e e d cellulose c o m p e t e d with the acetyl LDL for the scavenger receptor. Fig. 2, B, shows the almost c o m p l e t e absence of fluorescence w h e n cells were incubated with 2.5 txg of fluorescent acetyl LDL in the p r e s e n c e of 100 txg of solubilized I n t e r c e e d cellulose. Control incubations with an equal a m o u n t of a m m o n i u m hydroxide solution c o r r e s p o n d i n g to the I n t e r c e e d c o n c e n t r a t i o n did n o t decrease the fluorescence, suggesting that it was n o t the a m m o n i a but the
I n t e r c e e d cellulose that decreased the fluorescence. W h e n the fluorescent material was extracted with methanol and quantified, there was a d o s e - d e p e n d e n t decrease in cell-associated fluorescence in the presence of I n t e r c e e d cellulose (Table I). As a measure of scavenger r e c e p t o r activity, RAW macrophages were incubated with 25 b~g acetyl LDL in the presence or absence of soluble I n t e r c e e d cellulose (125 b~g) and 100 n m o l of 14C-labeled oleate. T h e radioactive oleate i n c o r p o r a t e d into the cholesterol esters was as shown in Table II. It was observed that I n t e r c e e d cellulose decreased the oleate i n c o r p o r a t i o n by 42% c o m p a r e d with the control. In the presence of I n t e r c e e d cellulose the thioglycol a t e q n d u c e d mouse peritoneal m a c r o p h a g e s p r o d u c e d less IL-113 c o m p a r e d with the control (Table III). Inter-
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A
B Fig. 2. RAW macrophages cultured in 24-well dishes were incubated with and without Interceed cellulose in presence of fluorescent Dil ace~l LDL (as described in Material and methods section) for 24 to 48 hours. A, Control cells had increased fluorescence as seen under fluorescent microscope. B, Cells incubated with Interceed cellulose had a dose-dependent decrease in fluorescence.
T a b l e I. A m o u n t of f l u o r e s c e n c e e x t r a c t e d f r o m DiI-labeled acetyl LDL RAW m a c r o p h a g e s
Cellsample Control TC-7 10 Ixg TC-7 20 Ixg TC-7 50 >g TC-7 100 ixg Ammonium hydroxide
l
bTuorescenceunits 162.4 lt2.0 55.0 44.0 30.0 187.0
Raw macrophages cultured in 24-well plates were incubated with 2.5 Ixg/ml DiI-acetyl LDL in presence or absence of increasing concentrations of Interceed cellulose (0 to 100 p,g). After 24 hours' incubation, amount of fluorescent-labeled acetyl LDL taken up by macrophages was quantitated after extraction with methanol and measured on a spectrofluorometer (Ex 520 nm and Em 570 nm). Table I is representative average of two individual experiments.
ceed cellulose s e e m e d to d e c r e a s e the IL-I[3 p r o d u c t i o n s t i m u l a t e d by lipopolysaccharide, as seen by lower a m o u n t s of IL-1[3 r e l e a s e d into the m e d i u m . This suggests t h a t I n t e r c e e d cellulose may b e able to r e d u c e t h e inflammatow response of the macrophages.
Comment I n t e r c e e d oxidized r e g e n e r a t e d cellulose is a n a d h e sion b a r r i e r a p p r o v e d for use to p r e v e n t p o s t o p e r a t i v e adhesions. Its m e c h a n i s m o f action is u n k n o w n , b u t it has b e e n t h o u g h t to f u n c t i o n as a physical barrier. It has b e e n o b s e r v e d in a n i m a l studies t h a t t h e r e is a c o m p l e t e dissolution o f I n t e r c e e d cellulose in t h e p e r i t o n e a l cavity after 1 to 2 days b u t it c o n t i n u e s to p r e v e n t a d h e sions.20.2~ This o b s e r v a t i o n suggests t h a t I n t e r c e e d cellulose has biologic activity. W o m e n with e n d o m e t r i o s i s are
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Table II. I n t e r c e e d cellulose inhibits i n c o r p o r a t i o n of I4C-labeled oleate into m a c r o p h a g e cholesterol esters in p r e s e n c e of acetyl LDL Incubation conditions Acetyl LDL (25 ~xg/ml) Acetyl LDL (25 ~xg/ml) plus Interceed cellulose (125 p~g/ml)
14C-labeled oleate incorporated in cholesterol ester (nmol) 7.2 4.2
RAW macrophages were cultured in 6-well plates and incubated with acetyl LDL (25 b~g/ml) with and without Interceed cellulose (125 Ixg/ml), in presence of 100 nmol of 14C-labeled oleate. Macrophages were incubated at 37 ° C for 24 hours. The 14C-labeled oIeate incorporated into cholesterol ester was quantitated as described in Material and Methods section. Table II is representative average of two individual experiments. felt to have an increased n u m b e r of m a c r o p h a g e s in the peritoneal cavity32'2~ These m a c r o p h a g e s may be responsible for the dissolution of I n t e r c e e d cellulose in vivo and may result in its increased biologic activity. As observed in our study, w h e n RAW m a c r o p h a g e s were i n c u b a t e d with increasing concentrations of I n t e r c e e d cellulose, the I n t e r c e e d cellulose completely dissolved ,vithin 48 hours. Macrophages play an i m p o r t a n t p r o i n f l a m m a t o r y role in various diseases. 24' 25 It is well known that macrophages play a vital role in the f o r m a t i o n of adhesions. 26 These cells take up various anionic c o m p o u n d s such as fucoidin, polydnosinic acid, oxidized LDL, etc., by its scavenger receptors. 11 ' 12 Studies have suggested that the uptake of these ligands by m a c r 0 p h a g e s may increase its p r o i n f l a m m a t o r y capacity. ~3-16 I n t e r c e e d cellulose is taken up by m a c r o p h a g e s by the scavenger receptors and thereby may p r e v e n t adhesions. To establish w h e t h e r I n t e r c e e d cellulose affects macrophage uptake of scavenger ligands, we also used fluorescent-labeled acetyl LDL. Fluorescent-labeled acetyl LDL is a ligand for scavenger receptors. As these ligands bind to receptors the cells take on a characteristic fluoresc e n c e u n d e r microscopy. The presence of I n t e r c e e d cellulose decreased the uptake of fluorescent-labeled LDL. As a measure of scavenger r e c e p t o r activity, radioactive oleate i n c o r p o r a t i o n into the cholesterol ester in m a c r o p h a g e s was studied. It was observed that I n t e r c e e d cellulose decreased the oleate incorporation. We n e x t studied the effect of I n t e r c e e d cellulose on cellular b i o c h e m i c a l production. Bacterial lipopolysaccharide is a known ligand for the scavenger receptor. IL-113 p r o d u c t i o n is known to be m e d i a t e d by the scave n g e r receptor. In the p r e s e n c e of lipopolysaccharide I n t e r c e e d cellulose s e e m e d to block the e x p e c t e d increase in IL-113 caused by lipopolysaccharide. In conclusion, we have shown that I n t e r c e e d cellulose affects the ability of known scavenger r e c e p t o r ligands to interact with macrophages. The primary interaction of
] 319
Table III. IL-I[3 p r o d u c t i o n by macrophages Sample Control Interceed cellulose (100 Ixg/ml) LPS (1 ixg/ml) LPS plus Interceed cellulose Ammonium hydroxide
IL-l[3 (pg) 134 116 155 118 137
± 11 _+ 8 2 37 -~ 8 + 21
Mouse peritoneal macrophages grown on 24-well plates were incubated with Interceed cellulose (10O Izg/ml) in presence or absence of 1 brg/ml lipopolysaccharide for 24 hours. At end of incubation, 100 Ixl of culture supernatant was used to measure IL-1[3 released with use of commercially available ELISA kit as explained in Material and Methods section. Table III is representative average of three individual experiments. LPS, Lipopolysaccharide.
I n t e r c e e d cellulose may be on activated macrophages and their scavenger receptors, which are i m p o r t a n t in p r o m o t i n g the growth of ectopic e n d o m e t r i u m and the f o r m a t i o n of adhesions. This study is the first to provide preliminary evidence that I n t e r c e e d oxidized regenerated cellulose may function as a biologic barrier in preventing adhesion formation.
REFERENCES
1. Menzies D, Ellis H. Intestinal obstruction from adhesions-how big is the problem? Ann R Coil Surg Ing 1990;72:60-3. 2. Ellis H. The cause and prevention of postoperative intraperitoneal adhesions. Surg Gynecol Obstet 1971;133:497511. 3. Ray NF, LarsenJW, Stillman RJ,Jacobs RJ. Economic impact of hospitalization for lower abdominal adhesiolysis in the United States in 1988. Surg Gynecol Obstet 1993;176:271-6. 4. Franklin RR. Reduction of ovarian adhesions by the use of Interceed: Ovarian Adhesion Study Group. Obstet Gynecol 1995;86:335-40. 5. Nordic Adhesion Prevention Study Group. The efficacy of Interceed(TC7)* for prevention of refmxnation of postoperative adhesions on ovaries, fallopian tubes, and fimbriae in microsurgical operations for fertility: a multicenter study. FertiI Steril 1995;63:709-14. 6. Mais V, Ajossa S, Marongiu D, Peiretti RF, Guerriero S, Melis GB. Reduction of adhesion reformation after laparoscopic endometriosis surgmT: a randomized trial with an oxidized regenerated cellulose absorbable barrier. Obstet Gynecol 1995;86:512-5. 7. Sekiba K. Use of Interceed (TC7) absorbable adhesion barrier to reduce postoperative adhesion reformation in infertility and endometriosis surgery: the Obstetrics and Gynecology Adhesion Prevention Committee. Obstet Gynecol 1992;79:518-22. 8. diZerega GS. Contemporary adhesion prevention. Fertil Steril 1994;61:219-35. 9. Fraser I, Hughes D, Gordon S. Divalent cation-independent macrophage adhesion inhibited by monoclonal antibody to murine scavenger receptor. Nature 1993;364:343-6. 10. Hughes DA, Fraser IP, Gordon S. Murine macrophage scavenger receptor: in vivo expression and function as receptor for macrophage adhesion in lymphoid and nonlymphoid organs. EurJ Immunol 1995;25:466-73. 11. Pearson AM, Rich A, Krieger M. Polynucleotide binding to macrophage scavenger receptors depends on the formation of base-quartet-stabilized four-stranded helices. J Biol Chem 1993;268:3546-54.
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12. Parthasarathy S. Modified lipoproteins in the pathogenesis of atherosclerosis. Austin (TX): RG Landes; 1994. 13. Palkama T, Majuri ML, Mattila P, Hurme M, Renkonen R. Regulation of endothelial adhesion molecules by" ligands binding to the scavenger receptor. Clin Exp Immunol 1993;92:353-60. 14. Palkama T. Induction of interleukin-1 production by ligands binding to the scavenger receptor in human monocytes and the THP-1 cell line. Imnmnology 1991;74:432-8. 15. Thomas CE,Jackson RL, Ohlweiler DF, Ku G. Multiple lipid oxidation products in low density lipoproteins induce interlenkin-1 beta release from human blood mononuclear cells. J Lipid Res 1994;35:417-27. 16. Ku G, Thomas CE, Akeson AL, Jackson RL. Induction of interleukin 1 beta expression from human peripheral blood monocyte derived macrophages by 9-hydroxyoctadecadienoic acid. J Biol Chem 1992;267:14183-8. 17. Santanam N, Parthasarathy S. Paradoxical actions of antioxidants in the oxidation of low density lipoprotein by peroxidases. J Ctin Invest 1995;95:2594-600. 18. Dejager S, Mietus Snyder M, Friera A, Pitas RE. Dominant negative mutations of the scavenger receptor: native receptor inactivation by expression of truncated variants. J Clin Invest 1993;99:894-902. 19. Parthasarathy S, Printz DJ, Boyd D, Joy L, Steinberg D. Macrophage oxidation of low density lipoprotein generates a modified form recognized by the scavenger receptor. Arteriosclerosis 1986;6:505-10. 20. Haney AF, Doty E. Comparison of the peritoneal cells elicited by oxidized regenerated cellulose (Interceed) and expanded polytetrafluoroethylene (Gore-Tex Surgical Membrane) in a nmrine model..~n J Obstet Gynecol 1992;166:1137-49: 91. Diamond MP, Linsky CB, Cunningham T, Kamp L, Pines E, DeCherney AH, et al. Synergistic effects of INTERCEED (TCT) and heparin in reducing adhesion formation in the rabbit uterine horn model. Fertil Steril 1991;55:389-94. 99 HalmeJ, Becker S, Wing R. Accentuated cyclic activation of peritoneal macrophages in patients with endometriosis. Am J Obstet Gynecol 1984;148:85-90. 23. Uda S. The role of the peritoneal macrophage on the infertility associated with endometriosis. Osaka City Med J 1989;38:11-26. 24. Nathan CF, Tsunawaki S. Secretion of toxic oxygen products by macrophages: regulatory cytokines and their effects on the oxidase. Ciba Found Syrup 1986;118:211-30. 25. Yamamoto N, Willett NP, Lindsay DD. Participation of serum proteins in the inflammation-primed activation of macrophages. Inflammation 1994;18:311-92. 26. Haney AF. Endometriosis, macrophages, and adhesions [review]. Prog Clin Biol Res 1993;381:19-44. Discussion Dl~. MICHAELP. DIAMOND, Detroit, Michigan. Prevention of adhesions are a major problem each of us faces in every surgical procedure we perform because of the potential consequences of pain, infertility, small bowel obstruction, and difficult reoperative procedures. Nonetheless, the frequency with which adhesions develop has often been underappreciated, in part because a second operative procedure is used to identify them, yet there is often no clinical reason to justify such a procedure. We have previously reported that among women undergoing laparotomy for infertility adhesions were identified in 91 of 106 or 86% of subjects at second-look laparoscopy in spite of application of microsurgical principles and use of the carbon dioxide laser. More recently, we have also demonstrated that after laparoscopic adhesiolyses adhesions developed in 66 of
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68, or 97%, of women. Thus laparoscopic surgery is not a panacea for prevention of adhesions. Unfortunately, these observations are not unique to infertility, patients and the procedures they undergo. A study of m e n and women undergoing colectomy identified that 94% of control subjects had adhesions to the anterior abdominal wall incision line. Thus clearly there is a place for adjuvants to try to reduce postoperative adhesion development and tile clinical consequences. Currently there are two products approved by the Food and Drug Administration for the reduction of postoperative adhesions, Interceed and Seprafilm, each which are regulated as devices on the basis of the concept that they separate potentially opposing surfaces during the brief time period of days during which reperitonealization occurs. Dr. Reddy and colleagues are to be congratulated for this challenging hypothesis, that Interceed cellulose exerts its efficacy solely by this barrier mechanism. It has been known that this material, which is composed of oxidized regenerated cellulose, gelates after placement. Interceed is degraded into oligomers of varying length that get smaller in time as has been shown by highpressure liquid chromatography. This process occurs by both enzymatic and macrophage-dependent processes. In studies in which lead-labeled Interceed has been ingested, a peritoneal macrophage scanning electron micrographs have shown Interceed breakdown products within the cytoplasm in vacuoles and associated with cellular membranes. This article has provided evidence that the breakdown products may be biologically active. In this study solubilized Interceed inhibited uptake of acetylated LDL and fucoidin. Because each of these molecules normally serves as potent activators of the inflammatory process, including the synthesis and secretion of growth factors and cytokines, their inhibition may lessen inflammatory adhesion development. The clinical significance of this observation is that, if Interceed's efficacy is due to the breakdown products rather than to action as a barrier, then benefit may be realized at other sites throughout the abdominopehdc cavity distant from where it has been placed. Furthe> more, variation in efficacy may be the result of expression of function at scavenger receptors. In closing, I would like to ask three questions. (1) What limitations should be considered in view of having performed these studies in RAW macrophages, a celt line derived from rodents, as opposed to individually ha> vested macrophages from humans and have you assessed this issue? (2) Have you had the opportunity to assess the effect of blocking the scavenger receptor with Interceed on inflammatory process markers such as growlh factor or cytokine secretion? I too have thought that Interceed's action may in part be as a biologically active molecule rather than solely as a barrier; however, I had envisioned other possible actions. This raises the question of which biologic activity is associated with its efficacy. I wonder whether you have had the opportunity to statistically
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correlate whether animals in which efficacy is shown exhibit blockage of the scavenger receptors. In other words, do you have thoughts on how you might try to differentiate Interceed's efficacy as a barrier as opposed to biologically active membrane affecting scavenger receptor or other mechanisms? Dr. Reddy (Closing). I am looking forward to comparing cultured RAW macrophages to freshly collected cells from donors. I anticipate that the freshly gathered macrophages will have a more intense response than will the cultured cells. The cultured macrophages probably manifest a blunted response. As far as animal studies, we hope
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to prove that Interceed functions in its soluble form in mice. We would do this by producing tissue injury and then inserting soluble and intact Interceed and then comparing the n u m b e r and type of adhesions at second look. We also plan to determine how Interceed affects IL-I~ production. We are considering experiments with tritiated proline suture to determine the effect of Interceed on collagen formation. By showing that Interceed affects the production of these biochemical substances we would have further evidence that Interceed functions as a biologic barrier.