Address correspondence to Arthur O. Tzianabos, Channing Laboratory, 181 Longwood Avenue, Boston, MA 02115. Phone: 617-525-2610; Fax: 617-731-1541; E-mail: atzianabos{at}channing.harvard.edu
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Abstract |
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Key Words: T cells adhesions interleukin-17 chemokines peritonitis
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Introduction |
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Investigations of the cellular mechanism of postsurgical adhesion formation have centered on the role of macrophages and PMN (1215). A role for T lymphocytes has not been postulated, although these cells play a critical role in coordinating and regulating chemotactic responses in a variety of inflammatory tissue diseases, including experimental autoimmune encephalomyelitis, inflammatory bowel disease, and acute lung injury (1621). Investigations in this area have clearly shown that T cellderived cytokines and chemokines are among the predominant factors governing appropriate inflammatory responses. Recent investigations have shown that IL-17, a proinflammatory cytokine produced almost exclusively by activated CD4+ T cells, induces the release of neutrophil-specific chemokines such as macrophage inflammatory protein (MIP)*-2 and cytokine-induced neutrophil chemoattractant (KC) and controls PMN trafficking in the peritoneal cavity (2224). Based on these data, we hypothesized that T cells play a central role in orchestrating the inflammatory process leading to the development of peritoneal adhesions.
In this report, we demonstrate that Th1 CD4+ ß T cells are critical to the development of postsurgical and postinfectious adhesion formation. Moreover, activated T cells home to the peritoneal cavity shortly after the induction of adhesions and become a dominant cell type at this site during adhesiogenesis. Finally, soluble mediators such as the T cellderived proinflammatory cytokine IL-17 and chemokines that bind to the CXC chemokine receptor 2 (CXCR2) have a direct role in the pathogenesis of adhesion formation.
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Materials and Methods |
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Rodent Model of Surgical Adhesion Formation.
Rats were anesthetized with a single intraperitoneal injection of 0.15 ml pentobarbital sodium (50 mg/ml; Abbott Laboratories). Mice were anesthetized with 0.15 ml 1:5 (vol/vol) diluted pentobarbital sodium solution (10 mg/ml). For adhesion induction, we used a modified rat model of abdominal adhesion formation originally described by Krause et al. (25). An anterior midline incision was made through the abdominal wall and peritoneum. The cecum was isolated and abraded until visibly damaged by scrubbing with a sterile dry 4 x 4 surgical gauze. Apposing areas of the abdominal wall were also abraded. The incision was closed in two layers with silk sutures. Animals were killed and examined for adhesion formation 6 d later by an observer blinded to the identity of the experimental groups. Each animal was evaluated according to the following standard scoring system, which has been widely used in this field (2629): 0, no adhesion; 1, one thin filmy adhesion; 2, more than one thin adhesion; 3, thick adhesion with focal point; 4, thick adhesion with plantar attachment or more than one thick adhesion with focal point; and 5, very thick vascularized adhesions or more than one plantar adhesion. The adhesion scores for the various groups were compared by the Mann-Whitney U test or the Kruskal-Wallis with Dunn's multiple comparisons test. Differences between experimental groups were considered significant at P < 0.05.
Mouse Model of Intraabdominal Sepsis.
We used a model of intraabdominal sepsis as previously described (30). C57BL/6 mice were injected intraperitoneally with 0.2 ml 1:5 (vol/vol) diluted cecal contents inoculum containing both aerobic (9 x 105 CFU/ml) and anaerobic bacteria (8 x 107 CFU/ml). This dose was shown to yield a sublethal infection in these animals (unpublished data). Animals were killed and examined for adhesion formation 6 d later.
CD3+ T Cell Depletion in Rats.
Lewis rats were randomly assigned to one of three groups and subjected to cecal abrasion surgery. Group 1 was treated with 100 µg of a CD3-specific mAb (G4.18; BD PharMingen) via the intraperitoneal route at the time of surgery, group 2 was treated with 100 µg of an isotype-matched IgG antibody, and group 3 was treated with the same volume of saline via the same route. FACS® analysis showed that treatment with the anti-CD3 mAb depleted >95% of T cells (unpublished data). Animals were killed 6 d later and their adhesions were scored as previously described.
ßTCR+ T Cell Depletion in Rats and Mice.
For studies with rats depleted of T cells bearing ßTCR, mAb R73 (BD PharMingen) specific for rat
ßTCR was used. Lewis rats were treated with 100 µg mAb R73 or an isotype-matched control antibody via the intracardiac route 24 h before surgery. For experiments with mice, C57BL/6 mice were treated with 300 µg TCR ß chainspecific mAb H57-597 (BD PharMingen) or an isotype-matched control antibody via the intraperitoneal route 4 d before surgery. All rats and mice underwent cecal abrasion surgery and were assessed for adhesion formation as previously described.
CD4+ or CD8+ T Cell Depletion in Mice.
CD4+ and CD8+ T cells were depleted with CD4-specific mAb GK1.5 (BD PharMingen) and CD8-specific mAb 53-6.7 (BD PharMingen), respectively. C57BL/6 mice were treated with 0.2 mg of these mAbs via the intraperitoneal route 48 h before surgery. An additional group was treated with isotype-matched control antibody. All animals underwent cecal abrasion surgery and were killed and assessed for adhesion formation 6 d later.
Adoptive CD4+ T Cell Transfer Experiments.
Splenic T cells from Stat4-/-, Stat6-/-, or wild-type mice were purified on a nylon wool column and then CD4+ T cells were purified on CD4+ T cell enrichment immunocolumns (Cedarlane). CD4+ T cellenriched populations (>95% CD4+) were transferred to ßTCR-/- mice (2.4 x 106 cells per mouse) via the intracardiac route 24 h before surgery. All recipient animals underwent cecal abrasion surgery and were killed and assessed for adhesion formation 6 d later.
Kinetics of Cellular Influx into the Peritoneal Cavity After Cecal Abrasion.
C57BL/6 mice underwent cecal abrasion for studies measuring the cellular influx into the peritoneal cavity after this procedure. A control group underwent laparotomy without cecal manipulation. Animals (n = 5) underwent peritoneal lavage with 1 ml PBS 6, 24, 48, and 72 h after surgery. Lavage fluid from each animal (25 µl) was smeared on a microscope slide and stained with a modified Giemsa stain. Slides were examined microscopically and monocytes/macrophages, lymphocytes, and PMN (per 200 cells) were enumerated. The remaining specimens were pooled for FACS® analysis and red blood cells were removed via lysis with NH4Cl. After preincubation with rat antimouse CD16/CD32 (BD PharMingen) to block Fc receptors, cells were stained with FITC- or PE-labeled isotype control antibodies or mAbs to CD3, CD19, CD25, and CD69. Stained cells were analyzed on a Coulter EPICS XLTM cytometer (Beckman Coulter), the CELLQuestTM (Becton Dickinson), and WinMDI 2.8 analysis software (http://facs.scripps.edul; Scripps Research Institute). The absolute number of peritoneal cells collected was determined by trypan blue staining and a hemacytometer. The absolute numbers of macrophages/monocytes, PMN, and lymphocytes were calculated by multiplying the total number of peritoneal cells by the percentage of each cell type identified by microscopic examination and dividing the result by 100. The numbers of T and B lymphocytes were calculated based on the number of lymphocytes and the percentage of CD3+ and CD19+ cells obtained from FACS® analysis. The percentages of CD69+ and CD25+ T cells were determined by FACS® analysis and the absolute cell number was calculated by multiplying these percentages by the total peritoneal cell number.
Kinetics of IL-17 and CXC Chemokines in Peritoneal Fluid of Mice Undergoing Cecal Abrasion.
C57BL/6 mice underwent the cecal abrasion procedure for studies measuring IL-17, KC, and MIP-2 in peritoneal fluid after this procedure. A control group underwent laparotomy without cecal manipulation. Animals (n = 5 at each time interval) underwent peritoneal lavage with 1 ml PBS 6, 24, 48, and 72 h after surgery. The peritoneal fluid was stored at -80°C until assayed for IL-17, KC, and MIP-2 with ELISA kits (R&D Systems) according to the manufacturer's protocols.
Intracellular FACS® Analysis for IL-17.
Intracellular staining and subsequent FACS® analyses were used to determine the cell types in the peritoneal cavity that were responsible for the production of IL-17. Groups of C57BL/6 mice underwent cecal abrasion surgery and were killed 0, 4, or 6 h later. Peritoneal lavage was performed on each animal. Mononuclear cells from each group of animals were pooled and red blood cells were removed using Lymphoprep media (Nycomed Pharma). Cells from each group were stained with an mAb specific for CD3e conjugated to cychrome (BD PharMingen), an mAb specific for CD4 conjugated to FITC, and the appropriate isotype controls. The cells were washed, fixed, and permeabilized using cytofix/cytoperm solution and 13 perm/wash solution (BD PharMingen). Intracellular staining was performed with PE-conjugated mAb specific for IL-17 (BD PharMingen) or a PE-conjugated isotype control. Stained cells were analyzed on a Coulter EPICS XLTM cytometer as previously described. Analyses were performed to determine the proportion of CD3+4+ T cells that produce IL-17 compared with the proportion of non-CD3+4+ T cells that produce this cytokine.
Generation of Murine IL-17 and CXCR2-specific Polyclonal Antibodies.
Polyclonal antibody to murine IL-17 was produced by the immunization of rabbits at multiple intradermal sites with recombinant mouse IL-17 (R&D Systems) mixed with complete Freund's adjuvant as previously described (31). The IgG fraction was purified by HiTrap protein G affinity chromatography per the manufacturer's instructions (Amersham Pharmacia Biotech). Eluted fractions from the column were concentrated using Centriprep YM-10 (Millipore) and buffer exchanged to PBS using PD-10 columns. Specific antibody was obtained after additional IL-17 affinity chromatography. The concentration of purified antibody was determined using a standard protein assay. Polyclonal antibody to murine CXCR2 was produced by the immunization of goats at multiple intradermal sites with murine CXCR2 peptide mixed with complete Freund's adjuvant as previously described (32, 33). The peptide sequence Met-Gly-Glu-Phe-Lys-Val-Asp-Lys-Phe-Asn-Ile-Glu-Asp-Phe-Phe-Ser-Gly has been shown to contain the ligand-binding portion of CXCR2. This peptide-specific antibody does not deplete PMN (34). Purified antibody was obtained after HiTrap and CXCR2 affinity chromatography of the antiserum as previously described.
Effect of IL-17 and CXCR2-specific Neutralizing Antibody Treatment on Adhesion Formation.
For neutralization experiments, C57BL/6 mice were injected with increasing doses of affinity-purified IL-17 or CXCR2-specific antibodies (10, 50, or 100 mg of antibody per animal) via the intraperitoneal route at the time of cecal abrasion surgery and 6 h thereafter. Control groups were given 50 mg affinity-purified rabbit or goat IgG. All groups of mice were killed after 6 d and assessed for adhesion formation.
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Results |
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Role of T Cells in Adhesions Induced by Intraabdominal Sepsis.
To address the role of T cells in adhesions induced by an infectious process, we investigated whether adhesions induced during intraabdominal sepsis would develop in ßTCR-/- mice. Wild-type and
ßTCR-/- mice were challenged intraperitoneally with a sublethal cecal contents inoculum. Wild-type mice developed adhesions after challenge (median score = 3.5; Fig. 2 E), whereas
ßTCR-/- mice did not (median score = 0, P < 0.001; Fig. 2 E).
CD4+ T Cells Are the Major T Cell Subset Mediating Adhesion Formation.
To further characterize the T cell phenotype that mediates surgical adhesion formation, mice were depleted of CD4+ or CD8+ T cells with a CD4-specific (GK1.5) or a CD8-specific mAb (53-6.7). Treatment with CD4-specific mAb resulted in a significant reduction in adhesion formation compared with control antibodytreated mice, whereas treatment with CD8-specific mAb had no such effect (Fig. 3
A). Mice deficient in CD4+ T cells had lower median adhesion scores than wild-type animals (P = 0.002), whereas CD8-/- mice had scores comparable with those of wild-type animals (Fig. 3 B). Similarly, CD4-/- mice challenged intraperitoneally with a sublethal cecal contents inoculum failed to develop adhesions (median score = 0), whereas wild-type animals developed adhesions (median score = 2, P = 0.018). These results clearly indicate that the CD4+ ß T cell is the main phenotype mediating surgical and postinfectious adhesion formation in rodents.
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To address this question, we performed microscopic cell counts and FACS® analyses to determine the temporal kinetics of peritoneal cellular infiltration in mice after cecal abrasion surgery. After peritoneal injury, PMN accumulated within 6 h in the peritoneal cavity, becoming a dominant cell type at this site (Fig. 5 A), whereas there was approximately a fourfold increase in the number of T cells at this time interval. Macrophages entered the peritoneal cavity at 24 h and predominated at 4872 h, whereas the number of T cells continued to rise. By 72 h after surgery, macrophages and T cells were the predominant cell types in the peritoneal cavity. FACS® analysis of the cellular infiltrate showed that T cells entering the peritoneal cavity at 6 h expressed the activation marker CD69 compared with control animals that underwent laparotomy without cecal abrasion (Fig. 5 B). The finding that T cells are rapidly activated after peritoneal injury suggests a role for these cells in producing soluble mediators that coordinate cellular trafficking.
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T Cells Play an Important Role in CXC Chemokine Production during Adhesiogenesis.
To demonstrate the role of T cells in the production of CXC chemokines induced by cecal abrasion, the levels of KC and MIP-2 in the peritoneal fluid of ßTCR-/- mice and littermate wild-type animals that underwent cecal abrasion surgery were compared. 6 h after surgery,
ßTCR-/- mice showed significantly lower levels of KC and MIP-2 than wild-type animals (P = 0.013 and 0.001; respectively, Fig. 6 C).
Neutralization of IL-17 Reduces Adhesion Formation after Cecal Abrasion.
To demonstrate the role of IL-17 produced by activated CD4+ T cells in the development of postsurgical adhesions, we tested the effect of neutralization of IL-17 with specific antibody on adhesion formation after cecal abrasion. Although the administration of increasing concentrations of IL-17specific IgG resulted in the dose-dependent blockade of adhesiogenesis, it did not have this effect in animals treated with normal rabbit IgG (Fig. 7 A).
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Discussion |
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Postoperative adhesion formation in the peritoneal and pelvic cavities is a fibrotic tissue disorder manifested by excessive deposition of fibrin during normal wound healing. It is currently believed that a breakdown in the balance between fibrin deposition (fibrinogenesis) and fibrin degradation (fibrinolysis) during wound healing leads to this surgical complication (911). Despite a long-term research effort in this area, there is still little understanding of the cellular host response governing this process.
Recent studies have clearly shown that T cells play a critical role in orchestrating early events that lead to different inflammatory host tissue responses, such as experimental autoimmune encephalomyelitis, idiopathic pulmonary fibrosis, progressive systemic sclerosis, myocarditis, hepatic fibrosis, experimental colitis, and granuloma formation (1719, 4347). Intraabdominal abscess formation is another example of a T cellmediated host defense mechanism. CD4+ T cell activation by the capsular polysaccharides of Bacteroides fragilis and Staphylococcus aureus mediates intraabdominal abscess formation in a rodent model (48). Data from these collective studies led us to hypothesize that T cells and T cellderived cytokines and chemokines mediate surgical adhesion formation. We addressed this question using T cell depletion and T cell transfer experiments in rats and similar experiments with knockout mice genetically deficient in specific T cell subsets. The significant reduction in adhesion formation after cecal abrasion in T celldepleted animals or ßTCR-/- mice indicated that T cells play a definite role in the development of adhesions.
A standard surgical scoring system was used to assess the development of adhesions in rodents. This scoring system is widely used because clinical and experimental experience shows that adhesion formation is difficult to assess by quantitative assays that measure fibrin deposition, which do not accurately reflect the organization and number of defined fibrinous structures in people or animals. Therefore, it is generally accepted that visual determination of the degree of disease is the superior method to assess adhesion formation. This method has been adopted by the American Society for Reproductive Medicine in the evaluation of adhesion formation in clinical trials.
Intraabdominal sepsis is a known cause of peritoneal adhesion formation. However, the mechanism by which infection induces adhesions in this scenario is not clear (9). To address this question, we developed an adhesion model that mimicked a bacterial contamination arising from a colonic source. The significant reduction of adhesion formation in ßTCR-/- and CD4-/- mice indicated that in addition to surgical adhesion formation, CD4+ T cells play a critical role in postinfectious adhesion formation.
Additional studies using depleting mAbs and knockout mice demonstrated that CD4+ T cells contribute to postsurgical adhesion formation. This finding was confirmed by adoptive transfer experiments documenting the ability of T cell populations enriched for CD4+ T cells to transfer the capacity for adhesion formation to ßTCR-/- mice. CD4+ T cells can be assigned to two subsets, Th1 and Th2, according to the cytokines they produce. Experiments with Stat4 and Stat6 knockout mice showed that Th1 CD4+ cells are responsible for the development of postsurgical adhesions.
Surgical trauma to the peritoneum elicits a rapid but transient influx of PMN into the peritoneal cavity with the subsequent accumulation of macrophages (1214). Our studies of the kinetics of peritoneal inflammatory cell infiltration after cecal abrasion revealed a striking influx of activated T cells homing to this site after trauma. The number of T cells continued to increase in the peritoneum of mice until the experiment was terminated 72 h after surgery. We also observed early PMN infiltration at 6 h and associated macrophage influx at 24 h that has been reported in previous investigations of the cellular response associated with adhesion formation (12, 13). The finding that T cells are also a major component of the local inflammatory response has not been reported and led us to investigate the role of the T cellderived proinflammatory cytokine IL-17 in this process.
IL-17 is produced by activated T cells and has been shown to induce the release of MIP-2 and KC and regulate PMN infiltration in T cellmediated inflammatory processes (22, 23). We found that a significant number of T cells entering the peritoneal cavity shortly after cecal abrasion expressed activation marker CD69. Increased levels of IL-17 were found in the peritoneal cavity after cecal abrasion surgery. Intracellular cytokine staining and FACS® analysis showed that T cells were the only cell type present in the peritoneal cavity after cecal abrasion surgery that produces IL-17. The administration of a neutralizing antibody specific for IL-17 significantly reduced adhesion formation. These data demonstrate the critical role of this T cellderived cytokine in adhesion formation.
Because IL-17 has been shown to elicit the CXC chemokines MIP-2 and KC from peritoneal mesothelial cells during inflammation, we investigated whether these chemokines play a role in adhesion formation. We found increased levels of both MIP-2 and KC after cecal abrasion. Furthermore, significantly lower levels of these chemokines in ß T celldeficient animals confirmed the role of T cells in the production of these chemokines associated with adhesion formation. The administration of an antibody specific for the receptor that binds these chemokines (CXCR2) significantly reduced adhesion formation.
Adhesion formation is a serious yet common host response that is difficult to prevent or treat. On the basis of our findings, we propose that activated CD4+ ß T cells with a Th1 phenotype home to the peritoneal cavity after peritoneal injury and orchestrate this host response, which leads to the development of adhesions. These data demonstrate for the first time that T cells play a role in this disease process and suggest a new strategy for preventing this type of postsurgical and postinfectious complication.
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Acknowledgments |
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This work was supported in part by National Institutes of Health/NIGMS grant 6M64805-01 to A.O. Tzianabos.
Submitted: January 7, 2002
Revised: April 2, 2002
Accepted: April 29, 2002
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Footnotes |
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References |
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