Functional Th1 cells are required for surgical adhesion formation in a murine model

Abstract

Tissue trauma in the peritoneal and pelvic cavities following surgery or bacterial infection results in adhesions that are a debilitating cause of intestinal obstruction, chronic pelvic pain, and infertility in women. We recently demonstrated that CD4(+) alphabeta T cells are essential for development of this process. Using a murine model of experimental adhesion formation, we now demonstrate that adhesion formation is characterized by the selective recruitment of Tim-3(+), CCR5(+), CXCR3(+), IFN-gamma(+) cells, indicating the presence of a Th1 phenotype. We further demonstrate that adhesion formation is critically dependent on the function of Th1 cells because mice genetically deficient for IFN-gamma, T-bet, or treated with Abs to the Th1-selective chemoattractant IL-16 show significantly less adhesion formation than wild-type mice. In addition, disrupting the interaction of the Th1-specific regulatory molecule Tim-3, with its ligand, significantly exacerbates adhesion formation. This enhanced response is associated with increases in the level of neutrophil-attracting chemokines KC and MIP-2, known to play a role in adhesiogenesis. These data demonstrate that the CD4(+) T cells orchestrating adhesion formation are of the Th1 phenotype and delineate the central role of T-bet, Tim-3, IFN-gamma, and IL-16 in mediating this pathogenic tissue response.

FIGURE 1

Histologic examination of a representative surgical adhesion in which a portion of the small intestine has become attached to the abdominal wall. A, H&E staining. Original magnification, ×100. The adhesion (Ad) comprises the small intestine (SI), inflammatory infiltrate (I), and the remains of the abdominal wall (AW), which are indicated. The box indicates the field under higher magnification in B. B, The same adhesion under higher magnification. Bar, 100 μm. Original magnification, ×400. The smooth muscle (SM) of the intestine and the inflammatory infiltrate (I) are indicated. These results are representative of surgical adhesions obtained from the intestine, cecum, and intestinal wall, examined for each animal, from 10 different C57BL/6J mice.

FIGURE 2

CD4⁺ T cells expressing Tim-3, CCR5, and CXCR3 are present at the site of surgical adhesions. Sections were stained with anti-CD4 conjugated with Alexa Fluor 488 (green) and anti-CCR5 (A), anti-CXCR3 (B), anti-Tim-3 (C), and anti-CCR3 (D), all conjugated with Alexa Fluor 594 (red). Adhesions were examined under confocal microscopy. Bars, 20 μm. Original magnification, ×670. Cells costaining green and red are evident when the two channels are merged. These are representative images from five different mice for each receptor.

FIGURE 3

Mice deficient in T-bet expression have reduced adhesion formation due to reduced homing of IFN-γ-producing CD4⁺ T cells. A, C57BL/6J and T-bet-deficient mice were subjected to cecal abrasion surgery and adhesion formation was assessed 6 days later. *, p = 0.0422; Mann-Whitney U test. The data are combined from three independent experiments. The bars indicate median adhesion scores. B and C, CD4⁺ T cells were purified from mesenteric LN (B) or peritoneal lavage cells (C) of T-bet^−/− or C57BL/6J mice 6 days after cecal abrasion surgery, and the number of IFN-γ-producing cells was determined by ELISPOT assay. The number of IFN-γ-producing cells per 1 × 10⁵ (B) or 5 × 10⁴ (C) input CD4⁺ T cells is indicated. No CD4⁺ T cells were present in peritoneal lavage samples of control mice and therefore could not be tested. The data were generated using 10 T-bet^−/− mice and 13 control mice.

FIGURE 4

IFN-γ-deficient mice do not form detectable adhesions. C57BL/6J mice and IFN-γ-deficient mice were subjected to cecal abrasion surgery and examined and scored 6 days later for adhesions. *, p < 0.0025; Mann-Whitney U test. The bars indicate median adhesion scores. The data were generated from seven IFN-γ^−/− and control mice, respectively.

FIGURE 5

Tim-3 blockade exacerbates adhesion formation in the peritoneal cavity of WT mice. C57BL/6J mice were treated with Tim-3Ig or a control human IgG1, 250 μg s.c. at −24, 0, and 24 h relative to cecal abrasion surgery. Six days after surgery, the mice were euthanized and scored for adhesions. *, p < 0.0001; Mann-Whitney U test. The bars indicate median adhesion scores. For these experiments, 10 mice received Tim-3 Ig and 12 mice received IgG control Ab.

FIGURE 6

Tim-3 blockade increases chemokine production in the peritoneal cavity of mice following cecal abrasion. C57BL/6J mice were treated with Tim-3Ig or a control human IgG1, 250 μg s.c., once at −24 h and once at the time of cecal abrasion surgery. The mice were euthanized 6 or 24 h after surgery. MIP-2 (A) and KC (B) levels in peritoneal lavage fluid were assessed by ELISAs specific for these chemokines. The data are expressed as average values ± SD and **, p < 0.05; Student’s unpaired t test; *, p < 0.10. The averages were obtained from four different mice for each group.

FIGURE 7

Tim-3 blockade increases IL-16 production in the peritoneal cavity of mice following cecal abrasion. A, Ten C57BL/6J mice were treated with either Tim-3Ig or a control human IgG1, 250 μg s.c., once at −24 h and once at the time of cecal abrasion surgery. The mice were euthanized 24 h after surgery and IL-16 levels were assessed in the peritoneal lavage fluid by ELISA. B, Skewed murine Th1 or Th2 cells along with isolated peritoneal macrophages were cultured at 1 × 10⁶ cells/ml for 24 h in the absence or presence of 5 μg of Tim-3 Ig, 1 μg of immobilized anti-CD3 Ab, or 1 μg of LPS (for the macrophage cultures only), at which time the cell supernatants were assessed for IL-16 protein by ELISA. The data are expressed as average values ± SD and *, p < 0.05; Student’s unpaired t test.

FIGURE 8

Adhesion formation requires expression of IL-16. Sixteen C57BL/6 mice were subjected to cecal abrasion surgery and treated with 100 μg of either anti-IL-16 or control Ab at 6 h, 24 h, 3 days, and 5 days after surgery. Six days after surgery, the mice were euthanized and scored for adhesions. *, p = 0.0022; Mann-Whitney U test. The bars indicate the median adhesion score.