Amelioration of 2,4,6-trinitrobenzene sulphonic acid induced colitis in angiotensinogen gene knockout mice

Abstract

Background: A number of recent studies have demonstrated a protective effect of renin-angiotensin system (RAS) antagonism against immune mediated diseases such as myocarditis, chronic allograft rejection, and antiglomerular basement membrane nephritis. To our knowledge, there has been no report on the immunological contribution of the RAS in colonic tissue.

Aims: We evaluated the direct effect of angiotensin II (AII) on the pathogenesis of immune mediated colitis using angiotensinogen deficient homozygous (Atg-/-) mice.

Subjects: 2,4,6-Trinitrobenzene sulphonic acid (TNBS) colitis was induced in Atg-/- and wild-type (Atg+/+) mice.

Methods: Levels of proinflammatory cytokines in the colon were determined by enzyme linked immunosorbent assay. Histological analysis was performed simultaneously.

Results: Although Atg-/- mice developed colitis, the degree was much milder than that in Atg+/+ mice (p<0.05). Colonic cytokine analysis showed that the production of proinflammatory cytokines (interleukin (IL)-1beta, interferon gamma (IFN-gamma)) was impaired in Atg-/- mice. Furthermore, expression of cytokines such as IL-4 and IL-10 in the colon was predominant in Atg-/- compared with Atg+/+ mice after TNBS instillation (p<0.005, p<0.01, respectively). Similarly, subcutaneous infusion of losartan suppressed colitis (p<0.05) and the production of proinflammatory cytokines (IL-1beta, IFN-gamma). These results indicate that the RAS is directly involved in the pathogenesis of TNBS colitis through regulation of proinflammatory and anti-inflammatory cytokines in the colon.

Conclusions: This study revealed that the RAS is involved in the immune system in the colon. Antagonism of the RAS is a potential prophylactic strategy for the treatment of human inflammatory bowel disease.

Figure 1

Changes in body weight after administration of phosphate buffered saline (PBS) or 2,4,6-trinitrobenzene sulphonic acid (TNBS). Results are shown for PBS instilled wild-type mice (Atg+/+) (n = 9) and angiotensinogen gene knockout mice (Atg−/−) (n = 7) and for TNBS instilled Atg+/+ (n = 13) and Atg−/− (n = 12) mice. Results are expressed as mean (SEM). *p<0.05, **p<0.01, ***p<0.001 versus PBS instilled Atg+/+ mice; †p<0.05, ††p<0.01 versus TNBS instilled Atg−/− mice.

Figure 2

Levels of cytokines in the colon assayed by ELISA. tumour necrosis factor α (TNF-α) (A), interleukin (IL)-1β (B), interferon γ (IFN-γ) (C), IL-4 (D), and IL-10 (E) levels were measured three days after administration of phosphate buffered saline (PBS) or 2,4,6-trinitrobenzene sulphonic acid (TNBS). Bars represent PBS instilled wild-type mice (Atg+/+) (n = 8), PBS instilled angiotensinogen gene knockout mice (Atg−/−) (n = 7), TNBS instilled Atg+/+ (n = 13), and TNBS instilled Atg−/− (n = 12), as indicated. To investigate the proinflammatory and anti-inflammatory cytokine balance, the IFN-γ/IL-4 ratio was also calculated (F). The IFN-γ level of each mouse was divided by the IL-4 level of the same mouse and the mean calculated. Results are expressed as mean (SEM). *p<0.05, **p<0.01, ***p<0.005.

Figure 3

Histological findings in the colon three days after administration of phosphate buffered saline (PBS) or 2,4,6-trinitrobenzene sulphonic acid (TNBS). (C, D) TNBS instilled wild-type mice (Atg+/+), (G, H) TNBS instilled angiotensinogen gene knockout mice (Atg−/−), (A, B) PBS instilled Atg+/+ (E, F), and PBS instilled Atg−/− mice. Magnification: A, C, E, G ×25; B, D, F, H ×100.

Figure 4

Histological damage score of the colon. Severity of inflammation was scored as described in materials and methods. Bars represent phosphate buffered saline (PBS) instilled wild-type mice (Atg+/+) (n = 5), PBS instilled angiotensinogen gene knockout mice (Atg−/−) (n = 4), 2,4,6-trinitrobenzene sulphonic acid (TNBS) instilled Atg+/+ (n = 13), and TNBS instilled Atg−/− (n = 9), as indicated. *p<0.05, **p<0.005.

Figure 5

Changes in body weight after administration of phosphate buffered saline (PBS) or 2,4,6-trinitrobenzene sulphonic acid (TNBS). The groups were: PBS instilled 0.9% normal saline (NS) treated (n = 5) mice, PBS instilled losartan (Lsr) treated (n = 5) mice, TNBS instilled NS treated (n = 7) mice, and TNBS instilled Lsr treated (n = 9) mice. Results are expressed as mean (SEM). *Significant difference versus PBS instilled NS treated mice; †significant difference versus PBS instilled Lsr treated mice.

Figure 6

Results of oral administration of losartan (Lsr) after 2,4,6-trinitrobenzene sulphonic acid (TNBS) instillation. Body weight change (A) and interleukin 10 (IL-10) levels in the colon (B) are shown. (A) The groups were: phosphate buffered saline (PBS) instilled 0.9% normal saline (NS) treated (n = 5) mice, PBS instilled Lsr treated (n = 5) mice, TNBS instilled NS treated (n = 8) mice, and TNBS instilled Lsr treated (n = 11) mice. Results are expressed as mean (SEM). *Significant difference versus PBS instilled NS treated mice; †significant difference versus PBS instilled Lsr treated mice;‡ significant difference versus TNBS instilled Lsr treated mice. (B) IL-10 levels in NS treated PBS instilled (n = 5), NS treated TNBS instilled (n = 8), Lsr treated PBS instilled (n = 5), and Lsr treated TNBS instilled (n = 11) mice, as indicated. *p<0.05.

Figure 7

Levels of cytokines in the colon in the subcutaneous administration study. tumour necrosis factor α (TNF-α) (A), interleukin (IL)-1β (B), interferon γ (IFN-γ) (C), IL-4 (D), and IL-10 (E) levels were assayed by ELISA three days after administration of phosphate buffered saline (PBS) or 2,4,6-trinitrobenzene sulphonic acid (TNBS). Bars represent 0.9% normal saline (NS) treated PBS instilled (n = 5), NS treated TNBS instilled (n = 7), losartan (Lsr) treated PBS instilled (n = 5), and Lsr treated TNBS instilled (n = 9) mice, as indicated. The IFN-γ/IL-4 ratio was also calculated (F). The IFN-γ level of each mouse was divided by the IL-4 level of the same mouse and the mean calculated. Results are expressed as mean (SEM). *p<0.05.

Figure 8

Histological findings in the colon three days after administration of phosphate buffered saline (PBS) or 2,4,6-trinitrobenzene sulphonic acid (TNBS) in the subcutaneous administration study. (A, B) 0.9% Normal saline (NS) treated PBS instilled mice, (C, D) NS treated TNBS instilled mice, (E, F) losartan (Lsr) treated PBS instilled mice, and (G, H) Lsr treated TNBS instilled mice. Magnification: A, C, E, G ×25; B, D, F, H ×100.

Figure 9

Histological damage score of the colon in the subcutaneous administration study. Severity of inflammation was scored as described in materials and methods. Bars represent 0.9% normal saline (NS) treated phosphate buffered saline (PBS) instilled (n = 5) mice, NS treated 2,4,6-trinitrobenzene sulphonic acid (TNBS) instilled (n = 7) mice, losartan (Lsr) treated PBS instilled (n = 5) mice, and Lsr treated TNBS instilled (n = 9) mice, as indicated. *p<0.05.