Beta7 integrin deficiency suppresses B cell homing and attenuates chronic ileitis in SAMP1/YitFc mice

Abstract

Lymphocyte recruitment to intestinal tissues depends on β(7) integrins. In this study, we studied disease severity and lymphocyte recruitment into the small intestine in SAMP1/YitFc mice, which develop chronic ileitis with similarity to human Crohn's disease. To assess the role of β(7) integrins in chronic ileitis, we generated SAMP1/YitFc lacking β(7) integrins (SAMP1/YitFc Itgb7(-/-)) using a congenic strain developed via marker-assisted selection. We analyzed ileal inflammation in SAMP1/YitFc and SAMP1/YitFc Itgb7(-/-) mice by histopathology and the distribution of T and B lymphocytes in the mesenteric lymph nodes (MLNs) by flow cytometry. Short-term (18 h) adoptive transfer experiments were used to study the in vivo homing capacity of T and B lymphocytes. In both young (<20 wk) and old (20-50 wk) SAMP1/YitFc Itgb7(-/-) mice, ileitis was reduced by 30-50% compared with SAMP1/YitFc mice. SAMP1/YitFc Itgb7(-/-) mice showed a dramatic 67% reduction in the size of their MLNs, which was caused by a 85% reduction in lymphocyte numbers and reduced short-term B cell homing. Flow cytometric analysis revealed a highly significant decrease in the percentage of B cells in MLNs of SAMP1/YitFc Itgb7(-/-) mice. Cotransfer of SAMP1/YitFc MLN B cells but not SAMP1/YitFc Itgb7(-/-) MLN B cells along with CD4(+) T cells resulted in exacerbated ileitis severity in SCID mice. Our findings suggest that β(7) integrins play an essential role in spontaneous chronic ileitis in vivo by promoting homing of disease-exacerbating B cells to MLNs and other intestinal tissues.

FIGURE 1

Genetic deficiency of β₇ integrin results in attenuation of spontaneous chronic ileitis in SAMP1/YitFc mice. Inflammatory indices at 8–19 and 20–50 wk assessed as flattening or widening of the normal mucosal villous architecture [villus distortion index (VI)] (A), neutrophilic infiltration to the LP [active inflammatory index (AI)] (B), lymphocyte, plasma cell, and macrophage infiltration [chronic inflammatory index (CI)] (C), and total inflammatory index (VI+AI+CI) (D). E, Representative histopathological micrograph from terminal ileum of SAMP1/YitFc (left panel) and SAMP1/YitFc Itgb7^−/− (right panel) in late ileitis. H&E, original magnification ×20. Data are mean ± SD (n = 23, 8–19-wk-old, and n = 15, 20–50-wk-old SAMP1/YitFc; and n = 15, 8–19-wk-old, and n = 10, 20–50-wk-old SAMP1/YitFc Itgb7^−/− mice). Scale bar, 100 μm. **p < 0.01 by two-tailed t test.

FIGURE 2

β₇ integrin deficiency results in reduction of cellular content and size of MLNs in SAMP1/YitFc mice. A, Representative photograph of the MLN from 28-wk-old SAMP1/YitFc and 32-wk-old SAMP1/YitFc Itgb7^−/− mice. B, Weights of MLNs from 24–36-wk-old SAMP1/YitFc and SAMP1/YitFc Itgb7^−/− mice. Absolute cell number of MLNs and spleen from <20-wk-old (C) and 24–36-wk-old (D) SAMP1/YitFc and SAMP1/YitFc Itgb7^−/− mice. Data are mean ± SD (n = 3–6). **p < 0.01; ***p < 0.001 by two-tailed t test.

FIGURE 3

Expanded B cell population in SAMP1/YitFc MLN attenuated in SAMP1/YitFc Itgb7^−/− mice. Spleen (left panels) and MLNs (right panels) were obtained from 30-wk-old SAMP1/YitFc and SAMP1/YitFc Itgb7^−/− mice. PE-CD19⁺ (B cells) and FITC-CD3⁺ (T cells) among all CD45⁺ cells after eliminating doublet cells by pulse width and dead cells by using aqua dead cell stain (A). Percentage of MLN B cells from SAMP1/YitFc and SAMP1/YitFc Itgb7^−/− mice in early (young) 12–19-wk-old and late (old) 24–36-wk-old stages of ileitis. Percentage of CD19⁺ B cells and CD3⁺ T lymphocytes from spleen (n = 8, 5) (B) and MLN (n = 6, 5) (C) of young mice (12–19 wk old) and spleen (n = 4) (D), MLNs (n = 4) (E), and PPs (n = 4–9) (F) of old SAMP1/YitFc mice and SAMP1/YitFc Itgb7^−/− mice determined by flow cytometry. G, Alterations in the number of CD19⁺ and CD3⁺ cells during progression of ileitis from 24–36-wk-old SAMP1/YitFc and SAMP1/YitFc Itgb7^−/− mice calculated by multiplying total lymphocyte numbers with B and T cell percentages; area of the circle represents MLN cell number. Data are mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001 by two-tailed t test.

FIGURE 4

In vivo homing of integrin Itgb7^−/− lymphocytes to the intestine-associated lymphoid tissues is impaired. A, A representative flow cytometry plot of the competitive homing experiment from a SAMP1/YitFc recipient mouse for CD19⁺ (B cells) and CD3⁺ (T cells) among all CD45⁺ cells after eliminating doublet cells by pulse width and dead cells by using aqua dead cell stain. Competitive homing experiment comparing SAMP1/YitFc with SAMP1/YitFc Itgb7^−/− lymphocytes. B, Total of 20 × 10⁶ SAMP1/YitFc Itgb7^−/− (CFSE) splenocytes and SAMP1/YitFc (CMTMR) splenocytes were mixed and injected into SAMP1/YitFcs. Homing indices for CD19⁺ B cells (open bars) and CD3⁺ T cells (black bars) measured at 18 h. Data are mean ± SD (n = 2). *p < 0.05; **p < 0.01; ^#p < 0.01 by two-tailed t test.

FIGURE 5

Cotransfer of MLN B cells from SAMP1/YitFc Itgb7^−/− mice results in less ileitis severity in the CD4⁺ T cells adoptive transfer model compared with cotransfer of MLN B cells from SAMP1/YitFc mice. A total of 5 × 10⁵ CD4⁺ T cells from pooled SAMP1/YitFc MLNs were injected i.p. into SCID mice either alone (n = 6) or in combination with 2 × 10⁶ SAMP1/YitFc Itgb7^−/− (n = 5) or SAMP1/YitFc (n = 6) MLN B cells. Data are mean ± SD. A, After 6 wk, SCID mice were killed, terminal ilea stained with H&E, and scored by a blinded pathologist. B, Representative H&E histopathological micrographs from terminal ileum of SCID recipient mice receiving MLN SAMP CD4⁺ T cells only or in combination with MLN B cells from SAMP1/YitFc or SAMP1/YitFc Itgb7^−/− mice. Scale bar, 100 μm; original magnification ×10. *p < 0.05; **p < 0.01 by one-tailed t test.