Aberrant T-cell ontogeny and defective thymocyte and colonic T-cell chemotactic migration in colitis-prone Galphai2-deficient mice

Abstract

Galphai2-deficient mice, which spontaneously develop colitis, have previously been reported to have an increased frequency of mature, single positive thymocytes compared to wild-type mice. In this study we further characterized the intrathymic changes in these mice before and during overt colitis. Even before the onset of colitis, Galphai2(-/-) thymi weighed less and contained fewer thymocytes, and this was exacerbated with colitis development. Whereas precolitic Galphai2(-/-) mice had unchanged thymocyte density compared to Galphai2(+/-) mice of the same age, this was significantly decreased in mice with colitis. Thymic atrophy in Galphai2(-/-) mice involved mainly the cortex. Using a five-stage phenotypic characterization of thymocyte maturation based on expression of CD4, CD8, TCRalphabeta, CD69 and CD62L, we found that both precolitic and colitic Galphai2(-/-) mice had significantly increased frequencies of mature single-positive CD4(+) and CD8(+) medullary thymocytes, and significantly reduced frequencies and total numbers of immature CD4(+) CD8(+) double-positive thymocytes compared to Galphai2(+/-) mice. Furthermore, cortical and transitional precolitic Galphai2(-/-) thymocytes showed significantly reduced chemotactic migration towards CXCL12, and a trend towards reduced migration to CCL25, compared to wild-type thymocytes, a feature even more pronounced in colitic mice. This impaired chemotactic migration of Galphai2(-/-) thymocytes could not be reversed by increased chemokine concentrations. Galphai2(-/-) thymocytes also showed reduced expression of the CCL25 receptor CCR9, but not CXCR4, the receptor, for CXCL12. Finally, wild-type colonic lamina propria lymphocytes migrated in response to CXCL12, but not CCL25 and, as with thymocytes, the chemokine responsiveness was significantly reduced in Galphai2(-/-) mucosal lymphocytes.

Figure 1

Differences in (a) thymic weight (mg), (b) thymocyte numbers and (c) cellular density (cells/mg) between Gαi2^+/– mice (n = 7), precolitic Gαi2^–/– mice (n = 6) and colitic Gαi2^–/– mice (n = 5) aged 5–11 weeks. Bars represent mean value ± SD where *P = 0·05, **P = 0·01.

Figure 2

Changes in thymus medulla and cortex area with age. Thymi from 6-, 10-, 13-, 18- and 21-week-old Gαi2^–/– (light bars) and Gαi2^+/– (dark bars) mice were fixed in 4% buffered formalin and the medullary and cortical area of two or three 5-μm H&E-stained cross-sections per thymic lobe was calculated. Bars represent mean area (mm²) of medulla or cortex in one lobe ± SD of three to five mice per group. *P = 0·05.

Figure 3

Frequency (a) and total number (b) of immature thymocytes in precolitic and colitic Gαi2^–/– mice compared to Gαi2^+/– mice. Developmental stages of thymocytes are shown as follows: 1–2, CD4⁺ CD8⁺ TCRαβ^low/– CD69^– (cortical); 3, CD4⁺ CD8⁺ TCRαβ⁺ CD69⁺ (transitional between medulla and cortex), 4, CD4⁺ CD8^– TCRαβ⁺ CD69⁺ CD62L^low/– (medullary); and 5, CD4⁺ CD8^– TCRαβ⁺ CD69^– CD62L^high (medullary) as determined by FACS analysis. Results are shown as mean values of: Gαi2^+/– (n = 14), precolitic Gαi2^–/– (n = 10) and colitic Gαi2^–/– (n = 6) ± SD using 5- to 7-week-old-mice.

Figure 4

Migration of thymocytes from Gαi2^+/– (n = 6) and precolitic Gαi2^–/– (n = 6) mice in response to chemokines during their maturation process. Values are shown as per cent migration of different thymocyte subpopulations in response to (a) CXCL12 (SDF-α) (50 nm), (b) CCL21 (SLC) (100 nm), (c) CCL25 (TECK) (200 nm), and (d) CCL19 (MIP-3β) (10 nm). Background migration (e) (migration to medium alone, in the absence of chemokine) shows spontaneous migration from both Gαi2^–/– and Gαi2^+/– thymocytes. Developmental stages shown are as follows: 1–2, CD4⁺ CD8⁺TCRαβ^low/– CD69^– (cortical); 3, CD4⁺ CD8⁺ TCRαβ⁺ CD69⁺ (transitional between medulla and cortex); 4, CD4⁺ CD8^– CD69⁺ CD62L^low/– (early medullary); and 5, CD4⁺ CD8^– CD69^– CD62L^high (late medullary). The cells were counted and stained for four-colour flow cytometry both before and after migration in response to the indicated chemokine as follows: anti-CD4-APC, anti-CD8-PerCP, anti-CD69-PE and anti-CD62L-FITC or anti-TCRαβ-FITC. Results are demonstrated as mean value (% of input) of fluorescence-positive cells ± SD from six independent experiments. Results in (a) to (d) represent total migration minus background migration. *P = 0·05; **P = 0·01 between migrated Gαi2^–/– and Gαi2^+/– thymocytes.

Figure 5

Frequencies (a,c) and median fluorescence intensity (MFI) (b,d) of CCR9 (a,b) and CXCR4 (c,d) expression on thymocytes in maturation stage 1–4 (defined on figure). Cells were stained for four-colour flow cytometry as follows: anti-CD4-APC, anti-CD8-PerCP, anti-CD69-PE and anti-CXCR4-FITC or anti-CCR9-FITC. Gαi2^+/– mice were used as controls (CCR9, n = 11; CXCR4, n = 17); pre-colitic Gαi2^–/– mice (CCR9, n = 4; CXCR4, n = 9) and colitic Gαi2^–/– mice (CCR9, n = 4; CXCR4, n = 6). Bars represent mean value ± SD where *P = 0·05 and **P = 0·01.

Figure 6

Migration of colonic lamina propria lymphocytes from Gαi2^+/+ (n = 8, two or three mice pooled in three different experiments) and precolitic Gαi2^–/– (n = 3) mice in response to CCL25 and CXCL12. Values are shown as differences between total chemokine-induced migration minus spontaneous migration of CD4⁺ T lymphocytes and B lymphocytes. Cells were counted and stained for two-colour flow cytometry with anti-CD4-APC and anti-CD45R/B220-PE both before and after migration in response to the indicated chemokine. Results are demonstrated as ratio ± SD from three independent experiments. *P = 0·05 between Gαi2^–/– and Gαi2^+/+ colonic lamina propria lymphocytes.