Suppressive role of B cells in chronic colitis of T cell receptor alpha mutant mice

Abstract

The role of antibodies (Abs) in the development of chronic colitis in T cell receptor (TCR)-alpha-/- mice was explored by creating double mutant mice (TCR-alpha-/- x immunoglobulin (Ig)mu-/-), which lack B cells. TCR-alpha-/- x Ig mu-/- mice spontaneously developed colitis at an earlier age, and the colitis was more severe than in TCR-alpha-/- mice. Colitis was induced in recombination-activating gene-1 (RAG-1-/-) mice by the transfer of mesenteric lymph node (MLN) cells from TCR-alpha-/- x Ig mu-/- mice. When purified B cells from TCR-alpha-/- mice were mixed with MLN cells before cell transfer, colitis did not develop in RAG-1-/- mice. Administration of the purified Ig from TCR-alpha-/- mice and a mixture of monoclonal autoAbs reactive with colonic epithelial cells led to attenuation of colitis in TCR-alpha-/- x Ig mu-/- mice. Apoptotic cells were increased in the colon, MLN, and spleen of TCR-alpha-/- x Ig mu-/- mice as compared to Ig mu-/- mice and TCR-alpha-/- mice. Administration of the purified Ig from TCR-alpha-/- mice into TCR-alpha-/- x Ig mu-/- mice led to decrease in the number of apoptotic cells. These findings suggest that although B cells are not required for the initiation of colitis, B cells and Igs (autoAbs) can suppress colitis, presumably by affecting the clearance of apoptotic cells.

Figure 1

(A) Mice were screened for the TCR-α and Igμ genotypes by PCR on tail DNA. In screening of Cα locus, the wild-type locus and the disrupted locus represent a 195- and a 276-bp fragment, respectively. The amplification of membrane exon of Cμ locus yields a 700- and a 900-bp fragment corresponding to the wild-type locus and the disrupted locus, respectively. The left lane indicates a molecular weight marker (bp). (B) Splenic cells (for detection of B cells) and MLN cells (for detection of T cells) from TCR-α^+/−, TCR-α^−/− , Igμ^−/−, and TCR-α^−/− × Igμ^−/− mice were analyzed by FACScan^®. TCR-α^−/− × Igμ^−/− mice show no mature B cells (B220⁺,sIgM⁺) and increased percentage of T cells, comprising CD3ε⁺TCR-β^low cells (TCR-α⁻β⁺ T cells expressing TCR-β chain without TCR-α chain on cell surface) and CD3ε⁺TCR-δ⁺ cells.

Figure 1

(A) Mice were screened for the TCR-α and Igμ genotypes by PCR on tail DNA. In screening of Cα locus, the wild-type locus and the disrupted locus represent a 195- and a 276-bp fragment, respectively. The amplification of membrane exon of Cμ locus yields a 700- and a 900-bp fragment corresponding to the wild-type locus and the disrupted locus, respectively. The left lane indicates a molecular weight marker (bp). (B) Splenic cells (for detection of B cells) and MLN cells (for detection of T cells) from TCR-α^+/−, TCR-α^−/− , Igμ^−/−, and TCR-α^−/− × Igμ^−/− mice were analyzed by FACScan^®. TCR-α^−/− × Igμ^−/− mice show no mature B cells (B220⁺,sIgM⁺) and increased percentage of T cells, comprising CD3ε⁺TCR-β^low cells (TCR-α⁻β⁺ T cells expressing TCR-β chain without TCR-α chain on cell surface) and CD3ε⁺TCR-δ⁺ cells.

Figure 2

Distal segments of large intestine from TCR-α^−/− and TCR-α^−/− × Igμ^−/− mice at 8 wk of age. The large intestine of TCR-α^−/− × Igμ^−/− mice is markedly thickened as compared to that of TCR-α^−/− mice.

Figure 3

The severity of colitis determined by histological examinations in TCR-α^−/−, Igμ^−/−, and TCR-α^−/− × Igμ^−/− mice at 4, 8, 12, and 20 wk of age maintained under specific pathogen-free conditions.

Figure 4

TCR-α^−/− × Igμ^−/− mice were intraperitoneally injected with PBS (open bar), purified Ig (2 mg, six times) from TCR-α^−/− (solid bar), TCR-α^+/− (dotted bar) mice, or a mixture of mAbs (autoAbs) reactive with colonic epithelial cells (hatched bar); and killed at 8 wk of age. The percentage of mice revealing no (normal) or severe colitis is shown. The results were obtained from groups of 10–16 mice.

Figure 5

Apoptotic cells in the colon (top) and spleen (bottom) of Igμ^−/−, TCR-α^−/−, and TCR-α^−/− × Igμ^−/− mice injected with PBS or Ig purified from sera of TCR-α^−/− mice were detected by TUNEL assay (×20 objective). All mice were 8 wk of age. The numbers in the right lower corner indicate the number of apoptotic cells per mm².

Figure 6

(A) Circulating self Ags from C57BL/6 (closed triangles), Igμ^−/− (closed squares), TCR-α^−/− (open squares), and TCR-α^−/− × Igμ^−/− mice with injection of PBS (closed circles) or Ig (open circles) purified from sera of TCR-α^−/− mice are quantified. 200 μl of sera (from various mice)/CFA were injected to C57BL/6 mice. On day 14 after immunization, the reactivity of sera from the immunized C57BL/6 mice against colonic epithelial antigens was determined by ELISA. (B and C) The reactivity of sera from the immunized C57BL/6 mice with sera of TCR-α^−/− × Igμ^−/− mice with (B) or without (C) Ig transfer was confirmed by immunohistochemical analysis using sections of colon (×40 objective) from RAG-1^−/− mice.

Figure 6

(A) Circulating self Ags from C57BL/6 (closed triangles), Igμ^−/− (closed squares), TCR-α^−/− (open squares), and TCR-α^−/− × Igμ^−/− mice with injection of PBS (closed circles) or Ig (open circles) purified from sera of TCR-α^−/− mice are quantified. 200 μl of sera (from various mice)/CFA were injected to C57BL/6 mice. On day 14 after immunization, the reactivity of sera from the immunized C57BL/6 mice against colonic epithelial antigens was determined by ELISA. (B and C) The reactivity of sera from the immunized C57BL/6 mice with sera of TCR-α^−/− × Igμ^−/− mice with (B) or without (C) Ig transfer was confirmed by immunohistochemical analysis using sections of colon (×40 objective) from RAG-1^−/− mice.