Augmentation of Valpha14 NKT cell-mediated cytotoxicity by interleukin 4 in an autocrine mechanism resulting in the development of concanavalin A-induced hepatitis

Abstract

The administration of concanavalin A (Con A) induces a rapid severe injury of hepatocytes in mice. Although the Con A-induced hepatitis is considered to be an experimental model of human autoimmune hepatitis, the precise cellular and molecular mechanisms that induce hepatocyte injury remain unclear. Here, we demonstrate that Valpha14 NKT cells are required and sufficient for induction of this hepatitis. Moreover, interleukin (IL)-4 produced by Con A-activated Valpha14 NKT cells is found to play a crucial role in disease development by augmenting the cytotoxic activity of Valpha14 NKT cells in an autocrine fashion. Indeed, short-term treatment with IL-4 induces an increase in the expression of granzyme B and Fas ligand (L) in Valpha14 NKT cells. Moreover, Valpha14 NKT cells from either perforin knock-out mice or FasL-mutant gld/gld mice fail to induce hepatitis, and hence perforin-granzyme B and FasL appear to be effector molecules in Con A-induced Valpha14 NKT cell-mediated hepatocyte injury.

Figure 1

Elevation of serum transaminase activities after intravenous injection of Con A. Serum transaminase levels in B6 wild-type (•), Vα14 NKT KO (□), and RAG KO (▵) mice were assessed at different times after Con A (0.5 mg) administration. Three mice were used in each group. The mean transaminase activities (Karmen units per liter [K.U./l]) of ALT (A) and AST (B) of triplicate samples with standard errors are depicted.

Figure 2

Restoration of Con A–induced hepatitis by Vα14 NKT cells in Vα14 NKT KO mice. (A) TCR-β/NK1.1 profiles of liver mononuclear cells of Vα14 NKT KO mice 9 h after adoptive transfer of 2 × 10⁷ spleen cells from Vα14 NKT mice are shown. The percentages of cells present in each area are shown in each panel. (B) The rearranged Vα14Jα281 gene was assessed by genomic PCR with specific primers detecting Vα14Jα281. Vα14 NKT KO mice were intrasplenically injected with the indicated numbers of freshly prepared whole spleen cells from Vα14 NKT mice (C) or Vα14 NKT cell line established from spleen cells (spl.) of Vα14 NKT mice (D). 1 h after cell transfer, the mice were administered Con A (0.5 mg; Stim.) intravenously. The serum ALT and AST activities were measured 8 h after Con A injection. Three mice were used in each group, and the serum samples were analyzed individually. The mean values of triplicate samples with standard errors are shown.

Figure 2

Restoration of Con A–induced hepatitis by Vα14 NKT cells in Vα14 NKT KO mice. (A) TCR-β/NK1.1 profiles of liver mononuclear cells of Vα14 NKT KO mice 9 h after adoptive transfer of 2 × 10⁷ spleen cells from Vα14 NKT mice are shown. The percentages of cells present in each area are shown in each panel. (B) The rearranged Vα14Jα281 gene was assessed by genomic PCR with specific primers detecting Vα14Jα281. Vα14 NKT KO mice were intrasplenically injected with the indicated numbers of freshly prepared whole spleen cells from Vα14 NKT mice (C) or Vα14 NKT cell line established from spleen cells (spl.) of Vα14 NKT mice (D). 1 h after cell transfer, the mice were administered Con A (0.5 mg; Stim.) intravenously. The serum ALT and AST activities were measured 8 h after Con A injection. Three mice were used in each group, and the serum samples were analyzed individually. The mean values of triplicate samples with standard errors are shown.

Figure 2

Restoration of Con A–induced hepatitis by Vα14 NKT cells in Vα14 NKT KO mice. (A) TCR-β/NK1.1 profiles of liver mononuclear cells of Vα14 NKT KO mice 9 h after adoptive transfer of 2 × 10⁷ spleen cells from Vα14 NKT mice are shown. The percentages of cells present in each area are shown in each panel. (B) The rearranged Vα14Jα281 gene was assessed by genomic PCR with specific primers detecting Vα14Jα281. Vα14 NKT KO mice were intrasplenically injected with the indicated numbers of freshly prepared whole spleen cells from Vα14 NKT mice (C) or Vα14 NKT cell line established from spleen cells (spl.) of Vα14 NKT mice (D). 1 h after cell transfer, the mice were administered Con A (0.5 mg; Stim.) intravenously. The serum ALT and AST activities were measured 8 h after Con A injection. Three mice were used in each group, and the serum samples were analyzed individually. The mean values of triplicate samples with standard errors are shown.

Figure 3

Development of Con A–induced hepatitis in Vα14 NKT mice. (A) Serum transaminase levels in B6 and Vα14 NKT mice 12 h after Con A administration (0.75 mg). All mice died within 24 h (†). The mean values of three samples with standard errors are shown. (B) Light micrographs of the liver with hematoxylin and eosin staining (×200 and ×400) are shown. The indicated mice were treated with 0.75 mg of Con A and killed 8 h later. Arrows indicate massive necrosis observed in the liver; arrowheads indicate mononuclear cell infiltration. WT, wild-type.

Figure 3

Development of Con A–induced hepatitis in Vα14 NKT mice. (A) Serum transaminase levels in B6 and Vα14 NKT mice 12 h after Con A administration (0.75 mg). All mice died within 24 h (†). The mean values of three samples with standard errors are shown. (B) Light micrographs of the liver with hematoxylin and eosin staining (×200 and ×400) are shown. The indicated mice were treated with 0.75 mg of Con A and killed 8 h later. Arrows indicate massive necrosis observed in the liver; arrowheads indicate mononuclear cell infiltration. WT, wild-type.

Figure 4

Production of IL-4 and IFN-γ from Con A–activated Vα14 NKT cells. Whole spleen cells from B6, Vα14 NKT KO, Vα14 NKT, and RAG KO mice (A) or the Vα14 NKT cell line and conventional T cell line (B) were stimulated in vitro with Con A (10 μg/ml). 24 h later, the amounts of IL-4 or IFN-γ in the culture supernatants were measured by ELISA. Mean values in triplicate samples with standard deviations are shown. n.d., not detectable.

Figure 5

Requirement of IL-4 produced by Vα14 NKT cells for Con A–induced hepatitis. (A) MACS-sorted NK1.1⁺ spleen cells (10⁷) from B6, IL-4 KO, or IFN-γ KO mice were transferred intrasplenically into Vα14 NKT KO mice. 1 h later, the mice were injected with Con A (0.5 mg). The serum activities of ALT and AST were assessed 8 h after Con A injection. (B) Induction of Con A–induced hepatitis by adoptive transfer of cells from the Vα14 NKT cell line (2 × 10⁶). Three mice were used in each group. The mean values in triplicate with standard errors are shown.

Figure 6

Augmentation of the cytotoxic activity of Vα14 NKT cells by short-term incubation with IL-4 in vitro. (A) Flow cytometric analysis of the expression of IL-4 and IFN-γRs on freshly isolated Vα14 NKT cells. (B) Spleen cells from Vα14 NKT mice were cultured for 8 h in the presence of 100 U/ml of IL-4 or 100 U/ml of IFN-γ. The cells were washed extensively and subjected to the standard ⁵¹Cr-release cytotoxic assay in triplicate on target TLR2 liver cells. (C) Augmented expression of mRNAs of granzyme B and FasL in Vα14 NKT cells upon stimulation with IL-4.

Figure 7

Requirement of FasL expression and perforin production from Vα14 NKT cells for Con A–induced hepatitis. MACS-sorted NK1.1⁺ spleen cells (10⁷) from B6, B6-gld/gld, or perforin KO mice were transferred intrasplenically into Vα14 NKT KO mice. 1 h later, the mice were injected with Con A (0.5 mg). 8 h later, the serum activities of ALT and AST were assessed. The mean values of triplicate samples with standard errors are shown.