Glucocorticoid-induced TNF receptor family-related protein ligand regulates the migration of monocytes to the inflamed intestine

Abstract

Glucocorticoid-induced TNF receptor family-related protein (GITR) regulates the function of both T cells and antigen-presenting cells (APCs), while the function of GITR ligand (GITR-L) is largely unknown. Here we evaluate the role of GITR-L, whose expression is restricted to APCs, in the development of enterocolitis. On injecting naive CD4(+) T cells, GITR-L(-/-)Rag(-/-) mice develop a markedly milder colitis than Rag(-/-) mice, which correlates with a 50% reduction of Ly6C(+)CD11b(+)MHCII(+) macrophages in the lamina propria and mesenteric lymph nodes. The same result was observed in αCD40-induced acute colitis and during peritonitis, suggesting an altered monocyte migration. In line with these observations, the number of nondifferentiated monocytes was approximately 3-fold higher in the spleen of GITR-L(-/-)Rag(-/-) mice than in Rag(-/-) mice after αCD40 induction. Consistent with the dynamic change in the formation of an active angiotensin II type 1 receptor (AT1) dimer in GITR-L(-/-) splenic monocytes during intestinal inflammation, the migratory capability of splenic monocytes from GITR-L-deficient mice was impaired in an in vitro transwell migration assay. Conversely, αGITR-L reduces the number of splenic Ly6C(hi) monocytes, concomitantly with an increase in AT1 dimers. We conclude that GITR-L regulates the number of proinflammatory macrophages in sites of inflammation by controlling the egress of monocytes from the splenic reservoir.

Keywords: GITR-L; TNFSF18; experimental colitis; macrophage.

Figure 1.

Reduced colitis in GITR-L^−/−Rag^−/− mice after transfer of CD4⁺CD45RB^hi T cells. A) Splenic CD4⁺CD45RB^hi T cells from B6 mice were transferred to GITR-L^−/−Rag^−/− or Rag^−/− mice (5×10⁵ cells/mouse). Mice were weighed weekly and euthanized after 6 wk. Values represent mean ± sd weight. B, C) DAI (B) and histology scores (C) were evaluated. D) Representative histology of panel C. Original view: ×10. E–G) MLN cells of the recipient mice were activated with PMA/ionomycin in the presence of GolgiPlug for 4 h, and stained for IFNγ (E) and IL-17A (F), or stained for FoxP3 directly (G). Each open or solid circle represents 1 mouse.

Figure 2.

Reduced infiltrating MØs in GITR-L^−/−Rag^−/− mice after transfer of CD4⁺CD45RB^hi T cells. Colitis was induced in GITR-L^−/−Rag^−/− or Rag^−/− mice by the transfer of CD4⁺CD45RB^hi T cells, as described in Fig. 1. A) Colon lamina propria cells represent 2 individual experiments; each experiment was a pool of 4–5 colons. B, C) Representative staining (B) and statistical results (C) of the MLN cells. D) Number of MLN cells. Each solid or open circle represents 1 mouse.

Figure 3.

GITR-L^−/−Rag^−/− mice are resistant to αCD40-induced colitis. A) αCD40 or control rat IgG2a (200 μg/mouse) was administered by i.p. injection into Rag^−/− or GITR-L^−/−Rag^−/− mice. Mice were weighed daily and normalized to their initial weight and euthanized at d 7. B, C) DAI (B) and histology scores (C) were determined. D) Representative histology of panel C. Original view: ×10. Values represent mean ± sd weight. Each circle or square represents 1 mouse. Mean and individual values of each group are indicated. Solid circle, Rag^−/− + IgG; open circle, Rag^−/− + αCD40; solid square, GITR-L^−/−Rag^−/− + IgG; open square, GITR-L^−/−Rag^−/− + αCD40.

Figure 4.

Reduced CD11b⁺ MØ infiltration in the absence of GITR-L on inducing colitis with αCD40. At 7 d after αCD40 induction, colon lamina propria cells Rag^−/− or GITR-L^−/−Rag^−/− mice (5 mice/group) were surface stained for CD11b and CD11c. A) Representative stainings. B) Combined result of 5 independent experiments. Frozen colon sections were immunohistochemistry stained for CD11b-expressing cells. C) Representative staining of IHC. Original view: ×10. D) Statistical results of panel C.

Figure 5.

Reduced CD11b⁺ MØs in peritoneal cavity of GITR-L^−/− mice after thioglycollate induction. Thioglycollate broth (4%, 2ml/mouse) was administered by i.p. injection into the peritoneal cavity of C57BL/6, GITR-L^−/−, and CX3CR1(GFP) reporter mice. Mice were euthanized at 24, 48, and 72 h after induction. Peritoneal cells were surface stained for CD11b, F4/80, CD86, CD40, CD11c, CD4, and Ly6C. CX3CR1 expression was judged by the GFP in CX3CR1(GFP) reporter mice. A) Representative staining. B) Statistics results; 5 mice/group. Values represent means ± sd. C) Expression levels of CX3CR1, CD86, CD40, CD11c, CD4, and Ly6C on the cell surface of CD11b⁻F4/80⁺ and CD11b⁺F4/80⁺ cells were compared by FACS. Light shaded areas represent CD11b⁻F4/80⁺ cells. Dark lines represent CD11b⁺F4/80⁺ cells. D) Resident cells from the peritoneal cavity of C57BL/6 and GITR-L^−/− mice were stained for CD11b⁺F4/80⁺ MØs.

Figure 6.

GITR-L deficiency causes retention of monocytes in the spleen during inflammation. A) Rag^−/−or GITR-L^−/−Rag^−/− mice were treated with αCD40-induced colitis or without αCD40 induction as control, as described in Fig. 3. Mice were euthanized at d 7, and numbers of splenocytes were counted. B) Representative staining results of splenic monocytes and gating strategy. C) Number of Ly6C^hi monocytes was evaluated. D, E) Number of Ly6C^hi monocytes in bone marrow of Rag^−/− or GITR-L^−/−Rag^−/− mice was evaluated 7 d after αCD40 induction. Values in graphs represent means ± sd. Each solid or open circle represents 1 mouse. NT, no treatment.

Figure 7.

GITR-L deficiency dynamically affects the formation of AT1 dimer and the migration of splenic monocytes. A, B) Lysates were prepared from total splenocytes of Rag^−/− and GITR-L^−/−Rag^−/− mice (A), or from FACS-purified splenic Lin⁻CD11b⁺Ly6C^hi monocytes and Lin⁻CD11b^loF4/80⁺ MØs of Rag^−/− mice (B), and separated in SDS-PAGE gel. AT1 protein levels were evaluated by Western blotting; each lane represents 1 mouse. C, D) Lysates of FACS-purified Lin⁻CD11b⁺Ly6C^hi monocytes from the spleen of Rag^−/− or GITR-L^−/−Rag^−/− mice without (C) or with αCD40 inducing colitis for 5 or 7 d (D) were compared for the protein level of AT1 dimer. E) Quantitation of the relative protein levels of AT1 dimer in D. AT1 dimer and β-actin were quantitated with ImageI64 and normalized to that of Rag^−/− mice at each time point. Lanes 1, 3: Rag^−/− monocytes; lanes 2, 4: GITR-L^−/−Rag^−/− monocytes. Bars represent means ± se of 3 independent experiments; 5 mice/group. Same membrane was reprobed for β-actin in all the Western blotting. F) Lin⁻CD11b⁺Ly6C^hi monocytes were FACS purified from the spleen of Rag^−/− or GITR-L^−/−Rag^−/− mice 5 d after αCD40 inducing colitis, and used for transwell migration assay in a 96-well microplate. After 3 h incubation, the number of cells that crossed the 5-μm² pore membrane was evaluated under microscope and normalized to the medium. G) Lin⁻CD11b⁺Ly6C^hi monocytes sorted from the spleen of naive B6 or GITR-L^−/− mice were used for transwell migration as in panel F. Bars in panels F and G represent means ± se of triplicates. Results are representative of 3 independent experiments.

Figure 8.

Anti-GITR-L forces the egress of monocytes from the spleen and induces the formation of AT1 dimer in MØs. A–D) Colitis was induced in Rag^−/−or GITR-L^−/−Rag^−/− mice by αCD40 in the presence αGITR-L or hamster IgG control (each antibody 200 μg/mouse). Mice were euthanized 7 d after induction. Splenocytes and colon lamina propria cells were isolated and stained for monocytes and MØs, respectively. A) Percentages of Lin⁻CD11b⁺Ly6C^hi monocytes in the spleen. B) Number of splenocytes. C) Histology score. D) Lin⁻CD11b⁺MHCII⁺ MØs in the colon lamina propria. Each open or solid circle represents 1 mouse in. Panel D is representative of 2 independent experiments. E) RNA was extracted from splenic Lin⁻CD11b⁺Ly6C^hi monocytes and Lin⁻CD11b⁺F4/80⁺ MØs of Rag^−/− mice 5 d after αCD40-inducing colitis. RT-PCR was used to detect the expression of GITR and GITR-L. Expression of housekeeping gene GAPDH were used for control. F) Anti-CD40 and αGITR-L synergize to induce formation of the AT1 dimer in MØs. At 96 h after thioglycollate induction, peritoneal MØs were isolated and stimulated with αCD40 in the presence or absence of αGITR-L for the indicated time points. Cell lysates were evaluated for AT1 dimer by Western blot. Same membrane was reprobed for β-actin. Results represent ≥3 independent experiments.