Endogenous matrix metalloproteinases 2 and 9 regulate activation of CD4+ and CD8+ T cells

Abstract

We reported that inhibiting matrix metalloproteinases (MMP), known to remodel the extracellular matrix, also down-regulated antigen-specific T-cell responses. However, the direct role of MMP2 and MMP9 in regulating intracellular function in T cells is unknown. Markers of cellular activation and cytokine profiles were examined in anti-CD3-stimulated wild-type C57BL/6 mouse-derived CD4(+) or CD8(+) T cells, or MMP2- or MMP9-deficient (-/-) mice. MMP-sufficient T cells were also treated with SB-3CT, a highly selective inhibitor of MMP2 and MMP9. The effect of MMP-specific inhibition on T cell-dependent, antigen-specific murine lung injury was examined in vivo. SB-3CT induced dose-dependent reductions in anti-CD3-stimulated T-cell proliferation. Although MMP2(-/-) cells were reduced 20%, anti-CD3-induced proliferation was down-regulated 80-85% in MMP9(-/-) or in SB-3CT-treated wild-type CD4(+) and CD8(+) T cells. Intracellular calcium flux was augmented in response to MMP inhibition or deficiency in the same cells, and IL-2 production was reduced in CD4(+) and CD8(+) MMP9(-/-) T cells. SB-3CT-mediated MMP2 and MMP9 inhibition abrogated antigen-specific CD8(+) T cell-mediated lung injury in vivo. MMPs, particularly MMP9, may function intracellularly to regulate T-cell activation. T cell-targeted MMP inhibition may provide a novel approach of immune regulation in the treatment of T cell-mediated diseases.

Figure 1.

Differential matrix metalloproteinase (MMP) 9 mRNA and protein expression in CD4⁺ and CD8⁺ T cells. Pure splenic (A) CD4⁺ and (B) CD8⁺ T cells were cultured in the absence or presence of anti-CD3 antibody (Ab; 1 μg/ml) for 72 hours. RNA was isolated, cDNA synthesized, and mRNA expression levels measured by quantitative RT-PCR. Data were normalized to β-actin. Data are representative of two separate experiments performed in triplicate. (C) Gelatin zymogram analysis of CD4⁺ and CD8⁺ T-cell lysates and supernatant. Data are representative of one of four separate experiments. (A) *P = 0.01; (B) *P = 0.003. MW, molecular weight; RQ, relative quantification.

Figure 2.

Broad-spectrum and specific MMP inhibition abrogated anti-CD3–induced T-cell proliferation. Pure splenic CD4⁺ T cells were treated with (A) CD4⁺, and (B) CD8⁺ T cells were treated with SB-3CT (–25 μM), and cultured in the presence of anti-CD3 Ab (0.5 μg/ml) for 72 hours. T-cell proliferation was measured by 3H thymidine incorporation. Data are representative of the mean (±SD) of three experiments performed in triplicate (*P < 0.001). (C) Cell viability (annexin V and propidium iodide [PI]) assessed in CD4⁺ and CD8⁺ T cells after 6-hour treatment with SB-3CT.

Figure 3.

MMP2- and MMP9-deficient (−/−) T cells display altered proliferative ability. Wild-type (Wt) and (A) MMP2^−/− CD4⁺, (B) MMP9^−/− CD4⁺, and (C) MMP9^−/− CD8⁺ T cells were cultured in the presence of anti-CD3 Ab (0.5 μg/ml) for 72 hours. T-cell proliferation was measured by 3H thymidine incorporation. Data are representative of the mean (±SD) of three separate experiments performed in triplicate. ^#P = 0.02; **P < 0.001.

Figure 4.

MMP deficiency or inhibition increases calcium flux. (A) CD4⁺ or (B) CD8⁺ T cells isolated from wild-type and MMP9^−/− mice. (C) CD8⁺ T cells were treated with SB-3CT (10 μM) or vehicle (DMSO + polyethylene glycol [PEG], diluted similarly in cRPMI). (A–C) Cells were cultured in calcium-free media and stimulated with anti-CD3 Ab (10 μg/ml). calcium flux was measured for 100 seconds in real time. Data are representative of one of three separate experiments performed in triplicate.

Figure 5.

MMP deficiency or inhibition alters cytokine transcript and protein expression. CD4⁺ T cells were isolated from wild-type, MMP2^−/−, or MMP9^−/− mice. (B, D, and F) CD4⁺ T cells were treated with SB-3CT (–20 μM). Cells were cultured in the presence or absence of anti-CD3 Ab (1 μg/ml). (A–B) nuclear factor of activated T cells (NFATc1), (C–D) IL-2, and (E–F) CD25 expression levels were measured by quantitative RT-PCR. (C–D) IL-2 protein expression was measured by cytometric bead assay. Data are representative of three separate experiments performed in triplicate. ^#P < 0.05; *P < 0.001.

Figure 6.

SB-3CT–treated, antigen-specific T cells (OT-I) display impairment in proliferative ability. (A) OT-I transgenic CD8⁺ T cells were treated with SB-3CT (–20 μM) or vehicle (DMSO + PEG, diluted similarly in CRPMI), and cultured in the presence of ovalbumin (OVA)-pulsed antigen-presenting cells (APCs) for 72 hours. Data are representative of two separate experiments performed in triplicate (^#P < 0.05; *P < 0.001). (B) At 7 days after adoptive transfer, bronchoalveolar lavage (BAL) fluid from the CC10-OVA (CC10) or nontransgenic (B6) mice was analyzed, and total cells present in the BAL quantitated. (C) Neutrophils were stained with GR1 and analyzed by means of flow cytometry. **P < 0.01 as compared with stimulated wild-type cells (n = 10 mice [CC10] per treatment group; n = 5 control mice [B6] per treatment group).

Figure 7.

Murine model of antigen-specific CD8⁺ effector T cell–mediated lung injury. (A) CD8⁺Thy1.1⁺ T cells were isolated from the lungs of mice after the adoptive transfer of SB-3CT (10 μM) or vehicle (DMSO + PEG, diluted similarly in CRPMI). (B) CD25 expression in CD8⁺Thy1.1⁺ T cells from the lungs of CC10-OVA mice (*P < 0.01; n = 9 mice [CC10] per treatment group; n = 5 control mice [B6] per treatment group). (C) CD8⁺ T cells were isolated from OT-1 transgenic mice and treated with SB-3CT or vehicle. Histology of the lung was evaluated by hematoxylin and eosin staining (magnification, 20×; n = 6 mice [CC10] per group). Panels 1a and 1b are representative of two fields of a CC10 mouse lung after adoptive transfer of vehicle-treated OT-I CD8⁺ T cells. Panels 2 and 3 are representative of two different CC10 mouse lung samples after adoptive transfer of SB-3CT–treated OT-I CD8⁺ T cells. (D) Histological scoring was performed blinded for all groups. **P = 0.03 as compared with vehicle-treated (CC10) group (unpaired t test with Welch's correction).