The combined action of mast cell chymase, tryptase and carboxypeptidase A3 protects against melanoma colonization of the lung

Abstract

Mast cell secretory granules are densely packed with various bioactive mediators including proteases of chymase, tryptase and CPA3 type. Previous studies have indicated that mast cells can affect the outcome of melanoma but the contribution of the mast cell granule proteases to such effects has not been clear. Here we addressed this issue by assessing mice lacking either the chymase Mcpt4, the tryptase Mcpt6 or carboxypeptidase A3 (Cpa3), as well as mice simultaneously lacking all three proteases, in a model of melanoma dissemination from blood to the lung. Although mice with individual deficiency in the respective proteases did not differ significantly from wildtype mice in the extent of melanoma colonization, mice with multiple protease deficiency (Mcpt4/Mcpt6/Cpa3-deficient) exhibited a higher extent of melanoma colonization in lungs as compared to wildtype animals. This was supported by higher expression of melanoma-specific genes in lungs of Mcpt4/Mcpt6/CPA3-deficient vs. wildtype mice. Cytokine profiling showed that the levels of CXCL16, a chemokine with effects on T cell populations and NKT cells, were significantly lower in lungs of Mcpt4/Mcpt6/Cpa3-deficient animals vs. controls, suggesting that multiple mast cell protease deficiency might affect T cell or NKT cell populations. In line with this, we found that the Mcpt4/Mcpt6/Cpa3-deficiency was associated with a reduction in cells expressing CD1d, a MHC class 1-like molecule that is crucial for presenting antigen to invariant NKT (iNKT) cells. Together, these findings indicate a protective role of mast cell-specific proteases in melanoma dissemination, and suggest that this effect involves a CXCL16/CD1d/NKT cell axis.

Keywords: CD1d; carboxypeptidase A3; chymase; mast cells; tryptase.

Figure 1. Multiple KO mice have higher tumor load in lungs compared to WT mice

0.25 million of B16F10 cells were injected i.v. into WT, single KO or into multiprotease-deficient (multiple KO) mice. On day 14 post injection, the numbers of tumor nodules were quantified A, B. Results are presented as medians (n = 5-15). C. Representative images of lungs from WT and multiple KO mice. Note the higher numbers of tumor nodules in multiple KO compared to WT lungs. D. Total RNA was isolated from lungs on day14 post injection and subjected to qPCR analysis of the expression of melanoma-specific genes (DCT, black bars; GP100, gray bars). Expression of genes was evaluated relative to GAPDH and normalized to naïve mice. Results are presented as mean ± SEM; n = 8. Statistical calculations were performed using the Mann Whitney test.

Figure 2. Histological analysis of lung tissue

On day 14 post B16F10 cell injection, lungs were paraffin imbedded, sectioned and stained with hematoxylin and eosin A. toluidine blue B. or for chloroacetate esterase activity C. (A) Arrows point to tumor areas. Note a larger tumor area in the section from multiple KO mice (right) in comparison with WT controls (left); original magnification x 200; bar = 50 μm. (B) Toluidine blue-positive MCs (marked by arrows) are present in lung sections from both WT (left) and multiple KO (right). Inserts show a higher magnification of the toluidine blue-stained MCs; note that MCs in lungs of multiple KO mice stain less intensely in comparison with WT MCs. (C) Chloroacetate esterase staining shows the presence of chymase-positive MCs in lung of both WT (left) and multiple KO (right) mice (marked by arrows): insets show larger magnifications of chloroacetate esterase-stained MCs; note that MCs in lungs from multiple KO mice stain less intensely with chloroacetate esterase, indicating lower content of chymase activity. Original magnification x 400; bars = 50 μm; bars in the inserts =10 μm.

Figure 3. Expression of angiogenesis markers in lungs of naïve and melanoma-carrying mice

Total RNA was isolated from lungs of naïve mice (black bars) and lungs of mice taken day 14 post i.v. injection of B16F10 melanoma cells (gray bars). Total RNA was subjected to qPCR analysis of angiogenesis markers. Expression of genes was evaluated relative to GAPDH and normalized to naïve mice. Results are presented as mean ± SEM; n = 5 (black bars), n = 7-8 (gray bars). Note that mice with tumors do not show increased expression of angiogenesis markers compared to naïve mice and that the groups with tumors show similar expression of the markers. Statistical calculations were performed using the Mann Whitney test.

Figure 4. Lower levels of CXCL16 in lungs of multiple KO mice

A. B16F10 melanoma cells were injected i.v. into either WT or multiple KO mice. On day14 post injection, mice were sacrificed, blood and lungs were collected (n = 3-4/genotype), and protein array (detecting 62 mouse proteins; setup indicated below the panel) analysis was performed on serum samples and lung homogenates as indicated. Squares represent CXCL16; note the lower amounts of CXCL16 in samples from multiple KO mice. B. Mouse CXCL16 ELISA was performed on lung homogenates (n = 9-10/genotype) and serum (n = 4-6/genotype) from naïve mice (black bars) or mice injected with B16F10 melanoma cells (gray bars). Results are presented as mean ± SEM. C. qPCR analysis of CXCL16 gene expression was performed on lung tissue from naïve (black bars, n = 4) and B16F10 melanoma-injected mice (gray bars, n = 8). Expression of genes was evaluated relative to GAPDH and normalized to naïve mice. Results are presented as mean ± SEM (Kruskal-Wallis test). D. qPCR analysis of ADAM10 and ADAM17 expression was performed on lung tissue from naïve (black bars; n = 4-5) and B16F10 melanoma-injected mice (gray bars; n = 8). Expression of genes was evaluated relative to GAPDH and normalized to naïve mice. Results are presented as mean ± SEM. Statistical calculations were performed using the Mann Whitney test.

Figure 5. Decreased numbers of CD1d-expressing lung cells in multiple KO mice

Representative dot plots and numbers of A. CD45⁺CD3⁺CD4⁺/CD8a⁺ lymphocytes, B. CD45⁺CD11c⁺F4/80⁺ (macrophages), C. CD45⁺CD3⁻NK1.1⁺ (NK cells) and D. CD45⁺CD1d⁺ cells in lungs of melanoma-carrying WT or multiple KO mice (day 14 post melanoma administration). The bars show the mean ± SEM of 10-11 individual mice per group pooled from two independent experiments. Mann-Whitney test; * p < 0.05.

Figure 6. Decreased expression of CD1d in multiple KO mice

A. qPCR analysis of CD3ε, CD4, CD8b1, Vα14-Jα281 and CD1d expression was performed on lung tissues from B16F10 melanoma-carrying WT or multiple KO mice (n = 6-8). Expression of genes was evaluated relative to GAPDH and normalized to naïve mice. Results are presented as mean ± SEM. Statistical calculations were performed using the Mann Whitney test. B. Correlation between the expression of CD1d with expression of melanoma-specific genes: DCT and GP100; Nonparametric Spearman correlation.