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Myeloid expression of angiotensin-converting enzyme facilitates myeloid maturation and inhibits the development of myeloid-derived suppressor cells.

Shen XZ, Okwan-Duodu D, Blackwell WL, Ong FS, Janjulia T, Bernstein EA, Fuchs S, Alkan S, Bernstein KE - Lab. Invest. (2014)

Bottom Line: Here, we show that ACE expression correlates with myeloid maturation in vitro.Forced ACE overexpression in monocytic cells reduces the generation of MDSCs.Thus, manipulating myeloid ACE activity can interfere with MDSC development and the maturation of myeloid cells.

View Article: PubMed Central - PubMed

Affiliation: 1] Division of Immunology, Department of Biomedical Science, Cedars-Sinai Medical Center, Los Angeles, CA, USA [2] Department of Pathology and Laboratory Medicine, Cedars-Sinai Medical Center, Los Angeles, CA, USA.

ABSTRACT
Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of immature myeloid cells which accumulate in cancer, infection and chronic inflammation. These cells suppress T-cell function and the immune response. Angiotensin-converting enzyme (ACE) is a peptidase that is now known to regulate aspects of myelopoiesis. Here, we show that ACE expression correlates with myeloid maturation in vitro. Forced ACE overexpression in monocytic cells reduces the generation of MDSCs. In vivo, mice with a genetic change resulting in myeloid cell ACE overexpression have reduced numbers of blood and splenic MDSCs in a tumor model and in a model of chronic inflammation induced by complete Freund's adjuvant. In contrast, ACE- mice produce large numbers of MDSCs during chronic inflammation. Macrophages from mice with myeloid ACE overexpressing are more pro-inflammatory and have more tumor-killing activity than cells from wild-type mice. Thus, manipulating myeloid ACE activity can interfere with MDSC development and the maturation of myeloid cells.

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MDSC development in vitro. a. WT BM cells were cultured with GM-CSF and either IL-6 or tumor-conditioned medium (TCM) for 4 days. The non-adherent and adherent cells were then harvested and separately co-cultured with anti-CD3 and anti-CD28 stimulated CFSE-labeled T cells for another 3 days. Typical T cell proliferation profiles are shown (line) compared to the original T cells (dotted). The non-adherent cells suppress T cell proliferation resulting in less dilution of CFSE. b. The expression of surface maturation markers F4/80, CD80 and CD86 was assessed on the non-adherent and adherent cells. c. ACE expression on the non-adherent and adherent cells. d. ACE expression on the non-adherent cells with and without culture in M-CSF. e. The surface expression of Ly6G and Ly6C was measured on non-adherent cells (left). Four populations were identified: Ly6C−Ly6G+, Ly6C+Ly6G+, Ly6C+Ly6G−, and Ly6C−Ly6G−. These populations were gated and sorted, and their immunosuppressive activities were assessed by their ability to inhibit T cell proliferation (right). f. The surface maturation markers MHC class II (I-Ab) and CD86 were evaluated on the gated cells. For all the experiments, the representative flow cytometry figures presented are representative of at least 3 independent experiments.
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Figure 1: MDSC development in vitro. a. WT BM cells were cultured with GM-CSF and either IL-6 or tumor-conditioned medium (TCM) for 4 days. The non-adherent and adherent cells were then harvested and separately co-cultured with anti-CD3 and anti-CD28 stimulated CFSE-labeled T cells for another 3 days. Typical T cell proliferation profiles are shown (line) compared to the original T cells (dotted). The non-adherent cells suppress T cell proliferation resulting in less dilution of CFSE. b. The expression of surface maturation markers F4/80, CD80 and CD86 was assessed on the non-adherent and adherent cells. c. ACE expression on the non-adherent and adherent cells. d. ACE expression on the non-adherent cells with and without culture in M-CSF. e. The surface expression of Ly6G and Ly6C was measured on non-adherent cells (left). Four populations were identified: Ly6C−Ly6G+, Ly6C+Ly6G+, Ly6C+Ly6G−, and Ly6C−Ly6G−. These populations were gated and sorted, and their immunosuppressive activities were assessed by their ability to inhibit T cell proliferation (right). f. The surface maturation markers MHC class II (I-Ab) and CD86 were evaluated on the gated cells. For all the experiments, the representative flow cytometry figures presented are representative of at least 3 independent experiments.

Mentions: Culture of mouse BM with granulocyte/macrophage colony stimulating factor (GM-CSF) and either IL-6 or tumor conditioned medium generates MDSCs.15, 16 A 4 day culture of WT BM using either cell culture condition produced a mixed population of adherent and non-adherent cells. Analysis with trypan blue and 7-amino-actinomycin D showed that both populations were composed of living cells. The two populations were then separated and each was co-cultured with stimulated T cells previously labeled with the fluorescent dye CFSE (Figure 1a). Our data showed that the co-culture of stimulated T cells with non-adherent BM cells suppressed T cell proliferation while an equivalent experiment in which adherent cells were co-cultured with stimulated T cells showed non-suppression. We also measured the surface markers of non-adherent and adherent cells and found significantly less surface expression of the maturation markers F4/80, CD80 and CD86 on non-adherent cells as compared to the adherent population (Figure 1b). Thus, the non-adherent cells are a relatively immature population capable of suppressing T cell proliferation. Interestingly, the non-adherent cells express much less ACE than the adherent cell population (Figure 1c).


Myeloid expression of angiotensin-converting enzyme facilitates myeloid maturation and inhibits the development of myeloid-derived suppressor cells.

Shen XZ, Okwan-Duodu D, Blackwell WL, Ong FS, Janjulia T, Bernstein EA, Fuchs S, Alkan S, Bernstein KE - Lab. Invest. (2014)

MDSC development in vitro. a. WT BM cells were cultured with GM-CSF and either IL-6 or tumor-conditioned medium (TCM) for 4 days. The non-adherent and adherent cells were then harvested and separately co-cultured with anti-CD3 and anti-CD28 stimulated CFSE-labeled T cells for another 3 days. Typical T cell proliferation profiles are shown (line) compared to the original T cells (dotted). The non-adherent cells suppress T cell proliferation resulting in less dilution of CFSE. b. The expression of surface maturation markers F4/80, CD80 and CD86 was assessed on the non-adherent and adherent cells. c. ACE expression on the non-adherent and adherent cells. d. ACE expression on the non-adherent cells with and without culture in M-CSF. e. The surface expression of Ly6G and Ly6C was measured on non-adherent cells (left). Four populations were identified: Ly6C−Ly6G+, Ly6C+Ly6G+, Ly6C+Ly6G−, and Ly6C−Ly6G−. These populations were gated and sorted, and their immunosuppressive activities were assessed by their ability to inhibit T cell proliferation (right). f. The surface maturation markers MHC class II (I-Ab) and CD86 were evaluated on the gated cells. For all the experiments, the representative flow cytometry figures presented are representative of at least 3 independent experiments.
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Related In: Results  -  Collection

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Figure 1: MDSC development in vitro. a. WT BM cells were cultured with GM-CSF and either IL-6 or tumor-conditioned medium (TCM) for 4 days. The non-adherent and adherent cells were then harvested and separately co-cultured with anti-CD3 and anti-CD28 stimulated CFSE-labeled T cells for another 3 days. Typical T cell proliferation profiles are shown (line) compared to the original T cells (dotted). The non-adherent cells suppress T cell proliferation resulting in less dilution of CFSE. b. The expression of surface maturation markers F4/80, CD80 and CD86 was assessed on the non-adherent and adherent cells. c. ACE expression on the non-adherent and adherent cells. d. ACE expression on the non-adherent cells with and without culture in M-CSF. e. The surface expression of Ly6G and Ly6C was measured on non-adherent cells (left). Four populations were identified: Ly6C−Ly6G+, Ly6C+Ly6G+, Ly6C+Ly6G−, and Ly6C−Ly6G−. These populations were gated and sorted, and their immunosuppressive activities were assessed by their ability to inhibit T cell proliferation (right). f. The surface maturation markers MHC class II (I-Ab) and CD86 were evaluated on the gated cells. For all the experiments, the representative flow cytometry figures presented are representative of at least 3 independent experiments.
Mentions: Culture of mouse BM with granulocyte/macrophage colony stimulating factor (GM-CSF) and either IL-6 or tumor conditioned medium generates MDSCs.15, 16 A 4 day culture of WT BM using either cell culture condition produced a mixed population of adherent and non-adherent cells. Analysis with trypan blue and 7-amino-actinomycin D showed that both populations were composed of living cells. The two populations were then separated and each was co-cultured with stimulated T cells previously labeled with the fluorescent dye CFSE (Figure 1a). Our data showed that the co-culture of stimulated T cells with non-adherent BM cells suppressed T cell proliferation while an equivalent experiment in which adherent cells were co-cultured with stimulated T cells showed non-suppression. We also measured the surface markers of non-adherent and adherent cells and found significantly less surface expression of the maturation markers F4/80, CD80 and CD86 on non-adherent cells as compared to the adherent population (Figure 1b). Thus, the non-adherent cells are a relatively immature population capable of suppressing T cell proliferation. Interestingly, the non-adherent cells express much less ACE than the adherent cell population (Figure 1c).

Bottom Line: Here, we show that ACE expression correlates with myeloid maturation in vitro.Forced ACE overexpression in monocytic cells reduces the generation of MDSCs.Thus, manipulating myeloid ACE activity can interfere with MDSC development and the maturation of myeloid cells.

View Article: PubMed Central - PubMed

Affiliation: 1] Division of Immunology, Department of Biomedical Science, Cedars-Sinai Medical Center, Los Angeles, CA, USA [2] Department of Pathology and Laboratory Medicine, Cedars-Sinai Medical Center, Los Angeles, CA, USA.

ABSTRACT
Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of immature myeloid cells which accumulate in cancer, infection and chronic inflammation. These cells suppress T-cell function and the immune response. Angiotensin-converting enzyme (ACE) is a peptidase that is now known to regulate aspects of myelopoiesis. Here, we show that ACE expression correlates with myeloid maturation in vitro. Forced ACE overexpression in monocytic cells reduces the generation of MDSCs. In vivo, mice with a genetic change resulting in myeloid cell ACE overexpression have reduced numbers of blood and splenic MDSCs in a tumor model and in a model of chronic inflammation induced by complete Freund's adjuvant. In contrast, ACE- mice produce large numbers of MDSCs during chronic inflammation. Macrophages from mice with myeloid ACE overexpressing are more pro-inflammatory and have more tumor-killing activity than cells from wild-type mice. Thus, manipulating myeloid ACE activity can interfere with MDSC development and the maturation of myeloid cells.

Show MeSH
Related in: MedlinePlus