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The role of macrophage migration inhibitory factor in mast cell-stimulated fibroblast proliferation and collagen production.

Ningyan G, Xu Y, Hongfei S, Jingjing C, Min C - PLoS ONE (2015)

Bottom Line: In the current study, we successfully generated a practical and reliable human mast cell culture system with bone marrow CD34+ hematopietic precursors.This positive regulatory effect of mast cell conditioned medium can be dampened by MIF antibody.Moreover, we strongly believe mast cell culture and differentiation model as well as corresponding genetic manipulation methodology will be helpful in characterizing novel mast cell based therapeutic targets.

View Article: PubMed Central - PubMed

Affiliation: Institute of Dermatology, Chinese Academy of Medical Sciences and Peking Union Medical College, Nanjing, China.

ABSTRACT
Current clinical and translational studies have shown that mast cell plays a pivotal role in multiple fibrotic diseases including scleroderma. However, the lack of mature human mast cell culture model exhibits a major obstacle for further dissection of cytokines and signaling molecules required for mast cell mediated fibrosis in various diseases. Macrophage Migration Inhibitory Factor is a mast cell released pro-inflammatory cytokine which is deregulated in scleroderma patients and is also involved in non-scleroderma related fibrosis. In the current study, we successfully generated a practical and reliable human mast cell culture system with bone marrow CD34+ hematopietic precursors. The derivative mast cell is normal in terms of both morphology and function as manifested by normal degranulation. More importantly, we were able to show mast cell conditioned medium as well as MIF supplementation augments fibroblast proliferation and collagen synthesis. This positive regulatory effect of mast cell conditioned medium can be dampened by MIF antibody. In addition, MIF-knockdown significantly inhibits pro-fibrotic activities of CD34+ hematopietic precursor derived mast cells. These data strongly suggest that mast cell released MIF is required for mast cell mediated fibrogenic activities. The current manuscript seems to be the first mechanistic report showing the significance of MIF in mast cell mediated fibrosis, which may pave the way for the development of potential MIF-targeted therapy for fibrotic diseases to a further extent. Moreover, we strongly believe mast cell culture and differentiation model as well as corresponding genetic manipulation methodology will be helpful in characterizing novel mast cell based therapeutic targets.

No MeSH data available.


Related in: MedlinePlus

Degranulation assay of derived MCs.A. A representative result of flow cytometry on original CD34+ hematopoietic precursors using anti-FcεRI and anti-CD117 antibodies. B. A representative result of flow cytometry on derived mast cells using anti-FcεRI and anti-CD117 antibodies. C. Quantification analysis of FcεRI/CD117 double positive population in original CD34+ hematopoietic precursors and derived mast cells. D. Mast cell-released histamine in different group was assessed by ELISA assay. E. Mast cell-released MIF was assessed by ELISA assay, mean±SD, n = 3.
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pone.0122482.g003: Degranulation assay of derived MCs.A. A representative result of flow cytometry on original CD34+ hematopoietic precursors using anti-FcεRI and anti-CD117 antibodies. B. A representative result of flow cytometry on derived mast cells using anti-FcεRI and anti-CD117 antibodies. C. Quantification analysis of FcεRI/CD117 double positive population in original CD34+ hematopoietic precursors and derived mast cells. D. Mast cell-released histamine in different group was assessed by ELISA assay. E. Mast cell-released MIF was assessed by ELISA assay, mean±SD, n = 3.

Mentions: As the next step, we felt imperative to test whether the derivative mast cells is functional. Degranulation is a cellular process that releases antimicrobialcytotoxic molecules from secretoryvesicles) or granules from inside of the cells. As for mast cells, to induce degranulation, antigens interact with IgE bound to high affinity Fc receptors (FcεRI) on cell surface. Then the mast cells release a series of inflammatory mediators including histamine, proteoglycans, serotonin, and serine proteases from its cytoplasmic granules [42]. In the current study, the expression of FcεRI in differentiated cells was examined by flow cytometry. Since CD117 (stem cell growth factor receptor or called c-kit) plays a vital role in survival, proliferation and differentiation of mast cells, we performed FcεRI/CD117 double staining experiments. As shown in Fig 3A, 3B, and 3C, only a very small portion (<2%) of the un-differentiated cells expressed both molecular markers. On the contrary, over 91% of the differentiated cells were positively stained for both FcεRI and CD117. More importantly, the results meant that the derivative mast cells express FcεRI which is required for degranulation. As a positive control, histamine was found to be secreted by the derivative mast cells (Fig 3D) given the presence of IgE and IgE antibody, which indicates that degranulation function was well preserved in these mast cells. As expected, we also found that MIF was secreted by these mast cells during degranulation (Fig 3E).


The role of macrophage migration inhibitory factor in mast cell-stimulated fibroblast proliferation and collagen production.

Ningyan G, Xu Y, Hongfei S, Jingjing C, Min C - PLoS ONE (2015)

Degranulation assay of derived MCs.A. A representative result of flow cytometry on original CD34+ hematopoietic precursors using anti-FcεRI and anti-CD117 antibodies. B. A representative result of flow cytometry on derived mast cells using anti-FcεRI and anti-CD117 antibodies. C. Quantification analysis of FcεRI/CD117 double positive population in original CD34+ hematopoietic precursors and derived mast cells. D. Mast cell-released histamine in different group was assessed by ELISA assay. E. Mast cell-released MIF was assessed by ELISA assay, mean±SD, n = 3.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4380314&req=5

pone.0122482.g003: Degranulation assay of derived MCs.A. A representative result of flow cytometry on original CD34+ hematopoietic precursors using anti-FcεRI and anti-CD117 antibodies. B. A representative result of flow cytometry on derived mast cells using anti-FcεRI and anti-CD117 antibodies. C. Quantification analysis of FcεRI/CD117 double positive population in original CD34+ hematopoietic precursors and derived mast cells. D. Mast cell-released histamine in different group was assessed by ELISA assay. E. Mast cell-released MIF was assessed by ELISA assay, mean±SD, n = 3.
Mentions: As the next step, we felt imperative to test whether the derivative mast cells is functional. Degranulation is a cellular process that releases antimicrobialcytotoxic molecules from secretoryvesicles) or granules from inside of the cells. As for mast cells, to induce degranulation, antigens interact with IgE bound to high affinity Fc receptors (FcεRI) on cell surface. Then the mast cells release a series of inflammatory mediators including histamine, proteoglycans, serotonin, and serine proteases from its cytoplasmic granules [42]. In the current study, the expression of FcεRI in differentiated cells was examined by flow cytometry. Since CD117 (stem cell growth factor receptor or called c-kit) plays a vital role in survival, proliferation and differentiation of mast cells, we performed FcεRI/CD117 double staining experiments. As shown in Fig 3A, 3B, and 3C, only a very small portion (<2%) of the un-differentiated cells expressed both molecular markers. On the contrary, over 91% of the differentiated cells were positively stained for both FcεRI and CD117. More importantly, the results meant that the derivative mast cells express FcεRI which is required for degranulation. As a positive control, histamine was found to be secreted by the derivative mast cells (Fig 3D) given the presence of IgE and IgE antibody, which indicates that degranulation function was well preserved in these mast cells. As expected, we also found that MIF was secreted by these mast cells during degranulation (Fig 3E).

Bottom Line: In the current study, we successfully generated a practical and reliable human mast cell culture system with bone marrow CD34+ hematopietic precursors.This positive regulatory effect of mast cell conditioned medium can be dampened by MIF antibody.Moreover, we strongly believe mast cell culture and differentiation model as well as corresponding genetic manipulation methodology will be helpful in characterizing novel mast cell based therapeutic targets.

View Article: PubMed Central - PubMed

Affiliation: Institute of Dermatology, Chinese Academy of Medical Sciences and Peking Union Medical College, Nanjing, China.

ABSTRACT
Current clinical and translational studies have shown that mast cell plays a pivotal role in multiple fibrotic diseases including scleroderma. However, the lack of mature human mast cell culture model exhibits a major obstacle for further dissection of cytokines and signaling molecules required for mast cell mediated fibrosis in various diseases. Macrophage Migration Inhibitory Factor is a mast cell released pro-inflammatory cytokine which is deregulated in scleroderma patients and is also involved in non-scleroderma related fibrosis. In the current study, we successfully generated a practical and reliable human mast cell culture system with bone marrow CD34+ hematopietic precursors. The derivative mast cell is normal in terms of both morphology and function as manifested by normal degranulation. More importantly, we were able to show mast cell conditioned medium as well as MIF supplementation augments fibroblast proliferation and collagen synthesis. This positive regulatory effect of mast cell conditioned medium can be dampened by MIF antibody. In addition, MIF-knockdown significantly inhibits pro-fibrotic activities of CD34+ hematopietic precursor derived mast cells. These data strongly suggest that mast cell released MIF is required for mast cell mediated fibrogenic activities. The current manuscript seems to be the first mechanistic report showing the significance of MIF in mast cell mediated fibrosis, which may pave the way for the development of potential MIF-targeted therapy for fibrotic diseases to a further extent. Moreover, we strongly believe mast cell culture and differentiation model as well as corresponding genetic manipulation methodology will be helpful in characterizing novel mast cell based therapeutic targets.

No MeSH data available.


Related in: MedlinePlus