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Distinguishing Mast Cell Progenitors from Mature Mast Cells in Mice.

Dahlin JS, Ding Z, Hallgren J - Stem Cells Dev. (2015)

Bottom Line: The maturation statuses of the cells were confirmed using three main strategies: (1) MCp, but not mature mast cells, were shown to be depleted by sublethal whole-body γ-irradiation. (2) The MCp were small and immature in terms of granule formation, whereas the mature mast cells were larger and had fully developed metachromatic granules. (3) The MCp had fewer transcripts of mast cell-specific proteases and the enzyme responsible for sulfation of heparin than mature mast cells.Moreover, isolated peritoneal MCp gave rise to mast cells when cultured in vitro.Using this strategy, mast cell maturation can be studied in vivo.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Biochemistry and Microbiology, Uppsala University , Uppsala, Sweden .

ABSTRACT
Mast cells originate from the bone marrow and develop into c-kit(+) FcɛRI(+) cells. Both mast cell progenitors (MCp) and mature mast cells express these cell surface markers, and ways validated to distinguish between the two maturation forms with flow cytometry have been lacking. Here, we show that primary peritoneal MCp from naïve mice expressed high levels of integrin β7 and had a low side scatter (SSC) light profile; whereas mature mast cells expressed lower levels of integrin β7 and had a high SSC light profile. The maturation statuses of the cells were confirmed using three main strategies: (1) MCp, but not mature mast cells, were shown to be depleted by sublethal whole-body γ-irradiation. (2) The MCp were small and immature in terms of granule formation, whereas the mature mast cells were larger and had fully developed metachromatic granules. (3) The MCp had fewer transcripts of mast cell-specific proteases and the enzyme responsible for sulfation of heparin than mature mast cells. Moreover, isolated peritoneal MCp gave rise to mast cells when cultured in vitro. To summarize, we have defined MCp and mature mast cells in naïve mice by flow cytometry. Using this strategy, mast cell maturation can be studied in vivo.

No MeSH data available.


Related in: MedlinePlus

(A) Peritoneal cells from naïve mice were analyzed by flow cytometry. The Lin−/lo c-kithi T1/ST2+ cells were divided into SSClo and SSChi cells. The SSChi cells contained mostly CD16/32int integrin β7−/lo/int cells, whereas the SSClo fraction (encircled with a dashed ellipse) was mainly characterized as CD16/32int integrin β7hi. Virtually all Lin−/lo c-kithi T1/ST2+ SSChi CD16/32int integrin β7−/lo/int and Lin−/lo c-kithi T1/ST2+ SSClo CD16/32int integrin β7hi cells in the peritoneum expressed FcɛRI. (B) Mice were exposed to 5 Gy whole-body γ-irradiation. After 7 days, cells from peritoneal lavage were extracted and analyzed by flow cytometry. The Lin−/lo c-kithi T1/ST2+ SSChi and the Lin−/lo c-kithi T1/ST2+ SSClo (encircled with a dashed ellipse) cells were gated. (C) The total number of cells per milliliter peritoneal fluid was assessed in naïve and γ-irradiated mice. (D) The number of Lin−/lo c-kithi T1/ST2+ SSClo CD16/32int integrin β7hi FcɛRI+ MCp per milliliter peritoneal fluid was quantified in naïve and γ-irradiated mice. (E) The number of Lin−/lo c-kithi T1/ST2+ SSChi CD16/32int integrin β7−/lo/int FcɛRI+ mature mast cells (MC) was quantified in naïve and γ-irradiated mice. The data in (C–E) are derived from three pooled experiments where both groups were evaluated in parallel. Each group consisted of nine animals. The means and SEM are shown. The frequency of the gated cells in each graph was calculated by dividing the gated cell numbers with the total number of peritoneal cells. MCp, mast cell progenitors; SSC, side scatter. Color images available online at www.liebertpub.com/scd
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f1: (A) Peritoneal cells from naïve mice were analyzed by flow cytometry. The Lin−/lo c-kithi T1/ST2+ cells were divided into SSClo and SSChi cells. The SSChi cells contained mostly CD16/32int integrin β7−/lo/int cells, whereas the SSClo fraction (encircled with a dashed ellipse) was mainly characterized as CD16/32int integrin β7hi. Virtually all Lin−/lo c-kithi T1/ST2+ SSChi CD16/32int integrin β7−/lo/int and Lin−/lo c-kithi T1/ST2+ SSClo CD16/32int integrin β7hi cells in the peritoneum expressed FcɛRI. (B) Mice were exposed to 5 Gy whole-body γ-irradiation. After 7 days, cells from peritoneal lavage were extracted and analyzed by flow cytometry. The Lin−/lo c-kithi T1/ST2+ SSChi and the Lin−/lo c-kithi T1/ST2+ SSClo (encircled with a dashed ellipse) cells were gated. (C) The total number of cells per milliliter peritoneal fluid was assessed in naïve and γ-irradiated mice. (D) The number of Lin−/lo c-kithi T1/ST2+ SSClo CD16/32int integrin β7hi FcɛRI+ MCp per milliliter peritoneal fluid was quantified in naïve and γ-irradiated mice. (E) The number of Lin−/lo c-kithi T1/ST2+ SSChi CD16/32int integrin β7−/lo/int FcɛRI+ mature mast cells (MC) was quantified in naïve and γ-irradiated mice. The data in (C–E) are derived from three pooled experiments where both groups were evaluated in parallel. Each group consisted of nine animals. The means and SEM are shown. The frequency of the gated cells in each graph was calculated by dividing the gated cell numbers with the total number of peritoneal cells. MCp, mast cell progenitors; SSC, side scatter. Color images available online at www.liebertpub.com/scd

Mentions: Cells from peritoneal lavage were analyzed with flow cytometry to investigate whether MCp are present at this site. A population of Lin−/lo c-kithi T1/ST2+ cells was found in naïve BALB/c mice (Fig. 1A) that constituted 1.59% of the peritoneal cells. This population could be divided into SSClo and SSChi cells. The Lin−/lo c-kithi T1/ST2+ SSClo cells expressed high levels of integrin β7, intermediate levels of CD16/32, and virtually all were positive for FcɛRI (Fig. 1A). The phenotype of these cells was similar to Lin− c-kithi T1/ST2+ integrin β7hi CD16/32hi MCp in naïve mouse blood [3]. The Lin−/lo c-kithi T1/ST2+ SSChi expressed intermediate levels of CD16/32. Further, they expressed no, low, or intermediate levels of integrin β7 and were positive for FcɛRI (Fig. 1A). Altogether, the Lin−/lo c-kithi T1/ST2+ SSChi CD16/32int integrin β7−/lo/int FcɛRI+ phenotype suggested that these cells were mature mast cells. The Lin−/lo c-kithi T1/ST2+ SSClo CD16/32int integrin β7hi FcɛRI+ and the Lin−/lo c-kithi T1/ST2+ SSChi CD16/32int integrin β7−/lo/int FcɛRI+ cells comprised 0.05% and 1.46% of the total peritoneal cells, respectively (Fig. 1A, small panels).


Distinguishing Mast Cell Progenitors from Mature Mast Cells in Mice.

Dahlin JS, Ding Z, Hallgren J - Stem Cells Dev. (2015)

(A) Peritoneal cells from naïve mice were analyzed by flow cytometry. The Lin−/lo c-kithi T1/ST2+ cells were divided into SSClo and SSChi cells. The SSChi cells contained mostly CD16/32int integrin β7−/lo/int cells, whereas the SSClo fraction (encircled with a dashed ellipse) was mainly characterized as CD16/32int integrin β7hi. Virtually all Lin−/lo c-kithi T1/ST2+ SSChi CD16/32int integrin β7−/lo/int and Lin−/lo c-kithi T1/ST2+ SSClo CD16/32int integrin β7hi cells in the peritoneum expressed FcɛRI. (B) Mice were exposed to 5 Gy whole-body γ-irradiation. After 7 days, cells from peritoneal lavage were extracted and analyzed by flow cytometry. The Lin−/lo c-kithi T1/ST2+ SSChi and the Lin−/lo c-kithi T1/ST2+ SSClo (encircled with a dashed ellipse) cells were gated. (C) The total number of cells per milliliter peritoneal fluid was assessed in naïve and γ-irradiated mice. (D) The number of Lin−/lo c-kithi T1/ST2+ SSClo CD16/32int integrin β7hi FcɛRI+ MCp per milliliter peritoneal fluid was quantified in naïve and γ-irradiated mice. (E) The number of Lin−/lo c-kithi T1/ST2+ SSChi CD16/32int integrin β7−/lo/int FcɛRI+ mature mast cells (MC) was quantified in naïve and γ-irradiated mice. The data in (C–E) are derived from three pooled experiments where both groups were evaluated in parallel. Each group consisted of nine animals. The means and SEM are shown. The frequency of the gated cells in each graph was calculated by dividing the gated cell numbers with the total number of peritoneal cells. MCp, mast cell progenitors; SSC, side scatter. Color images available online at www.liebertpub.com/scd
© Copyright Policy - open-access
Related In: Results  -  Collection

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f1: (A) Peritoneal cells from naïve mice were analyzed by flow cytometry. The Lin−/lo c-kithi T1/ST2+ cells were divided into SSClo and SSChi cells. The SSChi cells contained mostly CD16/32int integrin β7−/lo/int cells, whereas the SSClo fraction (encircled with a dashed ellipse) was mainly characterized as CD16/32int integrin β7hi. Virtually all Lin−/lo c-kithi T1/ST2+ SSChi CD16/32int integrin β7−/lo/int and Lin−/lo c-kithi T1/ST2+ SSClo CD16/32int integrin β7hi cells in the peritoneum expressed FcɛRI. (B) Mice were exposed to 5 Gy whole-body γ-irradiation. After 7 days, cells from peritoneal lavage were extracted and analyzed by flow cytometry. The Lin−/lo c-kithi T1/ST2+ SSChi and the Lin−/lo c-kithi T1/ST2+ SSClo (encircled with a dashed ellipse) cells were gated. (C) The total number of cells per milliliter peritoneal fluid was assessed in naïve and γ-irradiated mice. (D) The number of Lin−/lo c-kithi T1/ST2+ SSClo CD16/32int integrin β7hi FcɛRI+ MCp per milliliter peritoneal fluid was quantified in naïve and γ-irradiated mice. (E) The number of Lin−/lo c-kithi T1/ST2+ SSChi CD16/32int integrin β7−/lo/int FcɛRI+ mature mast cells (MC) was quantified in naïve and γ-irradiated mice. The data in (C–E) are derived from three pooled experiments where both groups were evaluated in parallel. Each group consisted of nine animals. The means and SEM are shown. The frequency of the gated cells in each graph was calculated by dividing the gated cell numbers with the total number of peritoneal cells. MCp, mast cell progenitors; SSC, side scatter. Color images available online at www.liebertpub.com/scd
Mentions: Cells from peritoneal lavage were analyzed with flow cytometry to investigate whether MCp are present at this site. A population of Lin−/lo c-kithi T1/ST2+ cells was found in naïve BALB/c mice (Fig. 1A) that constituted 1.59% of the peritoneal cells. This population could be divided into SSClo and SSChi cells. The Lin−/lo c-kithi T1/ST2+ SSClo cells expressed high levels of integrin β7, intermediate levels of CD16/32, and virtually all were positive for FcɛRI (Fig. 1A). The phenotype of these cells was similar to Lin− c-kithi T1/ST2+ integrin β7hi CD16/32hi MCp in naïve mouse blood [3]. The Lin−/lo c-kithi T1/ST2+ SSChi expressed intermediate levels of CD16/32. Further, they expressed no, low, or intermediate levels of integrin β7 and were positive for FcɛRI (Fig. 1A). Altogether, the Lin−/lo c-kithi T1/ST2+ SSChi CD16/32int integrin β7−/lo/int FcɛRI+ phenotype suggested that these cells were mature mast cells. The Lin−/lo c-kithi T1/ST2+ SSClo CD16/32int integrin β7hi FcɛRI+ and the Lin−/lo c-kithi T1/ST2+ SSChi CD16/32int integrin β7−/lo/int FcɛRI+ cells comprised 0.05% and 1.46% of the total peritoneal cells, respectively (Fig. 1A, small panels).

Bottom Line: The maturation statuses of the cells were confirmed using three main strategies: (1) MCp, but not mature mast cells, were shown to be depleted by sublethal whole-body γ-irradiation. (2) The MCp were small and immature in terms of granule formation, whereas the mature mast cells were larger and had fully developed metachromatic granules. (3) The MCp had fewer transcripts of mast cell-specific proteases and the enzyme responsible for sulfation of heparin than mature mast cells.Moreover, isolated peritoneal MCp gave rise to mast cells when cultured in vitro.Using this strategy, mast cell maturation can be studied in vivo.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Biochemistry and Microbiology, Uppsala University , Uppsala, Sweden .

ABSTRACT
Mast cells originate from the bone marrow and develop into c-kit(+) FcɛRI(+) cells. Both mast cell progenitors (MCp) and mature mast cells express these cell surface markers, and ways validated to distinguish between the two maturation forms with flow cytometry have been lacking. Here, we show that primary peritoneal MCp from naïve mice expressed high levels of integrin β7 and had a low side scatter (SSC) light profile; whereas mature mast cells expressed lower levels of integrin β7 and had a high SSC light profile. The maturation statuses of the cells were confirmed using three main strategies: (1) MCp, but not mature mast cells, were shown to be depleted by sublethal whole-body γ-irradiation. (2) The MCp were small and immature in terms of granule formation, whereas the mature mast cells were larger and had fully developed metachromatic granules. (3) The MCp had fewer transcripts of mast cell-specific proteases and the enzyme responsible for sulfation of heparin than mature mast cells. Moreover, isolated peritoneal MCp gave rise to mast cells when cultured in vitro. To summarize, we have defined MCp and mature mast cells in naïve mice by flow cytometry. Using this strategy, mast cell maturation can be studied in vivo.

No MeSH data available.


Related in: MedlinePlus