Allergy Asthma Immunol Res.  2018 May;10(3):268-277. 10.4168/aair.2018.10.3.268.

Immune Characterization of Bone Marrow-Derived Models of Mucosal and Connective Tissue Mast Cells

Affiliations
  • 1Department of Pediatrics, Mindich Child Health and Development Institute, Immunology Institute, Icahn School of Medicine at Mount Sinai, New York, NY, USA. sbenede@ucm.es

Abstract

PURPOSE
It is well appreciated that mast cells (MCs) demonstrate tissue-specific imprinting, with different biochemical and functional properties between connective tissue MCs (CTMCs) and mucosal MCs (MMCs). Although in vitro systems have been developed to model these different subsets, there has been limited investigation into the functional characteristics of the 2 major MC subsets. Here, we report the immunologic characterization of 2 MCs subsets developed in vitro from bone marrow progenitors modeling MMCs and CTMCs.
METHODS
We grew bone marrow for 4 weeks in the presence of transforming growth factor (TGF)-β, interleukin (IL)-9, IL-3, and stem cell factor (SCF) to generate MMCs, and IL-4, IL-3, and SCF to generate CTMCs.
RESULTS
CTMCs and MMCs differed in growth rate and protease content, but their immune characteristics were remarkably similar. Both subsets responded to immunoglobulin E (IgE)-mediated activation with signaling, degranulation, and inflammatory cytokine release, although differences between subsets were noted in IL-10. CTMCs and MMCs showed a similar toll-like receptor (TLR) expression profile, dominated by expression of TLR4, TLR6, or both subsets were responsive to lipopolysaccharide (LPS), but not poly(I:C). CTMCs and MMCs express receptors for IL-33 and thymic stromal lymphopoietin (TSLP), and respond to these cytokines alone or with modified activation in response to IgE cross-linking.
CONCLUSIONS
The results of this paper show the immunologic characterization of bone marrow-derived MMCs and CTMCs, providing useful protocols for in vitro modeling of MC subsets.

Keyword

Mucosal mast cells; connective tissue mast cells; mast cell subsets

MeSH Terms

Bone Marrow
Connective Tissue*
Cytokines
Immunoglobulin E
Immunoglobulins
In Vitro Techniques
Interleukin-10
Interleukin-3
Interleukin-33
Interleukin-4
Interleukins
Mast Cells*
Stem Cell Factor
Toll-Like Receptors
Transforming Growth Factors
Cytokines
Immunoglobulin E
Immunoglobulins
Interleukin-10
Interleukin-3
Interleukin-33
Interleukin-4
Interleukins
Stem Cell Factor
Toll-Like Receptors
Transforming Growth Factors

Figure

  • Fig. 1 Maturation and differentiation of MMCs and CTMCs from bone marrow progenitors. (A) Growth rates of MMCs and CTMCs. (B) Representative staining of FcεRI and c-kit expression after 2 or 4 weeks of culture with growth factors (left), and percentage of FcεRI+/c-kit+ cells after 1, 2, 3, or 4 weeks of culture with growth factors. (C) RT-PCR for MMCP-1, MMCP-2, MMCP-4, MMCP-5, MMCP-6, MMCP-7, MMCP-8, and CPA in MMCs and CTMCs after 2 or 4 weeks of culture of bone marrow progenitors from Balb/c mice with growth factors. Relative expression refers to data normalized to the housekeeping gene. Data are expressed has the percentage distribution of the total protease expression. Data represent the mean of 3 different experiments. (D) As in C, but using bone marrow from C57BL/6 mice and analyzed at 4 weeks. MMC, mucosal mast cell; CTMC, connective tissue mast cell; RT-PCR, real-time polymerase chain reaction; MMCP, mouse mast cell protease; CPA, carboxypeptidase. *P<0.05; †P<0.0001.

  • Fig. 2 Metachromatic staining of MMCs and CTMCs with toluidine blue or the chloroacetate esterase dyes. (A) Metachromatic staining with toluidine blue or (B) choroacetate esterase. Cytospins of MCs were prepared at 150 rpm for 5 minutes. Images are representative of 3 samples per condition. Data correspond to bone marrow-derived MCs obtained from Balb/c mice. MMC, mucosal mast cell; CTMC, connective tissue mast cell.

  • Fig. 3 Activation of MMCs and CTMCs after stimulation through FcεRI. (A) Intracellular expression of phospho-Syk, (B) surface expression of CD107a, and (C) degranulation, measured as percentage of β-hexosaminidase secretion, in sensitized MMCs and CTMCs with mouse IgE or anti-DNP IgE after 30 minutes of activation with α-IgE or DNP-HSA. (D) Percentage of β-hexosaminidase secreted by MMCs and CTMCs after stimulation with compound 48/80. Data correspond to bone marrow-derived MCs obtained from Balb/c mice. MMC, mucosal mast cell; CTMC, connective tissue mast cell; IgE, immunoglobulin E; DNP, 2,4-dinitrophenol; DNP-HAS, dinitrophenylated human serum albumin; MC, mast cell.

  • Fig. 4 Cytokine expression of MMCs and CTMCs after activation through FcεRI. (A) Cytokine mRNA expression expressed as fold change by RT-PCR in MMCs and CTMCs 2 hours after activation with α-IgE. Cytokine secretion from MMCs and CTMCs 2 (B) or (C) 24 hours after stimulation with anti-IgE or DNP-HSA. IL-33, IFN-γ, IL-17, and IL-4 were measured, but were below the level of detection. Data correspond to bone marrow derived MCs obtained from Balb/c mice. MMC, mucosal mast cell; CTMC, connective tissue mast cell; RT-PCR, real-time polymerase chain reaction; IgE, immunoglobulin E; DNP-HAS, dinitrophenylated human serum albumin; IL, interleukin; IFN, interferon; MC, mast cell. *P<0.05; †P<0.01; ‡P<0.001; §P<0.0001.

  • Fig. 5 TLRs on MMCs and CTMCs. (A) TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR11, TLR12, and TLR13 expression by MMCs and CTMCs, expressed as relative expression compared to housekeeping gene. IL-6 secretion from MMCs and CTMCs after stimulation with a dose range of LPS (B) or poly(I:C) (C) for 24 hours in the absence of anti-IgE. Data correspond to bone marrow derived MCs obtained from Balb/c mice. TLR, toll-like receptor; MMC, mucosal mast cell; CTMC, connective tissue mast cell; IL, interleukin; LPS, lipopolysaccharide; IgE, immunoglobulin E; MC, mast cell. *P<0.05.

  • Fig. 6 Effect of IL-33 and TSLP on MMCs and CTMCs. (A) ST2, TSLPR, and CD127 expression by MMCs and CTMCs in the absence of anti-IgE stimulation. IL-6, IL-13, and TNF-α secretion by MMCs and CTMCs after 24 hours of stimulation with 100 ng/mL IL-33 (B) or TSLP (C), alone or in the presence of anti-IgE. Showed data correspond to bone marrow-derived MCs obtained from Balb/c mice. Note the difference in scale between (B) and (C). IL, interleukin; TSLP, thymic stromal lymphopoietin; MMC, mucosal mast cell; CTMC, connective tissue mast cell; TSLPR, thymic stromal lymphopoietin receptor; IgE, immunoglobulin E; TNF, tumor necrosis factor; MC, mast cell. *P<0.05; †P<0.01; ‡P<0.001; §P<0.0001.


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