Mast cells express connexins on their cytoplasmic membrane

doi:10.1016/S0091-6749(99)70239-3

Journal of Allergy and Clinical Immunology

Volume 103, Issue 4, April 1999, Pages 656-662

https://doi.org/10.1016/S0091-6749(99)70239-3 Get rights and content

Abstract

Background: Because of the close association between mast cells and fibroblasts in the microenvironment and the importance of connexins (Cxs) in fibroblast communication with other cells, we hypothesized that mast cells also express Cxs, allowing them to similarly communicate with other cells through gap junctions. Objectives: We sought to identify the expression of Cxs (particularly Cx43, Cx32, and Cx26) by murine mast cells. Methods: The expression of Cxs was studied by RT-PCR, Northern blot analysis, Western blot analysis, flow cytometry, and confocal laser scanning microscopy. Results: In this report we demonstrate that murine bone marrow cultured mast cells and the growth factor–independent murine mast cell line C57, express Cx43 and Cx32 as assessed by RT-PCR, Northern blot analysis, Western blot analysis, and flow cytometry, but do not express Cx26. We also show, by confocal laser scanning microscopy, that Cx43 localizes to the cytoplasmic membrane of mast cells in a pattern similar to that seen in fibroblasts. Conclusions: Mast cells express Cx43 and Cx32, and Cx43 is associated with the cytoplasmic membrane, suggesting that mast cells have the potential to communicate with other cells in their microenvironment in part through gap junctions. (J Allergy Clin Immunol 1999;103:656-62.)

Section snippets

Materials

WEHI-3 conditioned medium (containing IL-3) (CollaborativeBiomedical Products, Bedford, Mass); Dulbecco’s modified Eagle’s medium (DMEM), RPMI-1640, HEPES, penicillin/streptomycin, L- glutamine, nonessential amino acids, and FCS (Biofluids Inc, Rockville, Md); Trizol, Superscript Reverse Transcription kit, and Taq polymerase (Life Technologies, Gaithersburg, Md); phorbol 12-myristate 13-acetate (PMA), DNP, 2-mercaptoethanol (2-ME), IgE, monoclonal anti-DNP antibody, clone SPE-7, propyl gallate,

RT-PCR

Total RNA was isolated from NIH-3T3 fibroblasts, C57 mast cells, and BMCMCs with Trizol according to the manufacturer’s instructions. Five micrograms of total RNA was used for reverse transcription of first-strand cDNA with the Superscript-RT kit. One tenth of the total cDNA was used to amplify Cx26, Cx32, and Cx43 mRNA with 30 cycles at 94°C for 1 minute, 60°C for 1 minute, and 72°C for 1 minute with a Perkin-Elmer 9600 automatic cycler (Perkin-Elmer, Norwalk, Conn). The following primers were

Mast cell Cx mRNA expression

To determine whether mast cells express Cx, we first examined Cx expression by RT-PCR. In addition to Cx43, which is expressed on monocytes and thymocytes, we also determined the expression of Cx32 and Cx26, which are transcribed in multiple tissues. As can be seen in Fig 1, A , BMCMCs and C57 mast cells express mRNA for Cx43 and Cx32.

. Mast cell Cx mRNA expression. A, RT-PCR for Cx43, Cx32, and Cx26. Cx43 (lanes 1 to 3 ), Cx32 (lanes 4 to 6 ), and Cx26 (lanes 7 to 9 ) were amplified from NIH-3T3

DISCUSSION

Gap junctions are channels that are composed of oligomerized Cxs, which allow intercellular movement of ions and molecules of less than 1 kd. In this study we report that murine mast cells express mRNA (Fig 1) and protein (Fig. 2, Fig. 3) for Cx43 and Cx32 but not Cx26. Furthermore, at least Cx43 immunoreactivity appeared to be expressed on the cytoplasmic membrane of mast cells, and its localization was similar with that seen in fibroblasts (Fig 4).

Information concerning the expression of Cxs

References (25)

S Adachi et al.
Necessity of extracellular domain of W (c-kit) receptors for attachment of murine cultured mast cells to fibroblasts
Blood
(1992)
DL Paul
New functions for gap junctions
Curr Opin Cell Biol
(1995)
JJ Cho et al.
Identification and categorization of inducible mast cell genes in a subtraction library
Biochem Biophys Res Commun
(1998)
C Prussin et al.
Detection of intracytoplasmic cytokine using flow cytometry and directly conjugated anti-cytokine antibodies
J Immunol Methods
(1995)
EC Beyer et al.
Evidence that the gap junction protein connexin-43 is the ATP-induced pore of mouse macrophages
J Biol Chem
(1991)
H Ginsburg et al.
Mast cell growth on fibroblast monolayers: two-cell entities
Immunology
(1982)
F Levi Schaffer et al.
Co-culture of interleukin 3-dependent mouse mast cells with fibroblasts results in a phenotypic change of the mast cells
Proc Natl Acad Sci U S A
(1986)
JR Gordon et al.
Promotion of mouse fibroblast collagen gene expression by mast cells stimulated via the FcϵRI. Role for mast cell-derived transforming growth factor beta and tumor necrosis factor-α
J Exp Med
(1994)
BL Gruber et al.
Human mast cells activate fibroblasts: tryptase is a fibrogenic factor stimulating collagen messenger ribonucleic acid synthesis and fibroblast chemotaxis
J Immunol
(1997)
E Razin et al.
Connective tissue mast cells in contact with fibroblasts express IL-3 mRNA. Analysis of single cells by polymerase chain reaction
J Immunol
(1991)

AS Kirshenbaum et al.

Effect of IL-3 and stem cell factor on the appearance of human basophils and mast cells from CD34+ pluripotent progenitor cells

J Immunol

(1992)

M Rottem et al.

The effects of stem cell factor on the ultrastructure of FcϵRI+ cells developing in IL-3-dependent murine bone marrow-derived cell cultures

J Immunol

(1993)

Cited by (33)

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By 2040 neurodegenerative diseases will become the world’s second leading cause of death after cardiovascular disease (WHO). Major efforts are required to elucidate the underlying molecular and cellular mechanisms of neurodegenerative diseases. Connexin and pannexin membrane channel proteins are conduits through which neuronal, glial, and vascular tissues interact. In the normal brain, this interaction underlies homeostasis, metabolic supply and neuroprotection. In models of neuroinflammation these channels present aberrant functioning. Validation of the molecular mechanisms by which these membrane channels influence neurodegeneration particularly in Alzheimer’s disease could lead to new and alternative therapeutic strategies targeting these channels.
The role of connexin and pannexin containing channels in the innate and acquired immune response
2018, Biochimica et Biophysica Acta - Biomembranes
Citation Excerpt :
Formation of GJs between eosinophils and tissue resident cells may provide another mechanism of interaction during inflammatory reactions, which could lead to limited and specific activation of communicating cells, as opposed to the broad activation obtained through the paracrine effects of released cytokines and chemokines [118]. Mast cells, also called mastocytes, express Cx43, Cx32 [123–125], and Panx-2 in myenteric and submucosal ganglia [126]. Cx43 and Cx32 expression have also been detected in murine bone marrow cultured mast cells and the mast cell line C57 [123].
Connexin (Cx) and pannexin (Panx) containing channels – gap junctions (GJs) and hemichannels (HCs) – are present in virtually all cells and tissues. Currently, the role of these channels under physiological conditions is well defined. However, their role in the immune response and pathological conditions has only recently been explored. Data from several laboratories demonstrates that infectious agents, including HIV, have evolved to take advantage of GJs and HCs to improve viral/bacterial replication, enhance inflammation, and help spread toxicity into neighboring areas. In the current review, we discuss the role of Cx and Panx containing channels in immune activation and the pathogenesis of several infectious diseases. This article is part of a Special Issue entitled: Gap Junction Proteins edited by Jean Claude Herve.
Expression and role of connexin-based gap junctions in pulmonary inflammatory diseases
2016, Pharmacology and Therapeutics
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If GJ channels are not closed during the inflammatory response, enhanced intercellular diffusion of signaling molecules and recruitment of neighboring cells may therefore contribute to the excessive inflammatory response of cystic fibrosis airway epithelium (Chanson et al., 2001; Huang et al., 2003; Dudez et al., 2008). Finally, although a precise role of Cx43 in inflammation associated with lung allergic diseases has not been investigated yet, Cx43 protein is expressed on the cytoplasmic membrane of murine bone marrow cultured mast cells and of a murine mast cell line, suggesting that mast cells have the potential to communicate with other cells in their microenvironment through GJs (Vliagoftis et al., 1999). As mast cells are key drivers of pathophysiological changes and tissue remodeling, associated with chronic allergic inflammation in asthma and other allergic disorders (Galli & Tsai, 2012), their communication with adjacent cells such as fibroblasts or smooth muscle cells through GJs may therefore play a role in the development and persistence of lung inflammation.
Connexins are transmembrane proteins that can generate intercellular communication channels known as gap junctions. They contribute to the direct movement of ions and larger cytoplasmic solutes between various cell types. In the lung, connexins participate in a variety of physiological functions, such as tissue homeostasis and host defence. In addition, emerging evidence supports a role for connexins in various pulmonary inflammatory diseases, such as asthma, pulmonary hypertension, acute lung injury, lung fibrosis or cystic fibrosis. In these diseases, the altered expression of connexins leads to disruption of normal intercellular communication pathways, thus contributing to various pathophysiological aspects, such as inflammation or tissue altered reactivity and remodeling.
The present review describes connexin structure and organization in gap junctions. It focuses on connexins in the lung, including pulmonary bronchial and arterial beds, by looking at their expression, regulation and physiological functions. This work also addresses the issue of connexin expression alteration in various pulmonary inflammatory diseases and describes how targeting connexin-based gap junctions with pharmacological tools, synthetic blocking peptides or genetic approaches, may open new therapeutic perspectives in the treatment of these diseases.
Connexins: Intercellular Signal Transmitters in Lymphohematopoietic Tissues
2015, International Review of Cell and Molecular Biology
Life-long hematopoietic demands are met by a pool of hematopoietic stem cells (HSC) with self-renewal and multipotential differentiation ability. Humoral and paracrine signals from the bone marrow (BM) hematopoietic microenvironment control HSC activity. Cell-to-cell communication through connexin (Cx) containing gap junctions (GJs) allows pluricellular coordination and synchronization through transfer of small molecules with messenger activity. Hematopoietic and surrounding nonhematopoietic cells communicate each other through GJs, which regulate fetal and postnatal HSC content and function in hematopoietic tissues. Traffic of HSC between peripheral blood and BM is also dependent on Cx proteins. Cx mutations are associated with human disease and hematopoietic dysfunction and Cx signaling may represent a target for therapeutic intervention. In this review, we illustrate and highlight the importance of Cxs in the regulation of hematopoietic homeostasis under normal and pathological conditions.
Intercellular interactions between mast cells and fibroblasts promote pro-inflammatory signaling
2013, Experimental Cell Research
Citation Excerpt :
Since the elevated co-culture-induced IL-8 was observed within 2 h of co-culture and as phase contrast microscopy showed mast cell attachment to fibroblasts, we examined intercellular communication between these cells by confocal microscopy. Fibroblasts were incubated with mast cells that had been labelled previously with calcein [74]. Live imaging showed time-dependent dye transfer from mast cells to fibroblasts, which was detected within 60 min after initiation of co-cultures.
The mechanisms that mediate acute exacerbations in chronic inflammatory diseases are not understood. As IL-8 is a potent chemoattractant for neutrophils in acute inflammatory lesions, we investigated the role of fibroblast-mast cell interactions in short-term IL-8 release. Human gingival fibroblasts were co-cultured with human mast cells (HMC). The concentration of IL-8 in co-culture medium was measured by ELISA. HMC-fibroblast co-cultures showed >8-fold higher IL-8 secretion than fibroblasts or HMC alone. Increased IL-8 secretion required HMC-fibroblast intercellular contact, was enhanced by serum and was blocked by the gap junction inhibitor β-glycyrrhetinic. Calcein-dye transfer showed intercellular, gap junction communication between HMC and fibroblasts that was dependent in part on hyaluronic acid on the cell surface of fibroblasts. IL-8 secretion by fibroblasts was strongly promoted by hyaluronic acid. Pre-treatment of HMC with thapsigargin provoked 15-fold higher IL-8 production by fibroblasts in co-cultures. Chemotaxis of mouse neutrophils was enhanced 2-fold in response to conditioned medium from HMC-fibroblast co-cultures. We conclude that mast cells adhere to fibroblasts and promote IL-8 secretion, which enhances neutrophil chemotaxis and the inflammatory response.
CD4<sup>+</sup>CD25<sup>+</sup> Regulatory T Cells Suppress Mast Cell Degranulation and Allergic Responses through OX40-OX40L Interaction
2008, Immunity
T regulatory (Treg) cells play a role in the suppression of immune responses, thus serving to induce tolerance and control autoimmunity. Here, we explored whether Treg cells influence the immediate hypersensitivity response of mast cells (MCs). Treg cells directly inhibited the FcɛRI-dependent MC degranulation through cell-cell contact involving OX40-OX40L interactions between Treg cells and MCs, respectively. When activated in the presence of Treg cells, MCs showed increased cyclic adenosine monophosphate (cAMP) concentrations and reduced Ca²⁺ influx, independently of phospholipase C (PLC)-γ2 or Ca²⁺ release from intracellular stores. Antagonism of cAMP in MCs reversed the inhibitory effects of Treg cells, restoring normal Ca²⁺ responses and degranulation. Importantly, the in vivo depletion or inactivation of Treg cells caused enhancement of the anaphylactic response. The demonstrated crosstalk between Treg cells and MCs defines a previously unrecognized mechanism controlling MC degranulation. Loss of this interaction may contribute to the severity of allergic responses.

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^☆: The confocal part of the study was supported by the Medical Research Council of Canada and the Alberta Heritage Foundation for Medical Research (AHFMR). Dr Moqbel is an AHFMR Senior Medical Scholar.

^☆☆: *Harissios Vliagoftis, MD, and Anne M. Hutson, BSc, shared equally in the essential nature of this paper.

^★: Reprint requests: Harissios Vliagoftis, MD, Pulmonary Research Group, Department of Medicine, 574 HMRC, University of Alberta, Edmonton, AB, T6G 2S2, Canada.

^★★: 1/1/96779

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Mast cells express connexins on their cytoplasmic membrane☆,☆☆,★,★★

Abstract

Section snippets

Materials

RT-PCR

Mast cell Cx mRNA expression

DISCUSSION

Blood

Curr Opin Cell Biol

Biochem Biophys Res Commun

J Immunol Methods

J Biol Chem

Mast cell growth on fibroblast monolayers: two-cell entities

Immunology

Co-culture of interleukin 3-dependent mouse mast cells with fibroblasts results in a phenotypic change of the mast cells

Proc Natl Acad Sci U S A

Promotion of mouse fibroblast collagen gene expression by mast cells stimulated via the FcϵRI. Role for mast cell-derived transforming growth factor beta and tumor necrosis factor-α

J Exp Med

Human mast cells activate fibroblasts: tryptase is a fibrogenic factor stimulating collagen messenger ribonucleic acid synthesis and fibroblast chemotaxis

J Immunol

Connective tissue mast cells in contact with fibroblasts express IL-3 mRNA. Analysis of single cells by polymerase chain reaction

J Immunol

Effect of IL-3 and stem cell factor on the appearance of human basophils and mast cells from CD34+ pluripotent progenitor cells

J Immunol

The effects of stem cell factor on the ultrastructure of FcϵRI+ cells developing in IL-3-dependent murine bone marrow-derived cell cultures

J Immunol