A functional comparison of canine and murine bone marrow derived cultured mast cells

Abstract

Disorders involving mast cells are extremely common in dogs, ranging from allergic diseases to neoplastic transformation resulting in malignant mast cell tumors. Relatively little is known regarding the basic biologic properties of normal canine mast cells, largely due to the difficulty in reliably purifying large numbers from canine skin. In vitro generated bone marrow derived cultured mast cells (BMCMCs) are routinely used in both human and murine studies as a ready source of material for in vitro and in vivo studies. We previously developed a technique to generate canine BMCMCs from bone marrow derived CD34+ cells and demonstrated that these cells exhibit the phenotypic properties characteristic of mast cells and release histamine in response to IgE cross-linking. The purpose of the following study was to characterize the functional properties of these canine BMCMCs and contrast these with the functional properties of murine BMCMCs. Our work demonstrates that both IL-4 and IL-10 promote canine BMCMC proliferation, possibly through upregulation of Kit expression, while TGFβ inhibits proliferation. The canine BMCMCs produce a variety of cytokines and chemokines in response to IgE cross-linking and chemical stimulation including IL-3, IL-4, IL-13, GM-CSF, RANTES, and MIP1α. Interestingly, the canine BMCMCs released significantly larger amounts of MCP-1 and tryptase and significantly smaller amounts of IL-6 following chemical stimulation and IgE cross-linking when compared to murine BMCMCs. Lastly, the canine BMCMCs produced larger amounts of active MMP9 than their murine counterparts. In summary, canine BMCMCs exhibit unique functional properties that distinguish them from murine BMCMCs and provide insight into the contribution of these cells to mast cell disorders in the dog.

Introduction

Disorders involving mast cells are common in dogs and include allergic diseases such as atopy as well as mast cell tumors (de Mora et al., 1993, Misdorp, 2004; von Ruedorffer et al., 2003), one of the most prevalent neoplasms found in this species. Furthermore, there is substantial evidence that many of these disorders exhibit particular breed predispositions suggesting that genetics plays a role in mast cell diseases (London and Seguin, 2003). Despite the abundance of mast cell disorders in dogs, relatively little is known regarding the biology of normal canine mast cells, particularly with respect to their responses to a variety of stimuli.

Historically, canine mast cell biology has been studied by purifying mast cells from canine skin (Brazis et al., 1998), or by evaluating cell lines derived from malignant mast cell tumors (Fang et al., 1996, Takahashi et al., 2001). With respect to canine skin derived mast cells, they have been shown to release histamine and TNFα through both IgE-dependent and -independent mechanisms which were enhanced by SCF stimulation (Brazis et al., 2000). Other studies have demonstrated that canine mast cells can respond to C-reactive protein (Fujimoto et al., 2003). Unfortunately, it is very difficult to obtain large numbers of mast cells from skin and in many cases, these populations are not pure. As such, detailed studies on the biologic function of skin derived canine mast cells are not possible.

Due to the historical difficulty in obtaining a large number of normal canine mast cells, the characterization of canine mast cell biology has relied up on evaluation of the functional properties of canine malignant mast cell lines. For example, both the BR and CM-MC canine mast cell lines failed to respond to IgE cross-linking (Garcia et al., 1998), but the CM-MC line released histamine in response to cross-linking of either IgG1 or IgG4 (Sato et al., 2004, Takahashi et al., 2001). These cell lines also produced TGFβ which inhibited their proliferation (Pennington et al., 1992). Furthermore, functional Kit, estrogen, and adenosine receptors were shown to be present on the cell lines (Auchampach et al., 1997, Larsen and Grier, 1989, Liao et al., 2002) and production of metalloproteinases MMP2, MMP9, IL-4, IL-5, FGFβ, PDGF, and PGD₂ could be demonstrated (Fang et al., 1999). However, the cell lines did not exhibit consistent cytokine expression profiles. Lastly, some receptors believed to be expressed on normal mast cells were not identified on the canine mast cell lines (Lin et al., 2006). These data suggest that while the malignant mast cell lines exhibit some functional properties that are seen in normal mast cells, they are probably not reliable for detailed studies aimed at dissecting the biology of these cells.

Human and mouse mast cells differentiated in vitro from bone marrow, cord blood or other peripheral blood have been used for several years to characterize basic aspects of mast cell biology (Dahl et al., 2002, Kinoshita et al., 1999, Mekori et al., 1993, Shimizu et al., 2002). These cell populations are considered a valuable resource as generating sufficient numbers of normal mast cells from either human or mouse tissues is extremely difficult, time consuming, and precludes certain studies, such as those that involve reconstitution of mast cell deficient mice. In vitro generated mast cells have been used to define the role of these cells in asthma (Stassen et al., 2000), automimmunity (Robbie-Ryan et al., 2003), as well as innate immune responses (Bidri et al., 1997). Furthermore, they have been instrumental in dissecting the regulation of mast cell mediator production such as proteases (chymases, tryptases), prostaglandins, and a variety of cytokines/chemokines. Recent work employing in vitro derived mast cells has defined their involvement in angiogenesis as it relates to both normal processes such as wound healing (Noli and Miolo, 2001), as well as pathologic processes such as neoplasia and fibrotic diseases (Theoharides and Conti, 2004).

Given the frequency of mast cell disease in the dog, it is important that the biology of normal canine mast cells be further explored to begin to identify those factors that ultimately lead to pathologic processes involving these cells. In a previous study, we developed a technique to generate canine mast cells from bone marrow derived CD34+ cells (Lin et al., 2006). These bone marrow derived cultured mast cells (BMCMCs) were found to contain chymase and tryptase and expressed typical cell surface markers including Kit, FcɛRI, and integrins. The canine BMCMCs were dependent on canine stem cell factor (SCF) for survival and proliferated and migrated in response to SCF. Lastly, cross-linking of cell surface bound IgE induced histamine and TNFα release. Therefore, the canine BMMCs possess phenotypic and functional properties similar to those described of mast cells directly isolated from canine skin. The purpose of the following study was to expand on this initial work and begin a detailed characterization of the functional properties of canine BMCMCs, employing murine BMCMCs for comparison.