CCR1 expression and signal transduction by murine BMMC results in secretion of TNF-α, TGFβ-1 and IL-6

Abstract

Chemokine receptors (CCRs) are important co-stimulatory molecules found on many blood cells and associated with various diseases. The expression and function of CCRs on mast cells has been quite controversial. In this study, we report for the first time that murine bone marrow-derived mast cells (BMMC) express messenger RNA and protein for CCR1. BMMC cultured in the presence of murine recombinant stem cell factor and murine IL-3 expressed CCR1 after 5-6 weeks. We also report for the first time that mBMMC(CCR1+) cells endogenously express neurokinin receptor-1 and intercellular adhesion molecule-1. To examine the activity of CCR1 on these BMMC, we simultaneously stimulated two receptors: CCR1 by its ligand macrophage inflammatory protein-1alpha and the IgE receptor FcepsilonRI by antigen cross-linking. We found that co-stimulation enhanced BMMC degranulation compared with FcepsilonRI stimulation alone, as assessed by beta-hexosaminidase activity (85 versus 54%, P < 0.0001) and Ca(2+) influx (223 versus 183 nM, P < 0.05). We also observed significant increases in mast cell secretion of key growth factors, cytokines and chemokine mediators upon CCR1-FcepsilonRI co-stimulation. These factors include transforming growth factor beta-1, tumor necrosis factor-alpha and the cytokine IL-6. Taken together, our data indicate that CCR1 plays a key role in BMMC function. These findings contribute to our understanding of mechanisms for immune cell trafficking during inflammation.

Role of CCL7 in Type I Hypersensitivity Reactions in Murine Experimental Allergic Conjunctivitis

“…CCL3 was reported to stimulate human mast cell degranulation in vitro (16) and murine mast cell degranulation in vitro and in vivo (7,17). We found that treatment of mast cells with CCL3 and Ag results in greater degranulation than does cross-linking of FcεRI alone (8,17). Mice in which CCL3 is deficient or neutralized fail to display typical allergic symptoms after ocular exposure to allergen.…”

Evidence That Formation of Vimentin·Mitogen-activated Protein Kinase (MAPK) Complex Mediates Mast Cell Activation following FcεRI/CC Chemokine Receptor 1 Cross-talk

“…To generate primary BMMCs, low density mononuclear cells (LDMNCs) were isolated from BALB/c mice (Jackson Laboratories) and cultured in RPMI1640 containing 10% FBS, 4 mM glutamine, 100 units/ml penicillin, 100 g/ml streptomycin, 0.1 mM non-essential amino acids, and 50 M 2-mercaptoethanol in the presence of 5 ng/ml of recombinant murine IL-3 (PeproTech) for 4 -6 weeks (12,17). The purity of BMMCs exceeded 80%, which was determined by flow cytometric analysis after staining c-Kit and Fc⑀RI on the cell surface.…”

“…Moreover, CCL3 synergistically enhanced Fc⑀RI-mediated degranulation and gene expression of cytokines and chemokines (e.g. IL-6 and CCL7/MCP-3) in a rat basophilic leukemia 2H3 cell line (RBL-2H3) expressing CC chemokine receptor 1 (CCR1), a receptor for CCL3, and bone marrow-derived murine mast cells (11)(12)(13)(14). These observations indicate that (i) the simultaneous engagement of Fc⑀RI and CCR1 is important for optimal activation of mast cells in vitro and physiologically relevant levels of mast cell activation in vivo, and (ii) there is a cross-talk between the Fc⑀RI-mediated and CCR1-mediated signaling cascades.…”

The role of TRB3 in mast cells sensitized with monomeric IgE

“…Culture medium was refreshed every 3 days, and 1 mg/mL of Geneticin (G-418 sulfate) was added to the medium of RBL-CCR1 cells as described previously (Toda et al, 2004;Fifadara et al, 2009). To generate primary BMMCs, low-density mononuclear cells (LDMNCs) were isolated from BALB/c mice (Charles Rivers) Laboratories, and cultured in RPMI1640 containing 10% fetal bovine serum, 4 mM glutamine, 100 units/ml penicillin, 100 μg/ml streptomycin, 0.1 mM non-essential amino acids, and 50 μM 2-mercaptoethanol in the presence of 5 ng/ml of recombinant murine IL-3 (PeproTech) for 4-6 weeks (Toda et al, 2004;Fifadara et al, 2009). The purity of BMMCs exceeded 80%, which was determined by flow cytometric analysis after staining c-Kit and FcεRI on the cell surface.…”