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- Materials and methods
Background: Mast cells play pivotal roles in IgE-mediated airway inflammation and other mast cell-mediated inflammation by activation and chemoattraction of inflammatory cells.
Objective: We investigated the intracellular signaling mechanisms regulating chemokine release from human mast cell line-1 (HMC-1) cells activated by stem cell factor (SCF) or tumor necrosis factor (TNF)-α.
Methods: Chemokine gene expressions were assessed by reverse transcription-polymerase chain reaction, while the releases of chemokines were determined by flow cytometry or enzyme-linked immunosorbent assay (ELISA). To elucidate the intracellular signal transduction regulating the chemokine expression, phosphorylated-extracellular signal-regulated kinase (ERK), phosphorylated-p38 mitogen-activated protein kinase (MAPK) and nuclear translocated nuclear factor (NF)-κB-DNA binding were quantitatively assessed by ELISA.
Results: Either SCF or TNF-α could induce release from HMC-1 cells of interleukin (IL)-8, monocyte chemoattractant protein (MCP)-1, regulated upon activation normal T-cell expressed and secreted (RANTES), and I-309, while SCF and TNF-α induced release of macrophage inflammatory protein (MIP)-1β and interferon-γ-inducible protein-10 (IP-10), respectively. Using various selective inhibitors for signaling molecules, we found that the inductions of IL-8, MCP-1, and I-309 were mediated by either SCF-activated ERK or TNF-α-activated p38 MAPK, while the induction of IP-10 by TNF-α was mediated by both activated p38 MAPK and NF-κB. The induction of RANTES by SCF or TNF-α was mediated by ERK and NF-κB, respectively, and SCF induced MIP-1β release was mediated by ERK.
Conclusion: The above results therefore elucidated the different intracellular signaling pathways regulating the release of different chemokines from SCF and TNF-α-activated mast cells, thereby shedding light for the immunopathological mechanisms of mast cell-mediated diseases.
Mast cells are central effector cells that cause immediate-type hypersensitivity. Upon allergen provocation, cross linkage of IgE bound on mast cells, via the high-affinity receptors triggers the release of an array of inflammatory mediators including histamine, neutral proteases and heparin sulphate, prostaglandins, and cysteinyl leukotrienes, as well as various cytokines and chemokines that are involved in recruitment and activation of mast cells and other leukocytes such as eosinophils (1, 2). Mast cells are also involved in both natural and acquired immunity (3) and T helper (Th)-mediated inflammation such as inflammatory bowel disease (4), and rheumatoid arthritis (5).
All the mast cell-mediated inflammatory reactions are characterized by an accumulation of mast cells in the inflammatory sites (6). For example, the selective microlocalization of mast cells within specific airway structures, such as the airway smooth muscle and submucosal glands, is important in the pathophysiology of inflammatory lung disease (7). Apart from the release of inflammatory mediators, mast cells also play their multiple immunopathological roles in many inflammatory disorders by releasing multiple chemokines to recruit different subtypes of leukocytes (1, 8).
In inflammatory cascades, human mast cells can response to stem cell factor (SCF) secreted by fibroblasts and epithelial cells, resulting in enhanced mast cell survival, delayed apoptosis, differentiation, migration and priming for mediator and chemokine release via SCF receptor (c-kit) (2, 8–10). It is known that pro-inflammatory cytokine tumor necrosis factor (TNF)-α is released from mast cells, macrophages and T cells via IgE-dependent mechanisms in allergic responses (11, 12). Tumor necrosis factor-α can stimulate TNF-αR1 receptors on the cell surface of mast cells for mediator release (2).
Previous studies have suggested that the transcription factor nuclear factor (NF)-κB is involved in the expression of many inflammatory cytokines and adhesion molecules of mast cells (13). Extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK) were found to play a role of adenosine A2B receptor-mediated interleukin (IL)-8 production and IgE-mediated IL-6 production in mast cells (14, 15). However, the intracellular mechanisms coordinated by MAPK and NF-κB for the release of various chemokines of mast cells in inflammatory response have not been well studied. In an attempt to further elucidate the immunological roles of mast cells in inflammation, the intracellular mechanisms for the release of a panel of chemokine IL-8, monocyte chemoattractant protein-1 (MCP-1), regulated upon activation normal T-cell expressed and secreted (RANTES), I-309, macrophage inflammatory protein-1 β (MIP-1β), and interferon-γ-inducible protein-10 (IP-10) from SCF and TNF-α-activated mast cells were investigated.
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- Materials and methods
The HMC-1 is a well established and widely used cell line exhibiting many characteristics of mast cells (16). As shown from other and our previous study, these cells can be activated by phorbol esters, calcium ionophore, SCF, and TNF-α. Human mast cell line-1 cells can therefore serve as a useful mast cell system for the study of the expression of chemokines during inflammation (1, 20). We showed in the present study that upon SCF and TNF-α stimulation, HMC-1 cells could induce the release a panel of chemokines including IL-8 for neutrophils, MCP-1 for Th2 cells, eosinophils and macrophages, RANTES for activated T cells and eosinophils, IP-10 for Th1 cells, MIP-1α for monocytes and natural killer (NK) cells, and I-309 for Th2 cells. Since we observed that SCF and TNF-α could not exhibit any significant effect on the proliferation of HMC-1 cells (data not shown), the induction of release of chemokines was not due to the increase of cell number.
Mast cells were found to be the abundant and major source of MIP-1β in the lymph nodes during hypersensitivity (21), and MIP-1β could profoundly recruit T cells from the circulation to lymph nodes during the initiation of the primary immune response (22). Previous studies have also shown that I-309, which is constitutively over expressed by mast cells, could markedly be elevated after stimulation via high-affinity Fcɛ receptor I (FcɛRI ) on mast cells for Th2 chemoattraction to trigger allergic inflammation (23, 24). Monocyte chemoattractant protein-1, which is also constitutively over expressed in HMC-1 cells, acts as the crucial chemokine in attracting eosinophils and macrophages in several inflammatory diseases including asthma and parasitic infestation (2). Interferon-γ-inducible protein-10 was shown to be induced by interferon (IFN)-α in mast cells during innate immune response against bacterial infection (25). Allergic dermatitis and other chronic inflammatory skin diseases have also been shown to be mediated by the Th1 cells recruited by IP-10, which is mainly derived from mast cells (26). Since TNF-α is a Th1 related pro-inflammatory cytokine, it is reasonable that TNF-α but not SCF could induce Th1 chemokine IP-10 release in mast cells. Interleukin-8 is responsible for recruitment and degranulation of neutrophil and granulocytopenia during inflammation (27, 28). Stem cell factor has been shown to induce airway hypersensitivity by the release of RANTES for the recruitment of activated T cells (29). Together, the above six chemokines are chemotactic factors of Th1 cells for cell-mediated immunity; Th2 cells for humoral immunity, macrophage, neutrophils and natural killer cells for innate immune response, and eosinophils for allergic inflammation. In conjunction with other mast cell-derived cytokines such as IL-1, IL-3, IL-4, IL-5, IL-6, IL-13, and TNF-α (2), our results therefore confirmed that mast cells can orchestrate differential immunological roles by the activation and chemoattraction of different leukocytes and immune effector cells in various mast cell-mediated inflammations.
Regarding the molecular regulatory mechanisms in HMC-1 cells, we applied several widely used specific inhibitors PD98059, SB203580, and BAY117082 to elucidate the intracellular signaling mechanisms regulating the induction of different chemokines. Following our previous publication for the toxicity test of different inhibitors on HMC-1 cells (20), we adopted the optimal concentrations of BAY117082 (30–100 μM), PD98059 (20–100 μM), and SB203580 (10–50 μM) with the highest inhibitory effect for chemokine release without any cell toxicity. Results indicated that SCF could induce ERK activity while TNF-α could induce both p38 MAPK and NF-κB activity (Figs 3 and 4). Moreover, we also observed that the selective inhibitors of c-Jun N terminal kinase (JNK) and Janus kinase (JAK) pathway, i.e., SP600125 and AG-490, respectively, had no effect in either SCF or TNF-α induced chemokine expression (data not shown), thereby indicating that there was no involvement of JNK- and JAK-signal transduction and activator of transcription pathways in chemokine expression in SCF- or TNF-α-activated HMC-1 cells. As shown in Figs 5 and 6, inductions of IL-8, MCP-1, and I-309 were mediated by either SCF-activated ERK or TNF-α-activated p38 MAPK, while the induction of IP-10 by TNF-α was mediated by both activated p38 MAPK and NF-κB. The induction of RANTES by SCF or TNF-α was mediated by ERK and NF-κB respectively, and SCF induced MIP-1β release was mediated by ERK. The above discrepancies of the intracellular signaling mechanisms of SCF and TNF-α induced expression of different chemokines may be due to the discrete activation of intracellular signaling pathways (i.e., SCF induced ERK and TNF-α induced p38 MAPK and NF-κB) and differential regulation of various transcription factors for the production of different chemokines. There are increasing evidences showing that MAPK, e.g., p38 MAPK, is required for NF-κB-dependent gene expression (30) and cross talk between discrete intracellular signaling pathways (31). Further experiments are required to confirm this type of regulatory mechanisms for the discrete responses upon different cytokine treatment for the release of various chemokines from mast cells.
It has been documented that HMC-1 cell has a mutated c-kit (SCF receptor) that is constitutively phosphorylated on tyrosine residues (32). We have performed additional experiments using selective c-kit inhibitors ST1571 (imatinib mesilate) and PP1, which are selective inhibitors of c-kit receptor tyrosine kinase and c-kit src tryrosine kinase, respectively (20). Results showed that both optimal effective concentration of STI571 (0.1 μM) and PP1 (5 μM) could not significantly inhibit the basal level of the release of all studied chemokines from HMC-1 cells (data not shown). These results therefore indicated that the constitutive phosphorylation on tyrosine residues of c-kit in HMC-1 cells cannot contribute to the high-basal level of protein expression of chemokines.
Apart from the studied signaling molecules, we speculate that there may be other signaling pathways such as phosphoinositide-3 kinase (PI3K)/Akt and various transcription factors contributing to the cytokine and chemokine gene expression and synthesis in HMC-1 cells. Protein kinase C (PKC) has recently been found to regulate the NF-κB-dependent transcription (33), therefore, the role of PKC in chemokine release also required further investigation. Regarding the protein release, there may be some other unidentified intracellular mechanisms that are responsible for the basal secretion and release of preformed chemokines via the export by the Golgi apparatus. Therefore, even the gene expression and protein synthesis are suppressed, the basal release of chemokines may not be affected in HMC-1 cells.
In view of the increasing prevalence of allergic diseases such as asthma, allergic rhinitis, and eczema worldwide (34), there is a need for novel and safe treatments of the underlying inflammation of these mast cell-mediated diseases (35). Apart from histamine release by degranulation, mast cells play differential roles in the inflammation by initiating and orchestrating immune responses by the release of various chemokines and cytokines via differential intracellular signal transduction pathways. In conjunction with our previous study regarding the cross-talk between different signaling pathways for the fine control of adhesion molecule expression on HMC-1 cells (20), our present results therefore provide further new insight that the activation of HMC-1 mast cell are under fine, diversified and complicated intracellular regulation. Because of recent advances in the application of p38 MAPK and NF-κB inhibitors as potential anti-inflammatory agents (36, 37), our study of molecular mechanisms of the release of HMC-1 cells derived chemokines may represent potential targets for pharmacologic intervention using bio-therapeutic inhibitors for treating diseases varying from mast cell-relate diseases to allergic asthma.