Tae Chul Moon

Advances in mast cell biology: new understanding of heterogeneity and function.

Mast cells are classically viewed as effector cells of IgE-mediated allergic diseases. However, over the last decade our understanding has been enriched about their roles in host defense, innate and adaptive immune responses, and in homeostatic responses, angiogenesis, wound healing, tissue remodeling, and immunoregulation. Despite impressive progress, there are large gaps in our understanding of their phenotypic heterogeneity, regulatory mechanisms involved, and functional significance. This review summarizes our knowledge of mast cells in innate and acquired immunity, allergic inflammation and tissue homeostasis, as well as some of the regulatory mechanisms that control mast cell development, phenotypic determination, and function, particularly in the context of mucosal surfaces.

Mast Cell Mediators: Their Differential Release and the Secretory Pathways Involved

Mast cells (MC) are widely distributed throughout the body and are common at mucosal surfaces, a major host–environment interface. MC are functionally and phenotypically heterogeneous depending on the microenvironment in which they mature. Although MC have been classically viewed as effector cells of IgE-mediated allergic diseases, they are also recognized as important in host defense, innate and acquired immunity, homeostatic responses, and immunoregulation. MC activation can induce release of pre-formed mediators such as histamine from their granules, as well as release of de novo synthesized lipid mediators, cytokines, and chemokines that play diverse roles, not only in allergic reactions but also in numerous physiological and pathophysiological responses. Indeed, MC release their mediators in a discriminating and chronological manner, depending upon the stimuli involved and their signaling cascades (e.g., IgE-mediated or Toll-like receptor-mediated). However, the precise mechanisms underlying differential mediator release in response to these stimuli are poorly known. This review summarizes our knowledge of MC mediators and will focus on what is known about the discriminatory release of these mediators dependent upon diverse stimuli, MC phenotypes, and species of origin, as well as on the intracellular synthesis, storage, and secretory processes involved.

Expression of DP2 (CRTh2), a Prostaglandin D2 Receptor, in Human Mast Cells

PGD2 has long been implicated in allergic diseases. Recent cloning of a second PGD2 receptor, DP2 (also known as CRTh2), led to a greater understanding of the physiological and pathophysiological implications of PGD2. PGD2 signaling through DP1 and DP2 mediates different and often opposite effects in many cell types of the immune system. Although mast cells (MC) are the largest source of PGD2 in the body, there is little information about their potential expression of DP2 and its functional significance. In this study, we show that tissue MC in human nasal polyps express DP2 protein, and that human MC lines and primary cultured human MC express mRNA as well as protein of DP2. By immunohistochemistry, we detected that 34% of MC in human nasal polyps expressed DP2. In addition, flow cytometry showed that 87% of the LAD2 human MC line and 98% of primary cultured human MC contained intracellular DP2. However, we could not detect surface expression of DP2 on human MC by single cell analysis using imaging flow cytometry. Blocking of endogenous PGD2 production with aspirin did not induce surface expression of DP2 in human MC. Two DP2 selective agonists, DK-PGD2 and 15R-15-methyl PGD2 induced dose-dependent intracellular calcium mobilization that was abrogated by pertussis toxin, but not by three DP2 selective antagonists. MC mediator release including degranulation was not affected by DP2 selective agonists. Thus, human MC express DP2 intracellularly rather than on their surface, and the function of DP2 in human MC is different than in other immune cells such as Th2 cells, eosinophils and basophils where it is expressed on the cell surface and induces Th2 cytokine and/or granule associated mediator release. Further studies to elucidate the role of intracellular DP2 in human MC may expand our understanding of this molecule and provide novel therapeutic opportunities.

Role of nitric oxide in mast cells: controversies, current knowledge, and future applications.

Mast cells (MC) are important effector cells in allergic disorders. Recenty, the role of MC in innate and adaptive immunity is gaining prominence. Nitric oxide is an important signaling molecule and its production in mast cell has been reported widely. However, controversy exists about whether MC produce NO. This review addresses the role of NO in MC biology and the reasons behind the controversy and discusses effects of NO in regulation of MC phenotype and function.

Protein Tyrosine Nitration of Aldolase in Mast Cells: A Plausible Pathway in Nitric Oxide-Mediated Regulation of Mast Cell Function

NO is a short-lived free radical that plays a critical role in the regulation of cellular signaling. Mast cell (MC)-derived NO and exogenous NO regulate MC activities, including the inhibition of MC degranulation. At a molecular level, NO acts to modify protein structure and function through several mechanisms, including protein tyrosine nitration. To begin to elucidate the molecular mechanisms underlying the effects of NO in MCs, we investigated protein tyrosine nitration in human MC lines HMC-1 and LAD2 treated with the NO donor S-nitrosoglutathione. Using two-dimensional gel Western blot analysis with an anti-nitrotyrosine Ab, together with mass spectrometry, we identified aldolase A, an enzyme of the glycolytic pathway, as a target for tyrosine nitration in MCs. The nitration of aldolase A was associated with a reduction in the maximum velocity of aldolase in HMC-1 and LAD2. Nuclear magnetic resonance analysis showed that despite these changes in the activity of a critical enzyme in glycolysis, there was no significant change in total cellular ATP content, although the AMP/ATP ratio was altered. Elevated levels of lactate and pyruvate suggested that S-nitrosoglutathione treatment enhanced glycolysis. Reduced aldolase activity was associated with increased intracellular levels of its substrate, fructose 1,6-bisphosphate. Interestingly, fructose 1,6-bisphosphate inhibited IgE-mediated MC degranulation in LAD2 cells. Thus, for the first time we report evidence of protein tyrosine nitration in human MC lines and identify aldolase A as a prominent target. This posttranslational nitration of aldolase A may be an important pathway that regulates MC phenotype and function.

Limited replication of influenza A virus in human mast cells

Mast cells are important in innate immunity and protective against certain bacterial infections. However, there is limited evidence that mast cells respond to viruses. As mast cells are abundant in mucosal tissues of the lung, they are in a prime location to detect and respond to influenza virus. In this study, we characterized for the first time the replication cycle of influenza A virus in human mast cells by measuring influenza A virus transcription, RNA replication, protein synthesis, and formation of infectious virus as compared to the replication cycle in epithelial cells. We detected the presence of influenza A viral genomic RNA transcription, replication, and protein synthesis in human mast cells and epithelial cells. However, there was no significant release of infectious influenza A virus from mast cells, whereas epithelial cells produce ~100-fold virus compared with the inoculating dose. We confirmed that influenza A virus infects human mast cells, begins to replicate, but the production of new virus is aborted. Thus, mast cells may lack critical factors essential for productive infection or there are intrinsic or inducible anti-influenza A mechanisms in mast cells.

Microenvironmental regulation of inducible nitric oxide synthase expression and nitric oxide production in mouse bone marrow-derived mast cells

In addition to its well‐known role in relaxation of vascular smooth muscle, NO modulates immune responses in a concentration‐ and location‐specific manner. For MC, it is well accepted that exogenous NO regulates their function. However, there are inconsistencies in the literature of whether MC express NOS and make NO. MC progenitors mature in peripheral tissues, but the factors that influence MC maturation and their specific phenotype, such as whether they express NOS, are not well understood. To study microenvironmental conditions that could be “permissive” for NOS expression, we cultured BMMC in various conditions—BMMCIL‐3, BMMCSCF/IL‐3, or BMMCSCF/IL‐4—for >3 weeks and examined NOS expression. We detected Nos2 mRNA in BMMCSCF/IL‐4 but not BMMCIL‐3 or BMMCSCF/IL‐3. After stimulation with IFN‐γ and/or LPS, NOS2 expression and NO production were detected in BMMCSCF/IL‐4 but rarely detected in BMMC cultured with other conditions. Confocal microscopic analysis showed that NOS2 expression induced by IFN‐γ colocalized in CD117+ BMMC. NO production, after activation with IFN‐γ and LPS in BMMCSCF/IL‐4, was abrogated by pretreatment with the NOS2‐specific inhibitor. In addition to NOS2 expression, BMMCSCF/IL‐4 were distinguished from BMMCIL‐3 in heparin and MMCP expression. Thus, MC progenitors that develop in SCF + IL‐4 can be induced to express NOS2 after receiving appropriate signals, such as IFN‐γ, and subsequently produce NO. Microenvironmental conditions during their development can influence whether MC are capable of NOS expression and of NO production.

Expression of nitric oxide synthases in leukocytes in nasal polyps

BACKGROUND Nitric oxide (NO) has various roles in airway physiology and pathophysiology. Monitoring exhaled NO levels is increasingly common to measure airways inflammation and inhaled NO studied for its therapeutic value in premature infants and adult respiratory distress syndrome. NO is produced by 3 isoforms of NO synthase (NOS1, 2, 3), and each can play distinct and perhaps overlapping roles in the airways. However, the distribution, regulation, and functions of NOS in various cells in the upper airways, particularly in leukocytes, are incompletely understood. OBJECTIVE To characterize the expression of NOS isoforms in leukocytes in normal middle turbinate tissues (MT) and in inflammatory nasal tissue (nasal polyps, NP). METHODS Normal MT tissue was collected from surgical specimens that were to be discarded. The NP samples were from surgical tissue archives of 15 patients with chronic rhinosinusitis. Isoforms of NOS in cells were identified by double immunostaining using NOS isoform-specific and leukocyte-specific (mast cell, eosinophil, macrophage, neutrophil, or T cell) antibodies. RESULTS The proportion of total cells below the epithelium that were positive for each isoform of NOS was higher in NP than in MT. Each isoform of NOS was found in all leukocyte populations studied, and there were significant differences in the percentage of leukocytes expressing NOS isoforms between MT and NP. CONCLUSION All isoforms of NOS are expressed in leukocytes in MT and NP, and their expression varies among leukocyte types. Our data provide a basis to investigate the regulation, cell distribution, and distinct functions of NOS isoforms in normal and inflamed nasal tissues.

Exogenous nitric oxide regulates cyclooxygenase-2 expression and prostaglandin D2 generation through p38 MAPK in mouse bone marrow-derived mast cells

Nitric oxide (NO) is an important signaling molecule that regulates MC function. However, the involvement of NO in an important lipid mediator, prostaglandin (PG) D(2) production by MC, is unclear. The role of NO in cyclooxygenase (COX)-2 expression and PGD(2) generation as well as IL-6 production in mouse bone marrow-derived MC (BMMC) was investigated using NO donors. Exogenous NO augmented COX-2 protein expression and increased COX-2-dependent PGD(2) generation in response to SCF, IL-10, and IL-1beta, or antigen activation in combination with IL-10 and IL-1beta after sensitization with IgE. The increased expression of COX-2 by NO donors was inhibited by hemoglobin. Moreover it was not affected by soluble guanylyl cyclase inhibitor, but reduced by the p38 MAPK inhibitor, SB202190. Downstream of p38 MAPK, NO donors augmented not only COX-2 mRNA transcription but also its stability. Exogenous NO also augmented IL-6 production by SCF, IL-10, and IL-1beta. These results show that exogenous NO can increase COX-2-dependent PGD(2) and IL-6 production by MC in inflammatory environments through the p38 MAPK pathway. Therefore, our novel observations suggest that the effect of NO on MC is not limited to the suppression of their activation as has been the emphasis previously, but can also augment certain MC responses.

Measurement of Nitric Oxide in Mast Cells with the Fluorescent Indicator DAF-FM Diacetate

The production of nitric oxide in mast cells has been difficult to measure due to the low amounts made by mast cells, as well as limitations in the specificity and sensitivity of the assays available. We present here a sensitive and specific 96-well plate-based method to directly measure NO using the cell-permeable fluorescent compound DAF-FM diacetate.