Laurent L. Reber

New models for analyzing mast cell functions in vivo

In addition to their well-accepted role as critical effector cells in anaphylaxis and other acute IgE-mediated allergic reactions, mast cells (MCs) have been implicated in a wide variety of processes that contribute to disease or help to maintain health. Although some of these roles were first suggested by analyses of MC products or functions in vitro, it is critical to determine whether, and under which circumstances, such potential roles actually can be performed by MCs in vivo. This review discusses recent advances in the development and analysis of mouse models to investigate the roles of MCs and MC-associated products during biological responses in vivo, and comments on some of the similarities and differences in the results obtained with these newer versus older models of MC deficiency.

Ser276 phosphorylation of NF-kB p65 by MSK1 controls SCF expression in inflammation.

Transcription of the mast cell growth factor SCF (stem cell factor) is upregulated in inflammatory conditions, and this is dependent upon NF-κB, as well as the MAP kinases p38 and ERK activation. We show here that the MAPK downstream nuclear kinase MSK1 induces NF-κB p65 Ser276 phosphorylation upon IL-1ß treatment, which was inhibited in cells transfected with a MSK1 kinase-dead (KD) mutant compared to the WT control. In addition, we show by ChIP experiments that MSK1 as well as MAPK inhibition abolishes binding of p65, of its coactivator CBP, and of MSK1 itself to the κB intronic enhancer site of the SCF gene. We show that interaction between NF-κB and CBP is prevented in cells transfected by a p65 S276C mutant. Finally, we demonstrate that both transfections of MSK1-KD and MSK1 siRNA - but not the WT MSK1 or control siRNA - downregulate the expression of SCF induced by IL-1ß. Our study provides therefore a direct link between MSK1-mediated phosphorylation of Ser276 p65 of NF-κB, allowing its binding to the SCF intronic enhancer, and pathophysiological SCF expression in inflammation.

A Beneficial Role for Immunoglobulin E in Host Defense against Honeybee Venom

Allergies are widely considered to be misdirected type 2 immune responses, in which immunoglobulin E (IgE) antibodies are produced against any of a broad range of seemingly harmless antigens. However, components of insect venoms also can sensitize individuals to develop severe IgE-associated allergic reactions, including fatal anaphylaxis, upon subsequent venom exposure. We found that mice injected with amounts of honeybee venom similar to that which could be delivered in one or two stings developed a specific type 2 immune response that increased their resistance to subsequent challenge with potentially lethal amounts of the venom. Our data indicate that IgE antibodies and the high affinity IgE receptor, FcεRI, were essential for such acquired resistance to honeybee venom. The evidence that IgE-dependent immune responses against venom can enhance survival in mice supports the hypothesis that IgE, which also contributes to allergic disorders, has an important function in protection of the host against noxious substances.

Different activation signals induce distinct mast cell degranulation strategies

Mast cells (MCs) influence intercellular communication during inflammation by secreting cytoplasmic granules that contain diverse mediators. Here, we have demonstrated that MCs decode different activation stimuli into spatially and temporally distinct patterns of granule secretion. Certain signals, including substance P, the complement anaphylatoxins C3a and C5a, and endothelin 1, induced human MCs rapidly to secrete small and relatively spherical granule structures, a pattern consistent with the secretion of individual granules. Conversely, activating MCs with anti-IgE increased the time partition between signaling and secretion, which was associated with a period of sustained elevation of intracellular calcium and formation of larger and more heterogeneously shaped granule structures that underwent prolonged exteriorization. Pharmacological inhibition of IKK-β during IgE-dependent stimulation strongly reduced the time partition between signaling and secretion, inhibited SNAP23/STX4 complex formation, and switched the degranulation pattern into one that resembled degranulation induced by substance P. IgE-dependent and substance P-dependent activation in vivo also induced different patterns of mouse MC degranulation that were associated with distinct local and systemic pathophysiological responses. These findings show that cytoplasmic granule secretion from MCs that occurs in response to different activating stimuli can exhibit distinct dynamics and features that are associated with distinct patterns of MC-dependent inflammation.

Selective ablation of mast cells or basophils reduces peanut-induced anaphylaxis in mice

BACKGROUND Studies with c-kit mutant mast cell (MC)-deficient mice and antibody-mediated depletion of basophils suggest that both MCs and basophils can contribute to peanut-induced anaphylaxis (PIA). However, interpretation of data obtained by using such approaches is complicated because c-kit mutant mice have several phenotypic abnormalities in addition to MC deficiency and because basophil-depleting antibodies can also react with MCs. OBJECTIVE We analyzed (1) the changes in the features of PIA in mice after the selective and inducible ablation of MCs or basophils and (2) the possible importance of effector cells other than MCs and basophils in the PIA response. METHODS Wild-type and various mutant mice were orally sensitized with peanut extract and cholera toxin weekly for 4 weeks and challenged intraperitoneally with peanut extract 2 weeks later. RESULTS Peanut-challenged, MC-deficient Kit(W-sh/W-sh) mice had reduced immediate hypothermia, as well as a late-phase decrease in body temperature that was abrogated by antibody-mediated depletion of neutrophils. Diphtheria toxin-mediated selective depletion of MCs or basophils in Mcpt5-Cre;iDTR and Mcpt8(DTR) mice, respectively, and treatment of wild-type mice with the basophil-depleting antibody Ba103 significantly reduced peanut-induced hypothermia. Non-c-kit mutant MC- and basophil-deficient Cpa3-Cre;Mcl-1(fl/fl) mice had reduced but still significant responses to peanut. CONCLUSION Inducible and selective ablation of MCs or basophils in non-c-kit mutant mice can significantly reduce PIA, but partial responses to peanut can still be observed in the virtual absence of both cell types. The neutrophilia in Kit(W-sh/W-sh) mice might influence the responses of these mice in this PIA model.

A Dissociated Glucocorticoid Receptor Modulator Reduces Airway Hyperresponsiveness and Inflammation in a Mouse Model of Asthma

The glucocorticoid receptor (GR) is a transcription factor able to support either target gene activation via direct binding to DNA or gene repression via interfering with the activity of various proinflammatory transcription factors. An improved therapeutic profile for combating chronic inflammatory diseases has been reported through selectively modulating the GR by only triggering its transrepression function. We have studied in this paper the activity of Compound A (CpdA), a dissociated GR modulator favoring GR monomer formation, in a predominantly Th2-driven asthma model. CpdA acted similarly to the glucocorticoid dexamethasone (DEX) in counteracting OVA-induced airway hyperresponsiveness, recruitment of eosinophils, dendritic cells, neutrophils, B and T cells, and macrophages in bronchoalveolar lavage fluid, lung Th2, Tc2, Th17, Tc17, and mast cell infiltration, collagen deposition, and goblet cell metaplasia. Both CpdA and DEX inhibited Th2 cytokine production in bronchoalveolar lavage as well as nuclear translocation of NF-κB and its subsequent recruitment onto the IκBα promoter in the lung. By contrast, DEX but not CpdA induces expression of the GR-dependent model gene MAPK phosphatase 1 in the lung, confirming the dissociative action of CpdA. Mechanistically, we demonstrate that CpdA inhibited IL-4–induced STAT6 translocation and that GR is essential for CpdA to mediate chemokine repression. In conclusion, we clearly show in this study the anti-inflammatory effect of CpdA in a Th2-driven asthma model in the absence of transactivation, suggesting a potential therapeutic benefit of this strategy.

Potential effector and immunoregulatory functions of mast cells in mucosal immunity.

Mast cells (MCs) are cells of hematopoietic origin that normally reside in mucosal tissues, often near epithelial cells, glands, smooth muscle cells, and nerves. Best known for their contributions to pathology during IgE-associated disorders such as food allergy, asthma, and anaphylaxis, MCs are also thought to mediate IgE-associated effector functions during certain parasite infections. However, various MC populations also can be activated to express functional programs—such as secreting preformed and/or newly synthesized biologically active products—in response to encounters with products derived from diverse pathogens, other host cells (including leukocytes and structural cells), damaged tissue, or the activation of the complement or coagulation systems, as well as by signals derived from the external environment (including animal toxins, plant products, and physical agents). In this review, we will discuss evidence suggesting that MCs can perform diverse effector and immunoregulatory roles that contribute to homeostasis or pathology in mucosal tissues.

Stem cell factor expression, mast cells and inflammation in asthma.

The Kit ligand SCF or stem cell factor (SCF) is a multipotent growth factor, acting as an important growth factor for human mast cells. SCF induces chemotaxis and survival of the mast cell, as well as proliferation and differentiation of immature mast cells from CD34+ progenitors. Additionally, SCF enhances antigen‐induced degranulation of human lung‐derived mast cells, and induces a mast cell hyperplasia after subcutaneous administration. SCF expression increases in the airways of asthmatic patients, and this is reversed after treatment with glucocorticoids. A role for SCF may thus be hypothesized in diseases associated with a local increase in the number and/or activation of mast cells, as occurring in the airways in asthma. SCF will be reviewed as a potential therapeutic target in asthma, to control the regulation of mast cell number and activation. We here report the main pathways of SCF synthesis and signalling, and its potential role on airway function and asthma.

Targeting mast cells in inflammatory diseases.

Although mast cells have long been known to play a critical role in anaphylaxis and other allergic diseases, they also participate in some innate immune responses and may even have some protective functions. Data from the study of mast cell-deficient mice have facilitated our understanding of some of the molecular mechanisms driving mast cell functions during both innate and adaptive immune responses. This review presents an overview of the biology of mast cells and their potential involvement in various inflammatory diseases. We then discuss some of the current pharmacological approaches used to target mast cells and their products in several diseases associated with mast cell activation.

Mast Cells Contribute to Bleomycin-Induced Lung Inflammation and Injury in Mice through a Chymase/Mast Cell Protease 4–Dependent Mechanism

Mast cells (MCs) are found in large numbers in lungs of patients with pulmonary fibrosis. However, the functions of MCs in lung fibrosis remain largely unknown. We assessed the role of MCs and MC protease 4 (MCPT4), the mouse counterpart of human MC chymase, in a mouse model of bleomycin (BLM)-induced lung injury. We found that levels of inflammation in the bronchoalveolar lavage and the lung, as well as levels of lung fibrosis, were reduced 7 d after intranasal delivery of BLM MC-deficient KitW-sh/W-sh mice compared with wild-type (WT) mice. Confirming the implication of MCs in these processes, we report that the levels of inflammation and fibrosis observed in KitW-sh/W-sh mice can be restored to those observed in WT mice after the adoptive transfer of bone marrow–derived cultured MCs into KitW-sh/W-sh mice. Additionally, we show that levels of inflammation and fibrosis are also reduced in MC chymase MCPT4-deficient mice as compared with WT mice at day 7, suggesting a role for MC-derived MCPT4 in these processes. Our results support the conclusion that MCs can contribute to the initial lung injury induced by BLM through release of the MCPT4 chymase.