A Mast Cell Degranulation Screening Assay for the Identification of Novel Mast Cell Activating Agents.

The development and use of vaccines and their ability to prevent infection/disease is a shining example of the benefit of biomedical research. Modern vaccines often utilize subunit immunogens that exhibit minimal immunogenicity and require the use of adjuvants to maximize the induction of protective immune responses. We recently described a novel class of vaccine adjuvants, mast cell (MC) activators, that exhibit safe and effective vaccine adjuvant activity when administered by intranasal or intradermal routes. A compound library containing 580 functionalized benzopyrans, a structural motif found in a diverse array of natural and designed bioactive compounds, was screened using a MC degranulation assay to identify novel MC activating compounds for future evaluation as novel vaccine adjuvants. This approach identified 12 novel MC degranulating compounds. Therefore, MC degranulation can be used to reliably detect novel compounds for evaluation as adjuvants for use in mucosal vaccine strategies.

The mast cell in innate and adaptive immunity.

Mast cells (MCs) were once considered only as effector cells in pathogenic IgE- and IgG-mediated responses such as allergy. However, developments over the last 15 years have suggested that MCs have evolved in vertebrates as beneficial effector cells that are involved in the very first inflammatory responses generated during infection. This pro-inflammatory environment has been demonstrated to be important for initiating innate responses in many different models of infection and more recently, in the development of adaptive immunity as well. Interestingly this latter finding has led to the discovery that small MC-activating compounds can behave as adjuvants in vaccine formulations. Thus, our continued understanding of the MC in the context of infectious disease is likely to not only expand our scope of the MC in the normal processes of immunity, but provide new therapeutic targets to combat disease.

Particulate allergens potentiate allergic asthma in mice through sustained IgE-mediated mast cell activation.

Allergic asthma is characterized by airway hyperresponsiveness, inflammation, and a cellular infiltrate dominated by eosinophils. Numerous epidemiological studies have related the exacerbation of allergic asthma with an increase in ambient inhalable particulate matter from air pollutants. This is because inhalable particles efficiently deliver airborne allergens deep into the airways, where they can aggravate allergic asthma symptoms. However, the cellular mechanisms by which inhalable particulate allergens (pAgs) potentiate asthmatic symptoms remain unknown, in part because most in vivo and in vitro studies exploring the pathogenesis of allergic asthma use soluble allergens (sAgs). Using a mouse model of allergic asthma, we found that, compared with their sAg counterparts, pAgs triggered markedly heightened airway hyperresponsiveness and pulmonary eosinophilia in allergen-sensitized mice. Mast cells (MCs) were implicated in this divergent response, as the differences in airway inflammatory responses provoked by the physical nature of the allergens were attenuated in MC-deficient mice. The pAgs were found to mediate MC-dependent responses by enhancing retention of pAg/IgE/FcεRI complexes within lipid raft­enriched, CD63(+) endocytic compartments, which prolonged IgE/FcεRI-initiated signaling and resulted in heightened cytokine responses. These results reveal how the physical attributes of allergens can co-opt MC endocytic circuitry and signaling responses to aggravate pathological responses of allergic asthma in mice.

Mast cells augment adaptive immunity by orchestrating dendritic cell trafficking through infected tissues.

Mast cells (MCs) are best known for eliciting harmful reactions, mostly after primary immunity has been established. Here, we report that, during footpad infection with E. coli in MC-deficient mice, as compared to their MC-sufficient counterparts, the serum antibody response is significantly diminished and less protective following passive immunization in a urinary tract infection (UTI) model in wild-type mice. MCs were found to recruit large numbers of dendritic cells (DCs) into the infected tissue site, which eventually migrated into draining lymph nodes (DLNs) during a prolonged time course. This pattern of trafficking was facilitated by MC-generated TNF, which increased the expression of E-selectin on local blood vessels. Antibody blockade of E-selectin inhibited DC recruitment into the site of infection and DLNs and consequently impaired the primary humoral immune response. Thus, during infection, resident MCs contribute to the primary protective adaptive response through recruitment of DCs from the circulation into infected sites.

The mast cell activator compound 48/80 is safe and effective when used as an adjuvant for intradermal immunization with Bacillus anthracis protective antigen.

We evaluated the safety and efficacy of the mast cell activator compound 48/80 (C48/80) when used as an adjuvant delivered intradermally (ID) with recombinant anthrax protective antigen (rPA) in comparison with two well-known adjuvants. Mice were vaccinated in the ear pinnae with rPA or rPA+C48/80, CpG oligodeoxynucleotides (CpG), or cholera toxin (CT). All adjuvants induced similar increases in serum anti-rPA IgG and lethal toxin neutralizing antibodies. C48/80 induced a balanced cytokine production (Th1/Th2/Th17) by antigen-restimulated splenocytes, minimal injection site inflammation, and no antigen-specific IgE. Histological analysis demonstrated that vaccination with C48/80 reduced the number of resident mast cells and induced an injection site neutrophil influx within 24h. Our data demonstrate that C48/80 is a safe and effective adjuvant, when used by the intradermal route, to induce protective antibody and balanced Th1/Th2/Th17 responses.

Mast cell activators: a new class of highly effective vaccine adjuvants.

Mast cells (MCs) have recently received recognition as prominent effectors in the regulation of immune cell migration to draining lymph nodes and lymphocyte activation. However, their role in the development of humoral immune responses is not clear. Here, we demonstrate that subcutaneous or nasal administration of small-molecule MC activators with vaccine antigens evokes large increases in antigen-specific serum immunoglobulin G (IgG) responses. These responses were MC dependent and correlated with increased dendritic cell and lymphocyte recruitment to draining lymph nodes. Nasal instillation of these formulations also evoked antigen-specific secretory IgA and provided protection against anthrax lethal toxin challenge in vitro and against vaccinia virus infection in vivo. Collectively, these results define the MC as an integral sensory arm of the adaptive immune system. Moreover, they highlight MC activators as a new class of vaccine adjuvants, capable of inducing protective antigen-specific immune responses through needle-free routes of administration.

Harboring of particulate allergens within secretory compartments by mast cells following IgE/FcepsilonRI-lipid raft-mediated phagocytosis.

Although much is known regarding the exocytic responses of mast cells following allergen/IgE-mediated activation, little is currently known of the fate of the activating allergens, many of which are particles. We have found that IgE-bound particulate allergens were phagocytosed by activated mast cells in a lipid raft-dependent manner. The nascent allergen-containing phagosomes were found to transform into granule compartments by acquiring VAMP7 and serotonin and exhibited the capacity to empty their contents upon mast cell activation. When allergen-harboring mast cells were stimulated, the intracellular allergens were expelled intact and shown to activate adjacent mast cells. This capacity of mast cells to phagocytose and retain whole and antigenically intact allergens could potentially contribute to the course of inflammatory diseases such as asthma.

In vivo models for studying mast cell-dependent responses to bacterial infection.

Mast cells are a critical component of host defense against bacterial infections. Activation of these cells during infection induces both innate and adaptive aspects of protective immunity needed for the elimination of the bacteria and survival of the host. These functional roles for the mast cell have been principally characterized using two in vivo models of acute bacterial infection featuring Gram-negative pathogens such as Escherichia coli. Here, we present basic protocols for the identification of mast cell-dependent biological functions during bacterial infection. These include the use of mast cell-deficient mice, the identification of mast cells in tissue, the culture of uropathogenic E. coli, and the basic analysis of mast cell-dependent functions in the peritoneal cavity and footpad models of bacterial pathogenesis.

IL-10 inhibits Fc epsilon RI expression in mouse mast cells.

FcepsilonRI expression and function is a central aspect of allergic disease. Using bone marrow-derived mouse mast cell populations, we have previously shown that the Th2 cytokine IL-4 inhibits FcepsilonRI expression and function. In the current study we show that the Th2 cytokine IL-10 has similar regulatory properties, and that it augments the inhibitory effects of IL-4. FcepsilonRI down-regulation was functionally significant, as it diminished inflammatory cytokine production and IgE-mediated FcepsilonRI up-regulation. IL-10 and IL-4 reduced FcepsilonRI beta protein expression without altering the alpha or gamma subunits. The ability of IL-4 and IL-10 to alter FcepsilonRI expression by targeting the beta-chain, a critical receptor subunit known to modulate receptor expression and signaling, suggests the presence of a Th2 cytokine-mediated homeostatic network that could serve to both initiate and limit mast cell effector function.

Mast cell-derived tumor necrosis factor induces hypertrophy of draining lymph nodes during infection.

Palpable swelling of regional lymph nodes is a common sequela of microbial infections but the mechanism responsible for the sequestration and subsequent coordination of lymphocyte responses within these dynamic structures remains poorly understood. Here we show that draining lymph nodes of mast cell-deficient mice did not demonstrate swelling after intradermal bacterial challenge. Testing of individual mast cell-derived products in this model indicated that tumor necrosis factor was the main mediator of nodal hypertrophy, whereas tryptase and histamine had no effect. After peripheral mast cell activation, both tumor necrosis factor concentrations and the recruitment of circulating T cells were increased within draining nodes. These results show a critical function for peripheral mast cell-derived tumor necrosis factor in regulating the hypertrophy of draining lymph nodes during infection.

Stat5 expression is critical for mast cell development and survival.

Interleukin-3 (IL-3) and stem cell factor (SCF) are important mast cell growth and differentiation factors. Since both cytokines activate the transcription factor signal transducer and activator of transcription 5 (Stat5), a known regulator of proliferation and survival, we investigated the effects of Stat5 deficiency on mast cell development and survival. Bone marrow-derived mast cell (BMMC) populations cultured from Stat5A/B-deficient mice survived in IL-3 + SCF, but not in either cytokine alone. These cells demonstrated reduced expression of Bcl-2, Bcl-x(L), cyclin A2, and cyclin B1, with increased apoptosis and delayed cell cycle progression during IL-3 or SCF culture. Finally, the absence of Stat5 resulted in loss of in vivo mast cell development, as judged by assessments of Stat5-deficient mice and transplantation of Stat5-deficient bone marrow cells to mast cell-deficient recipient mice. These results indicate that Stat5A and Stat5B are critical regulators of in vitro and in vivo mast cell development and survival.

Mast cell-restricted p70 Stat6 isoform is a product of selective proteolysis.

Signal transducer and activator of transcription (Stat)-6 is the principle Stat protein activated by interleukin (IL)-4. We defined a role for IL-4 in mast cell homeostasis through inhibiting expression of Kit and F(c)epsilonRI, and by inducing mast cell apoptosis. These effects required Stat6 expression. A molecular mechanism by which Stat6 directs these inhibitory actions in BMMC was potentially elucidated by the discovery of a carboxyl-truncated Stat6 isoform. Expression of this 70kDa isoform was unique to cultured mast cells and mast cell lines. Furthermore, this isoform lacked a carboxyl-transactivation domain, suggesting that it might behave as a dominant negative isoform. To better understand this truncated Stat6, we characterized its origins. Using Western blotting and electrophoretic mobility shift assay analysis, we assessed BMMC p70 Stat6 expression using standard and enhanced protease inhibitor cocktails. These experiments demonstrated that p70 Stat6 is derived by proteolysis during sample preparation, and has no cellular correlate. While some Stat family members are known to exist as naturally occurring truncated forms, p70 Stat6 does not appear to be such a case. Instead, the very high concentrations of proteases released during mast cell lysis result in selective proteolysis of the full-length Stat6, with p70 being the major degradation product.

Stat5: an essential regulator of mast cell biology.

Interleukin-3 (IL-3) and stem cell factor (SCF) are important mast cell growth and differentiation factors. Since both cytokines activate the transcription factor Stat5, a known regulator of proliferation and survival, we investigated the effects of Stat5 deficiency on mast cell development and survival. This article will review data presented at The Fourth International Workshop on Signal Transduction in the Activation and Development of Mast Cells and Basophils. The full set of data is now in preparation for publication. We find that the absence of Stat5 A and B results in a total loss of in vivo mast cell development. Bone marrow-derived mast cell (BMMC) populations can be cultured and maintained from Stat5-deficient mice in IL-3+SCF, but not in either cytokine alone. The absence of Stat5 resulted in aberrant control of Bcl-2, Bcl-x(L) and cyclin A2, with increased apoptosis and delayed cell cycle progression after IL-3 or SCF stimulation. These results indicate that Stat5 A and B are critical regulators of in vitro and in vivo mast cell biology.