Biomarkers for evaluation of mast cell and basophil activation

Mast cells and basophils play a pathogenetic role in allergic, inflammatory, and autoimmune disorders. These cells have different development, anatomical location and life span but share many similarities in mechanisms of activation and type of mediators. Mediators secreted by mast cells and basophils correlate with clinical severity in asthma, chronic urticaria, anaphylaxis, and other diseases. Therefore, effective biomarkers to measure mast cell and basophil activation in vivo could potentially have high diagnostic and prognostic values. An ideal biomarker should be specific for mast cells or basophils, easily and reproducibly detectable in blood or biological fluids and should be metabolically stable. Markers of mast cell and basophil include molecules secreted by stimulated cells and surface molecules expressed upon activation. Some markers, such as histamine and lipid mediators are common to mast cells and basophils whereas others, such as tryptase and other proteases, are relatively specific for mast cells. The best surface markers of activation expressed on mast cells and basophils are CD63 and CD203. While these mediators and surface molecules have been associated to a variety of diseases, none of them fulfills requirements for an optimal biomarker and search for better indicators of mast cell/basophil activation in vivo is ongoing.     

Regulation of mast cell responses in health and disease

Mast cells are multifunctional cells that initiate not only IgE-dependent allergic diseases but also play a fundamental role in innate and adaptive immune responses to microbial infection. They are also thought to play a role in angiogenesis, tissue remodeling, wound healing, and tumor repression or growth. The broad scope of these physiologic and pathologic roles illustrates the flexible nature of mast cells, which is enabled in part by their phenotypic adaptability to different tissue microenvironments and their ability to generate and release a diverse array of bioactive mediators in response to multiple types of cell-surface and cytosolic receptors. There is increasing evidence from studies in cell cultures that release of these mediators can be selectively modulated depending on the types or groups of receptors activated. The intent of this review is to foster interest in the interplay among mast cell receptors to help understand the underlying mechanisms for each of the immunological and non-immunological functions attributed to mast cells. The second intent of this review is to assess the pathophysiologic roles of mast cells and their products in health and disease. Although mast cells have a sufficient repertoire of bioactive mediators to mount effective innate and adaptive defense mechanisms against invading microorganisms, these same mediators can adversely affect surrounding tissues in the host, resulting in autoimmune disease as well as allergic disorders.     

Mast Cells and Immunological Skin Diseases

Mast cells play an important role in both adaptive and innate immunity, and a large body of literature demonstrates their functions in skin immunity. This article reviews the literature on the role of this cell type in the pathogenesis of a number of immunological skin diseases, including contact dermatitis, atopic dermatitis, immunobullous disease, scleroderma, and chronic graft-vs.-host disease. In all these diseases, mast cells are noted to increase in number and undergo degranulation in the affected skin, and in some cases, their specific mediators are detected. Elucidation of the contribution of mast cells to the pathogenesis of these diseases has been aided significantly by the use of animal models, especially mouse models. The studies of mast cell-deficient mice in conjunction with normal congenic mice have been particularly fruitful, although in some cases, such as contact dermatitis, a definitive conclusion has not been achieved despite extensive efforts. The role of mast cells in atopic dermatitis has also been suggested by studies of gene polymorphism, which have linked some of the mast cell-related genes to the disease. In the case of scleroderma and chronic graft-vs.-host disease, the function of mast cells in fibrosis is further supported by the ability of these cells and their mediators to induce activation and proliferation of fibroblasts. Therapies targeting mast cells may prove beneficial for treatment of these inflammatory and autoimmune diseases.     

Adapters in the organization of mast cell signaling

Summary:  Mast cells are pivotal in innate immunity and play an important role in amplifying adaptive immunity. Nonetheless, they have long been known to be central to the initiation of allergic disorders. This results from the dysregulation of the immune response whereby normally innocuous substances are recognized as non-self, resulting in the production of IgE antibodies to these 'allergens'. Preformed and newly synthesized inflammatory (allergic) mediators are released from the mast cell following allergen-mediated aggregation of allergen-specific IgE bound to the high-affinity receptors for IgE (FcεRI). Thus, the process by which the mast cell is able to interpret the engagement of FcεRI into the molecular events necessary for release of their allergic mediators is of considerable therapeutic interest. Unraveling these molecular events has led to the discovery of a functional class of proteins that are essential in organizing activated signaling molecules and in coordinating and compartmentalizing their activity. These so-called 'adapters' bind multiple signaling proteins and localize them to specific cellular compartments, such as the plasma membrane. This organization is essential for normal mast cell responses. Here, we summarize the role of adapter proteins in mast cells focusing on the most recent advances toward understanding how these molecules work upon FcεRI engagement.     

Serotonin release from rat brain mast cells in vitro.

Mast cells are primarily localized in connective tissues, where they secrete numerous mediators. They have also been identified in the mammalian central nervous system on the basis of their histochemical and morphological properties, but their role there remains unknown. A perfusion system was used to investigate in vitro mediator release from rat brain mast cells. Compound 48/80, the classic mast cell secretagogue of connective tissue mast cells, induced dose-dependent and non-cytotoxic release of serotonin, histamine and beta-hexosaminidase from mast cells in the rat thalamus and hypothalamus, but not in the cerebellum which was used as a negative control. Detailed studies were performed on thalamic mast cells, which were identified on the basis of metachromasia with Toluidine Blue and Safranin-positive staining with the Alcian Blue/Safranin technique. Their secretion was characterized by: (a) parallel release of serotonin, histamine and beta-hexosaminidase; (b) lack of dependence on extracellular calcium; (c) susceptibility to inhibition by disodium cromoglycate; and (d) lack of lactate dehydrogenase release. These results indicate that the morphology and secretory characteristics of thalamic mast cells resemble those of connective tissue mast cells. The ability of brain mast cells to secrete their mediators is discussed in the context of their possible involvement in brain pathophysiology.     

Buffy coat preparation is a convenient source of progenitors for culturing mature human mast cells

Mast cells are unique immune cells that release a spectrum of chemical mediators contributing to the inflammatory symptoms of allergic disorders. Mature mast cells have recently been cultured from CD34(+) progenitors isolated from fresh umbilical cord blood and adult peripheral blood. In the current study, we investigated whether buffy coat preparations, which are readily available from blood banks, could be used as a convenient and more abundant source of progenitors for culturing human mast cells. We were able to culture a homogeneous population of human mast cells from progenitor cells isolated from human buffy coat. Morphologically, our cultured mast cells contained abundant cytoplasmic granules which stained positively using antibodies against human mast cell tryptase and, to a lesser extent, with those against human mast cell chymase. Functionally, these cultured mast cells responded to anti-human-IgE by releasing histamine in a dose-dependent manner after sensitization with human IgE. Taken together, buffy coat preparations can be a convenient source for culturing human mast cells which are predominantly tryptase positive only and express functional high-affinity IgE receptors.     

Recent advances in the cellular and molecular biology of mast cells

The mast cell is now considered to play a pivotal role not only in allergic reactions but also in a number of inflammatory disorders. After immunological activation via the IgE receptor, the mast cell releases a variety of cytokines, lipid-derived mediators, amines, proteases and proteoglycans--all of which can regulate adjacent cells and the metabolism of the extra-cellular matrix of connective tissues. While it had been known for some time that mast cells differ in a number of properties in varied tissue sites, it was not known why or how this heterogeneity occurred. The development of in-vitro techniques to culture mast cells and the reconstitution of mast-cell-deficient mice are two major approaches that have facilitated analyses of how the tissue microenvironment regulates the phenotype of mast cells. In this review by Richard L. Stevens and K. Frank Austen, some of the recent findings on the molecular biology of mast cell secretory granule proteins and proteoglycans, and the interaction of mast cells with fibroblasts in the presence and absence of interleukin 3(IL-3) are highlighted.     

Mast cells in renal inflammation and fibrosis: Lessons learnt from animal studies

Mast cells are hematopoietic cells involved in inflammation and immunity and have been recognized also as important effector cells in kidney inflammation. In humans, only a few mast cells reside in kidneys constitutively but in progressive renal diseases their numbers increase substantially representing an essential part of the interstitial infiltrate of inflammatory cells. Recent data obtained in experimental animal models have emphasized a complex role of these cells and the mediators they release as they have been shown both to promote, but also to protect from disease and fibrosis development. Sometimes conflicting results have been reported in similar models suggesting a very narrow window between these activities depending on the pathophysiological context. Interestingly in mice, mast cell or mast cell mediator specific actions became also apparent in the absence of significant mast cell kidney infiltration supporting systemic or regional actions via draining lymph nodes or kidney capsules. Many of their activities rely on the capacity of mast cells to release, in a timely controlled manner, a wide range of inflammatory mediators, which can promote anti-inflammatory actions and repair activities that contribute to healing, but in some circumstances or in case of inappropriate regulation may also promote kidney disease.     

Novel Strategies to Target Mast Cells in Disease

Mast cells (MCs) are versatile effector cells of the immune system, characterized by a large content of secretory granules containing a variety of inflammatory mediators. They are implicated in the host protection toward various external insults, but are mostly well known for their detrimental impact on a variety of pathological conditions, including allergic disorders such as asthma and a range of additional disease settings. Based on this, there is currently a large demand for therapeutic regimens that can dampen the detrimental impact of MCs in these respective pathological conditions. This can be accomplished by several strategies, including targeting of individual mediators released by MCs, blockade of receptors for MC-released compounds, inhibition of MC activation, limiting mast cell growth or by inducing mast cell apoptosis. Here, we review the currently available and emerging regimens to interfere with harmful mast cell activities in asthma and other pathological settings and discuss the advantages and limitations of such strategies.     

Inhibition of rat mast cell arachidonic acid cyclooxygenase by dapsone

Dapsone (diaminodiphenylsulfone) has been used therapeutically for a variety of disorders in which mast cell participation has been demonstrated, including bullous pemphigoid and some form of necrotizing vasculitis. The mechanism of action of dapsone in these disorders is unknown but potentially relates to inhibition of mast cell activation and prevention of generation and/or release of mast cell mediators. Evidence for this possibility has been obtained in rat mast cells in which dapsone in a concentration-dependent manner prevented generation of prostaglandin D2 PGD2 from exogenous or endogenous arachidonic acid with 50% inhibition achieved at 1 and 0.2 to 0.4 mM, respectively. Dapsone inhibited cyclooxygenase conversion of arachidonic acid to PGD2 but not the GSH-dependent conversion of 14C-PGH2 to PGD2 by PGH-D isomerase in broken cell preparations. Dapsone prevented the immunologic generation of PGD2 from antigen-challenged rat mast cells but did not affect the release of histamine. Thus dapsone may exert some of its therapeutic effects by prevention of mast cell PGD2 generation.     

Estradiol augments while tamoxifen inhibits rat mast cell secretion.

Mast cells have been studied extensively for their involvement in allergic reactions, where they secrete numerous powerful mediators in response to immunoglobulin E and specific antigens. However, they are also triggered by neuropeptides, they have been found in close contact with neurons, and they are activated in diseases such as angioedema, interstitial cystitis and irritable bowel disease, the prevalence of which is much higher in women. When tested on purified rat peritoneal mast cells, 17 beta-estradiol augmented secretion of histamine and serotonin, starting at 1 microM and in a dose-dependent manner, whether stimulated by the mast cell secretagogue compound 48/80 or the neuropeptide substance P. However, 17 beta-estradiol did not augment mast cell secretion stimulated by immunoglobulin E and specific antiserum indicating that immunologic stimulation is under different regulation. Testosterone inhibited secretion induced by compound 48/80. Tamoxifen, an estrogen receptor antagonist used in the treatment of breast cancer, inhibited serotonin and histamine release from purified rat peritoneal mast cells triggered by compound 48/80 or substance P. Tamoxifen also inhibited the increase in intracellular free Ca2+ originating from an influx of extracellular Ca2+ in response to compound 48/80. Moreover, tamoxifen antagonized the synergistic effect of phorbol myristate and the cation ionophore A23187 on mast cell secretion, suggesting that tamoxifen's inhibition may be due to regulation of protein kinase C activity. Tamoxifen may, therefore, have a beneficial effect in other neuroimmunoendocrine disorders both through estrogen receptor blockade and inhibition of mast cell secretion.     

Mast cells in allergic asthma and beyond

Mast cells have been regarded for a long time as effector cells in IgE mediated type I reactions and in host defence against parasites. However, they are resident in all environmental exposed tissues and express a wide variety of receptors, suggesting that these cells can also function as sentinels in innate immune responses. Indeed, studies have demonstrated an important role of mast cells during the induction of life-saving antibacterial responses. Furthermore, recent findings have shown that mast cells promote and modulate the development of adaptive immune responses, making them an important hinge of innate and acquired immunity. In addition, mast cells and several mast cell-produced mediators have been shown to be important during the development of allergic airway diseases. In the present review, we will summarize findings on the role of mast cells during the development of adaptive immune responses and highlight their function, especially during the development of allergic asthma.     

Mast cells in mastocytosis and allergy – Important player in metabolic and immunological homeostasis

The role of mast cell (MC) activity in pathophysiology is complex and challenging and its clinical effects are difficult to predict. Apart from the known role of MCs in basic immunological processes and allergy, underlined is their importance in bone mineralization and in regulation of autoimmune reactions. Mast cell mediators, especially those released from mast cells in degranulation, but also those released constitutively, are important both in metabolic and immunological processes. Mastocytosis is a heterogeneous group of disorders characterized by accumulation of MC in one or more organs. There are scientific data indicating that mastocytosis patients are at increased risk of osteoporosis in the systemic form of the disease and children with cutaneous mastocytosis have a higher rate of hypogammaglobulinemia. Moreover, the origin of osteoporosis in patients with allergy is no longer considered as linked to steroid therapy only, but to the mast cell mediators’ activity as well. There are indications that osteoporosis symptoms in this group of patients may develop independently of the cumulative steroids’ dose. Thus, the influence of mast cells on metabolic and immunologic processes in allergic patients should be investigated. The assessment of mast cell activity and burden in mastocytosis may be used to guide clinical management of patients with allergy.     

Mast cells in allergy and infection: Versatile effector and regulatory cells in innate and adaptive immunity

Mast cells are widely distributed in tissues, particularly near surfaces exposed to the environment. Mast cells can be activated to secrete diverse mediators and cytokines by IgE and specific Ag and many other stimuli, including products derived from either pathogens or the host during innate immune responses. Although mast cells are best known for their role in IgE‐associated allergic disorders, mast cells can also exacerbate models of autoimmunity, enhance the sensitization and/or effector phases of certain cutaneous contact hypersensitivity responses, and increase inflammation and mortality during some severe bacterial infections. In other settings, however, mast cells can limit inflammation and tissue injury: mast cells promote host resistance in certain models of bacterial or parasite infection, limit pathology during some acquired immune responses to environmental Ag, including examples of severe contact hypersensitivity, and have adjuvant‐like properties that can enhance the development of protective immunity against pathogens. These and other findings suggest that mast cells occupy a critical niche at the interface of innate and acquired immunity, where, depending on circumstances that remain to be fully understood, mast cells may function to perturb or help to restore homeostasis (or both), with consequences that can either promote health or contribute to disease.     

The mast cell and allergic diseases: role in pathogenesis and implications for therapy

Mast cells have long been recognized for their role in the genesis of allergic inflammation; and more recently for their participation in innate and acquired immune responses. Mast cells reside within tissues including the skin and mucosal membranes, which interface with the external environment; as well as being found within vascularized tissues next to nerves, blood vessels and glandular structures. Mast cells have the capability of reacting both within minutes and over hours to specific stimuli, with local and systemic effects. Mast cells express the high affinity IgE receptor (Fc?RI) and upon aggregation of Fc?RI by allergen‐specific IgE, mast cells release and generate biologically active preformed and newly synthesized mediators which are involved in many aspects of allergic inflammation. While mast cells have been well documented to be essential for acute allergic reactions, more recently the importance of mast cells in reacting through pattern recognition receptors in innate immune responses has become recognized. Moreover, as our molecular understanding of the mast cell has evolved, novel targets for modulation have been identified with promising therapeutic potential.     

Roles of mast cells and basophils in innate and acquired immunity

There have been several recent advances in knowledge about mast cells and basophils in immune responses, of which some are particularly important: a role has been found for heparin in the storage of certain proteases and other mediators in mast cell cytoplasmic granules; an important role for mast cells in the development of several chronic aspects of an asthma model in mice has been discovered; and a new approach has been developed, based on the generation of mast cells from embryonic stem cells in vitro, to investigate mast cell function in vitro or in vivo.     

Roles and relevance of mast cells in infection and vaccination

In addition to their well-established role in allergy mast cells have been described as contributing to functional regulation of both innate and adaptive immune responses in host defense.Mast cells are of hematopoietic origin but typically complete their differentiation in tissues where they express immune regulatory functions by releasing diverse mediators and cytokines.Mast cells are abundant at mucosal tissues which are portals of entry for common infectious agents in addition to allergens.Here,we review the current understanding of the participation of mast cells in defense against infection.We also discuss possibilities of exploiting mast cell activation to provide adequate adjuvant activity that is needed in high-quality vaccination against infectious diseases.     

Mast cells and inflammation

Mast cells have long been recognized as potent producers of a large panel of biologically highly active mediators such as biogenic amines, arachidonic acid metabolites, cytokines and chemokines, but most of their biological functions have been elusive and speculative. By taking advantage of mast cell-deficient mice, the role of mast cells in a variety of experimental settings can now be studied in detail and such approaches have dramatically altered and enlarged our knowledge about mast cell biology and function. Herein we will focus on the role of mast cells in inflammatory reactions of diverse origin, such as delayed type hypersensitivity, atopy, immune complex-mediated inflammation and innate immune responses. From the current standpoint, there is no doubt that the most outstanding and beneficial feature of mast cells is their recently discovered ability to induce a life-saving inflammatory response rapidly upon encountering microbes and microbial constituents. Nevertheless, the picture is also emerging that mast cells are deeply involved in the induction and maintenance of a variety of severe allergic and autoimmune diseases. However, a deeper understanding of their activation and immune-modulatory capacity might open a new window for the development of curative strategies.     

Possible role of mast cell membrane structures in the enhancement of spleen cell proliferation

The role of mast cells and their mediators in the regulation of immune processes is an area of current interest. In previous studies, we demonstrated that rat peritoneal mast cells and their supernatants enhance the spontaneous and mitogen-induced proliferation of immunocytes. The results of this paper indicate that the enhancing factor in mast cell supernatants is not dependent on mast cell activation or on the release of mediators from within the mast cell. Instead it seems to be due to the passive diffusion of surface structures during mast cell culture. Pretreatment of mast cells with glutaraldehyde did not alter the ability of intact mast cells to enhance spleen cell proliferation. In contrast, mast cells lost their enhancing ability if they were pretreated with trypsin followed by glutaraldehyde fixation. Based on these data, we suggest that protein membrane structures on the mast cell surface are responsible for a contact cooperation between mast cells and spleen cells and that they cause the enhancing effect on spleen cell proliferation. This type of interaction may be responsible for the regulation of immune cell function during local immune reactions.

     

New aspects in thrombotic research: complement induced switch in mast cells from a profibrinolytic to a prothrombotic phenotype

Mast cells, strategically located in the vicinity of blood vessels, are multifunctional effector cells participating in the modulation of various inflammatory and cardiovascular disease processes by actively releasing a wide variety of vasoactive mediators. These cells have also been implicated in the regulation of thrombosis and the development and progression of atherosclerosis. By expressing enzymatically active tissue type-plasminogen activator (t-PA), human mast cells (MC) might play a role in endogenous fibrinolysis and extracellular matrix remodelling--both processes that are essential in the pathogenesis of cardiovascular disorders. However, when treated with the anaphylotoxin C5a, mast cells express the PA inhibitor 1 (PAI-1) in excess over t-PA. In context with studies suggesting a role for mast cells and components of the complement system in the development of cardiovascular disease our results lead to the hypothesis that mast cells by producing t-PA in a resting state and by expressingPAI-1 when activated by C5a might participate in the modulation of the balance between proteases and protease inhibitors regulating tissue injury and repair in these disease processes. In addition, C5a might upregulate PAI-1 in mast cells to prevent its own in activation by plasmin in an autocrine or paracrine fashion.