Ido Bachelet

Mast cells in allergy and beyond

Mast cells (MC) are highly granulated tissue dwelling cells, widely distributed throughout the body in connective tissues and on mucosal surfaces. They are derived from bone marrow progenitors that migrate into the blood and subsequently into the tissues, where they undergo final maturation. Mast cell proliferation, differentiation, survival and activation are regulated by stem cell factor, the ligand for the c-kit tyrosine kinase receptor, expressed on the mast cell surface. They release a large number of pro-inflammatory and immunoregulatory mediators after activation induced by either immunoglobulin E-dependent or immunoglobulin E-independent mechanisms. Mast cells have been most widely studied in the context of allergic reactions and parasite infections, but there is now compelling evidences that they are important players in innate and acquired immunity, wound healing, fibrosis, tumors and autoimmune diseases. This review will discuss current advances in these fields.

The Inhibitory Receptor IRp60 (CD300a) Is Expressed and Functional on Human Mast Cells

Mast cell-mediated responses are likely to be regulated by the cross talk between activatory and inhibitory signals. We have screened human cord blood mast cells for recently characterized inhibitory receptors expressed on NK cells. We found that IRp60, an Ig superfamily member, is expressed on human mast cells. On NK cells, IRp60 cross-linking leads to the inhibition of cytotoxic activity vs target cells in vitro. IRp60 is constitutively expressed on mast cells but is down-regulated in vitro by the eosinophil proteins major basic protein and eosinophil-derived neurotoxin. An immune complex-mediated cross-linking of IRp60 led to inhibition of IgE-induced degranulation and stem cell factor-mediated survival via a mechanism involving tyrosine phosphorylation, phosphatase recruitment, and termination of cellular calcium influx. To evaluate the role of IRp60 in regulation of allergic responses in vivo, a murine model of allergic peritonitis was used in which the murine homolog of IRp60, LMIR1, was neutralized in BALB/c mice by mAbs. This neutralization led to a significantly augmented release of inflammatory mediators and eosinophilic infiltration. These data demonstrate a novel pathway for the regulation of human mast cell function and allergic responses, indicating IRp60 as a candidate target for future treatment of allergic and mast cell-associated diseases.

2B4 (CD244) Is Expressed and Functional on Human Eosinophils

Eosinophils are present in parasitic, allergic, various immunological, and malignant disorders as well as in a variety of idiopathic hypereosinophilic syndromes. However, their exact role in some of these conditions remains elusive. They can be activated both in vivo and in vitro by various agonists, such as Igs, lipid mediators, and cytokines. By phenotyping the surface of the eosinophils, it may be possible to better define their function(s) in different pathophysiological settings. In the present work we screened eosinophils with a panel of Abs recognizing CD2 subfamily receptors usually present on a number of hemopoietic cells. We have demonstrated that human peripheral blood eosinophils, but not basophils or neutrophils, express NTB-A. In addition eosinophils express 2B4, CD84, CD58, and CD48, but not signaling lymphocytic activation molecule or CD2, on their surface (FACS). Cross-linking of 2B4 on eosinophils elicited a significant release of eosinophil peroxidase (30 min), IFN-γ, and IL-4 (18 h). Moreover, activation of eosinophils via 2B4 induced eosinophil-mediated cytotoxicity toward two malignant cell lines, i.e., mouse mastocytoma P815 and EBV-infected 721.221 B cell lines. Cross-linking of 2B4 on the surface of eosinophils or pervenadate treatment elicited ERK and tyrosine phosphorylation, respectively. Furthermore, we showed that eosinophils express slam-associated protein. The demonstration that human eosinophils express a functional 2B4 receptor indicates a broader role for these cells in health and disease.

Mast Cell Costimulation by CD226/CD112 (DNAM-1/Nectin-2) A NOVEL INTERFACE IN THE ALLERGIC PROCESS

Mast cells have critical effector functions in various immune reactions. In allergic inflammation, mast cells interact with tissue-infiltrating eosinophils, forming a regulatory unit in the late and chronic phases of the allergic process. However, the pathways and molecules within this unit are still largely undefined. Here, we show that human mast cells and eosinophils express DNAX accessory molecule 1 (DNAM-1, CD226) and its ligand Nectin-2 (CD112). CD226 synergizes with FcϵRI on mast cells, and its engagement augments degranulation through a pathway involving Fyn, linker of activation of T-cells, phospholipase C γ2, and CD18. This pathway is subject to negative interference by inhibitory receptors and is completely inhibited by linking IgE with IRp60 (CD300a) using a bispecific antibody. Moreover, blocking CD112 expressed on eosinophils using neutralizing antibodies normalized the hyperactivity resulting from IgE-dependent activation of mast cells co-cultured with eosinophils. Our findings demonstrate a novel interface between these two effector cells, implicating relevance for in vivo allergic states. Moreover, costimulatory responses might be a critical component in allergic reactions and may therefore become novel targets for anti-allergic therapy.

CD48 Is an Allergen and IL-3-Induced Activation Molecule on Eosinophils

Eosinophils are involved in a variety of allergic, parasitic, malignant, and idiopathic disorders by releasing a variety of factors including specific granule proteins, lipid mediators, and proinflammatory and immunoregulatory cytokines and chemokines. In addition, they interact with various cell types in the inflamed tissue. Yet, the mechanism of eosinophil activation is still poorly understood. Recently, we described the expression and function of the CD2-subfamily of receptors and especially 2B4 on human eosinophils. In this study we focus on CD48, the high-affinity ligand of 2B4. CD48 is a GPI-anchored protein involved in cellular activation, costimulation, and adhesion, but has not been studied on eosinophils. We demonstrate that human eosinophils from atopic asthmatics display enhanced levels of CD48 expression and that IL-3 up-regulates CD48 expression. Furthermore, cross-linking CD48 on human eosinophils triggers release of eosinophil granule proteins. Assessment of CD48 expression in a murine model of experimental asthma revealed that CD48 is induced by allergen challenge and partially regulated by IL-3. Additionally, anti-IL-3 reduces CD48 expression and the degree of airway inflammation. Thus, CD48 is an IL-3-induced activating receptor on eosinophils, likely involved in promoting allergic inflammation.

Suppression of Normal and Malignant Kit Signaling by a Bispecific Antibody Linking Kit with CD300a

Through its receptor Kit (CD117), stem cell factor (SCF) critically regulates human mast cell (MC) differentiation, survival, priming, and activation. The dominance of SCF in setting these parameters compels stringent contra-regulation to maintain a balanced MC phenotype. We have synthesized a library of bispecific Ab fragments to examine the effect of linking Kit with CD300a. In this study, we report that CD300a exerts a strong inhibitory effect on Kit-mediated SCF-induced signaling, consequently impairing MC differentiation, survival, and activation in vitro. This effect derives from Kit-mediated tyrosine phosphorylation of CD300a and recruitment of the SHIP-1 but not of SH2-containing protein phosphatase 1. CD300a inhibits the constitutive activation of the human leukemic HMC-1 cells but not their survival. Finally, CD300a abrogates the allergic reaction induced by SCF in a murine model of cutaneous anaphylaxis. Our findings highlight CD300a as a novel regulator of Kit in human MC and suggest roles for this receptor as a suppressor of Kit signaling in MC-related disorders.

Co-Culture of Mast Cells With Fibroblasts

Mast cell development, function, and survival are likely to be regulated by a complex interplay of cellular signaling. Usually, these signals derive from the cellular milieu associated with the specific mast cell environment in health or disease conditions. A major methodological issue in studying in vitro mast cells, as well as any other tissue dwelling cell, is the essential lack of all the tissue-derived signals. Because some of the signals can be unknown, the in vivo system they form is virtually impossible to mimic completely in vitro. The mast cell-fibroblast co-culture system partially overcomes this problem and is the main topic of this chapter. The experimental importance of mast cell-fibroblast co-culture for the mast cells derives mainly from two reasons: first, fibroblasts constitute a major cellular scaffold of the tissues where mast cells dwell in the body, and as such are one of the fundamental cells participating in mast cell regulation in vivo and, second, there is an analogy with the traditional model of allergic inflammation, where the late, chronic phase is characterized by mast cell-structural cell crosstalk and eventual fibrotic outcome. Therefore, the co-culture system can be viewed as a suitable tool to investigate mast cell-fibroblast crosstalk.

CD48 as a Novel Target in Asthma Therapy

CD48, a CD2-related surface molecule, emerges as a critical effector molecule in immune responses. CD48 has a striking array of biological functions, among them adhesion, pathogen recognition, cellular activation, and cytokine regulation. Still, it is surprising that this mysterious molecule has not yet met its proper use as a therapeutical target in exaggerated immune disorders like hematopoietic tumors, autoimmunity and allergic reactions. Recently, CD48 was investigated in our laboratory as an effector molecule in human eosinophil function and in asthma. In this review, we shall discuss the known aspects of CD48 biology and describe the recent advances regarding the role of CD48 in human disease. Moreover, we shall review inventions making use of CD48, and discuss recent patents and the potential of CD48 as a future target for the therapy of allergic and other diseases.

Eosinophil major basic protein activates human cord blood mast cells primed with fibroblast membranes by integrin‐β1

Mast cell (MC) – eosinophil (Eos) activating cross‐talk might be critical for the severity and chronicity of allergy. Among soluble mediators, eosinophil major basic protein (MBP), a hallmark of allergy, is particularly important because it was shown to activate specific MC subtypes. We previously demonstrated that MBP activates IgE‐desensitized rat MC and human lung and cord blood‐derived MC (CBMC) after priming with fibroblast membranal stem cell factor. However, a distinct mechanism for this activation was missing. Therefore, we aimed to investigate it.

Tryptase as an inflammatory marker in allergic disease and asthma.

Asthma is a chronic inflammatory disease of the airways, varying from occasional episodes of wheezing and shortness of breath, to an irreversible, life-threatening obstructive disease. While many cases are managed with relative ease, others do not respond to the traditional inhaled therapy or even to oral glucocorticosteroids. Although it cannot be cured as yet, asthma can be controlled if properly diagnosed. Usually, functional clinical parameters form the basis for estimation of the disease severity. In addition, the growing database of cytokine and mediator profiles have allowed their exploitation as molecular markers for processes underlying airway inflammation in asthma. Tryptase is a potent and versatile mediator in allergic inflammation, orchestrating both acute and chronic events by acting on a vast array of cells and tissue components. For more than a decade, tryptase has been used as a marker for allergic inflammation in asthma as well as in a variety of other airway diseases. In this review, the current advantages and disadvantages of the use of tryptase as an inflammatory marker in asthma will be discussed.