Dean D. Metcalfe

IgE, mast cells, basophils, and eosinophils

IgE, mast cells, basophils, and eosinophils are essential components of allergic inflammation. Antigen-specific IgE production, with subsequent fixation of IgE to FcepsilonRI receptors on mast cells and basophils, is central to the initiation and propagation of immediate hypersensitivity reactions. Mast cells, basophils, and eosinophils are central effector cells in allergic inflammation, as well as in innate and adaptive immunity. This review highlights what is known about these components and their roles in disease pathogenesis.

Tryptase Levels as an Indicator of Mast-Cell Activation in Systemic Anaphylaxis and Mastocytosis

Better methods are needed to assess mast-cell activation in vivo and to distinguish the activation of mast cells from that of basophils. Tryptase, a neutral protease selectively concentrated in the secretory granules of human mast cells (but not basophils), is released by mast cells together with histamine and serves as a marker of mast-cell activation. In 17 patients with systemic mastocytosis, concentrations of tryptase in plasma were linearly related to those of histamine (P less than 0.01). Eleven of the 17 patients had tryptase levels of 4 to 88 ng per milliliter, indicating ongoing mast-cell activation. In each of six patients who experienced corresponding anaphylactic reactions after penicillin, aspirin, or melon ingestion, a wasp sting, exercise, or antilymphocyte globulin injection, tryptase levels in serum ranged from 9 to 75 ng per milliliter, indicating mast-cell activation during each of these events. In contrast, serum tryptase levels were less than 5 ng per milliliter in all patients presenting with myocardial disease (n = 8, 6 with hypotension) or sepsis (n = 6, 3 with hypotension) and in the controls (n = 20). One patient had a myocardial infarction after anaphylaxis in response to a wasp sting and an elevated tryptase level of 25 ng per milliliter. Thus, the plasma or serum tryptase level is a diagnostic correlate of mast-cell-related events.

Standards and standardization in mastocytosis: Consensus Statements on Diagnostics, Treatment Recommendations and Response Criteria

Although a classification for mastocytosis and diagnostic criteria are available, there remains a need to define standards for the application of diagnostic tests, clinical evaluations, and treatment responses. To address these demands, leading experts discussed current issues and standards in mastocytosis in a Working Conference. The present article provides the resulting outcome with consensus statements, which focus on the appropriate application of clinical and laboratory tests, patient selection for interventional therapy, and the selection of appropriate drugs. In addition, treatment response criteria for the various clinical conditions, disease‐specific symptoms, and specific pathologies are provided. Resulting recommendations and algorithms should greatly facilitate the management of patients with mastocytosis in clinical practice, selection of patients for therapies, and the conduct of clinical trials.

Detection of intracytoplasmic cytokine using flow cytometry and directly conjugated anti-cytokine antibodies

Recently, there have been several reports demonstrating improvements in the flow cytometric detection of intracellular cytokines. These advances, although significant, have not yielded techniques that have easily been translated into broad use. To address this issue, we have coupled a fixation and permeabilization method with the use of directly labelled monoclonal anti-cytokine antibodies, providing both improved signal and simpler staining. The kinetics of in situ cytokine production in both CD4 and CD8 cells are shown for IL-2, IL-4, IL-5 and IFN-gamma. Based on these data, 6 h was chosen for optimal detection of this combination of cytokines. We show the specificity of this technique by blocking cytokine staining using a molar excess of recombinant cytokine. Additionally, unlabelled anti-cytokine antibodies are demonstrated to block specific staining of labelled antibody, providing an objective means to place statistical markers. Using such controls, we routinely detected as few as 0.1% false positive cells, allowing the flow cytometric detection of IL-5, which is below the threshold of detection of published methods. To further prove the specificity of staining, we stained using two anti-IL-5 mAbs known to recognize different epitopes and demonstrate that the same cells stain with both antibodies. Without permeabilization we could detect a fraction of cells with low intensity staining for cytokine. This staining was further examined using differential two color staining for intracellular and extracellular cytokine, clearly demonstrating no cells staining exclusively for extracellular cytokine, confirming a lack of passive transfer of cytokine to nearby cells. We show that cytokine flow cytometry is useful in examining the increased IL-5 production characteristic of eosinophilic states and that IL-5 production is limited to the CD27 negative subpopulation. These data illustrate the unique capability of cytokine flow cytometry to correlate cytokine expression with cell surface phenotype without cell separation. In summary, using directly conjugated anti-cytokine antibodies, cytokine flow cytometry becomes a specific and versatile technique for the assessment of complex cytokine production phenotypes in fresh ex vivo T cell subpopulations.

Assessment of the allergenic potential of foods derived from genetically engineered crop plants

This article provides a science-based, decision tree approach to assess the allergenic concerns associated with the introduction of gene products into new plant varieties. The assessment focuses on the source from which the transferred gene was derived. Sources fall into three general categories: common allergenic food proteins; less common allergenic foods or other known allergen sources; and sources with no history of allergenicity. Information concerning the amino acid sequence identity to known allergenic proteins, in vitro and/or in vivo immunologic assays, and assessment of key physiochemical properties are included in reaching a recommendation on whether food derived from the genetically modified plant variety should be labeled as to the source of the transferred gene. In the end, a balanced judgement of all the available data generated during allergenicity assessment will assure the safety of foods derived from genetically engineered crops. Using the approaches described here, new plant varieties generated by genetic modification should be introduced into the marketplace with the same confidence that new plant varieties developed by traditional breeding have been introduced for decades.

Mast cells in innate immunity

Summary: Mast cells are known to be the main effector cells in the elicitation of the IgE‐mediated allergic response. The specific location of mast cells within tissues that interface the external environment, and the extent of their functional capacity, including the ability to phagocytose and to produce and secrete a wide spectrum of mediators, have led investigators to propose a potential role for mast cells in innate immune responses. Certain microorganisms have been found to interact either directly or indirectly with mast cells. This interaction results in mast cell activation and mediator release which elicit an inflammatory response or direct killing leading to bacterial clearance. The in vivo relevance of these in vitro observations has been demonstrated by the use of complement‐deficient and/or mast cell‐deficient and mast cell‐reconstituted mice. It thus has been shown that both C3 and mast cell‐ and tumor necrosis factor‐a‐dependent recruitment of circulating leukocytes with bactericidal properties are crucial to a full response in certain models of acute infection. Modulation of mast cell numbers in vivo was also found to affect the host response against bacterial infection. Thus, mast cells do have a role in innate immunity in defined animal models of bacterial infection. Whether mast cells participate in innate immune responses in the protection of the human host against bacteria remains to be determined.

Characterization of novel stem cell factor responsive human mast cell lines LAD 1 and 2 established from a patient with mast cell sarcoma/leukemia; activation following aggregation of FcεRI or FcγRI

Two novel stem cell factor (SCF) dependent human mast cell lines, designated LAD 1 and 2, were established from bone marrow aspirates from a patient with mast cell sarcoma/leukemia. LAD 1 and 2 cells have the ultrastructural features of human mast cells, and express FcepsilonRI, CD4, 9, 13, 14, 22, 31, 32, 45, 64, 71, 103, 117, 132, CXCR4 (CD184), CCR5 (CD195); and intracytoplasmic histamine, tryptase and chymase. LAD 1 and 2 do not exhibit activating mutations at codon 816 of c-kit. Both LAD 1 and 2 release beta-hexosaminidase following FcepsilonRI or FcgammaRI aggregation. The availability of these cell lines offers an unparalleled circumstance to examine the biology of human mast cells.

Mast cells and mastocytosis

Mast cells have been recognized for well over 100 years. With time, human mast cells have been documented to originate from CD34+ cells, and have been implicated in host responses in both innate and acquired immunity. In clinical immunology, they are recognized for their central role in IgE-mediated degranulation and allergic inflammation by virtue of their expression of the high-affinity receptor for IgE and release of potent proinflammatory mediators. In hematology, the clinical disease of mastocytosis is characterized by a pathologic increase of mast cells in tissues, often associated with mutations in KIT, the receptor for stem cell factor. More recently, and with increased understanding of how human mast cells are activated through receptors including the high-affinity receptor for IgE and KIT, specific tyrosine kinase inhibitors have been identified with the potential to interrupt signaling pathways and thus limit the proliferation of mast cells as well as their activation through immunoglobulin receptors.

The alpha form of human tryptase is the predominant type present in blood at baseline in normal subjects and is elevated in those with systemic mastocytosis.

Tryptase, a protease produced by all mast cells, was evaluated as a clinical marker of systemic mastocytosis. Two sandwich immunoassays were evaluated, one which used the mAb G5 for capture, the other which used B12 for capture. The B12 capture assay measured both recombinant alpha- and beta-tryptase, whereas the G5 capture assay measured primarily recombinant beta-tryptase. G5 binds with low affinity to both recombinant alpha-tryptase and tryptase in blood from normal and nonacute mastocytosis subjects, and binds with high affinity to recombinant beta-tryptase, tryptase in serum during anaphylaxis, and tryptase stored in mast cell secretory granules. B12 recognizes all of these forms of tryptase with high affinity. As reported previously, during systemic anaphylaxis in patients without known mastocytosis, the ratio of B12- to G5-measured tryptase was always < 5 and approached unity (Schwartz L.B., T.R. Bradford, C. Rouse, A.-M. Irani, G. Rasp, J.K. Van der Zwan and P.-W.G. Van der Linden, J. Clin. Immunol. 14:190-204). In this report, most mastocytosis patients with systemic disease have B12-measured tryptase levels that are elevated (> 20 ng/ml) and are at least 10-fold greater than the corresponding G5-measured tryptase level. Most of those subjects with B12-measured tryptase levels of < 20 ng/ml had only cutaneous manifestations. The B12 assay for alpha-tryptase and beta-tryptase, particularly when performed in conjunction with the G5 assay for beta-tryptase, provides a more precise measure of mast cell involvement than currently available assessments, a promising potential screening test for systemic mastocytosis and may provide an improved means to follow disease progression and response to therapy.

Bone marrow stromal cells use TGF-β to suppress allergic responses in a mouse model of ragweed-induced asthma

Bone marrow stromal cells [BMSCs; also known as mesenchymal stem cells (MSCs)] effectively suppress inflammatory responses in acute graft-versus-host disease in humans and in a number of disease models in mice. Many of the studies concluded that BMSC-driven immunomodulation is mediated by the suppression of proinflammatory Th1 responses while rebalancing the Th1/Th2 ratio toward Th2. In this study, using a ragweed induced mouse asthma model, we studied if BMSCs could be beneficial in an allergic, Th2-dominant environment. When BMSCs were injected i.v. at the time of the antigen challenge, they protected the animals from the majority of asthma-specific pathological changes, including inhibition of eosinophil infiltration and excess mucus production in the lung, decreased levels of Th2 cytokines (IL-4, IL-5, and IL-13) in bronchial lavage, and lowered serum levels of Th2 immunoglobulins (IgG1 and IgE). To explore the mechanism of the effect we used BMSCs isolated from a variety of knockout mice, performed in vivo blocking of cytokines and studied the effect of asthmatic serum and bronchoalveolar lavage from ragweed challenged animals on the BMSCs in vitro. Our results suggest that IL-4 and/or IL-13 activate the STAT6 pathway in the BMSCs resulting in an increase of their TGF-β production, which seems to mediate the beneficial effect, either alone, or together with regulatory T cells, some of which might be recruited by the BMSCs. These data suggest that, in addition to focusing on graft-versus-host disease and autoimmune diseases, allergic conditions—specifically therapy resistant asthma—might also be a likely target of the recently discovered cellular therapy approach using BMSCs.