Evidence of kinetic proofreading through allergen specific IgE at the human mast cell IgE receptor
Authors
Charlotte Hjort, Department of Clinical Medicine, Aarhus University and Department of Respiratory Diseases and Allergy, Aarhus University Hospital, Denmark
Jesper Just, Department of Molecular Medicine, Aarhus University Hospital and Department of Clinical Medicine, Aarhus University, Denmark
Lars I. Andersson, Department of Respiratory Medicine, Karolinska University Hospital and Department of Medicine Huddinge, Karolinska Institute, Stockholm, Sweden
Craig Wheelock, The Institute of Environmental Medicine, Karolinska Institute, and Department of Respiratory Medicine, Karolinska University Hospital, Stockholm, Sweden
Sven-Erik Dahlén, The Institute of Environmental Medicine, Karolinska Institute, and Department of Respiratory Medicine, Karolinska University Hospital, Stockholm, Sweden
Peter Adler Würtzen, ALK Abello, Denmark
Lars Harder Christensen, ALK Abello, Denmark
Hans Jürgen Hoffmann*, Department of Clinical Medicine, Aarhus University and Department of Respiratory Diseases and Allergy, Aarhus University Hospital, Denmark
Contribution:
HJH, PAW and CH designed the study, CH performed mast cell experiments, JJ performed data analyses, LA, SED, DF and CW performed measurements, LHC contributed essential reagents. CH, JJ and HJH drafted the manuscript. All authors revised the manuscript and will approve the final version.
Funding/Acknowledgements:
CH was funded by the Frølich Foundation and Harboefonden. LHC is and PAW was employee of ALK Abelló.
Abstract (208 words)
Background
Activation of mast cells through IgE results in secretion and shedding of mast cell proteins and in vivo models suggest that these processes are governed by IgE antibody affinity.
Methods
We passively sensitized cultured primary human mast cells with recombinant human IgE clones with either high or low affinity for Der p 2, with a 200-fold affinity difference, and activated them with recombinant allergen. Activation was assessed by CD63 upregulation and PGD2 secretion. Supernatants collected from mast cells activated for 0, 3, 6 and 24 hours were assessed for PGD2 and inflammatory mediators on the OLINK platform at repeated time points.
Results
CD63 upregulation and PGD2 synthesis scaled with affinity, as did secretion of cytokines like IL-8 and IL-13. Secretion of chemokines like CCL3 and CCL4 appeared to depend less on affinity, whereas shedding of surface markers CD40, SLAMF4 and CD5, and secretion of intracellular markers SIRT2 and CASP-8, were elevated by stimulation through low affinity IgE compared with high affinity IgE, illustrating differential responses dependent on the affinity of IgE.
Conclusion
Cytokine secretion and shedding of surface receptors of sensitized, cultured primary human mast cells is differentially regulated depending on the affinity of IgE for the Der p 2 allergen and may shape the chronic response to repeated allergic activation.
Introduction (text 3113 words, 31 refs, 5 figures, 2 tables)
Signaling mediated by antibodies and their receptors through ITAM motifs has been shown to be subject to molecular editing through kinetic proofreading in mouse and rat models, resulting in distinct responses of myeloid effector cells depending on the affinity of the immunoglobulin-antigen interaction(1,2). Activation with low affinity antigen reduces leukotriene and cytokine (TNF-a, IL-6, IL-13) production but enhances production of chemokines CCL2, CCL3 and CCL4. This is reversed in cells activated with high affinity antigen (2). We hypothesize that human mast cells stimulated through high- and low affinity IgE respond similarly.
The generation of cytokines and chemokines from activated mast cells is a result of de novo synthesis and release of these soluble mediators. Ectodomain shedding of membrane proteins is another, more rapid, mechanism of control of membrane proteins that can lead to generation of soluble messengers and feedback loops (3). This process is, however, uncharted on mast cells during activation. CD5, CD6, CD40 (4) and SLAMF4 (5) are regulatory molecules expressed on mast cells. CD5 has not been identified in mast cells by immunohistochemistry (6), but activity at the CD5 promoters has been documented recently (7). Differential regulation of these surface markers may affect mast cell function significantly.
We have previously shown that, in the immediate allergic response, reactivity increases threefold and sensitivity (EC50) increases 15000-fold in cultured human mast cells (MC) sensitized with two low affinity IgE clones compared with two high affinity IgE clones specific for the house dust mite Dermatophagoides pteronyssinus class two allergen (Der p 2) (8,9). A tenfold increase in the affinity of IgE clones contributing to complex formation resulted in 19% increase in mast cell reactivity (9).
Here we present evidence using the major allergen Der p 2 and cloned pairs of IgE molecules specific for Der p 2 with affinity differing 200 fold (8,9), that a broad range of cytokine and chemokine expression as well as shedding of surface receptors of primary cultured human mast cells, depends on the affinity of IgE for allergen. This is the first documentation of molecular editing due to variation in affinity of the cellular response in human mast cells and suggests that measures of quantity of allergen specific IgE (sensitization) is inadequate without knowledge of the affinity of the interaction between IgE and its cognate allergen. In previous work, the significance of complexity and composition of IgE clones has been illustrated (8).
Methods