<div>Evidence of kinetic proofreading through allergen specific IgE at the
human mast cell IgE receptor</div><div>Authors</div><div>Charlotte Hjort, Department of Clinical Medicine, Aarhus University and
Department of Respiratory Diseases and Allergy, Aarhus University
Hospital, Denmark</div><div>Jesper Just, Department of Molecular Medicine, Aarhus University
Hospital and Department of Clinical Medicine, Aarhus University, Denmark</div><div>Lars I. Andersson, Department of Respiratory Medicine, Karolinska
University Hospital and Department of Medicine Huddinge, Karolinska
Institute, Stockholm, Sweden</div><div>Craig Wheelock, The Institute of Environmental Medicine, Karolinska
Institute, and Department of Respiratory Medicine, Karolinska University
Hospital, Stockholm, Sweden</div><div>Sven-Erik Dahlén, The Institute of Environmental Medicine, Karolinska
Institute, and Department of Respiratory Medicine, Karolinska University
Hospital, Stockholm, Sweden</div><div>Peter Adler Würtzen, ALK Abello, Denmark</div><div>Lars Harder Christensen, ALK Abello, Denmark</div><div>Hans Jürgen Hoffmann*, Department of Clinical Medicine, Aarhus
University and Department of Respiratory Diseases and Allergy, Aarhus
University Hospital, Denmark</div><div>Contribution:</div><div>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.</div><div>Funding/Acknowledgements:</div><div>CH was funded by the Frølich Foundation and Harboefonden. LHC is and PAW
was employee of ALK Abelló.</div><div>Abstract (208 words)</div><div>Background</div><div>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.</div><div>Methods</div><div>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
PGD<sub>2</sub> secretion. Supernatants collected from mast cells
activated for 0, 3, 6 and 24 hours were assessed for
PGD<sub>2</sub> and inflammatory mediators on the OLINK platform at
repeated time points.</div><div>Results</div><div>CD63 upregulation and PGD<sub>2</sub> 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.</div><div>Conclusion</div><div>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.</div><div>Introduction (text 3113 words, 31 refs, 5 figures, 2 tables)</div><div>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.</div><div>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.</div><div>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).</div><div>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).</div><div>Methods</div>