40.000. 30.000. 20.000. 10.000. -10.000. 0. N u m b e. r o. f m ig ra te d c e lls m edium. IL-8 anti-IL-8IgA1-lin. FcαRI1-lin. FcαRI8-CLIPS. FcαRI9-ox. Supporting ...
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European Journal of Immunology Supporting Information for DOI 10.1002/eji.201646782 Marieke H. Heineke, Lydia P.E. van der Steen, Rianne M. Korthouwer, J. Joris Hage, Johannes P.M. Langedijk, Joris J. Benschop, Jantine E. Bakema, Jerry W. Slootstra and Marjolein van Egmond Peptide mimetics of immunoglobulin A (IgA) and FcαRI block IgA-induced human neutrophil activation and migration
C 2017 The Authors. European Journal of Immunology published by WILEY-VCH Verlag
GmbH & Co. KGaA, Weinheim.
www.eji-journal.eu
Supporting Information Figure 1
A YRIGHYRFR
B
CQAIREAYL
LKFWNETDP
C EALPLAFTQ
LEDLLLGSE
WLQSQELPR
Supporting Figure 1. Model of interaction sites of IgA and FcaRI (A) Model of the structure of the complex of two IgA Fc heavy chains, interacting with two extracellular domains of FcαRI. (Two molecules of FcαRI form a complex with one IgA molecule). (B and C) Magnification of position of FcαRI sequences (B) and IgA sequences (C) involved in binding.
Supporting Information Figure 3
Number of migrated cells
40.000 30.000 20.000 10.000 0
n R I1 -li R n I8 -C Fc LIP S R I9 -o x
8
Fc
Fc
A 1li
L-
Ig
i-I
-8
an t
IL
m ed i
um
-10.000
Supporting Figure 3. Peptide mimetics do not block IL-8 induced chemotaxis Number of fluorescently labelled neutrophils which migrated to IL-8 in a chemotaxis chamber, either in the presence or absence of peptide mimetics. The number of migrated neutrophils was determined with a fluorimeter. Neutrophil migration to IL-8 is indicated with a dotted line. Medium and blocking IL-8 antibody (anti-IL-8) were used as controls. Experiments were performed three times in triplo. Mean ± SD of one representative experiments in triplo is shown. Statistical analysis: ANOVA
Supporting Information Figure 2
B 160
140
140
120
120
80 60
100 80 60
20
0
0
Ig
Fc
Ig
A 7o Ig
A 6o Ig
A 2C LI Ig PS A 3C Fc LI PS R I3 -C Fc LI PS R I4 -C Fc LI PS R I5 -C LI PS
20
A
40
x R I6 -o Fc x R I7 -o Fc x R I8 -o Fc x R I9 -o x
40
A
100
Ig
% migration
160
x
% migration
A
Supporting Figure 2. Oxidated or small peptide mimetics do not block IgA-induced migration in vitro Percentage of migration of fluorescently labelled neutrophil to IgA-coated beads, either in the presence or absence of peptide mimetics. The number of migrated neutrophils was determined with a fluorimeter. Neutrophil migration to IgA was normalized to 100% (dotted line). Neutrophils or beads were pre-incubated with (A) oxidated or (B) small cyclic peptides mimicking FcαRIsequences (white bars) or IgA-sequences (black bars). Data are representative of 3 independent experiments, performed in triplicates. Mean ± SD of one representative experiment is shown. Statistical analysis: ANOVA
