1. Neutrophils Self-Regulate Immune Complex-Mediated Cutaneous Inflammation through CXCL2 
Jackson LiangYao Li, Chun Hwee Lim, Fen Wei Tay, Chi Ching Goh, Sapna Devi, Benoit Malleret, Bernett Lee, Nadja Bakocevic, Shu Zhen Chong, Maximilien Evrard, Hideaki Tanizaki, Hwee Ying Lim, Bruce Russell, Laurent Renia, Francesca Zolezzi, Michael Poidinger, Veronique Angeli, Ashley L. St. John, John E. Harris, Hong Liang Tey, Suet Mien Tan, Kenji Kabashima, Wolfgang Weninger, Anis Larbi, Lai Guan Ng 
Journal of Investigative Dermatology 
Volume 136, Issue 2, Pages (February 2016)
DOI: /JID
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2. Figure 1
Neutrophil interaction with ICs during the RPA reaction. (a) Neutrophil counts in RPA-induced mouse ears or control ear (N = 3–9). (b) Representative photographs showing RPA-induced leakage. Green insets, pixel classification: leakage, white; no leakage, black. (c) Images from in vivo imaging during RPA. White square, region described in d. (d) Zoomed-in region of c, showing neutrophil behavior and IC formation. Yellow arrows, same IC-laden neutrophil. (e) Analysis of ICs and neutrophils from imaging data (mean of nine independent experiments), plotted against time (left) or against each other (right) (each dot represents one time point). (f) Flow cytometry analysis of live CD45+ cells present in RPA ear, showing high uptake of ICs by neutrophils. *P < 0.05, **P < 0.01, ***P < by an unpaired t-test. ICs, immune complexes; RPA, reverse passive Arthus. Scale bars = 50 μm (c), 10 μm (d).
Journal of Investigative Dermatology  , DOI: ( /JID )
Copyright © 2015 The Authors Terms and Conditions

3. Figure 2
Neutrophil depletion during the RPA reaction. (a) Representative photographs showing dye leakage in RPA ears in neutrophil-depleted mice. Green insets, pixel classification: leakage, white; no leakage, black. (b) Total amount of Evans blue dye in mouse ears measured at various time points after RPA induction, in neutrophil-depleted or nondepleted mice (N = 3 per group per time point). (c) Images from in vivo two-photon imaging of ear skin in neutrophil-depleted mouse at various time points after initiation of RPA. One of the three independent experiments is shown. *P < 0.05, **P < 0.01, ***P < (nondepleted vs. neutrophil-depleted in RPA ears); #P < 0.05, ##P < 0.01, ###P < 0.001, ####P < (RPA vs. control) by an unpaired t-test for each time point. RPA, reverse passive Arthus. Scale bars = 50 μm.
Journal of Investigative Dermatology  , DOI: ( /JID )
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4. Figure 3
Neutrophil expression of CXCL2 during the RPA reaction. (a) mRNA levels of neutrophils exhibiting high IC uptake (6-hour RPA) analyzed using NanoString technology, normalized to GAPDH. Data were compared with steady-state blood neutrophils (N = 3 each). (b) ELISA readings in supernatants of isolated neutrophils stimulated with or without IC for 3 hours in vitro (N = 5 per condition). n.d., below detection limit. (c) ELISA measurements in mouse ear homogenates (6-hour RPA) in neutrophil-depleted or nondepleted mice (N = 4 each). (d) Scatterplot showing correlation between neutrophils levels and protein levels of chemokine (protein levels correspond to those in c). (e) mRNA levels of various sorted major cell types in RPA ears (3 hours) measured with nanostring technology (N = 3) (mean ± s.d.). (f) Schematic diagram of the experimental setup for g. (g) qPCR assessment of CXCL1 when incubated with IC-stimulated neutrophils (N = 3 per condition). ***P < 0.001; ns, nonsignificant, unpaired t-test. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; ICs, immune complexes; RPA, reverse passive Arthus.
Journal of Investigative Dermatology  , DOI: ( /JID )
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5. Figure 4
Autocrine production of CXCL2 by neutrophils. (a) Uptake of fluorescent ICs by neutrophils incubated with neutralizing antibodies in vitro (N = 5). (b) ROS production by isolated neutrophils incubated with ICs for 30 minutes with neutralizing antibodies in vitro (N = 5). (c) qPCR assessment of CXCL2 mRNA levels in isolated neutrophils incubated with ICs in the presence of neutralizing antibodies (N = 5). (d) qPCR assessment of CXCL2 mRNA levels in isolated neutrophils induced by exogenous recombinant chemokine in the presence of preformed ICs (N = 5; one-way ANOVA). (e) Dose-response curve of CXCL1-induced or autocrine CXCL2 mRNA expression (N = 3; mean ± s.d.). (f) Surface CD11b expression of isolated neutrophils in the presence of CXCL1 or CXCL2 in vitro (N = 4). (g) Surface CXCR2 expression in the presence of CXCL1 or CXCL2 (N = 4) after 1 hour (unpaired t-test). *P < 0.05, **P < 0.01, ***P < 0.001; ns, nonsignificant. ICs, immune complexes; ROS, reactive oxygen species.
Journal of Investigative Dermatology  , DOI: ( /JID )
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6. Figure 5
Neutralization of CXCL1 and CXCL2 during the RPA reaction. (a, b) Total neutrophils (left) and Evans blue leakage (right) in the ears of (a) WT (N = 6) or CXCR2-KO mice (N = 5) (6-hour RPA); or in (b) neutralizing antibody-treated mice (intravenous delivery 30 minutes before initiating RPA). Data pooled from three independent experiments performed on different days and normalized against the mean IgG sample results obtained on the same day. (c, d) Representative images of (c) the mouse ear 1 hour after intradermal injection, or (d) surgically exposed mouse tibial muscle 90 minutes after topical application of exogenous recombinant chemokines into WT or CXCR2-KO mice. (e, f) Images of the mouse ear after interstitial delivery (intradermal injection) of (e) anti-CXCL1 or (f) anti-CXCL2 during the RPA reaction (one of three independent experiments shown each). SHG, blue; neutrophils, green; Evans blue, red; IC, white; scale bars = 50 μm. RPA, reverse passive Arthusa; SHG, second harmonic generation; WT, wild-type.
Journal of Investigative Dermatology  , DOI: ( /JID )
Copyright © 2015 The Authors Terms and Conditions