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Supplementary Materials

Supplementary Material for:

Complement C5a receptor is the key initiator of neutrophil adhesion igniting immune complex–induced arthritis

Yoshishige Miyabe, Chie Miyabe, Thomas T. Murooka, Edward Y. Kim, Gail A. Newton, Nancy D. Kim, Bodduluri Haribabu, Francis W. Luscinskas, Thorsten R. Mempel, Andrew D. Luster*

*Corresponding author. Email: aluster{at}mgh.harvard.edu

Published 20 January 2017, Sci. Immunol. 2, eaaj2195 (2017)
DOI: 10.1126/sciimmunol.aaj2195

This PDF file includes:

  • Fig. S1. In vivo imaging coadoptively transferred WT and CAR-KO neutrophils into WT mice with full-blown arthritis.
  • Fig. S2. In vivo imaging demonstrated that C5ar-KO and Ccr1-KO neutrophils detach from the endothelium in the joints of mixed BMC mice.
  • Fig. S3. Analysis of C5a expression in VWF+ endothelium of (A) aorta and coronary artery, (B) superior vein cava, (C) kidney, and (D) lung from WT mice on day 7 after AST.
  • Fig. S4. In vivo joint adhesion assay after the application of CA to the exposed joint of WT-LysM-GFP mice (A to D) and Blt1-KO-LysM-GFP and Ccr1-Cxcr2-DKO-LysM-GFP mice (E to G).
  • Fig. S5. LFA-1 and Mac-1 expression on neutrophils.
  • Fig. S6. C5a-induced neutrophil adhesion initiates IC-mediated inflammation.
  • Fig. S7. Representative gating strategy of Ly6G+ neutrophil.
  • Legend for movies S1 to S6

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Other Supplementary Material for this manuscript includes the following:

  • Table S1 Excel file containing tabulated data for Figs. 1 to 8.
  • Movie S1 (.mp4 format). In vivo joint imaging of WT-LysM-GFP and CAR-KO-LysM- GFP mice on day 7 after AST.
  • Movie S2 (.mp4 format). Dynamic transition of adoptively transferred WT and CAR-KO neutrophils from arrest to crawling on the endothelium of inflamed joints in WT mice on day 7 after AST.
  • Movie S3 (.mp4 format). Detachment of adoptively transferred neutrophils from the endothelium of inflamed joints in WT mice on day 7 after AST.
  • Movie S4 (.mp4 format). In vivo adhesion assay: Application of CXCL1, CXCL2, CCL3, C5a, LTB4 (1 nM), or PBS (control) to exposed joints of naïve WT-LysM-GFP mice.
  • Movie S5 (.mp4 format). In vivo adhesion assay: β2 integrins mediate C5a-induced neutrophil adhesion on joint endothelium.
  • Movie S6 (.mp4 format). In vivo adhesion assay: β2 integrin blockade caused C5a-activated neutrophils to detach from the joint endothelium.

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