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Evidence for a cross-talk between human neutrophils and Th17 cells
Blood Pelletier et al.
10.1182/blood-2009-04-216085
Supplemental materials for: Pelletier et al
Respiratory burst
Measurement of superoxide anion generation was performed by colorimetric assay (reduction of cytochrome c) in 96-well tissue culture plates as previously described.1
Quantitative RT-PCR
Real-time RT-PCR was performed exactly as described,2 using gene-specific primer pairs (purchased from Invitrogen) available in the public database RTPrimerDB (http://medgen.ugent.be/rtprimerdb/) under the following entry codes: CCL2 (3533), CXCL10 (3537), CXCL8 (3647), GAPDH (3539), PPIB (7786), CCL20 (3911), TNFα (3646), IL-1β (7760), IL-17A (7754), IL-17F (7788), IL-23R (7755), IL-22 (7759), IL-26 (7789), IL-17RA (7791), IL-17RC (7790), and Act1 (8116).
Confocal microscopy
Detection of CD161+, CD15+, and RORγt+ cells in gut samples was performed by laser confocal microscopy (LSM 510META; Carl Zeiss, Inc.) using FITC conjugated anti-CD161, -CD15 mAbs (BD Biosciences), rabbit polyclonal anti-RORγt Ab (Abcam Inc. Cambridge, USA), rabbit polyclonal IgG, and goat anti-rabbit Alexafluor 633 Ab (Molecular Probe-Invitrogen). Nuclei were counterstained with TO-PRO-3 (Invitrogen).
Measurement of soluble mediators
Cytokine concentrations in cell-free supernatants, cell lysates, and RA SF samples were measured by specific ELISA kits for: CCL2, CCL17, CCL20, CCL22, CXCL10, VEGF (R&D Systems), CXCL8, IFNγ, TNFα (Immunotools, Friesoythe, Germany), GM-CSF, and IL-17A (eBioscience, San Diego, CA, USA). The detection of lactoferrin was performed as previously described.3
Immunoblots
Cytoplasmic extracts prepared from neutrophils by nitrogen cavitation,4 as well as whole-cell extracts prepared from neutrophils, monocytes, and HBEC by hypertonic lysis with NP-40,2 were analyzed by western blot using anti–phospho-p44/42 ERK (Cell Signaling Technology, Inc. Danvers, USA), anti-Act1 (H-300, Santa Cruz Biotechnology, Inc. Heidelberg, Germany), and ERK1/2 (Santa Cruz) Abs.
Flow cytometry
Analysis of intracellular cytokine and surface antigen expression was performed using fluorochrome-conjugated anti-CD3, -CD11b, -CD16, -CD66b, -CCR3 (Biolegends), anti-CCR6, -IFNγ, -IL-4, –IL-8 (BD Biosciences), –IL-17A (eBioscience), –IL-17R, -CCR2 or -CXCR3 (R&D Systems). PMA and ionomycin, used for polyclonal stimulation, and brefeldin A were purchased from Sigma-Aldrich. Biotinylated anti–IL-17RC Ab was from R&D Systems, and PE-conjugated streptavidin was from Pharmingen. Apoptosis was detected using the Annexin–V-FLUOS staining kit (Roche, Mannheim, Germany).
REFERENCES
1. Vinante F, Marchi M, Rigo A, Scapini P, Pizzolo G, Cassatella MA. Granulocyte-macrophage colony-stimulating factor induces expression of heparin-binding epidermal growth factor-like growth factor/diphtheria toxin receptor and sensitivity to diphtheria toxin in human neutrophils. Blood. 1999;94:3169–3177.
2. Tamassia N, Le Moigne V, Calzetti F et al. The MyD88-independent pathway is not mobilized in human neutrophils stimulated via TLR4. J Immunol. 2007;178:7344–7356.
3. Merck E, Gaillard C, Scuiller M et al. Ligation of the FcR gamma chain-associated human osteoclast-associated receptor enhances the proinflammatory responses of human monocytes and neutrophils. J Immunol. 2006;176:3149–3156.
4. Borregaard N, Heiple JM, Simons ER, Clark RA. Subcellular localization of the b-cytochrome component of the human neutrophil microbicidal oxidase: translocation during activation. J Cell Biol. 1983;97:52–61.
Files in this Data Supplement:
- Figure S1. Highly purified neutrophils produce and release significant amounts of CCL2, CCL20, and CXCL10 (JPG, 280 KB)
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(A), highly purified neutrophils (> 99.7 %), obtained after immunomagnetic separation are devoid of any lymphocytes (lympho; CD3+ CD16− CCR3−), monocytes or eosinophils (CD16low CCR3+) that often contaminate Ficoll-Paque isolated granulocytes preparations (~ 95 %), as determined by selected labeling markers. One representative flow cytometric analysis is shown. (B), CXCL10, CCL2, CCL20, CCL17, and CCL22 concentrations (n = 5) in supernatants harvested from magnetically-purified neutrophils (5 × 106/mL) cultured for 21 h with IFNγ and/or LPS.
- Figure S2. Phenotypic characteristics of Th17, Th1, and Th2 clones (JPG, 142 KB)
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(A,B), Th17, Th1, and Th2 clones were evaluated by flow cytometry for intracellular IL-17A, IFNγ, and IL-4 expression (A), as well as CCR6, CCR2, and CXCR3 surface expression (B), to determine the percentages of positive cells. (C) A representative CCR6 and CCR2 surface coexpression by Th17 and Th1 clones showing that the majority of Th17 clones are positive for both CCR6 and CCR2, while Th1 clones are only positive for CCR2. FACS staining using CCR6 and CCR2 related isotype Abs are also shown.
- Figure S3. Preactivated neutrophils do not respond to IL-17A (JPG, 108 KB)
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(A–C), neutrophils were cultured for 21 h in the presence or absence of IFNγ plus LPS or TNFα, before being treated for 2 and 10 min (A), or 24 h (B,C), with or without 100 ng/ml IL-17A (A–C) or 100 nM fMLF (A). In (A), phospho-ERK1/2 (as well as ERK1/2) levels were evaluated by western blot. One representative out of three independent experiments with similar results is shown. In (B), CXCL8, CCL2, and CXCL10 levels were measured in cell-free supernatants (n = 3). In (C), the degree of apoptosis (as assessed by Annexin-V/PI staining) was evaluated after 45 h of total incubation (n = 4).
- Figure S4. Inflammatory and activated neutrophils do not express IL-17RC (JPG, 97 KB)
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(A) IL-17RA and IL-17RC surface expression was evaluated in neutrophils magnetically purified from the synovial fluid of RA patients. In (B and C), neutrophils were incubated for 21 h in the presence or absence of IFNγ, LPS or IFNγ plus LPS prior to evaluating IL-17RA and IL-17RC surface expression (B) and IL-17A levels in the supernatant by ELISA (n = 5) (C). CD4+ CCR6+ T cells stimulated in the presence or absence of PMA/ionomycin were used as positive controls for IL-17A release.
- Figure S5. Activated Th17 clones modulate apoptosis of cytokine-primed neutrophils through an IL-17–independent mechanism (JPG, 57.5 KB)
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Neutrophils were cultured for 21 h in the presence or absence of IFNγ plus LPS before being treated for 24 h with supernatants harvested from αCD3/αCD28-stimulated Th17 clones, in the presence or absence of anti–IL-17 and GM-CSF neutralizing Abs. The degree of apoptosis (as assessed by Annexin-V/PI staining) was evaluated after 45 h of total incubation (n = 4).
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