Data Supplement Plasmin Triggers Cytokine Induction in Human Monocyte-Derived Macrophages

Transcription

1 Data Supplement Plasmin Triggers Cytokine Induction in Human Monocyte-Derived Macrophages Qun Li, Yves Laumonnier, Tatiana Syrovets, and Thomas Simmet Methods Antibodies and Reagents Antibodies for immunoblotting: annexin A, SA (BD Biosciences, Bedford, MA), phospho-jak, JAK, JAK and phospho-stat-(tyr ) (Biosource, Nivelles, Belgium), phospho-jak and phospho-stat-(ser ) (Epitomics, Burlingame, CA), phospho-p and p, phospho-iκbα, phospho-erk/, ERK, and phospho-tyk (Cell Signaling, Beverly, MA), phospho-akt-(ser ) (Upstate, Charlottesville, VA), and actin (Chemicon International, Temecula, CA). Antibodies for flow cytometry: annexin A (Biodesign, Saco, ME), SA (RDI, Concord, MA), anti-cd and anti-cd (BD Biosciences), anti-cdc and anti-cd (DAKO, Hamburg, Germany), CD (BD Pharmingen). mouse and rabbit IgG and PE-conjugated donkey anti-mouse and anti-rabbit IgG F(ab) were from Dianova (Hamburg, Germany). Limulus amoebocyte lysate assay, and lipopolysaccharide (LPS; Escherichia coli serotype :B) were from Sigma (St. Louis, MO). Purified human plasmin (lot 9/,. CTA U/mg protein) tested for LPS was from Fluka (Deisenhofen, Germany); plasmin activity is given in Committee on Thrombolytic Agents (CTA) units/ml., The catalytic inhibitor of plasmin, D-Val-Phe-Lys chloromethyl ketone (VPLCK), the JAK inhibitor AG9, the p MAPK inhibitor SB and the MEK inhibitor U were from Calbiochem (San Diego, CA), recombinant human M-CSF was from Biovision (Mountain View, CA). Chemically pure acetyl--keto-β-boswellic acid was isolated as previously described. Cell Preparation and Culture Monocyte-derived human macrophages were differentiated from buffy coats for days with ng/ml M-CSF. Monocytes and neutrophils were isolated by Percoll gradient centrifugation. Macrophages were cultured in RPMI (Invitrogen, Carlsbad, CA),

2 supplemented with % FCS. The cells were stimulated with plasmin in lysine-free RPMI (Sigma). Analysis of Protein Expression For Western blot analysis and flow cytometry, macrophages were analyzed as described. The cytokine levels were quantified with ELISAs (R&D Systems, Minneapolis, MN). For the cleavage of annexin A, macrophages were either unstimulated or stimulated for min with. CTA U/mL plasmin or equivalent amounts of catalytically inactivated plasmin., Reverse Transcription and Polymerase Chain Reaction Analysis Total RNA from macrophages stimulated with. CTA U/mL plasmin or equivalent amounts of catalytically inactivated plasmin (VPLCK-plasmin) was isolated by Trizol (Invitrogen) and analyzed by semi-quantitative RT-PCR. Conditions for TNF-α were as described; for IL- the forward primer -TACATCCTCGACGGCATCTCA-, and the reverse primer -AGTTGTCATGTCCTGCAGCCA- with cycles of amplification (denaturation 9 C s, annealing C s and elongation C s) were used. PCR was performed in the linear range of amplification; GAPDH served as internal standard. The transcripts were identified by direct automated sequencing (Genetic Analyzer; Applied Biosystems). Inhibition of Annexin A and SA Expression by Antisense ODN For in vitro knockdown of annexin A and SA, μm of phosphorothioatemodified oligodeoxynucleotides (ODN) (Thermo Hybaid, Ulm, Germany) were applied to macrophage cultures every h for h as described. STAT and NF-κB family Transcription Factor Assays Human macrophages were stimulated with. CTA U/mL plasmin for min (STAT) or h (NF-κB), and nuclear extracts were prepared., Activation of STATα,, A, B and p, c-rel, RelB were determined in μg nuclear extract with DNA-binding

3 TransAM ELISAs for STAT and NF-κB family transcription factors (Active Motif, Carlsbad, CA). Data are expressed as amount of corresponding transcription factor in treated cells compared to unstimulated controls using - different nuclear extract preparations each. Statistical Analysis Values shown represent mean ± SEM where applicable. Statistical significances were calculated with the Newman-Keuls test. Differences were considered significant for P<.. References. Burysek L, Syrovets T, Simmet T. The serine protease plasmin triggers expression of MCP- and CD in human primary monocytes via activation of p MAPK and janus kinase (JAK)/STAT signaling pathways. J Biol Chem. ;:9-.. Laumonnier Y, Syrovets T, Burysek L, Simmet T. Identification of the annexin A heterotetramer as a receptor for the plasmin-induced signaling in human peripheral monocytes. Blood. ;:-9.. Büchele B, Simmet T. Analysis of different pentacyclic triterpenic acids from frankincense in human plasma by high-performance liquid chromatography and photodiode array detection. J Chromatogr B Analyt Technol Biomed Life Sci. ;9:-.. Colognato R, Slupsky JR, Jendrach M, Burysek L, Syrovets T, Simmet T. Differential expression and regulation of protease-activated receptors in human peripheral monocytes and monocyte-derived antigen-presenting cells. Blood. ;:-.. Syrovets T, Jendrach M, Rohwedder A, Schüle A, Simmet T. Plasmin-induced expression of cytokines and tissue factor in human monocytes involves AP- and IKKβ-mediated NF-κB activation. Blood. ;9:9-9.. Syrovets T, Büchele B, Krauss C, Laumonnier Y, Simmet T. Acetyl-boswellic acids inhibit lipopolysaccharide-mediated TNF-α induction in monocytes by direct interaction with IκB kinases. J Immunol. ;:9-.

4 Supplemental Data A P-JAK (fold activation) P-TYK (fold activation) P-STATTyr (fold activation) P-STATSer (fold activation) B P-p (fold activation) P-ERK/ (fold activation) C P-AKt (fold activation) P-IκBα (fold activation) Supplemental Figure I.

5 D P-AKt (fold activation) AG P-ERK/ (fold activation) Plasmin+AG9 (um) AG P-IκBα (fold activation) AG P-p (fold activation) AG Supplemental Figure I. Plasmin triggers activation of JAK/STAT, MAPK and NF-κB signaling pathways. Macrophages (x /assay) were stimulated with plasmin (. CTA U/mL) in serum-free RPMI for the indicated time. Whole cell lysates were separated and analyzed by densitometric scanning of Western immunoblots. A, Timedependent activation of JAK/STAT. Analysis of phospho-jak (P-JAK), phospho- TYK (P-TYK), phospho-stat-(tyr ) and phospho-stat-(ser ); JAK, TYK and actin served as loading controls. B, Time-dependent activation of MAPK. Analysis of phospho-p and phospho-erk/; p or ERK served as loading control. C, Timedependent activation of the NF-κB pathway. Analysis of phospho-akt-(ser ) and phospho-iκbα from whole cell lysates from macrophages stimulated with plasmin. D, Effects of the JAK inhibitor, AG9, on the plasmin-induced MAPK and NF-κB signaling pathways. Cells pretreated with μm AG9 for min were stimulated with. CTA U/mL plasmin for min. Analysis of phospho-erk/, phospho-akt-(ser ), phospho- IκBα, and phospho-p; non-phosphorylated proteins and actin served as loading controls. All results are mean ± SEM of at least independent experiments.

6 A TNF-α mrna/gapdh mrna..... Plasmin (. CTA U/mL) LPS ( µg/ml) h h h IL- mrna/gapdh mrna Plasmin (. CTA U/mL) LPS ( µg/ml) h h h B TNF-α mrna/gapdh mrna LPS.... Plasmin (CTA U/mL) IL- mrna/gapdh mrna LPS.... Plasmin (CTA U/mL) C TNF-α mrna/gapdh mrna IL- mrna/gapdh mrna Plasmin Plasmin-VPLCK. Plasmin Plasmin-VPLCK Supplemental Figure II. Plasmin triggers time- and concentration-dependent induction of TNF-α and IL- genes. A, Time-dependent and B, concentration-dependent stimulation of the TNF-α and IL- mrna expression. Macrophages were stimulated with plasmin (. CTA U/mL) or LPS ( μg/ml) for the indicated times and the mrna levels were analyzed by RT-PCR and densitometry. C, The gene induction depends on the proteolytic activity of plasmin. Macrophages were stimulated for h with plasmin (. CTA U/mL) or with equivalent amounts of catalytically inactivated plasmin; TNF-α and

7 IL- mrna were analyzed by RT-PCR and densitometry. GAPDH served as loading control. In each case, results are means ± SEM of independent experiments. Protein expression (fold)... Sense annexin A AS annexin A Sense SA AS SA. annexin A SA Supplemental Figure III. The annexin A heterotetramer is indispensable for the plasmin-stimulated release of cytokines by the macrophages. Specific antisense ODN decrease the expression of annexin A and SA. Macrophages were treated for h with μm of the corresponding ODN (sense or antisense). After h recovery in RPMI without lysine, the cells were lysed, the proteins were separated and analyzed by immunoblotting and densitometric scanning. Actin served as loading control. The data represent the mean percentage ± SEM of independent experiments.