Regulation of monocyte migration by amphoterin (HMGB1)‏ by Ari Rouhiainen, Juha Kuja-Panula, Erika Wilkman, Jukka Pakkanen, Jan Stenfors, Raimo K. Tuominen, Mauri Lepäntalo, Olli Carpén, Jaakko Parkkinen, and Heikki Rauvala Blood Volume 104(4):1174-1182 August 15, 2004 ©2004 by American Society of Hematology

Western blotting analysis of amphoterin and RAGE Western blotting analysis of amphoterin and RAGE. (A) Leukocytes were lysed with 1% SDS, and 10-g samples of total cellular protein or 40 ng recAtn were run under reducing conditions on SDS-PAGE and transferred to a nitrocellulose filter. Western blotting analysis of amphoterin and RAGE. (A) Leukocytes were lysed with 1% SDS, and 10-g samples of total cellular protein or 40 ng recAtn were run under reducing conditions on SDS-PAGE and transferred to a nitrocellulose filter. The filter was immunostained with anti-recAtn antibodies. PMN indicates polymorphonuclear leukocytes. (B) Monocyte lysates (40 μg protein) and recAtn (80 ng) were run in SDS-PAGE and transferred to nitrocellulose filters. The filters were immunostained with 5 affinity-purified antibodies raised against different amphoterin peptides (I-V). (C) Rat brain microglia lysates were analyzed in Western blotting with anti-recAtn and antipeptide II. Both antibodies recognized a single 30-kD band from the lysate. (D) sRAGE was purified from bovine lung acetone powder. Purified sRAGE migrated as a single band in 12% SDS-PAGE stained with Coomassie blue (lane i). sRAGE was detected by 4 different anti-RAGE antibodies in 10% to 20% SDS-PAGE and Western blotting experiment: anti-P300 (lane ii), anti-P301 (lane iii), anti-RAGE (lane iv), and anti-RAGE N-16 (lane v). (E) Western-blotting of monocyte RAGE. RAGE was detected from 1-hour amphoterin adherent human monocytes or rat leukocytes using anti-P300 (lane i), anti-RAGE (lane ii), or anti-P301 (lane iii). Lanes i-ii: human cells. Lane iii: rat cells. Ari Rouhiainen et al. Blood 2004;104:1174-1182 ©2004 by American Society of Hematology

Secretion of amphoterin from mononuclear cells. Secretion of amphoterin from mononuclear cells. (A-D) Freshly isolated peripheral blood leukocytes were double-immunostained in suspension (A-B) and after adhesion and culture on coverslips for 18 hours in the presence of 10% autologous serum (C-D). Staining for CD14 is shown in panels A and C and for amphoterin in panels B and D. Cells were fixed with 2% paraformaldehyde for 15 minutes and double-stained with anti-CD14 and TRITC-conjugated antimouse immunoglobulins followed by anti-recAtn and FITC-conjugated antichicken immunoglobulins. (E-G) Embolectomy sample taken within 24 hours of the onset of symptoms for lower-limb arterial occlusion was snap-frozen in liquid nitrogen. Frozen sections were fixed with cold acetone, incubated with anti-recAtn (E-F) or nonspecific chicken IgY (G), and detected with peroxidase-labeled antichicken IgY. Scale bar 20 μm (A-G). (H) Secretion of amphoterin from RAW 264.7 cells is induced by IFN-γ and PMA and inhibited with ABC-1 inhibitors DIDS and glyburide. Cells were treated with 20 ng/mL IFN-γ or 10 nM PMA, and medium samples (0, 7, or 10 hours) were collected. Medium (1.5 mL) was concentrated to 15 L and analyzed in anti-recAtn Western blotting. Amphoterin was detected from activated cell culture medium after 7 or 10 hours of culture. Secretion was dose dependently inhibited with DIDS and to a lower extent with glyburide. BSP did not inhibit secretion. Medium samples were analyzed for activity (in mU/mL) of LDH by enzyme activity measurement. The results shown are from a representative experiment of at least 3 experiments. IFN indicates IFN-γ. (I) Optical density (OD) of the amphoterin band in Western blotting of IFN-γ activated (7 hours) cell culture supernatants, and lactate dehydrogenase activity in supernatants was measured. OD of bands from noninhibited samples was defined as 1 (± SD, n = 3). Ari Rouhiainen et al. Blood 2004;104:1174-1182 ©2004 by American Society of Hematology

Adhesion and spreading of monocytes on amphoterin and extracellular matrix proteins. Adhesion and spreading of monocytes on amphoterin and extracellular matrix proteins. (A-C) Peripheral blood leukocytes were kept on coverslips coated with vitronectin (A), fibronectin (B), or recAtn (C) for 1 hour in serum-free medium. The coverslips were washed, fixed with 2% paraformaldehyde, and immunostained with anti-CD14 and TRITC-labeled antimouse immunoglobulins. Scale bar (A-C) 10 μm. (D) Measurement of amphoterin and fibronectin adherent monocyte areas. Monocytes were adhered to recAtn or fibronectin-coated plastic for 1 hour, and adherent cells were fixed. Digital microscopy pictures were taken, and cell areas were measured. Mean of areas was calculated. Amphoterin-adherent cells spread strongly compared with fibronectin-adherent cells, and spreading was inhibited with 100 g/mL sRAGE but not with sAMIGO. Error bars represent ± SD (n = 3). (E) Peripheral blood mononuclear cells in serum-free medium were incubated in microwells coated with recAtn, fibronectin (Fn), or albumin (BSA) for 1 hour, and the bound CD14+ cells were detected with anti-CD14 and ELISA. (F-G) Peripheral blood leukocytes were incubated on recAtn or Fn-coated coverslips for 1 hour in serum-free medium, and the bound cells were immunostained with anti-CD14 and TRITC-labeled second antibody. The number of CD14+ cells (F) and their proportion of total cells (G) were determined by fluorescence and phase-contrast microscopy. The mean and SEM of 3 different experiments (E) or 12 fields of 2 different experiments (F-G) are shown. Ari Rouhiainen et al. Blood 2004;104:1174-1182 ©2004 by American Society of Hematology

Detection of chromogranins B and C from monocytes and microglia, and amphoterin-induced up-regulation of chromogranin B. Cells were adhered to amphoterin or fibronectin for 1 or 20 hours. mRNA was isolated and amplified in RT-PCR using primers specific for ... Detection of chromogranins B and C from monocytes and microglia, and amphoterin-induced up-regulation of chromogranin B. Cells were adhered to amphoterin or fibronectin for 1 or 20 hours. mRNA was isolated and amplified in RT-PCR using primers specific for chromogranins B and C. (A) Amplified monocyte cDNA was analyzed using agarose gel electrophoresis and ethidium bromide staining. Bands of the expected size were obtained for both chromogranin B and C. +RT indicates reverse transcripted RNA; -RT, RNA without reverse transcription. (B) Amplified microglia cDNA from 1-hour fibronectin adherent or 20 hours of amphoterin or fibronectin-adherent microglia was analyzed. Bands specific for chromogranin B and C were detected in agarose gel electrophoresis. (C) Amphoterin induced chromogranin B up-regulation. Optical density of the RT-PCR bands was determined. Optical density of bands from cells adhering for 1 hour on fibronectin was defined as 1. Chromogranin B mRNA was strongly up-regulated in monocytes during 20 hours of adhesion to amphoterin but not to fibronectin. In addition, chromogranin B was up-regulated in amphoterin-adherent microglia. Chromogranin C mRNA was not up-regulated during adhesion. Results of RT-PCR are from 3 different experiments using monocytes from 3 different donors and from 4 different experiments using rat microglia from 4 different littermates. Bars represent ± SD. Ari Rouhiainen et al. Blood 2004;104:1174-1182 ©2004 by American Society of Hematology

Monocyte transendothelial migration is inhibited by anti-amphoterin and anti-RAGE antibodies, and by soluble fragments of RAGE. (A) Western blotting of monocyte and HUVEC lysates with the anti-Atn used in the migration assay. Monocyte transendothelial migration is inhibited by anti-amphoterin and anti-RAGE antibodies, and by soluble fragments of RAGE. (A) Western blotting of monocyte and HUVEC lysates with the anti-Atn used in the migration assay. Cells were lysed in hot reducing Laemmli sample buffer, and the lysates were analyzed in Western blotting on 10% to 20% gradient gel. recAtn was used as a standard. Western blotting experiments revealed specifically a 30-kD band. (B) Transendothelial migration assay of monocytes. Monocytes were added to upper chambers of HUVEC-coated Transwell filters. Monocytes were allowed to migrate for 3 hours. Various concentrations of antibodies were added to the upper well at the start of the experiment where indicated. Migrated monocytes were counted microscopically. Noninhibited migration was defined as 100%. Bars represent ± SD (n = 3). (C) Monocyte transendothelial migration is inhibited by sRAGE isolated from bovine lung. Monocytes were added to the upper wells of the Transwell chambers with various concentrations of sRAGE. Noninhibited migration was defined as 100%. Migration assay was done as in Figure 5B. (± SD, n = 3). (D) Monocyte transendothelial migration is inhibited by recombinant sRAGE-Ig and the RAGE V1 domain. Monocytes were added to the upper wells of the Transwell chambers with 40 g/mL sRAGE-Ig, V1-Ig, or Fc-control (Ig). Noninhibited migration was defined as 100%. Migration assay was done as in Figure 5B (± SD, n = 8 for noninhibited, Ig, and sRAGE-Ig wells, and n=4 for V1-Ig wells). (E) Various concentrations of anti-RAGE antibodies (anti-P300 and anti-P301) were added to upper well at the start of the experiment where indicated. Nonimmune IgG was used as a control. Migration assay was done as in Figure 5B. Noninhibited migration was defined as 100% (± SD, n = 4, *P < .00005). Ari Rouhiainen et al. Blood 2004;104:1174-1182 ©2004 by American Society of Hematology