Mast cells and neutrophils proteolytically activate chemokine precursor CTAP-III and are subject to counterregulation by PF-4 through inhibition of chymase and cathepsin G by Florian Schiemann, Tobias Alexander Grimm, Josef Hoch, Roland Gross, Buko Lindner, Frank Petersen, Silvia Bulfone-Paus, and Ernst Brandt Blood Volume 107(6):2234-2242 March 15, 2006 ©2006 by American Society of Hematology

Processing of CTAP-III by human skin mast cells and neutrophils. Processing of CTAP-III by human skin mast cells and neutrophils. Increasing concentrations of human skin mast cells were incubated with cross-linking goat α-IgE antiserum (3 μg/mL) or left untreated for 30 minutes at 37°C (A). For comparison, increasing concentrations of human neutrophils were either incubated with fMLP or left untreated under the same conditions (B). Immediately following the stimulation period, cell samples were split and the original cell suspensions (SUSP) as well as the cell-free supernatants (SN) prepared thereof were incubated with 3 μM CTAP-III for 30 minutes (A-B). Thereafter, 2 μL cell-free supernatant of each sample and standard preparations of CTAP-III and NAP-2 (50 ng per lane) were separated by SDS-PAGE, subjected to Western blotting, and immunochemically stained to visualize CTAP-III and potential degradation products with rabbit antiserum Rα-βTG and an IRDye 800-conjugated goat α-rabbit antiserum. Data from 1 representative experiment of 3 are shown. Florian Schiemann et al. Blood 2006;107:2234-2242 ©2006 by American Society of Hematology

Time course of mast cell–mediated NAP-2 formation as detected by immunoreactivity and biologic activity. Time course of mast cell–mediated NAP-2 formation as detected by immunoreactivity and biologic activity. A fixed concentration (1 × 104/mL) of either α-IgE–stimulated (□) or unstimulated (▪) MCs was incubated with 3 μM CTAP-III for increasing periods of time. Thereafter, 2 μL cell-free supernatant of each sample and standard preparations of CTAP-III and NAP-2 (50 ng per lane) were separated by SDS-PAGE, subjected to Western blotting, and immunochemically stained with rabbit antiserum Rα-βTG to visualize CTAP-III and potential degradation products (A). Migration of standard CTAP-III (CT) and NAP-2 (NA) is indicated. Data from 1 representative experiment of 3 are shown. For quantification of NAP-2 biologic activity, CTAP-III and derivates were isolated from recovered cell-free supernatants by immunoaffinity chromatography, and the amount of NAP-2–like activity formed was assessed by its neutrophil-stimulating capacity as determined by the elastase release assay against a standard of purified NAP-2 (B). Shown are means ± SD of data obtained in 3 independent experiments. In the approach using α-IgE–stimulated MCs, the initial velocity (Vi) of NAP-2 formation was calculated as the increase in NAP-2 concentration per minute on the basis of the first time point at which significantly elevated NAP-2-levels could be detected. Florian Schiemann et al. Blood 2006;107:2234-2242 ©2006 by American Society of Hematology

Inhibition of mast cell–and mast cell protease–mediated CTAP-III conversion. Inhibition of mast cell–and mast cell protease–mediated CTAP-III conversion. Increasing concentrations of purified natural neutrophil CathG and mast cell chymase were incubated with a fixed concentration of 3 μM CTAP-III for 30 minutes at 37°C in processing buffer. Subsequently, 2 μL of each sample was subjected to SDS-PAGE and Western blotting and thereafter stained with Rα-βTG and IRDye 800-conjugated goat α-rabbit antiserum. The amount of generated NAP-2 is shown as assessed by Li-cor quantification against a standard of NAP-2 run in parallel. Data represent mean ± SD from 3 independent experiments (A). Likewise, fixed concentrations of CathG (500 ng/mL) (B) and chymase (250 ng/mL) (C) were incubated with 3 μM CTAP-III under the same conditions in the absence or presence of inhibitors SBTI (1 μg/mL), aprotinin (1 μg/mL), or PMSF (2 mM) for 30 minutes. Correspondingly, human skin MCs (1 × 104/mL) prestimulated with 3 μg/mL α-IgE for 30 minutes were incubated with 3 μM CTAP-III in the absence or presence of inhibitors as indicated above (D). The Western blots shown in panels B-D were performed and developed using the primary and secondary antisera given in Figure 1. The migration of untreated CTAP-III (CT) and NAP-2 (NA) is indicated. For quantification of formed NAP-2, fluorescence of NAP-2-bands was determined by Li-cor analysis and compared with that of 50 ng standardized NAP-2 on the same blot. One representative experiment of 3 is shown. Florian Schiemann et al. Blood 2006;107:2234-2242 ©2006 by American Society of Hematology

Inhibitory impact of PF-4 on CTAP-III processing by mast cells and neutrophils. Inhibitory impact of PF-4 on CTAP-III processing by mast cells and neutrophils. Human skin MCs (1 × 104/mL) prestimulated with α-IgE (A) and human neutrophils (5 × 106/mL) (C) were incubated with 3 μM CTAP-III in the absence and in the presence of increasing dosages of PF-4 for 30 minutes at 37°C. For time-course studies, prestimulated MCs (B) and neutrophils (D) were incubated for increasing times with either 3 μM CTAP-III alone (▪) or in the presence of 4 μM PF-4 (□). To determine the amount of generated NAP-2, 2-μL samples of cell-free supernatants were subjected to SDS-PAGE, Western blotting, and immunochemical staining with rabbit antiserum Rα-βTG. NAP-2 was then quantified by Li-cor analysis against a standard of NAP-2 run in parallel (A-B). In panel A, results are given as the percentage of NAP-2 formed in the absence of PF-4. Data represent mean ± SD from 3 independent experiments with cells from different donors. Florian Schiemann et al. Blood 2006;107:2234-2242 ©2006 by American Society of Hematology

Effect of PF-4 on NAP-2 formation by purified chymotryptic proteases. Effect of PF-4 on NAP-2 formation by purified chymotryptic proteases. Purified human neutrophil cathepsin G (500 ng/mL), human mast cell chymase (250 ng/mL), or bovine pancreas chymotrypsin (200 ng/mL) was incubated with 3 μM CTAP-III alone or in the presence of 4 μM PF-4 for 30 minutes at 37°C. Thereafter, 2μL of each sample was subjected to SDS-PAGE and Western blotting and subsequently stained in sequence with Rα-βTG and IRDye 800–conjugated goat α-rabbit antiserum. NAP-2 was then quantified by Li-cor analysis against a standard of NAP-2 run in parallel. Data represent mean ± SD from 3 independent experiments. Florian Schiemann et al. Blood 2006;107:2234-2242 ©2006 by American Society of Hematology

Purified chymase and CathG cleave CTAP-III but not PF-4. Purified chymase and CathG cleave CTAP-III but not PF-4. Human chymase (Chy; 250 ng/mL) purified from mast cells (left panels) or human CathG (500 ng/mL) purified from neutrophil granulocytes (right panels) were incubated with either 3 μM CTAP-III (top row) or 4 μM PF-4 (bottom row) in processing buffer for 0 minutes (solid line) or 120 minutes (broken line) at 37°C. The products were separated, detected by their absorbance at 214 nm, and fractionated by reverse-phase HPLC using a linear gradient of acetonitrile in 0.1% TFA (dashed line) as described in “Materials and methods.” Fractions containing protein peaks eluting at retention times of 23.0 minutes, 22.2 minutes, 16.6 minutes, and 25.4 minutes were analyzed by ESI-FT-MS and identified by their respective mass of 9286 amu, 7623 amu, 1682 amu, and 7764 amu as CTAP-III, NAP-2, CTAP-III[1-15], and PF-4, respectively. One representative experiment of 3 is shown. Florian Schiemann et al. Blood 2006;107:2234-2242 ©2006 by American Society of Hematology

Impact of PF-4 on the cleavage of SP by purified chymase and CathG. Impact of PF-4 on the cleavage of SP by purified chymase and CathG. SP (10 μM) was incubated with the same enzyme concentrations as used previously for CTAP-III processing of either 250 ng/mL chymase (left panels) or 500 ng/mL CathG (right panels) for 0, 30, and 120 minutes in the absence (solid line) and in the presence (broken line) of PF-4 (4 μM) in processing buffer at 37°C. SP and its cleavage products were separated by reverse-phase HPLC using a linear acetonitrile gradient in 0.1% TFA (dashed line). Peptides as detected by their absorbance at 214 nm at retention times of 20.4 minutes, 13.7 minutes, 15.4 minutes, and 17.1 minutes were analyzed by ESI-FT-MS and identified by their respective mass of 1363 amu, 901 amu, 466 amu, and 1048 amu as full-size SP (SP[1-11], calculated mass of oxidized form, 1363 amu) and fragments SP[1-7] (901 amu), SP[8-11] (467 amu), and SP[1-8] (1046 amu). One representative experiment of 3 is shown. Florian Schiemann et al. Blood 2006;107:2234-2242 ©2006 by American Society of Hematology