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Originally published online as doi:10.1189/jlb.0404261 on August 17, 2004

Published online before print August 17, 2004
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(Journal of Leukocyte Biology. 2004;76:1010-1018.)
© 2004 by Society for Leukocyte Biology

Induction and antimicrobial activity of platelet basic protein derivatives in human monocytes

Andreas Schaffner*,{dagger},1, Charles C. King{ddagger}, Dominik Schaer{dagger} and Donald G. Guiney*

* Division of Infectious Diseases and
{ddagger} Department of Pediatrics, University of California San Diego; and
{dagger} Research Unit Med Klinik B, Department of Medicine,University of Zürich, Switzerland

1 Correspondence: Medizinische Klinik B, Universitätsspital AW 9, Rämistrasse 100, CH-8091 Zürich, Switzerland. E-mail.klinsar{at}usz.unizh.ch


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The antimicrobial activity of a number of chemokines has recently come into focus of research about innate immunity. We have previously shown that platelet basic protein (PBP), which gives rise to several antimicrobial peptides of platelets, is also expressed in human monocytes. In the present studies, we show that exposure of human monocytes to bacteria or microbial components (lipopolysaccharide and zymosan) induces a several-fold greater expression of derivates of PBP. Also, activation of proteinase-activated receptors (PARs) by thrombin or the synthetic peptide ligand SFLLRN of PAR-1 significantly increased PBP expression, presumably on the transcriptional level, as evidenced by higher mRNA levels. Derivates of PBP appeared to reach phago-lysosomes, as higher concentration was found in latex phagosomes isolated by a flotation method. By the gel-overlay technique, two bactericidal derivatives of PBP could be visualized, which were immunoreactive with anti-PBP antibody in Western blots. By matrix-assisted laser desorption/ionization time of flight and surface-enhanced laser desorption and ionization techniques, it was confirmed that the bands corresponded to PBP derivates. After immunofixation with a monoclonal antibody to PBP, the major peptide in zymosan-stimulated monocytes was identified to correspond by molecular weight to connective tissue-activating peptide III, which has been reported to be a major antimicrobial PBP derivate also in platelets. Our observations indicate that PBP and its derivates are constituents of the antimicrobial arsenal of human monocytes. Their increased expression after exposure to microorganisms allows a rapid host response to pathogens.

Key Words: macrophages • antimicrobial cationic peptides • ß-thromboglobulin • chemokines • receptors • proteinase-activated


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Antimicrobial peptides are phylogenetically ancient constituents of host defense and are expressed by immune and nonimmune cells of invertebrates and vertebrates [1 2 3 4 5 6 7 8 9 10 11 12 13 14 ]. Many classes of antimicrobial peptides have been identified [1 , 4 , 7 , 10 , 11 , 15 16 17 18 19 ]. Evidence for their importance in the network of host defense mechanisms is more or less indirect and based on their broad distribution in multicellular organisms and their diversity.

Leukocytes contribute many different species of antimicrobial peptides such as {alpha}-defensins, ß-defensins, lactoferrin, lysozyme, cathelicidins, and chemokines [20 21 22 23 24 25 26 ]. In search of novel, nonoxidative, antimicrobial systems of human mononuclear phagocytes, we have recently shown that monocytes express constitutively important amounts of platelet basic protein (PBP), a peptide that belongs to the CXC chemokine family and has potentially dual functions as signaling and as an antimicrobial effector molecule [12 , 13 , 27 , 28 ]. The functions of PBP depend on its derivatization, which is the result of limited proteolytic digestion [29 30 31 ]. PBP was previously believed to be expressed selectively only in the megakaryocyte cell lineage [32 ]. As PBP derivatives have not been shown to be secreted by monocytes and as the quantity of PBP was impressively high in human monocytes, we postulated that PBP has an intracellular function in monocytes, possibly as an antimicrobial peptide [26 ].

In differential display studies, we discovered that dexamethasone down-regulates a quantitatively important expression at a transcriptional level in human monocytes in vitro and in vivo. Further observations indicated that interferon-{gamma} (IFN-{gamma}) and short-term exposure to lipopolysaccharide (LPS) fail to increase expression of PBP [26 ]. In chickens, expression of a phylogenetically related CXC chemokine 9E3/cCAF [17 ] is induced by thrombin [33 , 34 ] through proteinase-activated receptors (PARs), which are also expressed on human monocytes [35 ].

In the present studies, we explored the effects of exposure of monocytes and macrophages to microorganisms, to thrombin, the PAR agonist SFLLRN, as well as other stimuli on the expression of PBP in monocytes and in in vitro-differentiated macrophages. Furthermore, we studied the antimicrobial activity of PBP derivatives from human monocytes and macrophages. Finally, we identified one of the antimicrobially active derivatives produced in monocytes and preliminarily studied the deposition of PBP derivatives in phago-lysosomes.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cells: monocytes
Human mononuclear phagocytes were isolated from heparinized blood (100 U/ml) as described [36 ]. In brief, after separation by Ficoll gradient (Ficoll-paque, Pharmacia Biotech Europe, Switzerland), three washes in Gey’s balanced salt solution (GBSS; Sigma Chemical Co., St. Louis, MO) mononuclear cells were suspended in Dulbecco’s modified Eagle’s medium (DMEM; Mediatech Cellgro, Herndon, VA), supplemented with 20% autologous fresh-frozen serum off the clot (complete DMEM) and seeded onto baked (220°C, 4 h), endotoxin-free, sterile, 90 mm-diameter, glass tissue-culture plates at a density of 3–4 x 107 cells per plate as described [26 ] or 100 µl of a suspension of 2 x 108 cells/ml on 12 mm glass coverslips in 24-cluster wells. In experiments without bacteria, DMEM was supplemented with 10 µg/ml gentamycin. Monocytes were obtained by glass adherence after 2 h at 37°C, 5% CO2, 98% humidity, and vigorous washing four times in warmed GBSS with a purity of >98%, as determined by Giemsa staining. Monocytes were incubated for 48 h prior to studies or for 8–10 days for in vitro differentiation into macrophages. Medium was changed after 24 h and in experiments with longer incubation times, every 72 h thereafter.

THP-1 cells
THP-1 cells from American Type Culture Collection (Manassas, VA) were stored in aliquots of a third passage in liquid nitrogen and cultured in RPMI (Mediatech Cellgro) supplemented with 10% fetal calf serum (Mediatech Cellgro) and used for experiments up to their 15th passage. For in vitro differentiation THP-1 cells were cultured for 18 h in the presence of 20 ng/ml phorbol myristate acetate (PMA; Sigma Chemical Co.).

Platelets
Platelets were isolated from blood drawn from the same donors into 10 mM sodium citrate. Blood was diluted 1:1 with Ca++-free phosphate-buffered saline (PBS) containing bovine serum albumin (BSA; Sigma Chemical Co.) and centrifuged for 7 min at 200 g at room temperature. The thrombocyte-enriched plasma was collected and supplemented with EDTA to a final concentration of 10 mM and centrifuged for 10 min at 500 g at room temperature. Supernatant was discarded, and the platelets in the pellet were lysed directly as described below. Platelet purity was almost 100%.

Bacteria
Salmonella enterica serovar Typhimurium strains used in this study consisted of the wild-type 14028 s [37 ], a virulent derivative expressing green fluorescent protein (GFP; 14028 s rpsM::gfp, kindly provided by Stanley Falkow, Stanford University, CA; ref. [38 ]) and an attenuated phoP mutant (14028 s phoP::Tn10; ref. [39 ]). Bacteria were cultured in Luria-Bertani broth. For cell infections, overnight cultures were washed two times in PBS and adjusted to a concentration of 107 cells/ml, followed by opsonization for 30 min in 50% normal human serum. In some experiments, bacterial cells were fixed for 2 h at 37ºC in 2.5% paraformaldehyde in PBS and then washed extensively in large volumes of PBS prior to use.

Reagents
PMA, SFLLRN (14 amino acid peptide), human thrombin, 2-morpholinoethanesulfonic acid monohydrate (MES), imidazole, 1,2-dioctanoyl-sn-glycerol (DOG), amastatin, calcium ionophore A23187, zymosan, 0.8 µm latex particles, GBSS, and urea were from Sigma Chemical Co. Zymosan was boiled three times in PBS, pH 7.4, for 10 min, washed in an additional change of PBS prior to use. Seakem GTG agarose was from BioWhittaker Molecular Applications (Rockland, ME). Recombinant hirudin (leptirudin) was from Hoechst (Kansas City, MO). Protease inhibitor cocktail without EDTA was from Roche Diagnostics (Rotkreuz, Switzerland). Acrylamid/bis-acrylamide 29:1 was from National Diagnostics (Atlanta, GA).

Isolation of phagosomes
Phagosomes containing latex particles were isolated according to the flotation method of Desjardins et al. [40 , 41 ]. In brief, monocytes were grown on 10 cm glass dishes in 10 ml complete DMEM, as described above, prior to challenge with a 10% suspension of latex particles, diluted 1:400 in complete medium and passaged several times through a 21-gauge needle to disrupt clumps. Cultures were washed after 45 min of incubation for phagocytosis and washed three times with prewarmed GBSS, and then incubated for a further 2 h for the formation of mature phago-lysosomes. Cells were then washed again twice with GBSS, then once in ice-cold homogenization buffer (250 mM sucrose, 3 mM imidazole, pH 7.2, protease inhibitor cocktail), and then scraped in 500 µl ice-cold homogenization buffer per dish. Monocytes from two dishes were homogenized in a motor-driven Dounce homogenizer on melting ice, unbroken cells were pelleted at 1200 rpm for 5 min at 4°C, and the supernatant containing the phagosomes was brought to a sucrose concentration of ~40% by adding an equal volume of 62% sucrose supplemented with protease inhibitor cocktail and 3 mM imidazole buffer. The phagosome-containing fraction was loaded on a 62% sucrose cushion and overlayered with 35%, 20%, and 10% sucrose with buffer and protease inhibitor prior to centrifugation at 100,000 g for 60 min in a SW40 rotor. The band with phagosomes, located between the 10% and 20% sucrose, was collected and pelleted at 45,000 g in 50 ml PBS with protease inhibitor cocktail at 4°C for 20 min and resuspended in distilled water with protease inhibitor prior to measurement of the protein content and Western blot analyses.

Antibodies
An antigen affinity-purified, polyclonal rabbit anti-human neutrophil-activating peptide-2 (NAP-2) antibody (Peprotech, Rocky Hill, NJ) and a monoclonal mouse anti-human NAP-2 antibody (Peprotech) were used throughout the studies. Both antibodies react with all derivatives of PBP as described previously [26 ]. For immunofluorescence studies, secondary antibodies were Alexa Fluor® 568 goat anti-mouse immunoglobulin G (IgG; H+L) and Alexa Fluor® 568 goat anti-rabbit IgG (H+L; Molecular Probes, Eugene, OR).

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis
For protein analysis, cells of one 90 mm glass tissue-culture plate were scraped in 1 ml PBS supplemented with protease inhibitor cocktail on ice. Lysis of cells was performed by four cycles of rapid freezing in liquid nitrogen and thawing. After centrifugation at 16,000 rpm in an Eppendorf microfuge, the supernatant was collected and used for SDS-PAGE on 15% Tris/Tricine mini-gels under reducing conditions (Gradipore, French Forest, Australia) according to standard protocols. After blotting onto 0.2 µm polyvinylidene difluoride (PVDF; BioRad, Hercules, CA), membranes were incubated with antigen affinity-purified, polyclonal rabbit anti-human NAP-2 antibody (Peprotech). Blots were developed with an enhanced chemiluminescence Western blotting detection system (Amersham Bioscience, Sunnyvale, CA).

Acid urea gels and Western blotting
For native protein electrophoresis of cationic peptides, cells were scraped into 1000 µl ice-cold, 5% acetic acid supplemented with protease inhibitor and extracted by stirring with magnetic bars overnight on melting ice in 1.5 ml tubes. Native protein electrophoresis was performed in 12% mini-urea gels according to the protocol of Lehrer et al. [42 ] on a Miniprotean II cell (BioRad) using 5% acetic acid as running buffer at a constant power of 150 V, resulting in a currant of ~35 mA per gel. Transfer on PVDF was accomplished in 0.7% acetic acid with a constant currant of 250 mA for 60 min in a Miniprotean II cell at 4°C. Membranes were washed in distilled water, equilibrated with a large volume of PBS, and developed as described above.

Killing assay
Gel overlay killing assays were performed by a modified method originally described by Lehrer et al. [42 ]. Per lane, 40 µg proteins in 5% acetic acid and 3 M urea were electrophoresed in 12% urea gels as described above. Gels were cut into three parts for parallel studies with Coomassie stain, Western blots, and overlay killing assays. Parts used in killing assays were equilibrated for 4 x 5 min in ~50 mL MES buffer, pH 6.0, in large petri dishes on a rotatory shaker to remove urea and acetic acid. Bacteria were grown to mid-log phase at 37°C in shaking Luria broth for 2.5–3 h, pelleted at room temperature, and resuspended in MES buffer, pH 6.0, supplemented with 3 gr/L D-glucose. Bacteria (2x108) were mixed with 20 ml 1.5% low endosmosis agarose in MES–glucose, pH 6.0, held at 42°C and poured immediately in thin layers in petri dishes. Equilibrated gels were overlayed for passive transfer of proteins and peptides on the agarose-containing bacteria and incubated for 3 h at 37°C in a humid chamber. After removal of gels, bacteria were overlayed with double-strength Luria broth supplemented with 1.5% agar noble (Difco, Detroit, MI) and incubated at 37°C overnight. Thereafter, plates were overlayed with 2.5% w/v paraformaldehyde in PBS, pH 7.4, for 30 min at 37°C, the feeding layer was removed, and agarose gels were stained with diluted Coomassie stain.

Immunofluorescence
For immunofluorescence, monocytes were cultured for 2 or 10 days on round 12 mm glass coverslips, fixed with 2.5% paraformaldehyde in PBS, pH 7.4, for 10 min at 37°C and after three washes with PBS, pH 7.3, permeabilized for 15 min at room temperature with 0.1% Triton X-100 (Sigma Chemical Co.) in PBS. Nonspecific sites were blocked for 1 h at room temperature with 10% goat serum in PBS supplemented with 0.1% saponin (GS)–PBS prior to incubation with the primary antibody (1 µg/ml in GS–PBS) for 1 h at room temperature followed by three washes in PBS with 5% BSA (Sigma Chemical Co.) and incubation with the secondary antibody [2 µg/ml in GS–PBS, Alexa Fluor® 568 goat anti-mouse IgG (H+L), Molecular Probes]. Coverslips were mounted with Vectashield mounting medium (Vector Laboratories, Burlingham, CA) and read on a Nikon Eclipse E 400 fluorescence microscope, using an excitation wavelength of 568 nm and read at 603 nm for enumeration of the fraction of positive cells.

Real-time reverse transcriptase-polymerase chain reaction (RT-PCR) for measurement of mRNA levels of monocytes
Total cellular RNA of monocyte cultures was isolated with the Qiagen RNAeasy mini kit (Qiagen, Basel, Switzerland) according to the manufacturer’s instructions. All RNA samples were treated with DNase I (Qiagen), and equal amounts of total RNA (4 µg total DNA-free RNA of each cellular preparation) were reverse-transcribed to cellular, total cDNA using Stratagene ProSTAR first strand synthesis kit (Stratagene, Amsterdam, Netherlands) according to the manufacturer’s instructions. The cDNA samples were amplified in the LightCycler real-time PCR system using the FastStart DNA Master SYBR green I kit (Roche Applied Science Technical Note Nos. LC 11/2000 and LC 10/200, Roche Diagnostics). To analyze transcription of pro-platelet basic protein (PPBP) in human monocytes and other human blood cells, sequence-specific primers and temperature cycling profiles were used as described [26 ]. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a housekeeping gene to correct for small differences in cDNA content. Equal amounts of total RNA/cDNA were processed in all experiments.

Matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry
Peptide masses were determined by MALDI-TOF mass spectrometry. Coomassie-stained peptides were excised, washed three times in 50% acetonitrile/50% NH4HCO3, and lyophilized. Peptides were subjected to in-gel reduction with 10 mM dithiothreitol at 56°C for 1 h and alkylation with 100 mM iodoacetic acid for 30 min at room temperature, followed by digestion with 0.5 µg trypsin (Roche Diagnostics). Isolated peptides were washed and concentrated in C18 ZipTips (Millipore, Bedford, MA) according to the manufacturer’s protocol, mixed with 4-{alpha} hydroxy cinnamic acid matrix, and spotted onto a platform. MALDI-TOF was performed on an Applied Biosystems DE STR mass spectrometer (Applied Biosystems, Foster City, CA). Each sample was spiked with an internal control for angiotension and renin tetradecapeptide. Data were analyzed using the Paws program (http://prowl.rockefeller.edu).

Surface-enhanced laser desorption/ionization (SELDI)-TOF
Peptides were also analyzed by SELDI. A PS10 protein chip was incubated with 1 µl monoclonal NAP-2 antibody (500 µg/ml) in PBS at 4°C for 18 h. Residual sites were blocked by washing the protein chip with 0.5 M ethanolamine, pH 8.0, for 15 min followed by a second 15-min wash in PBS containing 0.5% Triton X-100 (CalBiochem, La Jolla, CA). The PS20 chip was transferred to a Ciphergen bioprocessor (Ciphergen Biosystems, Fremont, CA), and an equal volume of sample (adjusted to 0.5 mg protein/ml) was mixed with PBS containing 1% BSA and 0.1% Triton X-100 and incubated at 4°C for 8 h. The sample was removed, and the chip was washed three times with PBS containing 0.5% Triton X-100, three times with PBS alone, and three times with Milli-Q H2O. The chip was allowed to air dry and then was spotted twice with 0.5 µl of a 10% dilution of saturated 4-{alpha} hydroxy cinnamic acid. The samples were analyzed in a PBS-II protein chip array reader (Ciphergen Biosystems) in positive ion mode with laser intensity of 205–210 µJ. Data were analyzed using the Ciphergen Biosystems ProteinChip software 3.0.2.

Statistics
Mean ± SD are given. For comparison of discrete values, t-test was applied; for discontinuous values, Man-U Whitney test using Instat® 3.0 (Graphpad Inc., San Diego, CA) was used. Where appropriate, Dunnett’s correction for comparison of multiple samples with one control was applied.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Induction of expression of PBP in monocytes and macrophages
The amount of PBP expressed by monocytes was studied by immunofluorescence and in some experiments, in parallel by Western blots of serial dilutions of cell lysates. Overnight exposure to LPS, bacteria, or zymosan of monocytes cultured in vitro for 2 days resulted in an increase of the percentage of cells staining with a monoclonal antibody (mAb) to PBP and its derivatives. In several experiments, we found that it was not important for stimulation by bacteria whether monocytes were challenged with wild-type Salmonella typhimurium or the attenuated mutant PhoP, which has less propensity to inhibit phago-lysosomal fusion [43 ], or even Salmonella fixed in 2.5% paraformaldehyde (not shown). Activation by PMA, 1,2-dioctanoyl-sn-glycerol, and the calcium ionophore A23187 had no effects on monocytes (Fig. 1 ). Next, we searched for an endogenous signal augmenting PBP expression. As IFN-{gamma} did not induce PBP expression in our previous experiments [26 ], and as in chicken fibroblasts, thrombin induces expression of the phylogenetically related CXC chemokine 9E3/cCAF [34 ], we studied in search of an endogenous signal that up-regulates PBP expression the effects of thrombin. The serine proteinase thrombin, by cutting the tethered autochthonous ligands of PARs, results in signal transduction. Similarly, synthetic peptides corresponding to the amino acid sequence of the natural ligands results in activation of PARs [44 ]. Thrombin as well as SFLLRN, a synthetic ligand of PARs, caused an increase in PBP expression, and the effect of thrombin was partially antagonized by recombinant hirudin (Fig. 2 ). Comparably to the studies with microbial components and bacteria, not only the fraction of positive cells increased but also the intensity of immunostains in the cells, in particular in the perinuclear area corresponding to the Golgi zone (Fig. 3C 3F and 3G ). Western blots confirmed a four- to ninefold up-regulation of protein expression after a challenge with Salmonella as well as stimulation by LPS or PAR activation by thrombin or SFLLRN (Figs. 1 and 2) . In chicken fibroblasts, PAR activation results in regulation of the expression of 9E3/cCAF through G-protein coupling and the Elk-2 pathway and regulates expression at a transcriptional level [45 , 46 ]. In accordance with this observation, levels of PBP–mRNA measured by RT-PCR were increased in the same order of magnitude as were protein levels in Western blots (Fig. 4 ).



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  		Figure 1. Studies regarding the induction of expression of PBP in monocytes by immunofluorescence and Western blot analyses by bacteria and microbial components calcium ionophore and PMA. Primary human monocytes cultured on coverslips for 24 h were treated for 24 h with various stimuli prior to immunostain with a mAb to human PBP and enumeration of the number of PBP-positive cells. Control, Control cells without stimuli; Zymosan, ~106 particles/ml; DOG, 5 µM; PMA, 20 ng/mL; Ionophor, calcium ionophore A23187, 2 µg/ml; Salmonella, 2 x 105 bacteria/mL; LPS, 100 ng/mL. Mean ± SEM from five experiments with three independent wells per experiment, and 1 ≥ 100 enumerated cells per well. P < 0.02 for the comparisons of control with zymosan, Salmonella, and LPS. (A and B) Two Western blots with polyclonal anti-PBP antibody from two independent experiments done in parallel with the immunofluorescence studies. Lanes 1a–c, Cell lysates from monocytes stimulated with 105 S. typhimurium/ml. Lanes 2a–c, Cell lysates from cells stimulated with 100 ng/ml of LPS. Lanes 3a–c, Cell lysates from unstimulated control cells. Lanes a–c were 1:4 dilutions of 1 µg protein per lane. Note at least a fourfold induction of expression of PBP in both experiments.

 


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  		Figure 2. Studies regarding the induction of expression of PBP in monocytes by immunofluorescence and Western blot analyses by agents stimulating proteinase-activated receptors. Primary human monocytes cultured on coverslips for 24 h were treated for the indicated times with thrombin or the synthetic PAR receptor-activating peptide SFLLRN prior to immunostain with a mAb to human PBP and enumeration of the number of PBP-positive cells. Control, Unstimulated control cells; Thrombin, human thrombin 13.8 U/ml for 24 h; Thrombin + Hirudin, 13.8 U/ml thrombin and 15 U/ml of the thrombin antagonist hirudin for 24 h; SFLLRN 4 hours, 100 µM SFLLRN for 4 h; SFLLRN, 100 µM SFLLRN for 24 h; Hirudin, 15 U/ml hirudin for 24 h. Mean ± SEM from triplicate experiments each with three independent wells per experiment and ≥100 enumerated cells per well. Control versus thrombin and versus SFLLRN < 0.01; thrombin versus thrombin + hirudin, P = 0.03. (A and B) Two Western blots with polyclonal anti-PBP antibody from two independent experiments done in parallel with the immunofluorescence studies. Lanes 1a–c, Cell lysates from control cells. Lanes 2a–d, Cell lysates from cells stimulated with 13.8 U/ml human thrombin. Lanes 3a–d, Cell lysates from cells stimulated with 100 µM SFLLRN. Lanes a–d were 1:3 dilutions of 1 µg protein per lane. Note an approximate ninefold induction of expression of PBP in both experiments.

 


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  		Figure 3. Immunfluorescence staining of PBP in monocytes and in vitro-differentiated, blood-derived macrophages. Red, Positive stain with a monoclonal mouse antibody to human PBP. Blue, 6-Diamidine-2'-phenylindole dihydrochloride stain for nuclei. (A) Unstimulated control monocytes. (B) Stimulation of monocytes for 24 h with 100 µM SFLLRN (x200). (C) Intense granular and patchy perinuclear fluorescence for PBP after stimulation with SFLLRN in monocytes cultured in vitro for 24 h (x1200). (D) Monocyte 24 h after ingestion of zymosan particles with a green autofluorescent center and a dark outer zone; in this preparation, the primary antibody to PBP but not the 2°C antibody was omitted. (E) Monocytes 24 h after ingestion of autofluorescent zymosan particles stained for PBP. Note the intimate contact between PBP staining and the phagocytosed particles (x1200). (F–K) Photomicrographs of the same in vitro-differentiated macrophage 24 h after phagocytosis of paraformaldehyde-fixed S. typhimurium expressing GFP. (F–H) Most PBP activity appears in a perinuclear zone corresponding to the hypogranular Golgi zone in the phase-contrast picture. (I) Green fluorescent bacteria. (K) Bacteria lodge in a zone that stains densely for PBP.

 


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  		Figure 4. Results of quantitative PBP–mRNA analysis by real-time PCR with and without stimulation with 200 µM SFLLRN. Human monocytes cultured for 48 h in vitro were stimulated 8 h prior to harvest of mRNA with SFLLRN or cultured without the stimulus. Levels of mRNA were measured by RT-PCR after reverse transcription to cDNA, and the small differences in cDNA were corrected by parallel measurements of the housekeeping gene GAPDH. Note the fivefold increase of mRNA levels after stimulation with SFLLRN. P < 0.01.

 
The pattern of baseline expression and response to stimuli differed to some extent in monocytes differentiated in vitro for 10 days prior to stimulation. Baseline expression as assessed by immunofluorescence was lower with a smaller fraction of positive cells, LPS had a lower stimulatory effect, and PMA increased the number of positive cells and the intensity of staining. Again, a challenge with zymosan or Salmonella induced expression as it did in monocytes (Fig. 5 ).



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  		Figure 5. Studies regarding the induction of expression of PBP in in vitro-differentiated, blood-derived macrophages by immunofluorescence. Primary human monocytes were cultured for 9 days in 20% autologous serum for differentiation into macrophages prior to stimulation for 24 h with various agents followed by immunostain with a mAb to human PBP and enumeration of the number of PBP-positive cells. Control, Unstimulated control cells; Salmonella, 2 x 105 bacteria/mL; LPS, 100 ng/mL; Zymosan, ~106 particles/ml; PMA, 20 ng/mL; Thrombin, human thrombin, 13.8 U/ml for 24 h; SFLLRN, 100 µM SFLLRN for 24 h; Thrombin + Hirudin, 13.8 U/ml thrombin and 15 U/ml thrombin antagonist hirudin for 24 h; Hirudin, 15 U/ml hirudin for 24 h. Mean ± SEM from triplicate experiments each with three independent wells per experiment and ≥100 enumerated cells per well. P < 0.05 for the comparison of stimulation with Salmonella, zymosan, PMA, thrombin, and SFLLRN with control cells as well as thrombin with thrombin + hirudin.

 
The human monoblast cell line THP-1, which proliferates in suspension, did not express PBP at a level detectable by immunostains. Induction of differentiation by exposure to 20 ng/ml PMA for 24 h resulted in glass-adherent, macrophage-like cells with an intense staining for PBP in >80% of cells (not shown), indicating that similar PMA effects on cell differentiation might occur during proliferation and differentiation of monocytes in vitro and helping to understand the different response of primary monocytes and differentiating macrophages (Figs. 1 and 5) .

Next, we turned to the question of whether PBP or its derivatives could reach the phago-lysosomal compartment. Immunostains of monocytes that had ingested zymosan particles or paraformaldehyde-fixed Salmonella showed an intimate proximity between stainable PBP and autofluorescent zymosan particles or bacteria (Fig. 3E 3F 3G 3H 3I 3J 3K) . When we purified phago-lysosomes after challenge of monocytes with 0.8 µM diameter latex beads by the flotation technique described by Desjardins et al. [40 , 41 ], we observed a higher fraction of PBP in the phago-lysosomal protein preparations compared with the fraction in total cell lysates (Fig. 6 ), even so phagocytosis of latex beads by itself did not increase PBP expression in immunofluorescence studies (not shown). It is of note that in such preparations, no contamination with cytoplasmatic proteins or organelles occurs [40 ] and that washing of the isolated phago-lysosomal fraction in 50 vol PBS did not decrease PBP.



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  		Figure 6. Western blot analysis of the phago-lysosomal fraction of monocytes after phagocytosis of latex particles and of unfractionated cells for PBP. Monocytes cultured for 36 h were challenged with 0.8 µ latex particles, washed, and incubated for maturation of phago-lysosomes, which were isolated after disruption of cells by flotation centrifugation. Lane 1, Lysate of unfractionated cells after disruption, 10 µg protein/lane. Lane 2, Lysate of unfractionated cells after disruption, 0.5 µg protein/lane. Lane 3, Phago-lysosomal fraction after washing in 50 vol PBS, 0.5 µg protein/lane. Lane 4, Phago-lysosomal fraction prior to washing in PBS, 0.5 µg protein/lane. Lane 5, Latex particles incubated for 30 min at 37°C in disrupted cells prior to flotation centrifugation, 0.5 µg protein/lane. Tric/tricine gel (15%) immunodetection with a polyclonal rabbit anti-PBP antibody. Note that the fraction of PBP is increased in phago-lysosomes when comparing lanes 2 and 3. Washing of phago-lysosomes in large volumes of PBS does not affect the amount of PBP in the preparation (lanes 3 and 4), and "dummy" latex particles do not bind PBP when incubated in cell lysates.

 
To confirm a postulated, antimicrobial function of PBP derivatives from monocytes, we studied antimicrobial activities of electrophoresed acid extracts of native proteins by the overlay technique originally described by Lehrer et al. [42 ] and correlated bands resulting in bacterial killing with Coomassie stains and Western blots. For this purpose, we used as indicator strain the attenuated mutant of S. typhimurium PhoP, which might be more susceptible to cationic antimicrobial peptides such as PBP [47 ] (Fig. 7 ). At least six distinct bands of bacterial clearing could be identified by this method. Two of these bands reacted in Western blots done on the same gels with anti-PBP antibodies (Fig. 8 ). An additional band, reactive in Western blots, provoked no clearing of bacteria under the conditions used in these studies. In contrast to the two other bands with bacterial killing, we also found no correlate in Coomassie stains (Fig. 8) , indicating a low quantity for this derivative, which was possibly insufficient for killing of bacteria. The PBP derivative, which was less mobile in the acid urea gel, corresponded in regard to mobility, reactivity with anti-PBP antibody, and antimicrobial activity to a major peptide band in electrophoresed acid extracts from platelets (Fig. 8) and PMA-differentiated THP-1 cells (Fig. 9 ). Extracts from THP-1 cells did not display four of the other bands that induced a zone of bacterial clearing with monocyte extracts, including the more mobile derivative of PBP.



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  		Figure 7. Gel overlay killing assay with electrophoresed acid extracts from monocytes and platelets. Monocytes cultured for 2 days in vitro were stimulated 24 h prior to protein harvest with zymosan particles. Acid extracts of monocytes (M; right lane) and platelets from the same donor (P; left lane) with 60 µg native protein were electrophoresed, and the gels were overlayed for passive transfer of peptides on agarose with 107 S. typhimurium PhoP for 3 h prior to removal of the gel, feeding, and incubation of the agarose for bacterial growth followed by fixation and Coomassie stain (for details, see Materials and Methods). Transfer of peptides caused killing of bacteria in five distinct bands with peptides extracted from monocytes and three bands with peptides extracted from platelets, all of the same mobility. An additional band with low mobility in both preparations is faint and blurry. The position of the removed gel is marked with black ink.

 


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  		Figure 8. Parallel analysis of Western blots, Coomassie stain, and bacterial killing of acid protein extracts from monocytes and platelets. All analyses of proteins were done on the same acid urea gel. Protein (20 µg) was applied to each lane. W, Western blot developed with a polyclonal rabbit antibody to PBP; C, Coomassie stain; K, bacterial killing in the gel overlay killing assay. A prominent band detected by anti-PBP antibody appears in Western blots of platelet and monocyte peptides visible in Coomassie stains and killing S. typhimurium PhoP. In monocytes, two additional, more mobile peptide bands result in bacterial killing, and the upper band is visible in the Coomassie stain and reacts with the anti-PBP antibody.

 


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  		Figure 9. Parallel analysis of Western blots, Coomassie stain, and bacterial killing of acid protein extracts from THP-1 cells differentiated with PMA and zymosan-stimulated monocytes. All analyses of proteins were done on the same acid urea gel. Protien (20 µg) was applied to each lane. C, Coomassie stain; W, Western blot developed with a polyclonal rabbit antibody to PBP; K, bacterial killing in the gel overlay killing assay. A prominent band of identical mobility is detected by anti-PBP antibody and appears in Western blots of THP-1 and monocyte peptides visible in Coomassie stains and killing S. typhimurium PhoP. In monocytes, five additional peptide bands result in bacterial killing, again (see Fig. 8 ) with a more mobile band reacting with anti-PBP antibody.

 
Finally, we attempted to identify the PPBP derivatives using mass spectrometry-based techniques. Proteins that aligned with regions on gels with enhanced bacterial killing, which also cross-reacted with the anti-PBP antibody, were subjected to limited trypsin digestion. Samples were analyzed by MALDI-TOF mass spectrometry to determine which PPBP derivative was present. Table 1 shows three peptides from PBP that were detected by MALDI-TOF and confirms that PBP or a processed peptide from PBP was responsible for the killing. One of the peptides detected, corresponding to amino acids 48–62 of PPBP, contained amino acids that are removed during the formation of the smaller PBP derivatives NAP-2 and TC1. To determine which PPBP derivative was present in the more mobile peptide band, we undertook SELDI analysis. Acid extracts from platelets and untreated, zymosan-stimulated or interleukin (IL)-4-treated monocytes were analyzed by specific immunoaffinity capture on ProteinChip arrays containing monoclonal antisera recognizing the NAP-2 fragment of PBP, which is part of all derivatives with known biological activity. Acid-extracted platelets failed to immunoprecipitate any peptides when a nonspecific antibody was bound to the ProteinChip (Fig. 10A ); however, a distinct peak was detected at 9287 Da when the NAP-2 antibody was used (Fig. 10B) . Upon examination of all known peptides derived from PBP, the mass of this peptide closely matched the calculated mass for CTAP-III (9291 Da) and was 182 Da from TC-2, the next closest PBP derivative. Immunoprecipitation of NAP-2 antibody-binding peptides from zymosan-stimulated monocytes also pulled down a peptide corresponding to CTAP-III (Fig. 10C) . Nonstimulated monocytes failed to bring down any NAP-2-binding peptides (Fig. 10D) . Increasing the amount of starting material from zymosan-stimulated monocytes increased the amount of peptide pulled down (Fig. 10E) but only unimportantly in the absence of the microbial stimulus (Fig. 10F) . The response to zymosan could be suppressed by IL-4, as IL-4-treated, -stimulated monocytes failed to show the increase in the amount of the pulled-down peptide, as expected from our previous observation that IL-4 decreases PBP expression in monocytes (Fig. 10G) [26 ]. Taken together, these data identify CTAP-III as one of the antimicrobial-active PBP derivatives in monocytes.


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  		Table 1. Tryptic Peptides from a Digest of a PPBP Derivative

 


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  		Figure 10. Zymosan-stimulated monocytes produce connective tissue activation peptide III (CTAP-III). SELDI spectra from acid-extracted lysates from primary human platelets (A and B) or monocytes (C–G) were incubated on PS10 ProteinChips (Ciphergen Biosystems) in the absence (A) or presence (B–G) of the PBP-immunoprecipitating antibody. Samples were prepared for SELDI analysis as described in Materials and Methods. (A) Mock immunoprecipitation from platelets; (B) PBP immunoprecipitation; (C) 100 µl zymosan-stimulated monocytes; (D) 100 µl unstimulated monocytes; (E) 200 µl zymosan-stimulated monocytes; (F) 200 µl unstimulated monocytes; (G) 200 µl IL-4 (100 U/ml)-treated, zymosan-stimulated monocytes.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
PBP extracted from blood platelets and several of its derivatives have been shown not only to have signaling functions as CXC chemokines but also to have antimicrobial activity against bacteria and fungi [13 , 30 , 48 , 49 ]. We have previously shown that expression of PBP is not limited to blood platelets but that monocytes constitutively express important amounts of PBP. We show in the present studies that PBP expression is several-fold up-regulated after phagocytosis of bacteria or exposure to microbial components such as zymosan or LPS in human monocytes. During in vitro differentiation of glass adherent monocytes for 10 days in 20% autologous serum to macrophages, the percentage of cells positive for PBP decreases, but after microbial challenge, the fraction of cells with intense immunostaining for PBP increases again to 40–45%, indicating that PBP is also expressed by mature macrophages during a host response to pathogens. We have previously shown that a short-term exposure of human mononuclear phagocytes to LPS, in contrast to our present findings with an exposure to LPS for ≥24 h, does not increase mRNA levels of PBP or the expression of the peptide. Accordingly, IL-1 and tumor necrosis factor {alpha} have no important effect on the expression of PBP (unpublished observation). Also, activation of monocytes by IFN-{gamma} did not increase PBP expression and even induced a decrease in PBP mRNA and PBP protein expression [26 ].

In search of an endogenous stimulus for PBP expression, we studied the role of an activation of monocytes and macrophages through the PAR family [44 , 50 ]. In these experiments, we found that activation of monocytes and macrophages by thrombin leads to a several-fold increase in PBP protein expression. Specificity for thrombin was confirmed by the antagonism of the antithrombin hirudin and by showing a comparable activating effect for SFLLRN, a synthetic 14-amino acid peptide, homologous in its sequence to some of the natural ligands of human PARs [44 ]. Increased protein expression was accompanied by comparably higher PBP–mRNA levels, an observation in accordance with the concept that PAR activation regulates mRNA transcription through G-protein coupling [46 ].

When monocytes or macrophages ingested autofluorescent zymosan particles or paraformaldehyde-fixed S. typhimurium expressing a GFP, we found an intense immunostaining for PBP in close proximity of phagocytosed particles. Furthermore, in phago-lysosomal cell fractions containing ingested latex particles, which were isolated by a sucrose flotation method that avoids contamination with cytoplasm or other cell organelles [40 , 41 ], we found an increase in the proportion of PBP compared with nonfractionated cells, indicating that PBP or its derivatives reach the phago-lysosomal compartment. For an antimicrobial peptide to have antimicrobial function, contact with the pathogen is one of the prerequisites. Another prerequisite is that the environmental conditions are favorable for their activity. Tang and collaborators [49 ] found that an acid environment, such as that found in the phago-lysosomal compartment, is important for the antimicrobial activity of PBP derivatives. Taken together, these observations make an antimicrobial role for PBP or its derivatives of human mononuclear phagocytes conceivable.

Finally, we demonstrated by a gel-overlay technique that human monocytes express PBP derivatives that are antimicrobially active. In Western blots of acid extracts from zymosan-activated monocytes, we identified two bands with antibacterial activity corresponding in Western blots to PBP derivatives. The less mobile band corresponded to a band identified in extracts from blood platelets and the human monocytic cell line THP-1. In zymosan-stimulated monocytes, a second, more mobile band was identified to be CTAP-III by MALDI-TOF and SELDI techniques.

In conclusion, these studies show that human mononuclear phagocytes express antimicrobially active derivatives of PBP that have the potential to reach the phago-lysosomal compartment. Exposure of mononuclear phagocytes to microbial components, such as LPS or zymosan, or phagocytosis of intact bacteria as well as thrombin induces an increased expression of PBP derivatives advancing these CXC chemokines to major cellular proteins. Phylogenetically, the development of the coagulation system and the immune system has common roots [15 , 51 , 52 ]. Our finding that thrombin, a protein of the coagulation cascade, activates host defense cells to express increased amounts of a CXC chemokine with antimicrobial activity, which is also expressed in large amounts by blood platelets that have main functions in hemostasis, can be viewed as further evidence for the coevolution of the immune and coagulation system. More important, these studies show that noxious stimuli, which result in activation of blood coagulation and thrombin formation [53 54 55 56 ], as well as microbial components and microorganisms, result in an increased expression of PBP. This rapid reaction, independent of the necessity to mount an adoptive immune response, appears meaningful in view of the proposed role of cationic, antimicrobial peptides including PBP and several other chemokines in innate immunity.

In any event, our studies point out that PBP derivatives are constituents of the antimicrobial arsenal of human mononuclear phagocytes regulated by direct microbial stimuli or by mediation of an activation of the coagulation cascade with formation of thrombin during sepsis and tissue damage.


   ACKNOWLEDGEMENTS
 
This work was supported in part by Grant 3200B0-102236/1 of the Swiss National Science Foundation.

Received April 30, 2004; revised June 28, 2004; accepted July 2, 2004.


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 DISCUSSION
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