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Originally published online as doi:10.1189/jlb.0603288 on December 12, 2003

Published online before print December 12, 2003
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(Journal of Leukocyte Biology. 2004;75:495-503.)
© 2004 by Society for Leukocyte Biology

Constitutive and regulated expression of platelet basic protein in human monocytes

Ahmed El-Gedaily, Gabriele Schoedon, Markus Schneemann and Andreas Schaffner1

Department of Medicine, University Hospital, Zürich, Switzerland

1 Correspondence: Department of Medicine AW9, Research Unit Medical Clinic B, University Hospital, CH-8091 Zürich, Switzerland. E-mail: klinsar{at}usz.unizh.ch


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Platelet basic protein (PBP) and several of its derivatives are known for their broad range of functions as signaling molecules and cationic antimicrobial peptides and were considered hitherto megakaryocyte- and platelet-specific. In search of glucocorticoid-regulated antimicrobial systems of monocytes, we found a 15-fold down-regulation of PBP mRNA by differential display. Regulation was confirmed in vivo even at low prednisone doses. Quantitative mRNA analyses confirmed down-regulation also for platelets. Western blotting and immunostains showed down-regulation at the protein level. Pro-PBP derivatives were in the size range of 7.5-14 kD and in immunostains, gave granular cytoplasmatic patterns. Interleukin (IL)-4 and IL-10 induced a similar down-regulation. Phagocytosis resulted in an increase of smaller derivatives in the range of 7.5 kD. Stimulation with interferon-{gamma} and lipopolysaccharide did decrease expression of PBP and affected derivatization. Expression of PBP and its derivatives is not restricted to the megakaryocytic cell lineage. PBP and some of its derivatives might contribute to the antimicrobial armamentarium of mononuclear phagocytes or have monokine functions. Our studies define PBPs as one among the many immunosuppressive targets of glucocorticoids.

Key Words: macrophages • antimicrobial cationic peptides • ß-thromboglobulin • glucocorticoids/immunology


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Platelet basic protein (PBP) derived from its precursor pro-PBP (PPBP) belongs to the family of CXC chemokines and is known as a major granular protein of platelets [1 2 3 4 ]. By post-translational cleavage, this small protein of ~14 kD gives rise to connective tissue-activating peptide-III (CTAP-III) [5 6 7 ], neutrophil-activating peptide-2 (NAP-2) [1 , 8 , 9 ], ß-thromboglobulin (ß-TG) [2 ], and two variants of thrombocidin (TC-1 and TC-2) [10 ] (Table 1) . Peptidases, some of which are derived from monocytes and neutrophil granulocytes, play a role in post-transcriptional processing of PBP [4 , 11 12 13 14 ]. The derivatives have an amazing functional diversity: NAP-2 is a neutrophil activator, CTAP-III is a mitogen for fibroblasts and has other metabolic effects, and PBP, CTAP-III, and TC-1 as well as TC-2 have antimicrobial activity. TCs and other derivatives of PPBP belong to the cationic antimicrobial peptides (CAMPs) and kill many bacteria and fungi [10 , 15 ]. Expression of PPBP has not yet been shown for mononuclear phagocytes [1 , 16 ]. To the contrary, NAP-2 was only detected in supernatants of lipopolysaccharide (LPS)-stimulated human monocytes in the presence of a platelet-release supernatant but not when the latter was omitted [1 ]. In addition, no PPBP mRNA was found in LPS-stimulated human alveolar macrophages [16 ]. Studies specifically addressing the question of expression of PPBP in monocytes have not been reported. Nevertheless, PPBP and its derivatives are viewed as megakaryocyte and platelet-specific peptides [4 ], and studies of the genomic regulatory elements responsible for its tissue-specific expression have been advanced [17 , 18 ]. The gene is located in a gene cluster comprising several CXC chemokines in the vicinity of platelet factor 4 in a locus considered as megakaryocyte-specific on human chromosome 4 [17 , 18 ]. Regulation of expression has been studied for a number of chemokines, but studies of the regulation of PPBP and its derivatives have focused on post-transcriptional processing, possibly in part as a result of difficulties in studying megakaryocytes [4 ].


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  		Table 1. PPBP and Its Derivatives

 
In previous studies, we found that glucocorticoids and the monocyte-deactivating interleukins (IL)-4 and IL-10 directly affect antimicrobial activity of mononuclear phagocytes, in particular, nonoxidative killing of fungi and bacteria [19 20 21 22 23 24 ]. Deactivation of antimicrobial activity by glucocorticoids was receptor-mediated and required treatment of monocytes for 36 h to be expressed [20 ]. These observations were compatible with the assumption of a repressive effect on genomic expression [25 ] of an antimicrobial principle, rather than an inductive effect [26 ]. As known nonoxidative, antimicrobial systems considered to contribute to the activity of mononuclear phagocytes [27 ] against microorganisms were not expressed in our human monocyte system (nitric oxide [28 ], {alpha}-defensins [29 ]) or were not affected by glucocorticoids (phagolysosomal fusion and acidification [22 ], ß-defensins [30 ]) or were considered unlikely to contribute to our observations (nutritional deprivation [31 ]), we started to search for hitherto-unknown antimicrobial systems in monocytes. To identify transcriptionally repressed gene products during the deactivation reaction of monocytes, we have conducted differential RNA display studies that identified PPBP as a target of a major repressive regulation by glucocorticoids, IL-4 and IL-10. As PPBP gives rise to CAMPs, we have conducted the following studies in blood-derived human mononuclear phagocytes as well as in human blood platelets.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Isolation of blood cells
Monocytes
Human mononuclear phagocytes were isolated from buffy coats of healthy donors (Swiss Red Cross Blood Bank, Zurich) or heparinized blood (10 U/ml) as described [24 ]. In brief, after separation by Ficoll gradient (Ficoll-Paque, Pharmacia Biotech Europe, Switzerland), three washes in Gey’s balanced salt solution (GBSS; Gibco Europe, Basel, Switzerland) mononuclear cells, suspended in Iscove’s modified Dulbecco’s medium (IMDM; Gibco Europe), supplemented with 20% pooled human serum (complete IMDM), were seeded onto baked (220°C, 4 h), endotoxin-free, sterile, 90-mm-diameter glass tissue-culture plates at a density of 1–2 x 107 cells/ml as described [32 ]. Mononuclear cells 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 further 24 h without or with a glucocorticoid receptor-saturating dose of dexamethasone (2.5x10-7M, Sigma Chemical Co., St. Louis, MO) with solvent controls as described previously [20 ] before isolation of total RNA (see below). LPS from Escherichia coli (Sigma Chemical Co.), interferon-{gamma} (IFN-{gamma}; 100 U/ml), IL-10 (5 ng/ml), and IL-4 (2 ng/ml), all from PeproTech (Rocky Hill, NJ), were used at a concentration shown previously to be maximally active on human monocytes [19 ].

Lymphocytes
Lymphocytes were isolated using the supernatant of mononuclear cells after glass adherence for 2 h as described above. Purity was at least 98% by Coulter counter analysis (ADVIA 120, Hematology System, Bayer, Leverkusen, Germany) with less than 0.1% platelets.

Neutrophils
Neutrophils were isolated by methocell gradient as described [33 ]. Methocell (15 ml) was over-layered with 35 ml buffy coat diluted 1:1 with GBSS and allowed to separate at 1 g at room temperature for 60–120 min until erythrocytes were completely sedimented. The supernatant was transferred to fresh 50 ml Falcon tubes containing 15 mL Ficoll-Paque and was centrifuged for 20 min at room temperature at 1475 rpm to sediment neutrophils. Purity was at least 98% as determined by Coulter analysis.

Platelets
Platelets were isolated from blood drawn into 10 mM sodium citrate. Blood was diluted 1:1 with Ca++-free GBSS containing 1% human serum albumin (Swiss Red Cross Blood Bank) and was centrifuged for 7 min at 200 g at room temperature. The thrombocyte-enriched plasma was collected and substituted 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 directly lysed for RNA extraction as described below. Platelet purity was almost 100%.

Monoblasts
Monoblasts were obtained by leukapheresis from a patient with acute monocytic/monoblastic leukemia. At the time of leukapheresis, the patient had 220,000 blasts/µL and <10,000 platelets/µl. Platelet-free, passaged monoblasts [34 ] were lysed for total RNA or protein preparation as described below.

Prednisone treatment of healthy volunteers
Six healthy volunteers (two females, four males; age range, 35–45 years) were treated with increasing doses of prednisone (25 mg/day, 50 mg/day, 100 mg/day) over 9 days. Each dose was given for 3 days. Heparinized blood (10 mL) was taken at baseline, at the third day after each prednisone dose, and 4 days after the end of the last dose (washout). Mononuclear cells were isolated by Ficoll density centrifugation as described above and were lysed for total RNA extraction without further separation of monocytes and lymphocytes. PPBP expression was measured in equal amounts of total RNA by real-time reverse transcriptase-polymerase chain reaction (RT-PCR) as described below. To control for the in vivo effect of prednisone, the glucocorticoid-induced expression of the monocyte-specific marker CD163 [35 , 36 ] was analyzed in the same samples. Results are given as ratios of PPBP–mRNA or CD163–mRNA to the monocyte-specific and nonregulated marker CD155, a poliovirus receptor [37 ].

Patients
Blood was obtained from eight patients after having received 7–12 days of treatment with glucocorticoids in a dose equivalent to at least 100 mg prednisone. The most frequent indication for corticosteroids was metastatic disease to the brain. From eight patients and in parallel from 10 normal volunteers, 8 ml blood was collected in standard tubes with sodium citrate for platelet separation as described above for quantitative RNA analysis.

RNA isolation
Total cellular RNA 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 stored frozen at –70°C in RNase-free tubes (Eppendorf Biopure Microtubes, Hamburg, Germany) until use.

Liquid-phase differential display RT-PCR
Equal amounts of total RNA (2 µg total DNA-free RNA of each cellular preparation) was 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 LightCyclerTM real-time PCR system using the FastStart DNA Master SYBR Green I kit (Roche Diagnostics, Rotkreuz, Switzerland), different combinations of primer T24 (oligo dT, Microsynth, Balgach, Switzerland), and a set of 13-mer arbitrary primers (H-AP set, Gene Hunter, Nashville, TN; see Table 2 ).


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  		Table 2. Primer Pairs Used in This Study

 
PCR products were labeled with the dye SYBR Green I, which fluoresces only when bound to double-stranded DNA. PCR reactions were set up in glass capillaries in a total volume of 20 µl containing Fast-StartTM reaction mix and enzyme, 3.3 mM MgCl2, 2 µl cDNA sample, water, and 1 µM each primer (see Table 2 ). Temperature cycling profiles were as follows: preincubation time, 10 min, 95°C; denaturation, 10 s, 95°C; annealing time, 5 s, 45°C; and elongation, 30 s, 72°C for 30 cycles, with acquisition of fluorescence at the end of each cycle. As negative control, complete reaction mixture without cDNA was cycled.

Differentially expressed genes were then directly detected by melting-curve analysis, comparing temperature profiles of amplification products obtained with the respective primer combinations. Each peak of a melting curve represents the melting point of a unique product and corresponds to the amount of this amplicon. Thus, up-, down-, or unregulated products generated with certain primer combinations can be easily discriminated.

Amplification products found to be regulated were directly collected from the glass capillaries by short centrifugation in an Eppendorf microfuge (Eppendorf Biopure Microtubes), gel-purified, and custom-sequenced (Microsynth). Sequences were then compared with sequences in the EMBL and GenBank® databases for nucleic acid homologies using the FASTA (ebi.ac.uk) and BLAST (ncbi.nih.gov) software.

Real-time RT-PCR
(Roche Applied Science technical note nos. LC 11/2000 and LC 10/2000.) To analyze transcription of PPBP in human monocytes and other human blood cells, sequence-specific primers were used (see Table 2 ; primers PPBP and PPBP2, which together, span the complete sequence of PPBP). Temperature cycling profiles were as described above with primer-dependent annealing temperature (Tm±5°C). GAPDH was used as a housekeeping gene to correct for small differences in cDNA content. Monocyte-specific genes CD163 (up-regulated expression by glucocorticoids) and CD155 (nonregulated) were used to control for the glucocorticoid effect and monocyte specificity, respectively (Table 2) . To control for platelet specificity, GPIIb-specific primers were used (Table 2) . Equal amounts of total RNA/cDNA were processed in all comparative experiments. Amounts of PPBP expressed were given as ratios of PPBP/CD155 in monocytes and PPBP/GPIIb or PPBP/GAPDH in thrombocytes.

Western blot analysis
For protein expression analysis, monocytes were isolated from heparinized blood and cultured as described above. Incubation with 2.5 x 10-7 M dexamethasone was for 36 h to ensure regulated protein expression. Cells of one 90 mm glass tissue-culture plate were scraped in 1 ml phosphate-buffered saline (PBS) containing a protease inhibitor cocktail (Roche Diagnostics). Lysis of blood cells was performed by four cycles of rapid freezing in liquid nitrogen and thawing. After centrifugation at 14,000 rpm in an Eppendorf microfuge (Eppendorf Biopure Microtubes), the supernatant was collected and used for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on 16.5% Tris/Tricine mini-gels under reducing conditions (Bio-Rad, Hercules, CA) according to standard protocols. Equal amounts of protein lysates (2 µg each preparation) were applied for SDS-PAGE. After blotting onto nitrocellulose, membranes were incubated with antigen affinity-purified, polyclonal rabbit anti-human NAP-2 antibody (Peprotech). Blots were developed with the enhanced chemiluminescence Western blotting detection system (Amersham Biosciences, Little Chalfont, UK). For measurement of PPBP and its derivatives, bands were quantitated on a Bio-Rad Model GS-700 imaging densitometer with Bio-Rad Multi-Analyst/PC Version 1.1. (Bio-Rad).

Cathepsin G digestion
For digestion with cathepsin G (Sigma Chemical Co.), lysis of cells was performed in 10 mM sodium phosphate, 150 mM sodium chloride, pH 7.4, without protease inhibitors. After lysis by freeze/thaw and centrifugation, the supernatant was filtered through a Millipore ultrafree filter unit, 30,000 nominal MW limit (Millipore, Bedford, MA), for removal of large proteins. Protease digestion reaction was performed in a total volume of 60 µL with 0.02 U cathepsin G at 37°C for 10 min and 60 min, respectively [11 ]. The reaction was stopped at the indicated time points by addition of 120 µl Tris/Tricine sample buffer and boiling at 95°C for 5 min.

Immunocytochemistry and immunofluorescence
Human mononuclear cells were isolated as described above and suspended in complete IMDM at a density of 1–2 x 107 cells/mL. Aliquots (100 µL) of this suspension were dispensed onto 12-mm Thermanox tissue-culture coverslips (Nalge Nunc International, Kopenhagen, Denmark) and allowed to adhere for 2 h at 37°C, 5% CO2, 98% humidity. After four times washing with warm GBSS, fresh, complete IMDM without and with dexamethasone (2.5x10-7M) was added, and the cells were incubated for 36 h. Then, coverslips were washed in warm, physiologic saline and air-dried overnight. After fixation in acetone/methanol 1:1 for 70 s, cells were immunochemically stained using murine anti-human NAP-2 monoclonal antibody (mAb; PeproTech) and the Dako murine APAAP system (Dakopatts, Carpinteria, CA), according to standard protocols, or the rabbit polyclonal anti-human NAP-2 antibody (PeproTech), the alkaline phosphatase-conjugated goat anti-rabbit secondary antibody (Jackson Immuno Research, Milan Analytica, Switzerland), and Dako FastRed alkaline substrate tablets (Dakopatts). For immunofluorescence, cells were cultured for 48 h on round 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 (GS-PBS) before 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% bovine serum albumin (Sigma Chemical Co.) and incubation with the secondary antibody [2 µg/ml in GS-PBS; Alexa Fluor® 568 goat anti-mouse immunoglobulin G (H+L), Molecular Probes, Eugene, OR]. Coverslips were mounted with Vectashield mounting medium (Vector Laboratories, Burlingham, CA) and read on a Nikon Eclipse E 400 fluorescence microscope with an excitation wavelength of 568 nm and read at 603 nm.

Challenge with Candida albicans
Human monocytes cultured on coverslips as described above for 24 h were challenged with heat-killed (2 h, 70°C) yeast cells from C. albicans suspended in complete IMDM at a ratio of two or five spores/monocyte. After 1 h for ingestion, coverslips were washed three times in prewarmed GBSS; fresh, prewarmed, complete IMDM was added, and the cells incubated for an additional 18 h before further analyses.

Statistics
Mean or median ± SD is given as indicated. For comparison of discrete values, t-test was applied; for discontinuous values, Mann-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.

All human studies were approved by our institutional ethical committee and executed in accordance with the Helsinki declaration.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
By liquid-phase differential display with random primers, we identified and confirmed by sequence analysis that transcription of the message for PPBP is importantly down-regulated by steroid receptor-saturating concentration of dexamethasone (Fig. 1A and 1B ). Differential display by this new methodology gave highly reproducible results with RNA from different blood donors. Real-time PCR with specific primers (Table 2) confirmed an ~15-fold down-regulation with monocytes from four donors (n=4, mean±SD: 16.4±4.9, P<0.01). The possibility that the message of PPBP was from contaminating platelets was excluded by probing our monocyte RNA preparation with primers specific for platelet GPIIb (Fig. 1C) . Furthermore in the granulocytic and lymphocytic fraction of blood, PPBP message was not detected (Fig. 1D) . Reversibility of regulation was studied in a time-kinetic experiment (Fig. 2 ) in which PPBP mRNA was shown to rise again to a presuppressive level after removal of dexamethasone. This documents synthesis of PPBP mRNA in human monocytes in vitro. To confirm down-regulation in vivo, we quantified PPBP–mRNA in monocytes from human volunteers taking oral prednisone and compared this repressive steroid effect with the inductive effect of prednisone [36 ] on the message of CD163 [35 ], a scavenger receptor. In these experiments because of the low blood volumes drawn, lymphocytes and monocytes were not separated. We therefore measured the ratio of PPBP mRNA to CD155 mRNA, which is nonregulated by dexamethasone and is monocyte/macrophage-specific. This precaution was taken to avoid a possible influence of changes in the composition of mononuclear cells by glucocorticoids in vivo that contribute to GAPDH mRNA. These studies confirmed the repressive effect of glucocorticoids on PPBP mRNA in vivo and contrasted it to the inductive effect on CD163 (Fig. 3 ). It is of note that the effects occurred already at intermediate, clinically used prednisone doses.



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  		Figure 1. Liquid-phase differential display, depending on quantitative melting-curve analysis of cDNA. (A) Melting-curve analysis of products generated by the random primer set AP primers 1–10 and oligo dT24 with RNA from human mononuclear phagocytes treated with 2 x 10-7M dexamethasone (MD) for 24 h and control monocytes (M). The strongly regulated products, marked M (untreated) and MD, were 492 bp long and 100% homologues with nucleotides 198–690 of PPBP. (B) Confirmation of dexamethasone regulation of PPBP mRNA levels was done with sequence-specific primers. (C) Analysis of macrophage RNA preparations for contamination with platelet RNA using platelet (P) GPIIb-specific primers. (D) Expression analysis of PPBP in equal amounts of total RNA from fractionated blood cells. L, Lymphocytes; N, neutrophils. Note that the analysis for N and L was identical. All curves from one representative experiment out of four experiments with RNA from four different blood donors. For primer sequences, see Table 2 .

 


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  		Figure 2. Ratio of PPBP/GAPDH mRNA ratios in human monocytes exposed in vitro for variable time periods to dexamethasone (Dexa). Analysis of the regulation of PPBP mRNA expression by transient exposure to 2.5 x 10-7 M dexamethasone for 18 h followed by a wash-out phase of 18 h or continuous exposure to dexamethasone for 36 h before measurement of the ratio of PPBP mRNA to GAPDH mRNA. Mean ± SD from one of two experiments, each with triplicate monocyte cultures that gave comparable results. Note resynthesis of the repressed PPBP mRNA after removal of dexamethasone.

 


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  		Figure 3. Ex vivo expression analysis of PPBP– and CD163–mRNA in monocytes from healthy volunteers treated with increasing doses of prednisone. The daily prednisone dose was 25 mg from baseline to day 3, 50 mg from days 3 to 6, and 100 mg from days 6 to 9. Primers for the receptor CD155, not regulated by prednisone, were used as monocyte-specific reference. Mean ratios ± SD from six volunteers at each time point. Note the reciprocal progress of the regulatory effect on PPBP (repressed) and CD163 (induced).

 
To study a possible effect of glucocorticoids on the regulation of PPBP–mRNA in platelets, we collected blood from patients receiving glucocorticoids in a dose equivalent to >=100 mg/day prednisone for at least 7 days. The longer exposure to the corticosteroids was chosen because of the half life of platelets of 9–12 days and the fact that transcriptional regulation of mRNA can no longer take place in the nucleus-free platelets after release from megakaryocytes. These studies confirmed repressive mRNA regulation by glucocorticoids also for platelets (Fig. 4 ).



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  		Figure 4. Effect of glucocorticoids on PPBP–mRNA levels in platelets in vivo. Ex vivo expression analysis of PPBP in platelets obtained from six normal volunteers and eight patients receiving a glucocorticoid dose equivalent to >=100 mg prednisone for >=7 days. Median, 25–75% range, and ± SD. P < 0.001.

 
PPBP protein was studied in monocytes by Western blot analyses and immunostains with mono- and polyclonal antibodies to NAP-2, a cleavage product of PPBP that shares part of the amino acid sequence with all derivatives of PPBP (Table 1) . By Western blot, using a polyclonal antibody raised to recombinant human NAP-2, products of variable size were identified ranging from 7.5 to 14 kD (Fig. 5 ). As the analyzed preparations of monocytes were highly purified even before washing, and granulocytes and lymphocytes do not express PPBP–RNA (Fig. 1) , contamination by platelet-derived PPBP or PPBP from another source appeared very unlikely. The possibility of contamination was further excluded by demonstrating PPBP and derivatives in passaged, platelet-free monoblasts from a patient with acute monocytic/monoblastic leukemia, which transcribed PPBP–mRNA and also expressed the protein (Fig. 5) . PPBP expression was further proven by digestion of macrophage lysates with cathepsin G, which cleaves PBP N-terminally to NAP-2 or TC-1 [11 , 14 ]. Monocytes did not reveal significant amounts of smaller cleavage products. By digestion of PPB with cathepsin G, a small peptide in the size of NAP-2 or TC-11 was generated from the parent molecule (Fig. 6 ). Thus, the antibody recognized a peptide cleaved by cathepsin, as expected for PBP, and produced an immunoreactive derivative of the expected size. Immunostains with a polyclonal antibody and mAb, which react with PBP, confirmed expression of PBP also by this method in monocytes (Fig. 7 ). By immunofluorescence, most intense activity was attributed to the golgi zone with a distinct granular pattern in the cytoplasm (Fig. 7F) . After challenge with heat-killed yeast cells from C. albicans, individual monocytes with ingested yeast displayed a stronger activity than neighboring cells (Fig. 7D) . Accordingly, we found higher PPBP mRNA and PBP levels in challenged cultures (Fig. 8 ), an effect that did not supersede down-regulation by dexamethasone. Furthermore, in experiments comparing peptide profiles after phagocytosis of boiled yeasts from C. albicans, a small derivative of PPBP of the same size as NAP-2 or TC-1 appeared in Western blots of cells challenged with fungi, indicating that exposure to microorganisms affects processing of PPBP (Fig. 9 ).



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  		Figure 5. Expression of PPBP and derivatives in monocytes, monocytes treated with dexamethasone, and passaged monocytic leukemia cells. Western blot analysis with a polyclonal anti-NAP-2 antibody. NAP2, 5 ng recombinant human NAP-2; MD, 2 µg protein from macrophages treated with 2 x 10-7 M dexamethasone for 36 h; M, 2 µg protein from control macrophages cultured for 36 h in parallel. Tc, 2 µg protein from thrombocytes; M5b, 2 µg protein from passaged leukemic monoblasts. Densitometric comparison of the bands in lanes MD and M showed a 4.42-fold down-regulation of immunoreactive PBP derivatives by dexamethasone.

 


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  		Figure 6. Digestion by cathepsin G of the protein reacting with antihuman-NAP-2 antibody produces identical products in monocytes and platelets. Western blot analysis with polyclonal antihuman-NAP-2 antibody of fragments after digestion with cathepsin G. Lanes 1–3, Protein from platelets; NAP2, recombinant human NAP-2; lanes 4–6, protein from monocytes. Lanes 1 and 4, Before digestion; lanes 2 and 5, after 10 min of digestion; lanes 3 and 6, after 60 min of digestion.

 


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  		Figure 7. Immunocytochemical staining of PPBP and derivatives in cultured monocytes and platelets. (A and B) 400x original magnification. Immunostain with polyclonal anti-NAP-2 antibody. (A) Control cells cultured for 36 h on coverslips; (B) monocytes treated with 2 x 10-7M dexamethasone for 36 h on coverslips. (C) Immunostain of freshly isolated platelets with monoclonal anti-NAP-2 antibody (1000x original magnification). (D) Immunostain with monoclonal anti-NAP-2 antibody in monocytes challenged with boiled yeast from C. albicans 18 h before fixation and staining. Note the strong reaction in the two cells with ingested yeast cells (1000x original magnification). (E) Immunofluorescence with the monoclonal anti-NAP-2 antibody shows an activity in most monocytes (200x original magnification). (F) With an intense staining in the golgi zone and a granular pattern throughout the cytoplasm (800x original magnification).

 


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  		Figure 8. Regulation of PPBP RNA and PBP protein levels by dexamethasone in monocytes after phagocytosis of boiled yeast from C. albicans. Mean ± SEM from triplicate experiments and duplicate wells. *, Duplicate experiments. Dexa, Dexamethasone, 2.5 x 10-7 M for 36 h before the measurements; Candida 2:1, challenge of monocytes with boiled yeast cells in a ratio of 2:1; Candida 5:1, in a ratio of 5:1 18 h before measurements. P < 0.01 for the comparison of Control with Dexa and Candida 2:1 with Candida 2:1 and Dexa. P < 0.01 for the comparison of Candida 2:1 combined with Candida 5:1 with Control.

 


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  		Figure 9. Western blot analysis with polyclonal anti-NAP-2 antibody of protein from monocytes challenged with boiled C. albicans for 10 h (lane 2) and control cells (lane 1). NAP2, Human recombinant NAP-2. By densitometry, up-regulation of all bands together was 1.55-fold and that of the 7.5-kD band, 2.89-fold. Results from one typical experiment out of two.

 
Finally, we extended our quantitative studies of regulated PPBP expression to the two monocyte/macrophage-deactivating cytokines IL-10 and IL-4 [24 ] and to LPS and IFN-{gamma}. IL-4 and IL-10 reduced PPBP mRNA levels and accordingly, PBP expression (Fig. 10 ). IFN-{gamma} and LPS did not increase PPBP mRNA levels in these experiments with stimulation times of 8 and 18 h (Fig. 11 ) nor in several experiments with exposures to these activating signals up to 7 days (not shown). To the contrary, in particular, IFN-{gamma} reduced PPBP mRNA after prolonged treatment (Fig. 11) , and the two activating signals IFN-{gamma} and LPS did not counteract down-regulation of PBP expression by dexamethasone. Western blot analyses of PPBP and its derivatives confirmed all regulations at the mRNA level and again, pointed to an influence of the activating agents IFN-{gamma} and LPS on derivatization (Fig. 12 ).



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  		Figure 10. Regulation of PPBP mRNA and PBP protein expression by dexamethasone, IL-4, and IL-10. Monocytes were treated with dexamethasone (Dexa; 2.5x10-7 M), IL-4 (20 U/ml), and IL-10 (20 U/ml) for 36 h before measurements. (A) Mean ± SEM from triplicate experiments and duplicate wells. (B) Mean from duplicate experiments. (A) P < 0.01 for the comparison of control with dexamethasone, IL-4, and IL-10.

 


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  		Figure 11. Expression analysis of PPBP mRNA in monocytes treated with LPS, IFN-{gamma}, and dexamethasone. Concentrations were 2.5 x 10-7 M dexamethasone (Dexa), 100 U/ml IFN-{gamma}, or 100 ng/ml endotoxin (LPS). Results are given as ratio with GAPDH (A and C) or the much less abundant but monocyte/macrophage-specific poliovirus receptor CD155 (B and D). (A and B) LPS or IFN-{gamma}, Treatment for 8 h; dexamethasone treatment, 18 h. Mean ± SEM from triplicate experiments with duplicate measurements. For all comparisons with and without dexamethasone, P < 0.01. LPS or IFN-{gamma} versus control, P > 0.05. (C and D) Treatment with all agents for 18 h; mean from duplicate measurements. Note progressive down-regulation of PPBP mRNA by longer exposure to IFN-{gamma} and LPS.

 


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  		Figure 12. Western blot analysis with polyclonal antihuman-NAP-2 antibody of soluble monocyte lysates from monocytes treated with IL-10, IL-4, IFN-{gamma}, or LPS. Protein (2 µg) was applied per lane. Lanes 1 and 7, Recombinant human NAP-2; lane 2, control monocytes; lane 3, monocytes treated for 18 h with 20 U/ml IL-4; lane 4, monocytes treated for 18 h with 20 U/ml IL-10; lane 5, monocytes treated for 18 h with 100 U/ml IFN-{gamma}; lane 6, monocytes treated for 18 h with 100 ng LPS. Note the congruence of the reduced density of protein bands with that of measured PPBP mRNA levels in Figures 10 and 11 .

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
These studies show for a first time that human mononuclear phagocytes transcribe and translate constitutively PPBP, which is processed intracellularly into several derivatives. Expression of PBP and its derivatives is therefore not restricted to the platelet/megakaryocyte lineage. Derivatives of PPBP have signaling and effector functions [38 ]. As monocytes do not release important amounts of the chemokine NAP-2 into the supernatant [11 ], even upon activation [1 ], it appears conceivable that PBP is synthesized for intracellular functions. Potent antimicrobial activity has been attributed to PBP and its derivatives [10 , 15 ]. The detection of various derivatives of PPBP in the size range of PBP, CTAP-III, TC-2, and TC-1 points to the possibility that antimicrobially active peptides such as PBP, CTAP-III, and TCs are formed in mononuclear phagocytes and contribute to their antimicrobial activity.

In platelets, PBP is stored in {alpha}-granules. It is presently unknown whether PBP reaches these platelet-specific organelles through the lysosomal compartment or directly through the microvesicular apparatus [39 ]. Immunostains of monocytes with antisera to PBP showed, in addition to an intense activity in the golgi zone, a distinct granular pattern indicating localization in subcellular organelles. A deployment through the lysosomal compartment would assist the idea that PBP can reach the phagolysosome of mononuclear phagocytes during phagolysosomal fusion. In any event, the phagolysosome would provide a milieu that provides proteases [40 , 41 ] for derivatization of PBP and a pH that is favorable to the antimicrobial activity of most derivatives [15 ].

PPBP mRNA is strongly repressed by glucocorticoids in monocytes and in platelets; accordingly, fewer PBP and derivatives are expressed in blood monocytes cultured with steroid receptor-saturating concentrations of dexamethasone in vitro. PPBP mRNA repressed by dexamethasone was resynthesized to starting levels after removal of dexamethasone, pointing to a transcriptional regulation, as expected from a steroid receptor-mediated effect [25 ] rather than an influence on RNA stability. It is of note in this context that transcription of PBP in monocytes is down-regulated already by intermediate doses of glucocorticoids in vivo in healthy volunteers. The two macrophage-deactivating cytokines studied here, IL-4 and IL-10, also strongly down-regulated PPBP mRNA levels and in parallel, protein expression. These deactivating effects cannot be explained through an effect through the nuclear factor-{kappa}B pathway, as LPS, a strong activator of this system [42 ], which readily induces synthesis of IL-1 and tumor necrosis factor {alpha} (TNF-{alpha}) in human mononuclear phagocytes, did not increase but decreased PPBP expression. This contrasts with the LPS- and TNF-{alpha}-induced chemokine IL-8 localized closely to PPBP on chromosome 4q [17 ]. Also, IFN-{gamma} lessened in all experiments, PPBP mRNA levels, and expression of PPBP. In this regard, regulation of PPBP expression is similar to its closely related [43 ] chemokine glutamate carboxypeptidase 2/CXC chemokine ligand 6 of the same gene cluster [44 ], which is also down-regulated by IFN-{gamma}.

After challenge of mononuclear phagocytes with C. albicans, expression of PPBP was increased in our experiments, and more of the smaller derivatives appeared on Western blots. This increase could be attributed to individual cells with ingested fungal cells, as estimated by immunostains. Also, treatment of monocytes with LPS or IFN-{gamma} affected the composition of derivatives visualized by Western blot, with a relative increase of smaller peptide species. This observation is not surprising, as phagocytosis, LPS, and IFN-{gamma} are known to affect expression or activity of proteases in mononuclear phagocytes [41 , 45 46 47 48 ], and it is probable that the observed effects on processing of PBP were mediated through this mechanism comparable with extracellular derivatization of PBP from platelets by induction of monocyte proteases [1 ].

Our observations indicate that platelets are also a target for the immunosuppressive effects of glucocorticoids. Platelets and their antimicrobial peptides [49 ] have recently come into focus as a host-defense system. Platelets attach to and, presumably through their antimicrobial peptides, kill hyphae from Aspergillus fumigatus [50 ], a fungus with a propensity for intravascular infections in patients receiving glucocorticoids or in bone marrow failure. Platelets and their antimicrobial peptides play a role in the control of bacterial growth in vegetations of bacterial endocarditis [51 ]. Antibodies to platelet antimicrobial peptides increase bacterial growth in vegetations of bacterial endocarditis [51 ]. The observation that glucocorticoids lessen PPBP expression in platelets might provide an explanation for the observation that dexamethasone promotes bacterial proliferation in experimental endocarditis, where phagocytes or serum factors are considered to be of little or no importance [52 ].

In conclusion, these studies show that transcription of PPBP and its derivatives is not limited to platelets among blood cells but in contrast to granulocytes and circulating lymphocytes, also takes place constitutively in mononuclear phagocytes. The ratio of PPBP– to GAPDH–mRNA was high and measured to be 1–6 in all our experiments with monocytes from more than 30 different blood donors, indicating that PPBP mRNA is abundant in monocytes. The strong expressional regulation of PPBP–mRNA and PBP in vitro and in vivo in monocytes and platelets defines PPBP and its derivatives as a target for immunosuppressive effects of glucocorticoids and the macrophage-deactivating IL-10 and IL-4. Other antimicrobially, active chemokines and antimicrobial peptides [53 ] might be targeted by macrophage-deactivating signals, with the potential to interfere with their activity and additive or synergistic effects [15 , 53 ]. Studies addressing intracellular trafficking of PBP, its processing, its capability to reach the phagolysosomal compartment, and its role as an antimicrobial peptide of mononuclear phagocytes or as monokine are warranted. As chemotactically active PBP derivatives have not been found to be secreted by monocytes, we speculatively propose that PBP and/or its derivatives belong to the nonoxidative armamentarium of mononuclear phagocytes.


   ACKNOWLEDGEMENTS
 
The Rubatto Foundation, the Liebermann Foundation, and in part, Grant 32-55180.98 of the Swiss National Science Foundation supported this work.

Received June 24, 2003; revised August 11, 2003; accepted August 28, 2003.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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