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Originally published online as doi:10.1189/jlb.1105634 on November 9, 2006

Published online before print November 9, 2006
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(Journal of Leukocyte Biology. 2007;81:465-473.)
© 2007 by Society for Leukocyte Biology

Proteinase-activated receptors induce nonoxidative, antimicrobial peptides and increased antimicrobial activity in human mononuclear phagocytes

Nadine Lippuner, Bernhard Morell, Andreas Schaffner1 and Dominik J. Schaer

Research Unit, Clinic for Internal Medicine, University Hospital Zürich, Zürich, Switzerland

1 Correspondence: Research Unit, Clinic for Internal Medicine, AW 9, University Hospital, CH-8091 Zürich, Switzerland. E-mail: klinsar{at}usz.unizh.ch


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ABSTRACT
 
As thrombin and SFLLRNPNDKYEPF (SFLLRN-14), a synthetic ligand, mainly of the proteinase-activated receptor-1 (PAR-1), induce in monocytes the synthesis and secretion of chemokines, the PAR pathway can be viewed as a mononuclear phagocyte-activating principle. Classically, antimicrobial activity of mononuclear phagocytes is the measure for activation. Here, we investigated whether thrombin or SFLLRN-14 increases the antimicrobial activity of human monocytes and compared these effects to those of IFN-{gamma}. Furthermore, we measured the effects of these agents on the secretion of reactive oxygen intermediates and the antimicrobial activity of acid peptide extracts from monocytes. Human monocytes were exposed to maximally active concentrations of thrombin, SFLLRN-14, and IFN-{gamma}. Human monocytes treated with thrombin or SFLLRN-14 and then challenged with Salmonella enterica serovar typhimurium, including its attenuated mutant phoP, or Listeria monocytogenes killed, within 3 h, significantly more bacteria than control cells, an effect comparable with or surpassing the effect of IFN-{gamma}. This finding establishes the proteinase-PAR pathway as a potent, alternate activation pathway of mononuclear phagocytes. Thrombin and SFLLRN-14 had no significant effects on the amount of H2O2 secreted by monocytes. This was in contrast to IFN-{gamma}, which as expected, increased the secretion of H2O2 by approximately fourfold. Thrombin and SFLLRN-14, but not IFN-{gamma}, however, significantly increased the antimicrobial activity of acid peptide extracts of monocytes in a radial diffusion assay. Taken together, these findings suggest that IFN-{gamma} and thrombin differentially regulate oxidative and nonoxidative killing systems of human monocytes.

Key Words: monocytes • oxidative killing • Salmonella


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INTRODUCTION
 
The antimicrobial activity of mononuclear phagocytes depends on oxidative and nonoxidative killing mechanisms [1 2 3 4 5 6 7 8 9 10 11 12 13 ], and various activating and deactivating signals affect killing of bacteria and fungi [10 , 14 , 15 ]. The classic macrophage-activating signal is IFN-{gamma}, which enhances reactive oxygen intermediate (ROI) secretion among other important effects [3 , 5 , 8 , 11 , 13 , 16 , 17 ]. Little is known about the regulation of nonoxidative killing systems in mononuclear phagocytes. Recent studies showed that thrombin and the proteinase-activated receptor-1 (PAR-1) agonist SFLLRNPNDKYEPF (SFLLRN-14) and other agonists acting through PAR-2 up-regulate in monocytes the expression of several chemokines, among others, platelet basic protein and its derivatives, platelet factor 4, and RANTES, which are antimicrobially active [18 19 20 21 22 ]. PARs are G-protein-coupled receptors that are activated by proteinases such as thrombin [23 , 24 ]. Proteolytic cleavage frees the amino terminal-tethered ligand to dock on a receptor domain [24 ]. Similarly, short, synthetic peptides corresponding to the amino acid sequence of the natural ligands result in activation of PARs without proteolytic cleavage and thereby act as PAR agonists. SFLLRN-14 acts mainly through PAR-1, one of the four identified PARs, and less so through PAR-2 [23 ].

The observation that macrophages can be activated through PARs to secrete larger amounts of antimicrobial chemokines prompted the present studies investigating whether activation of human monocytes by thrombin or SFLLRN-14 also results in the classical measure for increased macrophage activity, namely, an increased capacity to kill cellular targets [1 ]. Listeria monocytogenes and Salmonella enterica serovar typhimurium, facultative, intracellular pathogens, were chosen as test organisms because of their potential to escape from or survive in phagosomes [25 , 26 ] and the importance of oxidative as well as nonoxidative killing mechanisms in the elimination of these bacteria after phagocytosis [3 4 5 , 13 , 27 28 29 30 31 32 ]. The attenuated mutant of S. typhimurium phoP was chosen because of its increased susceptibility to cationic, antimicrobial peptides [33 ], which we presumed to be an important PAR-induced system. Furthermore, to get an estimate of the potential biologic relevance of PAR-inducible killing systems, we quantitatively compared the capacity of monocytes to kill these bacteria after activation through PARs with the activity after IFN-{gamma} treatment. Finally, we studied the effects of thrombin, SFLLRN-14, and IFN-{gamma} on the capacity of human monocytes to secrete H2O2 in response to PMA and measured the antimicrobial activity of acid extracts from monocytes treated with these agents.


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MATERIALS AND METHODS
 
Cells
Human mononuclear cells were isolated from heparinized blood (100 U/ml) obtained from normal volunteers or from buffy coats purchased from the Swiss National Red Cross [22 ] on a Ficoll-Paque gradient (Pharmacia Biotech Europe, Switzerland) after centrifugation at 270 g for 25 min at room temperature. Cells were collected and washed three times in Gey’s balanced salt solution (GBSS; Sigma Chemical Co., St. Louis, MO) for heparinized blood or PBS (pH 7.4, Sigma Chemical Co.) for isolation from buffy coats. Cells were suspended in DMEM (Sigma Chemical Co.), supplemented with 20% fresh-frozen autologous serum or IMDM (Sigma Chemical Co.) with 10% commercial human serum of the clot, respectively. In experiments examining H2O2 secretion, medium was supplemented with 10 µg/ml gentamicin. For bacterial killing assays, 4 x 106 cells/well were placed into 24-well cluster plates for measurements of H2O2 secretion; 4 x 106 cells in 100 µl medium were placed on baked (220°C, 4 h), endotoxin-free, round, 9 mm-diameter glass coverslips in 35 mm tissue-culture dishes; and for the production of acid peptide extracts, 1 x 107 cells/well were placed in six-well cluster plates. Monocytes were obtained by plastic or glass adherence after 2 h at 37°C, 5% CO2, 98% humidity, followed by four vigorous washings in prewarmed GBSS, giving a purity of >98% as determined by Giemsa staining. Average monocyte counts were 8000/well. Monocytes were incubated for 48 h prior to studies. Medium was changed after 24 h. Following treatment protocols were applied to monolayers: dexamethasone, 2.5 x 10–7 M, a steroid receptor saturating concentration [12 ] for 36 h; IFN-{gamma}, 200 U/ml for 18 h; and thrombin, 10 U/ml, and SFLLRN-14, 200 µM, both at a maximal stimulating concentration [21 ] for 18 h.

Bacteria
S. typhimurium strain 14028 s [34 ] and its attenuated mutant phoP (14028 s phoP::Tn10 [25 ]) were gifts from D. Guiney (Department of Medicine, UC50, La Jolla, CA); L. monocytogenes strain EGD was a gift from R. Zinkernagel (Department of Pathology, University of Zürich, Zürich, Switzerland). Salmonellae were cultured in Luria-Bertani (LB) broth and Listeria in trypticase soy broth. Overnight cultures (~2x109 bacteria/mL for Salmonella and ~5x108 for Listeria) were washed three times. For the challenge of monocyte monolayers, bacteria were washed in PBS and adjusted to a concentration of 4.5 x 107 bacteria/ml, followed by opsonization for 20 min in 50% fresh human serum at 37°C. For radial diffusion killing assays, bacteria were washed in HEPES-HCl, pH 6.0, supplemented with 0.3% (w/v) dextrose (HEPES-dextrose) and diluted in 2% (w/v) agarose (Bio-Rad, Hercules, CA) at 40°C in HEPES-dextrose to give a final concentration of ~107 bacteria/ml. The macrophage-inhibitory cytokine (MIC) of gentamicin was 5 µg/ml, was determined for S. typhimurium 14028 s by serial 3:4 dilutions in LB broth and complete cell culture medium, and was identical (5 µg/ml) in both.

Reagents
SFLLRN-14 (14 amino acid peptide: Ser-Phe-Leu-Leu-Arg-Asn-Pro-Asn-Asp-Lys-Tyr-Glu-Pro-Phe) was from Bachem (Switzerland) or Sigma-Aldrich (St. Louis, MO). The scrambled peptide Glu-Pro-Phe-Ser-Phe-Leu-Leu-Arg-Pro-Asn-Asp-Asn-Lys-Tyr, composed of the amino acids of SFLLRN-14, was from Bachem. Human thrombin, luminol, HRP, PMA, dexamethasone, MTT, and HEPES were from Sigma-Aldrich. IFN-{gamma} was from PeproTech (Rocky Hill, NJ) and LPS, from Escherichia coli, trypticase soy broth, and LB broth were from Difco Laboratories (Detroit, MI). Low endosmosis, low melting temperature agarose was from Bio-Rad.

Bacterial killing by intact monocytes
Antimicrobial activity was measured as a decrease in the number of CFU of cell-associated bacteria within 3 h after challenge, as described previously [3 , 35 ]. In brief, monolayers in 24 cluster wells were challenged with 4.5 x 106 opsonisized bacteria per well. After 15 min of incubation for phagocytosis, noningested bacteria were removed by three thorough washes with GBSS. CFU were counted subsequently (for baseline counts) and after incubation for 3 h in complete medium. In some experiments with Salmonellae, the medium was supplemented with the MIC of 5 µg/ml gentamicin. For quantitative cultures of bacteria, monocytes were lysed in 0.5 ml distilled water, and serial dilutions of the lysates were cultured on LB agar for 20 h at 37°C for counting CFU. Enumeration of bacterial colonies was always done at a dilution giving 10–102 colonies. Results are given as the logarithmic change in the number of CFU between baseline values and after incubation of infected mononuclear phagocytes for 3 h from quadruplicate wells per experiment. With all three strains, bacterial baseline counts were between 104 and 5 x 104 per monocyte monolayer.

Measurement of H2O2 secretion by chemiluminescence
H2O2 secretion was quantified by chemiluminescence in a luminol-HRP-amplified system as described previously [13 , 36 ]. Total photoemission was detected in a LKB 1251 luminometer (LKB Produkter, Bromma, Sweden), which converts photons with a photomultiplier tube into an electric current. This luminometer permits the automated, simultaneous, discontinuous measurement of up to 25 samples kept at 37°C with intermittent shaking. Luminol-amplified chemiluminescence primarily measures H2O2 with only minimal effect of other ROI generated during the respiratory burst. For the studies, 1.36 mg luminol was dissolved in 20 µl dimethyl sulfoxide and diluted directly in 50 ml GBSS containing 5 mg HRP. Coverslips with monolayers were washed by two passages through prewarmed GBSS, individually placed in cuvettes, and covered immediately with 1 ml luminol-HRP solution. After warming the cuvettes to 37°C for 10 min, baseline chemiluminescence was recorded for each sample. The respiratory burst was then triggered by the addition of PMA (final concentration, 175 ng/ml), using the system’s automated injection pump, which synchronizes the application of the trigger with subsequent measurements. The emission of photons was recorded discontinuously during 40 min until after peak chemiluminescence was reached. In each experiment, measurements were performed on four coverslips per treatment group as indicated. Results are given in percent mV of control cells. To evaluate an influence of variable cell numbers from individual coverslips, monolayers were lysed by exposure of monolayers to stromatolysin. Free nuclei were then counted in a Coulter Model S counter (Coulter Electronics, UK).

Analysis of myeloperoxidase (MPO) and MPO activity
For FACS analysis of intracellular MPO expression, macrophages were washed twice in PBS-A, fixed, and permeabilized with Fix & Perm reagent (Caltag Laboratories, Burlingame, CA). After incubation in 10% mouse serum to block nonspecific, IgG-binding sites, 1 x 105 cells were incubated with PE anti-MPO and FITC anti-CD14 (both Becton Dickinson, Basel, Switzerland) for 30 min. In parallel, cells were stained with nonspecific control antibodies (Becton Dickinson). After washing with PBS-A, the cells were analyzed using a FACSCalibur (Becton Dickinson), equipped with a 488-nm argon laser and CellQuest software. MPO expression on CD14+ macrophages is expressed as mean fluorescence and percent positive cells, respectively. MPO activity was measured in 96-well microcultures of monocytes cultured in vitro for 48 h by H2O2-dependent oxidation of diaminobenzidine using a commercial kit (Boehringer Mannheim, Germany).

Acid peptide extracts and measurement of antimicrobial activity in a radial diffusion assay
After culture of monocytes in six-well cluster plates for 48 h, monolayers were washed three times with GBSS to remove the culture medium containing 20% serum, and cells were scraped on ice in cold 5% acetic acid, frozen, thawed three times, and extracted for 2 h on melting ice with magnetic stirring bars. Cell debris was removed by centrifugation at 12,000 g, and the cleared supernatants were frozen at –80°C and lyophilized. Lyophilisates were resuspended in 1 mM HEPES-HCl, pH 6.0, and recentrifuged at 12,000 g, and protein concentrations were measured in a Nano-Drop® spectrophotometer by the Bradford micro-method (Bio-Rad) using BSA in HEPES-HCl, pH 6, as standard. For the antimicrobial assays, protein concentrations were adjusted to a concentration of 150 µg/ml. Antimicrobial activity was measured in a modified radial diffusion assay, originally described by Lehrer et al. [37 ]. In brief, 20 ml 2% agarose in HEPES-dextrose containing ~2 x 107 bacteria/mL were pored on a horizontally leveled surface into plastic covers for microtiter plates (Semadeni, Ostermundigen, Switzerland), resulting in a 3-mm layer, in which after solidification, wells were punched with the help of a template equipped with 2 mm-diameter steel needles. Each well was filled with 5 µl serial dilutions of acid peptide extracts, and plates were incubated for 3 h in a humified atmosphere for 3 h at 37°C, overlayed with 0.8% agar in brain heart infusion broth supplemented with 1% yeast extract (Difco Laboratories). After incubation overnight at 37°C, the agar overlay was removed and replaced with 0.5 mg/mL MTT in DMEM without phenol red and placed into a water-saturated atmosphere of 5% CO2 at 37°C for 30 min for the formation of blue, insoluble formazan by surviving bacteria. Plates were then fixed with 2.5% paraformaldehyde in PBS, pH 7.4, and photographed in a CRX gel doc system (Bio-Rad), and clearing zones were measured with the QuantityOne® software package with the included, automated quantification tool (Bio-Rad).

Statistical methods
Results are given as mean ± SEM or ± SD as appropriate. Mean values were compared by one way of variance (ANOVA) applying Dunnett’s post-test correction for comparison of multiple sets of data with one set of control data and the Bonferroni multiple comparison test for comparison of multiple values using the GraphPad Instat® software (GraphPad, San Diego, CA).


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RESULTS
 
Effects of IFN-{gamma}, thrombin, and SFLLRN-14 on the antibacterial activity of monocytes
In preliminary experiments, the best inoculum size was determined for measurements of the antimicrobial activity against S. typhimurium. Then, comparative experiments with cells exposed for 18 h to IFN-{gamma}, thrombin, and SFLLRN-14 were performed. Monocytes cultured for 2 days killed, after treatment with IFN-{gamma}, thrombin, or SFLLRN-14, significantly more bacteria compared with unstimulated monolayers (Fig. 1 ). The effect of the PAR-agonist SFLLRN-14 and thrombin surpassed the effect of IFN-{gamma} in several experiments, but the difference between the pooled mean values of the reduction in CFU was not statistically significant (P>0.05).


Figure 1
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  		Figure 1. Comparison of the reduction of CFU of S. typhimurium by human monocytes treated with SFLLRN-14, thrombin, or IFN-{gamma} or control cells. Monocytes were cultured for 18 h prior to exposure to SFLLRN-14 (200 µM), thrombin (10 U/ml), or IFN-{gamma} (200 U/ml) and then challenged with opsonisized S. typhimurium 14028 s. {Delta} log CFU: mean ± SEM of the difference between the log10 of CFU 15 min after challenge for phagocytosis and removal of nonphagocytized bacteria by three washes and after 3 h for bacterial killing. Results from six independent experiments with quadruplicate wells for each control group and each treatment group. Control cells compared with each of the three treatment groups, P < 0.01; treatment groups among themselves, P > 0.05. Monocytes were isolated from freshly drawn blood and cultured in autologous serum; medium was supplemented with a MIC of gentamicin after phagocytosis of bacteria to suppress extracellular bacterial growth. Note: A difference of 1 log10 corresponds to a difference of 90%.

To exclude the possibility that thrombin increased the anti-Salmonella activity by nonspecific effects on monocytes, the antimicrobial activity of monocytes treated with thrombin, heat-inactivated thrombin (95°C, 5 min), and control cells was compared. Thrombin-activated monocytes killed within 3 h were >80% of ingested bacteria (reduction by 0.895 log10±0.06; mean±SD from quadruplicate wells) compared with <25% killing by control cells (reduction by 0.24±0.05 log10; compared with thrombin P<0.01) as well as by monocytes treated with heat-inactivated thrombin (reduction 0.25±0.03 log10, compared with thrombin P<0.01, compared with control P>0.05).

To assure that the difference in the reduction of the number of CFU was not dependent on differences in the number of bacteria phagocytized by treated or control cells, we compared the number of bacteria 15 min after challenge and subsequent washing of monocyte monolayers (Table 1 ). By this measure, we found no indication that the treatments had a significant effect on the phagocytic rate.


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  		Table 1. Comparison of the Number of S. phimurium Associated to Monocyte Monolayers 15 min after Challenge and Removal of Noningested Bacteria by Three Washings

We have shown previously in dose-response studies that the maximal effect of SFLLRN-14 on the induction of chemokine expression in monocytes occurs at a concentration of 200 µM [22 ]. This concentration appears high at first, but all experiments were performed with medium containing 20% human serum with preserved proteolytic activity. Comparable dose-response curves have been reported by others for SFLLRN in the monocytic cell line U937 with no effects of a scrambled peptide of the same amino acid composition [38 ]. Nevertheless, we have conducted control experiments with a scrambled SFLLRN-14 peptide that had no effect on the anti-Salmonella activity of monocytes (Fig. 2 ), excluding a nonspecific peptide effect.


Figure 2
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  		Figure 2. Comparison of the reduction of CFU of S. typhimurium by human monocytes treated with SFLLRN-14, a scrambled peptide composed of the 14 amino acids of SFLLRN-14, and control cells. Monocytes were cultured for 18 h prior to exposure to SFLLRN-14 (200 µM), the scrambled peptide (200 µM), or the solvent (PBS) and then challenged with opsonisized S. typhimurium 14028 s. Srambled: Glu-Pro-Phe-Ser-Phe-Leu-Leu-Arg-Pro-Asn-Asp-Asn -Lys-Tyr, a scrambled peptide of SFLLRN-14. {Delta} log CFU: man ± SD of the difference between the log10 of CFU 15 min after challenge for phagocytosis and removal of nonphagocytized bacteria by three washes and after 3 h for bacterial killing. Results from two independent experiments with cells from two different donors and quadruplicate wells for each control group and each treatment group. For both experiments, control cells compared with the SFLLRN-14-treatment group, P < 0.01; scrambled peptide versus control cells, P > 0.5.

Next, we investigated whether thrombin-induced, antimicrobial activity also increases killing of L. monocytogenes and that of the attenuated mutant S. typhimurium phoP, which is more susceptible to cationic antimicrobial peptides and further, has an inferior capacity to hinder phagolysosomal fusion compared with the parent strain [28 ]. In these experiments Listeria and the phoP mutant were more susceptible to killing by monocytes than the wild-type S. typhimurium, and activation of monocytes resulted in a significantly increased killing rate (Fig. 3 ), thus confirming the results obtained with the wild-type of S. typhimurium. The increment in the killing rate of S. typhimurium phoP appeared, however, less-striking, most probably because of a much more efficient killing of ~90% of the bacteria, even by control cells. Therefore, a further increase in the killing activity by thrombin had to remain modest compared with killing of the wild-type.


Figure 3
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  		Figure 3. Killing of L. monocytogenes (LM), S. typhimurium wild-type (ST), and its attenuated mutant phoP by human monocytes treated with 10 U thrombin for 24 h prior to challenge or control cells. Results from two independent experiments. (A) Cells were prepared from a commercial buffy coat; (B) cells from heparinized blood cultured in autologous serum. Mean log10 ± SD from quadruplicate wells per group and time-point. P < 0.002 for each comparison between thrombin treatment and control for Listeria and S. typhimurium wild-type. For S. typhimurium phoP, P = 0.059 and 0.036. Gentamicin was omitted in these experiments. Note that control cells killed 50–60% of Listeria, 10–25% of the wild-type of Salmonella, and 90% (1 log10) of S. typhimurium phoP.

Effects of IFN-{gamma}, thrombin, SFLLRN-14, and LPS on the respiratory burst and MPO activity
To gain insight into the mechanisms of monocyte antimicrobial activation, we studied the effects of thrombin, SFLLRN-14, and IFN-{gamma} on the capacity of the phagocytes to mount a respiratory burst. To that end, H2O2 secretion upon stimulation with PMA was measured in activated and control phagocytes by HRP-luminol-amplified chemiluminescence. Exposure of monocytes cultured in vitro for 2 days to IFN-{gamma} resulted, as expected, in a fourfold increased capacity to secrete H2O2, whereas no significant response could be observed after exposure to LPS, SFLLRN-14, or thrombin (Fig. 4 ). As monocytes prepared from buffy coats were for unknown reasons more responsive to the applied stimuli compared with monocytes prepared from freshly drawn blood, results of experiments with cells from these two sources were analyzed separately (Fig. 4A and 4B) . Both large groups of independent experiments gave comparable results. Cell counts measured for each well after completion of H2O2 quantification were uniform and had no influence on the measured burst (not shown).


Figure 4
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  		Figure 4. H2O2 secretion upon PMA stimulation of blood-derived monocytes treated with IFN-{gamma}, LPS, thrombin, or SFLLRN-14. The oxidative burst of monocytes cultured in vitro for 2 days and treated for 18 h with IFN-{gamma} (100 U/ml), LPS (100 ng/ml), thrombin (10 U/ml), or SFLLRN-14 (200 µM) or untreated control cells was compared. (A) Results from 11 independent experiments with cells prepared from buffy coats and (B) results from 11 independent experiments with cells prepared from freshly drawn, heparinized blood and cultured in autologous serum. Results are given in percent of peak chemiluminescence (mV) measured in control cells as mean ± SEM combined from 11 independent experiments with quadruplicate measured monolayers in each experiment. P < 0.01 for the comparison of IFN-{gamma} with control cells; P > 0.05 for all other comparisons between treated and control cells.

As monocytes cultured in vitro for several days can lose MPO activity [39 ], we excluded the possibility that thrombin conserved this constituent of the halide-peroxidase system and thereby increased the oxidative, antimicrobial potency. To that end, expression and activity of MPO were compared in cells treated for 24 h with 10 U thrombin and untreated control cells cultured for the same amount of time without additives. Thrombin treatment reduced the percentage of MPO-positive cells from 34.3 ± 4% to 12.6 ± 1.5%, as well as the average intensity of fluorescence after staining with a mAb to human MPO, and MPO activity of monocyte cultures remained similar with and without thrombin treatment: 47.6 ± 19 arbitrary units compared with 45.4 ± 8.9 in thrombin-treated cells (mean±SD, n=5, P>0.5). Both observations excluded the possibility that thrombin enhances oxidative killing of bacteria by affecting MPO.

Effects of thrombin, SFLLRN-14, IFN-{gamma}, and dexamethasone on the antimicrobial activity of peptide extracts from human monocytes
We then turned to the analysis of acid peptide extracts from monocytes activated by thrombin, SFLLRN-14, or IFN-{gamma}, from control cells cultured without additives for the same time, as well as from cells deactivated by dexamethasone. We and others [21 , 37 ] have shown that such extracts from mononuclear phagocytes contain several antimicrobial peptides. A qualitative assessment of several extracts after acid urea gel resolution showed identical bands of bacterial killing in overlay assays as described by us previously [21 ]. The radial diffusion assay originally developed by Lehrer et al. [37 ] proved to give highly reproducible results. In contrast to the original procedure, we changed the buffer system in the agarose gel to an acid system with low molarity and devoid of phosphate anions, as several antibiotic peptides work best at low pH and are inhibited by phosphate [40 ]. In preliminary attempts to establish the system, we used MES buffers, pH 6–6.5, used by Tang et al. [40 ] but found that the bacterial strains under study were highly susceptible to the buffer and were killed by MES concentrations as low as 100 mM within 6 h. We therefore changed to HEPES-HCl, which had no antibacterial activity. An example of radial diffusion assays is given in Figure 5 .


Figure 5
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  		Figure 5. Radial diffusion assays measuring the antimicrobial potency of acid-extracted peptides from monocytes treated for 24 h with 200 µM SFLLRN-14, 2 x 10–7 M dexamethasone (DEXA), 10 U thrombin, and 200 U IFN-{gamma} and control cells cultured without additives. Wells were filled with the given amounts of peptides in 5 µl HEPES-dextrose. Surviving bacterial colonies converted MTT to dark blue-insoluble formazan, leaving a distinct clearing zone in the center without surviving bacteria. WT, Wild-type.

First, we examined the antimicrobial activity of acid peptide extracts from thrombin-treated monocytes and control cells. In three independent experiments, thrombin significantly increased the potency of extracts to kill Listeria and Salmonellae (Fig. 6A ).


Figure 6
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  		Figure 6. Comparison of the antimicrobial activity of acid peptide extracts from monocytes treated with various agents against Listeria, S. typhimurium phoP, and its parent strain (wild-type). (A) Peptides (0.75 µg) extracted from thrombin-activated and control cells. Results are given as mean ± SEM from triplicate experiments with duplicate measurements per experiment. Control versus thrombin, P < 0.02, for all three bacteria. Note the inferior susceptibility of the wild-type Salmonella compared with its attenuated mutant. (B) Comparison of the antimicrobial activity of 0.75 µg peptides extracted from SFLLRN-14-activated and control cells against Listeria, S. typhimurium phoP, and its parent strain (wild-type). Results are given as mean ± SEM from triplicate experiments with duplicate measurements per experiment. Control versus thrombin treatment, P < 0.003, for all three bacteria. (C) Comparison of the antimicrobial activity of 0.75 µg peptides extracted from IFN-{gamma} and dexamethasone-treated cells with extracts from control cells against Listeria, S. typhimurium phoP, and its parent strain (wild-type). Results are given as mean ± SEM from triplicate experiments with duplicate measurements per experiment. Control versus dexamethasone, P < 0.01, for Listeria and S. typhimurium phoP; P < 0.05, for the wild-type strain of Salmonella; control versus IFN-{gamma}, P < 0.05, for Listeria; P > 0.05, for the two strains of Salmonella.

To confirm that this increased, antimicrobial potency of extracts was mediated through PAR activation, we studied in the next experiments the effects of SFLLRN-14, the synthetic ligand of PAR-1 (Fig. 6B) . The results of these observations confirm that the increased antimicrobial activity of the extracts is indeed mediated through the PARs.

As we suspected, based on our previous observations that IFN-{gamma} does not increase or even reduce the amount of some antimicrobial peptides in monocytes [20 , 21 ], we investigated the effects of IFN-{gamma} on the antimicrobial activity of acid monocyte extracts and compared the activity with that of extracts from dexamethasone-deactivated macrophages (Fig. 6C) . Although dexamethasone significantly reduced the antimicrobial activity of acid-extractable peptides, IFN-{gamma} failed to increase the activity and even reduced it against L. monocytogenes and in tendency, also against the two strains of S. typhimurium.


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DISCUSSION
 
These studies show for a first time that human mononuclear phagocytes can be activated in vitro through thrombin and a synthetic PAR ligand to kill a larger fraction of pathogenic, facultative, intracellular bacteria than control cells. Thrombin treatment or a PAR-ligand-mediated activation resulted in an antimicrobial efficiency comparable with that of IFN-{gamma}-stimulated cells. This observation is well in line with prior reports that stimulation of monocytes with thrombin or synthetic PAR agonists activates monocytes to synthesize and secrete larger amounts of cytokines and chemokines [18 19 20 21 22 ], among them several chemokines with antimicrobial activity. Taken together, the proteinase-PAR-G-protein pathway appears to be an alternate macrophage activation principle, independent from lymphokines such as IFN-{gamma} or host defense cells such as lymphocytes. The finding that activation of mononuclear phagocytes through the PAR pathway includes increased antimicrobial activity [1 , 2 ] indicates that proteinases such as thrombin play a role in innate antimicrobial host defense by modulating mononuclear phagocyte function.

Phylogenetically, the coagulation and defense systems have coevolved inter-relatedly [41 ]. Wound-sealing and immobilization of invading microorganisms by clot formation, phagocytosis, and release of antimicrobial peptides are found to share common cells and mechanisms in urchins, worms, and other primitive invertebrates as well as in insects (reviewed in refs. [42 , 43 ]). In Drosophila, several proteolytic cascades are activated in response to invading microorganisms, resulting in clotting, antimicrobial active peptide formation, and intercellular signaling [43 ]. Noxious stimuli such as infection and trauma initiate the human coagulation cascade and result in the activation of proteinases such as thrombin and other coagulation factors. This response results in the synthesis and/or release of signaling peptides including the antimicrobially active chemokines shared by monocytes with the formed elements of the coagulation system—the blood platelets [40 , 44 ]. For platelets, the presence of plasma is a prerequisite to kill in in vitro assays [45 ], and proteinases derived from plasma enhance the capacity of monocytes to kill bacteria. Proteinases by their effect through PARs therefore appear to be an important signaling component of innate immunity.

IFN-{gamma} is known to activate the oxidative killing machinery of mononuclear phagocytes and antimicrobial activity against several microorganisms, including Salmonella [32 , 46 ] and Listeria [3 , 13 ]. The capacity to secrete ROIs such as H2O2 has been related to the competence of mononuclear phagocytes to kill microorganisms [8 , 9 , 13 ]. Other mechanisms contributing to killing of Salmonella, however, are also boosted by IFN-{gamma}, such as an enhanced fusion of phagosomes with lysosomes and early, nonoxidative killing [4 , 5 ]. To gain more insight into possible mechanisms of enhanced intracellular killing of bacteria, we measured the capacity of monocytes to secrete H2O2 after activation with thrombin, SFLLRN-14, or IFN-{gamma}. In contrast to IFN-{gamma}, an activation of human monocytes through PARs did not result in a significant enhancement of the capacity to secrete H2O2 in response to PMA in a large number of 22 independent experiments. It appears therefore plausible that although at least an important part of the activation of the antimicrobial activity of human monocytes by IFN-{gamma} depends on oxidative mechanisms, activation through PARs depends on other antimicrobial effector molecules. As activation of monocytes through PARs results in an up-regulation of the expression of several antimicrobial peptides [20 21 22 ], we studied the antimicrobial activity of acid peptide extracts from monocytes activated through PARs or IFN-{gamma}. IFN-{gamma} did not augment the activity of cell extracts against Salmonellae or even lessened that against Listeria. In contrast, activation through PARs resulted in an enhanced activity against all three test organisms. Deactivation by dexamethasone also decreased the antimicrobial activity of monocyte extracts, as expected, from several previous studies that showed a reduced expression of some antimicrobial peptides [20 21 22 , 46 ] and a reduced, nonoxidative killing by intact cells [3 , 12 , 13 , 35 ].

In conclusion, these studies show that stimulation of mononuclear phagocytes through PARs constitutes an alternate activation pathway, presumably by enhancing nonoxidative effector mechanisms. Proteinase-mediated enhancement of innate host defense is phylogenetically old and contributes to human innate immunity.


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ACKNOWLEDGEMENTS
 
This work was supported by Grant 32-102236 of the Swiss National Science Foundation, the Foundation for Research of the Medical Faculty, University of Zürich, and the HOLCIM Foundation.

Received November 6, 2005; revised September 12, 2006; accepted September 19, 2006.


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