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(Journal of Leukocyte Biology. 2001;70:96-102.)
© 2001 by Society for Leukocyte Biology

Stimulation of human neutrophils and monocytes by staphylococcal phenol-soluble modulin

W. Conrad Liles, Anni R. Thomsen, D. Shane O’Mahony and Seymour J. Klebanoff

Division of Allergy and Infectious Diseases, Department of Medicine, University of Washington, Seattle

Correspondence: W. Conrad Liles, M.D., Ph.D., Department of Medicine, Box 357185, HSB I-104, University of Washington, Seattle, WA 98195-7185. E-mail: foghorn{at}u.washington.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Modulins represent microbial products that stimulate cytokine production in host cells. The modulins responsible for gram-positive sepsis remain poorly understood. Staphylococci release a factor (or factors) that activates nuclear factor-{kappa}B and stimulates cytokine production in cells of macrophage lineage. This factor, termed phenol-soluble modulin (PSM), has been recently isolated from culture supernatant of Staphylococcus epidermidis. We examined the effects of PSM on proinflammatory properties of human neutrophils and monocytes in vitro. PSM activated the respiratory (oxidative) burst in neutrophils and primed neutrophils for enhanced respiratory burst activity in response to formyl-methionyl-leucyl-phenylalanine. PSM also stimulated neutrophil degranulation as reflected by increased surface expression of CD11b and CD18, which was accompanied by rapid shedding of L-selectin. Spontaneous apoptosis of both neutrophils and monocytes was inhibited by PSM. Furthermore, PSM also functioned as a chemoattractant factor for both neutrophils and monocytes. Thus, the proinflammatory properties of PSM resemble those of both lipopolysaccharide and bacterial chemotactic peptides. These findings suggest that PSM may play a role in the pathogenesis and systemic manifestations of sepsis caused by staphylococci.

Key Words: inflammation • phagocyte • chemotaxis • apoptosis • respiratory burst • degranulation


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Modulins represent microbial products that stimulate cytokine production in host cells [1 , 2 ]. The best-characterized modulin is lipopolysaccharide (LPS), which is involved in the development of sepsis syndrome in response to gram-negative bacterial infection. However, the modulins responsible for gram-positive sepsis are less well characterized. Staphylococci have been shown to release a factor or factors that activate the HIV-1 long terminal repeat in the transfected monocytic cell line THP-1 [3 ]. This factor, termed phenol-soluble modulin (PSM) based on its partitioning into the phenol layer on hot aqueous phenol extraction, has been recently isolated from the culture supernatant of Staphylococcus epidermidis [4 ]. PSM contains three strongly hydrophobic active polypeptides, designated PSM{alpha} (22-amino-acid polypeptide), PSMß (44-amino-acid polypeptide), and PSM{gamma} (25-amino-acid polypeptide), respectively [4 ].

The HIV-1 LTR contains two binding sites for the transcription factor nuclear factor-{kappa}B (NF-{kappa}B), which also is involved in the initiation of transcription for a large number of host defense-related genes and cytokines [5 , 6 ]. PSM was previously shown to activate NF-{kappa}B in THP-1 cells and induce cytokine synthesis [specifically, tumor necrosis factor-{alpha} (TNF{alpha}), interleukin (IL)-1ß, and IL-6 in THP-1 cells and monocytes] [4 ]. These findings suggest a possible role for PSM in the pathogenesis and clinical manifestations of gram-positive sepsis. In the study reported here, the effects of PSM on human neutrophils and monocytes were further investigated. We report that PSM stimulated multiple proinflammatory properties in phagocytes, including activation of the respiratory burst, inhibition of apoptosis, increased surface expression of CD11b and CD18, shedding of L-selectin, and induction of chemotaxis. These results provided additional evidence for PSM as an important modulator of the innate host response to infection by staphylococci.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Isolation of staphylococcal PSM
Staphylococcal PSM was isolated as described previously [4 ]. Briefly, S. epidermidis UW-3 (University of Washington Medical Center strain 3 [3 ]) was grown overnight on a shaker in 10 L of Iscove’s modified Dulbecco’s medium with N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) and L-glutamine (BioWhittaker, Inc, Walkersville, MD) and supplemented with 1% glucose. Bacteria were removed by centrifugation (6,000 g x30 min) and filtration. The resultant supernatant was concentrated by tangential and centrifugal ultrafiltration to approximately 20 mL. This preparation was dialyzed extensively against 0.4 M NaCl in 25-kDa-molecular-mass cutoff tubing, then against distilled water in 12-kDa-molecular-mass cutoff tubing. A 50-mL volume of buffer-saturated phenol (Gibco-BRL, Rockville, MD) was added to the product, followed by sufficient 1 M sodium acetate (pH 4.7) to bring the aqueous portion to 0.1 M. The mixture was converted to a single phase by agitation at 65°C for 1 h. After cooling and centrifugation for 15 min, the phenol layer was removed and saved. The aqueous layer was extracted twice with 25 mL of buffer-saturated phenol. The pooled phenol layers were extensively dialyzed against distilled water in 12-kDa-molecular-mass cutoff tubing at 4°C. This procedure resulted in a fine precipitate, which was vigorously mixed into the aqueous retentate. This PSM material was lyophilized and stored at -20°C until use.

Preparation of purified populations of normal human phagocytes
Venous blood was collected from healthy human volunteers using 0.2% K2 EDTA as an anticoagulant. Neutrophils were isolated by sequential sedimentation in dextran (Sigma, St. Louis, MO) in 0.9% sodium chloride, centrifugation in Histopaque-1077 (Sigma), and hypotonic lysis of erythrocytes, as previously described [7 , 8 ]. Preparations contained >97% polymorphonuclear leukocytes, of which >95% were neutrophils as determined by modified Wright’s staining (Diff-Quik Stain Set; Baxter, McGraw Park, IL). Platelet-depleted monocytes were isolated from peripheral blood mononuclear cells (PBMCs) by negative immunoselection using an indirect magnetic labeling system (Monocyte Isolation Kit; Mitenyi Biotec, Auburn, CA). In brief, the PBMC fraction was isolated from anticoagulated venous blood by centrifugation over Histopaque-1077. Platelets were depleted from the PBMCs by repeated washing in phosphate-buffered saline (PBS) containing 2 mM EDTA. Monocytes were isolated from the platelet-depleted PBMC fraction by negative immunoselection using the monocyte isolation kit and depletion column type BS (Miltenyi Biotec, Auburn, CA) according to the vendor’s instructions. The resulting preparations contained >95% mononuclear cells of which >95% were CD14+ [using monoclonal antibody (mAb) M{phi}P9-phycoerythrin (Becton Dickinson Immunocytometry Systems, San Jose, CA)] by immunofluorescence flow cytometry (fluorescein-activated cell sorter [FACS]).

Assay of luminol-enhanced chemiluminescence
Luminol-enhanced chemiluminescence was used as a sensitive measure of the human neutrophil respiratory burst as previously described [9 , 10 ]. In brief, purified human neutrophils (106) were preincubated for 15 min in a 0.5-mL volume of RPMI 1640 with 10 mM HEPES and L-glutamine in polystyrene chemiluminescence cuvettes (Analytical Luminescence Laboratory, San Diego, CA) at room temperature. At the start of the assay, 10 µM luminol (Sigma) and the designated concentration of PSM (30 ng/mL–3 µg/mL) were added to the reaction mixture. Luminol-enhanced chemiluminescence was recorded at 10-min intervals with a Monolight 1500 luminometer (Analytical Luminescence Laboratory, Sparks, MD). Chemiluminescence is reported as relative light units. For priming of the respiratory burst, neutrophils were preincubated with PSM (100 ng/mL) for 30 min prior to the addition of 100 nM formyl-methionyl-leucyl-phenylalanine (fMLP; Calbiochem, San Diego, CA).

Detection of cell surface CD11b, CD18, and L-selectin on neutrophils by FACS
Freshly isolated human neutrophils (5x106/mL) were maintained with and without PSM (100 ng/mL) in RPMI 1640 with 10 mM HEPES for 60 min at 37°C. At the end of this incubation period, cell surface expression of CD11b, CD18, and f48l-selectin (Leu-8) was assayed by direct immunofluorescence flow cytometry using saturating concentrations of commercially available fluorescence-labeled (i.e., either fluorescein isothiocyanate- or phycoerythrin-labeled) murine mAbs, as described previously [11 , 12 ]. In brief, the isolated cell preparations were suspended in ice-cold PBS, and aliquots (50 µL, 106 cells) were mixed with 50 µL of mAb diluted in PBS containing 0.1% bovine serum albumin and 0.1% sodium azide in wells of a 96-well polyvinyl microtiter assay plate (Costar, Cambridge, MA) kept on ice. Cells were stained for 45 min at 4°C, washed once with ice-cold PBS, then fixed with 1% paraformaldehyde in PBS and stored in the dark at 4°C before FACS. The following mAbs were obtained from Becton Dickinson: D12-PE (anti-CD11b), L130-fluorescein isothiocyanate (anti-CD18), and SK11-PE (anti-f48l-selectin). Simultaneous negative-control staining reactions were performed with appropriate irrelevant isotype-specific murine fluorescence-labeled immunoglobulin G controls (Becton Dickinson). FACS data were reported as mean fluorescence intensity (MFI) ratios. These values represent the MFI determined for each specific mAb divided by the MFI of the appropriate negative-control antibody [13 ].

Morphologic assessment of apoptosis
Isolated human neutrophils were maintained in vitro for 16 h in RPMI 1640 (supplemented with 10 mM HEPES, 0.2 mM L-glutamine, 25 U/mL of penicillin, and 25 mg/mL of streptomycin) at a concentration of 5 x 106 cells/mL in a 48-well cell culture cluster plate (Costar) at 37°C in a humidified CO2 incubator (5% CO2-95% air). PSM (100 ng/mL), LPS [1 µg/mL (derived from Escherichia coli 055:B5); Sigma], interferon-{gamma} (IFN-{gamma}) (1,000 U/mL; R & D Systems, Minneapolis, MN), granulocyte colony-stimulating factor (G-CSF) (100 ng/mL; Amgen, Thousand Oaks, CA), and granulocyte-macrophage colony-stimulating factor (GM-CSF) (100 ng/mL; Immunex, Seattle, WA) were added to the maintenance cultures as indicated. Isolated human monocytes were maintained at a concentration of 106 cells/mL under similar conditions in Teflon paraformaldehyde vials (Cole-Parmer, Vernon Hills, IL), with PSM (100 ng/mL), LPS (1 µg/mL), and IFN-{gamma} (1,000 U/mL) as indicated. At 0 and 16 h, aliquots of cells were removed, and cytospin (Shandon Southern Cytospin; Shandon, Pittsburgh, PA) samples were prepared. After Wright-Giemsa staining, the samples were scored as either apoptotic or nonapoptotic in a blinded fashion. Apoptotic cells were recognized as cells with diminished cell volume and fragmented, condensed chromatin [14 , 15 ]. Five-hundred cells were counted per sample, and the results are reported as the percentage of apoptotic cells.

DNA fragmentation assay
Isolated human neutrophils were maintained in vitro as described above for morphologic assessment of apoptosis. Aliquots were obtained at 0 and 24 h for quantitation of DNA fragmentation by determination of fractional solubilized DNA by diphenylamine assay as previously described [8 , 16 ]. In brief, 5 x 106 PBS-washed neutrophils were lysed in 0.5 mL of lysis buffer (5 mM Tris-HCl, 20 mM EDTA, 0.5% Triton X-100, pH 8.0), and the lysates were centrifuged (15,000 g) to separate high-molecular-weight DNA (pellet) and cleaved, low-molecular-weight DNA (supernatant). After precipitation with 0.5 N perchloric acid and the addition of diphenylamine reagent, DNA was quantitated by spectrophotometry. Results are reported as relative proportions (percentages) of soluble, low-molecular-weight DNA.

Analysis of phagocyte chemotaxis
PSM-induced chemotaxis of human neutrophils and monocytes was assessed by modifications of a previously described fluorescence-based assay using 96-well chemotaxis chambers containing polycarbonate filters with 8 µM pores (ChemoTx, Neuro Probe Inc, Gaithersburg, MD) [17 , 18 ]. Freshly isolated normal human neutrophils or monocytes (5 x106 cells/mL) suspended in RPMI 1640 containing 10% fetal calf serum were incubated at 37°C with 5 µg/mL of calcein AM (Molecular Probes, Eugene, OR), then washed twice with phenol red-free RPMI 1640 (Sigma) and suspended at 4 x 106 cells/mL in RPMI for the assay. The chamber wells were filled with 29 µL of various concentrations of PSM (from 100 pg/mL to 1 µg/mL) diluted in phenol red-free RPMI or with RPMI as a negative control. Chamber wells containing fMLP (100 nM) or 10% zymosan-activated serum (ZAS) served as positive controls for chemotaxis. ZAS was prepared by incubating human AB-type serum with 100 mg/mL of zymosan (ICN Pharmaceuticals, Inc., Cleveland, OH) for 30 min at 37°C. The filters were applied, and either neutrophils or monocytes (25 µL containing 105 cells) were placed directly onto the filters. All conditions were repeated in quadruplicate for each experiment. The chambers were incubated for 60 min at 37°C. At the end of the incubation period, nonmigrating cells on the origin (top) side of the filter were removed by washing and aspirating with excess phenol red-free RPMI and gentle wiping with a tissue. To determine the percentage of cells that migrated into the bottom chamber during the course of the experiment, the chemotaxis chamber was placed in a multiwell fluorescence plate reader (Cyto Fluor II; PerSeptive Biosystems, Framingham, MA), and fluorescence was determined in the bottom-read position (excitation, 485 nm; emission, 530 nm). The data were reported as a chemotaxis index, normalized to baseline migration of control neutrophils or monocytes.

Statistical analysis
Results were reported as the mean ± SD or SE as specified below, and statistical differences were determined by repeated-measures analysis-of-variance testing using Prism 2.0 software (GraphPad Software, San Diego, CA). Differences were not regarded as significant if P >= 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
PSM stimulated the neutrophil respiratory burst
As measured by luminol-enhanced chemiluminescence, PSM stimulated the inducible respiratory burst in neutrophils in a dose-dependent manner over a PSM range from 30 ng/mL to 3 µg/mL (Fig. 1 ). Inducible respiratory burst activity above baseline (control neutrophils without added PSM) was evident within 30 min of the exposure of neutrophils to PSM. This response was maximal at 60 min, then progressively declined over the ensuing 120 min.



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  		Figure 1. PSM stimulates the respiratory burst in neutrophils. Neutrophils (5 x 105/mL) were incubated as described in Materials and Methods under the conditions listed in the key. Respiratory burst activity was determined at the designated time points by assay of luminol-enhanced chemiluminescence. RLU, relative light units. The data represent mean values from six experiments involving independent donors.

 
PSM primes neutrophils for the inducible respiratory burst
Certain agents such as LPS can prime neutrophils for enhanced release of oxygen metabolites of the respiratory burst in response to a second stimulus [19 ]. PSM also effectively primed neutrophils for this response. When neutrophils were preincubated with PSM (100 ng/mL) for 30 min, the magnitude of the luminol-enhanced chemiluminescence response to fMLP (100 nM) was significantly increased (Fig. 2 ). Both PSM (100 ng/mL) and fMLP (100 nM) induced modest chemiluminescence responses when added separately to neutrophils. When fMLP (100 nM) was added to neutrophils after preincubation with PSM (100 ng/mL), maximal chemiluminescence was observed at 30 min. This maximal amount of chemiluminescence was significantly greater than the sum of the chemiluminescence responses stimulated by both PSM (100 ng/mL) and fMLP (100 nM) when these agents were used separately to stimulate neutrophils (P <0.05).



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  		Figure 2. PSM primes neutrophils for activation of the respiratory burst. Neutrophils, at a concentration of 5 x 105/mL, were incubated with PSM (100 ng/mL), fMLP (100 nM), PSM (100 ng/mL), or polymorphonuclear neutrophils (PMN) for 30 min prior to the addition of fMLP (100 nM), or without addition of a stimulus as described in Materials and Methods. Respiratory burst activity was determined at the designated time points by assay of luminol-enhanced chemiluminescence. RLU, relative light units. Data represent the mean ± SE from six experiments involving independent donors.

 
PSM rapidly increased CD11b/CD18 expression and decreased L-selectin expression on the cell surface of neutrophils
Activation of neutrophils alters the surface expression of major adhesion molecules, including CD11b/CD18 (Mac-1) and L-selectin. After their biosynthesis, a proportion of the ß-integrin CD11b/CD18 (Mac-1) heterodimers are stored in intracellular secondary and tertiary granules in granulocytes. The surface expression of CD11b/CD18 increased rapidly on neutrophils during the process of degranulation, induced by a wide variety of activating stimuli including chemotactic factors, phorbol esters, and cytokines [20 21 22 23 24 ]. This phenomenon does not require de novo protein synthesis and occurs as a result of translocation of granule-associated contents to the cell surface. In contrast, surface expression of L-selectin is rapidly down-regulated on activated neutrophils as a result of metalloprotease-mediated cleavage and subsequent shedding from the cell surface [25 26 27 28 ]. These changes in adhesion molecule expression are considered to play a physiologic role in neutrophil migration to inflammatory sites [29 ].

The effects of PSM on CD11b/CD18 and L-selectin expression were evaluated by FACS. PSM (100 ng/mL) rapidly up-regulated cell surface expression of CD11b and CD18 on neutrophils within 60 min (P <0.05) (Fig. 3 ). This enhanced expression of CD11b/CD18 was accompanied by significant down-regulation of L-selectin expression (P <0.05) (Fig. 3) . CD11b expression increased greater than fourfold, with the MFI ratio rising from a baseline value of 26.0 to 114.4. A threefold increase in CD18 expression was observed (MFI ratio baseline, 8.0; PSM, 24.7). Under the same conditions, PSM (100 ng/mL) induced an 80% decrease in L-selectin expression (MFI ratio baseline, 18.3; PSM, 3.6).



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  		Figure 3. PSM rapidly increases CD11b/CD18 expression and decreases L-selectin on the cell surface of neutrophils. Neutrophils were incubated at 37°C for 1 h with (filled bars) and without (open bars) PSM (100 ng/mL) as described in Materials and Methods. At the end of the incubation period, surface expression of CD11b, CD18, and L-selectin was determined by direct immunofluorescence flow cytometry (FACS). The results are reported as MFI ratios, which represent the specific mAb MFI divided by the MFI of an appropriate isotype control mAb MFI. Values represent the means ± SD from four experiments involving independent donors. Asterisk (*) denotes a significant difference from control (no PSM) neutrophils as determined by analysis of variance (P <0.05).

 
PSM delayed spontaneous apoptosis in neutrophils in vitro
Neutrophils rapidly undergo spontaneous apoptosis when maintained in vitro [14 , 16 , 30 31 32 33 ]. Although neutrophils appear to be committed to death via apoptosis, their life span and functional activity can be significantly increased with proinflammatory cytokines and bacterial products such as LPS [14 , 16 , 30 , 32 ]. Under the experimental conditions used for this study, 77.3 ± 5.9% (mean ± SD) of control neutrophils developed apoptotic morphology after an incubation period of 16 h. Consistent with previous reports, the proportion of neutrophils undergoing apoptosis was significantly decreased by treatment with GM-CSF (100 ng/mL; 44.7 ±3.5% apoptosis), G-CSF (100 ng/mL; 43.3 ±3.1% apoptosis), IFN-{gamma} (1,000 U/mL; 50.7 ±4.7% apoptosis), or LPS (1 µg/mL; 43.0±3.5% apoptosis) (P <0.05) (Fig. 4 ). Treatment with PSM (100 ng/mL) resulted in a comparable decrease in the extent of neutrophil apoptosis, with only 45.5 ± 4.0% of neutrophils developing apoptotic morphology during the 16-h incubation (P <0.05 compared with control neutrophils) (Fig. 4) .



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  		Figure 4. PSM inhibits spontaneous neutrophil apoptosis in vitro. Neutrophils were maintained in vitro for 16 h at 37°C with PSM (100 ng/mL), LPS (1 µg/mL), IFN-{gamma} (1,000 U/mL), G-CSF (100 ng/mL), or GM-CSF (100 ng/mL) and without a stimulus (control), as described in Materials and Methods. The percentage of cells undergoing apoptosis in each condition was determined morphologically after modified Wright staining of cytospin preparations. Values represent the means ± SD from six experiments performed with independent donors. Asterisk (*) denotes a significant difference from control neutrophils as determined by repeated-measures analysis of variance (P<0.05).

 
Apoptosis is characterized by cytoplasmic shrinkage, membrane blebbing, chromatin condensation, and DNA fragmentation. The latter feature is considered a hallmark of an apoptotic mode of death [34 ]. To confirm that PSM inhibited spontaneous neutrophil apoptosis, the development of DNA fragmentation in neutrophils was quantitated after maintenance in vitro for 24 h. At 0 h (baseline), freshly isolated neutrophils contained only 1.2 ± 0.8% (mean ±SD) of cleaved, soluble, low-molecular-weight DNA (data not shown). At 24 h, the proportion of cleaved low-molecular-weight DNA increased to 54.1 ± 3.2% in control neutrophils. Development of low-molecular-weight DNA during neutrophil maintenance in vitro was significantly inhibited by incubation with G-CSF (100 ng/mL), GM-CSF (100 ng/mL), IFN-{gamma} (1,000 U/mL), LPS (1 µg/mL), or PSM (100 ng/mL) (P <0.05). The percentage of low-molecular-weight DNA was reduced to 38.4 ± 2.1% by PSM, 35.7 ± 1.8% by LPS, 29.6 ± 3.0% by IFN-{gamma}, 35.9 ± 2.4% by G-CSF, and 35.5 pm 1.9% by GM-CSF (data not shown).

PSM delayed spontaneous apoptosis in monocytes in vitro
Normal human monocytes also undergo spontaneous apoptosis without requiring additional external stimuli when cultured in vitro [11 , 35 36 37 ]. The process of apoptosis in monocytes can be delayed in vitro by treatment with proinflammatory mediators such as LPS, GM-CSF, IFN-{gamma}, IL-1ß, TNF{alpha}, and CD40 ligand (CD154) [11 , 35 36 37 ]. To determine whether PSM affects monocyte apoptosis, the development of apoptotic morphology was assessed in monocytes maintained in culture for 16 h. At baseline, virtually no apoptosis was detected in freshly isolated human monocytes [0.4 ±0.3% (mean ±SD)] (data not shown). After 16 h in culture, 21.7 ± 1.6% of monocytes developed morphologic features of apoptosis. Treatment with PSM (100 ng/mL), LPS (1 µg/mL), or IFN-{gamma} (1,000 U/mL) significantly reduced the percentage of monocytes undergoing apoptosis during the culture period (P <0.05) (Fig. 5 ). The percentage of apoptotic cells was reduced to 7.7 ± 1.3% by PSM, 8.8 ± 1.3% by LPS, and 10.9 ± 1.7% by IFN-{gamma} (Fig. 5) .



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  		Figure 5. PSM inhibits spontaneous monocyte apoptosis in vitro. Monocytes were maintained in vitro for 24 h at 37°C with PSM (100 ng/mL), LPS (1 µg/mL), and IFN-{gamma} (1,000 U/mL) and without a stimulus (Control) as described in Materials and Methods. The percentage of cells undergoing apoptosis in each condition was determined morphologically after modified Wright’s staining of cytospin preparations. Values represent the means ± SD from six experiments performed with independent donors. Asterisk (*) denotes a significant difference from control monocytes as determined by repeated-measures analysis of variance (P<0.05).

 
PSM stimulated neutrophil chemotaxis
To determine whether PSM could function as a chemoattractant for neutrophils, the relative abilities of PSM, fMLP, and 10% ZAS to induce human neutrophil chemotaxis were compared in a fluorescence-based assay using 96-well chemotaxis chamber plates [17 , 18 ] (Fig. 6 ). PSM induced migration of neutrophils in a characteristic dose-dependent manner, with optimal neutrophil migration observed with PSM at a concentration of 1 ng/mL (Fig. 6) . PSM-induced migration of neutrophils above baseline [control (no stimulus)] was detected by 15 min and was maximal at 60 min (data not shown). At 1 h, 16.3 ± 1.1% (mean±SE) of neutrophils migrated under control conditions (no stimulus). PSM (1 ng/mL) induced migration of 37.4 ± 6.8% of neutrophils during the 1-h incubation period, yielding a chemotaxis index value of 2.3 ± 0.4 (mean ±SD) when normalized to the control value (P <0.05) (Fig. 6) . The magnitude of PSM-induced neutrophil chemotaxis was comparable to the levels of chemotaxis mediated by fMLP (100 nM; chemotaxis index, 2.7 ±0.4) and 10% ZAS (chemotaxis index, 3.0 ±0.4) (Fig. 6) .



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  		Figure 6. PSM mediates chemotaxis of neutrophils and monocytes. The effect of PSM on neutrophil and monocyte chemotaxis was assessed by a rapid fluorescence-based assay of neutrophil migration in vitro as described in Materials and Methods. Chemotaxis was measured during a 1-h incubation period. The data are reported as a chemotaxis index, normalized to baseline migration of control neutrophils or control monocytes, respectively. Values represent the means ± SD from four experiments performed with independent donors. Asterisk (*) denotes a significant difference from control (no PSM) neutrophils or control monocytes as determined by analysis of variance (P <0.05).

 
PSM stimulated monocyte chemotaxis
The ability of PSM to stimulate monocyte chemotaxis was also assessed. During the 1-h incubation period, 14.7 ± 0.4% (mean ±SD) of normal human monocytes migrated across the chamber filter. PSM stimulated monocyte chemotaxis in a dose-dependent manner, with maximal migration (chemotaxis index, 1.6 ±0.1) observed at a PSM concentration of 1 ng/mL (P <0.05) (Fig. 6) . The magnitude of monocyte migration was comparable to that induced by 10% ZAS (chemotaxis index, 1.5 ±0.2).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In a previous report, staphylococcal PSM was shown to activate NF-{kappa}B in the monocytic THP-1 cell line and stimulate production of proinflammatory cytokines (specifically TNF{alpha}, IL-1ß, and IL-6) from normal human monocytes [4 ]. The present study substantially extends these initial observations and demonstrates that PSM activated multiple inflammatory responses in normal human phagocytes. In neutrophils, PSM directly activated the respiratory burst and primed the cells for an enhanced respiratory burst in response to fMLP (Fig. 1 and 2) . PSM rapidly stimulated neutrophil degranulation, as reflected by increased surface expression of CD11b and CD18, and it caused concomitant shedding of L-selectin (Fig. 3) . The survival of both monocytes and neutrophils was prolonged in vitro by PSM via inhibition of spontaneous apoptosis (Fig. 4 and 5) . PSM also served as a potent chemoattractant for both monocytes and neutrophils, with PSM-mediated chemotactic activity comparable to that mediated by fMLP and 10% ZAS (Fig. 6) .

The staphylococcal PSM examined in this study was derived from Staphylococcus epidermidis, and infection with S. epidermidis is a recognized cause of sepsis and septic shock in humans and animals [38 39 40 41 42 43 44 45 ]. PSM was released spontaneously into the extracellular fluid during overnight staphylococcal culture and was detected in the supernatant fluid on vortexing of washed organisms [3 , 4 ]. This preparation was previously shown to contain three active polypeptides, designated PSM{alpha}, PSMß , and PSM{gamma} [4 ]. PSM{alpha} showed no close homologies in a standard BLAST search, whereas PSMß demonstrated partial homology to previously described gonococcal inhibitors 1, 2, and 3 from Staphylococcus hemolyticus and SLUSH polypeptides A, B, and C from Staphylococcus lugdunensis [4 , 46 , 47 ]. PSM{gamma} was completely homologous to the delta toxin of S. epidermidis [4 , 48 ].

Strains of Staphylococcus aureus also release factors that induce production of TNF{alpha} and IL-1ß by human monocytes [3 , 49 , 50 ]. A soluble proinflammatory component (or components) from S. aureus which induces cytokine production from human PBMCs via a CD14-dependent process has been described previously [51 , 52 ]. This factor differed from PSM in that it was distributed into the aqueous layer on hot phenol extraction. Moreover, PSM induced cytokine release from monocytes via a CD14-independent mechanism (data not shown). We have detected a phenol-soluble factor (or factors) released by S. aureus as well as other staphylococcal species which, like the S. epidermidis PSM described in this study, activates the HIV-1 long-terminal repeat in THP-1 cells [3 ]. Most strains of S. aureus release delta toxin, which is capable of inducing the release of cytokines from human monocytes [50 ]. However, it is not known whether a factor equivalent to PSM{alpha} or PSMß is produced and spontaneously released from S. aureus.

Although infections with staphylococci are well recognized as a clinical cause of sepsis, the pathogenesis of gram-positive sepsis remains unclear. Gram-positive bacteria can produce septic shock in humans, and gram-positive infections account for as many sepsis-related deaths as gram-negative infections each year in the United States [2 ]. However, the microbial products responsible for the pathophysiology of gram-positive sepsis have not been well characterized. With respect to staphylococci, most attention has focused on the potential roles of bacteria-derived lipoteichoic acid and peptidoglycan in this process. Our findings suggest that PSM may participate in the initiation of inflammation during staphylococcal infection. PSM exhibited biological properties characteristic of both LPS and chemotactic bacterial peptides such as fMLP, resulting in activation of multiple proinflammatory responses in neutrophils and monocytes. Like LPS, PSM stimulated production of cytokines by monocytes, inhibited apoptosis, prolonged survival in both neutrophils and monocytes, and primed neutrophils for the respiratory burst. Similar to bacterial peptides such as fMLP, PSM served as a chemotactic agent for both neutrophils and monocytes, directly activated the respiratory burst, and altered adhesion molecule expression on the surface of neutrophils. PSM was previously shown to be more effective than lipoteichoic acid in inducing cytokine production by monocytes [4 ]. Furthermore, PSM is rapidly shed or secreted by bacteria into extracellular fluid. These properties indicate that PSM may play an important role as a pathogen-derived mediator to initiate the host inflammatory response during staphylococcal infection in vivo.


   ACKNOWLEDGEMENTS
 
This work was supported by National Institutes of Health grant AI07763.

Received November 17, 2000; revised February 15, 2001; accepted February 19, 2001.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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