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
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
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
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
(22-amino-acid polypeptide), PSMß (44-amino-acid polypeptide), and
PSM
(25-amino-acid polypeptide), respectively [4
].
The HIV-1 LTR contains two binding sites for the transcription factor
nuclear factor-B (NF-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-B in THP-1 cells and induce cytokine synthesis
[specifically, tumor necrosis factor- (TNF), 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.
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
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
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- (IFN-) (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- (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.
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
 View larger version (36K): [in a new window] |
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.
|
 View larger version (31K): [in a new window] |
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.
|
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).
 View larger version (21K): [in a new window] |
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).
|
(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)
.
 View larger version (52K): [in a new window] |
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- (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).
|
(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-,
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-, IL-1ß, TNF, 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- (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- (Fig. 5)
.
 View larger version (42K): [in a new window] |
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- (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).
|
 View larger version (46K): [in a new window] |
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).
|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
B in the monocytic THP-1 cell line and stimulate production of
proinflammatory cytokines (specifically TNF, 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, PSMß , and PSM [4
]. PSM
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 was completely homologous to the delta toxin of S.
epidermidis [4
, 48
].
Strains of Staphylococcus aureus also release factors that
induce production of TNF 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 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.
Received November 17, 2000; revised February 15, 2001; accepted February 19, 2001.
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
B in the immune system Annu. Rev. Immunol. 12,141-179[Medline]B Annu. Rev. Cell Biol. 10,405-455-interferon on human neutrophils: protection from deterioration during storage Blood 80,225-234, and IL-1ß prevent programmed cell death (apoptosis) in human peripheral blood monocytes J. Immunol. 147,3408-3412This article has been cited by other articles:
 |
|
 |
|
  B. Fournier and D. J. Philpott Recognition of Staphylococcus aureus by the Innate Immune System Clin. Microbiol. Rev., July 1, 2005; 18(3): 521 - 540. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Suttmann, N. Lehan, A. Bohle, and S. Brandau Stimulation of Neutrophil Granulocytes with Mycobacterium bovis Bacillus Calmette-Guerin Induces Changes in Phenotype and Gene Expression and Inhibits Spontaneous Apoptosis Infect. Immun., August 1, 2003; 71(8): 4647 - 4656. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Li, J. D. Loike, J. A. Ember, P. P. Cleary, E. Lu, S. Budhu, L. Cao, and S. C. Silverstein The Bacterial Peptide N-Formyl-Met-Leu-Phe Inhibits Killing of Staphylococcus epidermidis by Human Neutrophils in Fibrin Gels J. Immunol., January 15, 2002; 168(2): 816 - 824. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
