# Staphylococcal Toxic Shock Syndrome Toxin-1 Induces  the Translocation and Secretion of High Mobility Group-1 Protein from Both Activated T Cells and Monocytes

Authors: Shirin Kalyan; Anthony W. Chow
Journal: Mediators of Inflammation
 (2008)

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## Abstract

High mobility group box-1 (HMGB-1) is a DNA-binding protein secreted by
activated monocytes and has been identified as a key late mediator of endotoxic shock. We investigated the regulation of HMGB-1 in human peripheral blood mononuclear cells (PBMCs) following stimulation with the staphylococcal superantigen, toxic shock syndrome toxin-1 (TSST-1), and found that TSST-1, like LPS, induced the secretion of HMGB-1 from human PBMC. However, unlike monocyte-driven sepsis caused by endotoxin, translocation and secretion of HMGB-1 mediated by TSST-1 was dependent on the presence of both activated T cells and monocytes. Furthermore, we show that nuclear HMGB-1 is released from TSST-1 stimulated T cells. This finding presents a basis for investigating the potential of targeting HMGB-1 for the treatment of toxic shock syndrome, and provides further insight on the role of HMGB-1 in the cross-talk between activated monocytes and T cells.


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## Body

## 1. Introduction

High mobility group box-1 (HMGB-1) protein is a
30 kDa nonhistone nuclear DNA binding protein that is shown to have an
extracellular role in inflammation, cell differentiation, adherence, and
motility [1–3]. Extracellular HMGB-1 was initially identified
as amphoterin, a heparin-binding protein promoting neurite outgrowth in the
perinatal rat brain [4]. HMGB-1 is now regarded as an endogenous
danger signal that is passively released by necrotic cells or actively secreted
by stimulated cells [5]. Wang et al. discovered that this pivotal
protein was a late mediator of endotoxin shock [6, 7]. Activated macrophages and monocytes secrete
this inflammatory molecule by a process requiring
acetylation of the protein, which permits its translocation from the nucleus to
secretory lysosomes [8]. Unlike TNFα and IFNγ that appear within the first 4–6 hours post-LPS treatment in mice, HMGB-1 serum levels rise between 16 and 32 hours
after LPS administration. Furthermore, neutralizing antibodies directed at
HMGB-1 rescued mice from lethal endotoxemia even when administered 24 hours
after sepsis initiation [7, 9]. For these reasons, HMGB-1 is viewed as an attractive therapeutic target for various inflammatory disorders including
endotoxic shock [10].Toxic shock syndrome toxin-1 (TSST-1) is a
superantigen (sAg) secreted by some strains ofS. aureus, and this sAg
was subsequently identified as the major
causative agent of toxic shock syndrome [11]. In contrast to LPS-induced sepsis, which is primarily mediated by stimulated monocytes, toxic shock
syndrome induced by sAg’s requires the cross-linking of Vβ-specific regions of the αβ T cell
receptor (TCR) to class II major histocompatibility complex (MHC II) molecules
on antigen presenting cells (APCs) [12]. In addition to massive T cell proliferation, this trimolecular interaction leads to the uncontrolled release of various proinflammatory cytokines
[13], which are pivotal to the pathogenesis of TSS. In the present
study, we sought to determine whether HMGB-1 also plays a central role in
TSST-1-induced hyperinflammatory responses. We found that TSST-1 mediates the translocation and subsequent secretion of intracellular HMGB-1 from resting human PBMC. Unlike previous studies that found that monocytes, but not T cells, released HMGB-1 upon stimulation with either LPS or TNFα
[6, 7, 14], we found that the
loss and subsequent secretion of intracellular HMGB-1 induced by TSST-1 was
dependent on the cooperative interaction of both T cells and monocytes, and
both cell types mobilized HMGB-1 upon TSST-1 treatment.


## 2. Materials and Methods

### 2.1. Toxin Purification

Recombinant TSST-1 was purified from culture supernatants ofS. aureus strain RN4220 previously transformed to carry the tst gene,
using both preparative isoelectric focusing and chromatofocusing
[15]. Toxin purity was assessed by silver staining after sodium dodecyl sulfate-polyacrylamide gel electrophoresis on 14% acrylamide gels, and LPS
activity was undetectable by the Limulus amoebocyte lysate gelation
(sensitivity limit, 10 pg/mL).


### 2.2. Preparation of Cells and Culture Conditions

Fresh human PBMCs from healthy donors were obtained by Ficoll-Paque PLUS (Amersham Biosciences
Corp., Piscataway, NJ, USA) density centrifugation, and cultured in 96-well
U-bottom plates at1.5×106 cells/mL in complete culture medium
consisting of RPMI 1640 (StemCell Technologies Inc., Vancouver, BC, Canada),
10% heat-inactivated fetal bovine serum (HyClone Laboratories Inc., Logan, Utah,
USA), 2 mM L-glutamine (StemCell), 25 mM Hepes buffer (StemCell), and 2 ug/mL
of polymyxin B sulphate (Sigma-Aldrich Corp., St. Louis, Mo, USA). For analysis
of secreted HMGB-1 in cell culture supernatants, PBMCs were plated in 24-well
flat-bottom plates in Opti-MEM I reduced serum medium (Gibco). THP-1 cells
(human monocytic cell line) were obtained from ATCC and kept in recommended
growth media until use, at which time the cells were plated at a concentration
of 1.5×106 cells in Opti-Mem I medium. T cell depletion of human
PBMC was performed using a column-free method of magnetic bead separation
(EasyCep from StemCell Technologies) by positive selection of T cells using an
anti-CD3 conjugated antibody according to manufacturer's directions. This
procedure left the monocytes negatively selected for and untouched for the purpose
of further experimental investigations.


### 2.3. Treatment of PBMC with TSST-1 or LPS

Purified PBMCs were plated in either 24-well or 96-well culture plates overnight at
37°C in 5% CO2 prior to stimulation with 1 nM TSST-1 or 500 ng/mL LPS (Sigma), where noted. Culture supernatants were collected at 24 hours
post-TSST-1 treatment, microcentrifuged at 800xg for 5 minutes, and frozen at –70°C until protein analysis.


### 2.4. Detection of HMGB-1 by Fluorescent Microscopy

PBMCs were first surface-stained for expression of CD3 with mouse
antihuman CD3 IgG (Pharmingen BD Biosciences, Mississauga, ON, Canada) followed by antimouse IgG antibody
conjugated to Alexa594  (Molecular Probes); cells were subsequently fixed using
Cytofix buffer (Pharmingen). For the intracellular  detection of HMGB-1, cells
were permeabilized with Cytoperm (Pharmingen) and incubated for 30 minutes
(4°C) with rabbit polyclonal anti-HMGB-1 antibodies (Orbigen BioCarta, San
Diego,Calif, USA) diluted in blocking buffer
(phosphate buffered saline, 3% FBS)  followed by a secondary antibody with Alexa488-conjugated
goat antirabbit IgG. The anti-HMGB-1 antibodies were raised against the peptide
sequence corresponding to amino acids 166–181 that were previously shown to be
specific for HMGB-1 and not HMGB-2 [16]. Surface expression of HMGB-1 on PBMC was established by culturing
cells (37°C, 5% CO2) directly on coverslips to allow attachment of
adherent cells overnight before stimulation with 1 nM of TSST-1. Cells were
then fixed and surface-stained as above (but without the permeabilization
step). Fluorescent-labeled PBMCs were subsequently stained with Hoechst 3342
nuclear dye (molecular probes), and mounted on slides using prolong antifade
reagent (molecular probes). Cells were visualized with an AxioPlan II
fluorescence microscope equipped with a CCD camera using Northern Eclipse
software (Epix) for acquisition of images. Images were taken with the 63x oil
immersion objective lens, and Adobe Photoshop 6.0 software was used for image
layout.


### 2.5. Flow Cytometric Analysis of Surface-Expressed HMGB-1 on Differentiated Cells

Surface-expressed HMGB-1 was analyzed by flow cytometry (FACSCalibur Flow
Cytometry System, BD BioSciences Pharmingen) using the anti-HMGB-1 antibody and
secondary Alexa488-conjugated antibody described above in conjunction with
phycoerythrin (PE)-conjugated anti-CD3 and anti-CD14 antibodies (BD BioSciences
Pharmingen), with a minimum of 10 000 events collected for each sample.


### 2.6. Western Blot Analysis of Secreted HMGB-1

PBMC or THP-1 culture supernatants were concentrated 10-fold from original volume using
Amicon Ultra centrifugal filters with a molecular weight cut-off of 10 kDa
(Millipore). Some culture supernatants
had been further prepared using the SDS-PAGE Clean-Up Kit (Amersham) according
to the manufacturer’s directions prior to running on a 12% polyacrylamide gel.
Western blotting was performed by semidry transfer of proteins (Trans-Blot SD Semi-Dry Electrophoretic Transfer
Cell, BioRad) onto an Immobilon-P PVDF membrane (Millipore) which had been
blocked for 1 hour at room temperature with 1% BSA, 0.5% Tween in Tris buffered
saline (TBS) prior to overnight incubation (at 4°C) with rabbit polyclonal
anti-HMGB-1 antibody. The membrane was subsequently incubated with antirabbit
IgG horse-radish-peroxidase (HRP)-conjugated secondary antibody for 1 hour at
room temperature on a shaker. Detection of HMGB-1 was performed using super signal
substrate (Pierce) and developed as well as analyzed using the Alpha Innotech
3400 Gel Documentation system (Alpha Innotech, Calif, USA).


### 2.7. Statistical Analysis

Statistical analysis was performed using Prism
3.0 software package (GraphPad). The proportion of PBMCs expressing HMGB-1
following treatment with TSST-1 or RPMI in different donors was compared by paired Student’st-test.
Differences were considered significant if P < .05.


## 2.1. Toxin Purification

Recombinant TSST-1 was purified from culture supernatants ofS. aureus strain RN4220 previously transformed to carry the tst gene,
using both preparative isoelectric focusing and chromatofocusing
[15]. Toxin purity was assessed by silver staining after sodium dodecyl sulfate-polyacrylamide gel electrophoresis on 14% acrylamide gels, and LPS
activity was undetectable by the Limulus amoebocyte lysate gelation
(sensitivity limit, 10 pg/mL).


## 2.2. Preparation of Cells and Culture Conditions

Fresh human PBMCs from healthy donors were obtained by Ficoll-Paque PLUS (Amersham Biosciences
Corp., Piscataway, NJ, USA) density centrifugation, and cultured in 96-well
U-bottom plates at1.5×106 cells/mL in complete culture medium
consisting of RPMI 1640 (StemCell Technologies Inc., Vancouver, BC, Canada),
10% heat-inactivated fetal bovine serum (HyClone Laboratories Inc., Logan, Utah,
USA), 2 mM L-glutamine (StemCell), 25 mM Hepes buffer (StemCell), and 2 ug/mL
of polymyxin B sulphate (Sigma-Aldrich Corp., St. Louis, Mo, USA). For analysis
of secreted HMGB-1 in cell culture supernatants, PBMCs were plated in 24-well
flat-bottom plates in Opti-MEM I reduced serum medium (Gibco). THP-1 cells
(human monocytic cell line) were obtained from ATCC and kept in recommended
growth media until use, at which time the cells were plated at a concentration
of 1.5×106 cells in Opti-Mem I medium. T cell depletion of human
PBMC was performed using a column-free method of magnetic bead separation
(EasyCep from StemCell Technologies) by positive selection of T cells using an
anti-CD3 conjugated antibody according to manufacturer's directions. This
procedure left the monocytes negatively selected for and untouched for the purpose
of further experimental investigations.


## 2.3. Treatment of PBMC with TSST-1 or LPS

Purified PBMCs were plated in either 24-well or 96-well culture plates overnight at
37°C in 5% CO2 prior to stimulation with 1 nM TSST-1 or 500 ng/mL LPS (Sigma), where noted. Culture supernatants were collected at 24 hours
post-TSST-1 treatment, microcentrifuged at 800xg for 5 minutes, and frozen at –70°C until protein analysis.


## 2.4. Detection of HMGB-1 by Fluorescent Microscopy

PBMCs were first surface-stained for expression of CD3 with mouse
antihuman CD3 IgG (Pharmingen BD Biosciences, Mississauga, ON, Canada) followed by antimouse IgG antibody
conjugated to Alexa594  (Molecular Probes); cells were subsequently fixed using
Cytofix buffer (Pharmingen). For the intracellular  detection of HMGB-1, cells
were permeabilized with Cytoperm (Pharmingen) and incubated for 30 minutes
(4°C) with rabbit polyclonal anti-HMGB-1 antibodies (Orbigen BioCarta, San
Diego,Calif, USA) diluted in blocking buffer
(phosphate buffered saline, 3% FBS)  followed by a secondary antibody with Alexa488-conjugated
goat antirabbit IgG. The anti-HMGB-1 antibodies were raised against the peptide
sequence corresponding to amino acids 166–181 that were previously shown to be
specific for HMGB-1 and not HMGB-2 [16]. Surface expression of HMGB-1 on PBMC was established by culturing
cells (37°C, 5% CO2) directly on coverslips to allow attachment of
adherent cells overnight before stimulation with 1 nM of TSST-1. Cells were
then fixed and surface-stained as above (but without the permeabilization
step). Fluorescent-labeled PBMCs were subsequently stained with Hoechst 3342
nuclear dye (molecular probes), and mounted on slides using prolong antifade
reagent (molecular probes). Cells were visualized with an AxioPlan II
fluorescence microscope equipped with a CCD camera using Northern Eclipse
software (Epix) for acquisition of images. Images were taken with the 63x oil
immersion objective lens, and Adobe Photoshop 6.0 software was used for image
layout.


## 2.5. Flow Cytometric Analysis of Surface-Expressed HMGB-1 on Differentiated Cells

Surface-expressed HMGB-1 was analyzed by flow cytometry (FACSCalibur Flow
Cytometry System, BD BioSciences Pharmingen) using the anti-HMGB-1 antibody and
secondary Alexa488-conjugated antibody described above in conjunction with
phycoerythrin (PE)-conjugated anti-CD3 and anti-CD14 antibodies (BD BioSciences
Pharmingen), with a minimum of 10 000 events collected for each sample.


## 2.6. Western Blot Analysis of Secreted HMGB-1

PBMC or THP-1 culture supernatants were concentrated 10-fold from original volume using
Amicon Ultra centrifugal filters with a molecular weight cut-off of 10 kDa
(Millipore). Some culture supernatants
had been further prepared using the SDS-PAGE Clean-Up Kit (Amersham) according
to the manufacturer’s directions prior to running on a 12% polyacrylamide gel.
Western blotting was performed by semidry transfer of proteins (Trans-Blot SD Semi-Dry Electrophoretic Transfer
Cell, BioRad) onto an Immobilon-P PVDF membrane (Millipore) which had been
blocked for 1 hour at room temperature with 1% BSA, 0.5% Tween in Tris buffered
saline (TBS) prior to overnight incubation (at 4°C) with rabbit polyclonal
anti-HMGB-1 antibody. The membrane was subsequently incubated with antirabbit
IgG horse-radish-peroxidase (HRP)-conjugated secondary antibody for 1 hour at
room temperature on a shaker. Detection of HMGB-1 was performed using super signal
substrate (Pierce) and developed as well as analyzed using the Alpha Innotech
3400 Gel Documentation system (Alpha Innotech, Calif, USA).


## 2.7. Statistical Analysis

Statistical analysis was performed using Prism
3.0 software package (GraphPad). The proportion of PBMCs expressing HMGB-1
following treatment with TSST-1 or RPMI in different donors was compared by paired Student’st-test.
Differences were considered significant if P < .05.


## 3. Results and Discussion

### 3.1. Secretion of
Cell-Surface Expressed HMGB-1 From Differentiated and Adherent Cells 24 Hours
Post-TSST-1 Stimulation

Some
differentiated as well as adherent cells express HMGB-1 on the cell surface, and this cell membrane-associated HMGB-1 has been referred to as
amphoterin to distinguish it from intracellular HMGB-1 [17]. Extracellular amphoterin expression has been well described
previously, and has been investigated using various methods including
subcellular fractionation, immunogold electron microscopy, and mRNA localization studies
[18]. We examined the surface expression of HMGB-1 (amphoterin) in human PBMC after culturing cells over a 48-hour period in 96-well U-bottom plates in
complete growth medium. After this incubation time, we identified a population
of differentiated cells that expressed high levels of HMGB-1 in the absence of
any further treatment with exogenous stimulants (Figure 1(a)).  Because they are cells in suspension, freshly purified PBMCs do not express amphoterin (i.e., cell-surface expressed HMGB-1). Expression of cell-surface-associated HMGB-1 took place over the 48-hour time course that PBMCs were cultured, and cells of monocyte lineage had become adherent. T cells, which were
identified by costaining with anti-CD3 and Alexa594-conjugated antimouse IgG
(red), did not express amphoterin since  they remain in suspension, and the
surface expression of HMGB-1 appears to be a property of adherent cells;
however, all cells express intracellular HMGB-1 (as shown in Figure 2). Our
observations corroborate with those of Rouhiainen et al. [18], who reported the accumulation of amphoterin in the extracellular
space of cells bearing process extensions, and HMGB-1 surface expression was
inhibited in cells without these processes. Following TSST-1 stimulation, there
was a significant loss of amphoterin from the cell surface from the population
of cultured adherent cells that had surface expression of HMGB-1, as observed
by fluorescent microscopy (Figures 1(a) and 1(b)), and quantified by flow
cytometric analysis (Figure 1(c)).
Flow cytometry also confirmed that the high-level surface expression of amphoterin was confined to a subpopulation of PBMC having a high forward scatter corresponding to the monocyte/macrophage
population (estimated to be between 3% and 12% of resting PBMC among the donors
tested). More than half of these amphoterin-expressing cells released HMGB-1
upon TSST-1 stimulation (7.3±3.69% prior to stimulation versus 3.3±1.32%
post- TSST-1 treatment; P < .05, paired Student's t-test from
4 different donors). Flow cytometric analysis, to our knowledge, has not been utilized previously for quantifying the surface expression of
HMGB-1, and we found it to be a useful tool to evaluate the change in
extracellular HMGB-1 expression.Secretion of membrane-associated HMGB-1 in the extracellular milieu following TSST-1
treatment. Extracellular expression of HMGB-1 on adherent PBMC in
complete growth medium (a) or after TSST-1 treatment. (b) Anti-HMGB-1: green
fluorescence (Alexa488); nucleus: blue (Hoechst 3342 nuclear dye); anti-CD3:
red (Alexa594). Close-up views from
representative fields are depicted in the left panels. (c) Flow cytometry
analysis of surface-expressed HMGB-1 in PBMC 24 hours following TSST-1
stimulation. Left panel, resting or PBMC treated; right panel, 24 hours after
TSST-1 stimulation. Surface HMGB-1 expression significantly decreased after
TSST-1 stimulation (12.0% versus 4.3% in the donor shown; 7.3±3.69% prior to
stimulation versus 3.3±1.32% post-TSST-1 treatment from 4 different donors; P<.05, paired Student's t-test).
(a)(b)(c)Nuclear
translocation of HMGB-1 in human PBMC following TSST-1 stimulation. Human PBMCs were treated with either 1 nM TSST-1 or RPMI medium for 10 hours,
and the intracellular expression of HMGB-1 was detected by confocal fluorescent
microscopy. Close-up views from representative fields are depicted in the right
panels. (a) Resting (RPMI) PBMC, intracellular HMGB-1 (green) was seen to be
localized primarily in the nucleus (blue). (b) At 10 hours following
stimulation with 1 nM TSST-1, HMGB-1 was seen to translocate into the
extracellular space. (c) Fluorescent microscopy image of resting T cells, and (d)
TSST-1 stimulated T cells in the process of actively translocating intracellular HMGB-1 10 hours following toxin
treatment. T cells were detected by staining with antihuman CD3
antibodies conjugated to Alexa594 (red). (e) Detection of HMGB-1 by immunoblot
in the culture supernatant of human PBMC 24 hours after treatment with 1 nM
TSST-1 or RPMI medium.
(a)
Resting PBMC(b)
TSST-1 stimulated PBMC(c)
Resting T cell(d)
TSST-1 stimulated T cells(e)


### 3.2. Translocation and Secretion of Intracellular HMGB-1 From Human PBMCs 10 Hours Post-TSST-1 Stimulation

To investigate the potential change in
intracellular HMGB-1 expression upon TSST-1 exposure, human PBMCs were treated
with 1 nM of TSST-1 for 10 hours, and then subsequently evaluated for the
translocation of intracellular HMGB-1 by fluorescence microscopy (Figure2). As reported previously [19], HMGB-1 in resting PBMC is expected to be
localized primarily in the nucleus (Figure 2(a)). However, 10 hours after
treatment with 1 nM TSST-1, a significant proportion of PBMCs had decreased
their intracellular stores of HMGB-1 (Figure 2(b)). Previous studies that used
either LPS or the inflammatory cytokines IFNγ or TNFα [6, 7] as stimulants found that only macrophages, but
not T cells, translocated and subsequently secreted nuclear HMGB-1. However, we
found that T lymphocytes in human PBMCs also underwent a change in
intracellular HMGB-1 expression following TSST-1 treatment (Figures 1(c) and 1(d)).To verify that
HMGB-1 was actively secreted from TSST-1 treated PBMCs, we performed a Western
blot analysis of cell culture supernatants collected 24 hours post-TSST-1
treatment (Figure2(e)).


### 3.3. Requirement of Activated T Cells in TSST-1-Induced Nuclear Translocation and Secretion of HMGB-1 in Human PBMC

Undifferentiated and nonadherent monocytes in suspension do not express cell-surface-associated
HMGB-1. However, during the first steps in differentiation, monocytes actively
transport this molecule to their cell surface, and HMGB-1 is subsequently
secreted into the extracellular milieu upon further stimulation [17]. To further investigate the requirement of T
cells for nuclear translocation and secretion of HMGB-1 following TSST-1
stimulation, we treated the human monocytic cell line, THP-1, with either
TSST-1 (1 nM), LPS (500 ng/mL), or leaving them untreated in RPMI medium. THP-1 cells express
basal levels of both MHC Class II required for TSST-1 activation and CD14
required for LPS-induced activation [20]. As shown in Figure 3, TSST-1 treatment failed
to induce surface expression of HMGB-1 on undifferentiated THP-1 cells, in
contrast to stimulation with LPS. We confirmed this observation in primary cells
using human PBMCs that were markedly depleted of their T cell pool that was stimulated
with either TSST-1 or LPS (Figure 3(a)). Western blot analysis also verified
that THP-1 cells and T cell-depleted PBMC secreted detectable amounts of HMGB-1
upon LPS stimulation, but not TSST-1 treatment (Figure 3(b)). Collectively,
these results confirm our previous studies which established that both
monocytes and T cells are required for the secretion of TNFα and IL-1β from human PBMC stimulated with highly purified TSST-1 [13].Requirement of T cells for
HMGB-1 secretion following TSST-stimulation. (a) THP-1 cells or T
cell-depleted human PBMCs
were stimulated with either TSST-1 (1 nM), LPS (500 ng/mL), or RPMI medium for
24 hours, and surface expression of HMGB-1 was examined by fluorescence
microscopy. (b) The secretion of HMGB-1 in supernatants of THP-1 cells (left
panel) or T cell-depleted PBMC (right panel) 24 hours following treatment with
LPS, TSST-1, or RPMI medium control was examined by immunoblot. rHMGB-1 (15 ng)
was used as a positive control.
(a)(b)


## 3.1. Secretion of
Cell-Surface Expressed HMGB-1 From Differentiated and Adherent Cells 24 Hours
Post-TSST-1 Stimulation

Some
differentiated as well as adherent cells express HMGB-1 on the cell surface, and this cell membrane-associated HMGB-1 has been referred to as
amphoterin to distinguish it from intracellular HMGB-1 [17]. Extracellular amphoterin expression has been well described
previously, and has been investigated using various methods including
subcellular fractionation, immunogold electron microscopy, and mRNA localization studies
[18]. We examined the surface expression of HMGB-1 (amphoterin) in human PBMC after culturing cells over a 48-hour period in 96-well U-bottom plates in
complete growth medium. After this incubation time, we identified a population
of differentiated cells that expressed high levels of HMGB-1 in the absence of
any further treatment with exogenous stimulants (Figure 1(a)).  Because they are cells in suspension, freshly purified PBMCs do not express amphoterin (i.e., cell-surface expressed HMGB-1). Expression of cell-surface-associated HMGB-1 took place over the 48-hour time course that PBMCs were cultured, and cells of monocyte lineage had become adherent. T cells, which were
identified by costaining with anti-CD3 and Alexa594-conjugated antimouse IgG
(red), did not express amphoterin since  they remain in suspension, and the
surface expression of HMGB-1 appears to be a property of adherent cells;
however, all cells express intracellular HMGB-1 (as shown in Figure 2). Our
observations corroborate with those of Rouhiainen et al. [18], who reported the accumulation of amphoterin in the extracellular
space of cells bearing process extensions, and HMGB-1 surface expression was
inhibited in cells without these processes. Following TSST-1 stimulation, there
was a significant loss of amphoterin from the cell surface from the population
of cultured adherent cells that had surface expression of HMGB-1, as observed
by fluorescent microscopy (Figures 1(a) and 1(b)), and quantified by flow
cytometric analysis (Figure 1(c)).
Flow cytometry also confirmed that the high-level surface expression of amphoterin was confined to a subpopulation of PBMC having a high forward scatter corresponding to the monocyte/macrophage
population (estimated to be between 3% and 12% of resting PBMC among the donors
tested). More than half of these amphoterin-expressing cells released HMGB-1
upon TSST-1 stimulation (7.3±3.69% prior to stimulation versus 3.3±1.32%
post- TSST-1 treatment; P < .05, paired Student's t-test from
4 different donors). Flow cytometric analysis, to our knowledge, has not been utilized previously for quantifying the surface expression of
HMGB-1, and we found it to be a useful tool to evaluate the change in
extracellular HMGB-1 expression.Secretion of membrane-associated HMGB-1 in the extracellular milieu following TSST-1
treatment. Extracellular expression of HMGB-1 on adherent PBMC in
complete growth medium (a) or after TSST-1 treatment. (b) Anti-HMGB-1: green
fluorescence (Alexa488); nucleus: blue (Hoechst 3342 nuclear dye); anti-CD3:
red (Alexa594). Close-up views from
representative fields are depicted in the left panels. (c) Flow cytometry
analysis of surface-expressed HMGB-1 in PBMC 24 hours following TSST-1
stimulation. Left panel, resting or PBMC treated; right panel, 24 hours after
TSST-1 stimulation. Surface HMGB-1 expression significantly decreased after
TSST-1 stimulation (12.0% versus 4.3% in the donor shown; 7.3±3.69% prior to
stimulation versus 3.3±1.32% post-TSST-1 treatment from 4 different donors; P<.05, paired Student's t-test).
(a)(b)(c)Nuclear
translocation of HMGB-1 in human PBMC following TSST-1 stimulation. Human PBMCs were treated with either 1 nM TSST-1 or RPMI medium for 10 hours,
and the intracellular expression of HMGB-1 was detected by confocal fluorescent
microscopy. Close-up views from representative fields are depicted in the right
panels. (a) Resting (RPMI) PBMC, intracellular HMGB-1 (green) was seen to be
localized primarily in the nucleus (blue). (b) At 10 hours following
stimulation with 1 nM TSST-1, HMGB-1 was seen to translocate into the
extracellular space. (c) Fluorescent microscopy image of resting T cells, and (d)
TSST-1 stimulated T cells in the process of actively translocating intracellular HMGB-1 10 hours following toxin
treatment. T cells were detected by staining with antihuman CD3
antibodies conjugated to Alexa594 (red). (e) Detection of HMGB-1 by immunoblot
in the culture supernatant of human PBMC 24 hours after treatment with 1 nM
TSST-1 or RPMI medium.
(a)
Resting PBMC(b)
TSST-1 stimulated PBMC(c)
Resting T cell(d)
TSST-1 stimulated T cells(e)


## 3.2. Translocation and Secretion of Intracellular HMGB-1 From Human PBMCs 10 Hours Post-TSST-1 Stimulation

To investigate the potential change in
intracellular HMGB-1 expression upon TSST-1 exposure, human PBMCs were treated
with 1 nM of TSST-1 for 10 hours, and then subsequently evaluated for the
translocation of intracellular HMGB-1 by fluorescence microscopy (Figure2). As reported previously [19], HMGB-1 in resting PBMC is expected to be
localized primarily in the nucleus (Figure 2(a)). However, 10 hours after
treatment with 1 nM TSST-1, a significant proportion of PBMCs had decreased
their intracellular stores of HMGB-1 (Figure 2(b)). Previous studies that used
either LPS or the inflammatory cytokines IFNγ or TNFα [6, 7] as stimulants found that only macrophages, but
not T cells, translocated and subsequently secreted nuclear HMGB-1. However, we
found that T lymphocytes in human PBMCs also underwent a change in
intracellular HMGB-1 expression following TSST-1 treatment (Figures 1(c) and 1(d)).To verify that
HMGB-1 was actively secreted from TSST-1 treated PBMCs, we performed a Western
blot analysis of cell culture supernatants collected 24 hours post-TSST-1
treatment (Figure2(e)).


## 3.3. Requirement of Activated T Cells in TSST-1-Induced Nuclear Translocation and Secretion of HMGB-1 in Human PBMC

Undifferentiated and nonadherent monocytes in suspension do not express cell-surface-associated
HMGB-1. However, during the first steps in differentiation, monocytes actively
transport this molecule to their cell surface, and HMGB-1 is subsequently
secreted into the extracellular milieu upon further stimulation [17]. To further investigate the requirement of T
cells for nuclear translocation and secretion of HMGB-1 following TSST-1
stimulation, we treated the human monocytic cell line, THP-1, with either
TSST-1 (1 nM), LPS (500 ng/mL), or leaving them untreated in RPMI medium. THP-1 cells express
basal levels of both MHC Class II required for TSST-1 activation and CD14
required for LPS-induced activation [20]. As shown in Figure 3, TSST-1 treatment failed
to induce surface expression of HMGB-1 on undifferentiated THP-1 cells, in
contrast to stimulation with LPS. We confirmed this observation in primary cells
using human PBMCs that were markedly depleted of their T cell pool that was stimulated
with either TSST-1 or LPS (Figure 3(a)). Western blot analysis also verified
that THP-1 cells and T cell-depleted PBMC secreted detectable amounts of HMGB-1
upon LPS stimulation, but not TSST-1 treatment (Figure 3(b)). Collectively,
these results confirm our previous studies which established that both
monocytes and T cells are required for the secretion of TNFα and IL-1β from human PBMC stimulated with highly purified TSST-1 [13].Requirement of T cells for
HMGB-1 secretion following TSST-stimulation. (a) THP-1 cells or T
cell-depleted human PBMCs
were stimulated with either TSST-1 (1 nM), LPS (500 ng/mL), or RPMI medium for
24 hours, and surface expression of HMGB-1 was examined by fluorescence
microscopy. (b) The secretion of HMGB-1 in supernatants of THP-1 cells (left
panel) or T cell-depleted PBMC (right panel) 24 hours following treatment with
LPS, TSST-1, or RPMI medium control was examined by immunoblot. rHMGB-1 (15 ng)
was used as a positive control.
(a)(b)


## 4. Conclusion

Various cell types have recently been identified as contributing to the extracellular
pool of HMGB-1, including human umbilical vein endothelial cells [21], platelets [18], pituicytes, and macrophages [3]. Our finding that
HMGB-1 is secreted by T cells following TSST-1 stimulation may parallel the
observation made by Semino et al. [22] who found that distinct NK cell subsets
secreted HMGB-1 which served to differentiate autologous dendritic cells, and
this event was modulated by environmental stimuli. HMGB-1 was shown to induce
monocytes to differentiate into dendritic cells that specifically polarized T
cells to give a Th1 response [19], a feature characteristic of the effect of
TSST-1 stimulation on human PBMC. We previously demonstrated that the cytokine
secretion and costimulatory molecule expression by human PBMC following TSST-1
stimulation follows a bimodal pattern [23], with the first phase peaking at ~3 hours
poststimulation, and a second burst at ~24 hours poststimulation. In light of
the secretion of HMGB-1 into the extracellular milieu at this later time point,
it will be worthwhile to determine whether the second inflammatory burst and
the upregulation of costimulatory molecules, such as CD86, CD40, as well as
HLA-DR (which peaked at 48 hours) [23], are in direct response to the secretion of HMGB-1 that is
considered the late mediator of sepsis [24].The best studied ligand for extracellular HMGB-1 is RAGE (receptor of advanced
glycation end products) which is upregulated on activated macrophages as well
as endothelial cells [25]. Previous studies have aimed to determine how
superantigen-activated T cells adhere to vascular endothelial cells and induce
vascular injury [26]. It was also shown that rHMGB-1 elicited proinflammatory responses
on endothelial cells [25]. It would, therefore, be of interest to further investigate the
role of HMGB-1 and RAGE in the inflammatory response and vascular endothelial
injury mediated by TSST-1. It is anticipated that the model of TSST-1-induced inflammation, which
necessitates the bridging of Vβ2-specific T cells and MHC class II molecules
bearing antigen presenting cells, will provide a useful means to study the role of HMGB-1 in T cell-monocyte interactions, Th1
polarization, and the ensuing immune response leading to tissue injury.
Finally, this study also provides a rationale to investigate the
potential to target HMGB-1 for therapy for inflammatory disorders induced by
TSST-1, such as toxic shock syndrome, as it is currently being investigated for
gram negative sepsis [9]. It should be noted that the inflammatory
response to different staphylococcal exotoxins varies [27], and it would be of clinical interest to
investigate the role and regulation of HMGB-1 in the context of other
superantigens and its potential as a primary target for superantigen-mediated
diseases.



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*Source: 512196-2008-11-04.xml*