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 [ 13 ] . extracellular hmgb-1 was initially identified 
 as amphoterin , a heparin - binding protein promoting neurite outgrowth in the 
 perinatal rat brain . hmgb-1 is now regarded as an endogenous 
 danger signal that is passively released by necrotic cells or actively secreted 
 by stimulated cells . 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 . unlike 
 tnf and ifn that appear within the first 46 
 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 . toxic shock syndrome toxin-1 ( tsst-1 ) is a 
 superantigen ( sag ) secreted by some strains of s. aureus , and this sag 
 was subsequently identified as the major 
 causative agent of toxic shock syndrome . 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 v-specific 
 regions of the  t cell 
 receptor ( tcr ) to class ii major histocompatibility complex ( mhc ii ) molecules 
 on antigen presenting cells ( apcs ) . in addition to massive t cell proliferation , this trimolecular interaction leads to the uncontrolled release of various proinflammatory cytokines 
 , 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 tnf 
 [ 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 . recombinant tsst-1 was purified from culture supernatants of s. aureus strain rn4220 previously transformed to carry the tst gene , 
 using both preparative isoelectric focusing and chromatofocusing 
 . 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 ) . 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 at 1.5  10 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 . 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  10 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 . purified pbmcs were plated in either 24-well or 96-well culture plates overnight at 
 37c 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 70c until protein analysis . 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 
 ( 4c ) 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 166181 that were previously shown to be 
 specific for hmgb-1 and not hmgb-2 . surface expression of hmgb-1 on pbmc was established by culturing 
 cells ( 37c , 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 . 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 . 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 4c ) 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 ) . the proportion of pbmcs expressing hmgb-1 
 following treatment with tsst-1 or rpmi in different donors was compared by paired student 's t - test . 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 . 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 
 . 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 . , 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 . 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 ( figure 2 ) .  
 as reported previously , 
 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 ifn or tnf 
 [ 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 ( figure 2(e ) ) . 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 . 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 . 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 tnf and il-1 from human pbmc stimulated with highly purified tsst-1 . various cell types have recently been identified as contributing to the extracellular 
 pool of hmgb-1 , including human umbilical vein endothelial cells , platelets , pituicytes , and macrophages . our finding that 
 hmgb-1 is secreted by t cells following tsst-1 stimulation may parallel the 
 observation made by semino et al .   who found that distinct nk cell subsets 
 secreted hmgb-1 which served to differentiate autologous dendritic cells , and 
 this event hmgb-1 was shown to induce 
 monocytes to differentiate into dendritic cells that specifically polarized t 
 cells to give a th1 response , 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 , 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 ) , are in direct response to the secretion of hmgb-1 that is 
 considered the late mediator of sepsis . 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 . previous studies have aimed to determine how 
 superantigen - activated t cells adhere to vascular endothelial cells and induce 
 vascular injury . 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 v2-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 . it should be noted that the inflammatory 
 response to different staphylococcal exotoxins varies , 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 .
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 .