preparation of recombinant tsg-6 proteins  full - length 
  recombinant human ( rh)tsg-6 was expressed in drosophila s2 cells and 
  purified as described previously 
  ( 39 ) .
link_tsg6 ( the link 
  module of human tsg-6 , corresponding to residues 36133 of the 
  pre - protein ( 1 ) ) and cub_c_tsg6 
  ( the cub domain and c - terminal 27 amino acids ; i.e. residues 
  129277 of the human pre - protein ) were expressed in e. coli , 
  purified , and characterized as described in day et al . 
 
effect of tsg-6 on rankl - induced human osteoclast 
  formation  to determine the effects of tsg-6 on rankl - induced human 
  osteoclast formation , human peripheral blood mononuclear cells ( pbmcs ) were 
  isolated from healthy male volunteers ( age range 2535 years ) as 
  described in sabokbar and athanasou 
  ( 41 ) .
briefly , blood was 
  collected in edta - treated tubes and diluted 1:1 in -minimum essential 
  medium ( mem ) ( invitrogen ) , layered over histopaque ( sigma - aldrich ) , 
  then centrifuged ( 693  g ) , washed , and resuspended in 
  mem with 10% ( v / v ) heat - inactivated fetal calf serum ( invitrogen ) . 
 
pbmcs were counted after lysis of red cells with 5% ( v / v ) acetic acid and 
  seeded ( at 5  10 cells / well ) into 96-well tissue culture 
  plates containing either dentine slices or coverslips .
after 2 h of 
  incubation , dentine slices and coverslips were removed from the wells and 
  washed vigorously in mem to remove non - adherent cells before transfer 
  into 24-well tissue culture plates containing 1 ml / well mem 
  supplemented with 10% ( v / v ) fetal calf serum , 10 mm l - glutamine , 
  antibiotics ( 100 iu / ml penicillin and 10 g / ml streptomycin ) , 25 ng / ml 
  macrophage colony - stimulating factor ( m - csf ; r&d systems europe ) , and 50 
  ng / ml soluble ( s)rankl ( peprotech ) . after the addition of rhtsg-6 ( 30.1 kda 
  ( 39 ) ) at 050 ng / ml or 
  equimolar concentrations of link_tsg6 ( 10.9 kda 
  ( 17 ) ) or cub_c_tsg6 ( 16.8 kda 
  ( 12 ) ) , cultures were 
  maintained for up to 21 days , during which time the entire culture medium 
  containing all factors was replenished every 23 days .
osteoclast formation was assessed cytochemically by determining the number 
  of multinucleated tartrate - resistant acid phosphatase - positive cells after 14 
  days in culture .
cells on coverslips were fixed in 10% ( v / v ) formalin and 
  stained using napthol as - bi as a substrate in the presence of 1.0 m 
  acetate - tartrate ; the reaction was stopped with 0.5 m naf .
bone 
  resorptive activity , an indicator of osteoclast activation , was determined by 
  the measurement of resorption lacunae on dentine slices .
after 21 days in 
  culture dentine slices were removed from the wells , rinsed in pbs , placed in 
  1.0 m nh4oh overnight , and then sonicated for 510 
  min ; this resulted in complete removal of cells from the dentine slice , 
  permitting examination of its surface .
the slices were washed in distilled 
  water , stained with 0.5% ( w / v ) aqueous toluidine blue , ph 5.0 , and examined by 
  light microscopy .
tartrate - resistant acid phosphatase staining was analyzed 
  using imagej software 
  ( rsb.info.nih.gov/ij ) , 
  whereas for lacunar resorption the data were expressed as the mean percentage 
  area resorbed from four dentine slices per treatment .
tsg-6 mice were generated as described 
  previously ( 27 ) and 
  back - crossed into the balb / c background 
  ( 35 ) .
age - matched pairs of 
  female tsg-6 and wild type ( wt ) balb / c mice ( between 18 
  and 25 weeks of age ) were used for experiments .
all procedures carried out on 
  animals were approved by the institutional animal care and use committee ( rush 
  university medical center , chicago , il ) .
bone marrow cells from the long bones 
  of tsg-6 mice were isolated as previously described 
  ( 42 ) .
briefly , mice were 
  killed by co2 inhalation , and the femora and tibiae were 
  aseptically removed and dissected free of adherent soft tissue .
the bone ends 
  were cut , and the marrow cavity was flushed out into a petri dish by slowly 
  injecting mem at one end of the bone using a sterile 21-gauge needle . 
  the bone marrow suspension
was carefully agitated with a plastic pasteur 
  pipette to obtain a homogeneous suspension of cells , and these were incubated 
  for 2 h at a density of 5  10 cells / ml in 96-well plates 
  containing dentine slices .
non - adherent cells were removed from the dentine 
  slices ( by vigorous washing in mem ) , which were then transferred to 
  24-well plates containing mem with 10% ( v / v ) heat - inactivated serum and 
  antibiotics ( as described above ) .
cultures on dentine slices were maintained 
  in the presence of 25 ng / ml m - csf ( r&d systems europe ) and 30 ng / ml murine 
  srankl ( peprotech ) for 10 days during which the culture media and factors were 
  replenished every 23 days .
micro - computerized tomography ( ct ) analysis of long bones from 
  tsg-6-deficient and wt mice 
micro - ct analysis of the long bones in 
  tsg-6-deficient and wt mouse femurs was performed to determine any differences 
  in trabecular bone ; four pairs of age - matched male mice ( 2428 weeks ) 
  were analyzed .
briefly , plastic - wrapped knees were mounted vertically on a 
  skyscan 1172 micro - ct scanner and scans ( 6.77 m with a voxel resolution of 
  6.9 m ) were performed using a 20100-kv microfocus x - ray 
  source with a 10-megapixel digital x - ray camera .
the images obtained were 
  subjected to three - dimensional angular resampling ( using skyscan 3d - creator 
  software ) , and morphometric parameters were calculated for trabecular regions 
  of interest using a marching cubes model 
  ( 43 ) as described previously 
  ( 44 ) .
mouse 
  knee joints were prepared for confocal immunohistochemistry essentially as 
  described in plaas et al . 
 
briefly , mouse legs were 
  fixed in 10% ( v / v ) neutral - buffered formalin for 48 h , skin and muscle tissue 
  were removed , and joints were decalcified in 5% ( w / v ) edta in pbs for 14 days . 
 
thin sections ( 4 
  m ) were deparaffinized , rehydrated , and exposed to a rabbit anti - tsg-6 
  polyclonal antibody ( prepared in collaboration with dr .
j. d. sandy ( rush 
  university , chicago , il ) and affinity bioreagents ( golden , co ) against the 
  peptide asvtaggfqik , affinity - purified , and shown to recognize recombinant 
  murine tsg-6 ( r&d systems ) and rhtsg-6 
  ( 39 ) by western blotting ; see 
  supplemental fig .
1 ) at 10 g / ml ( igg ) in 1.5% ( v / v ) goat serum in pbs for 
  30 min , washed in pbs for 5 min , and then incubated with alexafluor-568 goat 
  anti - rabbit igg ( molecular probes ; 1:250 in pbs ) for 1 h at room temperature . 
 
sections were co - stained for ha using 5 g / ml biotinylated bovine cartilage 
  ha - binding protein / link protein complexes ( isolated in the a1a1d6 fraction 
  after cscl gradient centrifugation ) followed by alexafluor-488-streptavidin 
  ( molecular probes ) as described in plaas et al . 
 
nuclei were stained with 
  toto-3 ( molecular probes ; 1:500 in pbs ) , and sections were examined by 
  confocal microscopy as detailed previously 
  ( 45 ) .
murine mc3t3-e1 osteoblastic cells ( european 
  collection of cell cultures ) 
  ( 46 ) and mba-15.4 
  pre - osteoblastic marrow - derived cells 
  ( 47 ) were seeded on 24-well 
  plates ( 1.25  10 cells / ml ) in dulbecco 's modified eagle 's 
  medium supplemented with 10% ( v / v ) heat - inactivated fetal bovine serum 
  ( invitrogen ) , 10 mm l - glutamine , and antibiotics ( 100 iu / ml 
  penicillin and 10 g / ml streptomycin ) .
differentiation into osteoblasts was 
  induced with 100 ng / ml bmp-2 ( r&d systems europe ) , and the effects of 
  adding rhtsg-6 ( 010000 ng / ml ) or molar equivalents of link_tsg6 or 
  cub_c_tsg6 proteins were determined .
cells were cultured for 7 days in a 
  humidified atmosphere with 5% ( v / v ) co2 at 37 c , during which 
  time the culture media ( including all factors ) was replaced every 72 h. cells 
  were washed in pbs and freeze - thawed 3 times , and alkaline phosphatase ( alp ) 
  was released by scraping cells into 50 l of 0.2% ( v / v ) nonidet p-40 
  ( fluka ) and subsequent sonication ( 5 s at 6 watts ) .
alp activity ( pmol/l 
  of material ) was determined using the fluorescent substrate 4-methyl 
  umbelliferyl phosphate ( sigma - aldrich ) .
briefly , 10 l of cell lysate was 
  diluted in 50 l of 0.16% ( v / v ) nonidet p-40 , 10 mm tris , ph 
  8.0 , in the wells of a nunclon delta ( nunc ) plate , and 100 l of 0.2 
  mm methyl umbelliferyl phosphate was added .
plates were incubated 
  at 37 c for 45 min , and the reaction was stopped by adding 100 l of 
  0.6 m na2co3 to each well .
fluorescence was 
  measured using an excitation wavelength of 360 nm and an emission wavelength 
  of 450 nm ( with a 435 nm cutoff ) on a spectramax gemini microplate 
  spectrofluorometer .
the yield of alp was calculated by the preparation of a 
  standard curve of 01000 pmol of 4-methyl umbelliferyl phosphate ( sigma ) 
  on the same plate and standardized against the total amount of protein / well 
  ( in g ) , determined using a bca protein assay kit ( pierce ) .
tsg-6 binding elisa  the bmp and srankl binding activities of 
  tsg-6 and its individual domains were determined colorimetrically using 
  elisas .
all dilutions , incubations , and washes were performed in standard 
  assay buffer ( sab ; pbs containing 0.2% ( v / v ) tween 20 ) and at room temperature 
  unless otherwise indicated .
bmps ( -2 , -13 , and -14 from peprotech ; -3 , -4 , -5 , 
  -6 , and -7 from r&d systems europe ) or srankl ( peprotech ) were coated 
  overnight on polysorp ( nunc ) microtiter plates ( 030 pmol / well ) in pbs 
  at room temperature . after this and
all wells were blocked for 90 min at 37 c in sab 
  containing 1% ( w / v ) bovine serum albumin and then incubated for 4 h with 
  rhtsg-6 , link_tsg6 , or cub_c_tsg6 at 2 or 5 pmol / well for the bmp and srankl 
  assays , respectively .
bound tsg-6 and cub_c_tsg6 were compared by incubation 
  for 45 min with a rabbit polyclonal antiserum ( rah-1 ) 
  ( 48 ) diluted 1:4000 followed 
  by incubation with alkaline phosphatase - conjugated goat anti - rabbit igg 
  ( 1:2000 ; sigma - aldrich ) for an additional 45 min .
bound link_tsg6 and tsg-6 
  were compared using the monoclonal antibody q75 
  ( 49 ) at 1.25 g / ml followed 
  by alkaline phosphatase - conjugated goat anti - rat igg ( 1:2000 ; sigma - aldrich ) , 
  where these were incubated for 45 min each .
disodium p - nitrophenyl 
  phosphate ( sigma - aldrich ) at 1 mg / ml in 0.05 m tris - hcl , 0.1 
  m nacl , 5 mm mgcl26h2o , ph 
  9.3 , was added , and absorbance values at 405 nm were measured after 30 or 60 
  min of incubation ( for bmp and srankl elisas , respectively ) and corrected 
  against control wells ( i.e. those containing no immobilized 
  protein ) .
srankl binding elisa  tsg-6 or its individual protein domains 
  ( 030 pmol / well ) were coated overnight onto microtiter plates , blocked , 
  and washed as described above .
all wells were incubated with 30 ng / well 
  biotinylated - srankl ( peprotech ) for 4 h , and bound srankl was detected with 
  extravidin alkaline phosphatase ( sigma - aldrich ) as described in mahoney et 
  al .
( 19 ) except that 
  absorbance values at 405 nm were measured after a 60-min development time .
surface plasmon resonance  all experiments were performed on 
  a biacore 2000 system in hepes - ep running buffer ( 10 mm hepes , ph 
  7.4 , 150 mm nacl , 3 mm edta , 0.005% ( v / v ) surfactant 
  p20 ) at a constant flow rate of 10 l / min .
bmps or srankl ( 0.370.5 
  m in 40 l of 10 mm sodium acetate , ph 4.0 ) were 
  coupled onto commercially available cm5 sensor chips preactivated with 
  n - hydroxysuccinimide and 1-ethyl-3-(3-dimethylaminopropyl ) 
  carbodiimide .
after immobilization of these proteins , binding analyses were 
  performed using 010 m rhtsg-6 , link_tsg6 , or cub_c_tsg6 , 
  where 100 l of the protein solution was passed over the sensor chip 
  surface , and maximum equilibrium binding was determined 600 s after the start 
  of the injection .
scatchard analysis was performed to determine the validity 
  of the 1:1 langmuir association / dissociation model , and data were analyzed 
  with origin software ( microcal ) using a sum of least squares iterative 
  improvement method .
statistical analysis  all data were normalized and expressed 
  as percentages relative to controls .
each set of experiments was repeated at 
  least three times unless otherwise stated . for in vitro work
, 
  differences between groups were analyzed using the unpaired student 's 
  t test or a tukey - kramer multiple comparisons test .
p < 
  0.01 was considered significant . for the histomorphometric analysis of distal 
  femurs ,
a non - parametrical kruskall - wallis test was used ; p < 0.05 
  was considered significant .
effect of tsg-6 on srankl - induced osteoclast formation and 
  activity  human pbmcs were incubated with m - csf and srankl for 14 
  days on coverslips to allow visualization of newly differentiated osteoclasts 
  or for 21 days on dentine slices to assay for activity in the form of lacunar 
  resorption .
the addition of full - length rhtsg-6 to these cultures resulted in 
  a dose - dependent inhibition of osteoclast - mediated dentine erosion , with an 
  ic50 value of 15 ng / ml ( 0.5 nm ) , as 
  illustrated in figs . 1 , a and 
  b. there was no apparent reduction in the number or the 
  size of the multinucleated osteoclasts formed in tsg-6-treated cultures , as 
  assessed by staining of cells grown on coverslips for tartrate - resistant acid 
  phosphatase ( fig .
1c ) ; 
  in contrast , lacunar resorption was markedly reduced in the presence of tsg-6 
  ( fig .
overall 
  these findings suggest that tsg-6 affects osteoclast activation rather than 
  differentiation . a comparison of the effects of rhtsg-6 with equimolar 
  quantities of link_tsg6 and cub_c_tsg6 on srankl - induced lacunar resorption 
  ( see fig .
2 ) revealed that the 
  isolated cub_c domain has little effect on osteoclast function .
in contrast , 
  the link module retains significant activity , although this is less than that 
  of the intact protein .
a , human 
  pbmcs were cultured with srankl and m - csf in the absence or presence of 
  rhtsg-6 ( 050 ng / ml ) for 21 days on dentine slices .
data 
  are expressed as mean percentage resorption ( n = 9 )  s.e .
, 
  compared with rankl / m - csf alone , where   and   = 
  p < 0.01 , p < 0.001 , respectively .
b and 
  c , human pbmcs were cultured with srankl and m - csf on dentine slices 
  ( b ) or coverslips ( c ) for 21 or 14 days , respectively , in 
  the absence ( - ) or presence ( + ) of rhtsg-6 ( 50 ng ) .
osteoclast formation or 
  activation was assessed by determining the extent of lacunar resorption 
  ( b ) or the number of tartrate - resistant acid phosphatase - positive 
  multinucleated cells ( c ) , respectively .
tsg-6 inhibits rankl - mediated osteoclastogenesis . a , human 
  pbmcs were cultured with srankl and m - csf in the absence or presence of 
  rhtsg-6 ( 050 ng / ml ) for 21 days on dentine slices .
data 
  are expressed as mean percentage resorption ( n = 9 )  s.e .
, 
  compared with rankl / m - csf alone , where   and   = 
  p < 0.01 , p < 0.001 , respectively .
b and 
  c , human pbmcs were cultured with srankl and m - csf on dentine slices 
  ( b ) or coverslips ( c ) for 21 or 14 days , respectively , in 
  the absence ( - ) or presence ( + ) of rhtsg-6 ( 50 ng ) .
osteoclast formation or 
  activation was assessed by determining the extent of lacunar resorption 
  ( b ) or the number of tartrate - resistant acid phosphatase - positive 
  multinucleated cells ( c ) , respectively .
cells isolated from the marrow of long bones of 
  tsg-6 mice or wild type balb / c animals ( wt ) were 
  cultured on dentine slices in the presence of m - csf and srankl for 10 days .
in 
  the case of the cells derived from tsg-6-deficient mice there was 
  substantially elevated osteoclast - mediated lacunar resorption compared with 
  controls ( fig .
3a ) , 
  which is consistent with the severe arthritis and tissue damage reported in 
  these animals ( 35 ) .
figure 2.full-length tsg-6 and its isolated link module inhibit rankl - induced 
  lacunar resorption .
rhtsg-6 ( 050 ng / ml ) or equimolar concentrations 
  of link_tsg6 ( link ) or cub_c_tsg6 ( cub_c ) were added to cultures of human 
  pbmcs in the presence of srankl and m - csf .
data are expressed as the mean 
  percentage of resorption with rankl alone ( n = 12 )  s.e . , 
  where   and   = p < 0.01 and p 
  < 0.001 , respectively .
full - length tsg-6 and its isolated link module inhibit rankl - induced 
  lacunar resorption .
rhtsg-6 ( 050 ng / ml ) or equimolar concentrations 
  of link_tsg6 ( link ) or cub_c_tsg6 ( cub_c ) were added to cultures of human 
  pbmcs in the presence of srankl and m - csf .
data are expressed as the mean 
  percentage of resorption with rankl alone ( n = 12 )  s.e . , 
  where   and   = p < 0.01 and p 
  < 0.001 , respectively .
micro - ct was used to determine the histomorphometric 
  parameters of the distal femurs from the knees of tsg-6 
  mice and wt controls ( both on a balb / c background ) .
preliminary analysis on 
  two pairs of mice indicated that the tsg-6 null animals had approximately 
  twice the bone mass of controls ( data not shown ) .
more detailed comparisons on 
  four different pairs confirmed that the percentage trabecular volume ( bv / tv ) 
  of the tsg-6-deficient animals ( 17% ) is significantly greater than that 
  of the wt mice ( 13% , p = 0.02 ) . tsg-6
knock - out animals also 
  show significant increases in trabecular number ( tb / n ) and trabecular 
  thickness ( tb / th ) but not trabecular separation ( tb / sp ) ( see 
  table 1 ) .
3b , which 
  compares three - dimensional reconstructions of the trabecular bone network for 
  the tsg-6 null and wt animals .
taken together these findings show that 
  tsg-6 mice have increased bone mass compared with wild 
  type littermates , which is contrary to what we had expected based on the 
  effects of tsg-6 on osteoclastic resorption described above and suggests a 
  role for tsg-6 in normal bone homeostasis that might involve modulation of 
  osteoblast differentiation / activity .
table 1histomorphometric parameters for the trabecular regions of distal femurs 
  from tsg-6/ and wt micemicetrabecular volume ( bv / tv)trabecular number ( tb / n)trabecular thickness ( tb / th)trabecular separation ( tb / sp ) wt 12.8 ( 0.81 ) 
   2.23 ( 0.13 ) 
   0.057 ( 0.001 ) 
   0.288 ( 0.019 ) 
  tsg-6 
  17.0 
  ( 0.42 ) 
  2.66 
  ( 0.04 ) 
  0.064 
  ( 0.002 ) 
  0.256 ( 0.012 ) 
  aaverage values shown ( n = 4  s.e.)bmean values were compared using a non - parametrical kruskall - wallis test and 
  found to differ significantly from wt ( p  0.02 ) histomorphometric parameters for the trabecular regions of distal femurs 
  from tsg-6/ and wt mice average values shown ( n = 4  s.e . )
mean values were compared using a non - parametrical kruskall - wallis test and 
  found to differ significantly from wt ( p  0.02 ) immunolocalization of tsg-6 in wild type mouse knee 
  joints  immunohistochemistry of wild type murine knee joints 
  ( fig .
4 ) with anti - tsg-6 
  antibody showed immunoreactive tsg-6 to be associated with cells in the 
  superficial and midzones of the articular cartilage ( panels i and 
  ii ) and the lower region of hypertropic cartilage in the epiphyseal 
  growth plate ( panel ii ) as well as with meniscal fibrochondrocytes 
  ( panel iv ) . in the synovium and periosteum only a few positively 
  stained cells were detected , where these have a flattened or fusiform shape 
  ( panels iv and v ) .
notably , the strongest immunostaining was 
  associated with the cells in the epiphyseal and metaphyseal bone marrow of 
  both the femur and tibia ( panels i , ii , iii , and v ) , at the 
  margins of bone marrow and trabecular bone , and in the extracellular matrix of 
  calcified cartilage adjoining the trabecular bone ( indicated by 
  asterisks in panels ii and iii ) .
figure 3.tsg-6 mice exhibit elevated osteoclast resorptive 
  activity in response to rankl / m - csf but greater trabecular bone mass in the 
  absence of challenge .
a , osteoclast precursors from the long bone 
  marrow of tsg-6 and wild type balb / c ( wt ) mice 
  were cultured on dentine slices in the absence ( media ) or presence of 
  m - csf and/or murine srankl for 10 days before the determination of lacunar 
  resorption .
data are plotted as mean percentage resorption ( n = 4 ) 
   s.e .
lacunar resorption was significantly greater in the 
  presence of cells isolated from tsg-6 mice as compared 
  with wt ; p = 0.03 for cultures containing srankl alone and p 
  = 0.004 for those with m - csf and srankl .
b , three - dimensional models 
  of the trabecular bone were generated after micro - ct scanning of the hind limb 
  distal femurs of wt ( upper panel ) and tsg-6 
  ( lower panel ) male mice .
tsg-6 mice exhibit elevated osteoclast resorptive 
  activity in response to rankl / m - csf but greater trabecular bone mass in the 
  absence of challenge .
a , osteoclast precursors from the long bone 
  marrow of tsg-6 and wild type balb / c ( wt ) mice 
  were cultured on dentine slices in the absence ( media ) or presence of 
  m - csf and/or murine srankl for 10 days before the determination of lacunar 
  resorption .
data are plotted as mean percentage resorption ( n = 4 ) 
   s.e .
lacunar resorption was significantly greater in the 
  presence of cells isolated from tsg-6 mice as compared 
  with wt ; p = 0.03 for cultures containing srankl alone and p 
  = 0.004 for those with m - csf and srankl .
b , three - dimensional models 
  of the trabecular bone were generated after micro - ct scanning of the hind limb 
  distal femurs of wt ( upper panel ) and tsg-6 
  ( lower panel ) male mice .
 images were taken at 10 ( panels i and 
  ii ; scale bars = 400 m ) and 63 ( panels ii , 
  iv , and v ; scale bars = 100
tsg-6 immunoreactivity is shown as red , ha as green , and 
  nuclei as blue fluorescence .
bm , bone marrow ; fcc , 
  femoral condyle cartilage ; gp , epiphyseal growth plate cartilage ; 
  men , meniscus ; po , periosteum ; scb , subcondral 
  bone ; syn , synovium ; tpc , tibial plateau cartilage ; 
  cl , cruciate ligament . immunolocalization of tsg-6 in normal mouse knee joints .
sections 
  were immunostained for tsg-6 and ha , as described under  experimental 
  procedures . 
images were taken at 10 ( panels i and 
  ii ; scale bars = 400 m ) and 63 ( panels ii , 
  iv , and v ; scale bars = 100 m ) magnifications . 
 
tsg-6 immunoreactivity is shown as red , ha as green , and 
  nuclei as blue fluorescence .
bm , bone marrow ; fcc , 
  femoral condyle cartilage ; gp , epiphyseal growth plate cartilage ; 
  men , meniscus ; po , periosteum ; scb , subcondral 
  bone ; syn , synovium ; tpc , tibial plateau cartilage ; 
  cl , cruciate ligament . effect of tsg-6 on bmp-2-induced osteoblast 
  differentiation  as a model of bmp-2-induced osteoblast 
  differentiation , we measured the production of alp 
  ( 50 ) by two murine 
  pre - osteogenic cell lines ( i.e. stromal mba-15.4 cells 
  ( fig .
5b ) ) cultured in the presence of bmp-2 with and without 
  the addition of tsg-6 proteins . the full - length protein caused a 
  dose - dependent reduction in bmp-2-induced alp activity , with essentially 
  complete inhibition of differentiation seen at 33 and 6.6 nm tsg-6 
  for the mba-15.4 and mc3t3.e1 cell lines , respectively .
in contrast , neither 
  link_tsg6 nor cub_c_tsg6 had significant effects on alp release by these cells 
  ( fig .
interactions of tsg-6 with bmps and srankl  the interactions 
  of tsg-6 with bmp-2 and other bmps were investigated using elisas . as 
  illustrated in fig .
6 , bmp-2 
  and the structurally and functionally related bmp-4 , -5 , -6 , -7 , -13 , and -14 
  all interact with tsg-6 , whereas there is no apparent binding to the more 
  distantly related bmp-3 .
further analysis of bmp-2 and two other 
  representative bmps ( i.e. bmp-13 and -14 ) revealed that link_tsg6 and 
  cub_c_tsg6 ( fig .
7 ) have a 
  lower level of binding to these immobilized proteins compared with full - length 
  tsg-6 .
surface plasmon resonance experiments , where tsg-6 or its individual 
  domains were flowed over bmp - coupled chips , allowed determination of the 
  affinities for these interactions .
the dissociation constants for tsg-6 
  binding to bmp-2 , -13 , and -14 are all 0.2 m 
  ( table 2 ) . in the case of 
  bmp-2 ,
its interactions with the link module and cub_c domain are both 
  11-fold weaker , suggesting that bmp-2 interacts with a composite binding 
  surface on tsg-6 that involves both these domains . similarly , bmp-13 and -14 
  have lower affinities for the isolated domains of tsg-6 compared with the 
  full - length protein . however , these bmps both bind more strongly to the link 
  module than the cub_c domain , perhaps indicating that the link module makes a 
  greater contribution to the interaction surfaces in these cases .
table 2dissociation constants for the interactions of tsg-6 and its isolated 
  domains with bmps and srankl , determined by surface plasmon resonanceproteinkd tsg-6kd link_tsg6kd cub_c_tsg6 m m m bmp-2 
   0.220 2.43 
   2.54 
   bmp-13 
   0.236 
   2.84 
   6.88 
   bmp-14 
   0.184 
   1.10 
   10.8 
 
  srankl 
 
  1.97 
 
  8.25 
 
  aeach experiment was performed in duplicate with the average values shown 
  here ; all interactions conformed to a 1:1 langmuir modelbdissociation constants are expressed to three significant figuresca value could not be obtained due to the low level of signal ( response 
  units ) detected , combined with nonspecific binding to the sensor chip dissociation constants for the interactions of tsg-6 and its isolated 
  domains with bmps and srankl , determined by surface plasmon resonance each experiment was performed in duplicate with the average values shown 
  here ; all interactions conformed to a 1:1 langmuir model dissociation constants are expressed to three significant figures a value could not be obtained due to the low level of signal ( response 
  units ) detected , combined with nonspecific binding to the sensor chip we also investigated the interactions tsg-6 , link_tsg6 , and cub_c_tsg6 with 
  srankl by elisa and surface plasmon resonance .
8 , a and 
  b , full - length tsg-6 binds well to immobilized srankl , 
  whereas equivalent amounts ( 5 pmol / well ) of link_tsg6 or the cub_c domain show 
  less binding .
similarly , when srankl was incubated with plates coated with the 
  tsg-6 proteins ( fig . 
 
8c ) , the extent of binding to link_tsg6 and cub_c_tsg6 
  was lower than that seen for the full - length protein .
these data suggest that , 
  like bmp-2 , -13 , and -14 , srankl binds to a composite interaction surface 
  involving both the link and cub_c domains of tsg-6 .
consistent with this , 
  surface plasmon resonance analysis revealed that full - length tsg-6 bound to 
  immobilized srankl with an 4-fold higher affinity than the isolated link 
  module ( i.e. about 2 and 8 m , respectively ) , although 
  we were unable to obtain a kd value for cub_c_tsg6 ( see 
  table 1 ) .
the murine 
  pre - osteoblast cell lines mba-15.4 ( a ) and mc3t3-e1 ( b ) were 
  cultured for 7 days with bmp-2 in the absence or presence of rhtsg-6 ( at 
  010000 ng / ml ) or molar equivalents of link_tsg6 or cub_c_tsg6 .
alp 
  activity was measured , and values were plotted as the mean alp activity as a 
  percentage of control ( n = 8  s.e . ) where the addition of 
  bmp-2 alone was normalized to 100% ;   and   = 
  p < 0.01 and p < 0.001 , respectively , compared with 
  bmp-2 alone .
mba-15.4 cells ( a ) were more responsive to bmp-2 than 
  mc3t3-e1 cells ( b ) , with alp activity up - regulated by 272 and 37% , 
  respectively .
 , data for control cells with no added rhtsg-6 , link_tsg6 , 
  or cub_c_tsg6 are shown as 0 pm protein ; data from cells with added 
  tsg-6 proteins are plotted on a log scale .
the murine 
  pre - osteoblast cell lines mba-15.4 ( a ) and mc3t3-e1 ( b ) were 
  cultured for 7 days with bmp-2 in the absence or presence of rhtsg-6 ( at 
  010000 ng / ml ) or molar equivalents of link_tsg6 or cub_c_tsg6 .
alp 
  activity was measured , and values were plotted as the mean alp activity as a 
  percentage of control ( n = 8  s.e . ) where the addition of 
  bmp-2 alone was normalized to 100% ;   and   = 
  p < 0.01 and p < 0.001 , respectively , compared with 
  bmp-2 alone .
mba-15.4 cells ( a ) were more responsive to bmp-2 than 
  mc3t3-e1 cells ( b ) , with alp activity up - regulated by 272 and 37% , 
  respectively .
 , data for control cells with no added rhtsg-6 , link_tsg6 , 
  or cub_c_tsg6 are shown as 0 pm protein ; data from cells with added 
  tsg-6 proteins are plotted on a log scale . as described above , bmp-2 and srankl
both bind with higher affinity to the 
  intact tsg-6 protein compared with its isolated domains . in addition
, the 
  full - length protein has more potent biological effects than the link_tsg6 or 
  cub_c_tsg6 domains ( see figs . 2 
  and 5 ) . together
this provides 
  evidence that tsg-6-mediated inhibition of osteoblastogenesis and of 
  osteoclastic resorption is likely to be mediated via its direct interaction 
  with bmp-2 and rankl , respectively .
values are 
  plotted as the mean absorbance at 405 nm ( n = 4  s.e . ) after 
  a 30-min development time .
values are 
  plotted as the mean absorbance at 405 nm ( n = 4  s.e . ) after 
  a 30-min development time .
although it is well established that tsg-6 is protective in various 
  experimental models of arthritis 
  ( 3135 ) , 
  the exact mechanism(s ) by which its effects are mediated remains unclear . here 
 
we have identified two novel functions for tsg-6 ; it inhibits both 
  srankl - induced bone resorption by osteoclasts and bmp-2-induced osteoblast 
  differentiation , which may have a key role in protecting joint tissues during 
  disease and in the regulation of bone homeostasis .
our functional and ligand binding studies suggest that these activities of 
  tsg-6 are mediated through its interactions with rankl or bmp-2 ( and possibly 
  other bmps ) , respectively , where both of these are likely to require composite 
  binding surfaces involving the link module and cub_c domain of tsg-6 . in this 
  respect ,
the new binding partners reported here differ from most other tsg-6 
  ligands ( e.g. ha , heparin , bikunin , pentraxin-3 , and 
  thrombospondin-1 ) , which have binding sites within the link module 
  ( 22 ) . at present , the only 
  known ligand that binds outside this region of tsg-6 is fibronectin , which 
  interacts with the cub_c domain 
  ( 12 ) . in this study
we have shown that osteoclasts derived from 
  tsg-6 mice give rise to a marked elevation in dentine 
  resorption compared with those from wild type controls , which is consistent 
  with the severe bone erosion seen in tsg-6 mice with 
  proteoglycan - induced arthritis 
  ( 35 ) .
these experiments also 
  demonstrate that tsg-6 is produced by bone marrow - derived cells ( e.g. 
  pre - osteoclasts and/or stromal cells ) in response to stimulation with 
  srankl / m - csf and , thus , that tsg-6 has an autocrine and/or paracrine function 
  in this tissue .
rankl , the major regulator of osteoclast differentiation 
  ( 51 , 
  52 ) , is expressed on 
  osteoblasts as well as on dendritic cells and t cells 
  ( 53 , 
  54 ) in response to 
  calciotropic factors such as prostaglandin e2 , il-1 , and tnf 
  ( 55 ) .
the combination of il-1 
  or tnf with il-17 is particularly potent at inducing rankl expression , 
  e.g. in synoviocytes 
  ( 56 ) .
rankl binds to its 
  receptor rank on mononuclear osteoclast precursors 
  ( 57 ) , where this interaction 
  not only induces osteoclast differentiation but also stimulates the bone 
  resorbing activity of mature osteoclasts 
  ( 58 ) .
we have shown here that 
  tsg-6 inhibits rankl - induced bone resorption by human osteoclasts in a 
  dose - dependent manner .
our data suggest that tsg-6 acts at the point of 
  osteoclast activation , since we observed inhibition of dentine erosion , but no 
  reduction in osteoclast number or size when tsg-6 was added to pbmc cultures . 
 
it seems reasonable to suggest that tsg-6 might mediate this effect by binding 
  directly to rankl , thereby blocking its association with rank ; however , 
  further studies are required to demonstrate this definitively .
figure 7.bmps exhibit tighter binding to full - length tsg-6 than to its link and 
  cub_c domains .
the interactions of full - length rhtsg-6 (  ) with 
  immobilized bmp-2 , -13 , or -14 were compared with link_tsg6 (  ) 
  ( a ) or cub_c_tsg6 (  ) ( b ) .
protein binding was detected 
  using antibodies specific for the link module or cub_c domain as appropriate . 
 
values are plotted as the mean absorbance at 405 nm ( n = 8  
  s.e . ) after 60-min ( a ) or 30-min ( b ) development times .
bmps exhibit tighter binding to full - length tsg-6 than to its link and 
  cub_c domains .
the interactions of full - length rhtsg-6 (  ) with 
  immobilized bmp-2 , -13 , or -14 were compared with link_tsg6 (  ) 
  ( a ) or cub_c_tsg6 (  ) ( b ) .
protein binding was detected 
  using antibodies specific for the link module or cub_c domain as appropriate . 
 
values are plotted as the mean absorbance at 405 nm ( n = 8  
  s.e . ) after 60-min ( a ) or 30-min ( b ) development times . at present ,
osteoprotegerin ( opg ) , a soluble decoy receptor for rankl , is 
  the only known antagonist of the rankl / rank interaction that can effectively 
  inhibit osteoclast maturation and activation in vitro 
  ( 59 ) .
furthermore , studies on 
  rats with antigen - induced arthritis showed rankl ( expressed on the surface of 
  synovial effector t cells ) to be the key mediator of joint damage and bone 
  erosion , where treatment with opg provided protection against these effects 
  ( 60 ) .
the ability of tsg-6 to 
  inhibit rankl - induced osteoclast activity , possibly in a manner similar to opg 
  ( i.e. via a direct interaction with rankl ) , thus makes it a potential 
  model for the development of therapeutics .
the dissociation constant observed 
  here for the tsg-6/srankl interaction ( 2 m ) is 3 orders of 
  magnitude higher than that reported for the binding of opg to rankl 
  ( 61 ) .
however , as noted by 
  schneeweis et al . 
  ( 61 ) , the tight binding 
  observed for opg / rankl ( kd = 10 nm ) is likely 
  to be due the formation of 1:1 complexes between homodimers of opg and 
  homotrimers of rankl , i.e. leading to a high avidity 
  ( 61 ) .
interestingly , a 
  truncated form of opg containing the rankl - binding site but lacking the 
  dimerization domain bound rankl with a kd of 3 
  m .
our study utilized monomeric srankl , and it is possible that 
  the interaction of tsg-6 with trimeric rankl might have a considerably higher 
  affinity ; however , this remains to be investigated . in the lacunar resorption 
  assay used here , srankl ( mr = 20 kda ) is present at a 
  concentration of 50 ng / ml ( 2.5 nm ) , and we observed that rhtsg-6 
  inhibited dentine erosion with an ic50 of 15 ng / ml ( 0.5 
  nm ) . by comparison , human recombinant opg ( r&d systems ; 43.5 
  kda ) was found to have an ic50 value of 0.25 nm in 
  this assay.6 this is 
  consistent with a previous investigation of osteoclast formation from 
  arthrosplasty - derived macrophages , showing that human opg inhibited lacunar 
  resorption with an ic50 of between 50 and 100 ng / ml ( 1.5 
  nm ) ( 62 ) .
overall 
  this indicates that tsg-6 has a similar potency to opg in the in 
  vitro inhibition of osteoclastic resorption .
the 
  interactions of rhtsg-6 , link_tsg6 , or cub_c_tsg6 with srankl were compared 
  where either the srankl ( a and b ) or tsg-6 proteins 
  ( c ) were immobilized on microtiter plates .
absorbance values at 405 
  nm were measured after 20 min ( a ) , 30 min ( b ) , or 60 min 
  ( c ) and are plotted as mean values ( n = 8  
  s.e . ) .
the 
  interactions of rhtsg-6 , link_tsg6 , or cub_c_tsg6 with srankl were compared 
  where either the srankl ( a and b ) or tsg-6 proteins 
  ( c ) were immobilized on microtiter plates .
absorbance values at 405 
  nm were measured after 20 min ( a ) , 30 min ( b ) , or 60 min 
  ( c ) and are plotted as mean values ( n = 8  
  s.e . ) .
our observation that unchallenged tsg-6 mice have 
  higher trabecular bone mass than wild type controls might at first glance 
  appear to be at odds with the inhibitory effect of tsg-6 on bone erosion 
  described above .
however , we hypothesize that , although tsg-6 inhibits 
  osteoclast activity at inflammatory sites , it also has a pivotal role in 
  normal bone homeostasis . this is supported by our finding that tsg-6 is 
  expressed in normal joint tissue ( e.g. in bone marrow ) and that it 
  significantly reduces the production of alp ( a marker of differentiation ) by 
  osteoblast precursors stimulated with bmp-2 .
63 ) have also described 
  inhibition of bmp-2-mediated osteoblastogenesis after overexpression of tsg-6 
  in human mesenchymal stem cells ( hmsc ) or by the addition of partially 
  purified recombinant tsg-6 proteins to hmsc cultures ; binding of tsg-6 to 
  bmp-2 was detected in these systems by immunoprecipitation .
the authors 
  proposed that the link module of tsg-6 is responsible for inhibition of 
  osteoblast differentiation via interaction with bmp-2 , based on 
  semiquantitative experiments using deletion mutants where , for example , 
  protein lacking the link module was found to be inactive .
this conclusion is 
  inconsistent with our results that were obtained using defined amounts of 
  pure , highly characterized protein preparations 
  ( 1214 , 
  18 ) .
( 63 ) report little or no 
  inhibition of alp expression or immunoprecipitation of bmp-2/tsg-6 complex 
  with their deletion mutant lacking the link module , their data also show a 
  substantial reduction in these effects ( compared with full - length tsg-6 ) with 
  the cub deletion mutant .
( 63 ) are 
  consistent with bmp-2 binding to a surface that involves both the link and cub 
  modules of tsg-6 .
the ability of tsg-6 to interact with members of the tgf/bmp 
  superfamily closely related to bmp-2 
  ( 64 ) , namely bmp-4 , -5 , -6 , 
  -7 , -13 , and -14 , further supports the hypothesis that tsg-6 has a role in 
  bone homeostasis .
bmp-2 , -6 , -7 , and -9 are important in inducing the 
  differentiation of mesenchymal stem cells into osteoblasts 
  ( 65 , 
  66 ) , whereas most other bmps 
  are able to stimulate osteogenesis in mature osteoblasts 
  ( 6771 ) . 
 
tsg-6 might also have regulatory roles in other contexts , e.g. bmp-4 
  has been implicated in tooth development 
  ( 72 ) , bmp-2 and -4 are 
  required for limb patterning 
  ( 73 ) , and bmp-2 is essential 
  for the initiation of fracture repair 
  ( 74 ) . in this regard tsg-6 may 
  function like some of the known bmp antagonists , i.e. those of the 
  chordin family , noggin ( 75 ) , 
  gremlin ( 76 ) , and brorin 
  ( 77 ) , as well as sost 
  ( 78 ) , gdf3 
  ( 79 ) , and the recently 
  discovered suppressors of mineralization such as asporin 
  ( 80 ) , nov 
  ( 81 ) , and osteoclast 
  inhibitory lectin ( 82 ) . 
 
interestingly , we have shown that tsg-6 does not bind to bmp-3 , an antagonist 
  of osteogenic bmps that blocks differentiation of osteoprogenitor cells into 
  osteoblasts ( 83 ) .
in addition to its role in osteogenesis , bmp-2 can induce cartilage 
  formation and is expressed at elevated levels around lesions ( e.g. in 
  osteoarthritis ) , suggesting that it might contribute to cartilage repair 
  ( 84 ) . a recent study ,
looking 
  at the effects of bmp-2 overexpression in murine knee joints , has shown that 
  this protein promotes matrix turnover in cartilage with increased proteoglycan 
  synthesis and aggrecan degradation 
  ( 85 ) . furthermore , blocking of 
  bmp-2 activity ( by gremlin ) in il-1-damaged cartilage gave rise to an overall 
  decrease in proteoglycan content .
it is not yet known whether tsg-6 , which is 
  expressed by chondrocytes in response to il-1 
  ( 28 , 
  29 ) , can inhibit 
  bmp-2-mediated functions in cartilage and if so whether this contributes to 
  its protective effects in arthritis .
the significantly higher bone mass that we have observed in the present 
  study in tsg-6 mice compared with wild type animals 
  provides compelling evidence that tsg-6 has an in vivo role in the 
  regulation of bone formation .
although we have demonstrated that tsg-6 can 
  inhibit bmp-2-mediated osteoblast differentiation in vitro , we can not 
  rule out that tsg-6 may also affect the activities of other modulators of 
  osteoblastogenesis ( e.g. l - ascorbate and 
  1,25-(oh)-2-vitamin d3 ( 86 , 
  87 ) ) , which will require 
  further investigation . in this study
we have shown that tsg-6 is expressed in mouse knee joints , 
  where the strongest tsg-6 immunostaining was associated with the cells in the 
  epiphyseal and metaphyseal bone marrow and at the margins of bone marrow and 
  trabecular bone .
our in vitro cell culture experiments are also 
  consistent with tsg-6 being produced by bone marrow - derived cells in mice .
opg 
  and rankl have been shown to co - localize in the bone marrow lining cells , 
  osteoblasts , and newly embedded osteocytes at sites of bone remodeling in rats 
  ( 88 ) .
in addition , in rat 
  tibiae , alp activity ( a marker of bmp-2-mediated differentiation ) has been 
  detected on osteoblasts and some bone marrow fibroblastic stromal cells , in 
  particular those cells closest to the bone surface 
  ( 89 ) .
these observations are 
  indicative that tsg-6 is present within the bone marrow at similar 
  localizations to bmp-2 , rankl , and opg during bone remodeling .
the above data have led us to hypothesize that tsg-6 might coordinately 
  regulate the activities of osteoclasts and osteoblasts , thus having a key role 
  in bone turnover .
the molecular basis for these opposing functions and how 
  they are differentially controlled remains to be determined .
what is clear is 
  that tsg-6 is a novel regulator of bone cell biology with potent effects on 
  two processes that are central to both normal physiology and articular joint 
  disease .
 tsg-6 is an inflammation - induced protein that is produced at pathological 
  sites , including arthritic joints . in animal models of arthritis , 
 tsg-6 
  protects against joint damage ; this has been attributed to its inhibitory 
  effects on neutrophil migration and plasmin activity . 
 here we investigated 
  whether tsg-6 can directly influence bone erosion . 
 our data reveal that tsg-6 
  inhibits rankl - induced osteoclast differentiation / activation from human and 
  murine precursor cells , where elevated dentine erosion by osteoclasts derived 
  from tsg-6-/- mice is consistent with the very severe 
  arthritis seen in these animals . 
 however , the long bones from unchallenged 
  tsg-6-/- mice were found to have higher trabecular mass 
  than controls , suggesting that in the absence of inflammation tsg-6 has a role 
  in bone homeostasis ; we have detected expression of the tsg-6 protein in the 
  bone marrow of unchallenged wild type mice . 
 furthermore , we have observed that 
  tsg-6 can inhibit bone morphogenetic protein-2 ( bmp-2)-mediated osteoblast 
  differentiation . 
 interaction analysis revealed that tsg-6 binds directly to 
  rankl and to bmp-2 ( as well as other osteogenic bmps but not bmp-3 ) via 
  composite surfaces involving its link and cub modules . 
 consistent with this , 
  the full - length protein is required for maximal inhibition of osteoblast 
  differentiation and osteoclast activation , although the isolated link module 
  retains significant activity in the latter case . 
 we hypothesize that tsg-6 has 
  dual roles in bone remodeling ; one protective , where it inhibits rankl - induced 
  bone erosion in inflammatory diseases such as arthritis , and the other 
  homeostatic , where its interactions with bmp-2 and rankl help to balance 
  mineralization by osteoblasts and bone resorption by osteoclasts . 
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
Supplementary Material