proteinase - activated receptors ( pars ) belong to a family of g - protein - coupled receptors with seven transmembrane domains activated via proteolytic cleavage by serine proteinases .
a total of four pars have been identified and cloned . among them , par-1 [ 2 , 3 ] , par-3 , and par-4   are targets for thrombin , trypsin , and cathepsin g , whereas par-2 is resistant to thrombin but can be activated by trypsin , mast cell tryptase [ 6 , 7 ] , neutrophil elastase , and insect - derived proteinase . pars are expressed by various cells involved in inflammatory and immunological responses , such as vascular endothelial cells , epithelial cells , mast cells , t cells , monocyte , eosinophils , and neutrophils [ 10 , 11 ] . in these cells ,
activation of pars affects their main functions such as proliferation , degranulation , and release of inflammatory mediators [ 10 , 11 ] . in our previous study , we have showed the expression of par-1 , par-2 , and par-3 on t cells , and thrombin- , trypsin- , and tryptase - induced interleukin ( il-6 ) release from t cells .
it has also been reported that cytoplasmic free calcium and phospholipase c and protein kinase c activation are increased in t - leukemic cell lines following stimulation with thrombin or the thrombin receptor agonist peptide .
thrombin and thrombin receptor agonist also enhanced cd69 expression and il-2 productions by cross - linking t cell receptors in both jurkat t cells and peripheral blood lymphocytes .
we , therefore , anticipated that thrombin , trypsin , and tryptase might induce tnf release from t cells through pars .
tnf is a major proinflammatory cytokine that is thought to be important in the pathogenesis of asthma , food allergy , ocular allergy , and atopic dermatitis .
it has been reported that the increased number of tnf expressing cells and levels of tnf is observed in the bronchoalveolar lavage ( bal ) and in the airways of asthmatics .
inhaled tnf increases airway responsiveness to methacholine in asthmatic subjects associated with a sputum neutrophilia .
since pars , tnf , and t cells all play roles in inflammation , we believe , there must be some linkages between them .
the aim of the present study is to investigate roles of thrombin , tryptase , trypsin , elastase , and agonist peptides of pars in the secretion of tnf from purified human t cells and subtypes of t cells .

 human thrombin , trypsin ( specific activity : 10,000  baee  u / mg protein ) , soybean trypsin inhibitor ( sbti ) , and bovine serum albumin ( bsa , fraction v ) were purchased from sigma ( st louis , mo , usa ) .
recombinant hirudin and human neutrophil elastase ( specific activity : 20 meo - suc - ala - ala - pro - val - pna u / mg protein ) were obtained from calbiochem ( san diego , ca , usa ) .
recombinant human lung  tryptase ( specific activity : 1,000  n cbz - l - lysine thiobenzyl ester u / mg protein ) was from promega ( madison , wi , usa ) .
agonist peptides of pars , and their reverse forms , and par-2 antagonist peptide fsllry - nh2 were synthesized in cl bio - scientific inc .
the sequences of the active and reverse peptides were par-1 , sfllr - nh2 and rllfs - nh2 , tfllrn - nh2 and nrllft - nh2 ; par-2 , sligkv - nh2 and vkgils - nh2 as well as trans cinnamoyl ( tc)-ligrlo - nh2 and tc - olrgil - nh2 ; par-3 , tfrgap - nh2 and pagrft - nh2 .
rpmi 1640 and newborn calf serum ( ncs ) were obtained from gibco ( carlsbad , ca , usa ) .
pe - conjugated mouse anti - human cd3 monoclonal antibody , pe - conjugated goat - anti rabbit igg , and tnf opteia elisa kits were purchased from bd pharmingen ( san jose , ca , usa ) .
trizol reagent and sybr green i stain were purchased from invitrogen ( carlsbad , ca , usa ) .
cellular activation of signaling kits for extracellular signal - regulated kinase ( erk ) , 2-(2-diamino)-3-methoxyphenyl-4h-1-benzopyran-4-one ( pd98059 ) , akt , pi3k , and p38 2-(4-morpholinyl)-8-phenyl-4h-1-benzopyran-4-one ( ly294002 ) was purchased from cell signaling technology ( beverly , ma , usa ) .
exscript rt reagent kit and sybr premix ex taq ( perfect real time ) were obtained from takara ( dalian , china ) .
rabbit anti - human par-1 and rabbit anti - huamn par-2 polyclonal antibodies were purchased from santa cruz biotechnology ( santa cruz , ca , usa ) .
fitc - conjugated mouse anti - human cd4 monoclonal , pe - conjugated mouse anti - human cd8 monoclonal , percp - cy5.5-conjugated mouse anti - human tnf monoclonal , fitc - conjugated mouse anti - human ifn monoclonal , pe - conjugated mouse anti - human il-4 monoclonal , apc - conjugated mouse anti - human cd25 monoclonal , and apc - conjugated mouse anti - human il-17 monoclonal antibodies were purchased from ebioscience .
all other reagents were of analytic grade and obtained from sigma ( st louis , mo , usa ) .
human t cells were isolated from peripheral blood mononuclear cells ( pbmcs ) by a macs system with t cell isolation kit i according to the manufacturer 's protocol . in brief
, pbmcs were isolated from fresh blood donated by healthy volunteers , 100  ml from each individual per visit .
the informed consent from each volunteer and agreement with the ethical committee of the first affiliated hospital of nanjing medical university were obtained . after being separated from red blood cells by ficoll - paque density gradient ,
pbmcs were collected and incubated with microbead - linked anti - cd3 monoclonal antibody for 15  min at 8c .
cd3 + t cells were separated from other cells by passing through a magnetic cell separation system . for purity analysis
, the cells were resuspended in pbs and incubated with pe - conjugated monoclonal antibody against human cd3 for 1  h. the purity of t cells was consistently more than 95% and cell viability was more than 98% .
the purified cd3 + t cells were then used for the further cell challenge tests .
t cells were cultured in 24-well culture plates at a density of 5  10cells / well in rpmi 1640 medium containing 10% ncs at 37c for 2  h with 5% co2 , respectively .
the culture supernatants were then removed and cells were washed twice with fresh serum - free rpmi 1640 medium at 300  g for 10  min . for challenge experiments ,
cells were exposed to various doses of thrombin ( 0.013.0  g / ml , 1  u = 0.5  g , 1  u / ml = 5.6  nm , u = nih unit ) , trypsin ( 0.010.3  g / ml , 1  g / ml = 42  nm ) , tryptase ( 0.252.0  g / ml , 1  g / ml = 7.4  nm ) , and elastase ( 0.010.3  g / ml , 1  g / ml = 34  nm , 1  u / ml = 1700  nm ) with or without their inhibitors ; and to agonist peptides of par-1 , par-2 and par-3 ( all at 0.1100  m ) and their reverse peptides , respectively , for 16  h before the culture , supernatants were harvested and stored at 40c till use .
quantitative expression of tnf mrnas in t cells was determined by real - time pcr following the manufacture 's protocol . briefly , after synthesizing cdna from the total rna by using exscripttm rt reagent kit , real - time pcr was performed by using sybr premix ex taq on the abi prism 7000 sequence detection system ( perkin elmer applied systems , foster city , ca , usa ) .
each reaction contains 12.5  l of 2  sybr green master mix , 1  l of 10  m of primers , 1  l of the cdna , to a total volume of 25  l .
the thermal cycling conditions included an initial denaturation step at 50c for 2  min , 95c for 10  min ; 40 cycles at 95c for 15  s , annealing temperatures at 60c for 30  s , and extension at 72c for 30  s. the sequences of pcr primers for tnf and -actin were 5-ccccagggacctctctctaatc-3 ( forward ) and 5-ggtttgctacaacatgggctaca-3 ( reverse ) ; 5-aggggccggactcgtcatact-3 ( forward ) , and 5-ggcggcaacaccatgtaccct-3 ( reverse ) , respectively .
consequently , at the end of the pcr cycles , specificities of the amplification products were controlled by dissociation curve analysis .
expression of mrna in each sample was finally determined after correction with -actin expression . the gene specific threshold cycle ( ct ) for each sample ( ct ) was corrected by subtracting the ct for the housekeeping gene -actin .
untreated controls were chosen as the reference samples , and the ct for all experimental samples was subtracted by the ct for the control samples ( ct ) .
t cells were preincubated with 50  m of pd98059 , 20  m of ly294002 , or medium alone for 30  min before adding thrombin 3.0  g / ml , trypsin 0.3  g / ml , or medium alone for 30  min , 2  h , or 6  h. the cells were lysed in a buffer containing 20  mm of tris - hcl ( ph 7.4 ) , 137  mm of nacl , 10% glycerol , 1% triton x-100 , 2  mm of edta , 25  mm of -glycerophosphate , 2  mm of sodium pyrophosphate , and 0.5  mm of dithiothreitol at 4c for 30  min .
cell debris was removed by centrifugation of the lysate at 12,000  g for 10  min .
the supernatants were mixed with equal volumes of 2x sodium dodecyl sulphate ( sds ) sample buffer and heated to 100c for 10  min .
an equal volume of sample was fractionated by sds - page on a 10% acrylamide gel and transferred onto polyvinylidene difluoride ( pvdf ) membranes with a bio - rad transfer system , according to the manufacturer 's instructions . after blocking nonspecific binding sites with 5% bsa in tbst ( 50  mm of tris , 0.15  m of nacl , 0.1% tween 20 , ph 7.6 ) for 1  h
, membranes were probed with phospho - erk1/2 , phospho - akt , phospho - p38 , or phospho - pi3k antibodies at 4c overnight , followed by incubation with hrp - conjugated secondary antibodies .
immunoreactive bands were visualized by using enhanced chemiluminescence reagents according to the manufacturer 's protocol .
densitometry analysis of immunoblots was carried out using quantity one software ( bio - rad , usa ) .
the levels of tnf in culture supernatants were measured with opteia elisa kits according to the manufacturer 's instructions .
the plates were read on a plate reader ( molecular devices , menlo park , ca ) with the softmax data analysis program .
the minimum detectable concentration of tnf was 2.2  pg / ml . to test the par1 and 2 expressions after treatment of trypsin and thrombin
, isolated t cells were pelleted by centrifugation at 450  g for 10  min after cells were stimulated with thrombin 3.0  g / ml , trypsin 0.3  g / ml , or medium alone for 16  h. for par1 and par2 staining , cells were incubated with rabbit anti - human par1 or par2 antibodies at 37c for 1  h. after washing , cells were incubated with pe - conjugated goat anti - rabbit igg antibody 37c for 45  min .
after washing , cells were analyzed on a fluorescence - activated cell sorting ( facs ) arial flow cytometer with celldevia software ( bd biosciences , usa ) . to test the secretion of tnf from subtypes of t cells
, isolated t cells were pelleted by centrifugation at 450  g for 10  min and then fixed and permeabilized by using a cell fixation / permeabilization kit ( bd pharmingen ) . briefly
, thoroughly resuspended cells were added in 100  l of bd cytofix / cytoperm solution and incubated for 20  min at 4c .
cells were then incubated with fluorescence labeled anti - human cd4 , cd8 , cd25 , tnf , ifn , il-4 , and il-17 monoclonal antibodies or isotope control , respectively ( at a final concentration of 4  g / ml ) at 4c for 30  min .
after washing , cells were analyzed on a fluorescence - activated cell sorting ( facs ) arial flow cytometer with celldevia software ( bd biosciences , usa ) .
the purity of t cells was consistently more than 95% ( date was shown in supplementary material , figure s1 ) .
it has been shown that thrombin , trypsin , and tryptase can induce proinflammatory cytokine il-6 release from t cells , but little is known of serine proteinase - induced tnf release from t cells . here , we showed that thrombin at concentrations of 1.0 and 3.0  g / ml provoked tnf release from t cells following 16  h incubation period in a dose - dependent manner .
approximately up to 2.5-fold increase in tnf release was observed when t cells were incubated with thrombin for 16  h. at 6  h following incubation , data ( not shown ) on both basal and induced tnf release from t cells were inconsistent .
this is most likely due to the limitation of the assay sensitivity and relatively low secretion of tnf .
par-1 agonist peptides , sfllr - nh2 at the concentration of 100  m and tfllrn - nh2 at the concentration of 5  m , induced a significant release of tnf at 16  h following incubation .
however , rllfs - nh2 , a reverse peptide of sfllr - nh2 , and nrllft - nh2 , a reverse peptide of tfllrn - nh2 , had little effect on release of tnf from t cells ( figure 1(a ) ) .
hirudin , a specific thrombin inhibitor , was able to inhibit thrombin - induced secretion of tnf .
approximately up to 82.4% inhibition of thrombin - induced secretion of tnf was observed when 3.0  g / ml of thrombin and 10  u / ml of hirudin were added to t cells for 16  h. hirudin alone at the concentrations tested had little effect on tnf secretion from t cells .
sch 79797 , a par-1 antagonist at the concentration of 1  m , inhibited 89% thrombin - induced tnf release from t cells ( figure 1(a ) ) .
similarly , trypsin at the concentration of 0.3  g / ml induced 2.3-fold increase in tnf release from t cells at 16  h ( figure 1(b ) ) .
however , tryptase at the concentrations up to 2  g / ml and elastase at the concentrations up to 6  u / ml had little effect on tnf release from t cells ( data not shown ) .
inhibitors of trypsin , sbti at the concentrations of 10 and 30  g / ml , eliminated 0.3  g / ml trypsin - induced tnf release by a value up to 94.8 and 94.2% , respectively .
sbti alone at the concentrations tested had little effect on tnf secretion from t cells .
sch 79797 , a par-1 antagonist at the concentration of 1  m , inhibited 96.8% trypsin - induced tnf release from t cells ( figure 1(b ) ) .
sligkv , an agonist peptide of par-2 and tfrgap - nh2 , an agonist peptide of par-3 at the concentrations up to 100  m , did not appear to have any effect on tnf release from t cells ( data not shown ) . in order to confirm the findings above
, we investigated the influence of the serine proteinases on the expression of tnf mrna in t cells .
it was found that the expression of tnf mrna was upregulated when t cells were incubated with thrombin at 1 and 3  g / ml for 2 and 6  h. the maximum enhanced expression of tnf mrna was 4.2-fold over baseline control ( figure 2(a ) ) after 6  h incubation .
hirudin , a specific thrombin inhibitor at the concentration of 3  u / ml , completely abolished thrombin - induced upregulated expression of tnf mrna after 6  h incubation ( figure 2(b ) ) .
trypsin at the concentration of 0.3  g / ml also induced increased expression of tnf mrna by a value up to approximately 4.0-fold in t cells ( figure 2(a ) ) , which was completely blocked by sbti ( figure 2(b ) ) .
similarly , sch 79797 at the concentration of 1  m inhibited both thrombin- and trypsin - induced upregulated expression of tnf mrna in t cells by a value up to 72 and 72.5% , respectively ( figure 2(b ) ) .
sfllr - nh2 at the concentration of 100  m and tfllrn - nh2 at the concentration of 5  m significantly increase the expression of tnf mrna at 2 and 6  h following incubation ( figure 2(a ) ) .
but rllfs - nh2 , a reverse peptide of sfllr - nh2 , and nrllft - nh2 , a reverse peptide of tfllrn - nh2 , had little effect on expression of tnf mrna in t cells ( data not shown ) .
at the same time , neither thrombin nor trypsin showed obvious effect on the expression of par-1 and par-2 ( data not shown ) .
it is wellknown that there are numerous subtypes of t cells and each of them has distinctive functions .
we , therefore , investigated subtypes of t cells by flow cytometer analysis in order to determine the subtypes that upregulate tnf in response to trypsin or thrombin .
the results showed that trypsin and thrombin induced upregulated expression of tnf in cd4 + t cells , but not cd8 + t cells , following 16  h incubation period . among cd4 + t cells , trypsin and thrombin enhanced tnf expression in il-4 + or cd25 + t cells , but not in ifn+ or il-17 + t cells .
sch 79797 was able to inhibit enhanced tnf expression induced by trypsin and thrombin ( figures 3(a ) and 3(b ) ) . in order to examine signal transduction pathways of thrombin and trypsin
, t cells were preincubated with pd98059 , ly294002 , or medium alone for 30  min before adding thrombin 3.0  g / ml , trypsin 0.3  g / ml , or medium alone for 16  h. following 16  h incubation period , pd98059 an inhibitor of mapk pathway , and ly294002 , an inhibitor of pi3k , completely blocked thrombin- and trypsin - induced release of tnf ( figure 4(a ) ) . furthermore , pd98059 inhibited thrombin- and trypsin - induced upregulation of expression of tnf mrna by a value up to 91.2 and 98.6% , and ly294002 eliminated thrombin- and trypsin - induced expression of tnf mrna by 95.5 and 83.2% in t cells following 6  h incubation ( figure 4(b ) ) .
3  g / ml ) induced enhanced phosphorylation of erk1/2 in t cells following 0.5 , 2 , and 6  h incubation periods .
however , thrombin and trypsin did not significantly affect phosphorylation of p38 in t cells following 0.5 , 2 , and 6  h incubation periods ( date was shown in supplementary material , figure s2 ) .
pd98059 was able to completely block thrombin- and trypsin - induced phosphorylation of erk1/2 when it was preincubated with t cells for 30  min .
thrombin at a concentration of 3  g / ml and trypsin at a concentration of 0.3  g / ml induced significantly increased phosphorylation of akt in t cells following 0.5 , 2 , and 6  h incubation periods .
however , thrombin and trypsin did not significantly affect phosphorylation of pi3k in t cells following 0.5 , 2 , and 6  h incubation periods ( date was shown in supplementary material , figure s3 ) .
ly294002 was able to block thrombin- and trypsin - induced phosphorylation of akt when it was incubated with t cells for 30  min .
we discovered in the present study that serine proteinases thrombin and trypsin , but not tryptase induced tnf release from human t cells .
since tnf is a potent proinflammatory cytokine , our observation is likely to add some novel information for , understanding of actions of serine proteinases in causing inflammation . as little as 1.0  g / ml of thrombin was able to induce significant tnf release from t cells , suggesting this proteinase is a potent secretagogue of tnf .
this concentration of thrombin should be easily achieved in blood , particularly when the processes of platelet aggregation and coagulation are initiated .
inhibition of thrombin - induced tnf release by a specific inhibitor of thrombin and hirudin indicates that action of thrombin on t cells was dependent on the enzymatic activity of this serine proteinase .
there are 3 receptors for thrombin on cells , including par-1 , par-3 , and par-4 [ 2 , 3 ] .
since par-1 agonist peptides sfllr - nh2 , and tfllrn - nh2 , but not par-3 agonist peptide tfrgap - nh2 were capable of stimulating tnf release , a par-1 antagonist sch 79797   almost completely abolished thrombin - induced tnf release from t cells , and purified human t cells do not express par-4 ; the action of thrombin on t cells is most likely through activation of par-1 .
our previous report which found thrombin - induced il-6 secretion from human peripheral blood t cells may support our current findings . while little information is available on induction of tnf release from t cells by trypsin , the ability of trypsin to stimulate il-6 secretion from t cells   may support the anticipation that trypsin is capable of inducing cytokine release from t cells .
as little as 0.3  g / ml of trypsin was able to provoke tnf secretion from t cells proved that it is a potent stimulus of tnf release .
as for thrombin , inhibitor of trypsin sbti was able to inhibit trypsin - induced tnf release from t cells , indicating that an intact catalytic site is required for the serine proteinase to stimulate tnf release .
since par-1 is one of three receptors of trypsin , par-1 agonist peptides sfllr - nh2 and tfllrn - nh2 are capable of stimulating tnf release from t cells , and sch 79797 almost completely abolished trypsin - induced tnf release from t cells , the action of trypsin on t cells is most likely through activation of par-1 .
since par-2 agonist peptide sligkv - nh2 and tryptase are not capable of stimulating tnf release from t cells , the action of trypsin on t cells is not likely through activation of par-2 .
trypsin- and thrombin - induced upregulated expression of tnf was observed in cd4 + , il-4 + or cd25 + t cells , indicating that il-4 + , and cd25 + t cells are major sources of tnf .
while little information on the relationship between cd25 + t cells and tnf is available , a study which found that the percentage of cd4(+)cd25(+ ) t cells were significantly high , but the percentage of foxp3(+ ) cells were low in allergic rhinitis patients , and that il-4 , il-5 , and tnf levels in nasal lavage fluids were high indicates that the increased tnf release may be from cd4(+)cd25(+ ) , nonregulatory t cells .
we believe that the current study is the first work that demonstrates coexpression of cd25 and tnf in the subtype of cd4(+ ) t cells .
similarly , we clearly found that il-4 + t cells express enhanced tnf , though little information on co - expression of il-4 and tnf in t cells is available .
this finding implicates that trypsin and thrombin may be involved in the inflammation through induction of tnf release from il-4 + or cd25 + t cells .
it was demonstrated that nickel - specific cd4 + t cell lines   and th17 cells   corelease il-17 and tnf , but trypsin- and thrombin - induced tnf release appears not from il-17 + t cells as tnf expression in il-17 + t cells was not upregulated by these two proteinases .
mapk / erk pathway is the signaling pathway that is most likely involved in the thrombin- and trypsin - induced tnf release from highly purified t cells , as pd98059 , an inhibitor of mapk / erk pathway , almost completely blocked thrombin- and trypsin - provoked phophorylation of erk and tnf release . while little information on signaling pathways associated with par-1 signaling in purified t cells is available , the previous reports that par-1 agonists activated mapk / erk and p38 mapk signaling pathways in dermal   and cardiac fibroblasts   may support our current observation that mapk / erk pathway is the signaling pathway that is most likely involved in the thrombin- and trypsin - induced tnf release .
in addition , pi3k / akt signaling pathway seems also to be involved in thrombin and trypsin induced tnf secretion , as ly294002 an inhibitor of pi3k / akt signaling pathway partially diminished thrombin and trypsin induced tnf secretion and completely abolished thrombin and trypsin provoked phosphorylation of akt .
this finding is in the same line with the report , which showed that thrombin stimulated enhance pi3k activity in hamster embryonic fibroblasts , but different from our previous report , which showed that thrombin did not enhanced pi3k activity in human dermal fibroblasts .
the discrepancy between these studies may be due to the difference in cell origin and species .
tnf is a member of a growing family of peptide mediators comprising at least 19 cytokines , including lymphotoxin- , fas ligand , and cd40 ligand .
the family is now considered as central mediators of a broad range of biological activities in protective immune responses against a variety of infectious pathogens . on the other hand
, tnf also exerts host - damaging effects in sepsis and autoimmune disease [ 30 , 31 ] .
these findings indicate that tnf is one of key mediators of inflammation ; therefore , our current study is of importance in understanding tnf - related inflammation and the mechanism of proteinase - induced cytokine production in t cells .
in conclusion , it is discovered in the present study that serine proteinases thrombin and trypsin are potent stimuli of tnf secretion from highly purified t cells .
their actions on t cells depend on their enzymatic activities and are likely through activation of par-1 .
stimulation of tnf secretion from t cells by serine proteinases further proved that these proteinases are actively involved in the pathogenesis of inflammation and regulation of immune response in man .
 serine proteinases have been recognized as playing an important role in inflammation via proteinase activated receptors ( pars ) . 
 however , little is known about the influence of serine proteinases and pars on tnf secretion from highly purified t cells . 
 we challenged t cells from human peripheral blood with serine proteinases and agonist peptides of pars and measured the levels of tnf in culture supernatants by elisa . 
 the results showed that thrombin and trypsin , but not tryptase , stimulated approximately up to 2.5-fold increase in tnf release from t cells following 16 h incubation . 
 proteinase inhibitors and par-1 antagonist sch 79797 almost completely abolished thrombin- and trypsin - induced tnf release from t cells . 
 agonist peptides of par-1 , but not par-2 induced tnf release from t cells . 
 moreover , trypsin- and thrombin - induced upregulated expression of tnf was observed in cd4 + , il-4 + , or cd25 + t cells , but not in ifn+ or il-17 + t cells . 
 the signaling pathways mapk / erk and pi3k / akt are involved in the thrombin- and trypsin - induced tnf release from t cells . in conclusion , 
 thrombin and trypsin can induce tnf release from il-4 + and cd25 + t cells through activation of par-1 and therefore contribute to regulation of immune response and inflammation of the body . 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion
5. Conclusions