the vitamin k - dependent serine protease protein c ( pc ) is activated upon binding of thrombin to thrombomodulin ( tm ) which is supported by the endothelial protein c receptor ( epcr ) . the pc - tm - epcr complex activates protease activated receptor 1 ( par-1 ) , and as a consequence activated protein c ( apc ) elicits potent anti - inflammatory and cytoprotective effects besides its common anticoagulatory properties [ 1 , 2 ] . activation of par-1 inhibits nf-b translocation to the nucleus which results in a reduced production of proinflammatory cytokines and expression of cell adhesion molecules such as intercellular adhesion molecule 1 ( icam-1 ) and vascular cell adhesion molecule 1 ( vcam-1 )   and thereby blocks leukocyte recruitment . the cascade of leukocyte recruitment plays a crucial role in the immune defense during inflammation . the capture of free flowing leukocytes is followed by leukocyte rolling along the endothelial layer , triggering the activation of the 2-integrins lfa-1 and mac-1 which interact with different endothelial ligands such as icam-1 and rage , the receptor for advanced glycation end products [ 5 , 6 ] . this promotes firm adhesion to the inflamed endothelium and finally leads to leukocyte transmigration [ 4 , 7 ] . previous in vitro and in vivo studies have shown that leukocyte recruitment can be blocked by apc in various models of inflammation [ 8 , 9 ] . there is increasing evidence that this holds true for the zymogen protein c [ 1012 ] . we previously demonstrated that protein c concentrate gets sufficiently activated in vivo , blocks leukocyte adhesion and transmigration in different mouse models of inflammation , and improves survival during systemic inflammation . numerous reports stress the anti - inflammatory properties of pc in inflammatory conditions and diseases beyond sepsis [ 13 , 14 ] ; however , the mechanisms of action are only partially uncovered . the pattern recognition receptor rage , a strong activator of the proinflammatory transcription factor nf-b , serves as a signaling molecule in the innate immune system and is thus involved in various inflammatory diseases [ 15 , 16 ] . rage - deficient mice showed decreased mortality compared to wild - type mice in mouse models of systemic inflammation [ 17 , 18 ] . besides its signaling function , rage controls leukocyte recruitment as a direct ligand for the 2-integrin mac-1 [ 5 , 6 , 19 ] . thus , rage cooperates with icam-1 in mediating leukocyte recruitment during inflammation depending on the stimulus [ 5 , 6 ] . in this study we raised the question whether rage collaborates with icam-1 in mediating anti - inflammatory properties of pc , particularly the capacity to block leukocyte recruitment . therefore , we studied the effect of pc on leukocyte recruitment in rage and icam-1 mice using intravital microscopy of cremaster muscle venules in short - term ( trauma , fmlp , and cxcl-1 ) and long - term ( tnf- ) inflammation models . to increase the clinical relevance we observed leukocyte recruitment and survival after inflammatory stimulation with lps during lps - induced acute lung injury or lps - induced endotoxemia in these groups . in addition , we analyzed the expression of adhesion molecules in response to pc treatment . rage and icam-1 mice were kindly provided from peter nawroth ( university heidelberg ) generated as described earlier and backcrossed for at least 15 generations into c57bl/6 background [ 20 , 21 ] . all mice were maintained at the central animal facility of the university of heidelberg , germany . all animal experiments were approved by the animal care and use committee of the regierungspraesidium karlsruhe , germany ( az g85/11 ) . human protein c concentrate ( ceprotin ) was kindly provided from baxter ( unterschleiheim , germany ) , dissolved as indicated in the drug data sheet to an isotonic working solution of 100  u / ml protein c ( 1  u = 4  g pc ) , containing 8  mg / ml human serum albumin for stabilizing reasons . isotonic human serum albumin ( sigma - aldrich , taufkirchen , germany ) at 8  mg / ml served as the control solution . pc solution and control solution were further dissolved in normal saline to 200  l and were administered intraperitoneally . in all experiments , pc was administered at 100  u / kg referring to 400  g / kg . human activated protein c ( enzyme research laboratory , swansea , uk ) was diluted in normal saline to a working solution of 100  g / ml and was systemically injected into mice at 24  g / kg / h , 3  h before intravital microscopy . in some intravital experiments , fmlp ( 1  m , n - formyl - l - methionyl - l - leucyl - l - phenylalanine ; sigma , taufkirchen , germany ) was added to the superfusion buffer to induce additional leukocyte adhesion as described in [ 22 , 23 ] . in certain experiments , recombinant murine cxcr2 chemokine cxcl-1 ( keratinocyte - derived chemokine kc ; peprotech , london , uk ) was injected systemically at a dose of 600  ng / mouse . in designated in vivo experiments , recombinant murine tnf ( r&d systems , wiesbaden , germany ) was applied intrascrotally at 500  ng per mouse . to investigate the plasma apc concentration during pc therapy , mice were first anesthetized by intraperitoneal ( i.p . ) injection of ketamine ( 125  mg / kg body weight ; pfizer , karlsruhe , germany ) and xylazine ( 12.5  mg / kg body weight ; alverta , neumuenster , germany ) . briefly , mice were injected with 100  u / kg of human protein c into the tail vein . as positive controls , 50  mu human -thrombin ( hemochrom diagnostica , essen , germany ) was additionally injected 10 minutes prior to blood sampling in some experiments . 30 minutes after pc , blood was drawn as a final blood sample by heart puncture into 0.38% sodium citrate and 50  mm benzamidine hcl . human activated protein c was captured from these plasma samples using the hapc1555 antibody ( kindly provided by c. t. esmon , oklahoma medical research foundation , oklahoma city , usa ) , which is a highly specific mouse monoclonal antibody against human apc , developed by standard techniques . due to the antibodies capacity for capturing from plasma , the activity of the captured human pc was determined using a chromogenic substrate ( pca , american diagnostic ) . as recently reported , we used the cremaster muscle models of trauma- and tnf-induced inflammation . briefly , after anaesthesia by i.p . injection of ketamine and xylazine ( see above ) mice were placed on a heating pad to maintain body temperature during surgical preparation and intravital microscopy . intravital microscopy was conducted on an upright microscope ( leica , wetzlar , germany ) with a saline immersion objective ( sw40/0.75 numerical aperture , zeiss , jena , germany ) . the surgical preparation of the cremaster muscle was conducted as described previously ( trauma - induced inflammation ) . depending on the experimental setting , cxcl-1 chemokine ( kc ) , tnf , or fmlp was applied as stated above in order to induce additional leukocyte adhesion . microscopic observation of cremaster muscle venules of 2040  m diameter was recorded via a ccd camera ( cf8/1 ; kappa , gleichen , germany ) on a panasonic s - vhs recorder . the number of adherent leukocytes ( firm adhesion for > 30  s ) was assessed as adherent cells per mm vessel surface area . in a separate set of experiments , cremaster muscle whole mounts were obtained as described before   and analyzed for intra- and extravascular leukocytes using a leica dmrb upright microscope and a 63/0.75  na oil immersion objective ( both from leica , wetzlar , germany ) . to induce acute pulmonary inflammation we adapted the described model of lps - induced ali . briefly , lps from e. coli 0111:b4 ( 10  g lps/50  l pbs ; sigma - aldrich , taufkirchen , germany ) was instilled intratracheally during anaesthetic inhalation of isoflurane ( baxter , unterschleiheim , germany ) to trigger neutrophil infiltration into the lung . 100  u pc / kg was administered intravenously 0.5  h after lps installation in wt mice or rage- or icam-1-deficient mice to dissect the role of rage and icam-1 . injection as already mentioned , the trachea was cannulated and a bronchoalveolar lavage ( bal ) of the right lung was performed using a rinse solution containing pbs and protease inhibitor solution ( protease inhibitor cocktail , sigma - aldrich , taufkirchen ) to harvest infiltrated cells . for leukocyte differentiation , cells were coated on microscopic slides using a shandon cytospin centrifuge ( thermo fisher scientific , waltham , usa ) , stained with giemsa / may grnwald solution , and analyzed on a leica dmrb upright microscope and a 100/0.75  na oil immersion objective ( both from leica , wetzlar , germany ) . the expression of mac-1 and lfa-1 was assessed on murine bone marrow - derived neutrophils using flow cytometry . after red blood cell lysis , leukocytes were treated with pc ( 5  u/10 leukocytes , 3  h at 37c ) . next , cells were incubated with fitc - conjugated rat anti - mac-1 mab m1/70 , ( ebioscience , san diego , usa ) , fitc - conjugated rat anti - lfa-1 mab m17/4 ( ebioscience , san diego , usa ) , or respective fitc - conjugated isotype control antibodies ( ebioscience , san diego , usa ) to detect anti - mac-1 and anti - lfa-1 signals . mac-1 and lfa-1 expression were assessed on 10.000 cells within the neutrophil cluster defined by forward - side scatter analysis using lsrii with diva software package ( becton dickinson , san jose , usa ) and compared to their respective isotype controls . for flow cytometric analysis of intracellular rage and surface icam-1 expression of endothelial cells we used cultured murine aortic endothelial cells ( maecs ) . maecs were grown to near confluence in greiner 6-well plates ( greiner , frickenhausen , germany ) and incubated with tnf at 25  ng / ml for 4  h at 37c . pc pretreatment with 5  u/10 cells was initiated 3  h before tnf stimulation . cells were then harvested with accutase ( paa , clbe , germany ) and washed with pbs containing 1% bovine serum albumin ( bsa ) . for intracellular rage staining , cell permeability was achieved by incubation with pbs containing 0.1% saponin and 5% powdered milk ( both from carl roth , karlsruhe , germany ) for 30  min at 4c . after washing , cells were incubated with fitc - labeled polyclonal rabbit anti - rage antibody ( bioss , woburn , massachusetts , usa ) or isotype control ( santa cruz , heidelberg , germany ) for 45  min on ice . for icam-1 staining , cells were incubated with a fitc - conjugated anti - mouse cd54 mab or isotype control antibody ( both from ebioscience , germany ) for 45  min on ice . the antibody signal was detected on 10.000 cells using the four - decade facs scan lsrii with diva software package . injection of 40  mg / kg lps ( escherichia coli ; serotype 055:b5 sigma , taufkirchen , germany ) which was reconstituted in 100  l of sterile pbs , as reported previously   with modification . 100  u / kg pc or control solution was administered i.p . at 30 minutes and 8 and 24 hours after lps challenge . in the first group , survival was observed for 14 days in wt mice and rage- and icam-1-deficient mice . in the second group , mice were perfused with saline and lungs were harvested 24  h after lps injection . after fixation in pfa ( 4% ) they were prepared for paraffin - embedded sections . sections were performed at 3  m thickness and finally stained with h&e ( haematoxylin and eosin staining ) for microscopic evaluation . all statistical analyses were performed using prism 4 ( graphpad , la jolla , usa ) . to compare the mortality of pc - treated and control mice during lethal endotoxemia log - rank test of kaplan - meier survival distribution was used . statistical significance between groups and treatments were compared with one - way anova followed by a multiple pairwise comparison test ( newman - keuls - test ) . first , we showed that mice injected with zymogen pc were able to significantly activate pc . nevertheless , pc and thrombin coinjected mice reached even higher levels of pc activation ( see supplementary figure 1 available online at http://dx.doi.org/10.1155/2014/743678 ) . during trauma - induced inflammation , surgical preparation provokes firm leukocyte arrest mostly mediated via the 2-integrins lfa-1 and mac-1 interacting with icam-1 and rage , respectively [ 5 , 6 ] . to elucidate the role of rage and icam-1 in the context of pc - induced inhibition of leukocyte recruitment , leukocyte adhesion in postcapillary venules of surgically prepared cremaster muscles of wild - type ( wt ) , rage , and icam-1 mice microvascular and hemodynamic parameters did not vary significantly between the treatment groups and genotypes ( supplementary table 1 ) . in line with our previous findings , application of 100  u pc / kg in wt mice resulted in a profoundly inhibited leukocyte adhesion compared to control mice ( figure 1(a ) ) . as observed previously during trauma - induced inflammation , both rage and icam-1 mice showed significantly reduced numbers of adherent cells compared to wt control mice . interestingly , pc treatment did not lead to a further inhibition of leukocyte adhesion in both knockout mice . we then asked whether activated pc exerts a stronger anti - inflammatory potential than pc and compared both treatments in rage and wt mice . however , apc and pc showed similar inhibitory effects on leukocyte adhesion in that model ( supplementary figure 2 ) . since the integrin ligands rage and icam-1 regulate leukocyte recruitment in a stimulus - dependent manner , we used further proinflammatory stimuli in the trauma model . first , local superfusion with fmlp ( n - formyl - l - methionyl - l - leucyl - l - phenylalanine , 1  m ) , a potent chemoattractant and integrin activator [ 5 , 22 ] , induced additional leukocyte adhesion in cremaster muscle venules of wt and rage mice , but not of icam-1 mice . pc treatment significantly blocked fmlp - stimulated leukocyte adhesion in wt and rage mice , while there was no pc effect on leukocyte adhesion in icam-1 mice ( figure 1(b ) ) . second , mice were injected with the cxc chemokine cxcl1 , also known as keratinocyte - derived chemokine ( kc ) , for triggering additional leukocyte adhesion in exteriorized cremaster muscle venules [ 5 , 22 ] . pc blocked cxcl1-induced increase of leukocyte adhesion in wt and rage mice but not in icam-1 mice ( supplementary figure 3 ) . for this purpose , giemsa staining of whole mounts of fmlp - stimulated cremaster muscles , obtained after the respective intravital microscopic experiment , was performed ( figure 1(c ) ) . in line with leukocyte adhesion , fmlp - induced leukocyte transmigration of wt and rage mice these results indirectly indicate that pc - induced inhibition of leukocyte recruitment may at least in part involve icam-1 and rage . to investigate whether rage and icam-1 are involved in the mediation of anti - inflammatory properties of pc after tnf stimulation , we measured leukocyte adhesion in 3  h - tnf-treated cremaster muscle venules of wt , rage , and icam-1 mice with and without pc therapy ( 100  u / kg 3  h ) . consistent with earlier findings , pc profoundly blocked leukocyte adhesion in wt mice ( figure 2(a ) ) . under controlled conditions , leukocyte adhesion of rage - deficient mice was significantly impaired compared to wt mice , whereas the number of adherent leukocytes in icam-1-deficient mice was comparable to those of wt mice ( figure 2(a ) ) . similar to wt mice , pc administration significantly reduced leukocyte adhesion in icam-1 mice , while the number of adherent leukocytes was not further decreased in response to pc treatment in rage mice . in accordance , experiments with additional superfusion with fmlp ( 1  m , 5  min ) in 3  h - tnf-stimulated cremaster muscle venules showed a significant pc - induced inhibition of leukocyte adhesion in wt and icam-1 mice , but not in rage mice ( figure 2(b ) ) . next , giemsa - stained whole mounts of tnf- and fmlp - stimulated cremaster muscles were used to investigate leukocyte transmigration . as depicted in figures 2(c ) and 3(a)3(f ) , the genotype - specific effect of pc on leukocyte adhesion did also translate into leukocyte extravasation . representative micrographs of giemsa - stained whole mounts illustrate the inhibitory effect of pc on leukocyte transmigration in wt ( figure 3(a ) versus figure 3(b ) ) and icam-1 mice ( figure 3(c ) versus figure 3(d ) ) which is not detectable in rage mice ( figure 3(e ) versus figure 3(f ) ) . taken together , these results suggest that after long - term proinflammatory stimulation pc - induced inhibition of leukocyte recruitment is dependent on rage , but not on icam-1 . to address the question whether rage and icam-1 , which are known to be involved during lung inflammation [ 30 , 31 ] , mediate pc - induced inhibition of leukocyte recruitment in a disease relevant model of acute lung inflammation , we investigated neutrophil emigration during lps - induced acute lung injury ( ali ) in wt , icam-1 , and rage mice in response to pc . in contrast , the number of bronchoalveolar neutrophils was considerably reduced after pc therapy ( figure 4 ) . in line with previous reports [ 30 , 32 ] , the number of transmigrated neutrophils into acutely inflamed lungs was significantly reduced in icam-1 and rage control mice compared to wt control mice . in contrast to rage mice , pc treatment did further diminish neutrophil emigration in icam-1 mice ( figure 4 ) , suggesting that the potential of pc to inhibit leukocyte recruitment into the lung is rather related to rage than to icam-1 . since pc is known to improve survival during systemic inflammation , we asked whether rage and icam-1 contribute to that improvement of survival . we used an established mouse model of lethal endotoxemia by intraperitoneal injection of escherichia coli lps ( 40  mg / kg ) followed by treatment with 100  u / kg pc or control solution ( human albumin ( 8  mg / ml ) ) after 0.5 , 8 , and 24 hours . pc treatment significantly improved survival in wt and icam-1 mice compared to respective control mice ( about 20% versus 70% and 20% versus 45% , resp . , rage mice were protected against lps - induced endotoxemia ( 45% survival ) ( figure 5(b ) ) . notably , pc treatment did not further improve survival in rage - deficient mice ( both about 40% survival , figure 5(b ) ) . next , we determined leukocyte infiltration into the lung after 24 hours of lps - induced sepsis . pc treatment evidently reduced leukocyte emigration into the lungs of wt mice ( figures 6(a ) and 6(b ) ) and icam-1 mice ( figures 6(c ) and 6(d ) ) . in rage mice leukocyte infiltration was markedly reduced compared to wt control mice ( figures 6(e ) and 6(a ) ) ; however , pc treatment did not alter leukocyte emigration into the lung in the absence of rage ( figures 6(e ) and 6(f ) ) . these results point towards rage as an important mediator of anti - inflammatory effects of pc , even during systemic inflammation . to explore the underlying mechanisms of the observed pc effects , we investigated the capacity of pc to regulate expression of 2-integrins of neutrophils and rage and icam-1 on endothelial cells using flow cytometry . as depicted in supplementary figure 4 , lfa-1 and mac-1 expression of neutrophils were not altered in response to pc . however , intracellular accumulation of rage was reduced in tnf-stimulated murine aortic endothelial cells ( maec ) after pc treatment ( figure 7 ) . in addition , pc was able to downregulate icam-1 expression after pc pretreatment of freshly prepared endothelial cells ( figure 8(a ) ) . these cells were only stimulated by preparation procedures and resemble the surgical preparation of the trauma in vivo model . as depicted in figure 8(b ) , icam-1 expression was similarly downregulated by pc on tnf-stimulated endothelial cells which is surprising in light of our in vivo results during tnf-induced inflammation . these results stress the role of pc for the regulation of inflammatory response particularly of the endothelium . the receptor for advanced glycation end products is a multiligand receptor and is involved in a variety of inflammatory conditions . rage activates the proinflammatory nf-b pathway , but also acts as a ligand for the leukocyte expressed 2-integrin mac-1 [ 5 , 6 , 15 , 19 ] . interestingly , in vivo experiments using the models of trauma- and tnf-induced inflammation of murine cremaster muscles revealed that rage and icam-1 act together in mediating leukocyte adhesion in a stimulus - dependent manner [ 5 , 6 ] . this study provides evidence that rage also cooperates with icam-1 in mediating anti - inflammatory properties of pc . established murine inflammation models were used to dissect how the described pc - induced inhibition of leukocyte recruitment   is related to rage and icam-1 [ 5 , 6 , 23 ] . during trauma - induced inflammation , pc requires both rage and icam-1 , which reportedly act in concert in that model [ 5 , 6 ] in order to inhibit leukocyte adhesion , as there was no detectable effect of pc in rage and icam-1 mice . in this model , additional short - term administration of the leukocyte chemoattractant fmlp stimulates icam-1 to become more important for mediation of leukocyte adhesion and transmigration . consequently , icam-1 is predominantly involved in pc - induced inhibition of leukocyte recruitment , whereas rage is dispensable in that early phase of inflammation . in contrast , icam-1 is no longer a key molecule for leukocyte recruitment during tnf-induced inflammation of cremaster muscles , which is consistent with previous studies [ 5 , 6 , 23 ] . accordingly , it is not a relevant adhesion molecule targeted by anti - inflammatory functions of pc in that model , which was confirmed by effective pc - induced inhibition of leukocyte recruitment despite the absence of icam-1 . in context with our present and previous findings that rage is crucial for mediation of leukocyte recruitment during tnf-induced inflammation we argue that rage might also be critically involved in mediation of the anti - inflammatory potential of pc after tnf stimulation . the finding that pc did not block leukocyte recruitment in rage mice after tnf stimulation supports this hypothesis . next , we studied whether rage and icam-1 are linked to the anti - inflammatory pc pathway in more disease relevant mouse models : lps - induced acute lung injury ( lps - ali ) [ 34 , 35 ] and lps - induced lethal endotoxemia . based on our results during lps - induced ali , rage and icam-1 play a crucial role in leukocyte recruitment during lung inflammation , a finding that has been described in earlier studies [ 30 , 32 , 37 , 38 ] . since pc treatment had no effect on bronchoalveolar neutrophil emigration in rage mice but insignificantly reduced the number of neutrophils in bal of icam-1 mice , we suggest that rage , which is abundantly expressed in the lung [ 31 , 39 ] , plays a dominant role for mediating anti - inflammatory effects of pc during acute lung inflammation . in line with earlier reports [ 18 , 21 ] , rage - deficient mice showed an improved survival during lps - induced lethal endotoxemia . however , icam-1 deficiency was not beneficial for survival in our experimental setting , which is in contrast to xu et al . . the different lps serotype used in their study may explain the contrary results ( 0127:b7 versus 055:b5 in our study ) . while pc treatment was effective to improve survival of wt mice and icam-1-deficient mice , it failed in rage - deficient mice . moreover , mortality during lps - induced lethal endotoxemia correlated with leukocyte infiltration into the lungs , indicating that the anti - inflammatory potential of pc might be linked to rage even during systemic inflammation . however , it should be noted that rage is most likely not the only mediator of anti - inflammatory effects of pc in that model . taken together , these results provide indirect evidence that rage is predominantly involved in the anti - inflammatory pc pathway , particularly during persistent and prolonged inflammatory stimulation , while icam-1 is more relevant during brief and mild inflammation . thus , the findings of this study could stimulate the development of course - specific therapeutic approaches to treat inflammatory diseases involving icam-1 and rage [ 15 , 16 , 33 , 40 ] . to explore how pc interferes with rage and icam-1 , intracellular rage and extracellular icam-1 expression of endothelial cells were investigated in response to pc . indeed , the discovered pc - induced downregulation of intracellular rage in tnf-stimulated endothelial cells may explain some of the anti - inflammatory effects of pc observed in our in vivo inflammation models . consistent with previous studies [ 3 , 41 ] we also found that protein c strongly downregulates endothelial icam-1 expression . notably , pc - induced icam-1 downregulation was detectable during both mild ( cell preparation ) and strong ( tnf ) proinflammatory stimulation . the latter finding is in line with our recent observation , but in contrast to our in vivo results shows that icam-1 plays only a minor role for pc - induced effects during tnf-induced inflammation . however , the technical setup of the in vitro and in vivo experiments was different . further downstream nf-b and erk 1/2 mitogen - activated protein kinase ( mapk ) might be involved during pc dependent regulation of rage and icam-1 . this hypothesis is supported by the fact that nf-b and mapk are linked to both rage [ 42 , 43 ] and icam-1 [ 4446 ] on the one hand and are known mediators of endothelial cytoprotective protein c signaling on the other hand . with regard to our in vivo results it is important to discuss other potential mechanisms of action of pc like leukocyte - driven effects . as previously reported , activated pc may interfere with the 2-integrins lfa-1 and mac-1 [ 9 , 36 ] , which are ligands of icam-1 and rage , respectively . although pc did not change mac-1 and lfa-1 expression of neutrophils , we can not completely exclude influences of pc on 2-integrins in our study . furthermore , pc might also interact with other rage ligands , as described for hmgb1 [ 4850 ] . likewise , dinarvand et al . demonstrated in their very recent study that the inorganic polyphosphate induced proinflammatory rage signaling can be blocked by activated pc . this study further supports our hypothesis of interplay between the rage and pc pathway during inflammation . in addition to those observations , it is possible that rage could be involved in the activation of pc . consistent with previous findings we showed that the zymogen pc can be activated by mice in our experimental setting . although similar anti - inflammatory effects of treatment with zymogen pc and activated pc in both wt and rage knockout mice might argue against the role of rage during pc activation , further thorough investigation of rage - dependent pc activation is needed . this , however , is beyond the scope of this paper and should be performed in future studies . the anti - inflammatory effect of pc is mediated through and acts on rage and icam-1 dependent on stimulus . moreover , the ability of pc to induce its anti- inflammatory effect depends on the role of rage in leukocyte recruitment . in turn , pc treatment improved survival during lps - induced endotoxemia in a rage - dependent manner . these results may contribute to a better understanding of the immunoregulation by rage and the protein c pathway and eventually stimulate further studies .
by binding 2-integrins both icam-1 and the receptor for advanced glycation end products ( rage ) mediate leukocyte recruitment in a stimulus - dependent manner . using different inflammatory mouse models we investigated how 
 rage and icam-1 are involved in anti - inflammatory functions of protein c ( pc ; ceprotin , 100  u / kg ) . 
 we found that , depending on the stimulus , rage and icam-1 are cooperatively involved in pc - induced inhibition of leukocyte recruitment in cremaster models of inflammation . during short - term proinflammatory stimulation ( trauma , fmlp , and cxcl1 ) , 
 icam-1 is more important for mediation of anti - inflammatory effects of pc , whereas rage plays a major role after longer proinflammatory stimulation ( tnf ) . 
 in contrast to wt and icam-1/ mice , pc had no effect on bronchoalveolar neutrophil emigration in rage/ mice during lps - induced acute lung injury , suggesting that rage critically mediates pc effects during acute lung inflammation . 
 in parallel , pc treatment effectively blocked leukocyte recruitment and improved survival of wt mice and icam-1-deficient mice in lps - induced endotoxemia , but failed to do so in rage - deficient mice . 
 exploring underlying mechanisms , we found that pc is capable of downregulating intracellular rage and extracellular icam-1 in endothelial cells . 
 taken together , our data show that rage and icam-1 are required for the anti - inflammatory functions of pc .