severe sepsis is still the leading cause of death in surgical intensive care units and can be held responsible for approximately 9% of deaths per year in usa and germany . in the course of ongoing sepsis ,
, the lung can either be the source of infection or is remotely affected by systemic inflammation as bacterial degradation products reach this organ via the bloodstream . in both cases ,
an extensive release of proinflammatory cytokines and chemokines induces migration of neutrophils , fibroproliferation , and reorganization of lung tissue .
an accompanying disruption of the alveolar - capillary interface and consecutive leakage of protein - rich fluid into the interstitial and alveolar space causes hypoxemia and reduces lung compliance .
acute lung injury ( ali ) or its severe form , the acute respiratory distress syndrome ( ards ) , constitute clinical manifestations of the pathological process and develop in 40% of the patients suffering from sepsis .
toll - like receptors ( tlrs ) act as pivotal signalling proteins of the inflammatory response during sepsis .
gram - negative bacteria release the tlr4 ligand lipopolysaccharide ( lps )   whereas the cell wall component lipoteichoic acid ( lta ) from gram - positive bacteria is primarily bound by tlr2 .
both , tlr2 and tlr4 are localized in the cell membrane while tlr9 is localized in lysosomes and recognizes bacterial dna from both gram - positive as well as gram - negative bacteria .
bacterial dna contains cpg - odn motifs , which are unmethylated cg dinucleotides prevalent in bacterial but not in mammalian dna .
tlrs are differentially expressed in many organs including heart and lung as well as various cell types .
it has been shown that tlrs are involved in a number of inflammatory diseases of the lung , such as allergic asthma , autoimmune lung injury , and pneumonia .
bacterial components induce the expression of inflammatory mediators such as proinflammatory cytokines and chemokines , which are highly relevant for the pathogenesis of pulmonary inflammation and injury [ 1214 ] . both attract and activate immune cells infiltrating the lung .
also , during sepsis , they participate in disrupting the alveolo - capillary structure , rupture of basal membranes , and interstitial matrix remodelling .
the integrity of the extracellular matrix ( ecm ) is controlled by a dynamic equilibrium of synthesis and local degradation .
matrix metalloproteinases ( mmps ) are the main physiological mediators of ecm degradation , which are also upregulated under pathological conditions like pulmonary inflammation or sepsis .
they are primarily regulated by a class of endogenous inhibitors , tissue inhibitors of mmps ( timps ) .
there has been a significant gain of knowledge about the high complexity of organ - specific tlr signalling in the lung .
this organ exhibits a tlr pattern , which derives in part from parenchymatous cells as well as from immune cells like neutrophils .
hence , it may be speculated that the inflammatory response depends on specific tlr stimulation , and thus , various virulence factors might induce different inflammatory responses .
it can be hypothesized that different organs express specific tlr patterns , which serve their need in defense against pathogens
. an accurate characterization of tlr expression and signalling as well as the investigation of consecutively induced inflammatory mediators   is essential for understanding a differential inflammatory response to varying stimuli
. it might be the fundament for developing efficient strategies for diagnosis and treatment of sepsis .
however , the number of studies comparing the influence of different tlrs in vivo is rare . in particular
, a comparison of ligands for tlr2 , -4 , and -9 in vivo in the lung has not yet been performed .
therefore , we analysed the influence of three different tlr ligands ( lta , lps , and cpg - odn ) on the expression of tlrs , cytokines , chemokines , mmps , and timps in the murine lung .
also , the subsequent activities of mpo , mmp-2 , and -9 and the level of tnf- protein in pulmonary tissue were investigated .
the stimuli were applied remotely ( intraperitoneal , i.p . ) to simulate the progression of sepsis .
lipopolysaccharide ( lps ) was from escherichia coli ( e. coli , 0  :  111 , sigma aldrich chemie gmbh , munich , germany ) . lipoteichoic acid ( lta ) was kindly provided by s. morath , university of konstanz , germany ( charge mgm5 - 10 ) and prepared as described before .
the lps contamination of the lta preparations was less than 1  eu / mg as determined by the limulus amoebocyte lysate assay ( charles river , charleston , sc ) .
an immunostimulatory cpg - odn ( 1668 thioat , sequence : gctagacgttagcgt ) was purchased from tib - molbiol ( tib - molbiol gmbh , berlin , germany ) .
158 male c57bl/6 mice 12 weeks of age from charles river ( sulzfeld , germany ) were incorporated in the study .
mice were housed in pathogen - free cages with free access to water and standard rodent chow .
injections of pbs or different tlr ligands , that is , equipotent doses ( see below ) of either lta ( 15  mg / kg ) , lps ( 20  mg / kg ) , or 1668 thioat ( 1  nmol / g ) .
30  min before stimulation with cpg - odn , mice received 1  mg / kg d - galactosamine ( d - galn ; roth , karlsruhe , germany ; in control experiments d - galn alone did not induce an inflammatory response ; data not shown ) . at the end of the experiments ,
mice were sacrificed under anaesthesia with isoflurane 2.5  vol.% ( forene , abbott gmbh , wiesbaden , germany ) .
the animals were handled according to the principles of laboratory animal care ( nih publication no .
85 - 23 , revised 1996 ) , and animal procedures were approved by the local committee for animal care .
( n = 3/group ) were stimulated with lps ( 20  mg / kg i.p .
, according to ) , cpg - odn ( 1  nmol / g i.p .
, according to ) . to determine potency of the applied concentrations of the three tlr ligands , undiluted serum from stimulated mice was tested in vitro on fibroblast cultures .
the test of serum was chosen , as during a remote inflammation virulence factors are transported to the lung via the blood stream .
briefly , 2  h after stimulation , serum was taken , and murine fibroblast tumor cells were incubated with this serum and stained to determine viability .
binding of tnf- and - to surface receptors initiates lysis in certain types of cells .
the tnf activity assay employs tnf - sensitive , actinomycin d - treated murine l929 fibroblasts to quantify tnf activity .
murine fibroblast tumor cells were grown in rpmi 1640 medium containing 10% fetal calf serum ( fcs ) , 5  mm l - glutamin ,    25  mm hepes , 5  mm sodium pyruvate , and 100  u penicillin and streptomycin , respectively . a 96-well plate containing 5  10 cells per well was incubated over night in a humidified incubator ( 37c , 93% o2 , 7% co2 ) .
medium was removed and fresh serum or medium or tnf- standard ( rtnf , sigma aldrich , munich , germany ) were added .
10  l of 1  :  50 diluted actinomycin d ( sigma aldrich ) was given to each well and incubated over night .
after removal of the supernatant , cells were fixed with 5% formalin for 10  min .
100  l of 0.05% crystal violet ( sigma aldrich ) in 20% ethanol was added to each well followed by destaining with tab water , and , finally , plates were dried .
absorbance was measured at 590  nm after dilution of the stain with 100  l methanol per well .
increased staining and absorbance corresponded to increased l929 fibroblast viability and decreased lytic effects of tnf .  
pulmonary protein extracts were prepared using the ne - per nuclear and cytoplasmic extraction reagents ( pierce biotechnology , rockford , il , usa ) according to the manufacture 's protocol and as published previously .
the tissue weight was determined , and the appropriate amount of cer i ( 10-fold excess over the weight of tissue ) containing 0.5  mg / ml benzamidine ( roche , basel , switzerland ) , 2  g / ml aprotinin ( roche ) , 2  g / ml leupeptin ( roche , basel , switzerland ) , and 0.75  mm pmsf ( roche , basel , switzerland ) was added .
cer ii was added , incubated again for 1  min on ice , and centrifuged for 5  min at 13,200  u / min ( 16,110  g ) and 4c .
the supernatant was transferred to a prechilled tube and used as cytoplasmic fraction for analysis of zymographic activity .
after vortexing and incubation according to manufacturer 's protocol , the sample was centrifuged again for 10  min at 16,110  g. the supernatant was immediately transferred to prechilled tubes and used as nuclear extract for determination of nfb - dna binding activity .
the protein concentration was assessed by use of a bicinchoninic acid assay kit ( pierce biotechnology , rockford , il , usa ) according to the manufacturer 's protocol .
nfb activity was evaluated by electrophoretic mobility shift assay ( emsa ) 2  h after stimulation ( n = 3/group ) .
nuclear extracts used in supershift and competition experiments were harvested from snap frozen lungs as described above .
, santa cruz ca , usa ) nfb : 5-agt tga ggg gac ttt ccc agg c-3 ( sc-2505 ) ) were end - labelled with ( -32p ) atp ( amersham , freiburg , germany ) .  
binding reactions ( 25  l total ) were performed by incubating 20  g of nuclear extracts for 30  min at room temperature with 4  mm tris - cl ( ph 7.9 ) , 12  mm hepes , 1  mm dtt , 60  mm kcl , 10% glycerol , 1  mm edta , 2  mg poly(di - dc)-poly(di - dc ) , and 20,000  cpm of the labelled nfb oligonucleotide .
the specificity of the dna - protein binding was determined by competition with a 50-fold molar excess of the unlabeled nfb .
the dna - protein complexes were electrophoresed for 2  h at 30  ma in a 4% polyacrylamide gel in 0.5% tris - borate - edta running buffer .
the gels were dried for 1  h , exposed overnight to imaging plates , and scanned with a phosphoimager ( fla3000 , fuji , dsseldorf , germany ) .
( n = 46/group ) were analysed with ribonuclease protection assay ( rpa ) 4  h after stimulation . for rpa , lungs were flash - frozen in liquid nitrogen and kept at 80c .
the tissue was homogenized and total rna was extracted by guanidinium thiocyanate method as described elsewhere .
the mrna levels of mmp-1 , -2 , -3 , -8 , -9 , and timps 14 per 20  g rna sample were analysed with mmmp-1 multiprobe template set ( bd biosciences pharmingen , san diego , ca , usa ) .
chemokine mrna of lymphotactin , rantes ( regulated on activation and expressed / secreted by t cells ) , macrophage inflammatory protein ( mip)-1 , mip-1 , mip-2 , macrophage chemotactic peptide ( mcp)-1 , t - cell activation protein ( tca)-3 , eotaxin , and interferon - inducible protein ( ip)-10 was detected with mck-5c multiprobe template set ( bd biosciences pharmingen , san diego , ca , usa ) .
mrna expression of proinflammatory cytokines il-12p35 , il-12p40 , tnf- , il-1 , il-1 , il-6 , and ifn as well as receptor expression of tlr2 and tlr4 were determined with custom - made template sets ( bd biosciences , heidelberg , germany ) .
signals were quantified densitometrically with aida software v3.5 ( raytest , straubenhardt , germany ) and normalized to ribosomal housekeeping gene l32 .
tlr1 , -6 , and -9 gene expression ( n = 6/group ) was determined with rt - qpcr 4  h after stimulation .
tnf- mrna was monitored with the same technique 0 , 2 , 4 , and 6  h after tlr - ligand application .
the taqman gene expression assays ( applied biosystems , foster city ca , usa ) for murine tlr1 ( comm .  : 
tnf- protein expression ( n = 4/group ) was determined with enzyme linked immunosorbent assay ( elisa ; bd biosciences , san jose , ca , usa ) 0 , 2 , 4 , and 6  h after stimulation . for protein isolation ,
pulmonary tissue was homogenized and incubated on ice for 5  min in 1  ml elisa buffer containing protease inhibitors ( roche , complete mini no .
11836153 ) , pbs , triton x-100 ( 1  l / ml , sigma aldrich , munich , germany ) , phenylmethylsulfonyl - fluoride ( pmsf , 250  mm in isopropanol , 1  l / ml , roche , basel , switzerland ) .
samples were incubated on ice for 20  min , homogenized , and centrifuged 15  min at 4c and 13,110  g. the supernatant was used for measuring intrapulmonary tnf- protein levels in a microplate reader ( expert 96 , asys hitech , eugendorf , austria ) . for zymographic activity of pro and active forms of mmp-2 and -9 in lung tissue ( n = 3/group ) , 60  g protein were mixed with 2x tris - glycine sds sample buffer , and loaded on 10% polyacrylamide gels ( sds - page ) copolymerized with gelatin ( each 0.3  mg / ml type a from porcine skin and type b from bovine skin ; sigma aldrich , munich , germany ) . following 90  min of electrophoresis at 125  v ,
gels were washed with 2.5% triton x-100 ( sigma aldrich ) for 3  20 min to remove sds .
afterwards , gels were incubated for 48  h at 37c in developing buffer ( 50  mm tris hcl , 0.2  m nacl , 5  mm cacl2 , 0.02% brij ) .
( sigma aldrich ) in a mixture of water  :  methanol  :  acetic acid ( 5  :  5  :  1  v / v ) , destained in 45% methanol , and 3% acetic acid in water ( v / v ) . areas of protease activity were detected as transparent bands against blue background .
zymograms were scanned , and signals were quantified using aida software ( aida image analyser , raytest gmbh , straubenhardt , germany ) . for comparison , the zymographic activity of the three virulence factors 4  h after stimulation was normalized to their baseline activity ( 0  h , data not shown ) . after 4  h of tlr ligand stimulation , lungs were taken and flash frozen in liquid nitrogen ( n = 5/group ) .
tissue was homogenised on ice in 1  ml of 0.5% hexadecyltrimethyl ammonium bromide ( htab ; h-5882 , sigma ) in 50  mm potassium phosphate buffer ( 1  ml buffer/50  mg tissue )
. 1  ml of the homogenate was transferred into a tube and centrifuged at 5,000  rpm for 4  min .
7  l of the supernatant were mixed with 200  l o - dianisidine solution ( 16,7  mg o - dianisidine ( d-3252 , sigma ) in 90  ml ddh2o , 10  ml 50  mm potassium phosphate buffer , and 50  l 1% h2o2 ) .
the change in absorbance with time was continuously recorded at 450  nm using a kinetic microplate reader .
one - way anova was used to determine significant differences in mrna - expression ( n = 4 ) , elisa ( n = 4 ) , and zymographic assay ( n = 3 ) between the different stimulation groups . when appropriate , bonferroni post hoc testing was performed .
statistics were calculated using prism 5.0 ( graphpad software inc . , san diego , ca , usa ) .
to equilibrate the potency of the different applied tlr ligands a tnf activity assay was applied
. viability of tnf - sensitive fibroblasts exposed to serum from animals treated by one of the three tlr ligands dropped significantly in all groups ( lta : 57  13.7% , lps : 40  2.4% , cpg - odn : 42  5.1% ; p < .05 versus pbs serum control ) 2  h after incubation .
hence , nfb transcriptional activation was measured 2  h after stimulation with the three tlr ligands using an emsa .
no major visual differences were revealed following lps and cpg - odn stimulation ( figure 1 ) .
lta , however , induced a markedly lower nfb activity than the other two ligands .  
sensitivity of pulmonary tissue to lps , cpg - odn and lta may be regulated by the level of specific tlr expression .
therefore , we monitored mrna expression of tlr1 , 2 , 4 , 6 , and 9 4  h after stimulation .
lta application resulted in a twofold increase of tlr1 , 2 , and 6 expression but did not reach the level of significance ( figure 2 ) , neither tlr4 nor tlr9 mrna was changed .
lps acted more strongly elevating tlr2 and 6 expressions significantly versus control and tlr4 versus lta - treated animals .
tnf- has been shown to be an early inflammatory marker , which is often upregulated transiently .
therefore , we monitored the time course of pulmonary tnf- mrna expression 0 , 2 , 4 , and 6  h after virulence factor challenge ( table 1 , n = 4/group ) .
lps challenge , however , raised mrna expression significantly at all investigated time points . under baseline conditions ,
2  h after stimulation , all virulence factors significantly increased tnf- protein expression ( table 1 ) . at the later time points , a significant enhancement of tnf- protein
therefore , these mediators were investigated in the lung 4  h after stimulation using rpa ( figure 3 ) .
il-1 , tnf- , il-6 , il-12p35 , il-12p40 , and ifn- , whereas cpg - odn only elevated tnf- and il-12p40 mrna .   analysis of lungs from lps - treated mice revealed a marked and significant increase in all monitored chemokines ( lymphotactin , rantes , mcp-1 , mip-1 , mip-1 , mip-2 , tca-3 , eotaxin and ip-10 , figure 4 ) as compared to all other groups .
however , neither lta nor cpg - odn stimulation caused any significant changes in chemokines . 
					
myelop - eroxidase ( mpo ) activity was assayed as a marker of neutrophil infiltration into the lung .
mpo activity was increased 9- to 16-fold in all stimulated groups 4  h after tlr ligand application ( figure 5 ) . with a 16-fold induction compared to the control group ,
this value differed significantly from lta- and cpg - stimulated mice which showed a 9- and 11-fold increase .
interestingly , doubling of the standard lta dose did not further raise mpo activity . during inflammation
consequently , 4  h after simulation total mmp mrna expression was significantly enhanced by all three virulence factors with the greatest overall stimulus being cpg - odn ( figure 6 ) .
lta induced a significant increase in mmp-2 and mmp-9 as well as a 5-fold elevation in mmp-3 and 18-fold elevation in mmp-8 .
lps stimulation significantly enhanced mmp-1 , mmp-3 , mmp-8 , and mmp-9 while cpg - odn raised mmp-2 and mmp-8 significantly as well as mmp-1 , mmp-3 , and mmp-9 nonsignificantly .  
furthermore , lps significantly enhanced timp-1 and -3 expressions whereas lta and cpg - odn did not induce any changes in single timp expressions .
interestingly , the unchanged timp-2 expression was relatively high in all groups ( control : 57.78  14.79 ; lta : 57.08  2.63 ; lps : 25.38  1.68 ; cpg - odn : 42.56  2.80 ) compared to timp-1 and -3 .  
since the stoichiometry between the gene expression of mmps and their physiological antagonist ( timps ) has been shown to be important for the regulation of the extracellular matrix remodelling , we calculated the ratios of mmps to timps ( table 2 ) . due to the up - regulation of the total mmp mrna expression in all stimulated groups ,
overall mmp / timp ratio was significantly elevated 4-fold by lta , 5-fold by lps , and 8-fold by cpg - odn . to expand the results on mmp and timp mrna expression ,
accordingly , we detected an induction of mmp-2 and mmp-9 in their precursory ( pro ) and active form ( not shown ) . to compare effects of different virulence factors
, we normalized zymographic activity ( 4  h ) to baseline activity ( 0  h ) ( figure 7 ) .
cpg - odn challenge significantly increased mmp-2 activity compared to lps and lta application . here
with the present study , we characterized differential pulmonary inflammatory responses to various virulence factors .
studies comparing the influence of different tlr stimuli on pulmonary tissue were mainly performed in vitro .
therefore , we decided to compare lta , lps , and cpg - odn challenge using remote stimulation in vivo to simulate the initiation of sepsis . for characterization of the inflammatory cascade we monitored a variety of endpoints like tlr expression , nfb activation , expression of various cytokines and chemokines , and mpo activity as well as expression of mmp / timps . in the lung ,
this broad combination of tlr stimuli and endpoints detected in vivo has not yet been performed .   to provide comparability of results , we defined equipotent concentrations for lta , lps , and cpg - odn via a tnf activity assay .
after stimulation with lps we observed a strong proinflammatory reaction with increased nfb activation , enhanced cytokine and chemokine expression , and a differential response in mmp and timp expression .
the equipotent dose of cpg - odn initiated mediator expression to a lesser degree . here , cytokine and chemokine expression remained a multiple below values evoked by lps .
interestingly , stimulation with an equipotent dose of lta had a markedly lesser impact on the induction of cytokines and chemokines in lung tissue .
significant variations only occurred in tnf- expression , mpo activity , and mmp / timp regulation .   in order to understand the differential answers to tlr stimulation better
, we investigated the expression of tlr1 , 2 , 4 , 6 , and 9 .
lps induced an increased expression of tlr2 , 4 , and 6 whereas cpg - odn elevated solely the tlr6 expression .
induction of tlr2 and 4 by lps in pulmonary tissue has already been observed by others .
interestingly , pulmonary tlr4 mrna expression did not reveal a strong regulation after lps challenge .
in contrast , tlr2 expression was very sensitive to lps , as it was increased more than 10-fold . in this context
, it is interesting that also tlr6 is upregulated by lps and cpg - odn challenge , as lta signalling depends on heterodimerization of tlr2 and tlr6 or tlr2 and tlr1 , and cd36 .
this up - regulation of tlr2 and tlr6 might sensitize the lung in an ongoing inflammation to gram - positive stimuli . in accordance with our results , it has been shown that bronchial epithelium regulates its sensitivity to recognize microbes by managing tlr expression levels .
bronchial epithelial cells could be stimulated in vitro only marginally by gram - positive bacteria bearing known tlr2 ligands or with lta alone while gram - negative bacteria were easily recognized .
as mucosal surfaces are prone to contact with pathogenic , as well as nonpathogenic microbes , immune recognition principles have to be tightly regulated to avoid uncontrolled permanent activation . hence , airway epithelium displays a low sensitivity to inhaled gram - positive bacteria whereas gram - negative bacteria , which are rarely found in airways , do easily induce epithelial activation . a strong immune response following a first hit with lps and a moderate induction caused by first hit with lta
might be expected in the lung . however , due to the observed up - regulation of tlr2 by gram - negative challenge sensitivity to lta may be elevated during a second hit .
cpg - odn - dependent - up - regulation of tlr6 detected here may further support the sensitization to lta during ongoing inflammation .
however , significant up - regulation of pulmonary tlr9 expression was not demonstrated here and is in accordance with recent results from our group .  
cytokine release evoked by tlr stimulation on lung tissue has been investigated in different settings , such as cultured pulmonal cells or by application of tlr ligands to the airway side [ 12 , 14 ] . to our knowledge , this study compares for the first time a challenge with different tlr ligands applied systemically .
tnf- taken from the plasma was used to determine equipotent doses of remote stimuli . on the other hand ,
tnf- mrna and protein were investigated in pulmonary tissue to compare the inflammatory response of the lung from 06  h after stimulation with each tlr - ligand . here , lps led to a significant increase of tnf- mrna and protein at all time points while lta and cpg - odn elevated its expression only at the earliest time point ( 2  h ) .
these results confirm the well - known observation that tnf- is an early inflammatory marker . apart from tnf-
, lps also induced all other investigated proinflammatory cytokines at 4  h , cpg - odn raised only il12p40 while lta failed to elevate any of the other inflammatory markers .
these findings further support the above - mentioned low sensitivity of the lung to gram - positive stimuli , which may be overcome by direct intrapulmonary application [ 28 , 29 ] .
we found a significant up - regulation of il-12p40 in vivo in pulmonary tissue by cpg - odn and lps .
this transfers the results from albrecht et al .   to the in vivo situation , as they demonstrated an induction of il-12p40 mainly by cpg - odn in transfected macrophages and dendritic cells .
il-12p40 bridges the gap between innate and adaptive immunity because it is potently regulated in antigen - presenting cells , thereby activating the adaptive t - helper ( th1 ) cells .  
these immune cells are attracted into the site of inflammation by chemokines . in our study
this is in accordance with the observed high mpo activity after tlr4 stimulation , as monocytes and neutrophils attracted by chemokines exclusively release mpo .
lps - dependent infiltration of polymorphonuclear neutrophils into pulmonary vasculature , lung interstitium , and alveolar space as well as bronchoalveolar lavage fluid has already been demonstrated by others . in our setting of remote stimulation , cpg - odn , and
, this seems to be in contrast to the literature [ 28 , 32 , 33 ] .
however , this difference between our results and those of others may be attributed to the diverse mode of administration ( local versus systemical ) .  
mmps play a decisive role in the repair of the alveolar epithelium during pulmonary inflammation by cleaving components of the extracellular matrix ( ecm ) .
however , excessive expression of mmps can also destroy the ecm and initiate further inflammation and changes in pulmonary architecture .
mmp-2 and -9 can be produced by various resident cells in the lung   or might stem from stimulated immune cells .
similar to mmps , the defined role of their inhibitors is also not fully understood in sepsis .
some studies indicate that high timp-1 levels are associated with severe forms of the disease and might , therefore , serve as possible prognostic markers of poor survival . despite low mrna levels of cytokines and chemokines in the lta group , mmp-2 and -9
were significantly induced in lung tissue as a sign of inflammatory affection . however , timp-1 expression was low and remained unaffected by lta .
a high mmp9/timp1 ratio has been shown to be associated with a less severe progression of different lung diseases [ 3739 ] .
timp-3 may act in a similar manner as timp-3-deficient mice were protected from sepsis - induced pulmonary inflammation and remodelling .  
the present study provides detailed information on inflammatory events in the lung after in vivo challenge with equipotent doses of lta , lps , and cpg - odn in a murine model of sepsis .
these different remote stimuli resulted in specific inflammatory responses in pulmonary tissue indicating an organ - specific immune modulation .
lta induced the lowest number of inflammatory mediators , which was associated with a mild progression of lung remodelling .
as gram - positive bacteria are commonly inhaled , the respective immune response of the lung has to be strictly controlled to avoid autodestructive inflammation .
on the other hand , gram - negative bacteria enter the respiratory tract much more rarely , which may explain the stronger inflammatory response to lps in our setting .
however , an ongoing infection with gram - negative bacteria might sensitize the lung towards gram - positive stimuli as tlr2 and tlr6 expressions are upregulated by lps .
in addition , cpg - odn that induces the inflammatory cascade via tlr9 is released by both gram - positive and gram - negative bacteria .
thus , in case of a bacterial sepsis , the inflammatory response in the lung will never be induced by exclusive activation of tlr2 or tlr4 but always by costimulation of tlr9 .
our findings indicate that the lung may be more susceptible to systemic inflammation caused by gram - negative stimuli than to one elicited by gram - positive stimulation .
 
 objective . to determine whether systemically administered tlr ligands differentially modulate pulmonary inflammation 
 . 
 methods . 
 equipotent doses of lps ( 20  mg / kg ) , cpg - odn ( 1668-thioat 1  nmol / g ) , or lta ( 15  mg / kg ) were determined via tnf activity assay . 
 c57bl/6 mice were challenged intraperitoneally . pulmonary nfb activation ( 2  h ) and gene expression / activity of key inflammatory mediators ( 4  h ) were monitored . 
 
 results . all tlr ligands induced nfb . 
 lps increased the expression of tlr2 , 6 , and the cytokines il-1 , tnf- , il-6 , and il-12p35/p40 , cpg - odn raised tlr6 , tnf- , and il12p40 . 
 lta had no effect . additionally , lps increased the chemokines mip-1/ , mip-2 , tca-3 , eotaxin , and ip-10 , while cpg - odn and lta did not . 
 myeloperoxidase activity was highest after lps stimulation . 
 mmp1 , 3 , 8 , and 9 were upregulated by lps , mmp2 , 8 by cpg - odn and mmp2 and 9 by lta . 
 timps were induced only by lps . 
 mmp-2/-9 induction correlated with their zymographic activities . conclusion . 
 pulmonary susceptibility to systemic inflammation was highest after lps , intermediate after cpg - odn , and lowest after lta challenge . 
 
1. Introduction
2. Material and Methods
3. Results
4. Discussion
 Conflict of Interests