monocytes / macrophages play a critical role in managing innate and adaptive immunity - including inflammatory processes by secreting proinflammatory molecules ( eg . tumor necrosis factor ( tnf)- , and nitric oxide ( no ) ) .
the activation of macrophages and monocytes is mediated by activation of various receptors including toll like receptor-(tlr- ) 4 and their counter molecules such as lipopolysaccharide ( lps ) derived from bacteria or virus . in parallel , the activation of these cells triggers various cellular responses such as cell migration , adhesion , extravasation , and infiltration to induce effective movement of these cells into inflamed tissue by adhesion molecules such as 1 ( cd18 ) or 2 ( cd29 ) integrins and their ligands such as vascular cell adhesion molecule-(vcam- ) 1 or intercellular adhesion molecule-(icam-)1 .
the molecular interaction between surface receptors and counter molecules seen in various cellular inflammatory responses generates a series of complex signaling events composed of numerous intracellular enzymes such as phosphoinositide-3-kinase ( pi3k ) , phosphoinositide - dependent kinase 1 ( pdk1 ) , akt ( protein kinase b ) , and mitogen - activated protein kinases ( mapks ) such as extracellular signal - regulated kinase ( erk ) , c - jun n - terminal kinase ( jnk ) , and p38 [ 4 , 5 ] linked to actin cytoskeleton rearrangement for modulating cellular activation or the proinflammatory gene expression by mediating with transcription factors like nf-b and ap-1 .
recently , inflammatory responses by monocytes and macrophages were reported to be a critical pathological event in triggering various acute or chronic diseases such as septic shock , cancer , autoimmune diseases , cardiovascular diseases , obesity , and diabetes [ 7 , 8 ] .
it is therefore considered that development of promising regulators of monocyte / macrophage - mediated inflammatory responses without side effects could be useful for prevention of , or as the therapeutic remedy for , various inflammation - mediated diseases .
cinnamaldehyde ( ca ; figure 1(a ) ) , a major bioactive compound isolated from the leaves of cinnamomum osmophloeum kaneh [ 10 , 11 ] , has been known to trigger apoptosis through mitochondrial permeability transition in human promyelocytic leukemia hl-60 cells , by activating the proapoptotic bcl-2 family proteins .
treatment of cultured mouse splenocytes with ca in a dose - dependent manner blocked the proliferation of lymphocytes induced by concanavalin a and lps .
this compound was also found to suppress nf-b activation within macrophage - like raw264.7 cells .
it has been demonstrated that ca is capable of blocking inducible nitric oxide synthase ( inos ) and no production by mediation of nf-b activation blockade in lps - stimulated raw264.7 cells .
moreover , the production of pge2 was also reduced by ca exposure in cultured rat cerebral microvascular endothelial cells .
these results strongly suggested that ca can be applied as an anti - inflammatory drug .
however , the pharmacological target and inhibitory mechanism of ca , and its activity on various cellular events such as cell adhesion and migration commonly seen in the functional activation of monocytes / macrophages , have not been examined yet .
thus , in this study , we investigated the detailed regulatory roles of ca on monocyte / macrophage - mediated immune responses and its potential target enzyme .
ca was kindly supplied from the aging tissue bank ( pusan national university , busan , south korea ) .
lps , phorbol 12-myristate 13-acetate ( pma ) , fitc - dextran , 1,4-dithiolthreitol ( dtt ) , l - cysteine , and tnf- were obtained from sigma chemical co. ( st . louis , mo ) .
raw264.7 and tlr4-expressing hek293 cells were purchased from american type culture collection ( rockville , md ) and invivogen ( san diego , ca ) .
phospho - specific antibodies to p85 , pdk1 , akt , and ib  were obtained from cell signaling ( beverly , ma ) .
cell - cell adhesion - inducing antibodies to cd29 ( mem 101a , purified igg1 ) , cd43 ( 161 - 46 , ascites , igg1 ) , and p5d2 were used as reported previously [ 18 , 19 ] . antibodies to costimulatory ( cd80 , cd86 , cd40 , and cd69 ) and adhesion ( cd29 , cd43 , and cd18 ) molecules were from bd biosciences ( san diego , ca ) . antibodies to pattern recognition receptors ( dectin-1 , tlr2 , tlr4 , sr , and cr3 ) were purchased from serotec ( raleigh , nc ) .
suntide , a peptide sequence derived from akt ( protein kinase b ) , was synthesized by peptron ( daejeon , south korea ) .
raw264.7 and tlr4-expressing hek293 cells were cultured in rpmi1640 medium supplemented with 10% heat - inactivated fetal bovine serum ( gibco , grand island , ny ) , glutamine , and antibiotics ( penicillin and streptomycin ) , at 37c with 5%  co2 .
raw264.7 cells ( 2  10 cells / ml ) were incubated with lps ( 1  g / ml ) in the presence or absence of ca for 24  h. supernatants were assayed for no and tnf-   contents using griess reagent . since raw264.7 cells are not easily transfected with certain types of dna constructs , tlr4-expressing hek293 cells ( 1  10 cells / ml )
were used to be transfected with 1  g of plasmid with nf-b and -galactosidase by using the calcium phosphate method in a 12-well plate .
luciferase assays were performed using the luciferase assay system ( promega ) . to measure the phagocytic activity of raw264.7 cells , a previously reported method was used with slight modifications .
raw264.7 ( 5  10 ) cells pretreated with ca were resuspended in 100  l pbs containing 1% human serum and incubated with fitc - dextran ( 1  mg / ml ) at 37c and 0c for 30  min .
the incubation was stopped by the addition of 2  ml ice - cold pbs containing 1% human serum and 0.02% sodium azide .
the cells were washed three times with cold pbs - azide and analyzed by flow cytometry .
briefly , u937 cells maintained in complete rpmi1640 medium ( supplemented with 100  u / ml of penicillin 100 g / ml of streptomycin , and 10% fbs ) were preincubated with ca for 1  h at 37c and further incubated with aggregation - inducing ( agonistic ) antibodies ( 1  g / ml ) in a 96-well plate . after a 50  minute incubation ,
cell - cell clusters were determined by homotypic cell - cell adhesion assay using a hemocytometer   and analyzed with an inverted light microscope equipped with a cohu high - performance ccd ( diavert ) video camera .
for the cell - fibronectin adhesion assay , ca - treated u937 cells ( 5  10 cells / well ) were seeded on a fibronectin - coated ( 50  g / ml ) plate and incubated for 3  h . after removing unbound cells with pbs
, the attached cells were treated with 0.1% crystal violet for 15  min . the od value at 540  nm was measured by a spectramax 250 microplate reader .
raw264.7 cells ( 2  10 cells / ml ) were incubated with ca for 30  min . after scratching the cultured cells with a pipette
, the cells were further incubated for 48  h. the images of the cells in culture were obtained using an inverted phase contrast microscope attached to a video camera .
surface levels of adhesion molecules ( cd29 , cd43 , and cd18 ) in u937 and   co - stimulatory molecules ( cd69 , cd80 , cd40 , and cd86 ) and pattern recognition receptors ( dectin-1 , tlr2 , tlr4 , sr , and cr3 ) in raw264.7 cells were determined by flow cytometric analysis as reported previously .
stained cells were analyzed on a facscan device ( becton - dickinson , san jose , ca ) .
the total rna from the ca and lps - treated raw264.7 cells was prepared by adding trizol reagent ( gibco brl ) , according to manufacturer 's protocol .
the primers ( bioneer , daejeon , south korea ) were used as previously reported .
raw264.7 cells ( 5  10 cells / ml ) were washed 3 times in cold pbs with 1  mm sodium orthovanadate and lysed in lysis buffer ( 20  mm tris - hcl , ph 7.4 , 2  mm edta , 2  mm ethyleneglycotetraacetic acid , 50  mm -glycerophosphate , 1  mm sodium orthovanadate , 1  mm dithiothreitol , 1% triton x-100 , 10% glycerol , 10  g / ml aprotinin , 10  g / ml pepstatin , 1  mm benzimide , and 2 mm pmsf ) for 30  min with rotation at 4c .
the lysates were clarified by centrifugation at 16,000  g for 10  min at 4c and stored at 20c until needed .  
proteins were separated on 10% sds - polyacrylamide gels and transferred by electroblotting to polyvinylidene difluoride ( pvdf ) membrane .
membranes were blocked for 60  min in tris - buffered saline containing 3% bovine serum albumin , 20  mm naf , 2  mm edta , and 0.2% tween 20 at room temperature .
the membrane was incubated for 60  min with specific primary antibody at 4c , washed 3 times with the same buffer , and incubated for additional 60  min with horse radish peroxidase-(hrp- ) conjugated secondary antibody .
the total and phosphorylated levels of p85 , pdk1 , akt , ib , and -actin were visualized using the ecl system ( amersham , little chalfont , buckinghamshire , uk ) . 

 cyano-4-hydroxycinnamic acid ( 20  mg ) ( bruker daltonics , bremen , germany ) was dissolved in 1  ml acetone  :  ethanol ( 1  :  2 , v / v ) , and 0.5  l of the matrix solution was mixed with an equivalent volume of sample .
mass spectra were first calibrated in the closed external mode using the peptide calibration standard ii ( bruker daltonics ) , sometimes using the internal statistical mode to achieve maximum calibration mass accuracy .
the student 's t - test and one  way anova were used to determine the statistical significance between values of the various experimental and control groups .
monocytes / macrophages are the prime immune cells managing inflammatory responses , which contribute to development of number of diseases such as cancer , diabetes , and atherosclerosis [ 30 , 31 ]
. this view led us to develop novel immunoregulatory drugs based on the functional activation of monocytes and macrophages without side effects to prevent such diseases . in this context , medicinal plants that have traditionally been used for long time
are considered as attractive biopharmaceutical candidates . with this goal , therefore , we have attempted to develop macrophage function regulators using naturally occurring compounds or plants for a decade .  
the regulatory effect of ca on lps - induced macrophage immune responses was initially examined . upon nontoxic concentrations ( 0 to 40  m ) ( figure 1(b ) ) , ca strongly suppressed the production of no ( figure 2(a ) ) and the surface upregulation of costimulatory ( cd80 and cd69 ) and pattern recognition ( tlr2 and cr3 ) molecules ( figure 2(b ) ) . moreover , ca protected cells from lps - induced cytotoxicity and apoptosis , mainly induced by the no produced ( figure 2(c ) ) .
the inhibition of no release occurred at the transcriptional levels , according to figure 3 .
thus , ca blocked mrna expression of inos as well as other proinflammatory cytokines such as tnf- and il-1 as much as 80 to 95% ( figure 3 ) . because transcriptional downregulation of inflammatory mediators by ca has been reported to inhibit nf-b activation [ 16 , 32 ] , reporter gene assay for nf-b and immunoblotting analysis of upstream signaling were further conducted . as figure 4(a ) shows , ca blocked nf-b - mediated luciferase activity induced by lps treatment , similar to previous papers [ 15 , 32 ] .
this compound suppressed the phosphorylation of ib , akt , and pdk1 but not p85 , a regulatory subunit of pi3k ( figure 4(b ) ) , suggesting that the pharmacological target of ca may be pi3k or pdk1 in lps - mediated macrophage immune responses . unlike lps - induced inflammatory responses , fitc - dextran - induced phagocytic uptake of raw264.7 cells , a major response found in innate immunity , was not negatively modulated by this compound ( figure 5 ) , which suggests that ca can not regulate all macrophage functions but modulates
meanwhile , ca also strongly suppressed cell - cell adhesions induced by proaggregative antibodies to cd29 and cd43 up to 85% ( figures 6(a ) and 6(b ) ) .
however , adhesion of u937 cells to fibronectin , an extracellular matrix protein acting as a cd29 ligand , was not suppressed by this compound ( figure 6(c ) ) , indicating that adhesion between cells but not cell and extracellular matrix could be blocked by ca .
however , ca did not suppress the surface levels of adhesion molecules such as cd18 , cd29 , and cd43 even at 40  m ( figure 6(d ) ) , while ca diminished the migration of raw264.7 cells in an in vitro wound healing assay , compared to normal ( figure 6(e ) ) .
considering that cell - fibronectin adhesion only requires simple activation of cd29 , and while intracellular signaling ( erk , p38 , and protein kinase c ) and actin cytoskeleton change are important factors in cell - cell adhesion events [ 24 , 25 ] , ca seems to modulate intracellular signaling events rather than the blockade of a direct interaction between cd29 and fibronectin . in particular , these signaling events targeted to ca seem to be involved in modulating cell migration commonly seen in both cell - cell adhesion ( figure 6(a ) ) and wound healing assays ( figure 6(e ) ) .
cd29-mediated cell - cell adhesion is an essential phenomenon for survival and activation of immune cells , particular for an interaction between antigeny presenting cells ( apc ) and t lymphocytes or nk cells .
therefore , antiaggregative effect of ca may contribute to the regulation of monocyte / macrophage roles as apc requiring their adhesion responses . similarly , since pi3k / akt inhibition by ly294002 and wortmannin suppressed cd29-mediated cell - cell adhesion , ca inhibition of adhesion and migration events seems to occur at the level of pi3k / pdk1 , as in the case of lps signaling .    since thiol compounds such as dtt and l - cysteine are reported to block ca inhibition , we finally examined whether this pattern can be observed under the same conditions .
as expected , pretreatment of l - cysteine ( 200  m ) or dtt ( 300  m ) before ca treatment abrogated the inhibitory activity of ca in both no production ( figure 7(a ) ) and nf-b - mediated luciferase activity ( figure 7(b ) ) , suggesting that thiolation is the major chemical mechanism of ca inhibition .
up to date , we have not been able to locate the exact thiolation site on the target protein by ca . however , recent findings revealing that an adduct formation of hydroquinone with the sulfhydryl group of cys-310 in akt is able to block the phosphorylation of both thr-308 and ser-473   seem to suggest that a target cysteine sequence of pi3k or pdk1 can serve as a thiolation site affecting their phosphorylation and activation . indeed , we failed to detect an adduct formation between ca and suntide , a peptide fragment containing cysteine-310 designed according to akt amino acid sequence ( figure 7(c ) ) .   because identification of ca target thiolation site is an important step to understand the exact molecular mechanism of ca inhibition , we are currently undertaking further analysis using other peptide sequences containing cysteine residues from pi3k and pdk1 . in conclusion
, we found that ca was able to suppress the production of no and upregulation of surface levels of costimulatory molecules such as the surface upregulation of both costimulatory ( cd80 and cd69 ) and pattern recognition molecules ( tlr2 and cr3 ) .
in addition , ca also blocked both cell migration and cell - cell adhesion induced by cd29 and cd43 , but not cell - fibronectin adhesion .
the ca inhibition was likely due to the inhibition of pi3k and pdk1 , important for nf-b activation of signaling components , according to immunoblotting analysis .
in particular , l - cysteine and dtt strongly interfered ca - mediated inhibition of no production and nf-b activation .
therefore , our results suggest that ca can act as a strong regulator of monocyte / macrophage - mediated immune responses , possibly by the induction of thiolation at cysteine residues in the target enzyme ( pdk1 or pi3k ) . to prove a detailed inhibitory mechanism , identification of molecular targets of ca
 cinnamaldehyde ( ca ) has been known to exhibit   anti - inflammatory and anticancer effects . 
 although numerous pharmacological effects have been demonstrated , regulatory effect of ca on the functional activation of monocytes and macrophages has not been fully elucidated yet . to evaluate its monocyte 
 / macrophage - mediated immune responses , macrophages activated by lipopolysaccharide ( lps ) , and monocytes treated with proaggregative antibodies , and extracellular matrix protein fibronectin were employed .   
 ca was able to suppress both the production of nitric oxide ( no ) and upregulation of surface levels of costimulatory molecules ( cd80 and cd69 ) and pattern recognition receptors ( toll - like receptor 2 ( tlr2 ) and complement receptor ( cr3 ) ) . 
 in addition , ca also blocked cell - cell adhesion induced by the activation of cd29 and cd43 but not cell - fibronectin adhesion . 
 immunoblotting analysis suggested that ca inhibition was due to the inhibition of phosphoinositide-3-kinase ( pi3k ) and phosphoinositide - dependent kinase ( pdk)1 as well as nuclear factor-(nf- ) b activation . 
 in particular , thiol compounds with sulphydryl group , l - cysteine and dithiothreitol ( dtt ) , strongly abrogated ca - mediated no production and nf-b activation . 
 therefore , our results suggest that ca can act as a strong regulator of monocyte / macrophage - mediated immune responses by thiolation of target cysteine residues in pi3k or pdk1 . 
1. Introduction
2. Materials and Methods
3. Results and Discussion