BACKGROUND
migration is an important feature of leukocytes which represents a pre-requisite for normal leukocyte function in physiological processes such as protection against infection or foreign antigens.
under pathological conditions, leukocytes infiltrate tissue which locally produces pro-inflammatory substances and chemokines.
this process is known as leukocyte recruitment.
the normal ability for active movement is an important pre-requisite for the effective recruitment of leukocytes from the microvascular circulation  <cit> .
the blockade of leukocyte migration can abrogate the process of leukocyte recruitment and inflammation.
therefore, the modulation of leukocyte migration has been proposed as a potential therapeutic approach for the treatment of several inflammatory diseases such as psoriasis, eczema, multiple sclerosis and arthritis  <cit> .
dependent on the tissue structure, two types of non-directed leukocyte migration can be identified: two- and three-dimensional haptokinesis.
two-dimensional leukocyte haptokinesis can be found on two-dimensional surfaces, such as inner vessel walls, the peritoneum and the pleura.
three-dimensional haptokinetic migration occurs in the tissue which contains cellular or fibrillar extracellular matrix components promoting leukocyte movement  <cit> .
prevailing random haptokinetic leukocyte migration becomes directional , if a gradient of chemotacic agent is present in the tissue.
chemotaxis is induced by soluble, freely diffusing compounds that lead to preferential signaling and actin-rich protrusions at the leading edge.
in contrast to chemotaxis, haptotaxis represents the directed migration toward chemoattractants which are immobilized on tissue structures, such as interstitial collagens or stromal cell network  <cit> .
nonsteroidal anti-inflammatory drugs  are one of the most widely used anti-inflammatory substances.
the major anti-inflammatory effect of nsaid is the inhibition of cyclooxygenase  enzyme system which blocks prostaglandin synthesis and leads in consequence, to reduction of inflammation, pain and fever  <cit> .
it has also been shown that cox inhibition by nsaid reduces migratory activity of leukocytes which therefore leads to a reduction of leukocyte recruitment into the inflamed tissue.
although the effect of some nsaid such as aspirin, indometacin or ketoprofen on leukocyte migration was investigated in several previous studies  <cit> , these studies were mainly focused on the changes of migration of polymorphonucelar leukocytes  and did not analyze effects of nsaid at the single leukocyte level.
in the present study, we studied the effect of three different nsaid, ketoprofen, diclofenac and sc- <dig> on the haptokinesis and haptotaxis of pmns and activated lymphocytes at the single cell level in vitro and in vivo using time-lapse microscopy.
the study was in particular aimed to identify the effective concentrations of nsaid which can suppress leukocyte migration.
furthermore, the toxic potential of these nsaid and the role of ca2+ were analyzed.
ketoprofen and diclofenac are known to inhibit both cox- <dig> and cox- <dig> whereas sc- <dig> belongs to the group of cox-1-selective inhibitors.
methods
leukocytes preparation
polymorphonuclear leukocytes  or lymphocytes were isolated from human venous peripheral blood using density gradient centrifugation .
microscopic examination showed that at least 90% of isolated polymorphonuclear leukocytes were represented by neutrophils.
for the 3d haptotaxis, isolated human pmns or lymphocytes were used immediately after isolation.
in some experiments, lymphocytes were activated for  <dig> days at 37°c using  <dig>  μg/ml of concanavalin a  in iscove's modified dmem supplemented with 10% fcs .
to study inhibitory effects on leukocyte migration, the following nsaid were used: ketoprofen , diclofenac , sc- <dig> .
analysis of leukocyte haptokinesis
leukocyte migration in collagen matrix in vitro was analyzed using modified 96-wells collagen matrix assay as previously described  <cit> .
for this assay,  <dig> μl/well of collagen mixture was prepared and polymerized for  <dig> min at 37°c.
<dig> μl of leukocyte suspension  containing different concentrations of nsaid was layered upon the collagen gel.
plates were incubated at 37°c for  <dig> min to allow migrating leukocytes to invade into collagen gel.
the suspension of non-invading leukocytes were washed using multi-reagent washer .
<dig> μm/well of calcein am  were added and incubated for  <dig> min.
the fluorescence was measured using multilable counter .
the number of invaded leukocytes was calculated using reference concentration of leukocytes and was expressed as a percentage of migrating leukocytes to total leukocyte number.
for time-lapse microscopy,  <dig> ×  <dig> cells in  <dig> μl of pbs were added to the collagen mixture in a petri plate prior the polymerization.
digital time-lapse microscopy was performed after application of different concentrations of ketoprofen to the gel as previously described  <cit> .
leukocyte movement was recorded for  <dig> min.
subsequently, gel preparation was washed with pbs and recorded for further  <dig> min.
six experiments for each group were performed.
analysis of leukocyte haptotaxis
leukocytes invasion into the collagen matrix was analyzed.
for this aim,  <dig> μl of collagen matrix mixture were prepared on ice as previously described  <cit> .
additionally, this mixture contained il- <dig>  or sdf- <dig>  and ketoprofen and filled into the glass square tube .
collagen matrix was allowed to polymerize for  <dig> min at 37°c.
<dig> μl of leukocyte suspension  was added upon collagen matrix.
the tube was fixed in the upright position and incubated at 37°c for  <dig> hours for neutrophils or  <dig> hours for lymphocytes.
the distance of leukocyte invasion into the matrix was recorded using microscope and measured using histo software .
intravital microscopy
five male lysegfp-ki mice  were used.
these mice allow an excellent visualization of egfp-expressing neutrophils using fluorescence microscopy.
to induce local leukocyte activation and extravasation,  <dig> μl of il- <dig>  was injected onto the auricle.
an excessive amount of ketoprofen gel  was applied on the skin of the auricle before or  <dig> min after il- <dig> injection.
blood vessels were visualized using intravenous injection of  <dig>  mg of tritc-labelled albumin .
time-lapse intravital microscopy of auricle was performed using fluorescence microscope  combined with image deconvolution .
the speed and the fraction of migrating leukocytes were measured using capimage software .
all experiments were approved by the local committee of animal care .
toxicity assays
potential cytotoxic effect of nsaid on pmns was measured using calcein am assay  as described in manufacturer's instruction.
in brief, leukocytes were incubated with different concentrations of nsaid for  <dig> min at 37°c and loaded with  <dig> μm calcein am.
vital cells metabolised calcein and produced fluorescent signal which was measured using fluorimeter  and expressed as a percent of the non-treated control.
three experiments for every substance were performed.
i measurement
ca2+ was measured as previously described by nobiling and bührle  <cit> .
in brief, isolated leukocytes were incubated in collagen gel for  <dig> min and were loaded with  <dig> μm of fura- <dig> am .
intracellular ca2+  signal was recorded at alternately  <dig> and  <dig> nm wavelength for  <dig> sec at three stages: at baseline, after application of  <dig> and  <dig> mm ketoprofen and after removal of ketoprofen by washing.
i concentration was calculated as 340/ <dig> nm ratio and expressed in μm.
statistical analysis
statistical analysis was performed using spss software .
all data are given as mean ± sd.
to study differences between the groups, wilcoxon or mann-whitney-u-test were used, if appropriate.
p <  <dig>  was considered as significant.
RESULTS
nsaid inhibit leukocyte migration in vitro
the influence of different concentrations of nsaid on leukocyte haptokinesis in collagen matrix was studied using a 96-well calcein assay.
neutrophils or activated lymphocytes showed an active migration in the collagen matrix.
there were no inhibiting effects on leukocyte migration of low concentrations of nsaid.
we found that all used nsaid  inhibited migration of both neutrophils and lymphocytes in a dose-dependent manner in vitro .
depending on the selecting drug and on the leukocyte population , the dose of  <dig> - <dig>  mm led to complete blockade of leukocyte migration .
ketoprofen showed the highest ic <dig> on leukocyte migration whereas diclofenac had the lowest ic <dig> .
toxicity assay showed that only highest concentrations of ketoprofen  and diclofenac  led to significant reduction of the cell number.
<dig> mm diclofenac destroyed almost all leukocytes after  <dig> min incubation .
no reduction of leukocyte number was found after incubation with sc- <dig>
inhibition of leukocyte migration by nsaid is reversible
since all nsaid showed a similar inhibiting effect on leukocyte migration, further experiments using time-lapse microscopy were focused on the effects of ketoprofen.
we found that the percent of migrating leukocytes in collagen gel was  <dig> ± 6%, whereas the mean speed of leukocyte migration was  <dig>  ±  <dig>  μm/min.
migrating leukocytes showed a multipolar polarized shape and stable random movement  over a long time period.
leukocyte migration in control group was stable .
the concentration of  <dig>  mm ketoprofen significantly decreased speed, whereas a concentration of  <dig> mm caused a total blockade of leukocyte migration .
these effects were found immediately after application of nsaid.
washing-out of ketoprofen led to a recovery of leukocyte migration, although the speed of movement after recovery did not achieve its baseline value .
nsaid blocked leukocytes invasion in vitro and prevented leukocyte recruitment in vivo
to study leukocyte haptotaxis, we analyzed leukocyte invasion into the collagen matrix with or without chemokine.
in this assay, leukocytes showed a superficial invasion into the collagen matrix, if no chemoattractants were added to the collagen.
il- <dig> or sdf- <dig> induced a strong directed invasion of both neutrophils and lymphocytes into the collagen matrix .
interestingly, addition of  <dig>  and  <dig> mm ketoprofen to the matrix inhibited leukocyte penetration in a dose-dependent manner whereas  <dig> mm ketoprofen prevented leukocyte invasion into the matrix .
intravital microscopy showed that the cutaneous injection of il- <dig> induced a strong intravascular activation and extravasation of leukocytes, which infiltrated cutaneous tissue within several minutes .
interestingly, the local application of ketoprofen gel to the auricle led to the lost of leukocyte polarization and the blockade of leukocyte migration , which was observed within several minutes after ketoprofen application.
if ketoprofen gel was applied before il- <dig> injection, leukocyte recruitment and infiltration were almost absent .
ketoprofen decreases intracellular i in migrating human granulocytes in vitro
leukocytes of control experiments showed a well-polarised shape and a constant i signal.
ketoprofen concentration of 2- <dig> mm caused a rapid leukocyte depolarisation .
no significant changes of i were found after addition of  <dig> mm ketoprofen .
interestingly, the increase of ketoprofen concentration to  <dig> mm led to a dramatic decrease of i signal.
the higher the initial value of i, the sharper was its decline .
the washing-out of ketoprofen led to a partial restoration of i signal .
discussion
in the present study, the effect of three different nsaid, ketoprofen, diclofenac, sc- <dig> on leukocyte haptotaxis and haptokinesis was analyzed.
we found that all used nsaid inhibited leukocyte migration of in a dose-dependent manner both in vivo and in vitro.
at the single cell level, nsaid inhibited both leukocyte speed and the fraction of migrating leukocytes.
this effect occurred immediately after nsaid application and was partially restored after removal of inhibiting agent.
interestingly, inhibiting effect of nsaid was not dependent on the type of leukocytes, since migration of both pmns and lymphocytes was inhibited by equivalent concentrations.
the inhibitory effect of ketoprofen was reproducible in calcein-based assay and in time-lapse microscopy.
interestingly, inhibiting effect of nsaid on leukocyte migration is reversible.
as shown in the present study, leukocytes re-store their migration if nsaid concentration decreases.
results of the present study correspond well with previous results which analyzed the effect of some nsaid such as aspirin, indomethacin or ketoprofen on leukocyte migration  <cit> .
these studies showed that nsaid inhibit chemotaxis of polymorphonuclear leukocytes .
in contrast to previous studies, we analyzed both pmn and t cell migration, studied haptotactic movement and showed changes of leukocyte migration at the single cell level in vivo and in vitro.
we also showed that  <dig> mm ketoprofen stopped leukocyte invasion into the il- <dig> or sdf-1-containing matrix in vitro.
this effect was also studied in vivo using time-lapse intravital microscopy.
il- <dig> and sdf- <dig> are two important chemokines which control leukocyte chemo- and haptotaxis by binding to the cxcl <dig> and to cxcl <dig> chemokine receptor, respectively  <cit> .
leukocyte recruitment and infiltration were effectively prevented if ketoprofen was locally applied in vivo prior the pro-inflammatory stimulation.
we believe that the repellent action of nsaid is the direct consequence of their blocking effect on leukocyte migration both in vivo and in vitro.
ketoprofen blocked leukocyte migration for at least one hours.
the potential resuming of leukocyte movement after the longer exposure to ketoprofen were not analyzed.
previous studies showed that nsaid inhibit cox at a concentration of 10-5-10-3m.
the inhibition of cox is a major, but not a single mechanism of potential nsaid effects.
in the present study, the initial inhibition of leukocyte migration was found at nsaid concentrations beginning from 10- <dig> μm and the ic <dig> varied from  <dig> to  <dig> μm.
this concentrations is 10-fold higher than the highest known ic <dig> required for cox inhibition for diclofenac and sc- <dig> whereas this difference for ketoprofen is 500-fold  <cit> .
therefore, we believe that the direct inhibition of leukocyte migration through nsaid cannot only represent a cox-dependent phenomenon, but other mechanisms may participate in the nsaid action on leukocyte migration.
the data of the present study correspond well with results of previous studies.
these studies demonstrated that nsaid have an inhibiting effect on chemotaxis of pmns at high concentrations in vitro which was independent of their potency as prostaglandin inhibitors  <cit> .
in contrast to these studies, we showed that not only chemotactic, but also haptokinetic migratory activity of single leukocytes can be blocked by nsaid.
furthermore, almost all previous studies focused on the changes of migration of polymorphonuclear leukocytes, whereas the effect of nsaid on lymphocyte migration was poorly investigated.
results of the present study demonstrated that nsaid have similar inhibiting effects on migration of both pmns and lymphocytes and that lymphocyte movement can be stopped by nsaid.
other previous studies showed that low concentrations of nsaid inhibit cox production by activated mast cells and macrophages and reduce inflammation-induced leukocyte infiltration  <cit> .
although cox inhibition by low concentrations of nsaid can reduce leukocyte infiltration in inflamed tissue, this effect can be produced by rather different mechanism than the direct inhibition of leukocyte migration.
we believe that cox-related effects of nsaid are not a major factor for the direct inhibition of leukocyte migration.
furthermore, we propose that other possible mechanisms of nsaid action such as inhibition of intracellular atp  <cit> , calcium metabolism  <cit>  or i-dependent myosin ii activation  <cit>  can be responsible for the inhibition of leukocyte migration.
the results of the present study confirmed the important role of ca2+ signaling in nsaid action and showed that the stop of leukocyte migration by ketoprofen was accompanied by the dramatic decrease of i and by the cytotoxic effect whereas partial inhibition of leukocyte migration did not cause a significant reduction of i and did not induce cell toxicity.
concentrations of nsaid, which are required for blockade of leukocyte migration, are high.
as showed in previous studies, high nsaid concentration cause toxic side effects, if these substances are systemically applied  <cit> .
however, nsaid do not induce systemic toxic side effects after topic application as gel or salve formulation  <cit>  or after intraarticular injection.
previous studies showed that ketoprofen has a high percutaneous penetration  <cit> .
the present study showed that the topic application of concentrated ketoprofen gel  rapidly blocked migration of cutaneous leukocytes.
we believe that the suppression of leukocyte migration can represent one of the pharmacological effects of nsaid after the topic application in clinical use.
it is known that other anti-inflammatory drugs such as dexamethasone suppress leukocyte migration  <cit> .
it binds to its intracellular receptor and inhibits gene expression by binding to negative regulatory promoter regions or through protein/protein interactions  <cit> .
the effect of nsaid corresponds well with effect of dexamethasone.
as shown in the present study, ketoprofen can block sdf- <dig> and il-8-mediated leukocyte migration.
in accordance with our results, previous studies demonstrated that dexamethasone augmented intracellular chemokine signaling and inhibited sdf-1-induced chemotaxis of resting t cells  <cit> .
furthermore, dexamethasone also suppressed il-8-induced chemotaxis of pmn through inhibition of endogenous chemokine production  <cit> .
it can be proposed that inhibition of endogenous chemokine production by ketoprofen or other nsaid can also participate in the inhibition of leukocyte migration.
CONCLUSIONS
in summary, our results demonstrate that ketoprofen, diclofenac and sc- <dig> have a direct suppressive effect on haptotaxis and haptokinesis of polymorphonuclear leukocytes and lymphocytes.
this effect is dose-dependent and partially reversible after removal of inhibiting agent.
this process is probably induced by cox-dependent and cox-independent mechanisms whereas complete blockade, but not the partial inhibition of migration is accompanied by changes of ca2+signaling.
these results indicate a possible discrete mechanism of nsaid action which may be identified in further experiments.
the findings of the present study may have an important clinical implications since the blockade of leukocyte migration can also be expected after local application of nsaid in humans.
authors' contributions
sp: most experiments in vitro, manuscript preparation; ap and vk: design and data analysis of leukocyte migration experiments in vitro; rn: design, control and data aquisition of ca2+ measurement, js: data analysis, preparation of the manuscript; er: idea and concept, study design, haptotaxis experiments, intravital microscopy, manuscript preparation.
all authors read and approved the final manuscript.
supplementary material
additional file 1
time-lapse intravital microscopy of leukocyte extravasation in mouse cutaneous tissue .
cutaneous inflammation was induced by injection of il- <dig>  neutrophils express egfp and appear in green.
blood vessels are labelled using intravenous injection of tritc-labelled albumin.
real-time recording of  <dig> min was compressed to time-lapse sequence of  <dig> sec.
control experiment; il- <dig> induces an immediate extravasation and migration of leukocytes from the venule.
click here for file
additional file 2
time-lapse intravital microscopy of leukocyte extravasation in mouse cutaneous tissue .
cutaneous inflammation was induced by injection of il- <dig>  neutrophils express egfp and appear in green.
blood vessels are labelled using intravenous injection of tritc-labelled albumin.
real-time recording of  <dig> min was compressed to time-lapse sequence of  <dig> sec.
application of ketoprofen gel prior injection of il- <dig> blocks intravascular leukocyte adhesion and extravasation.
click here for file
acknowledgements
we thank dr. t. graf for providing of lysegfp-ki mice.
we thank c. bernardi for an excellent technical assistance.
we thank dr. w. gross for the help in "histo" software.

 migration is an important feature of leukocytes which represents a pre-requisite for normal leukocyte function in physiological processes such as protection against infection or foreign antigens. under pathological conditions, leukocytes infiltrate tissue which locally produces pro-inflammatory substances and chemokines. this process is known as leukocyte recruitment. the normal ability for active movement is an important pre-requisite for the effective recruitment of leukocytes from the microvascular circulation. the blockade of leukocyte migration can abrogate the process of leukocyte recruitment and inflammation. therefore, the modulation of leukocyte migration has been proposed as a potential therapeutic approach for the treatment of several inflammatory diseases such as psoriasis, eczema, multiple sclerosis and arthritis. dependent on the tissue structure, two types of non-directed leukocyte migration can be identified: two- and three-dimensional haptokinesis. two-dimensional leukocyte haptokinesis can be found on two-dimensional surfaces, such as inner vessel walls, the peritoneum and the pleura. three-dimensional haptokinetic migration occurs in the tissue which contains cellular or fibrillar extracellular matrix components promoting leukocyte movement. prevailing random haptokinetic leukocyte migration becomes directional, if a gradient of chemotacic agent is present in the tissue. chemotaxis is induced by soluble, freely diffusing compounds that lead to preferential signaling and actin-rich protrusions at the leading edge. in contrast to chemotaxis, haptotaxis represents the directed migration toward chemoattractants which are immobilized on tissue structures, such as interstitial collagens or stromal cell network. nonsteroidal anti-inflammatory drugs are one of the most widely used anti-inflammatory substances. the major anti-inflammatory effect of nsaid is the inhibition of cyclooxygenase enzyme system which blocks prostaglandin synthesis and leads in consequence, to reduction of inflammation, pain and fever. it has also been shown that cox inhibition by nsaid reduces migratory activity of leukocytes which therefore leads to a reduction of leukocyte recruitment into the inflamed tissue. although the effect of some nsaid such as aspirin, indometacin or ketoprofen on leukocyte migration was investigated in several previous studies, these studies were mainly focused on the changes of migration of polymorphonucelar leukocytes and did not analyze effects of nsaid at the single leukocyte level. in the present study, we studied the effect of three different nsaid, ketoprofen, diclofenac and sc- on the haptokinesis and haptotaxis of pmns and activated lymphocytes at the single cell level in vitro and in vivo using time-lapse microscopy. the study was in particular aimed to identify the effective concentrations of nsaid which can suppress leukocyte migration. furthermore, the toxic potential of these nsaid and the role of ca2+ were analyzed. ketoprofen and diclofenac are known to inhibit both cox- and cox- whereas sc- belongs to the group of cox-1-selective inhibitors. methods leukocytes preparation polymorphonuclear leukocytes or lymphocytes were isolated from human venous peripheral blood using density gradient centrifugation. microscopic examination showed that at least 90% of isolated polymorphonuclear leukocytes were represented by neutrophils. for the 3d haptotaxis, isolated human pmns or lymphocytes were used immediately after isolation. in some experiments, lymphocytes were activated for days at 37°c using μg/ml of concanavalin a in iscove's modified dmem supplemented with 10% fcs. to study inhibitory effects on leukocyte migration, the following nsaid were used: ketoprofen, diclofenac, sc-. analysis of leukocyte haptokinesis leukocyte migration in collagen matrix in vitro was analyzed using modified 96-wells collagen matrix assay as previously described. for this assay, μl/well of collagen mixture was prepared and polymerized for min at 37°c. μl of leukocyte suspension containing different concentrations of nsaid was layered upon the collagen gel. plates were incubated at 37°c for min to allow migrating leukocytes to invade into collagen gel. the suspension of non-invading leukocytes were washed using multi-reagent washer. μm/well of calcein am were added and incubated for min. the fluorescence was measured using multilable counter. the number of invaded leukocytes was calculated using reference concentration of leukocytes and was expressed as a percentage of migrating leukocytes to total leukocyte number. for time-lapse microscopy, × cells in μl of pbs were added to the collagen mixture in a petri plate prior the polymerization. digital time-lapse microscopy was performed after application of different concentrations of ketoprofen to the gel as previously described. leukocyte movement was recorded for min. subsequently, gel preparation was washed with pbs and recorded for further min. six experiments for each group were performed. analysis of leukocyte haptotaxis leukocytes invasion into the collagen matrix was analyzed. for this aim, μl of collagen matrix mixture were prepared on ice as previously described. additionally, this mixture contained il- or sdf- and ketoprofen and filled into the glass square tube. collagen matrix was allowed to polymerize for min at 37°c. μl of leukocyte suspension was added upon collagen matrix. the tube was fixed in the upright position and incubated at 37°c for hours for neutrophils or hours for lymphocytes. the distance of leukocyte invasion into the matrix was recorded using microscope and measured using histo software. intravital microscopy five male lysegfp-ki mice were used. these mice allow an excellent visualization of egfp-expressing neutrophils using fluorescence microscopy. to induce local leukocyte activation and extravasation, μl of il- was injected onto the auricle. an excessive amount of ketoprofen gel was applied on the skin of the auricle before or min after il- injection. blood vessels were visualized using intravenous injection of mg of tritc-labelled albumin. time-lapse intravital microscopy of auricle was performed using fluorescence microscope combined with image deconvolution. the speed and the fraction of migrating leukocytes were measured using capimage software. all experiments were approved by the local committee of animal care. toxicity assays potential cytotoxic effect of nsaid on pmns was measured using calcein am assay as described in manufacturer's instruction. in brief, leukocytes were incubated with different concentrations of nsaid for min at 37°c and loaded with μm calcein am. vital cells metabolised calcein and produced fluorescent signal which was measured using fluorimeter and expressed as a percent of the non-treated control. three experiments for every substance were performed. i measurement ca2+ was measured as previously described by nobiling and bührle. in brief, isolated leukocytes were incubated in collagen gel for min and were loaded with μm of fura- am. intracellular ca2+ signal was recorded at alternately and nm wavelength for sec at three stages: at baseline, after application of and mm ketoprofen and after removal of ketoprofen by washing. i concentration was calculated as 340/ nm ratio and expressed in μm. statistical analysis statistical analysis was performed using spss software. all data are given as mean ± sd. to study differences between the groups, wilcoxon or mann-whitney-u-test were used, if appropriate. p < was considered as significant.  nsaid inhibit leukocyte migration in vitro the influence of different concentrations of nsaid on leukocyte haptokinesis in collagen matrix was studied using a 96-well calcein assay. neutrophils or activated lymphocytes showed an active migration in the collagen matrix. there were no inhibiting effects on leukocyte migration of low concentrations of nsaid. we found that all used nsaid inhibited migration of both neutrophils and lymphocytes in a dose-dependent manner in vitro. depending on the selecting drug and on the leukocyte population, the dose of - mm led to complete blockade of leukocyte migration. ketoprofen showed the highest ic on leukocyte migration whereas diclofenac had the lowest ic. toxicity assay showed that only highest concentrations of ketoprofen and diclofenac led to significant reduction of the cell number. mm diclofenac destroyed almost all leukocytes after min incubation. no reduction of leukocyte number was found after incubation with sc- inhibition of leukocyte migration by nsaid is reversible since all nsaid showed a similar inhibiting effect on leukocyte migration, further experiments using time-lapse microscopy were focused on the effects of ketoprofen. we found that the percent of migrating leukocytes in collagen gel was ± 6%, whereas the mean speed of leukocyte migration was ± μm/min. migrating leukocytes showed a multipolar polarized shape and stable random movement over a long time period. leukocyte migration in control group was stable. the concentration of mm ketoprofen significantly decreased speed, whereas a concentration of mm caused a total blockade of leukocyte migration. these effects were found immediately after application of nsaid. washing-out of ketoprofen led to a recovery of leukocyte migration, although the speed of movement after recovery did not achieve its baseline value. nsaid blocked leukocytes invasion in vitro and prevented leukocyte recruitment in vivo to study leukocyte haptotaxis, we analyzed leukocyte invasion into the collagen matrix with or without chemokine. in this assay, leukocytes showed a superficial invasion into the collagen matrix, if no chemoattractants were added to the collagen. il- or sdf- induced a strong directed invasion of both neutrophils and lymphocytes into the collagen matrix. interestingly, addition of and mm ketoprofen to the matrix inhibited leukocyte penetration in a dose-dependent manner whereas mm ketoprofen prevented leukocyte invasion into the matrix. intravital microscopy showed that the cutaneous injection of il- induced a strong intravascular activation and extravasation of leukocytes, which infiltrated cutaneous tissue within several minutes. interestingly, the local application of ketoprofen gel to the auricle led to the lost of leukocyte polarization and the blockade of leukocyte migration, which was observed within several minutes after ketoprofen application. if ketoprofen gel was applied before il- injection, leukocyte recruitment and infiltration were almost absent. ketoprofen decreases intracellular i in migrating human granulocytes in vitro leukocytes of control experiments showed a well-polarised shape and a constant i signal. ketoprofen concentration of 2- mm caused a rapid leukocyte depolarisation. no significant changes of i were found after addition of mm ketoprofen. interestingly, the increase of ketoprofen concentration to mm led to a dramatic decrease of i signal. the higher the initial value of i, the sharper was its decline. the washing-out of ketoprofen led to a partial restoration of i signal. discussion in the present study, the effect of three different nsaid, ketoprofen, diclofenac, sc- on leukocyte haptotaxis and haptokinesis was analyzed. we found that all used nsaid inhibited leukocyte migration of in a dose-dependent manner both in vivo and in vitro. at the single cell level, nsaid inhibited both leukocyte speed and the fraction of migrating leukocytes. this effect occurred immediately after nsaid application and was partially restored after removal of inhibiting agent. interestingly, inhibiting effect of nsaid was not dependent on the type of leukocytes, since migration of both pmns and lymphocytes was inhibited by equivalent concentrations. the inhibitory effect of ketoprofen was reproducible in calcein-based assay and in time-lapse microscopy. interestingly, inhibiting effect of nsaid on leukocyte migration is reversible. as shown in the present study, leukocytes re-store their migration if nsaid concentration decreases. results of the present study correspond well with previous results which analyzed the effect of some nsaid such as aspirin, indomethacin or ketoprofen on leukocyte migration. these studies showed that nsaid inhibit chemotaxis of polymorphonuclear leukocytes. in contrast to previous studies, we analyzed both pmn and t cell migration, studied haptotactic movement and showed changes of leukocyte migration at the single cell level in vivo and in vitro. we also showed that mm ketoprofen stopped leukocyte invasion into the il- or sdf-1-containing matrix in vitro. this effect was also studied in vivo using time-lapse intravital microscopy. il- and sdf- are two important chemokines which control leukocyte chemo- and haptotaxis by binding to the cxcl and to cxcl chemokine receptor, respectively. leukocyte recruitment and infiltration were effectively prevented if ketoprofen was locally applied in vivo prior the pro-inflammatory stimulation. we believe that the repellent action of nsaid is the direct consequence of their blocking effect on leukocyte migration both in vivo and in vitro. ketoprofen blocked leukocyte migration for at least one hours. the potential resuming of leukocyte movement after the longer exposure to ketoprofen were not analyzed. previous studies showed that nsaid inhibit cox at a concentration of 10-5-10-3m. the inhibition of cox is a major, but not a single mechanism of potential nsaid effects. in the present study, the initial inhibition of leukocyte migration was found at nsaid concentrations beginning from 10- μm and the ic varied from to μm. this concentrations is 10-fold higher than the highest known ic required for cox inhibition for diclofenac and sc- whereas this difference for ketoprofen is 500-fold. therefore, we believe that the direct inhibition of leukocyte migration through nsaid cannot only represent a cox-dependent phenomenon, but other mechanisms may participate in the nsaid action on leukocyte migration. the data of the present study correspond well with results of previous studies. these studies demonstrated that nsaid have an inhibiting effect on chemotaxis of pmns at high concentrations in vitro which was independent of their potency as prostaglandin inhibitors. in contrast to these studies, we showed that not only chemotactic, but also haptokinetic migratory activity of single leukocytes can be blocked by nsaid. furthermore, almost all previous studies focused on the changes of migration of polymorphonuclear leukocytes, whereas the effect of nsaid on lymphocyte migration was poorly investigated. results of the present study demonstrated that nsaid have similar inhibiting effects on migration of both pmns and lymphocytes and that lymphocyte movement can be stopped by nsaid. other previous studies showed that low concentrations of nsaid inhibit cox production by activated mast cells and macrophages and reduce inflammation-induced leukocyte infiltration. although cox inhibition by low concentrations of nsaid can reduce leukocyte infiltration in inflamed tissue, this effect can be produced by rather different mechanism than the direct inhibition of leukocyte migration. we believe that cox-related effects of nsaid are not a major factor for the direct inhibition of leukocyte migration. furthermore, we propose that other possible mechanisms of nsaid action such as inhibition of intracellular atp, calcium metabolism or i-dependent myosin ii activation can be responsible for the inhibition of leukocyte migration. the results of the present study confirmed the important role of ca2+ signaling in nsaid action and showed that the stop of leukocyte migration by ketoprofen was accompanied by the dramatic decrease of i and by the cytotoxic effect whereas partial inhibition of leukocyte migration did not cause a significant reduction of i and did not induce cell toxicity. concentrations of nsaid, which are required for blockade of leukocyte migration, are high. as showed in previous studies, high nsaid concentration cause toxic side effects, if these substances are systemically applied. however, nsaid do not induce systemic toxic side effects after topic application as gel or salve formulation or after intraarticular injection. previous studies showed that ketoprofen has a high percutaneous penetration. the present study showed that the topic application of concentrated ketoprofen gel rapidly blocked migration of cutaneous leukocytes. we believe that the suppression of leukocyte migration can represent one of the pharmacological effects of nsaid after the topic application in clinical use. it is known that other anti-inflammatory drugs such as dexamethasone suppress leukocyte migration. it binds to its intracellular receptor and inhibits gene expression by binding to negative regulatory promoter regions or through protein/protein interactions. the effect of nsaid corresponds well with effect of dexamethasone. as shown in the present study, ketoprofen can block sdf- and il-8-mediated leukocyte migration. in accordance with our results, previous studies demonstrated that dexamethasone augmented intracellular chemokine signaling and inhibited sdf-1-induced chemotaxis of resting t cells. furthermore, dexamethasone also suppressed il-8-induced chemotaxis of pmn through inhibition of endogenous chemokine production. it can be proposed that inhibition of endogenous chemokine production by ketoprofen or other nsaid can also participate in the inhibition of leukocyte migration.  in summary, our results demonstrate that ketoprofen, diclofenac and sc- have a direct suppressive effect on haptotaxis and haptokinesis of polymorphonuclear leukocytes and lymphocytes. this effect is dose-dependent and partially reversible after removal of inhibiting agent. this process is probably induced by cox-dependent and cox-independent mechanisms whereas complete blockade, but not the partial inhibition of migration is accompanied by changes of ca2+signaling. these results indicate a possible discrete mechanism of nsaid action which may be identified in further experiments. the findings of the present study may have an important clinical implications since the blockade of leukocyte migration can also be expected after local application of nsaid in humans. authors' contributions sp: most experiments in vitro, manuscript preparation; ap and vk: design and data analysis of leukocyte migration experiments in vitro; rn: design, control and data aquisition of ca2+ measurement, js: data analysis, preparation of the manuscript; er: idea and concept, study design, haptotaxis experiments, intravital microscopy, manuscript preparation. all authors read and approved the final manuscript. supplementary material additional file 1 time-lapse intravital microscopy of leukocyte extravasation in mouse cutaneous tissue. cutaneous inflammation was induced by injection of il- neutrophils express egfp and appear in green. blood vessels are labelled using intravenous injection of tritc-labelled albumin. real-time recording of min was compressed to time-lapse sequence of sec. control experiment; il- induces an immediate extravasation and migration of leukocytes from the venule. click here for file additional file 2 time-lapse intravital microscopy of leukocyte extravasation in mouse cutaneous tissue. cutaneous inflammation was induced by injection of il- neutrophils express egfp and appear in green. blood vessels are labelled using intravenous injection of tritc-labelled albumin. real-time recording of min was compressed to time-lapse sequence of sec. application of ketoprofen gel prior injection of il- blocks intravascular leukocyte adhesion and extravasation. click here for file acknowledgements we thank dr. t. graf for providing of lysegfp-ki mice. we thank c. bernardi for an excellent technical assistance. we thank dr. w. gross for the help in "histo" software.
the results of the present study may have important clinical implications since blockade of leukocyte migration can be achieved after topical application of nsaid. the potential direct effect of nsaid on leukocyte migration was poorly investigated. nsaid induced an immediate inhibiting effect on leukocyte migration both in vitro and in vivo. the inhibition of leukocyte migration by nsaid was partially re-stored after removal of inhibiting agent. only complete blockade of leukocyte migration was accompanied by a strong reduction of i. nonsteroidal anti-inflammatory drugs represent a one of the most widely used anti-inflammatory substances. using time-lapse microscopy and 96-well fluorescence-based assay, we studied the effect of three different nsaid, ketoprofen, diclofenac and sc- on leukocyte haptokinesis and haptotaxis in vivo and in vitro.
 nonsteroidal anti-inflammatory drugs represent a one of the most widely used anti-inflammatory substances. their anti-inflammatory effects are mainly based on inhibition of cyclooxygenase. the potential direct effect of nsaid on leukocyte migration was poorly investigated. using time-lapse microscopy and 96-well fluorescence-based assay, we studied the effect of three different nsaid, ketoprofen, diclofenac and sc- on leukocyte haptokinesis and haptotaxis in vivo and in vitro.  nsaid induced an immediate inhibiting effect on leukocyte migration both in vitro and in vivo. this effect was dose-dependent and was not restricted to a specific type of leukocytes. the inhibition of leukocyte migration by nsaid was partially re-stored after removal of inhibiting agent. only complete blockade of leukocyte migration was accompanied by a strong reduction of i.  nsaid strongly supress leukocyte migration. the results of the present study may have important clinical implications since blockade of leukocyte migration can be achieved after topical application of nsaid. 