endothelial and epithelial monolayers and their associated basement membranes form physical and functional barriers separating distinct tissue compartments .
these barriers are frequently breached , e.g. , by immune cells as part of their homeostatic or inflammatory trafficking , by tumor cells during metastatic spread , or during tissue reorganizations in development .
first , often the migrating cell is guided toward the barrier by extracellular guidance cues such as chemokines .
second , adhesion of the migrating cell to the barrier is mediated by sequential cell - cell interactions , as exemplified in the leukocyte extravasation cascade ( reymond et  al .
third , transmigration across the barrier usually occurs via entry portals , which may either be developmentally regulated ( nourshargh et  al . , 2010 ,
seifert and lehmann , 2012 ) or induced by the migrating cell itself ( nourshargh et  al .
, 2010 , vestweber , 2015 ) . despite their apico - basal asymmetry , endothelia , and epithelia
are often penetrated in both directions : e.g. , neutrophils usually extravasate blood vessels from luminal to interstitial but can also transmigrate in the reversed direction ( intravasation ) ( woodfin et  al . ,
likewise , tumor cells intravasate blood vessels upon leaving the primary tumor and extravasate to colonize distant organs ( reymond et  al . , 2013 ) .
hence , rather than acting as pre - patterned gradients , which stereotypically guide the cells across the barrier , the directional cues governing transmigration might act locally and dynamically .
lymphatic endothelial cells ( lecs ) of the lymphatic capillaries form a barrier for intravasating leukocytes and tumor cells .
dendritic cells ( dcs ) enter these vessels on their way from the periphery to the lymph nodes , where dcs activate lymphocytes as part of the adaptive immune response ( frster et  al . , 2008 ) .
dcs are guided from the interstitium toward the lymphatic capillaries by gradients of the chemokine ccl21 ( weber et  al . , 2013 ) , which is homeostatically secreted by the lecs ( nakano and gunn , 2001 ) .
after arrival at the lymphatic capillary , dcs penetrate the basement membrane ( pflicke and sixt , 2009 ) and the lec junctions ( baluk et  al .
the molecular mechanisms directing dcs into the vascular lumen , which we investigate here , are largely unclear .
dcs are attracted to the vicinity of lymphatic capillaries via gradients of ccl21 ( weber et  al . , 2013 ) .
notably , ccl21 immunostainings in non - permeabilized tissues show a very faint interstitial signal compared to a strong signal localizing to lecs in permeabilized tissues ( johnson and jackson , 2010 , tal et  al . , 2011 , weber et  al . , 2013 ) .
these depots are observed as perinuclear puncta ( johnson and jackson , 2010 , weber et  al . ,
2013 ) , which co - stain for the golgi marker golph4 ( figure  1a ) and disperse upon golgi - endoplasmic reticulum ( er ) disrupting brefeldin a treatment ( weber et  al . , 2013 ) .
in addition to these trans - golgi depots , ccl21 locates to intracellular vesicles ( figures 1a and s1a ) ( johnson and jackson , 2010 ) , which might represent chemokine en route to homeostatic secretion or stores for  on demand  release . to test if intravasation of dcs changes ccl21 distribution , we layered in  vitro - generated dcs on mouse ear explants . in this setup ,
dcs enter the tissue within minutes and migrate along gradients of interstitial ccl21 to ultimately intravasate into the dermal lymphatic vessels ( pflicke and sixt , 2009 , weber et  al .
, 2013 , weber and sixt , 2013 ) ( figure  1b ) . in lymphatic capillaries , which had been entered by many dcs
, we found that perinuclear ccl21 depots were dispersed and overall diminished ( figures 1c and 1d ) .
this change in ccl21 distribution was not observed at sites where few dcs intravasated or in ears loaded with dcs deficient for the ccl21 receptor ccr7 ( figure  s1b ) .
ccr7 dcs can not read the interstitial ccl21 gradient and therefore do not interact with the lymphatic capillaries ( frster et  al .
, 2013).figure  1dendritic cell entry into mouse dermal lymphatic capillaries induces mobilization of ccl21(a ) mouse dermal lymphatic capillary stained for lyve1 ( green ) , golph4 ( red ) , ccl21 ( white ) , and nuclei ( dapi , blue ) .
zoom - in of boxed area is shown below.(b ) tamra - labeled dcs ( red ) and lyve1-stained lymphatic capillaries ( green ) after 3  hr 30 invasion .
yellow lines indicate the plane of orthogonal section , and yellow arrow highlights a transmigration event.(c ) lyve1 ( green ) , ccl21 ( white ) , and nuclei ( dapi , blue ) of the ear dermis after 3  hr 30 in presence or absence ( control ) of tamra - labeled dcs ( red ) .
white arrows indicate dispersion of ccl21 ( yellow arrow in control).(d ) dot blot graph shows ratio of signal in high intensity ccl21 depots to ccl21 in other areas of lecs .
columns represent mean values  sd of control ( n  = 3 ) and  + dc ( n  = 5 ) samples of 300  m long lymphatic vessel stretches.(e ) transmission electron micrograph of ccl21 staining of dermis .
black arrows indicate overlapping lec tips at cell - cell junctions , yellow arrow detachment of lecs from interstitial extracellular matrix , white arrow rearrangement of collagen bundles , and blue arrow a silver - amplified ccl21 immunogold label.(f ) quantification of extracellular ccl21 staining at sites of dc - associated tissue alterations ( see e ) compared to intact area in same sample or sample devoid of dcs .
bar graph shows mean  sd of two ( dc ) and four ( + dc ) independent ear samples .
scale bars , 10  m ( a ) ; 50  m ( b and c ) ; 10  m in zoom - in images ; and 500  nm ( e).see also figure  s1 .
dendritic cell entry into mouse dermal lymphatic capillaries induces mobilization of ccl21 ( a ) mouse dermal lymphatic capillary stained for lyve1 ( green ) , golph4 ( red ) , ccl21 ( white ) , and nuclei ( dapi , blue ) .
( b ) tamra - labeled dcs ( red ) and lyve1-stained lymphatic capillaries ( green ) after 3  hr 30 invasion .
yellow lines indicate the plane of orthogonal section , and yellow arrow highlights a transmigration event .
( c ) lyve1 ( green ) , ccl21 ( white ) , and nuclei ( dapi , blue ) of the ear dermis after 3  hr 30 in presence or absence ( control ) of tamra - labeled dcs ( red ) .
( d ) dot blot graph shows ratio of signal in high intensity ccl21 depots to ccl21 in other areas of lecs .
columns represent mean values  sd of control ( n  = 3 ) and  + dc ( n  = 5 ) samples of 300  m long lymphatic vessel stretches .
black arrows indicate overlapping lec tips at cell - cell junctions , yellow arrow detachment of lecs from interstitial extracellular matrix , white arrow rearrangement of collagen bundles , and blue arrow a silver - amplified ccl21 immunogold label .
( f ) quantification of extracellular ccl21 staining at sites of dc - associated tissue alterations ( see e ) compared to intact area in same sample or sample devoid of dcs .
bar graph shows mean  sd of two ( dc ) and four ( + dc ) independent ear samples .
scale bars , 10  m ( a ) ; 50  m ( b and c ) ; 10  m in zoom - in images ; and 500  nm ( e ) . to study
whether mobilization of ccl21 upon dc intravasation was associated with extracellular ccl21 enrichment , we employed immunometal transmission electron microscopy . in line with the immunofluorescence analysis ( figure  1a ) ,
intracellular ccl21 was found enriched in the nuclear periphery ( figure  s1c ) and , more sparsely , in intracellular vesicles of control samples ( figure  s1d ) .
extracellular ccl21 was mostly detected within 1  m distance of the basolateral side of lecs with no evidence of ccl21 gradients extending from the basolateral to the luminal side of the lecs ( figure  1e ) . in samples exposed to dcs ,
local presence of dcs was associated with poorly organized interstitial collagen and partial detachment of the lecs from the interstitium ( figures 1e , s1e , and s1f ) .
importantly , these signatures of dc intravasation were associated with extracellular  accumulations of basolateral , but not luminal , ccl21 ( figure  1e-1f ) .
these accumulations were most prominent at lec - lec junctions of detached endothelia ( figure  1e ) , whereas sheer proximity of dcs seemed insufficient to discharge chemokine ( figure  s1 g ) .
based on these findings , we hypothesize that contact between dcs and lecs triggers acute ccl21 secretion .
triggered exocytosis of chemokine is reminiscent of exocytotic targeting of the adhesion molecule pecam to areas of lymphocyte transmigration at the blood endothelium ( mamdouh et  al .
, 2003 ) . it might provide local guidance or act as a landmark for following dcs , which have been shown to intravasate with increased efficiency ( martin - fontecha et  al . , 2003 ) .
to directly study ccl21 secretion , we established a traceable  in  vitro setup , where we lentivirally expressed variants of ccl21 in human dermal lecs , which recapitulated localization to the golgi secretory pathway ( compare figures 2a and s1a ) ( de jong et  al . , 2005 , de jong et  al . , 2008 ) .
secretion of ccl21 into the cell culture supernatant was dependent on the n - terminal signaling peptide ( figures 2a and 2b ) .
surprisingly , exclusion of the ccl21n - mcherry from the secretory pathway led to nuclear localization , possibly owing to a potential bipartite nuclear localizing signal in mature ccl21 ( aa  2150 ) .
the loss of the heavily cationic c terminus ( ccl21c ) did not affect localization to the golgi secretory pathway ( figure  2a ) but instead prevented binding of ccl21 to the cell culture dish surface ( figure  s2a ) and lead to increased ccl21 in the cell culture supernatant ( figure  2b ) .
this was in line with previous findings , showing that the c terminus immobilizes ccl21 to charged surfaces and glycosaminoglycans ( hirose et  al .
, 2002).figure  2ccl21-positive vesicles localize to golgi - secretory pathway and traffic along microtubules(a ) giantin ( white ) and nuclear staining ( dapi , blue ) of ccl21-mcherry ( red ) , ccl21n - mcherry , or ccl21c - mcherry expressing lecs .
insets show golgi localized mcherry signal ( yellow arrows ) with or without staining of golgi marker giantin.(b ) quantification of mcherry intensities in supernatants of ccl21-mcherry , ccl21n - mcherry , and ccl21c - mcherry expressing lecs .
dot blot graph shows a mean  sd of pooled data from two independent experiments , n  = 4 for each construct .
intensities are normalized to media background , which is set as 1.(c ) basolateral non - directed tracks of tirf - imaged ccl21-mcherry - positive vesicles are indicated with white , directed tracks heading toward golgi area with green , directed tracks toward cell periphery with red , the golgi apparatus with a dashed yellow line , and the boundaries of cell contact surface with dashed white line .
yellow arrow indicates a white trajectory representing random movement within a confined area.(d ) tirf imaging of ccl21c - mcherry ( red ) and egfp - tubulin- ( green ) expressing primary lec .
white arrow highlights one ccl21c - mcherry positive vesicle ( red ) throughout the image series .
scale bars , 10  m ( a ) ; 2  m ( c and d).see also figure  s2 .
ccl21-positive vesicles localize to golgi - secretory pathway and traffic along microtubules ( a ) giantin ( white ) and nuclear staining ( dapi , blue ) of ccl21-mcherry ( red ) , ccl21n - mcherry , or ccl21c - mcherry expressing lecs .
insets show golgi localized mcherry signal ( yellow arrows ) with or without staining of golgi marker giantin .
( b ) quantification of mcherry intensities in supernatants of ccl21-mcherry , ccl21n - mcherry , and ccl21c - mcherry expressing lecs .
dot blot graph shows a mean  sd of pooled data from two independent experiments , n  = 4 for each construct .
( c ) basolateral non - directed tracks of tirf - imaged ccl21-mcherry - positive vesicles are indicated with white , directed tracks heading toward golgi area with green , directed tracks toward cell periphery with red , the golgi apparatus with a dashed yellow line , and the boundaries of cell contact surface with dashed white line .
( d ) tirf imaging of ccl21c - mcherry ( red ) and egfp - tubulin- ( green ) expressing primary lec .
white arrow highlights one ccl21c - mcherry positive vesicle ( red ) throughout the image series .
scale bars , 10  m ( a ) ; 2  m ( c and d ) . to trace the intracellular trafficking routes of ccl21 , we  performed real - time total internal reflection fluorescence ( tirf ) microscopy .
tracking of ccl21-mcherry - positive vesicles showed two modes of behavior : some vesicles rapidly advanced along linear trajectories , whereas others moved randomly within confined areas ( figure  2c ) .
linear tracks connected golgi area and the lec periphery ( figure  2c ) , matching the distribution pattern of microtubules ( figure  s2b ) .
accordingly , ccl21 was microtubule - associated as revealed by immunostaining of untagged ccl21 and ccl21c ( figure  s2b ) and live imaging of ccl21c - mcherry ( figure  2d ; movie s1 ) .
microtubule disruption by nocodazole abolished the component of directed movement ( movie s1 ) confirming that ccl21 is transported along microtubules , which is analogous to the transport of insulin in secretory pancreatic beta - cells ( boyd et  al .
similarly to insulin , some of the linear trajectories of ccl21 vesicles were directed toward the cell periphery and some toward the golgi area , suggesting regulation of vesicle dwell time at the lec periphery ( zhu et  al . , 2015 ) .
tirf imaging revealed that occasionally ccl21-mcherry vesicles were discharged into the extracellular space . regulated discharge of plasma membrane - docked vesicles is often initiated by calcium ( ca ) binding to synaptotagmins , which triggers the actual membrane fusion event ( sdhof , 2012 ) .
accordingly , the ca ionophore ionomycin triggered robust ccl21-mcherry and ccl21c - mcherry secretion as directly observed with  tirf microscopy as a sudden and transient increase in ccl21 intensity ( figures 3a and 3d ; movie s2 ) .
such flashes are due to the exponential increase in tirf signal once the quantum of fluorescent probe enters the glass - water interface below the cell ( axelrod , 1981 ) , which is then followed by rapid dilution of the radially diffusing probe .
bulk mcherry signal in the lec culture supernatant was increased by ionomycin ( figure  3b ) .
further , basal secretion seemed at least partially dependent on ca as chelation of intraendothelial ca with bapta - am causing a 30% reduction in ccl21c - mcherry secretion ( figure  3c).figure  3regulation of ccl21 secretion(a ) tirf microscopy of ccl21-mcherry vesicles ( white ) of a single lec after addition of 5  m ionomycin .
yellow arrows indicate vesicles at time point 0 , secretion events are highlighted by white arrows .
see movie s2.(b ) quantification of mcherry intensities of ccl21c - mcherry expressing lec culture supernatant subsequent to 6 control or 5  m ionomycin treatment .
bar graph shows mean  sd of pooled samples from 3 independent experiments , n  = 5 ( control ) and 6 ( ionomycin).(c ) quantification of mcherry intensities of culture supernatants of ccl21c - mcherry expressing lecs 4  hr after washout of 1  hr long control or 10  m bapta - am treatment .
bapta - am is a cell - permeant ca chelator , which is trapped in cells and  binds intracellular calcium via carboxylic acid functional groups thus severely decreasing intracellular - free calcium .
bar graph shows mean  sd of pooled samples from three independent experiments , n  = 7 for each condition
. intensities of ( b )  and ( c ) are normalized to media background , which is set as 1.(d ) quantification of pooled data from all tirf experiments on single vesicle secretion .
data points show secretion frequency in control , 5  m ionomycin- , or 500  ng / ml latrunculin b - treated ccl21-mcherry or ccl21c - mcherry expressing single lecs as indicated in the figure .
the dot blot graph shows mean  sd secretion frequency in pooled data of single cells from 6 independent experiments , cell number ( n ) is indicated in the figure.(e ) data points show ccl21-mcherry vesicle speed , displacement , and duration in the tirf imaging plane ( basolateral membrane ) in control and 13 latrunculin b ( 500  ng / ml)-treated single lecs , which displayed highest ccl21 secretion ( see figure  3d latrunculin b column ) .
the dot blot graph shows mean  sd of vesicle tracks ( 51513/cell ) in pooled data of single cells ( n  = 12 for control and 13 for latrunculin b ) of three independent experiments .
regulation of ccl21 secretion ( a ) tirf microscopy of ccl21-mcherry vesicles ( white ) of a single lec after addition of 5  m ionomycin .
yellow arrows indicate vesicles at time point 0 , secretion events are highlighted by white arrows .
( b ) quantification of mcherry intensities of ccl21c - mcherry expressing lec culture supernatant subsequent to 6 control or 5  m ionomycin treatment .
bar graph shows mean  sd of pooled samples from 3 independent experiments , n  = 5 ( control ) and 6 ( ionomycin ) .
( c ) quantification of mcherry intensities of culture supernatants of ccl21c - mcherry expressing lecs 4  hr after washout of 1  hr long control or 10  m bapta - am treatment .
bapta - am is a cell - permeant ca chelator , which is trapped in cells and  binds intracellular calcium via carboxylic acid functional groups thus severely decreasing intracellular - free calcium .
bar graph shows mean  sd of pooled samples from three independent experiments , n  = 7 for each condition
. intensities of ( b )  and ( c ) are normalized to media background , which is set as 1 .
( d ) quantification of pooled data from all tirf experiments on single vesicle secretion .
data points show secretion frequency in control , 5  m ionomycin- , or 500  ng / ml latrunculin b - treated ccl21-mcherry or ccl21c - mcherry expressing single lecs as indicated in the figure .
the dot blot graph shows mean  sd secretion frequency in pooled data of single cells from 6 independent experiments , cell number ( n ) is indicated in the figure .
( e ) data points show ccl21-mcherry vesicle speed , displacement , and duration in the tirf imaging plane ( basolateral membrane ) in control and 13 latrunculin b ( 500  ng / ml)-treated single lecs , which displayed highest ccl21 secretion ( see figure  3d latrunculin b column ) .
the dot blot graph shows mean  sd of vesicle tracks ( 51513/cell ) in pooled data of single cells ( n  = 12 for control and 13 for latrunculin b ) of three independent experiments . see movie s3 .
ionomycin did not trigger secretion of all plasma membrane - associated vesicles ( movie s2 ) .
some of these vesicles might not be plasma membrane - docked , but rather sequestered within the cortical actin meshwork , which has to be penetrated before the vesicles can directly contact the membrane ( nakata and hirokawa , 1992 , oheim and sthmer , 2000 , giner et  al . , 2007 ) . when we visualized the cortical actin meshwork  of lecs with lifeact - egfp , lateral movements similar to that of ccl21-mcherry vesicles were apparent ( movie s3 ) .
actin disassembly by latrunculin b substantially increased the frequency of secretion events ( figure  3d ; movie s3 ) , likely  because of enhanced contact between vesicles and secretory machinery .
accordingly , the lecs displaying latrunculin b - induced secretion showed increased movement of vesicles in the basolateral plane as well as in z - direction ( revealed by disappearance of vesicles from the tirf field ) ( figure  3e ; movie s3 ) .
these results suggest that the cortical actin meshwork controls ccl21 secretion by restricting membrane - docking of vesicles .
the finding that ca influx triggers ccl21 secretion in lecs ( figures 3a , 3b , and 3d ; movie s2 ) together with our in  situ finding that dcs cause release of ccl21 ( figures 1e and 1f ) suggested that dc - lec interactions might induce ca signals in lecs similar to ca signals in blood endothelia when being transmigrated by polymorphonuclear leukocytes ( huang et  al . ,
while ca signaling can be induced in several ways , we were struck by our in  situ observation of dcs probing lecs with a blunt foot - like processes ( figures 4a and 4b ) , which were reminiscent of lymphocyte processes physically penetrating blood endothelium ( carman et  al . , 2007 , shulman et  al . , 2011 ) .
dc protrusions considerably deformed lecs and even detached them locally from the underlying extracellular matrix ( figures 4a , 4b , s1e , and s1f ) .
this suggested that dcs exert pushing forces on lecs , which might promote communication via extended cell - cell contacts . to investigate whether direct interaction was required for ca signals , lecs were grown to confluency , and dcs
setting , dcs effectively transmigrated the monolayer ( maddaluno et  al . , 2009 ) as revealed by phase contrast live cell imaging ( figure  4c ; movie s4 ) .
transmigration was dependent on the secretion of ccl21 , as non - infected  or ccl21n - mcherry - infected lecs were only occasionally penetrated by dcs ( figure  s2d ; movie s4 ) .
importantly , dcs interacting with the apical side of lecs and dcs in transit through the monolayer induced an increase in ca concentration in lecs as revealed by time - lapse imaging of a ca sensor ( figure  4d ; movie s5 ) .
at the population level , lecs showed a low basal frequency of ca transients , which was increased 4.5-fold upon addition of dcs ( figure  4e ) . unlike dcs
, dc - conditioned media did not induce an increase in ca concentration ( figure  4e ) excluding a major role for secreted factors .
further , pre - treatment of dcs with mycalolide b , an irreversible actin depolymerizing agent ( hori et  al . , 1993 , saito et  al . , 1994 ) , which prevents formation of actin - based cell protrusions , did not abolish ca transients ( figure  4e ) .
these results suggest that molecular engagements at the dc - lec interface are sufficient triggers .
earlier , dc expressed 2 integrins ( lfa-1 ) and its lymphatic endothelial ligand icam have been shown to be necessary for  in  vivo and in  vitro transmigration of inflamed but not homeostatic endothelia , where icam is not expressed ( johnson et  al .
, 2006 , johnson and jackson , 2010 , vigl et  al . , 2011 ,
accordingly , 2 integrins were dispensable for the dc entry into the dermal lymphatic capillaries as shown before ( figure  s2e ) ( lmmermann et  al . , 2008 ) . at sites of intravasated 2 deficient ( itgb2 ) dcs
, ccl21 depots were dispersed from golgi similar to wild - type dc samples ( figures s2e and s2f ) .
thus , other molecular interactions at the dc - lec interface , such as the plexin1a - semaphorin3a axis ( takamatsu et  al . , 2010 ) , l1 ( maddaluno et  al . , 2009 ) , or yet unidentified factors might trigger the signal.figure  4endothelial ca signaling facilitates dc transmigration in  vitro(a ) transmission electron micrograph at a site of dc - lec interaction at ear dermis lymphatic capillary .
interstitium ( i ) , lumen ( l ) , lec , and dc are marked.(b ) immunoelectron micrograph of whole mount preparation labeled for ccl21 ( silver amplified gold particles ) at a site of dc - lec interaction in ear dermis .
the yellow arrows in ( a ) and ( b ) indicate the endothelial detachment from interstitium.(c ) phase contrast imaging of an in  vitro transmigration event from the apical to basolateral side of the lec monolayer .
white arrow points at leading edge , yellow arrow at cell body , and blue arrow at trailing edge of transmigrating dc .
see movie s4.(d ) epifluorescence imaging of oregon green bapta - am - treated lecs ( green ) and tamra - labeled dcs ( red ) .
blue arrow indicates dc leading edge and yellow arrow lec displaying a transient ca peak upon dc contact . below the image
, the kymograph and the corresponding line graph of the oregon green bapta - am intensity in the lec ( highlighted with yellow arrow ) are shown .
see movie s5.(e ) quantification of lecs displaying ca peaks upon addition of fresh media , dcs , dc - conditioned media , or mycalolide b - treated dcs .
fresh media and dc samples represent eight independent experiments ( n  = 18 and 19 , respectively ) , of which two experiments included mycalolide b - treated dcs ( n  = 6 ) , and three experiments included conditioned media samples ( n  = 4 ) .
data are normalized to average of dcs sample , which is set as 1.(f ) quantification of dc transmigration on control or 10  m bapta - am ( b - am)-treated ccl21-mcherry or ccl21c - mcherry expressing monolayers .
dot blot graph shows mean  sd of pooled samples from three independent experiments , n  = 7 for bapta - am - treated ccl21c - mcherry expressing lecs and eight for all other conditions .
scale bars , 500  nm ( a and b ) ; 50  m ( c ) ; and 10  m ( d).see also figure  s2 .
endothelial ca signaling facilitates dc transmigration in  vitro ( a ) transmission electron micrograph at a site of dc - lec interaction at ear dermis lymphatic capillary .
interstitium ( i ) , lumen ( l ) , lec , and dc are marked .
( b ) immunoelectron micrograph of whole mount preparation labeled for ccl21 ( silver amplified gold particles ) at a site of dc - lec interaction in ear dermis .
the yellow arrows in ( a ) and ( b ) indicate the endothelial detachment from interstitium .
( c ) phase contrast imaging of an in  vitro transmigration event from the apical to basolateral side of the lec monolayer .
white arrow points at leading edge , yellow arrow at cell body , and blue arrow at trailing edge of transmigrating dc .
( d ) epifluorescence imaging of oregon green bapta - am - treated lecs ( green ) and tamra - labeled dcs ( red ) .
blue arrow indicates dc leading edge and yellow arrow lec displaying a transient ca peak upon dc contact . below the image
, the kymograph and the corresponding line graph of the oregon green bapta - am intensity in the lec ( highlighted with yellow arrow ) are shown .
( e ) quantification of lecs displaying ca peaks upon addition of fresh media , dcs , dc - conditioned media , or mycalolide b - treated dcs .
fresh media and dc samples represent eight independent experiments ( n  = 18 and 19 , respectively ) , of which two experiments included mycalolide b - treated dcs ( n  = 6 ) , and three experiments included conditioned media samples ( n  = 4 ) .
( f ) quantification of dc transmigration on control or 10  m bapta - am ( b - am)-treated ccl21-mcherry or ccl21c - mcherry expressing monolayers .
dot blot graph shows mean  sd of pooled samples from three independent experiments , n  = 7 for bapta - am - treated ccl21c - mcherry expressing lecs and eight for all other conditions .
scale bars , 500  nm ( a and b ) ; 50  m ( c ) ; and 10  m ( d ) .
to directly investigate whether lymphatic endothelial ca signaling was necessary for the transendothelial migration of dcs , we selectively chelated endothelial ca with bapta - am .
we compared the responses to both ccl21-mcherry and ccl21c - mcherry , because full - length ccl21-mcherry accumulates on the cell culture dish surface ( figure  s2a ) , which is not seen in  vivo ( figure  1e ) .
thus , we presumed that the truncated chemokine more faithfully mimics the in  vivo situation .
both ccl21-mcherry and ccl21c - mcherry supported transmigration but ca chelation reduced the number of transmigration events only in ccl21c - mcherry expressing monolayers ( 62.4% reduction , figure  4f ; movie s6 ) , which lack the unphysiological accumulation of ccl21 ( see figure  s2a ) and thus lack a prepatterned ccl21 gradient across the monolayer ( similarly to the tissue context , figure  1e ) .
these results suggest that dcs induce a transient ca signal in lecs , which triggers acute ccl21 secretion and possibly enables opening of the endothelial junctions , as described for blood endothelia ( huang et  al . , 1993 ) . although the decisive role of chemokines in leukocyte extravasation is well established , it has remained unclear if and how chemokines act during the actual transmigration process .
our finding that ccl21 is acutely secreted upon contact between dcs and lecs extend data by shulman et  al .
( 2011 ) , which suggested that the chemokine ccl2 is released by blood endothelia in response to interactions with extravasating lymphocytes .
while we show that a direct dc - lec contact is sufficient to trigger chemokine release even in the absence of cytoskeletal force generation ( figure  4e ) , we found that applying mechanical pushing forces on lecs , and thus increasing lec plasma membrane tension ( apodaca , 2002 ) , can also trigger ca flux and ccl21 release in  vitro ( figures s2g  s2i ) .
this was in line with the observation that transmigrating dcs considerably deformed lecs in dermal tissue ( figures 4a and 4b ) and might suggest that molecular and mechanical triggers perpetuate the intravasation process in concert .
earlier , long range ccl21 gradients were shown to guide dcs to the vicinity of lymphatic vessels ( weber et  al . , 2013 ) .
however , lec penetration could not be explained by a simple concentration gradient across the endothelium as intra - luminal ccl21 concentrations are lower than those on the abluminal basement membrane ( weber et  al . , 2013 , russo et  al . ,
the acute chemokine release that we demonstrate exposes the leading edge of dcs to a burst of very steep ccl21 gradient , which will likely  motivate  the cell to locally push into the endothelium . at sites of loose button - like lec - lec junctions ( baluk et  al . , 2007 ) , physical pushing might open the junction and allow entry from the confined interstitial environment into the lymphatic capillary lumen where physical resistance is lower ( figure  4 g ) .
in addition , local chemokine release might boost transmigration by attracting more transmigrating cells toward the specific entry portal , or provide local guidance and thereby determine the actual site of penetration . taken together ,
our data show that the barrier cells take a more active role in their penetration than previously anticipated .
wild - type c57bl/6j ( charles river laboratories ) , ccr7 ( frster et  al . , 1999 ) and itgb2 ( wilson et  al . , 1993 ) male and female mice were bred and maintained according to the local rules ( institutional review board approval 66018/3-ii/3b/2010 ) .
mice were sacrificed at the age of 68  weeks for the ear preparation and at the age of 812  weeks for the bone marrow extraction .
ears of sacrificed mice were prepared as previously described ( weber et  al . , 2013 ) .
ear sheets were fixed with 4% paraformaldehyde ( pfa ) in pbs at room temperature for 25 or prepared for the dc lymphatic capillary intravasation assay as follows : the ventral half of the ear was mounted between a 0.5  ml microcentrifuge tube lid and the body of the tube , of which the lower part was cut off .
the 1.5  cm well formed by the cut tube was filled with 200  l of r10 with or without 50,000 ( figures 1b1f ) or 100,000 ( figures s2e and s2f ) tamra - labeled dcs .
unattached dcs were washed off with r10 after 30 incubation at  + 37c and 5% co2 and incubated in r10 for additional 13  hr ( figures 1b1f ) . as itgb2 dcs are slower in invading ear explants , itgb2 dcs  and wild - type controls were kept on the ear explants for 7  hr ( i.e. , for the whole duration of the experiment ) ( figures s2e and s2f ) .
the experiments were terminated by washing the ear once with pbs followed by fixation with 4% pfa in pbs at room temperature for 25. for the quantification of extra - lec ccl21 , we used two independent samples devoid of dcs ( control ) and four independent samples with dcs ( + dcs ) .
all extracellular ccl21-immunometal particles within 1  m distance to the interstitial ( basolateral ) side of the lymphatic vessel endothelial cell were quantified .
the particles were classified based on their location either in the intact area or in the area displaying dc - associated tissue alterations ( i.e. , disorganized collagen bundles and/or local detachment of lecs from the interstitial extracellular matrix ) . finally , the particle number was normalized to the corresponding endothelial length .
the bulk secretion of ccl21 was measured by analyzing mcherry intensities in  the culture supernatant . to measure the amounts of ccl21-mcherry , ccl21n - mcherry , and ccl21c - mcherry ( figure  2b ) ,
culture supernatant was collected 48  hr after full confluency of the lec culture and the last media change .
for the experiments shown in figure  3 , we used ccl21c - mcherry because it has low affinity to culture substrate ( figure  s2a ) , and the changes in secretion should be immediately reflected in the mcherry intensity in the supernatant . here
, culture supernatant was collected subsequent to 6 5  m ionomycin ( molecular probes ) , 4  hr after washout of 1  hr 10  m bapta - am ( life technologies , b6769 ) treatment or corresponding control ( dmso ) treatments .
results were normalized to the background fluorescence of the mv2 culture media . for the information on measurement and quantification ( figure  2b ) ,
ionomycin ( final concentration 5  m , molecular probes ) was added to the  ccl21-mcherry expressing lecs and imaging was started 10  s later  whereas latrunculin b ( 500  ng / ml , merck millipore 428020 ) and nocodazole ( 500  nm , sigma , m1404 ) were added on the lecs 30 prior to imaging .
averages of all the tracks / cell , which lasted for longer than 2.5 s , are shown in figure  3e .
only events in which the vesicles were observed prior to the  flash  and vesicle - like signal was severely decreased or totally absent after the flash were quantified as a secretion event ( figure  3d ) .
activated dcs were centrifuged ( 300rcf , 5 ) and resuspended in mv2 ( promocell ) culture media .
a total of 40,000 dcs ( ccl21c - mcherry monolayers ; figures 4f and s2d ; movie s4 ) were added on 1.5  cm well of days 45 confluent lecs . due to more efficient transmigration of dcs  on ccl21-mcherry monolayers in comparison to ccl21c - mcherry monolayers ,
the acquired movies were quantified for the number  of dcs beneath the monolayer at 1  hr for ccl21-mcherry or at 3  hr for ccl21c - mcherry expressing lec monolayers ( figure  4f ; movie s6 ) .
for quantification of transmigration efficiency on non - infected or ccl21-mcherry full - length or mutant construct expressing lec monolayers ( figure  s2d ) , a percentage of transmigrated dc of all dcs was determined . for the ca - sensor assays , fresh media , dc - conditioned media , or either tamra - labeled non - treated or mycalolide b - treated dcs were carefully added on lec monolayers treated for 50 with 10  m oregon green bapta - am .
the time - lapse imaging was started after 10. for the cell population analysis ( figure  4e ) , imaging lasted for 10 and the number of lecs displaying transient ca peaks was quantified from acquired videos .
all the ca sensor experiments were carried out in the absence of serum or added growth factors .
prism5 and prism6 software ( graphpad softwares ) was used to test the normality of the data ( dagostino and pearson omnibus normality test or kolmogorov - smirnov normality test ) and for subsequent parametrical t test ( two - tailed with welch s correction ) or non - parametrical mann - whitney tests .
figure  legends indicate number of independent experiments and biological replicates ( n ) used for statistical analysis . generation and labeling of dcs , ear sheet staining , preparation of transmission electron microscopy samples , construction of lentiviral expression plasmids , virus production , lec culture and infections ,
lec staining , microscopy setups , mechanical pushing of the lecs , image analysis , and identification of putative nuclear localizing signal in ccl21 are outlined in the supplemental experimental procedures .
 summarytrafficking cells frequently transmigrate through epithelial and endothelial monolayers . how monolayers cooperate with the penetrating cells to support their transit 
 is poorly understood . 
 we studied dendritic cell ( dc ) entry into lymphatic capillaries as a model system for transendothelial migration . 
 we find that the chemokine ccl21 , which is the decisive guidance cue for intravasation , mainly localizes in the trans - golgi network and intracellular vesicles of lymphatic endothelial cells . upon dc transmigration , 
 these golgi deposits disperse and ccl21 becomes extracellularly enriched at the sites of endothelial cell - cell junctions . 
 when we reconstitute the transmigration process in  vitro , we find that secretion of  ccl21-positive vesicles is triggered by a dc contact - induced calcium signal , and selective calcium chelation in lymphatic endothelium attenuates transmigration . 
 altogether , our data demonstrate a chemokine - mediated feedback between dcs and lymphatic endothelium , which facilitates transendothelial migration . 
Introduction
Results and Discussion
Experimental Procedures
Author Contributions