studies on asthmatic patients and animal models of allergy have demonstrated that th2 cells produce cytokines il-4 , il-5 , and il-13 and thus contribute to development of asthma characterized by airway inflammation , mucus hypersecretion , and airway hyperresponsiveness .
however , the mechanisms through which nave t cells differentiate to th2 cells in response to allergens remain unclear .
dcs are a group of cells which possess the ability in dictating nave cd4 + t cells differentiation to either th1 or th2 cells depending on ambient microenvironment [ 1 , 2 ] .
it is still an enigma how antigen - specific th2 cells get skewed polarization and how they maintain a dominant status in allergy .
recent studies have showed that dcs express t cell immunoglobulin mucin domain ( tim)4 that ligates tim1 on th2 cells to promote th2 cells development .
tim is a new family of cell surface proteins that are potentially involved in the regulation of effector t cell responses .
accumulated data suggest that several tim molecules play critical roles in the regulation of th1 and th2 immune responses . among them
, dc - derived tim4 has been found to be able to drive cd4 + t cells into th2 cells [ 4 , 5 ] .
it has also been observed that the expression of tim4 on dcs could be up - regulated dramatically upon activation [ 6 , 7 ] .
however , little is known about the actions of allergens in tim4 upregulation on dcs .
cockroaches have been identified to induce allergy in different regions of the world [ 811 ] .
the most common cockroach species , which are frequently found in homes , are b. germanica and periplaneta americana ( p. americana ) [ 12 , 13 ] .
over the past decade , seven allergens of b. germanica ( bla g 1 , bla g 2 , bla g 4 , bla g 5 , bla g 6 , bla g 7 , and bla g 8) have been cloned [ 1417 ] . among them , bla g 7 is a tropomyosin and has a highly cross - reactive pan - allergen to serum ige . since the structure of bla g 7 is very similar to per a 7 ( a tropomyosin from p. americana ) , which has been previously reported to be able to activate the secretion of th2 cytokine , il-4 , and il-13 , from p815 mast cell line [ 18 , 19 ] , we anticipated that bla g 7 may also stimulate th2 cytokine release from other immune cells .
as dcs are the most powerful antigen presenting cells , which could induce th2 polarization and intestinal allergy , the initiation factors through which dcs trigger cd4 + t cell differentiation remain unknown .
therefore , in this study , we hypothesized that bla g 7 might affect dcs by promoting tim4 expression in dcs and evoke cd4 + t cells to differentiate into th2 cells subsequently .
exscript rt reagent kit and sybr premix ex taq ( perfect real time ) were obtained from takara ( dalian , china ) .
pe - conjugated mouse antihuman il-4 , cd80 , cd86 , and fitc - conjugated mouse antihuman ifn- antibodies were obtained from ebioscience ( los angeles , ca , usa ) .
antihuman i-b , phospho - i-b , nf-b , and phospho - nf-b antibodies were purchased from cell signaling technology inc .
anti - tim4 antibody was purchased from r&d systems ( san diego , usa ) .
gm - csf , and il-4 was purchased from peprotech ( rocky hill , usa ) .
rbla g 7 and its monoclonal antibody were prepared in our laboratory as shown previously .
most of other reagents such as salt and buffer components were of analytical grade and obtained from sigma - aldrich ( st .
peripheral blood mononuclear cells ( pbmcs ) from 6 healthy volunteers were isolated by ficoll centrifugation .
cd14 + monocytes were then purified by cd14 + magnetic microbeads ( miltenyi biotec , germany ) according to the manufacturer 's instruction .
they were cultured in rpmi-1640 supplemented with 10% fetal bovine serum , antibiotics , gm - csf ( 10  ng / ml ) , and il-4 ( 5  ng / ml ) . the further addition of the cytokines was carried out every other day .
after 5 days of culture , immature dcs were harvested for use .   for challenge experiments ,
cells were exposed to various concentrations ( 101000  ng / ml ) of rbla g 7 with or without anti - tim4 antibody ( 10  ng / ml ) , or 100  ng / ml of lps ( as positive control ) . at 48  h following incubation ,
the culture plates were centrifuged at 450  g for 10  min at 25c . after the supernatants being collected and stored at 80c ,
the cell pellet containing approximately 5  10 matured dcs was collected for western - blot , flow cytometry , and real - time pcr analyses .
some dcs were pulsed with 100  ng / ml lps and 1  g / ml rbla g 7 for 24  h , washed , and then cultured with cd4 + t cells for 2 days . in order to investigate the influence of cd80 and cd86 molecules on rbla g
7-activated dcs induced polarization of cd4 + t cells , rbla g 7-activated dcs were preincubated with cd80 and cd86 blocking antibodies respectively , for 30  min at 37c before being cocultured with cd4 + t cells .
cd4 + t cells were purified from the pbmc ( donated by 6 healthy volunteers ) with cd4 + magnetic microbeads according to the manufacturer 's instruction .
isolated cd4 + t cells were then mixed with rbla g 7-activated dcs at a ratio of 10  :  1 with or without tim4 blocking antibody ( 10  ng / ml ) .
the cocultured two types of cells were maintained in rpmi-1640 culture medium supplemented with 10% fbs and antibiotics for 48  h at 37c .
following centrifugation , the supernatant was collected , and levels of ifn- and il-13 in the supernatant were measured by elisa .
the cell pellet was resuspended , and intracellular expression levels of ifn- and il-4 were measured by flow cytometry analysis . after being challenged with 10 , 100 and 1000  ng / ml rbla g 7 or medium alone for 48  h and challenged with 1000  ng / ml rbla g 7 ( or medium alone ) for 20  min , 40  min , and 60  min , the purified dcs were lysed as previously described .
densitometry analysis of immunoblots was carried out by using quantity one software ( bio - rad , usa ) .
the relative level of phosphor - i-b and nf-b was expressed as the ratio to i-b and nf-b , and tim4 was expressed as the ratio to gapdh , an internal control .
the cdna was prepared by exscript rt reagent kit according to the manufacturer 's instruction and then was amplified by pcr with specific primers .
briefly , real - time pcr was performed by using sybr premix ex taq on a sequence detection system ( eppendorf , germany ) .
the thermal cycling conditions included an initial denaturation step at 95c for 30  s , followed by 40 cycles of 5  s at 95c and 30  s at 60c .
primer sequences for human tim4 , il-12 , and il-13 and gapdh are summarized in table 1 .
dcs were pelleted by centrifugation at 450  g for 5  min and then fixed and permeabilized by using a cell fixation / permeabilization kit ( bd pharmingen ) . briefly
, thoroughly resuspended cells were added in 100  l of bd cytofix / cytoperm solution and incubated for 30  min at 4c .
dcs were then incubated with pe - conjugated cd80 , and cd86 and cd4 + t cells were incubated with fitc - conjugated ifn- and pe - conjugated il-4 monoclonal antibody or isotope control , respectively ( at a final concentration 4  g / ml ) , at 4c for 30  min .
after washing , cells were analyzed on a fluorescence - activated cell sorting ( facs ) arial flow cytometer with flowjo software ( treestar , san carlos , ca , usa ) .
levels of il-13 and il-12p70 ( excell china ) in the culture supernatant of dcs or il-13 and ifn- in the culture supernatant of t cells were measured by elisa kits according to manufacturer 's instructions .
data are expressed as mean  sem for the indicated number of independently performed duplicated experiments . statistical significance between means
previous reports indicated that dcs express tim4 that plays a critical role in inducing peripheral th2 polarization [ 4 , 21 ] .
however , the factors in upregulating tim4 expression in dcs remain unknown . as shown in western blot , the expression of tim4 in dcs
was significantly increased in response to rbla g 7 in a dose - dependent manner ( figures 1(a ) and 1(b ) ) .
similarly , tim4 mrna expression in human dcs was enhanced by up to approximately 28.2-fold assessed by realtime pcr analysis when the cells were cultured with various concentrations of rbla g 7 ( figure 1(c ) ) . once being added at the same time with rbla g 7 , anti - bla g 7 antibody was able to diminish rbla g 7 induced tim4 protein and mrna expression though it itself induced modest tim4 expression in dcs ( figure 1 ) .
cd80 and cd86 mediate the necessary costimulatory signals to t cells , which results in th1/th2 cell polarization .
it has also been reported that enhanced expression of cd80 and cd86 was observed on human monocyte - derived dendritic cell 's ( modcs ) during maturation , and we therefore investigated expression of cd80 and cd86 on dcs in the present study .
rbla g 7 induced approximately 2.4- and-2.2 fold upregulated expression of cd80 and cd86 on immature dcs , indicating the maturation of these immature dcs .
the phenotypic changes induced by rbla g 7 were comparable to those elicited by lps ( 100  ng / ml ) , suggesting that rbla g 7 is a potent stimulus of dc maturation and activation . once being added at the same time with rbla g 7 , anti - bla g 7 antibody was able to diminish rbla g 7 induced cd80 and cd86 expression ( figure 2 ) .  
il-13 is a well - known th2 cytokine , which contribute 's greatly to the development of allergic inflammation .
it has been shown that il-13 is not only secreted from th2 cells , but also released from basophils   and epithelial cells .
however , little is known about the influence of allergen on il-13 secretion from dcs . in the present study ,
rbla g 7 induced a dose - dependent secretion of il-13 from dcs , which was partially inhibited by tim4 blocking antibody ( figure 3(c ) ) , and cd80 and cd86 blocking antibodies ( figure 3(e ) ) .
rbla g 7 also up - regulated the expression of il-13 mrna in dcs ( figure 3(d ) ) . in the parallel experiments , rbla g 7 at the doses tested failed to show any effect on il-12 protein ( figure 3(a ) ) or mrna expression ( figure 3(b ) ) in dcs .
therefore , we examined i-b and nf-b activation in dcs in the presence of rbla g 7 .
the results revealed that rbla g 7 triggered phosphorylation of i-b and nf-b in dcs initiated at 20  min following incubation and lasted for 40 and 60  min , respectively ( figure 4 ) .
induction of th2 polarization is one of the crucial steps of sensitization process in allergy .
we found that coculture of rbla g 7-activated dcs with isolated cd4 + t cells induced th2 polarization of helper t - cells as shown by enhanced ratio of il-4 + cells versus ifn-+ cells ( 14.23  0.82 ) , which was approximately 7-fold higher than the ratio for immature dcs ( 2.082  0.46 ) ( figures 5(a ) and 5(b ) ) .
moreover , coculture of rbla g 7-activated dcs with isolated cd4 + t cells resulted in the elevated level of il-13 in the culture supernatant ( figure 5(c ) ) .
since dc - derived tim4 has been reported to drive cd4 + t cells into th2 cells [ 4 , 5 ] , it may mediate rbla g 7-induced th2 polarization .
indeed , tim4 antibody reduced rbla g 7-activated dcs induced il-4 expression of cd4 + t cells by approximately 48% ( figure 6(a ) ) , significantly diminished the ratio of il-4 + versus ifn-+ cells ( figure 6(b ) ) , and decreased the il-13 level in the culture supernatant of cocultured rbla g 7-activated dcs and isolated cd4 + t cells ( figure 6(c ) ) .
in addition , cd80 and cd86 antibodies also inhibited rbla g 7-induced th2 polarization when they were added to rbla g 7-activated dcs for 30  min before co - culturing with isolated cd4 + t cells ( figure 6 ) .
although bla g 1   and bla g 2   were employed as markers of b. germanica , little is known about the profile of bla g 7 in proinflammatory actions . in the present study
, we found for the first time that bla g 7 could potently induce tim4 expression on dcs .
since activated dcs highly express tim4   which plays a critical role in t cell proliferation   and th2 cell development , and dcs are mainly localized at the interface between epithelium and environment , and we believe that inhalant allergen bla g 7 might initiate the sensitization process of allergy by activating dcs [ 30 , 31 ] . on the other hand , bla g 7 could also induce dcs maturation assessed by the levels of cd80 and cd86 .
as cd80 and cd86 have been found to mediate the necessary costimulatory signals to evoke th2 polarization   involved in allergy , cd80 and cd86 upregulation induced by rbla g 7 in dcs may contribute to the subsequent allergic responses .
a recent study demonstrated that a p. americana allergen per a 10 induced significant cd86 upregulation on dcs , and provoked significantly low il-12 but high il-4 and il-5 release from dcs .
another report also confirmed that cd11c+ mhcii+ dcs within mediastinal lymph nodes expressed maturation markers such as cd80 and cd86 , which supports our observation .  
however , unlike per a 10 and fungal allergens , rbla g 7 was found to only downregulate the level of il-12 mrna , but not il-12 secretion from dcs .
since il-13 serves as a foremost cytokine   and provides a first and crucial signal for class switching of b cells to produce ige , our finding implicates that rbla g 7 may induce b cell to produce ige   without activating th2 cells .
the previous studies have demonstrated that grass pollen and birch pollen were able to induce il-13 production from human dcs , and that diesel - enriched particulate enhanced il-13 secretion from human dcs   may support our view above . moreover , our findings that antibodies against tim4 and cd86 , but not cd80 blocked rbla g 7-induced il-13 production in dcs , indicated that the effects of rbla g 7 are via a tim4 and cd86 dependent mechanism .
although high levels of cd80 and cd86 represent the dcs maturity , expression of cd80 and cd86 under the same stimulation respond differently as shown in the present study .
similar observation was reported previously that reduced expression of cd80 and cd86 was observed in the majority of dcs in vitro while a significantly increased cd86 expression was found in a subpopulation of dcs exposed to maxadilan .
induction of phosphorylation of i-b followed by nf-b activation by rbla g 7 indicates that nf-b translocation into nucleus may also be involved in maturation of dcs and il-13 release from dcs .
the previous report that nf-b1 expression within dcs is required to promote optimal th2 responses following exposure to eggs   may support our observation above .
although dcs from allergic individuals preferentially induce a th2-type response , the mechanism underlying by which nave cd4 + t cells develop to th2 cells and how this process is out of control is a critical point to elucidate the mechanism of allergy .
using the current experiment model , we demonstrated for the first time that cockroach allergen bla g 7 preferably induces nave cd4 + t cells to become th2 cells via dcs .
since antibodies against tim4 , cd80 , and cd86 blocked rbla g 7-activated - dcs induced il-13 release from cd4 + t cells , the actions of rbla g 7 on il-13 secretion from cd4 + t cells are likely via tim4 , cd80 , and cd86 dependent signaling pathways in dcs .
however , the antibodies against tim4 , but not cd80 and cd86 , blocked rbla g 7-activated - dcs induced il-4 upregulation in cd4 + t cells indicates that the actions of rbla g 7 on il-4 upregulation are likely via a tim4 , but not cd80 and cd86 pathway in dcs .
obviously , the involvement of different signaling pathways of dcs in the induction of il-13 and il-4 upregulation is still required to be further studied .   in conclusion
, we demonstrated in this study that cockroach allergen rbla g 7 induces dc - dictated th2 polarization of cd4 + t cells through tim4 , cd80 , and cd86 dependent mechanisms . since
th2 cells are pivotal factors in allergy , bla g 7 seems likely to play an important role in the development of cockroach allergy .
 as one of the most common sources of indoor aeroallergens worldwide , cockroach is important in causing rhinitis and asthma while the mechanisms underlying remain obscure . since t helper ( th ) type 2 polarization plays an important role in the pathogenesis of allergic diseases , we investigated the effect of bla g 7 , a pan - allergen from blattella germanica ( b. germanica ) , on th polarization which is controlled by monocyte - derived dendritic cells ( dcs ) . challenged by recombinant bla g 7 ( rbla g 7 ) , immature dcs obtained from human exhibited upregulated levels of tim4 , cd80 , and cd86 and increased il-13 secretion 
 . cocultured with cd4 + t cells , challenged dcs increased the ratio of il-4 + versus ifn-+ of cd4 + t cells , suggesting a balance shift from th1 to th2 . 
 moreover , antibodies against tim4 , cd80 , and cd86 reversed the enhancement of il-4+/ifn-+ ratio and alleviated the il-13 release induced by rbla g 7 , indicating that the th2 polarization provoked by rbla g 7 challenged dcs is via tim4- , cd80- , and cd86-dependent mechanisms . in conclusion , the present findings implied a crucial role of bla g 7 in the development of cockroach allergy and highlighted an involvement of dcs - induced th2 polarization in cockroach allergy . 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion