the allergic response is a complex process characterized by an aberrant immune response to inhaled environmental allergens . cd4 + lymphocytes with a th2 phenotype may play some important roles in the development of allergic rhinitis , and the suppression of th2 lymphocytes could have the potential to be new therapeutic targets for the treatment of allergic rhinitis . the cd4 + cd25 + regulatory t ( treg ) cells have been shown to suppress both th1 and th2 responses in vitro [ 13 ] . the most specific marker for treg cells is foxp3 as identified transcription factor that is essential for the development of treg cells .   impaired skin infiltration of cd4+cd25+foxp3 + t cells was observed in acute atopic dermatitis lesions , suggesting a dysregulated control of inflammation by treg cells . with regard to the local foxp3-expressing cells in allergic nasal mucosa , conflicting results have been reported regarding difference in the levels of foxp3-expressing cells in between allergic and nonallergic nasal mucosa . in the present study , we evaluated the number of foxp3 + cells and the frequencies of foxp3 + t lymphocytes in perennial allergic and nonallergic nasal mucosa by dual - immunofluorescence methods . human inferior turbinates were obtained after turbinectomy from 26 patients with nasal obstruction refractory to medical therapy . informed consent was obtained from all patients and this study was approved by the ethics committee of sapporo medical university . all were nonsmokers , and 14 patients had perennial allergy against mites as defined by questionnaire and cap test ( pharmacia , uppsala , sweden ) . subject without allergies ( nonallergic rhinitis : control group ) had to satisfy the following criteria : ( 1 ) no history of allergic diseases , ( 2 ) no detectable , specific ige antibodies against 4 major inhalant allergens ( rast score < class 1 ) , and ( 3 ) total serum ige levels below the general population mean . all medications , including glucocoriticoids , were prohibited for at least 3 weeks prior to the study . for immunohistochemistry of foxp3 , rabbit anti - human foxp3 polyclonal antibody ( catalog # 623802 , biolegend , co , usa ) was used at 1  :  40 dilutions . for immunohistochemistry of t - bet , rabbit anti - human t - bet polyclonal antibody ( catalog#sc-21003 , santa cruz biotechnology inc . for immunohistochemistry of gata3 , goat anti - human gata-3 polyclonal antibody ( catalog#sc-22206 , santa cruz biotechnology inc . ) was used at 1  :  40 dilutions . to identify the t lymphocytes , anti - cd3 monoclonal antibody ( f7.2.38 clone , dako corporation , carpinteria , ca , usa ) after microwave treatment ( 10 minutes at 500 watt in citrate buffer ) , the deparaffinized sections were incubated in blocking solution ( 10% normal goat serum in pbs ) for 30 minutes before incubation in primary antibody . then , the sections were incubated with anti - cd3 monoclonal antibody and anti - foxp3 polyclonal antibody or anti- t - bet polyclonal antibody for overnight at 4c , washed in pbs , and were incubated for 30 minutes with alexa fluor 594-labelled goat anti - mouse igg ( diluted 1  :  50 ; molecular probes , or , usa ) and alexa fluor 488-labelled goat anti - rabbit igg ( diluted 1  :  50 ; molecular probes ) . for double staining of cd3 and gata-3 , after microwave treatment ( 10 minutes at 500 watt in citrate buffer ) , the deparaffinized sections were incubated in blocking solution ( 10% normal rabbit serum in pbs ) for 30 minutes before incubation in primary antibody . then , the sections were incubated with anti - cd3 monoclonal antibody and anti - gata3 polyclonal antibody for overnight at 4c . after washing with pbs , the sections were incubated with alexa flour 594-labelled rabbit anti - mouse igg ( diluted 150 ; molecular probes ) and alexa flour 488-labelled rabbit anti - goat igg ( diluted 1  :  50 ; molecular probes ) . after washing with pbs , the sections were mounted with vectashield mounting medium with 4,6 diamidino-2-phenylindole ( dapi ) ( vector laboratories inc , burlingame , ca , usa ) and examined under olympus bx51 microscope , dp70 ccd camera ( olympus optical co. , tokyo , japan ) . all images were processed with dp controller and dp manager software ( olympus optical co ) for image analysis . negative controls were obtained by replacing primary antibodies by mouse igg1 and rabbit immunoglobulin fraction ( dako ) . for double immunofluorescence staining , the slides were counted using a microscope equipped with an eyepiece graticule . a total of 6 fields ( 1  mm each ) from each slide were counted by placing the upper edge of the grid at the epithelium . the number of foxp3+cd3 + cells , t - bet+cd3 + cells , or gata-3+cd3 + cells in cd3 + cells in allergic and nonallergic nasal mucosa was evaluated by 2 blinded investigators . results are expressed as the number of foxp3-positive cells per square millimeter ( mm ) and the percentage of foxp3+cd3+cells , t - bet+cd3+cells , or gata-3+cd3 + cells in cd3 + cells . linear regression analysis was used to determine the relationship between the percentage of foxp3 + cells in cd3 + cells and the ratio of gata3 + cd3 + cells / t - bet+ cd3 + cells in allergic nasal mucosa . as shown in figure 1 , immunoreactivity for foxp3 was significantly detected in nucleus of some cd3 + cells . similarly , immunoreactivity for t - bet ( figure 2(a ) ) and gata3 ( figure 2(b ) ) was significantly detected in some of cd3 + cells in both allergic and nonallergic nasal mucosa . specificity of the staining was also confirmed by the absence of labeling with normal mouse igg1 and rabbit immunoglobulin ( data not shown ) . as shown in figure 3 , no significant difference between the group was found in terms of the number of foxp3+cd3 + cells ( allergic rhinitis : 12.5  4.4 , nonallergic rhinitis 10.6  4.0 , p = 0.23 ) . next , we compared percentage of t - bet+ cd3 + cells / cd3 + cells , gata3+cd3 + cells / cd3 + cells , and foxp3+cd3 + cells / cd3 + cells in between allergic and nonallergic nasal mucosa . as shown in figure 4 , there are no significantly differences in the percentage of t - bet+ t cells and foxp3 + t cells among two groups . however , the percentage of gata3 + t cells are significantly higher in allergic nasal mucosa than those in nonallergic nasal mucosa ( allergic rhinitis : 3.9  1.8% , nonallergic rhinitis 1.1  0.6% ,   p = 0.003 ) . finally , we compared foxp3+cd3 + cells to th2/th1 ratio in allergic nasal mucosa . as shown in figure 5 , the proportion of foxp3+cd3 + cells in allergic nasal mucosa tend to be correlated positively with gata3+/t - bet+ cells ratio ( r = 0.56 , p = 0.04 ) . the slope of the regression line for percentage of foxp3-positive t cells against th2/th1 ratio was 0.77 . in this study , the average number of foxp3 + cells in allergic mucosa is higher than that in nonallergic mucosa . however , the difference between the groups is not statistically significant due to high standard deviation in both groups ( allergic rhinitis : 12.5  4.4 , nonallergic rhinitis 10.6  4.0 , p = 0.23 ) . with respect to the number of foxp3 + t cells in nasal mucosa , conflicting results have been reported regarding the difference between in allergic and in nonallergic nasal mucosa . demonstrated that the number of foxp3 + cells in the nasal mucosa of patients with allergic rhinitis was significantly increased compared with healthy subjects , and that seasonal increases in foxp3 + cells in nasal biopsy specimens compared with baseline values were observed in both placebo- and immunotherapy - treated patients with grass pollen allergy . in addition , nasal challenge with pollen extract increased the number of foxp3 + t cells at the inflammatory site in pollen - allergic patients . these reports suggest that local allergen exposure induces the increases of local foxp3 + t cells . on the contrary , another study has demonstrated that nasal secretions of patients with allergic rhinitis had significantly lower foxp3 mrna compared to nonallergic controls . from these observations , further studies will be necessary to determine whether the upregulation or downregulation of local t - regulatory cells may exist in patients with allergic rhinitis . in the present study , we focused on the local th1/th2 balance and regulatory t cells , and our study demonstrates a positive correlation between foxp3+cd3 + cells / cd3 + cells and gata3 + cd3 + cells / t - bet+ cd3 + cells ratio . it has been well known that il-4 is one of the most important cytokine on the local th1/th2 balance , because il-4-induced gata3 is associated with th2-type responses and il-4 has already been shown to negatively regulate the development of nave t cells into th1 or the il-17-producing t cells ( th17 ) . it is well known that foxp3 + t cells accumulate in a variety of human inflammatory disorders . one possibility , of course , is that the accumulation of foxp3 + cells , at least in part , depends on local proliferation induced by tgf- and other cytokines . with respect to the effect of il-4 on the induction of foxp3 , this observation may support our present data . on the other hand , mantel et al . whether or not th2 cytokines such as il-4 directly affect the accumulation of the regulatory t cells is still unclear and needs to be clarified . there have been several reports regarding the circulating regulatory t cells by mean of flow cytometory . it was found in peripheral blood of pollen - sensitized children that treg cells increase during the pollen season . circulating der p1-specific cd4+cd25foxp3 + treg cells obtained from atopic patient inhibited antigen - induced proliferation of t cells . another study found that circulating foxp3+/cd4 + ratio was significantly low in patients with asthma and atopic dermatitis . whether these treg cells directly contribute to allergic inflammation or not remains an important research question . allergen - specific immunotherapy , which consists of repeated subcutaneous or sublingual administration of allergen , has been widely used and proved effective for the management of allergic rhinitis . the mechanism of the therapeutic effect of allergen - specific immunotherapy is not completely understood but might involve the activation and expansion of treg cells . it is well established that foxp3 acts as master transcription factor for naturally occurring regulatory t cells ( ntreg ) development and function . several mechanisms involving treg in tolerance induction after allergen - specific immunotherapy have been documented . such mechanisms include increased capacity of treg to suppress th1 and th2 cells , induction of il-10 and tgf - beta , decreased allergen - stimulated t cell proliferation , or suppression of effector cells . the induction of a tolerant state in peripheral t cells represents an essential step in allergen - specific immunotherapy . peripheral t cell tolerance is characterized mainly by generation of allergen - specific treg cells and suppressed proliferative and cytokine responses against the major allergen . it is initiated by the autocrine action of il-10 and tgf- , which are increasingly produced by the antigen - specific tr1 cells . it was shown that grass pollen immunotherapy increased the expression of mucosal and peripheral t - cell il-10   and tgf- . it has been shown that the increased number of foxp3+cd25 + treg in nasal mucosa after grass pollen immunotherapy correlated with clinical efficacy and suppression of seasonal allergic inflammation , thus supporting the role of treg in the induction of allergen - specific tolerance in human subjects . from these reports , we suppose that the exposure of allergen locally or generally may increase the number of treg cells in nasal mucosa of perennial allergic rhinitis patients . so , further studies on nasal mucosa from the perennial rhinitis patients who received immunotherapy will be necessary to clarify the roles of immunotherapy on the local treg cells and th1-th2 balance in perennial allergic rhinitis . in conclusion , we have demonstrated a positive correlation with gata3+cd3 + cells / t - bet+cd3 + cells ratio and foxp3+cd3 + cells / cd3 + cells ratio , suggesting that treg cells are operative on th1-th2 balance in perennial allergic nasal mucosa . our present findings should be of considerable interest for understanding the role of regulatory t cells on upper airway diseases such as allergic rhinitis .
regulatory t cells ( treg ) play some important roles in allergic rhinitis . 
 the most specific marker for treg is foxp3 , a recently identified transcription factor that is essential for treg development . in order to clarify the levels of treg in allergic nasal mucosa 
 , we studied the relationship between foxp3-expressing cells and th1-th2 balance in nasal mucosa by means of immunohistochemistry . 
 human turbinates were obtained after turbinectomy from 26 patients ( 14 patients with perennial allergic rhinitis and 12 patients with nonallergic rhinitis ) . to identify the cells expressing the foxp3 protein , 
 double immunostaining was performed by using anti - foxp3 antibody and anti - cd3 antibody . 
 there was no significant difference in the percentage of foxp3+cd3 + cells among cd3 + cells in the nasal mucosa of two groups . 
 the proportion of foxp3+cd3 + cells tend to be correlated positively with gata3+cd3 + cells / t - bet+cd3 + cells ratio ( r = 0.56 , p = 0.04 ) . 
 a positive correlation with gata3+cd3+/t - bet+cd3 + ratio and foxp3+cd3+/cd3 + ratio suggests the role of local regulatory t cells as a minimal control of the chronic allergen exposure in nasal mucosa .