BACKGROUND
cd4+ t helper type  <dig>  cytokines such as il- <dig>  il- <dig> and il- <dig> play a critical role in inducing allergy and asthma.
these cytokines act on multiple cells types to initiate and propagate the hallmark features of asthma such as pulmonary inflammation, periodic narrowing of airways and mucus hypersecretion  <cit> .
experiments with mice deficient in these cytokines and studies in asthma patients have confirmed these findings  <cit> .
also, the fact that th <dig> cells are required in this disease setting has been demonstrated by using il-4-/- mice and adoptive transfer studies  <cit> .
apart from th <dig> cells, il- <dig> and il- <dig> are also secreted by natural killer  t cells, basophils, mast cells, macrophages and activated eosinophils .
il- <dig> and il- <dig> share receptor chains and signaling proteins.
binding of either cytokine to the type i or type ii receptor complex leads to the phosphorylation of signal transducer and activator of transcription factor   <dig>  <cit> .
polymorphisms in the il4ra and stat <dig> genes have been linked to increased risk of asthma  <cit> .
there is ample evidence that il- <dig> signaling through il-4rα and stat <dig> is important for th <dig> differentiation and for ige class-switching in b cells  <cit> .
furthermore, mucus hypersecretion, goblet cell hyperplasia and airway hyperresponsiveness  were completely abolished in il-4rα-/- or stat6-/- mice  <cit> .
we have previously shown that apart from th <dig> cells, il-4rα expression on a population of cd11b+ cells contributed to the severity of lung inflammation and eosinophil recruitment  <cit> .
although these signaling molecules have been studied extensively, there are conflicting reports in the literature regarding the roles of il-4rα and stat <dig> in modulating specific features of airway inflammation.
some studies have shown that there was no eosinophil recruitment in stat6-/- mice  <cit> , while other groups including us contend that lung eosinophilia and inflammation are only partially dependent on stat <dig>  <cit> .
recently it has been established that il- <dig> and il- <dig> can promote differentiation of alternatively activated macrophages  .
during type ii inflammation, aams as well as epithelial cells produce certain characteristic factors such as arginase  <dig>  chitinase- like mammalian proteins  and found in inflammatory zone  proteins.
four different sub-types of fizz proteins have been reported in the literature- fizz1- <dig>  fizz <dig> was originally discovered in the bronchoalveolar lavage  fluid in a mouse model of asthma  <cit> .
elevated levels of fizz <dig> and ym <dig> mrna or protein have since been detected in parasite infection models  <cit> , allergic lung inflammation  <cit> , allergic peritonitis  <cit> , bleomycin-induced lung fibrosis  <cit>  and hypoxia-induced pulmonary hypertension  <cit> .
interestingly, the promoter regions of both fizz <dig> and ym <dig> have functional binding sites for stat <dig>  <cit> , which explains how il- <dig> and il- <dig> can induce expression of these proteins.
our group has shown previously using in vitro studies, that fizz <dig>  ym <dig> and arginase  <dig> mrna are preferentially upregulated by il- <dig> and to a lesser extent by il- <dig>  <cit> .
loss of stat <dig> signaling results in a significant reduction in fizz <dig> and ym <dig> mrna levels in different model systems  <cit> .
however, the effect of il-4rα or stat <dig> on fizz1/ym <dig> protein induction in an asthma model has not yet been studied.
it is well established that signaling through the il- <dig> pathway is important for th <dig> cell differentiation and cytokine production .
il- <dig> signaling through the il-4r/stat <dig> axis upregulates gata <dig>  the th <dig> master transcription factor.
adoptive transfer of wild type th <dig> cells into mice is an effective tool to bypass the role of stat <dig> and il-4rα in th <dig> cell differentiation, while allowing the study of these signaling molecules in the effector phase of asthma.
most groups use in vitro-generated th <dig> effectors for this purpose.
however, this protocol involves several rounds of in vitro priming and expansion using large quantities of il- <dig>  also, it is becoming evident that in vivo th <dig> differentiation is more complex and that certain signals may be missing when cells are differentiated in vitro .
it is now known that tslp, il- <dig> and il- <dig> family cytokines can be involved in induction or maintenance of the th <dig> phenotype  <cit> .
in order to avoid using in vitro t cell activation, we isolated ovalbumin -specific cd4+ t cells from either unimmunized do <dig> xrag2-/- mice to get naïve cd4+ t cells or do <dig> xrag2-/- mice immunized with ova/alum to get in vivo-primed cd4+ t cells.
previous dogma led to the assumption that it would be difficult to immunize tcr transgenic mice  <cit>  as t cells in these mice undergo reduced proliferation.
however, recent reports indicate that it is possible to immunize tcr transgenic mice and that t cell proliferation and cytokine production are independent of each other  <cit> .
in this study, we found that in vivo primed cd4+ t cells underwent reduced proliferation in a lymphopenic environment when compared to naïve cd4+ t cells.
using this modified asthma model with in vivo generated effector t cells, we demonstrate that although there was a reduction in recruitment of inflammatory cells and eosinophils in absence of il-4rα or stat <dig>  significant amounts of eosinophils were still present both in the bal and also the lung tissue of stat6xrag2-/- and il-4rαxrag2-/- mice.
interestingly, ym <dig> protein was expressed by both epithelial cells and macrophages, while fizz <dig> protein was only expressed by epithelial cells in rag2-/- mice.
we further show that fizz <dig> and ym <dig> protein expression in the lung was completely dependent on signaling through the il-4rα and stat <dig>  thus it appears that while il-4/il- <dig> signaling through il-4rα and stat <dig> is essential for induction of aam genes, lung inflammation and eosinophilia are only partially dependent on this pathway.
RESULTS
activation and proliferation status of naïve versus in vivo primed t cells
since it has been suggested that th <dig> differentiation in vivo is quite complex  <cit>  and cannot necessarily be mimicked by in vitro priming of t cells, we tested the possibility of using either naïve or in vivo primed cd4+ do <dig> + t cells.
first, we determined if the in vivo primed t cells were capable of secreting more th <dig> cytokines as a result of ova/alum immunization of d <dig> xrag2-/- mice.
splenocytes isolated from either unimmunized  or immunized d <dig> xrag2-/- mice were stimulated with pma and ionomycin and secretion of il- <dig> into the cell culture supernatant was measured by elisa.
we found that, just one round of priming with ova/alum was sufficient to induce robust secretion of th <dig> cytokines, il- <dig> and il- <dig> .
the amount of il- <dig> and il- <dig> produced by cells isolated from immunized mice  was significantly higher than that of naïve cells .
next we compared the ability of the naïve and in vivo primed cd4+ t cells to be activated upon ova/alum priming.
since differentiation of cd4+ t cells can often be accompanied by cell proliferation, we also monitored t cell proliferation using 5-bromo-2-deoxyuridine  incorporation.
in order to test this, we transferred naïve or in vivo primed t cells into stat6xrag2-/- mice by tail vein injection, followed by immunization with ova/alum using a protocol shown in figure 2a.
one group of mice was injected with brdu.
splenocytes were harvested on day  <dig> and analyzed by flow cytometry.
we found that although the total number of splenocytes isolated from these two groups was similar , the number of do <dig> + cd4+ t cells in the in vivo primed transfer group was double that of the naïve transfer group-  <dig> % vs.  <dig> % ;  <dig> cells vs.  <dig> cells .
however the percentage of naïve do <dig> + cd4+ t cells that were proliferating, as measured by brdu incorporation was higher when compared to the in vivo primed do <dig> + cd4+ t cells- 22% vs. 10% .
we also looked at cell surface activation markers such as cd <dig>  expressed by these cells.
the majority of cells in both groups on day  <dig> were cd44hi, although expression of this protein was slightly higher in the group receiving in vivo primed t cells .
interestingly, although only 10% of the cd4+ t cells in the primed t cell transfer group and 22% of these cells in the naïve t cell transfer group were brdu+, cd44hi expression by these cells in the two groups of mice was 97% and 82% respectively.
this suggests that even cells that did not divide in both groups were expressing high levels of cd <dig> and were activated.
cell proliferation studies were conducted using the protocol mentioned in figure  <dig> and materials and methods.
briefly, naïve or in vivo primed cd4+ t cells were adoptively transferred into stat6xrag2-/- mice on day  <dig>  followed by ova/alum immunization of day  <dig>  mice were treated with brdu i.p for  <dig> days.
on day  <dig>  splenocytes were harvested, single cell suspensions were prepared and total cell numbers were counted .
cells were stained with fluorochrome conjugated antibodies and analyzed by flow cytometry.
lymphocytes were gated based on forward and side scatter parameters.
the cd4+ do <dig> + population in each transfer group was gated based on double expression of cd <dig> and kj <dig> by each cell .
brdu and cd <dig> expression in these gated cells was examined .
the numbers/percentages in columns 2- <dig> were determined by facs analysis.
<dig>  events  were collected for each tube/analyte.
another set of stat6xrag2-/- mice that received either naïve or in vivo primed do <dig>  t cells were immunized twice with ova/alum within a span of  <dig> week .
the activation status of the adoptively transferred t cells and the amount of il- <dig> produced by these cells was monitored.
the number of spleen cells recovered at this stage  from the naïve t cell transfer group was twice as much as the number recovered from spleens of mice that had received in vivo primed t cells .
moreover, the percentages of naïve do <dig> + cd4+ t cells  in the spleen were also higher when compared to that of the in vivo primed do <dig> + cd4+ t cells .
this result correlates well with the brdu incorporation data and indicates that naïve do <dig> + cd4+ t cells proliferate more in a lymphopenic environment, which leads to greater accumulation of these cells in spleens of mice at a later time.
this accumulation of t cells may be due to homeostatic proliferation.
however, the percentage of cells expressing cd <dig>  and il- <dig>  was similar in both the naïve and in vivo primed groups on day  <dig> .
t cell activation studies were conducted using the protocol mentioned in figure  <dig> and materials and methods.
briefly, naïve or in vivo primed cd4+ t cells were adoptively transferred into stat6xrag2-/- mice on day  <dig>  followed by ova/alum immunization of days  <dig> and  <dig>  four days later, splenocytes were harvested, single cell suspensions were prepared and cells were counted .
splenocytes were stimulated with pma and ionomycin for  <dig> h, followed by facs staining and analysis.
lymphocytes were gated based on forward and side scatter parameters.
the cd4+ do <dig> + population in each transfer group was gated based on double expression of cd <dig> and kj <dig> by each cell .
cd <dig> expression in these gated cells was examined .
the numbers/percentages in columns  <dig> and  <dig> were determined by facs analysis.
<dig>  events  were collected for each tube/analyte.
effect of stat <dig> and il-4rα on lung inflammation, eosinophilia and mucus production
considering that naïve do <dig> + cd4+ t cells were proliferating more in a lymphopenic environment and since we wanted to focus on the effector functions of il- <dig> and il- <dig> but not their role in priming naïve t cells, we chose the in vivo primed do <dig> + cd4+ t cells for all further experiments.
several groups including ours have shown that il- <dig> and il- <dig> signaling through il-4rα and stat <dig> plays an important role in inducing and exacerbating eosinophilic inflammation and mucus production in the lungs  <cit> .
since some of these studies were conducted using in vitro generated th <dig> effectors, we examined whether similar responses would be observed using in vivo primed t cells.
furthermore, although similar studies have been conducted with stat6-/- mice or il-4rα-/- mice alone  <cit> , no head to head comparisons between mice deficient in stat <dig> or il-4rα have been made.
to tease out the precise roles played by these signaling molecules, we conducted allergic inflammation studies on rag2-/-, stat6xrag2-/- and il-4rαxrag2-/- mice using our model of transferring in vivo primed t cells .
the degree of airway inflammation, eosinophil recruitment and mucus production in the lungs was analyzed in the three groups of mice.
as reported earlier  <cit> , priming with alum alone did not induce eosinophilia and airway inflammation  and served as a negative control.
upon enumerating the cellular composition in the bal, we found that the total number of cells recovered from ova treated rag2-/- mice was significantly higher  than the number of cells recovered from ova treated stat6xrag2-/- and il-4rαxrag2-/- mice .
.
among the different cell types  found in the bal, a 2- <dig> fold reduction in the numbers and percentages of eosinophils was seen in stat6xrag2-/- and il-4rαxrag2-/- mice when compared to rag2-/- mice challenged with ova .
in each case, the numbers of eosinophils, macrophages and lymphocytes present in the ova treated mice were much greater than the alum treated mice .
h&e stained lung sections of ova treated rag2-/- mice demonstrated severe lung inflammation  and most of the cellular infiltrate was composed of eosinophils .
multinucleated giant cells  were also present in large numbers.
in contrast, in absence of stat <dig> and il-4rα only minor cuffing of the airways and blood vessels was observed .
eosinophil recruitment into the lung although reduced, was not completely abolished in stat6xrag2-/- and il-4rαxrag2-/- mice .
pas staining on the above lung sections indicated that mucus production by epithelial cells was fully dependent on stat <dig> and il-4rα .
this is not surprising as it known that mucus production is mainly driven by il- <dig> mediated stat <dig> activation  <cit> .
the differences in eosinophil counts in the bal  in the three mouse strains were recapitulated in the lung tissue.
the number of eosinophils recruited to the airways and blood vessels in ova primed and challenged stat6xrag2-/- and il-4rαxrag2-/- mice was significantly reduced but not completely absent .
a corresponding increase in mononuclear cell infiltration was observed in these mice.
surprisingly, the number of mononuclear cells in stat <dig> deficient mice was higher than in il-4rα deficient mice.
it is possible that this is linked to the presence of increased numbers of myeloid progenitor cells that have been reported in stat6-/- mice  <cit> .
however, we found significantly higher eosinophils in the lung parenchyma in stat6xrag2-/- and il-4rαxrag2-/- mice, when compared to rag2-/- mice .
taken together, these results suggest that in vivo primed cd4+ t cells can induce robust allergic lung inflammation in mice.
in this model, stat <dig> and il-4rα expression are only partially required for inducing pulmonary inflammation and eosinophilia.
chemokine and cytokine profile in the bal in presence or absence of stat <dig> and il-4rα
il- <dig> and il- <dig> signaling can induce production of many chemokines by different cell types.
eotaxin- <dig>  and eotaxin- <dig>  are eosinophil chemoattractive proteins that are predominantly produced by epithelial cells in mice , upon il- <dig> or il- <dig> stimulation  <cit> .
previous studies have shown that induction of eotaxin, eotaxin  <dig> and tarc mrna in the lungs of ova-challenged mice was stat <dig> dependent  <cit> .
we determined the quantities of eotaxin, tarc and mouse je/ccl <dig> secreted into the bal .
using our model of in vivo primed t cell transfer and ova-induced allergic lung inflammation, we further show that significantly elevated levels of eotaxin and tarc protein were found in rag2-/- mice when compared head to head with stat6xrag2-/- and il-4rαxrag2-/- mice.
a similar trend is seen in the case of je/ccl <dig> production.
since eotaxin plays an important role in eosinophil trafficking, the reduced amount of eotaxin found in the bal of stat6xrag2-/- and il-4rαxrag2-/- mice could explain the lower numbers of eosinophils present around the airways in mice .
as th <dig> cytokines have been implicated in allergic lung inflammation, we evaluated il- <dig>  il- <dig> and il- <dig> secretion into the lungs and analyzed the contribution of stat <dig> and il-4rα head to head in this process, using our in vivo primed t cell model.
since we provided wt ova-specific t cells to all three groups of mice, these cells would be able to produce th <dig> cytokines.
we found that upon priming and challenge with ova, both rag2-/- and stat6xrag2-/- mice secreted similar amounts of il- <dig> and il- <dig> into the bal .
however, significantly higher levels of il- <dig> were present in the bal of il-4rαxrag2-/- mice when compared to the other two groups .
although not significant, il- <dig> secretion in these mice followed a similar trend.
it is published that binding of il- <dig> to the il-4r complex induces internalization and uptake of the cytokine  <cit> .
thus, in mice deficient in il-4rα, absence of the il-4r on cell surfaces may be preventing the internalization of il- <dig> and il- <dig>  thus increasing the concentration of these cytokines in the bal.
similar results were obtained by other groups when antibodies against the il-4rα chain or il-13rα <dig> were used  <cit> .
in case of il- <dig>  increasing amounts of this cytokine was detected in the three mouse strains, with the lowest quantity of il- <dig> present in the bal of rag2-/- mice, intermediate levels in stat6xrag2-/- mice and the highest in il-4rαxrag2-/- mice .
studies have shown that when in vitro generated th <dig> effectors were adoptively transferred into stat6-/- mice, there was a dramatic increase in il- <dig> secretion in the bal  <cit> .
the authors speculated that this difference was due to decreased consumption of il- <dig> by eosinophils.
in our model also, we find that there is a correlation between the degree of eosinophilic inflammation in mice and the amount of il- <dig> present in the bal.
thus the lower levels of il- <dig> found in the bal fluid in rag2-/- mice may be explained by increased consumption of this cytokine by eosinophils recruited into the lungs .
migration of th <dig> cells into the lungs is independent of stat <dig> expression
previous studies have shown that stat <dig> expression was necessary for th <dig> cell trafficking into the lung upon inhalation of ovalbumin.
mathew et.
al.
reported that in the absence of stat <dig>  less antigen specific th <dig> cells migrated into the lungs  <cit> .
to check if this was true in our studies, lung sections were stained with antibodies to cd <dig> to identify t cells.
since all mice were on a rag deficient background, the only cd3+ t cells present in the lungs were the ova-specific t cells that we adoptively transferred.
as evident from figure 4a, absence of stat <dig> or il-4rα did not block migration of antigen specific t cells into the lungs of mice.
when the cd3+ cells in these mice were quantified, we found that significantly greater numbers of t cells were recruited in the lungs of il-4rαxrag2-/- mice when compared to rag2-/- mice and a similar trend was seen in stat6xrag2-/- .
thus when the t cells express stat <dig> or il-4rα themselves, deficiency of those proteins in lung resident cells does not affect t cell trafficking.
effect of stat <dig> and il-4rα on fizz <dig> and ym <dig> protein expression
liu et.
al reported that induction of fizz <dig> transcripts was stat <dig> dependent in a bleomycin-induced lung fibrosis model  <cit> .
ym <dig> mrna was also upregulated in a stat <dig> dependent manner in a mouse model of allergic peritonitis  <cit> .
however, the expression patterns of these aam proteins by epithelial cells and macrophages have not been studied in allergic lung inflammation.
moreover, we have observed a disconnect between the amounts fizz <dig> mrna and protein induced by il- <dig> stimulated macrophages in vitro  <cit> .
thus, we examined the expression profile of fizz <dig> and ym <dig> protein in our model and investigated the role of stat <dig> and il-4rα in upregulation of these proteins.
serial lung sections from ova sensitized and challenged rag2-/-, stat6xrag2-/- and il-4rαxrag2-/- mice were stained with antibodies against both ym <dig> and fizz <dig> by immunohistochemistry .
lung epithelial cells in rag2-/- mice stained strongly for fizz <dig>  and ym <dig> .
however, macrophages from these mice were positive for only ym <dig> but not fizz <dig> .
multinucleated giant cells present in the lungs of rag2-/- mice also expressed ym <dig> .
in comparison, no fizz <dig> or ym <dig> protein was produced by epithelial cells  or macrophages  in mice deficient in il-4rα or stat <dig>
to quantify the amount of fizz <dig> and ym <dig> protein that was produced by each mouse strain, we analyzed the expression pattern of these proteins secreted into bal fluid by western blotting.
total protein present in the bal fluid samples from rag2-/-, stat6xrag2-/- and il-4rαxrag2-/- mice was first quantitated; more total protein was recovered from rag2-/- bal when compared to mice lacking stat <dig> or il-4rα .
generally, the amount of total protein present in bal correlates with the degree of inflammation seen in mice.
in order to compare the quantities of fizz <dig> and ym <dig> present in the different mouse strains, equal amounts of total bal protein from rag2-/-, stat6xrag2-/- and il-4rαxrag2-/- mice were used.
the bal protein samples were resolved by polyacrylamide gel electrophoresis, transferred onto a membrane and probed with antibodies to ym <dig> or fizz <dig>  similar to the immunohistochemistry study, large amounts of fizz <dig> and ym <dig> were secreted into the bal in rag2-/- mice, but this was greatly reduced in the absence of stat <dig> and il-4rα .
densitometry analysis of the blots revealed that the differences seen were significant .
these results demonstrate that stat <dig> activation through il-4rα signaling is required for expression of fizz <dig> protein in lung epithelial cells and ym <dig> protein in macrophages and epithelial cells during allergic lung inflammation.
effect of stat <dig> and il-4rα on airway remodeling
one characteristic feature of asthma is airway remodeling, which involves an increase in airway smooth muscle mass and enhanced collagen deposition.
it has been reported that both eosinophils and aam products such as fizz <dig> and ym <dig> can cause lung fibrosis and smooth muscle thickening  <cit> .
thus, we analyzed the amount of collagen deposition and airway smooth muscle thickness in rag2-/-, stat6xrag2-/- and il-4rαxrag2-/- mice.
masson's trichrome staining of representative lung sections from each mouse strain revealed that greater quantities of collagen  was present around the airways  and blood vessels  in rag2-/- mice, when compared with mice lacking stat <dig> and il-4rα .
quantification of the collagen staining using image analysis software showed that the differences were significant .
furthermore, the thickness of the smooth muscle layer around the airways  was also significantly reduced in absence of stat <dig> and il-4rα .
the airway smooth muscle layer was identified by h&e staining of lung sections  and the diameter of the muscle layer was measured at three different points in each airway examined, using image j software  <cit>  .
discussion
although research on the cytokines il- <dig> and il- <dig> over the past decade has substantially increased our understanding of their contribution to the pathophysiology of asthma, the extent to which the signaling pathways they activate play a role in airway inflammation and alternative activation of macrophages have not yet been determined.
in this study, we used stat <dig> and il-4rα deficient mice on a rag2-/- background to examine the role of the il-4/il- <dig> pathway in inducing the aforementioned features of allergic lung disease.
since th <dig> cells are indispensable in this disease setting, we provided t cells exogenously.
previously, most groups used in vitro generated th <dig> effectors for this purpose  <cit> .
here, we developed a model wherein in vivo primed ovalbumin-specific cd4+ t cells were adoptively transferred into various recipient mice, followed by immunization and challenge with ova.
we examined whether naïve cd4+ t cells or in vivo primed cd4+ t cells isolated from do <dig> x rag2-/- would be more suitable for this asthma model.
we found that in vivo primed t cells proliferated less when compared to naïve t cells as suggested by the following results: i. lower levels of brdu incorporation in cells; ii.
the number of cd4+do <dig> + cells recovered from lymphopenic mice was half of that recovered from the naïve t cell transfer group.
this increased proliferation seen in the naïve t cell transfer group may be due to homeostatic proliferation.
it has been demonstrated, that naïve t cells from tcr transgenic mice undergo slow homeostatic proliferation in lymphopenic mice, which is dependent on il- <dig>  <cit> .
it has been proposed that entry into the cell cycle  and clonal expansion is necessary for t cell differentiation  <cit> .
also, several groups have shown that tcr transgenic mice that have high frequency of antigen-specific t cells show only weak proliferation upon tcr ligation and that the t cells become anergic or die of apoptosis  <cit> .
however, a study conducted by laouar et.
al.
<cit>  and our studies have shown that when t cells from tcr transgenic mice were activated in vivo with specific peptide/antigen, these cells express cell surface activation markers such as cd <dig> and secrete effector cytokines, in spite of proliferating less .
why these transgenic t cells showed reduced proliferation is not known exactly, but it is hypothesized that at high cell frequency, there may be increased competition for growth factors, limited access to peptide/mhc complexes and also limited lymphoid space for expansion.
the other difference between our study and the ones where transgenic t cells became anergic/apoptotic is the method of immunization: we used ovalbumin complexed with an adjuvant  instead of using the antigen alone as was done previously.
thus, our results clearly show that in vivo primed cd4+ t cells from do <dig>  transgenic mice can be used to induce the hallmark features of asthma in mice.
this effect is not restricted to one transgenic mouse strain; similar results were obtained when ot-ii mice were used .
in mice that lack stat <dig> or il-4rα, th <dig> cell differentiation is impaired but they have normal th <dig> cell differentiation.
in order to track the exogenous in vivo primed t cells that we were transferring into these mice and to prevent interference of th <dig> cells, we used stat <dig> or il-4rα deficient mice on a rag2-/- background for our asthma experiments.
rag2-/- mice were used as controls.
in this study, we tested the ability of in vivo primed cd4+ t cells as opposed to in vitro generated th <dig> effectors to support allergic lung inflammation.
we found that in the absence of stat <dig> and il-4rα, mice developed less pulmonary inflammation, reduced perivascular and peribronchial cuffing and decreased eosinophilia than our control mice.
mucus production in these mice was abrogated.
this was expected since it has been conclusively shown that mucus production is dependent on stat <dig> activation by il- <dig> signaling  <cit> .
however, both stat6xrag2-/- and il-4rαxrag2-/- mice that were primed and challenged with ova were able to recruit significantly higher numbers of eosinophils when compared to alum primed mice.
several studies have shown the importance of these signaling molecules in asthma, but the roles of il-4rα and stat <dig> in modulating specific features of airway inflammation were unclear.
here we show that stat <dig> and il-4rα are only partially required for eosinophil recruitment to the lung.
our data matches with what was observed by kuperman et.
al.
<cit>  but is in apparent contradiction to that shown by mathew et.
al.
<cit> .
furthermore, in contrast to the latter's finding, we observe that there is no defect in t cell recruitment into the lung in absence of stat <dig> or il-4rα.
differences in the experimental set up may explain these discrepancies: we and kuperman et.
al.
used two priming steps with ova/alum in the asthma protocol, which was omitted by the other group.
recently it was demonstrated that alum stimulates the innate immune system by activating the nalp <dig> inflammasome, leading to secretion of pro-inflammatory cytokines such as il-1β, il- <dig> and il- <dig>  <cit> .
these cytokines play important roles in initiation and amplification of th <dig> responses including th <dig> cell proliferation and eosinophilia  <cit> .
alum also induces production of il- <dig> and il- <dig> in t cells  <cit> .
eosinophil migration and recruitment into the lungs depend on several factors.
il- <dig> controls differentiation, activation and survival of eosinophils in the bone marrow and is essential for their mobilization into the lungs .
eosinophil migration into the lung is mediated by adhesion to the vascular endothelium via vcam- <dig>  <cit> .
chemokines produced by airway epithelial cells and macrophages recruit eosinophils into the airways  <cit> .
eotaxin- <dig>  and eotaxin- <dig>  are eosinophil chemoattractive proteins, mainly produced by airway epithelial cells  <cit>  upon il- <dig> or il- <dig> stimulation  <cit> .
this was further substantiated by munitz et.
al.
when they demonstrated that eotaxin secretion in mice is completely dependent on the il-13rα <dig> chain and thus the type ii il-4/il- <dig> receptor .
in our studies, we show that eotaxin secretion in the bal is markedly reduced in the absence of stat <dig> or il-4rα.
this might explain the lower levels of eosinophils seen around the airways and blood vessels in stat6xrag2-/- and il-4rαxrag2-/- mice in comparison to rag2-/- mice.
however, in absence of eotaxin, il- <dig> may play a major role in recruitment of eosinophils in stat6xrag2-/- and il-4rαxrag2-/- mice.
the higher levels of il- <dig> found in the bal in these mice points to this direction.
other chemokines such as rantes, monocyte chemoattractant proteins  or macrophage inflammatory protein -1α could also play a role in this process.
interestingly, we found that greater numbers of eosinophils were present in the lung parenchyma in mice deficient in stat <dig> and il-4rα.
it is possible that in absence of eotaxin, eosinophils are unable to reach the airways and remain in the parenchyma.
in previous studies using wt and stat6-/- mice, th <dig> cytokine production was higher in wt mice in comparison to mice lacking stat <dig>  <cit> .
this is because stat <dig> is required for th <dig> cell differentiation.
since we provided wt effector t cells to all the groups of mice, they were capable of producing th <dig> cytokines.
when we measured the amounts of il- <dig> and il- <dig> in the bal in allergen challenged mice, we found that significantly higher amounts of these cytokines were present in il-4rαxrag2-/- mice than rag2-/- and stat6xrag2-/- mice.
studies have shown that binding of il- <dig> to the il-4r complex induces internalization and uptake of this cytokine  <cit> , analogous to that observed with binding of il- <dig> to the il2r complex  <cit> .
moreover, other groups have found that il- <dig> concentration in the bal was significantly increased when antibodies against the il-4rα chain were injected into mice, compared to control mice  <cit> .
similarly, more il- <dig> was found in the bal in il-13rα1-/- mice  <cit> .
therefore, based on our findings and published literature we hypothesize that the absence of il-4rα on cell surfaces may be preventing the internalization of il- <dig> and il- <dig>  thus increasing the concentration of these cytokines in the bal in il-4rαxrag2-/- mice.
our data also demonstrated that more il- <dig> was secreted into the bal when mice lacked stat <dig> or the il-4rα chain.
the higher concentration of il- <dig> found in stat6xrag2-/- mice in this model are consistent with the results reported by mathew et.
al.
<cit> .
they had observed that when in vitro generated th <dig> effectors were adoptively transferred into stat6-/- mice, there was a dramatic increase in il- <dig> secretion in the bal  <cit> .
the authors speculated that this difference was due to decreased consumption of il- <dig> by eosinophils.
in our model, since the stat6xrag2-/- and il-4rαxrag2-/-mice have significantly lower levels of eosinophils in both the bal and lung tissue , it is possible that the enhanced cytokine level in the bal in these mice is due to reduced consumption.
we did not see any significant differences in ifnγ levels in the three strains of mice.
il- <dig> is another cytokine that has been implicated in asthma in humans and mice .
in our asthma model, il- <dig> levels in the bal were below detection limits in all  <dig> mouse groups.
as our understanding of the roles of il- <dig> and il- <dig> increases, it is becoming clear that in addition to their action on t cells, b cells, eosinophils, epithelial cells, these cytokines can also stimulate macrophages such that they become alternatively activated.
instead of expressing inos like the classically activated macrophages, these cells produce proteins such as arginase, fizz and ym1/ <dig> among others .
it has now been established that il- <dig> and il- <dig> can also induce expression of the same group of proteins in airway and alveolar epithelial cells.
as mentioned earlier, copious amounts of fizz <dig> and ym <dig> have been detected in the bal of allergen challenged mice  <cit> .
in addition, upregulated levels of fizz <dig> and ym <dig> mrna have also been found in parasite infection models  <cit> , allergic lung inflammation and allergic peritonitis  <cit> , bleomycin-induced lung fibrosis  <cit>  and hypoxia-induced pulmonary hypertension  <cit> .
stutz et.
al.
<cit>  demonstrated using the bmnot cell line that the fizz <dig> promoter contains functional binding sites for stat <dig> and c/ebp.
they further showed that stat <dig> and c/ebp cooperated together to get optimal fizz <dig> expression.
welch et.
al.
<cit>  showed similar results for the ym <dig> promoter region using epithelial and macrophage cell lines: ym <dig> has several stat <dig> binding sites in its promoter region.
additional studies have indicated that induction of fizz <dig> or ym <dig> transcripts are stat <dig> dependent in bleomycin-induced lung fibrosis and allergic peritonitis model systems respectively  <cit> .
in this article, we show that fizz <dig> and ym <dig> protein expression is induced upon allergen challenge in rag2-/- mice.
we demonstrate for the first time in an allergic lung inflammation model that expression of these aam proteins is completely dependent on signaling through il-4rα and stat <dig>  furthermore, epithelial cells in control mice expressed both fizz <dig> and ym <dig> but macrophages expressed only ym <dig>  this was surprising to us since we have shown previously that il- <dig> induces a robust increase in fizz <dig> transcript levels in bone marrow macrophages  within the first  <dig> hours of stimulation  <cit> .
we failed to see fizz <dig> protein even when macrophages were stimulated with il- <dig> in vitro for  <dig> hours.
however, macrophages infected with nematodes or francisella tularensis have been reported to express fizz <dig> protein  <cit> .
the absence of this protein in macrophages in our studies could be due to decreased mrna stability or posttranslational modification of the fizz <dig> mrna, leading to its degradation.
more studies are required to elucidate the molecular basis of this finding.
the functions of the aam proteins in allergic lung inflammation and other diseases are still under debate and may differ depending on the disease setting and signaling pathways involved.
fizz <dig> has been shown to have a protective effect in parasite infection studies.
two groups have shown independently that fizz1-/- mice display significantly higher inflammation in the lung and liver, characterized by increased granuloma formation and extensive fibrosis when infected with schistosoma mansoni or nippostrongylus brasiliensis  <cit> .
it was demonstrated that fizz <dig> is required for worm expulsion and also for suppressing excessive th <dig> inflammation.
on the other hand, fizz <dig> has been implicated in exacerbating pulmonary inflammation, vascular remodeling, collagen and extracellular matrix deposition leading to fibrosis in hypoxia-induced pulmonary hypertension and bleomycin-induced lung fibrosis models  <cit> .
ym <dig> which is a secreted chitinase-like lectin binds to chitin but does not degrade it.
it is postulated that ym <dig> is involved in worm egg degradation and tissue repair  <cit>  and may also be involved in tissue remodeling  <cit> .
more importantly, ym <dig> can act as an eosinophil chemoattractant  <cit> .
the reduced expression of fizz <dig> and ym <dig> in the absence of stat <dig> or il-4rα may be functionally linked to the reduced inflammation and eosinophilia seen in stat6xrag2-/- and il-4rαxrag2-/- mice.
moreover, we found that there was a significant decrease in collagen deposition and airway smooth muscle thickness in mice lacking stat <dig> and il-4rα.
enhanced inflammation and eosinophilia as well as aam products can contribute to airway remodeling.
studies have shown that depletion of the eosinophil lineage protected both humans and mice from deposition of collagen, extracellular matrix proteins and increases in smooth muscle mass  <cit> .
it was recently shown that another chitinase-like protein brp- <dig>  was highly upregulated in alveolar macrophages and epithelial cells upon ova sensitization and challenge  <cit> .
in the absence of brp <dig> there were reduced antigen-specific th <dig> responses including il- <dig> induced tissue inflammation and fibrosis.
based on these data and our earlier finding that il- <dig> dramatically increases aam gene expression in macrophages  <cit> , we are inclined to think that elevated expression of aam proteins including fizz <dig> and ym <dig> increases the severity of lung pathology.
CONCLUSIONS
in summary, our data demonstrates that in vivo primed cd4+ t cells are able to support allergic lung inflammation.
moreover, stat <dig> and il-4rα play a major role in a range of th <dig> responses but the extent to which these signaling proteins control various aspects of allergic lung disease is variable.
our study establishes that stat <dig> and il-4rα are necessary for fizz <dig> and ym <dig> protein induction but are only partially responsible for the recruitment of eosinophils and pulmonary inflammation.
further research is required to tease out the other pathways that are contributing to the severity of allergic lung inflammation.

 cd4+ t helper type cytokines such as il- il- and il- play a critical role in inducing allergy and asthma. these cytokines act on multiple cells types to initiate and propagate the hallmark features of asthma such as pulmonary inflammation, periodic narrowing of airways and mucus hypersecretion. experiments with mice deficient in these cytokines and studies in asthma patients have confirmed these findings. also, the fact that th cells are required in this disease setting has been demonstrated by using il-4-/- mice and adoptive transfer studies. apart from th cells, il- and il- are also secreted by natural killer t cells, basophils, mast cells, macrophages and activated eosinophils. il- and il- share receptor chains and signaling proteins. binding of either cytokine to the type i or type ii receptor complex leads to the phosphorylation of signal transducer and activator of transcription factor. polymorphisms in the il4ra and stat genes have been linked to increased risk of asthma. there is ample evidence that il- signaling through il-4rα and stat is important for th differentiation and for ige class-switching in b cells. furthermore, mucus hypersecretion, goblet cell hyperplasia and airway hyperresponsiveness were completely abolished in il-4rα-/- or stat6-/- mice. we have previously shown that apart from th cells, il-4rα expression on a population of cd11b+ cells contributed to the severity of lung inflammation and eosinophil recruitment. although these signaling molecules have been studied extensively, there are conflicting reports in the literature regarding the roles of il-4rα and stat in modulating specific features of airway inflammation. some studies have shown that there was no eosinophil recruitment in stat6-/- mice, while other groups including us contend that lung eosinophilia and inflammation are only partially dependent on stat. recently it has been established that il- and il- can promote differentiation of alternatively activated macrophages. during type ii inflammation, aams as well as epithelial cells produce certain characteristic factors such as arginase chitinase- like mammalian proteins and found in inflammatory zone proteins. four different sub-types of fizz proteins have been reported in the literature- fizz1- fizz was originally discovered in the bronchoalveolar lavage fluid in a mouse model of asthma. elevated levels of fizz and ym mrna or protein have since been detected in parasite infection models, allergic lung inflammation, allergic peritonitis, bleomycin-induced lung fibrosis and hypoxia-induced pulmonary hypertension. interestingly, the promoter regions of both fizz and ym have functional binding sites for stat, which explains how il- and il- can induce expression of these proteins. our group has shown previously using in vitro studies, that fizz ym and arginase mrna are preferentially upregulated by il- and to a lesser extent by il-. loss of stat signaling results in a significant reduction in fizz and ym mrna levels in different model systems. however, the effect of il-4rα or stat on fizz1/ym protein induction in an asthma model has not yet been studied. it is well established that signaling through the il- pathway is important for th cell differentiation and cytokine production. il- signaling through the il-4r/stat axis upregulates gata the th master transcription factor. adoptive transfer of wild type th cells into mice is an effective tool to bypass the role of stat and il-4rα in th cell differentiation, while allowing the study of these signaling molecules in the effector phase of asthma. most groups use in vitro-generated th effectors for this purpose. however, this protocol involves several rounds of in vitro priming and expansion using large quantities of il- also, it is becoming evident that in vivo th differentiation is more complex and that certain signals may be missing when cells are differentiated in vitro. it is now known that tslp, il- and il- family cytokines can be involved in induction or maintenance of the th phenotype. in order to avoid using in vitro t cell activation, we isolated ovalbumin -specific cd4+ t cells from either unimmunized do xrag2-/- mice to get naïve cd4+ t cells or do xrag2-/- mice immunized with ova/alum to get in vivo-primed cd4+ t cells. previous dogma led to the assumption that it would be difficult to immunize tcr transgenic mice as t cells in these mice undergo reduced proliferation. however, recent reports indicate that it is possible to immunize tcr transgenic mice and that t cell proliferation and cytokine production are independent of each other. in this study, we found that in vivo primed cd4+ t cells underwent reduced proliferation in a lymphopenic environment when compared to naïve cd4+ t cells. using this modified asthma model with in vivo generated effector t cells, we demonstrate that although there was a reduction in recruitment of inflammatory cells and eosinophils in absence of il-4rα or stat significant amounts of eosinophils were still present both in the bal and also the lung tissue of stat6xrag2-/- and il-4rαxrag2-/- mice. interestingly, ym protein was expressed by both epithelial cells and macrophages, while fizz protein was only expressed by epithelial cells in rag2-/- mice. we further show that fizz and ym protein expression in the lung was completely dependent on signaling through the il-4rα and stat thus it appears that while il-4/il- signaling through il-4rα and stat is essential for induction of aam genes, lung inflammation and eosinophilia are only partially dependent on this pathway.  activation and proliferation status of naïve versus in vivo primed t cells since it has been suggested that th differentiation in vivo is quite complex and cannot necessarily be mimicked by in vitro priming of t cells, we tested the possibility of using either naïve or in vivo primed cd4+ do + t cells. first, we determined if the in vivo primed t cells were capable of secreting more th cytokines as a result of ova/alum immunization of d xrag2-/- mice. splenocytes isolated from either unimmunized or immunized d xrag2-/- mice were stimulated with pma and ionomycin and secretion of il- into the cell culture supernatant was measured by elisa. we found that, just one round of priming with ova/alum was sufficient to induce robust secretion of th cytokines, il- and il-. the amount of il- and il- produced by cells isolated from immunized mice was significantly higher than that of naïve cells. next we compared the ability of the naïve and in vivo primed cd4+ t cells to be activated upon ova/alum priming. since differentiation of cd4+ t cells can often be accompanied by cell proliferation, we also monitored t cell proliferation using 5-bromo-2-deoxyuridine incorporation. in order to test this, we transferred naïve or in vivo primed t cells into stat6xrag2-/- mice by tail vein injection, followed by immunization with ova/alum using a protocol shown in figure 2a. one group of mice was injected with brdu. splenocytes were harvested on day and analyzed by flow cytometry. we found that although the total number of splenocytes isolated from these two groups was similar, the number of do + cd4+ t cells in the in vivo primed transfer group was double that of the naïve transfer group- % vs. %; cells vs. cells. however the percentage of naïve do + cd4+ t cells that were proliferating, as measured by brdu incorporation was higher when compared to the in vivo primed do + cd4+ t cells- 22% vs. 10%. we also looked at cell surface activation markers such as cd expressed by these cells. the majority of cells in both groups on day were cd44hi, although expression of this protein was slightly higher in the group receiving in vivo primed t cells. interestingly, although only 10% of the cd4+ t cells in the primed t cell transfer group and 22% of these cells in the naïve t cell transfer group were brdu+, cd44hi expression by these cells in the two groups of mice was 97% and 82% respectively. this suggests that even cells that did not divide in both groups were expressing high levels of cd and were activated. cell proliferation studies were conducted using the protocol mentioned in figure and materials and methods. briefly, naïve or in vivo primed cd4+ t cells were adoptively transferred into stat6xrag2-/- mice on day followed by ova/alum immunization of day mice were treated with brdu i.p for days. on day splenocytes were harvested, single cell suspensions were prepared and total cell numbers were counted. cells were stained with fluorochrome conjugated antibodies and analyzed by flow cytometry. lymphocytes were gated based on forward and side scatter parameters. the cd4+ do + population in each transfer group was gated based on double expression of cd and kj by each cell. brdu and cd expression in these gated cells was examined. the numbers/percentages in columns 2- were determined by facs analysis. events were collected for each tube/analyte. another set of stat6xrag2-/- mice that received either naïve or in vivo primed do t cells were immunized twice with ova/alum within a span of week. the activation status of the adoptively transferred t cells and the amount of il- produced by these cells was monitored. the number of spleen cells recovered at this stage from the naïve t cell transfer group was twice as much as the number recovered from spleens of mice that had received in vivo primed t cells. moreover, the percentages of naïve do + cd4+ t cells in the spleen were also higher when compared to that of the in vivo primed do + cd4+ t cells. this result correlates well with the brdu incorporation data and indicates that naïve do + cd4+ t cells proliferate more in a lymphopenic environment, which leads to greater accumulation of these cells in spleens of mice at a later time. this accumulation of t cells may be due to homeostatic proliferation. however, the percentage of cells expressing cd and il- was similar in both the naïve and in vivo primed groups on day. t cell activation studies were conducted using the protocol mentioned in figure and materials and methods. briefly, naïve or in vivo primed cd4+ t cells were adoptively transferred into stat6xrag2-/- mice on day followed by ova/alum immunization of days and four days later, splenocytes were harvested, single cell suspensions were prepared and cells were counted. splenocytes were stimulated with pma and ionomycin for h, followed by facs staining and analysis. lymphocytes were gated based on forward and side scatter parameters. the cd4+ do + population in each transfer group was gated based on double expression of cd and kj by each cell. cd expression in these gated cells was examined. the numbers/percentages in columns and were determined by facs analysis. events were collected for each tube/analyte. effect of stat and il-4rα on lung inflammation, eosinophilia and mucus production considering that naïve do + cd4+ t cells were proliferating more in a lymphopenic environment and since we wanted to focus on the effector functions of il- and il- but not their role in priming naïve t cells, we chose the in vivo primed do + cd4+ t cells for all further experiments. several groups including ours have shown that il- and il- signaling through il-4rα and stat plays an important role in inducing and exacerbating eosinophilic inflammation and mucus production in the lungs. since some of these studies were conducted using in vitro generated th effectors, we examined whether similar responses would be observed using in vivo primed t cells. furthermore, although similar studies have been conducted with stat6-/- mice or il-4rα-/- mice alone, no head to head comparisons between mice deficient in stat or il-4rα have been made. to tease out the precise roles played by these signaling molecules, we conducted allergic inflammation studies on rag2-/-, stat6xrag2-/- and il-4rαxrag2-/- mice using our model of transferring in vivo primed t cells. the degree of airway inflammation, eosinophil recruitment and mucus production in the lungs was analyzed in the three groups of mice. as reported earlier, priming with alum alone did not induce eosinophilia and airway inflammation and served as a negative control. upon enumerating the cellular composition in the bal, we found that the total number of cells recovered from ova treated rag2-/- mice was significantly higher than the number of cells recovered from ova treated stat6xrag2-/- and il-4rαxrag2-/- mice.. among the different cell types found in the bal, a 2- fold reduction in the numbers and percentages of eosinophils was seen in stat6xrag2-/- and il-4rαxrag2-/- mice when compared to rag2-/- mice challenged with ova. in each case, the numbers of eosinophils, macrophages and lymphocytes present in the ova treated mice were much greater than the alum treated mice. h&e stained lung sections of ova treated rag2-/- mice demonstrated severe lung inflammation and most of the cellular infiltrate was composed of eosinophils. multinucleated giant cells were also present in large numbers. in contrast, in absence of stat and il-4rα only minor cuffing of the airways and blood vessels was observed. eosinophil recruitment into the lung although reduced, was not completely abolished in stat6xrag2-/- and il-4rαxrag2-/- mice. pas staining on the above lung sections indicated that mucus production by epithelial cells was fully dependent on stat and il-4rα. this is not surprising as it known that mucus production is mainly driven by il- mediated stat activation. the differences in eosinophil counts in the bal in the three mouse strains were recapitulated in the lung tissue. the number of eosinophils recruited to the airways and blood vessels in ova primed and challenged stat6xrag2-/- and il-4rαxrag2-/- mice was significantly reduced but not completely absent. a corresponding increase in mononuclear cell infiltration was observed in these mice. surprisingly, the number of mononuclear cells in stat deficient mice was higher than in il-4rα deficient mice. it is possible that this is linked to the presence of increased numbers of myeloid progenitor cells that have been reported in stat6-/- mice. however, we found significantly higher eosinophils in the lung parenchyma in stat6xrag2-/- and il-4rαxrag2-/- mice, when compared to rag2-/- mice. taken together, these results suggest that in vivo primed cd4+ t cells can induce robust allergic lung inflammation in mice. in this model, stat and il-4rα expression are only partially required for inducing pulmonary inflammation and eosinophilia. chemokine and cytokine profile in the bal in presence or absence of stat and il-4rα il- and il- signaling can induce production of many chemokines by different cell types. eotaxin- and eotaxin- are eosinophil chemoattractive proteins that are predominantly produced by epithelial cells in mice, upon il- or il- stimulation. previous studies have shown that induction of eotaxin, eotaxin and tarc mrna in the lungs of ova-challenged mice was stat dependent. we determined the quantities of eotaxin, tarc and mouse je/ccl secreted into the bal. using our model of in vivo primed t cell transfer and ova-induced allergic lung inflammation, we further show that significantly elevated levels of eotaxin and tarc protein were found in rag2-/- mice when compared head to head with stat6xrag2-/- and il-4rαxrag2-/- mice. a similar trend is seen in the case of je/ccl production. since eotaxin plays an important role in eosinophil trafficking, the reduced amount of eotaxin found in the bal of stat6xrag2-/- and il-4rαxrag2-/- mice could explain the lower numbers of eosinophils present around the airways in mice. as th cytokines have been implicated in allergic lung inflammation, we evaluated il- il- and il- secretion into the lungs and analyzed the contribution of stat and il-4rα head to head in this process, using our in vivo primed t cell model. since we provided wt ova-specific t cells to all three groups of mice, these cells would be able to produce th cytokines. we found that upon priming and challenge with ova, both rag2-/- and stat6xrag2-/- mice secreted similar amounts of il- and il- into the bal. however, significantly higher levels of il- were present in the bal of il-4rαxrag2-/- mice when compared to the other two groups. although not significant, il- secretion in these mice followed a similar trend. it is published that binding of il- to the il-4r complex induces internalization and uptake of the cytokine. thus, in mice deficient in il-4rα, absence of the il-4r on cell surfaces may be preventing the internalization of il- and il- thus increasing the concentration of these cytokines in the bal. similar results were obtained by other groups when antibodies against the il-4rα chain or il-13rα were used. in case of il- increasing amounts of this cytokine was detected in the three mouse strains, with the lowest quantity of il- present in the bal of rag2-/- mice, intermediate levels in stat6xrag2-/- mice and the highest in il-4rαxrag2-/- mice. studies have shown that when in vitro generated th effectors were adoptively transferred into stat6-/- mice, there was a dramatic increase in il- secretion in the bal. the authors speculated that this difference was due to decreased consumption of il- by eosinophils. in our model also, we find that there is a correlation between the degree of eosinophilic inflammation in mice and the amount of il- present in the bal. thus the lower levels of il- found in the bal fluid in rag2-/- mice may be explained by increased consumption of this cytokine by eosinophils recruited into the lungs. migration of th cells into the lungs is independent of stat expression previous studies have shown that stat expression was necessary for th cell trafficking into the lung upon inhalation of ovalbumin. mathew et. al. reported that in the absence of stat less antigen specific th cells migrated into the lungs. to check if this was true in our studies, lung sections were stained with antibodies to cd to identify t cells. since all mice were on a rag deficient background, the only cd3+ t cells present in the lungs were the ova-specific t cells that we adoptively transferred. as evident from figure 4a, absence of stat or il-4rα did not block migration of antigen specific t cells into the lungs of mice. when the cd3+ cells in these mice were quantified, we found that significantly greater numbers of t cells were recruited in the lungs of il-4rαxrag2-/- mice when compared to rag2-/- mice and a similar trend was seen in stat6xrag2-/-. thus when the t cells express stat or il-4rα themselves, deficiency of those proteins in lung resident cells does not affect t cell trafficking. effect of stat and il-4rα on fizz and ym protein expression liu et. al reported that induction of fizz transcripts was stat dependent in a bleomycin-induced lung fibrosis model. ym mrna was also upregulated in a stat dependent manner in a mouse model of allergic peritonitis. however, the expression patterns of these aam proteins by epithelial cells and macrophages have not been studied in allergic lung inflammation. moreover, we have observed a disconnect between the amounts fizz mrna and protein induced by il- stimulated macrophages in vitro. thus, we examined the expression profile of fizz and ym protein in our model and investigated the role of stat and il-4rα in upregulation of these proteins. serial lung sections from ova sensitized and challenged rag2-/-, stat6xrag2-/- and il-4rαxrag2-/- mice were stained with antibodies against both ym and fizz by immunohistochemistry. lung epithelial cells in rag2-/- mice stained strongly for fizz and ym. however, macrophages from these mice were positive for only ym but not fizz. multinucleated giant cells present in the lungs of rag2-/- mice also expressed ym. in comparison, no fizz or ym protein was produced by epithelial cells or macrophages in mice deficient in il-4rα or stat to quantify the amount of fizz and ym protein that was produced by each mouse strain, we analyzed the expression pattern of these proteins secreted into bal fluid by western blotting. total protein present in the bal fluid samples from rag2-/-, stat6xrag2-/- and il-4rαxrag2-/- mice was first quantitated; more total protein was recovered from rag2-/- bal when compared to mice lacking stat or il-4rα. generally, the amount of total protein present in bal correlates with the degree of inflammation seen in mice. in order to compare the quantities of fizz and ym present in the different mouse strains, equal amounts of total bal protein from rag2-/-, stat6xrag2-/- and il-4rαxrag2-/- mice were used. the bal protein samples were resolved by polyacrylamide gel electrophoresis, transferred onto a membrane and probed with antibodies to ym or fizz similar to the immunohistochemistry study, large amounts of fizz and ym were secreted into the bal in rag2-/- mice, but this was greatly reduced in the absence of stat and il-4rα. densitometry analysis of the blots revealed that the differences seen were significant. these results demonstrate that stat activation through il-4rα signaling is required for expression of fizz protein in lung epithelial cells and ym protein in macrophages and epithelial cells during allergic lung inflammation. effect of stat and il-4rα on airway remodeling one characteristic feature of asthma is airway remodeling, which involves an increase in airway smooth muscle mass and enhanced collagen deposition. it has been reported that both eosinophils and aam products such as fizz and ym can cause lung fibrosis and smooth muscle thickening. thus, we analyzed the amount of collagen deposition and airway smooth muscle thickness in rag2-/-, stat6xrag2-/- and il-4rαxrag2-/- mice. masson's trichrome staining of representative lung sections from each mouse strain revealed that greater quantities of collagen was present around the airways and blood vessels in rag2-/- mice, when compared with mice lacking stat and il-4rα. quantification of the collagen staining using image analysis software showed that the differences were significant. furthermore, the thickness of the smooth muscle layer around the airways was also significantly reduced in absence of stat and il-4rα. the airway smooth muscle layer was identified by h&e staining of lung sections and the diameter of the muscle layer was measured at three different points in each airway examined, using image j software. discussion although research on the cytokines il- and il- over the past decade has substantially increased our understanding of their contribution to the pathophysiology of asthma, the extent to which the signaling pathways they activate play a role in airway inflammation and alternative activation of macrophages have not yet been determined. in this study, we used stat and il-4rα deficient mice on a rag2-/- background to examine the role of the il-4/il- pathway in inducing the aforementioned features of allergic lung disease. since th cells are indispensable in this disease setting, we provided t cells exogenously. previously, most groups used in vitro generated th effectors for this purpose. here, we developed a model wherein in vivo primed ovalbumin-specific cd4+ t cells were adoptively transferred into various recipient mice, followed by immunization and challenge with ova. we examined whether naïve cd4+ t cells or in vivo primed cd4+ t cells isolated from do x rag2-/- would be more suitable for this asthma model. we found that in vivo primed t cells proliferated less when compared to naïve t cells as suggested by the following results: i. lower levels of brdu incorporation in cells; ii. the number of cd4+do + cells recovered from lymphopenic mice was half of that recovered from the naïve t cell transfer group. this increased proliferation seen in the naïve t cell transfer group may be due to homeostatic proliferation. it has been demonstrated, that naïve t cells from tcr transgenic mice undergo slow homeostatic proliferation in lymphopenic mice, which is dependent on il-. it has been proposed that entry into the cell cycle and clonal expansion is necessary for t cell differentiation. also, several groups have shown that tcr transgenic mice that have high frequency of antigen-specific t cells show only weak proliferation upon tcr ligation and that the t cells become anergic or die of apoptosis. however, a study conducted by laouar et. al. and our studies have shown that when t cells from tcr transgenic mice were activated in vivo with specific peptide/antigen, these cells express cell surface activation markers such as cd and secrete effector cytokines, in spite of proliferating less. why these transgenic t cells showed reduced proliferation is not known exactly, but it is hypothesized that at high cell frequency, there may be increased competition for growth factors, limited access to peptide/mhc complexes and also limited lymphoid space for expansion. the other difference between our study and the ones where transgenic t cells became anergic/apoptotic is the method of immunization: we used ovalbumin complexed with an adjuvant instead of using the antigen alone as was done previously. thus, our results clearly show that in vivo primed cd4+ t cells from do transgenic mice can be used to induce the hallmark features of asthma in mice. this effect is not restricted to one transgenic mouse strain; similar results were obtained when ot-ii mice were used. in mice that lack stat or il-4rα, th cell differentiation is impaired but they have normal th cell differentiation. in order to track the exogenous in vivo primed t cells that we were transferring into these mice and to prevent interference of th cells, we used stat or il-4rα deficient mice on a rag2-/- background for our asthma experiments. rag2-/- mice were used as controls. in this study, we tested the ability of in vivo primed cd4+ t cells as opposed to in vitro generated th effectors to support allergic lung inflammation. we found that in the absence of stat and il-4rα, mice developed less pulmonary inflammation, reduced perivascular and peribronchial cuffing and decreased eosinophilia than our control mice. mucus production in these mice was abrogated. this was expected since it has been conclusively shown that mucus production is dependent on stat activation by il- signaling. however, both stat6xrag2-/- and il-4rαxrag2-/- mice that were primed and challenged with ova were able to recruit significantly higher numbers of eosinophils when compared to alum primed mice. several studies have shown the importance of these signaling molecules in asthma, but the roles of il-4rα and stat in modulating specific features of airway inflammation were unclear. here we show that stat and il-4rα are only partially required for eosinophil recruitment to the lung. our data matches with what was observed by kuperman et. al. but is in apparent contradiction to that shown by mathew et. al.. furthermore, in contrast to the latter's finding, we observe that there is no defect in t cell recruitment into the lung in absence of stat or il-4rα. differences in the experimental set up may explain these discrepancies: we and kuperman et. al. used two priming steps with ova/alum in the asthma protocol, which was omitted by the other group. recently it was demonstrated that alum stimulates the innate immune system by activating the nalp inflammasome, leading to secretion of pro-inflammatory cytokines such as il-1β, il- and il-. these cytokines play important roles in initiation and amplification of th responses including th cell proliferation and eosinophilia. alum also induces production of il- and il- in t cells. eosinophil migration and recruitment into the lungs depend on several factors. il- controls differentiation, activation and survival of eosinophils in the bone marrow and is essential for their mobilization into the lungs. eosinophil migration into the lung is mediated by adhesion to the vascular endothelium via vcam-. chemokines produced by airway epithelial cells and macrophages recruit eosinophils into the airways. eotaxin- and eotaxin- are eosinophil chemoattractive proteins, mainly produced by airway epithelial cells upon il- or il- stimulation. this was further substantiated by munitz et. al. when they demonstrated that eotaxin secretion in mice is completely dependent on the il-13rα chain and thus the type ii il-4/il- receptor. in our studies, we show that eotaxin secretion in the bal is markedly reduced in the absence of stat or il-4rα. this might explain the lower levels of eosinophils seen around the airways and blood vessels in stat6xrag2-/- and il-4rαxrag2-/- mice in comparison to rag2-/- mice. however, in absence of eotaxin, il- may play a major role in recruitment of eosinophils in stat6xrag2-/- and il-4rαxrag2-/- mice. the higher levels of il- found in the bal in these mice points to this direction. other chemokines such as rantes, monocyte chemoattractant proteins or macrophage inflammatory protein -1α could also play a role in this process. interestingly, we found that greater numbers of eosinophils were present in the lung parenchyma in mice deficient in stat and il-4rα. it is possible that in absence of eotaxin, eosinophils are unable to reach the airways and remain in the parenchyma. in previous studies using wt and stat6-/- mice, th cytokine production was higher in wt mice in comparison to mice lacking stat. this is because stat is required for th cell differentiation. since we provided wt effector t cells to all the groups of mice, they were capable of producing th cytokines. when we measured the amounts of il- and il- in the bal in allergen challenged mice, we found that significantly higher amounts of these cytokines were present in il-4rαxrag2-/- mice than rag2-/- and stat6xrag2-/- mice. studies have shown that binding of il- to the il-4r complex induces internalization and uptake of this cytokine, analogous to that observed with binding of il- to the il2r complex. moreover, other groups have found that il- concentration in the bal was significantly increased when antibodies against the il-4rα chain were injected into mice, compared to control mice. similarly, more il- was found in the bal in il-13rα1-/- mice. therefore, based on our findings and published literature we hypothesize that the absence of il-4rα on cell surfaces may be preventing the internalization of il- and il- thus increasing the concentration of these cytokines in the bal in il-4rαxrag2-/- mice. our data also demonstrated that more il- was secreted into the bal when mice lacked stat or the il-4rα chain. the higher concentration of il- found in stat6xrag2-/- mice in this model are consistent with the results reported by mathew et. al.. they had observed that when in vitro generated th effectors were adoptively transferred into stat6-/- mice, there was a dramatic increase in il- secretion in the bal. the authors speculated that this difference was due to decreased consumption of il- by eosinophils. in our model, since the stat6xrag2-/- and il-4rαxrag2-/-mice have significantly lower levels of eosinophils in both the bal and lung tissue, it is possible that the enhanced cytokine level in the bal in these mice is due to reduced consumption. we did not see any significant differences in ifnγ levels in the three strains of mice. il- is another cytokine that has been implicated in asthma in humans and mice. in our asthma model, il- levels in the bal were below detection limits in all mouse groups. as our understanding of the roles of il- and il- increases, it is becoming clear that in addition to their action on t cells, b cells, eosinophils, epithelial cells, these cytokines can also stimulate macrophages such that they become alternatively activated. instead of expressing inos like the classically activated macrophages, these cells produce proteins such as arginase, fizz and ym1/ among others. it has now been established that il- and il- can also induce expression of the same group of proteins in airway and alveolar epithelial cells. as mentioned earlier, copious amounts of fizz and ym have been detected in the bal of allergen challenged mice. in addition, upregulated levels of fizz and ym mrna have also been found in parasite infection models, allergic lung inflammation and allergic peritonitis, bleomycin-induced lung fibrosis and hypoxia-induced pulmonary hypertension. stutz et. al. demonstrated using the bmnot cell line that the fizz promoter contains functional binding sites for stat and c/ebp. they further showed that stat and c/ebp cooperated together to get optimal fizz expression. welch et. al. showed similar results for the ym promoter region using epithelial and macrophage cell lines: ym has several stat binding sites in its promoter region. additional studies have indicated that induction of fizz or ym transcripts are stat dependent in bleomycin-induced lung fibrosis and allergic peritonitis model systems respectively. in this article, we show that fizz and ym protein expression is induced upon allergen challenge in rag2-/- mice. we demonstrate for the first time in an allergic lung inflammation model that expression of these aam proteins is completely dependent on signaling through il-4rα and stat furthermore, epithelial cells in control mice expressed both fizz and ym but macrophages expressed only ym this was surprising to us since we have shown previously that il- induces a robust increase in fizz transcript levels in bone marrow macrophages within the first hours of stimulation. we failed to see fizz protein even when macrophages were stimulated with il- in vitro for hours. however, macrophages infected with nematodes or francisella tularensis have been reported to express fizz protein. the absence of this protein in macrophages in our studies could be due to decreased mrna stability or posttranslational modification of the fizz mrna, leading to its degradation. more studies are required to elucidate the molecular basis of this finding. the functions of the aam proteins in allergic lung inflammation and other diseases are still under debate and may differ depending on the disease setting and signaling pathways involved. fizz has been shown to have a protective effect in parasite infection studies. two groups have shown independently that fizz1-/- mice display significantly higher inflammation in the lung and liver, characterized by increased granuloma formation and extensive fibrosis when infected with schistosoma mansoni or nippostrongylus brasiliensis. it was demonstrated that fizz is required for worm expulsion and also for suppressing excessive th inflammation. on the other hand, fizz has been implicated in exacerbating pulmonary inflammation, vascular remodeling, collagen and extracellular matrix deposition leading to fibrosis in hypoxia-induced pulmonary hypertension and bleomycin-induced lung fibrosis models. ym which is a secreted chitinase-like lectin binds to chitin but does not degrade it. it is postulated that ym is involved in worm egg degradation and tissue repair and may also be involved in tissue remodeling. more importantly, ym can act as an eosinophil chemoattractant. the reduced expression of fizz and ym in the absence of stat or il-4rα may be functionally linked to the reduced inflammation and eosinophilia seen in stat6xrag2-/- and il-4rαxrag2-/- mice. moreover, we found that there was a significant decrease in collagen deposition and airway smooth muscle thickness in mice lacking stat and il-4rα. enhanced inflammation and eosinophilia as well as aam products can contribute to airway remodeling. studies have shown that depletion of the eosinophil lineage protected both humans and mice from deposition of collagen, extracellular matrix proteins and increases in smooth muscle mass. it was recently shown that another chitinase-like protein brp- was highly upregulated in alveolar macrophages and epithelial cells upon ova sensitization and challenge. in the absence of brp there were reduced antigen-specific th responses including il- induced tissue inflammation and fibrosis. based on these data and our earlier finding that il- dramatically increases aam gene expression in macrophages, we are inclined to think that elevated expression of aam proteins including fizz and ym increases the severity of lung pathology.  in summary, our data demonstrates that in vivo primed cd4+ t cells are able to support allergic lung inflammation. moreover, stat and il-4rα play a major role in a range of th responses but the extent to which these signaling proteins control various aspects of allergic lung disease is variable. our study establishes that stat and il-4rα are necessary for fizz and ym protein induction but are only partially responsible for the recruitment of eosinophils and pulmonary inflammation. further research is required to tease out the other pathways that are contributing to the severity of allergic lung inflammation. 
however, the precise roles played by the il-4/il- receptors and stat in inducing aam protein expression and modulating specific features of airway inflammation are still unclear. moreover, specific aam proteins ym and fizz were expressed by epithelial cells, while macrophages expressed only ym in rag2-/- mice. we further show that fizz and ym protein expression in the lung was completely dependent on signaling through the il-4rα and stat consistent with the enhanced inflammation and aam protein expression, there was a significant increase in collagen deposition and smooth muscle thickening in rag2-/- mice compared to mice deficient in il-4rα or stat although there was reduced inflammation in mice lacking il-4rα or stat significant amounts of eosinophils were still present in the bal and lung tissue. these results establish that transfer of in vivo primed cd4+ t cells can induce allergic lung inflammation. we then used these in vivo generated effector t cells in an asthma model. since th differentiation and activation plays a pivotal role in this disease, we explored the possibility of developing an asthma model in mice using t cells that were differentiated in vivo. further studies are required to identify other proteins and signaling pathways involved in airway inflammation. we found that both the naïve and in vivo primed t cells expressed similar levels of cd and il- however, in vivo primed t cells underwent reduced proliferation in a lymphopenic environment when compared to naïve t cells. cd4+ t helper type cells, their cytokines il- il- and il- and the transcription factor stat are known to regulate various features of asthma including lung inflammation, mucus production and airway hyperreactivity and also drive alternative activation of macrophages. furthermore, while il-4/il- signaling through il-4rα and stat is essential for aam protein expression, lung inflammation and eosinophilia are only partially dependent on this pathway. in this study, we monitored the activation and proliferation status of adoptively transferred allergen-specific naïve or in vivo primed cd4+ t cells.
 cd4+ t helper type cells, their cytokines il- il- and il- and the transcription factor stat are known to regulate various features of asthma including lung inflammation, mucus production and airway hyperreactivity and also drive alternative activation of macrophages. however, the precise roles played by the il-4/il- receptors and stat in inducing aam protein expression and modulating specific features of airway inflammation are still unclear. since th differentiation and activation plays a pivotal role in this disease, we explored the possibility of developing an asthma model in mice using t cells that were differentiated in vivo.  in this study, we monitored the activation and proliferation status of adoptively transferred allergen-specific naïve or in vivo primed cd4+ t cells. we found that both the naïve and in vivo primed t cells expressed similar levels of cd and il- however, in vivo primed t cells underwent reduced proliferation in a lymphopenic environment when compared to naïve t cells. we then used these in vivo generated effector t cells in an asthma model. although there was reduced inflammation in mice lacking il-4rα or stat significant amounts of eosinophils were still present in the bal and lung tissue. moreover, specific aam proteins ym and fizz were expressed by epithelial cells, while macrophages expressed only ym in rag2-/- mice. we further show that fizz and ym protein expression in the lung was completely dependent on signaling through the il-4rα and stat consistent with the enhanced inflammation and aam protein expression, there was a significant increase in collagen deposition and smooth muscle thickening in rag2-/- mice compared to mice deficient in il-4rα or stat  these results establish that transfer of in vivo primed cd4+ t cells can induce allergic lung inflammation. furthermore, while il-4/il- signaling through il-4rα and stat is essential for aam protein expression, lung inflammation and eosinophilia are only partially dependent on this pathway. further studies are required to identify other proteins and signaling pathways involved in airway inflammation. 