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Cellular FLIP Long Form-Transgenic Mice Manifest a Th2 Cytokine Bias and Enhanced Allergic Airway Inflammation ¹

Wenfang Wu^*, Lisa Rinaldi, Karen A. Fortner^*, Jennifer Q. Russell^*, Jürg Tschopp, Charles Irvin and Ralph C. Budd^2,*

^* Immunobiology Program and Vermont Lung Center, Department of Medicine, University of Vermont College of Medicine, Burlington, VT 05405; and Institute of Biochemistry, University of Lausanne, Epalinges, Switzerland

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Cellular FLIP long form (c-FLIP_L) is a caspase-defective homologue of caspase-8 that blocks apoptosis by death receptors. The expression of c-FLIP_L in T cells can also augment extracellular signal-regulated kinase phosphorylation after TCR ligation via the association of c-FLIP_L with Raf-1. This contributes to the hyperproliferative capacity of T cells from c-FLIP_L-transgenic mice. In this study we show that activated CD4⁺ T cells from c-FLIP_L-transgenic mice produce increased amounts of Th2 cytokines and decreased amounts of Th1 cytokines. This correlates with increased serum concentrations of the Th2-dependent IgG1 and IgE. The Th2 bias of c-FLIP_L-transgenic CD4⁺ T cells parallels impaired NF-B activity and increased levels of GATA-3, which contribute, respectively, to decreased IFN- and increased Th2 cytokines. The Th2 bias of c-FLIP_L-transgenic mice extends to an enhanced sensitivity to OVA-induced asthma. Taken together, these results show that c-FLIP_L can influence cytokine gene expression to promote Th2-driven allergic reaction, in addition to its traditional role of blocking caspase activation induced by death receptors.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Naive CD4⁺ T cells can differentiate into two types of effector cells defined by their cytokine phenotype: Th1 and Th2 cells (1, 2). IFN- is the predominant Th1 cytokine and activates macrophages, neutrophils, and CD8⁺ T cells to promote cell-mediated immune responses (3, 4, 5). Th2 cells produce IL-4, IL-5, IL-6, IL-10, and IL-13, which promote, among other functions, B cell proliferation, maturation, and Ab production, thus augmenting the humoral immune response (4, 5, 6, 7). The differentiation of CD4⁺ T cells into Th1 or Th2 cells is tightly regulated. The loss of balance between these two can provoke various inflammatory conditions; asthma and allergic disorders are related to an increased Th2 response, whereas many infectious and autoimmune diseases manifest a prominent Th1 cytokine profile (4, 8, 9, 10, 11).

The factors regulating CD4⁺ T cell cytokine differentiation patterns are not fully understood. IL-4 and IL-6 are strong Th2-promoting cytokines (12, 13), whereas IL-12 is the counterpart that stimulates Th1 differentiation (12). The transcription factors GATA-3 and T-bet are expressed exclusively in, respectively, polarized Th2 cells and Th1 cells (14, 15, 16). GATA-3 is expressed at negligible levels in naive CD4⁺ T cells. Under Th2-skewing conditions, GATA-3 expression is rapidly induced and autoactivates its own expression in a Stat-6-independent manner (17, 18). In vitro studies also demonstrated that GATA-3 is sufficient for directing Th2 development (19, 20). In a similar manner, T-bet is a transcription factor that promotes Th1 differentiation (16, 21). It is proposed that GATA-3 and T-bet can counter-regulate each other, maintaining the Th polarization. Although T-bet is the predominant Th1-specific transcription factor for CD4⁺ T cells, there are certainly additional transcription factors, most notably NF-B, that contribute to IFN- production, as demonstrated most convincingly by the impairment of the production of this cytokine in mice expressing a dominant-negative IB transgene (22). Other factors that influence the Th1/Th2 balance include costimulatory signals, the type of APC, and Ag affinity or dose (23).

We (24) and others (25, 26) recently reported that caspase activity is required for the initiation of T cell proliferation, and that overexpression of the caspase-8-related cellular FLIP long form (c-FLIP_L)³ in T cells resulted in their hyperproliferation (27). FLIP is an antiapoptotic molecule that inhibits the death cascade by all known death receptors (28). Mammalian cells express two c-FLIP splice variants, a short form, c-FLIP_S, and a long form, c-FLIP_L (29, 30). c-FLIP_L contains two death effector domains and a caspase-like domain bearing significant homology to caspase-8 (31). c-FLIP_L lacks the critical Cys and His residues, however, that are located in the catalytic domain of caspase-8, rendering c-FLIP_L enzymatically inert. As such, c-FLIP_L can bind to the adaptor protein, Fas-associated death domain protein, via their mutual death effector domains, competing with caspase-8 recruitment and thus blocking the Fas-triggered death signal pathway (31). In addition, c-FLIP_L promotes activation of the mitogen-activated protein kinase extracellular signal-regulated kinase (ERK) by binding to Raf-1 (32). This may contribute to the hyperproliferative capacity of T cells seen in c-FLIP_L-transgenic (c-FLIP_L-Tg) mice (27). c-FLIP_L can also influence signaling of the NF-B pathway by its ability to recruit receptor-interacting protein (RIP), TNF receptor-associated factor 1 (TRAF1), and TRAF2 (32).

As the ERK and NF-B pathways are involved with the transcriptional regulation of several cytokine genes, we have examined the influence of overexpressed c-FLIP_L on cytokine production by T cells. We observed that CD4⁺ T cells from c-FLIP_L-Tg (c-FLIP_L-transgenic) mice are Th2 biased, and this is regulated at the transcriptional level. This is probably explained by the collective findings in c-FLIP_L-Tg CD4⁺ T cells of decreased NF-B leading to diminished IFN- production as well as increased levels of GATA-3 contributing to the elevated Th2 cytokine expression. The c-FLIP_L-Tg mice also develop greater allergic airway hypersensitivity to OVA, manifesting greater OVA-specific IgE serum levels and lung inflammation. These results extend the known functions of c-FLIP_L from merely a death receptor blocker to one that can also profoundly influence signaling of cytokine gene expression.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
c-FLIP_L-transgenic mice

c-FLIP_L is overexpressed in the T cell compartment as previously reported (27). c-FLIP_L-Tg mice were bred with C57BL/6 mice from The Jackson Laboratory (Bar Harbor, ME) at University of Vermont College of Medicine animal facility.

CD4⁺ T cell purification

Spleen cells after hemolysis were combined with lymph node cells, followed by negative selection to enrich for CD4⁺ cells. Cells were incubated with Abs to CD8 (Tib105), MHC class II (3F12), NK1.1 (PK136), and CD11b (BD PharMingen, San Diego, CA) for 30 min. Samples were washed and then incubated with goat anti-rat/mouse IgG-labeled magnetic beads (Polysciences, Warrington, PA) for 45 min, followed by magnetic field separation to remove CD8⁺ cells, B cells, NK cells, and macrophages. CD4⁺ T cell purity was confirmed by flow cytometry.

Cytokine ELISA

Purified CD4⁺ T cells were cultured at 10⁶/ml with plastic-bound anti-CD3 (10 µg/ml; 145-2C11) and soluble anti-CD28 (1/500 dilution of ascites; 37.51) for 24, 48, and 72 h. Quantification of cytokines (IL-2, IL-4, IL-5, and IFN-) in cell culture supernatants was performed using a sandwich ELISA as previously described (33). In IL-4-neutralization experiments, anti-IL-4 (11B11) was added at 10 or 20 µg/ml. In IFN--compensation experiments, IFN- (R&D Systems, Minneapolis, MN) was added at 200 or 500 U/ml.

RNA preparation and RNase protection assay (RPA)

Total RNA was prepared from cultured CD4⁺ T cells using Ultraspec (Biotecx, Houston, TX) according to the manufacturer’s recommendation. Cytokine RNA levels were determined by RPA using the RiboQuant multiprobe kit (BD Biosciences, San Jose, CA). Five micrograms of total RNA was hybridized overnight with a ³²P-labeled RNA probe that had been synthesized from the multicytokine template set, after which free probe and other ssRNA were digested with RNase.

Naive and memory CD4⁺ T cell purification

Purified CD4⁺ T cells were stained with anti-CD4-FITC and anti-CD44-PE. Naive T cells were sorted as CD4⁺CD44^low, whereas memory T cells were sorted as CD4⁺CD44^high. In separate experiments, naive CD4⁺CD44^low cells were also obtained by depletion of CD44^high cells using magnetic beads.

Gel mobility shift assay

Nuclear extracts were obtained as previously described (34). Binding reactions were performed using 2 µg of nuclear protein in the presence of 10,000 cpm of the specific ³²P end-labeled, double-stranded oligonucleotide for 20 min at 4°C. The oligonucleotides were as follows: AP-1, 5'-GTCGACGTGAGTCAGCGCGC-3'; and NF-B, 5'-GATCAGAGGGGACTTTCCGAG-3'.

AP-1- and NF-B-luciferase reporter mice and luciferase activity

AP-1-luciferase transgenic mice carry the luciferase gene driven by four human collagenase 12-O-tetradecanoylphorbol-13-acetate-responsive elements, which have high affinity for the AP-1 complex, in the context of the rat minimal prolactin promoter (35). The NF-B-luciferase reporter mice have the luciferase gene controlled by two copies of B sequences from the Ig enhancer (36). Purified CD4⁺ T cells were activated with anti-CD3 (10 µg/ml) and anti-CD28 (1/500 dilution of ascites). At 24, 48, and 72 h after activation, cells were harvested, washed with PBS, and lysed. The lysates were then analyzed using luciferin (Promega, Madison, WI) and measured in a luminometer for 10 s. Two measurements were made for each sample. Results are presented as the average of two measurements with background subtracted.

ERK and NF-B activation assays

CD4⁺ T cells were resuspended at 10⁷/ml in the medium. Anti-CD3 Ab (30 µg/ml) and 80 µg/ml goat anti-hamster Ab (Caltag Laboratories, Burlingame, CA) were added and incubated for 15 min at 37°C. Positive controls for activation were obtained using PMA (10 ng/ml; Sigma-Aldrich, St. Louis, MO) and ionomycin (250 ng/ml; Calbiochem, San Diego, CA) for 15 min. Cell lysates were prepared and used in Western blots for phospho-ERK and phospho-IB as described below.

Western blot analysis

Whole cell lysates or nuclear extracts, as indicated, were prepared from c-FLIP_L-Tg CD4⁺ T cells and littermate control CD4⁺ T cells. Whole cell lysates were made as previously described (24). Abs used were: phospho-specific p44/42 mitogen-activated protein kinase (ERK1/ERK2), total p44/42 mitogen-activated protein kinase (ERK1/ERK2), phospho-specific IB, total IB (all from New England Biolabs, Beverly, MA), GATA-3 (Santa Cruz Biotechnology, Santa Cruz, CA), and T-bet (a gift from Dr. L. Glimcher, Harvard University School of Public Health, Boston, MA). Proteins were detected with HRP-conjugated secondary Abs and developed by chemiluminescence.

Determination of serum Ig concentration

The concentrations of Ig subclasses in mouse sera were determined using isotype-specific Abs with a sandwich ELISA protocol. mAbs to mouse IgG1, IgG2a, IgE, IgM, and standards were obtained from BD Biosciences.

OVA-induced airway hypersensitivity

Mice were sensitized with 0.1 ml of 200 µg/ml OVA (grade V; Sigma-Aldrich) in 22.5 mg/ml aluminum hydroxide Imject alum solution (Pierce, Rockford, IL) i.p. on days 0 and 14. Sham-immunized mice received PBS/alum. On days 21, 22, and 23, mice were challenged by exposure to an aerosol of 1% OVA in PBS for 30 min. Mice were studied 48 h later to allow maximal development of eosinophilia and airway hyper-responsiveness.

OVA-specific IgE

Ninety-six-well microtiter plates were coated with OVA (5 µg/ml) in 0.1 M NaHCO₃ at 4°C overnight. Sera were diluted at 1/10 in PBS/1% BSA and tested for OVA-specific IgE with a sandwich ELISA protocol. Color development was read at 450 nm.

Bronchoalveolar lavage (BAL)

BAL cells were collected by lavaging whole lung three times with 1 ml of PBS. The recovery of each lavage was 0.7 ml. Cells from the three lavages were combined and counted with a hemocytometer. For cytospins, 2 x 10⁴ cells were centrifuged onto glass slides at 800 rpm. Cytospins were stained using the Hema3 kit (Biochemical Sciences, Swedesboro, NJ), and differential cell counts were performed on 300 cells.

Lung histopathology

Whole lungs were inflation-fixed by instillation of 4% paraformaldehyde in PBS via the trachea at 30 cm H₂O pressure, then immersed in the same fixative for 24 h. Tissue was embedded in paraffin, and 10-µm sections were stained with H&E for routine histology or periodic acid-Schiff (PAS) for mucus secretion.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
CD4⁺ T cells from c-FLIP_L-Tg mice are Th2 biased

Having previously observed that c-FLIP_L can influence TCR-mediated activation of the ERK and NF-B pathways, we were interested to determine how it might influence cytokine gene expression. Freshly prepared CD4⁺ T cells from c-FLIP_L-Tg mice or their non-Tg littermate controls were stimulated with anti-CD3 and anti-CD28 for up to 3 days. Culture supernatants from days 1, 2, and 3 were analyzed for cytokine production by ELISA. The expression of cytokine mRNA from day 2 cell cultures was determined by RPA. Higher levels of the Th2 cytokines IL-4 and IL-5 and lower levels of the Th1 cytokine IFN- were detected in culture supernatants from c-FLIP_L-Tg CD4⁺ T cells compared with non-Tg control CD4⁺ T cells, whereas IL-2 production showed no difference (Fig. 1A). These results were highly consistent over four experiments. The differences in the amounts of cytokine protein were confirmed at the mRNA level by RPA (Fig. 1B). Each of the Th2 cytokines (IL-4, IL-5, IL-10, and IL-13) had significantly higher mRNA expression in c-FLIP_L-Tg CD4⁺ T cells. In agreement with the protein level, IFN- mRNA expression in c-FLIP_L-Tg CD4⁺ T cells was reduced, whereas there was no significant difference in IL-2 mRNA expression between c-FLIP_L-Tg and wild-type control CD4⁺ T cells.

View larger version (20K): [in this window] [in a new window]   FIGURE 1. c-FLIPL-Tg CD4+ T cells are Th2-biased. Purified CD4+ T cells from c-FLIPL-Tg (FLIP) or non-Tg littermate control (NLC) mice were stimulated with anti-CD3 and anti-CD28 for the indicated times. A, The concentrations of cytokines IL-4, IL-5, IFN-, and IL-2 in the culture supernatants were determined by ELISA. B, Total RNA was prepared from day 2 cell cultures and analyzed by RPA.

Increased IL-4 and decreased IFN- produced by c-FLIP_L-Tg CD4⁺ T cells are independently regulated.

Th1 and Th2 cytokines can negatively regulate each other’s production to maintain a polarized phenotype. The decreased IFN- production by c-FLIP_L-Tg CD4⁺ T cells might thus result from increased IL-4 production or vice versa. To address these two possibilities, we performed studies using IL-4 neutralization and IFN- compensation. For IL-4 neutralization, CD4⁺ T cells from c-FLIP_L-Tg or control mice were stimulated and cultured with or without neutralizing anti-IL-4 mAb, and supernatants were taken on day 2 for measurement of IFN-. Production of IFN- by c-FLIP_L-Tg CD4⁺ T cells was somewhat increased in the presence of the neutralizing anti-IL-4 mAb, but not to the level observed with wild-type CD4⁺ T cells in the presence of equivalent IL-4 inhibition (Fig. 2A). RNA was also prepared from the same cell cultures and assayed by RPA. The mRNA levels of all Th2 cytokines (IL-4, IL-5, IL-10, and IL-13) were decreased in the presence of anti-IL-4 Ab (Fig. 2A), in agreement with the view that IL-4 is a pivotal Th2-promoting cytokine (12). At the same time, IFN- and IL-2 RNA levels were enhanced with IL-4 blocking, although the defect of IFN- production by c-FLIP_L-Tg CD4⁺ T cells persisted (Fig. 2A). The continued decrease in IFN- production was not the result of incomplete blocking of IL-4. Concentrations of anti-IL-4, even 20 µg/ml, did not completely reverse the decreased IFN- production of c-FLIP_L-Tg CD4⁺ T cells (data not shown). The defective production of IFN- was thus partly the result of an intrinsic defect in c-FLIP_L-Tg CD4⁺ T cells.

View larger version (33K): [in this window] [in a new window]   FIGURE 2. Increased IL-4 production and decreased IFN- production by c-FLIPL-Tg CD4+ T cells are independent events. A, CD4+ T cells from c-FLIPL-Tg (FLIP) or non-Tg littermate control (NLC) mice were stimulated with anti-CD3 and anti-CD28, in the absence or the presence of anti-IL-4 mAb (10 µg/ml). Supernatants were taken after 48 h and assayed for IFN-. Similar results were obtained with even higher concentrations of anti-IL-4 (20 µg/ml). Total RNA was prepared from the same cell cultures and subjected to RPA. IFN- expression was quantified by phosphorimager analysis and normalized to L32 plus GAPDH. B, Same conditions as described in A, except 200 U/ml IFN- instead of anti-IL-4 was used. Supernatants from 48-h culture were assayed for IL-4, and RNA was analyzed by RPA. Similar results were obtained with 500 U/ml IFN- (data not shown).

Th2 bias of c-FLIP_L-Tg CD4⁺ T cells exists even at the naive stage

As FLIP is an antiapoptotic molecule, one possibility is that c-FLIP_L-Tg mice have a larger proportion of memory CD4⁺ T cells than their non-Tg littermate controls. This would potentially contribute to the high level of production of certain cytokines. However, c-FLIP_L-Tg mice did not manifest a higher proportion of memory CD44^high CD4⁺ T cells (Fig. 3A), nor did they have any increased cellularity of lymphoid organs (27). Nevertheless, we determined cytokine production in both naive and memory CD4⁺ T cell subsets by sorting the CD4⁺ T cells into CD44^high (memory) and CD44^low (naive) subsets. These were then stimulated with anti-CD3 and anti-CD28 for 2 or 3 days, and culture supernatants were assayed for cytokine production. As anticipated, there was more IL-4 and less IFN- produced by c-FLIP_L-Tg CD4⁺ memory cells (Fig. 3B, right panels). In addition, naive c-FLIP_L-Tg CD4⁺ T cells produced more IL-4 and less IFN- than non-Tg naive CD4⁺ T cells (Fig. 3B, left panels). These findings were consistent in four experiments, and although modest compared with the cytokine levels in memory CD4⁺ T cells, they indicate that the Th2 bias of c-FLIP_L-Tg CD4⁺ T cells is already becoming established in naive T cells.

View larger version (33K): [in this window] [in a new window]   FIGURE 3. The Th2 bias of c-FLIPL-Tg CD4+ T cells is already present at the naive stage. A, The c-FLIPL-Tg mice contain a normal proportion of memory CD4+ T cells. Purified CD4+ T cells from lymph node and spleen were stained for the expression of CD4 and CD44. Numbers indicate the percentage of CD4+CD44high T cells in the boxed region. B, Th2 bias exists in both naive and memory c-FLIPL-Tg CD4+ T cells. Purified CD4+ T cells were sorted into CD44high (memory) or CD44low (naive) populations and stimulated for 2 or 3 days with anti-CD3 and anti-CD28. IL-4 and IFN- levels in the supernatants were measured by ELISA.

To better understand how a death receptor inhibitor might bias cytokine production, we examined signal pathways that influence cytokine gene expression after TCR ligation. Our previous studies have demonstrated that the expression of c-FLIP_L in Jurkat T cells resulted in increased ERK activation upon TCR ligation (32). AP-1 is an important transcription factor for IL-4 gene expression and is partially regulated by ERK activity. We thus examined the ability of nuclear protein to bind an AP-1 consensus site using EMSA. AP-1 binding capacity was very similar between c-FLIP_L-Tg and control CD4⁺ T cells on days 1 and 2 after activation, but by day 3, AP-1 binding was consistently stronger in c-FLIP_L-Tg CD4⁺ T cells (Fig. 4A).

View larger version (30K): [in this window] [in a new window]   FIGURE 4. Increased AP-1 activity and ERK activation in c-FLIPL-Tg CD4+ T cells. A, CD4+ T cells from c-FLIPL-Tg mice (F) or non-Tg littermates (N) were not stimulated (NS) or were stimulated for the times indicated with anti-CD3 and anti-CD28. Nuclear extracts were prepared and tested for AP-1 binding activity by EMSA. B, CD4+ T cells from c-FLIPL-Tg x AP-1-luciferase double-Tg mice or control AP-1-luciferase mice were stimulated for the times indicated with anti-CD3 and anti-CD28. Cells were harvested and lysed, and luciferase activity was measured. C, CD4+ T cells from c-FLIPL-Tg mice or non-Tg littermates were not stimulated (NS) or were stimulated by cross-linked anti-CD3 (CD3) or PMA plus ionomycin (P+I) for 15 min. Whole-cell lysates were prepared and analyzed for phosphorylated ERK and total ERK by Western blot.

Decreased NF-B activation in c-FLIP_L-Tg CD4⁺ T cells

The NF-B pathway is involved with the regulation of several cytokine genes, including IFN- (38, 39). c-FLIP_L binds to the adaptor proteins RIP, TRAF1, and TRAF2, which influence NF-B activation (32). We thus examined the effects of overexpressed c-FLIP_L on the NF-B signaling pathway. EMSA results revealed two bands of NF-B binding activity in CD4⁺ T cells from both strains of mice. Slightly decreased NF-B binding was observed with c-FLIP_L-Tg samples compared with littermate control after 1- or 2-day stimulation (Fig. 5A). This closely corresponded to decreased NF-B activity of CD4⁺ T cells from c-FLIP_L-Tg x NF-B-luciferase mice on days 1 and 2 after CD3 activation (Fig. 5B). Similarly, Western blot analysis revealed that both nonstimulated and activated c-FLIP_L-Tg CD4⁺ T cells manifested slightly less phospho-IB and proportionally more total IB than non-Tg control CD4⁺ T cells (Fig. 5C).

View larger version (31K): [in this window] [in a new window]   FIGURE 5. Decreased NF-B activity in c-FLIPL-Tg CD4+ T cells. A, Nuclear extracts prepared as described in Fig. 4A were tested for NF-B binding activity by EMSA. B, CD4+ T cells from c-FLIPL-Tg x NF-B-luciferase mice or control NF-B-luciferase mice were stimulated, and luciferase activity was measured. C, Whole cell lysates were prepared from either nonstimulated or stimulated cells as described in Fig. 4C. Phosphorylated IB and total IB were analyzed by Western blot. Ponceau S staining shows equivalent loading of protein in each lane.

Consensus and nonconsensus GATA-3 elements have been identified in the IL-4 locus that significantly increase trans-activation of the IL-4 gene (40). GATA-3 can also directly regulate IL-5 and IL-13 expression and inhibit the production of IFN- (19, 41, 42). By contrast, T-bet expression strongly correlates with IFN- production (16). We thus examined GATA-3 and T-bet protein expression in c-FLIP_L-Tg CD4⁺ T cell nuclear extracts. GATA-3 expression was significantly increased in resting c-FLIP_L-Tg CD4⁺ T cells and persisted through the first day of T cell activation. Conversely, T-bet expression was decreased in c-FLIP_L-Tg CD4⁺ T cells (Fig. 6A). Given the earlier finding that naive c-FLIP_L-Tg CD4⁺ T cells were already Th2-biased, we examined whether there were higher levels of GATA-3 in naive c-FLIP_L-Tg CD4⁺ T cells before activation. Indeed, purified naive c-FLIP_L-Tg CD4⁺ T cells showed higher expression of GATA-3 as well as lower expression of T-bet compared with wild-type naive CD4⁺ T cells (Fig. 6B). Thus, the propensity toward a Th2 program appears to be already established in naive c-FLIP_L-Tg CD4⁺ T cells.

View larger version (23K): [in this window] [in a new window]   FIGURE 6. Elevated levels of GATA-3 and decreased T-bet in naive c-FLIPL-Tg CD4+ T cells. A, Nuclear extracts from nonstimulated (NS) or CD3/CD28-stimulated total CD4+ T cells were probed for the expression of GATA-3 or T-bet by Western blot (N, non-Tg littermate control; F, c-FLIPL-Tg). B, Nuclear extracts from purified naive CD4+ T cells were immunoblotted for the expression of GATA-3 and T-bet.

The production of different Ig subclasses is differentially regulated by Th1 and Th2 cytokines. IFN- induces the IgG2a and IgG3 isotypes, whereas IL-4 promotes Ig class switching to IgG1 and IgE (8). To assess whether the Th2 bias in vitro of c-FLIP_L-Tg CD4⁺ T cells has an in vivo correlate, we measured serum Ig isotype concentrations by ELISA. The mean levels of serum IgG1 and IgE were statistically (p < 0.05) increased in c-FLIP_L-Tg mice compared with non-Tg littermate control mice, whereas the concentrations of IgG2a and IgM were not significantly different between the two strains of mice (Fig. 7). These data are consistent with the view that c-FLIP_L is involved in Th2 differentiation in vivo, leading to Th2 cytokine-dependent IgG1 and IgE production.

View larger version (23K): [in this window] [in a new window]   FIGURE 7. Increased serum IgG1 and IgE concentrations in c-FLIPL-Tg mice. Serum samples were obtained from 8- to 12-wk-old non-Tg littermate control (NLC) and c-FLIPL-Tg (FLIP) mice. The amounts of IgG1, IgG2a, IgE, and IgM subclasses were determined by ELISA using isotype-specific Abs. Squares indicate individual mice, and the bar indicates the mean titer. Differences were statistically significant for IgE and IgG1 (*, p < 0.05).

Human and murine studies of allergic asthma have demonstrated a clear Th2 cytokine response. To further explore the in vivo significance of the Th2 bias observed with c-FLIP_L-Tg CD4⁺ T cells, we examined a murine model of asthma in c-FLIP_L-Tg mice. The c-FLIP_L-Tg mice and their non-Tg littermate controls were sensitized to OVA and then exposed to nebulized OVA as described in Materials and Methods. Sera from OVA-sensitized c-FLIP_L-Tg mice contained higher levels of OVA-specific IgE than OVA-sensitized non-Tg littermates (Fig. 8A).

View larger version (20K): [in this window] [in a new window]   FIGURE 8. Increased OVA-specific IgE serum levels and airway cellularity of c-FLIPL-Tg mice in OVA-induced airway inflammation. Mice were immunized with PBS/alum (control) or OVA/alum (OVA sensitized) and subsequently received nebulized OVA according to the protocol detailed in Materials and Methods. A, OVA-specific IgE serum levels were determined by ELISA. Points indicate individual mice. B, The total and differential cell counts in the BAL are shown (*, p < 0.05). Results shown are representative of four experiments.

Lung histology revealed the same trend as seen in BAL. As shown for one of four experiments in Fig. 9A, the lungs of sham-immunized non-Tg littermate control mice were devoid of visible inflammation, consistent with the low cell counts in the BAL. This was also true of sham-immunized c-FLIP_L-Tg mice. OVA-sensitized littermate control mice showed moderate inflammation, typical of their C57BL/6 background, whereas the lungs of OVA-sensitized c-FLIP_L-Tg mice manifested marked inflammation, with striking leukocyte infiltration around airways and blood vessels. Most of these infiltrating leukocytes were lymphocytes and eosinophils. The epithelium along the airway was also severely fragmented in the c-FLIP_L-Tg mice. Another frequent histologic feature of asthma is mucus overproduction by the airway epithelium. This is believed to be stimulated to a large extent by IL-13 (45). IL-13 mRNA levels were greatly increased in c-FLIP_L-Tg CD4⁺ T cells. Lung sections were stained with PAS to assess mucus production. As shown in Fig. 9B, there was a dramatic increase in mucus production along the airways of OVA-sensitized c-FLIP_L-Tg mice compared with OVA-sensitized littermate controls.

View larger version (50K): [in this window] [in a new window]   FIGURE 9. Increased OVA-induced airway inflammation and mucus production in c-FLIPL-Tg mice. Lung tissue sections from the indicated groups of mice were prepared as described in Materials and Methods. A, H&E staining of lung sections (magnification, x200). B, PAS staining of lung sections. Note magenta staining of mucus (arrow).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

The c-FLIP_L overexpression enhances phosphorylation and activation of ERK in Jurkat T cells after TCR ligation via the recruitment of Raf-1 to c-FLIP_L (32). ERK was also rapidly phosphorylated after TCR triggering of c-FLIP_L-Tg CD4⁺ T cells in the current study. As the activation of the Ras/ERK pathway is required for IL-4R function (e.g., STAT-6 phosphorylation) and the differentiation of CD4⁺ Th2 cells (46), increased ERK activation in c-FLIP_L-Tg CD4⁺ T cells may be partly involved in their Th2 bias. ERK activation promotes c-Fos expression, a component of the AP-1 complex (47). This concurs with the increased AP-1 binding and transcriptional activity of activated c-FLIP_L-Tg CD4⁺ T cells. Thus, the enhanced ERK phosphorylation in c-FLIP_L-Tg CD4⁺ T cells may be linked to the increased AP-1 activity and Th2 bias of these cells.

NF-B is another important cytokine transcription factor activated by TCR ligation and influenced by c-FLIP_L (48). Kataoka et al. (32) observed that c-FLIP_L could activate NF-B either spontaneously in 293 human embryonic kidney cells or in Jurkat T cells after TCR stimulation. However, Wajant et al. (49) found that overexpression of c-FLIP_L inhibited death receptor-induced NF-B activation. These disparate findings might reflect the cell type and particularly the levels of c-FLIP_L expression. An additional regulatory factor in NF-B regulation in c-FLIP_L-Tg T cells is probably caspase-8. Recently, we observed that c-FLIP_L can bind to and activate pro-caspase-8 (50). The augmented caspase-8 activity that could accompany increased c-FLIP_L expression may serve to cleave and inactivate molecules that are important to NF-B activation. In this regard it is worth noting that RIP, an adaptor protein in NF-B activation, associates with c-FLIP_L and is cleaved by caspase-8 (50). Cleaved RIP acts as a dominant inhibitor of NF-B and could therefore be more pronounced in c-FLIP_L-Tg CD4⁺ T cells. In this regard, we observed cleavage of RIP after activation of wild-type T cells, and this was present even in resting T cells from c-FLIP_L-Tg mice (J. Q. Russell, unpublished observations). Some of the T cell aberrations in the c-FLIP_L-Tg mice might be explained in part by diminished NF-B activity, and they resemble certain aspects of the dominant repressor IB-Tg mice. Both mice manifest a partial selective loss of CD8⁺ T cells as well as defective IFN- production of CD4⁺ T cells (22, 51).

In addition to the described general transcription factors that influence both Th1 and Th2 cytokine gene transcription, there are specific transcription factors influencing the differentiation of Th precursor cells. T cells overexpressing GATA-3 or T-bet develop into, respectively, Th2 or Th1 cells (15, 16). GATA-3 and T-bet work as complementary transcriptional regulators of cytokine genes and facilitate the stabilization of a polarized phenotype. GATA-3 can be induced by IL-4 via a STAT-6-dependent process (19). GATA-3 can also self-regulate through a positive feedback loop, thus promoting Th precursor cells along a Th2 developmental pathway (17). Several GATA-3 binding sites have been found within the IL-4/IL-13 gene locus that help activate IL-4/IL-13 gene expression. GATA-3 is generally expressed at low levels in primary resting T cells and increases rapidly with activation (17, 18). The c-FLIP_L-Tg CD4⁺ T cells revealed higher levels of GATA-3 expression even before stimulation. This was already true within purified naive CD4⁺ T cells, accompanied by decreased levels of T-bet. This could provide a plausible explanation for the Th2 bias of the c-FLIP_L-Tg CD4⁺ T cells.

A cardinal feature of allergic asthma is a predominant Th2 cytokine profile. This is evidenced by the IL-4 induction of IgE class switching, IL-5-induced eosinophilia, and IL-13-regulated airway mucus production (52). The increased levels of IL-4, IL-5, and IL-13 observed in vitro in c-FLIP_L-Tg CD4⁺ T cells were reflected in vivo by the development of aggravated asthma, including up-regulated serum levels of OVA-specific IgE, increased infiltration of leukocytes (especially eosinophils in BAL and lung tissue), and severely damaged airway epithelium as well as goblet cell metaplasia and hypersecretion of mucus.

Our studies expend the functions of c-FLIP_L beyond that of an inhibitor of death receptors. Given the ability of c-FLIP_L to associate with Raf-1, RIP, and TRAF1/2, the current observations reveal new signaling capabilities for c-FLIP_L that profoundly affect the effector function of CD4⁺ T cells. Determining whether death receptors are involved with this function of c-FLIP_L and exactly what complexes associate with c-FLIP_L will be central to further understanding this process. Furthermore, determining the regulation of c-FLIP_L expression in normal CD4⁺ T cells is pivotal to understanding more fully the regulation of their effector function.

	Acknowledgments

 
We thank Colette Charland for technical assistance with flow cytometry, and Dr. Karen Fortner for helpful comments during the preparation of the manuscript.

	Footnotes

 
¹ This work was supported by grants from the National Institutes of Health (AI36333 and AI45666 (to R.C.B.) and National Center for Research Resources COBRE PZO-15557 (to C.I.)).

² Address correspondence and reprint requests to Dr. Ralph C. Budd, Immunobioloy Program, University of Vermont College of Medicine, Given Medical Building, D305, 89 Beaumont Drive, Burlington, VT 05405-0068. E-mail address: ralph.budd{at}uvm.edu

³ Abbreviations used in this paper: c-FLIP_L, cellular FLIP long form; BAL, bronchoalveolar lavage; c-FLIP_S, cellular FLIP short form; ERK, extracellular signal-regulated kinase; PAS, periodic acid-Schiff; RIP, receptor-interacting protein; RPA, RNase protection assay; Tg, transgenic; TRAF, TNF receptor-associated factor.

Received for publication December 8, 2003. Accepted for publication February 5, 2004.

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