Differential regulation of VLA-2 expression on Th1 and Th2 cells: a novel marker for the classification of Th subsets

Kotaro Sasaki1, Takemasa Tsuji1, Takafumi Jinushi1, Jyunko Matsuzaki1, Takeshi Sato1, Kenji Chamoto1, Yuji Togashi1, Toshiaki Koda1 and Takashi Nishimura1

1 Division of Immunoregulation, Section of Disease Control, Institute for Genetic Medicine, Hokkaido University, Sapporo 060-0815, Japan

Correspondence to: T. Nishimura; E-mail: tak24{at}imm.hokudai.ac.jp
Transmitting editor: T. Saito


    Abstract
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
We found that Th1 cells derived from ovalbumin (OVA)-specific TCR transgenic (DO11.10) mice showed significantly higher levels of VLA-2 (CD49b/CD29) expression than Th2 cells. In the early days (until 6 days) during induction of Th1 or Th2 cells, the expression of VLA-2 was gradually increased on both Th subsets. Thereafter, VLA-2 expression was further up-regulated on Th1 cells until 13 days, while a significant decrease of VLA-2 was observed in Th2 cells, resulting in a marked difference of expression at day 13. Up-regulation of VLA-2 on Th1 cells was not impaired in IFN-{gamma}–/– Th cells nor blocked by anti-IL-12 mAb treatment on wild-type Th cells, suggesting that up-regulation of VLA-2 on Th1 cells occurs in an IFN-{gamma}- and IL-12-independent manner. In contrast, Th cells cultured under IL-4-depleted Th2 conditions abrogated the down-regulation of VLA-2 expression, suggesting that down-regulation of VLA-2 expression on Th2 cells was dependent on IL-4. The finding that STAT6–/– Th2 cells did not show any down-regulation of VLA-2 expression and expressed the same levels of VLA-2 as Th1 cells indicated a critical role for the IL-4 receptor/STAT6 signaling pathway in IL-4-depedent down-regulation of VLA-2 on Th2 cells. Stimulation of Th1 cells by VLA-2 ligands such as collagen type I or agonistic mAb provided co-stimulation for anti-CD3 mAb-induced IFN-{gamma} production. However, these ligations had little effect on the IL-4 production of Th2 cells. Together, these results indicate that VLA-2 is a novel functional marker that dissociates Th1 from Th2 cells, and thus might be useful for therapeutic monitoring of Th1-dependent immune diseases such as rheumatoid arthritis or Crohn’s disease.

Keywords: CD49b, CD29, integrin, IL-4, STAT6


    Introduction
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Immune systems are largely regulated by cytokines differentially produced by two distinct Th subsets, Th1 and Th2 cells. Th1 cells, that produce IFN-{gamma} and IL-2, activate macrophages and cytotoxic T lymphocytes to promote cell-mediated immunity against intracellular pathogens, while Th2 cells producing IL-4, IL-5, IL-9 and IL-13 play an important role in the regulation of humoral immunity against extracellular pathogens (1,2). The imbalance between Th1 and Th2 immunity has recently been suggested to be critical for the outcome of various immune diseases such as inflammatory autoimmune diseases, infectious diseases and allergy (3,4). Thus, identification and characterization of selective cell-surface molecules on Th1 or Th2 cells is of great importance for monitoring immune diseases and understanding their pathogenesis. Recently, some surface molecules have been proposed to be efficient in distinguishing Th1 and Th2 cells. For example, it has been reported that chemokine receptors such as CCR5 and CXCR3 are preferentially expressed on Th1 cells, whereas CCR3, CCR4 and CCR8 are mainly expressed on Th2 cells (511). In addition, active ligands of P- and E-selectin are dominantly expressed on Th1 cells (12). The expression patterns of these chemokine receptors and selectin ligands seem to explain the distinct tissue distribution profiles between the two cell subsets. Other cell-surface molecules such as IL-12Rß (13,14), IL-18R (15), LAG-3 (16), Tim-3 (17) or ST2L (18), CRTH2 (19) and CD30 (20) have also been identified as markers for the classification of Th1 or Th2 cells.

In this report, we have for the first time demonstrated that VLA-2 (CD49b/CD29) could serve as a novel cell-surface marker for Th1/Th2 cell dissociation. VLA-2 is a heterodimeric surface molecule composed of non-covalently bound {alpha}2 (CD49b) and ß1 (CD29) integrin chains that mediate specific cell adhesion towards extracellular matrix (ECM) components such as collagen type I and IV (21). VLA-2 expressed on T cells has been shown to be involved in T cell activation, cell anchorage on collagen, signal transmission for cell activation, proliferation and survival (2227). Consistent with the literature, we found that stimulation of Th1/Th2 subsets with VLA-2 ligands combined with immobilized anti-CD3 mAb caused marked production of IFN-{gamma} by Th1 cells, while a negligible effect was observed for IL-4 production by Th2 cells. Our findings show that VLA-2 (CD49b/CD29) is a novel functional marker for the classification of Th1 from Th2 cells and thus may be useful for the diagnosis or monitoring of the Th1/Th2 imbalance involved in various immune diseases.


    Methods
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Mice
Ovalbumin (OVA)323–339-specific I-Ad-restricted DO11.10 TCR{alpha}ß transgenic mice (BALB/c background) were donated by Dr Kenneth M. Murphy [Department of Pathology, Washington University School of Medicine St Louis, MO (28)]. IFN-{gamma}–/– C57BL/6 mice were kindly provided by Dr Y. Iwakura (Institute of Medical Science, University of Tokyo, Tokyo, Japan) and then backcrossed to DO11.10 transgenic mice for more than six generations, generating IFN-{gamma}–/– DO11.10 transgenic mice. STAT6–/– (BALB/c background) mice were kindly provided by Tularik (San Francisco, CA). BALB/c mice were purchased from Charles River Japan (Yokohama, Japan). All mice were female and were used at 5–10 weeks of age.

Generation of Th1 and Th2 cells
Th1 and Th2 cells were induced from FACS Vantage (Becton Dickinson, San Jose, CA)-sorted CD4+CD45RB+ naive Th cells from DO11.10, IFN-{gamma}–/–DO11.10, wild-type BALB/c and STAT6–/– BALB/c mice respectively. Purified CD4+CD45RB+ cells were stimulated with 5 µg/ml OVA323–339 peptide (for transgenic Th cells) or 2 µg/ml soluble anti-CD3 mAb (for non-transgenic Th cells) in the presence of mitomycin C-treated BALB/c spleen cells as feeder cells, 20 U/ml IL-12, 1 ng/ml IFN-{gamma}, 50 µg/ml anti-IL-4 mAb and 100 U/ml IL-2 for Th1 development. Th cell development in the IFN-{gamma}-depleted Th1 condition was performed using IFN-{gamma}–/– DO11.10 and IFN-{gamma}–/– BALB/c as feeder cells. Th1 cells in the IL-12-depleted condition were induced using 50 µg/ml anti-IL-12 mAb without adding IL-12 to the Th1 cytokine condition mentioned above. Th cells in the IL-4-depleted Th2 condition were induced by adding anti-IFN-{gamma} mAb, anti-IL-12 mAb and anti-IL-4 mAb with 100 U/ml IL-2. Th2 cells were induced from the same naive Th cells in the presence of 1 ng/ml IL-4, 10 µg/ml anti-IFN-{gamma} mAb, 50 µg/ml anti-IL-12 mAb and 100 U/ml IL-2. At 48 h, cells were re-stimulated under the same conditions. Cytokines were added for the whole culture period. In some experiments, IL-12 and IFN-{gamma} were added only for the initial 5 days and then removed. At day 8 post-stimulation, cells were collected to measure intracellular IFN-{gamma} and IL-4 expression by flow cytometry after intracellular staining as previously described (29).

Cytokines, mAb and antigens
IL-12 was donated by Genetics Institute (Cambridge, MA). Anti-IL-12 mAb (C15.1 and C15.6) were a gift from Dr G. Trinchieri (Wistar Institute of Anatomy and Biology, Philadelphia, PA). Phorbol myristate acetate, Brefeldin A and recombinant murine IL-4 were purchased from Wako Pure Chemical (Osaka, Japan). Anti-IL-4 mAb (11B11) was purchased from ATCC (Rockville, MD). Phycoerythrin (PE)–anti-CD4 mAb, PE–anti-CD49b mAb (HM{alpha}2), PE–DX5 mAb, FITC–anti-CD45RB mAb, FITC–anti-CD29 mAb (HMß1), purified anti-CD3 mAb, purified anti-CD49b mAb, purified anti-CD29 mAb, control hamster Ig for the control towards HM{alpha}2 and HMß1, purified anti-FcR{gamma} mAb, and recombinant mouse IFN-{gamma} and anti-IFN-{gamma} mAb (R4-6A2) were purchased from PharMingen (San Diego, CA). OVA323–339 peptide was supplied by Dr H. Tashiro (Fujiya, Hadano, Japan).

Flow cytometric analysis
CD49b and CD29 expression on Th1 and Th2 cells was determined each day by three-color flow cytometry after staining with PE-conjugated anti-CD49b mAb (HM{alpha}2) or PE–DX5 mAb, FITC–anti-CD29 mAb (HMß1) and PerCP–anti-CD4 mAb performed in accordance with the manufacturer’s instructions. CD49b and CD29 expression on naive Th cells was measured by three-color flow cytometry after preincubated with anti-Fc{gamma}R mAb followed by staining with PE–anti-CD49b mAb or PE–DX5 mAb, FITC–anti-CD45RB mAb and PerCP–anti-CD4 mAb for the measurement of CD49b expression or PE–anti-CD45RB, FITC–anti-CD29 and PerCP– anti-CD4 mAb for the measurement of CD29 expression.

RT-PCR
Total RNA from cultured Th1 and Th2 cells at day 13 was isolated with the Isogen kit (NipponGene, Toyama, Japan) according to the manufacturer’s recommendations. Expression of CD49b, CD29 and CD25 mRNA in Th1 and Th2 was determined by RT-PCR.

As an internal control, we also measured mRNA levels of ß-actin. The cDNA mixture was synthesized from 5 µg of total RNA by reverse transcription using oligo(dT) primer and Superscript II reverse transcriptase (Invitrogen Corp., Carlsbad, CA) in a total reaction volume of 20 µl. PCR was performed using 2 µl of cDNA mixture, 1 µl of each primer set and Taq DNA polymerase (Sigma, St Louis, MO) in a total volume of 20 µl. Nucleotide sequences of the forward (-F) and reverse (-R) primers were as follows: CD49b-F, 5'-AGA ACC CAC TCC TGT ATC TGA C-3'; CD49b-R, 5'-GAG TTC TGT GGT CTC ATC CAT C-3'; CD29-F, 5'-GGG AGG CAC TGT GAA TGT AGC-3'; CD29-R, 5'-TCC TGT GCA CAC GTG TCT TTC-3'; CD25-F, 5'-AAC GGC ACC ATC CTA AAC TG-3'; CD25-R, 5'-GGC AGG AAG TCT CAC TCT CG-3'; ß-actin-F, 5'-GTG ATG GTG GGA ATG GGT CAG-3'; ß-actin-R, 5'-TTT GAT GTC ACG CAC GAT TTC C-3'. The amplification protocol consisted of denaturation for 30 s at 94°C, annealing for 30 s at 55°C and extension for 1 min at 72°C for a total of 28–35 cycles, using a GeneAmp PCR system model 9700 (Perkin Elmer, Boston, MA). The PCR products were visualized with ethidium bromide staining under UV light following electrophoresis on 4% agarose S (Wako Pure Chemical) gels.

Measurement of IFN-{gamma} and IL-4 production
TC 96-well plates (060715; Nunc, Rochester, NY) were coated with 50 µl of a suboptimal dose of anti-CD3 mAb (5 and 2 µg/ml respectively) in PBS for 2 h at 37°C. After removal of anti-CD3 solution, 10 µg/ml of ECM solution (collagen type I or BSA as a control) in double-distilled water or 10 µg/ml of anti-VLA-2 mAb (HM{alpha}2 and HMß1) or control hamster mAb in PBS was subsequently coated for 2 h at 37°C, and then washed once with double-distilled water or PBS respectively and used for assays. Cells were harvested at day 10–14 after stimulation, washed twice with 1% BSA in serum-free RPMI and then cell suspension in 1% BSA in serum-free RPMI was added to each well at 1 x 105/well. After 8 or 12 h incubation at 37°C respectively supernatants were collected, and IFN-{gamma} and IL-4 levels were measured by ELISA.


    Results
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
VLA-2 as a novel marker for Th1/Th2 cells
When investigating functional molecules expressed on mature Th1 and Th2 cells induced from CD4+CD45RB+ naive Th cells, we incidentally found that VLA-2, which consisted of CD49b and CD29, was highly expressed on Th1 cells compared with Th2 cells (Fig. 1A). To understand how VLA-2 expression is regulated during differentiation of Th1 and Th2 cells, we next examined the kinetics of CD49b/CD29 expression on both Th subsets. Although CD49b was undetectable on naive T cells, its expression gradually increased on both Th cells cultured under either Th1 or Th2 cytokine conditions until day 6. Thereafter, however, CD49b expression was consistently up-regulated on Th1 cells, while a marked decrease was observed on Th2 cells. As a result, fully mature Th1 cells expressed higher levels of CD49b compared with mature Th2 cells at day 13, i.e. CD49b was found to be expressed on >92% of Th1 cells, but <11% of Th2 cells. The different expression levels of CD49b between Th1 and Th2 cells were also defined using the DX5 mAb that has recently been shown to recognize CD49b (30), although the expression was shown to be lower than that of HM{alpha}2 staining, consistent with this previous report (Fig. 1B and C).



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Fig. 1. Kinetics of CD49b/CD29 expression on Th1 and Th2 cells. Th1 and Th2 cells were induced from naive Th cells as described in Methods. Th1 and Th2 cells were stained with PE–anti-CD49b mAb (HM{alpha}2), PE–DX5 mAb, FITC–anti-CD29 mAb (HMß1) and PerCP–anti-CD4 mAb each day since day 4. (A) Double-color flow cytometric analysis of the expression of CD49b ({alpha}2 integrin) and CD29 (ß1 integrin) on Th1 and Th2 cells at day 13 after primary antigen challenge. (B) Histogram analysis of CD49b, CD29 and DX5 expression on Th1 (Thick line), Th2 (solid line) and control (dotted line) cells at day 0, 4, 5, 7, 10 and 13. (C) Changes of CD49b and CD29 expression on Th1 and Th2 cells. The results represent mean ± SD of three independent experiments.

 
A similar regulation pattern was also confirmed for CD29 expression on Th1 and Th2 cells. During the early days of culture (until 6 day), CD29 expression on both Th subsets rapidly increased and its elevation levels were much higher than for CD49b expression. Almost all cells stained highly with anti-FITC–CD29 mAb (>97%) at day 4–6 on both Th1 and Th2 cells. However, CD29 expression on Th2 cells began to decrease thereafter and finally only <16% of Th2 cells were CD29+. In contrast, most Th1 cells sustained high levels of CD29 expression until day 13. These results clearly demonstrated that the expression of VLA-2, which consisted of CD49b and CD29, is differently regulated in Th1 and Th2 cells, and the VLA-2 molecule might serve as a novel cell-surface marker for the dissociation of Th1 from Th2 cells.

As described above, antigen stimulation rapidly up-regulated VLA-2 expression on Th cells for the initial 6 days of culture under both Th1 and Th2 cytokine conditions. These data suggested that a continuous activation signal through TCR may counteract against IL-4 effects on VLA-2 expression. To verify this hypothesis, we examined the kinetics of VLA-2 expression on repeatedly anti-CD3-stimulated Th cells cultured under either Th1 or Th2 cytokine conditions. As clearly illustrated in Fig. 2, it was demonstrated that repeated anti-CD3 stimulation counteracted IL-4-induced down-modulation of VLA-2 expression on Th2 cells and rapidly increased CD49b expression on Th2 cells to levels comparable to that of Th1 cells. However, once anti-CD3 stimulation stopped, Th2 cells soon showed decreased VLA-2 expression, although Th1 cells continued to express high levels of VLA-2. These data supported our idea that VLA-2 serves as a marker for the classification of Th subsets even after repeated TCR-mediated activation.



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Fig. 2. TCR stimulation counteracts against the IL-4 effect on VLA-2 down-modulation. Naive Th cells were cultured under Th1- or Th2-inducing conditions without re-stimulation (diamonds) or re-stimulated with anti-CD3 mAb at 4 days (triangles), at 4 and 6 days (circles) or at 4, 6 and 8 days (squares). The expression of CD49b (A) or CD29 (B) on Th1 cells (closed symbols) or Th2 cells (open symbols) was determined by FACS as indicated.

 
Difference in CD49b/CD29 mRNA expression between Th1 and Th2 cells
To examine whether these differences of expression could be seen at the mRNA level, we next carried out RT-PCR on RNA extracted from Th1 and Th2 cells at day 13 after antigen challenge. Then, CD49b, CD29 and CD25 mRNA expression on Th1 and Th2 cells was compared. Th1 showed higher mRNA expression of both CD49b (6.0-fold increase) and CD29 (3.6-fold increase) compared with Th2 cells, while CD25 (IL-2R{alpha} chain) is equally expressed on both Th cell types. Some visible levels of CD29 expression on Th2 cells may reflect the slight, but significant, protein expression on Th2 cells (Fig. 3). These data further reinforce that VLA-2 is highly expressed on Th1, rather than Th2, cells even at the mRNA level.



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Fig. 3. Differential mRNA expression of CD49b and CD29 on Th1 and Th2 cells. Expression of CD49b, CD29 and CD25 mRNA levels were determined by RT-PCR at day 13 after stimulation with antigen. As a control, ß-actin mRNA levels of the cells were determined.

 
Differential regulation mechanisms of VLA-2 expression on Th1 and Th2 cells
To elucidate the mechanism underlying the differential VLA-2 expression on Th1 and Th2 cells, naive T cells from DO11.10 mice were subjected to culture with OVA under various cytokine conditions. As shown in Fig. 4, Th cells cultured under Th1 cytokine conditions in the presence of anti-IL-12 mAb (IL-2, IFN-{gamma}, anti-IL-4 mAb and anti-IL-12 mAb) showed some down-modulation of CD49b expression compared with that of Th1 cells during the early phase of activation (until day 8 post-stimulation); however, these differences were not obvious at day 13, suggesting that CD49b expression was intact even in the absence of IL-12 (Fig. 4A). These results were also confirmed in the case of CD29 expression, i.e. CD29 expression of Th cells cultured under IL-12-depleted Th1 conditions as described above was highly positive and almost comparable to the level of Th1 cells (Fig. 4A).



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Fig. 4. IL-4 is essential for down-modulation of CD49b/CD29 expression on Th2 cells. (A) Naive Th cells were stimulated with antigen under Th1 cytokine conditions (IL-2, IFN-{gamma}, IL-12 and anti-IL-4 mAb) (open diamonds), Th2 cytokine conditions (IL-2, IL-4, anti-IFN-{gamma} and anti-IL-12 mAb) (open squares), IL-12-depleted Th1 cytokine conditions (IL-2, IFN-{gamma}, anti-IL-12 and anti-IL-4 mAb) (closed circles) or IL-4-depleted Th2 conditions (IL-2, anti-IFN-{gamma}, anti-IL-12 and anti-IL-4 mAb) (closed triangles). Then CD49b/CD29 expression was determined by flow cytometry. (B) Th1 and Th2 cells were induced from naive Th cells derived from IFN-{gamma}–/– DO11.10 transgenic mice under various conditions as follows: IFN-{gamma}-depleted Th1 cytokine conditions (IL-2, IL-12 and anti-IL-4 mAb) (closed diamonds), both IFN-{gamma}- and IL-12-depleted cytokine conditions (IL-2, anti-IL-12 and anti-IL-4 mAb) (closed triangles) and Th2 cytokine conditions (IL-2, IL-4, anti-IFN-{gamma} and anti-IL-12 mAb) (closed squares). Then, CD49b/CD29 expression was measured. Wild-type DO11.10 Th cells cultured under Th1 (open diamonds) or Th2 (open squares) cytokine conditions are also shown. All results represents mean ± SD of at least three independent experiments.

 
To verify the possible role of IFN-{gamma} on the expression of VLA-2 on Th cells, further experiments were performed using IFN-{gamma}–/– DO11.10 transgenic mice. IFN-{gamma}–/– Th cells were cultured with OVA peptide plus Th1 cytokine conditions in the absence of IFN-{gamma} (IFN-{gamma}-depleted Th1 condition) or both IFN-{gamma} and IL-12 (IFN-{gamma}- and IL-12-depleted Th1 condition). As shown in Fig. 4(B), Th cells cultured under the IFN-{gamma}-depleted Th1 cytokine condition expressed the same levels of CD49b and CD29 as Th1 cells derived from wild-type DO11.10 transgenic mice. Moreover, IFN-{gamma}–/– Th cells cultured in the absence of both IL-12 and IFN-{gamma} also showed up-regulation of both CD49b and CD29 to similar levels as Th1 cells (Fig. 4B). As expected, Th2 cells induced from IFN-{gamma}–/– DO11.10 transgenic naive cells showed a similar reduction of CD49b and CD29 expression to that of Th2 cells derived from wild-type DO11.10 transgenic mice. These results clearly showed that neither IFN-{gamma} nor IL-12 were involved in VLA-2 up-regulation during Th1 differentiation. In contrast, however, Th cells cultured under the IL-4-depleted Th2 cytokine condition (IL-2, anti-IL-12 mAb, anti-IFN-{gamma} mAb and anti-IL-4 mAb) showed up-regulation of VLA-2 expression, suggesting that IL-4 is a critical factor that negatively regulates VLA-2 expression during maturation of Th2 cells. Thus, the distinct expression of VLA-2 on Th1 and Th2 cells appeared to be derived from differential regulation of VLA-2 expression during differentiation of Th1 or Th2 cells.

Differential expression level of VLA-2 between Th1 and Th2 cells is derived from down-regulation of VLA-2 expression on Th2 cells in a STAT6-dependent manner
STAT6 is one of the transcription factors activated by IL-4/IL-4 receptor (IL-4R) ligation (31). Numerous studies have revealed that STAT6 is essential for Th2 development (3234). To examine whether reduction of VLA-2 expression on Th2 cells might be dependent on STAT6 or other signaling pathways of the IL-4R downstream, Th1 and Th2 cells were induced from wild-type BALB/c and STAT6–/– BALB/c mice, and the kinetics of CD49b/CD29 expression on Th1 and Th2 cells were examined. In contrast to wild-type mice, IL-4-mediated VLA-2 down-modulation was totally abrogated and VLA-2 expression was highly up-regulated in STAT6–/– mouse-derived Th2 cells as well as in Th1 cells (Fig. 5). Intracellular staining was performed at day 8 following re-stimulation by plate-bound anti-CD3 mAb. STAT6-deficient Th1 cells produced large amounts of IFN-{gamma} comparable to that of wild-type Th1 cells (76%), while Th2 cytokine-conditioned STAT6- deficient Th cells showed little production of either IL-4 or IFN-{gamma} (0.1 and 1.2% respectively), excluding the possibility that lack of reduction of VLA-2 expression on STAT6–/– Th2 cells was not due to the adverse polarization into Th1 cells. Taken together, these results clearly showed the dynamic down-regulation of VLA-2 in Th2 cells was controlled by the IL-4R/STAT6-dependent signaling pathway.



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Fig. 5. STAT6 is essential for IL-4-induced down-modulation of CD49b/CD29. Naive Th cells from wild-type (dotted lines) and STAT6–/– (solid lines) BALB/c mice were stimulated with anti-CD3 mAb and cultured under Th1 (diamonds) or Th2 (squares) cytokine conditions, and CD49b and CD29 expression measured as described in Methods. (A) Double-color flow cytometric analysis of the expression of CD49b and CD29 on Th1 and Th2 cells derived from wild-type BALB/c or STAT6–/– BALB/c mice at day 13 of culture. (B) Percentage of CD49b+ or CD29+ cells in Th1 and Th2 cells at each day.

 
VLA-2 highly expressed on Th1 cells acts as co-stimulatory molecule for anti-CD3 mAb-induced IFN-{gamma} production
It has been shown that collagen type I provides co-stimulation to T cells when co-immobilized with anti-CD3 mAb, and these ligations synergistically enhance T cell proliferation and inflammatory cytokine production that is likely mediated by VLA-1 and/or VLA-2 (24,26). Therefore, we examined whether VLA-2 ligand (collagen) exhibited the potent co-stimulatory effects on anti-CD3 mAb-induced activation of Th1 or Th2 cells. Th1 and Th2 cells were stimulated with collagen type I co-immobilized with a suboptimal dose of anti-CD3 mAb. As shown in Fig. 6(A), Th1 cells produced enhanced levels of IFN-{gamma} (5.7-fold increase) by stimulation with collagen type I and anti-CD3 mAb, whereas Th2 cells showed no enhanced IL-4 secretion in response to the same stimulation, suggesting that VLA-2 highly expressed on Th1, but not Th2, cells acts as functional co-stimulatory molecule on TCR-mediated T cell activation.



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Fig. 6. Functional expression of VLA-2 on Th1, but not Th2, cells accelerated the co-stimulatory effect of collagen type I against anti-CD3-induced activation. (A) Th1 and Th2 cells were harvested, and added into the wells coated with a suboptimal dose of anti-CD3 mAb and collagen type I or BSA. (B) Th1 and Th2 cells were similarly added to wells coated with a suboptimal dose of anti-CD3 mAb and anti-integrin {alpha}2 (anti-CD49b) mAb or anti-integrin ß1 (anti-CD29) mAb or both antibodies or control hamster mAb. After incubation at 37°C, supernatants were collected, and IFN-{gamma} production of Th1 cells and IL-4 production of Th2 cells measured by ELISA. The results represent mean ± SD of duplicate samples. Similar results were obtained in three different experiments.

 
To further confirm the co-stimulatory effects mediated by VLA-2 on Th1, but not on Th2, cells each type of Th cell was stimulated with either anti-CD49b ({alpha}2 integrin) mAb, anti-CD29 (ß1 integrin) mAb or both mAb co-immobilized with a suboptimal dose of anti-CD3 mAb. As expected, Th1 cells showed markedly enhanced IFN-{gamma} production by stimulation with anti-CD3 mAb combined with anti-CD49b (7.7-fold increase; P < 0.005), anti-CD29 (15.7-fold increase; P < 0.005) or both mAb (12.3-fold increase; P < 0.05). Such a significant synergistic co-stimulatory effect was not demonstrated when anti-CD3 mAb was co-immobilized with control hamster mAb. On the other hand, Th2 cells showed only a slight increase in IL-4 production by anti-CD49b (1.1-fold increase; NS), anti-CD29 (1.7-fold increase; NS) or both mAb (2.1-fold increase; NS) compared with control hamster mAb stimulation (Fig. 6B). When stimulated with collagen or immobilized antibodies alone, neither Th1 nor Th2 produced detectable cytokines (data not shown). Collectively, these results strongly suggested that mature Th1, but not Th2, cells could be provided with co-stimulation via a functional VLA-2 molecule.


    Discussion
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Integrins have been known to mediate T cell attachment to specific ECM components, and subsequently transmit information about the microenvironment in which the cell resides through multiple signaling cascades that lead to transendothelial migration, proliferation and cytokine production (outside-in signaling) (2426,35). On the other hand, signaling through TCR (36), chemokine receptors (3739), cytokine receptors (3841) and integrin itself (35) leads to modulation of affinity/avidity and/or expression of integrin (inside-out signaling). Here, we initially demonstrate that mature Th1 and Th2 cells exhibit distinct VLA-2 expression profiles because of their differential regulation mechanism of VLA-2 expression (Figs 1 and 2). Although naive Th cells do not express VLA-2, Th1 cells acquired high levels of VLA-2 expression during maturation in an IFN-{gamma}- and IL-12-independent manner (Fig. 4). The critical factor for inducing up-regulation of VLA-2 expression on Th1 cells remains unclear, but some cytokines secreted in the culture combined with TCR-mediated antigen stimulation may induce inside-out signaling essential for high VLA-2 expression. During Th2 cell differentiation, the expression of VLA-2 is up-regulated until 6 days, but markedly decreased thereafter. Although repeated anti-CD3 stimulation of Th2 cells counteracts the IL-4 effect on VLA-2 down-modulation, once anti-CD3 stimulation stopped, Th2 cells soon showed decreased VLA-2 expression while Th1 cells continued to express high levels of VLA-2 (Fig. 2). The disappearance of VLA-2 from the surface of Th2 cells was not demonstrated when STAT6–/– mouse-derived Th2 cells were used for the experiment (Fig. 5). This finding strongly suggested that cell-surface expression of VLA-2 on Th2 cells is down-modulated by a IL-4/IL-4R-mediated STAT6-dependent signaling pathway. This type of inside-out signaling essential for down-regulation of VLA-2 is induced by Th2-inducing conditions, but not by Th1 cell-inducing conditions. Indeed, Th2 cells derived from STAT6–/– mice or those induced in the presence of anti-IL-4 mAb expressed high levels of VLA-2 as well as Th1 cells. Therefore, sustained up-regulation of VLA-2 on Th1 cells might be due to the failure of inducing a negative signal essential for the down-modulation of VLA-2 in Th1-inducing culture conditions, in contrast to Th2-inducing conditions. However, it still remains unclear whether IL-4R/STAT6 signaling directly modulates transcription of CD49b/CD29 or Th2 differentiation resulted in the down-regulation of CD49b/CD29 via Th2-specific cytokines or intracellular factors. Interestingly, we incidentally found that the expression of VLA-2 on Th1 cells was down-modulated by IL-4 if we used Th1 cells that were cultured with IL-12 and IFN-{gamma} (in addition to antigen, IL-2 and anti-IL-4) for only the initial 5 days, and without IL-12 and IFN-{gamma} for the following days. Although such Th1 cells exhibited STAT6 phosphorylation in response to IL-4, IL-4 treatment did not affect the cytokine production profile of Th1 cells (data not shown). These data strongly suggested that down-regulation of VLA-2 expression is not a consequence of Th2 differentiation, but IL-4 directly contributes to the down-regulation via the IL-4R/STAT6 signaling pathway.

On the other hand, even excess amounts of IL-4 could not inhibit VLA-2 expression on Th1 cells which were cultured with IL-12 and IFN-{gamma} during the whole culture period. This may be due to the blockage of IL-4R/STAT6 signaling by some molecules such as SOCS5, which is an intracellular molecule selectively induced in Th1 cells via the IL-12R/STAT4 signaling pathway and blocks STAT6 phosphorylation induced by IL-4-induced activated Jak1 (42).

Interestingly, Lorentz et al. (43) found that stem cell factor-induced human mast cell adhesion on collagen type I and fibronectin was strongly reduced by 2 weeks pre-incubation with IL-4, which might be mediated by affinity/avidity changes of ß1 integrins with the expression unaltered. Furthermore, Clissi et al. (44) also reported that up-regulation of ß1 integrin-mediated adhesion induced by chemokines was impaired on human Th2 cells. In addition, we found that long-term cultured human Th2 cell lines that were induced from naive Th cells also showed a reduction of CD49b expression compared to the Th1 cell line (our unpublished data). These findings together with the present data strongly imply the existence of a IL-4-dependent signaling cascade that modulates integrins from inside to outside by down-regulating affinity/avidity and/or expression.

As clearly shown in Fig. 6, highly expressed VLA-2 on Th1, but not Th2, cells appeared to be functionally important, because stimulation of Th1 or Th2 cells with the ligands of VLA-2 caused a selective activation of Th1 cells to produce IFN-{gamma}, but not of Th2 cells after long-term culture. Interestingly, VLA-2 ligation also synergistically activated Th2 cells to produce IL-4 if early polarized Th2 cells, which expressed sufficient levels of VLA-2, were used for the experiments (data not shown). These results suggested that the failure of long-term cultured Th2 cells to respond to VLA-2 ligation is likely due to the lack of the VLA-2 molecule, but not because of an impairment of VLA-2-mediated signaling in Th2 cells.

VLA-2 integrin has recently been shown to play indispensable roles in chemokine-induced monocyte migration into alveoli (45), and in the murine model of delayed-type hypersensitivity, contact hypersensitivity and arthritis (46). Furthermore, intestinal Th cells expressing ß1 integrin at high levels were observed in patients with active Crohn’s disease, which showed synergistic proliferation and inhibition of apoptosis with CD3/ß1 integrin co-stimulation (47). More interestingly, these diseases are generally known to be Th1 dependent (46,4855). Therefore, VLA-2 expressed on Th1 cells may play a critical role for Th1-induced pathogenesis. In this paper, we also documented that repeated anti-CD3 stimulation caused sustained VLA-2 expression even on Th2 cells; however, once TCR-mediated signaling stopped, Th2 cells lose VLA-2 expression, while Th1 cells keep the high level of expression. Such differential expression of VLA-2 may contribute at the re-stimulation stage of memory Th1 and Th2 cells, and may account for the Th1-dominant chronic inflammatory diseases through the following mechanisms. Re-stimulated memory Th1, but not Th2, cells swiftly migrate into the site of inflammation through ß1 integrin and receive co-stimulation by collagens (type I and IV) that are physiological ligands of VLA-2, which are abundant in the tissues. Consequently, Th1 cells are rescued from activation-induced cell death (27,56), and then strongly activated, proliferate and produce Th1 cytokines such as IFN-{gamma} or tumor necrosis factor-{alpha} that may accelerate the Th1-dependent pathogenesis during immune diseases.

Further studies are needed to clarify the relevance of VLA-2 in Th1-dominant diseases. However, our findings will at least provide a novel method of monitoring mature Th1/Th2 cells, which will be useful for the diagnosis of Th1-dependent immune diseases.


    Acknowledgements
 
We thank Dr Michiko Kobayashi (Genetics Institute, Cambridge, MA) and Takuko Sawada (Shinogi Pharmaceutical Institute, Osaka, Japan) for their kind donation of IL-12 and IL-2 respectively. This work was supported in part by a grant-in-aid for Science Research on Priority Areas and Millennium Project from the Ministry of Education, Culture, Sports, Science and Technology.


    Abbreviations
 
ECM—extracellular matrix

IL-4R—IL-4 receptor

PE—phycoerythirn

OVA—ovalbumin


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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