Introduction

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IL-5 is a lymphokine produced primarily by activated T cells and enhances proliferation, differentiation and survival of eosinophils (Sanderson, 1992; Sanderson et al., 1988). It is also a selective chemoattractant for eosinophils and a potent activator of eosinophil functions, such as cytotoxicity and mediator release (Lopez et al., 1988). Upon activation, eosinophils secrete newly formed lipid mediators and several tissue damaging granule proteins, both of which are involved in the development of allergic inflammation. (Corrigan and Kay, 1992; Gleich, 1990; Gleich and Adolphson, 1986). Activated T cells expressing IL-5 mRNA were found in increased numbers in the bronchial mucosa of asthmatic patients (Hamid et al., 1991) and further increased upon antigen challenge (Robinson et al., 1993). Administration of anti-IL-5 neutralizing antibody abrogated the allergic eosinophilic inflammation in experimental asthma models (Chand et al., 1992; Kaminuma et al., 1997; Van Oosterhout et al., 1993). Taken together, IL-5 seems to be the key cytokine involved in allergic diseases associated with eosinophilic inflammation.

Accumulating evidence suggests that cAMP-elevating agents such as PGE₂ affect T cell function (Chouaib et al., 1985; Goodwin and Ceuppens, 1983; Minakuchi et al., 1990; Oppenheimer-Marks et al., 1994) and various inflammatory responses (Goodwin and Ceuppens, 1983; Moore and Willoughby, 1995; Teixeira et al., 1993). Intracellular cAMP regulates cytokine production of human T cells (Bastin et al., 1990; Mary et al., 1987). We previously reported that IL-5 production by PBMC of asthmatic patients was suppressed by a type 4 PDE inhibitor (Kaminuma et al., 1996). The suppression of IL-5 production was mediated by increase of intracellular cAMP caused by the inhibition of cAMP-PDE activity. These findings suggest that cAMP-elevating agents such as type 4 PDE inhibitors may be effective drugs for the management of allergic diseases associated with eosinophilic inflammation.

However, several conflicting findings have been reported regarding the action of cAMP. Snijdewint et al. (1993) reported that PGE₂ enhanced IL-5 production by human peripheral blood lymphocytes stimulated with anti-CD2 antibody plus anti-CD28 antibody. Watanabe et al. (1994) also reported that PGE₂ enhanced IL-5 production by human T cell clones stimulated with phorbol ester plus Ca⁺⁺ ionophore. The effects of intracellular cAMP on IL-5 production, therefore, differ depending on the nature of the activation signal.

Recently, it was reported that IL-5 production by human T cells was affected by a T cell growth factor, IL-2 (Mori et al., 1996; Watanabe et al., 1994). To further delineate the mechanisms of cAMP action on human T cell IL-5 synthesis, we used an allergen-specific T cell line and analyzed the effects of cAMP on IL-5 synthesis induced by two distinct signals. Our study clearly demonstrated that IL-5 production by antigen-reactive human T cell line was induced by IL-2 and further enhanced by cAMP-elevating agents, whereas TCR-induced IL-5 production was suppressed. It was also suggested that inhibition of IL-2 production was involved in the suppressive effects of cAMP on IL-5 production.

	Materials and methods

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Materials. Df was purchased from Torii Pharmaceutical Co. (Tokyo, Japan). PGE₂, forskolin and db-cAMP were from Sigma (St. Louis, MO), H-89 was from Seikagaku Kogyo (Tokyo, Japan), rhIL-2 was from Peprotech (London, UK), anti-human IL-2 monoclonal antibody was from R & D Systems (Mineapolis, MN), monoclonal anti-human CD3 antibody (OKT3) was from Ortho (Raritan, NJ), purified rat anti-mouse/human IL-5 monoclonal antibody and biotinylated rat anti-human IL-5 monoclonal antibody were from Pharmingen (San Diego, CA), ISOGEN was from Nippongene (Tokyo, Japan), Moloney leukemia virus reverse transcriptase and AmpliTaq polymerase were from Perkin-Elmer Cetus (Norwalk, CT) and AIM-V medium was from Gibco BRL (Gaithersburg, MD).

Human T cell line. Df-reactive T cell line was established from PBMC of allergic individuals as described previously (Mori et al., 1995b). Briefly, PBMC (2 × 10⁶/ml) were cultured with Df antigen (10 µg/ml) in AIM-V medium for 10 days, and nonadherent cells were recovered. Then 2 × 10⁵ live cells were cultured in 24-well culture plates with antigen and 2500 rad-irradiated autologous PBMC (2 × 10⁶/ml). Fresh medium containing 10 U/ml rhIL-2 was added once a week. The antigenic stimulation was repeated every 2 to 3 wk.

Measurement of intracellular cAMP. T cells passaged more than five times were harvested at least 10 days after the last antigenic stimulation. These cells were layered onto Ficoll-Paque and centrifuged. The interface was recovered, washed twice and resuspended in fresh medium. The resulting preparation usually consisted of more than 98% CD3⁺ cells, as determined by flow cytometry. Cells (10⁷/ml) were incubated with each test compound at 37°C for 10 to 360 min. The reaction was stopped by adding a 2-fold volume of ice cold 100% ethanol and the fluid was transferred to a centrifuge tube. The remaining precipitate in culture wells was further washed with ice cold 65% ethanol and also included in the corresponding tube. After drying these extracts in a vacuum oven, cAMP was measured with Biotrak cAMP EIA system (Amersham, Buckinghamshire, UK).

Stimulation of T cell line. Cells (10⁵/ml) were stimulated through TCR or with rhIL-2 for 24 hr. For TCR-stimulation, wells were preincubated with 10 µg/ml OKT3 in 0.05 M carbonate-bicarbonate buffer (pH 9.6) at 4°C overnight, and washed with fresh medium three times before use. After the designated culture period, the supernatant was collected and kept frozen at -70°C until assay. IL-5 was measured by EIA using purified rat anti-mouse/human IL-5 monoclonal antibody as a capture antibody and biotinylated rat anti-human IL-5 monoclonal antibody as a detecting antibody as described previously (Kaminuma et al., 1996). The range of detection of the assay system was 0.02 to 10 ng/ml.

mRNA expression in T cell line. Gene expression of IL-2 and IL-5 was analyzed by the reverse transcription-PCR method, as reported previously (Mori et al., 1995a). Briefly, RNA was extracted from the pelleted cells essentially following the one-step acid guanidinium isothiocyanate/phenol chloroform extraction method (Chomczynski and Sacchi, 1987) using ISOGEN. cDNA was synthesized from 1 µg of cytoplasmic RNA using random primers and murine Moloney leukemia virus reverse transcriptase. PCR was performed using the following primers (Clontech, Palo Alto, CA). Expected sizes of PCR amplification products were 305, 294 and 838 bp for IL-2, IL-5, and -actin, respectively.

Statistics. All data are presented as mean ± S.E. Statistical analysis was performed by Student's t test and values of P < .05 were considered to be statistically significant.

	Results

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Increase of intracellular cAMP in human T cell line by PGE₂ and forskolin. The first experiment was conducted to determine the effect of PGE₂ and forskolin on intracellular cAMP level in a human T cell line. Cells were incubated with PGE₂ (1 µM) or forskolin (10 µM) for the designated time periods, and then the reaction was stopped by the addition of ice cold 100% ethanol. The concentration of cAMP in the resulting supernatants was measured by EIA. As shown in figure 1, intracellular cAMP level rapidly increased after incubation with PGE₂ or forskolin, reached maximum at 10 min and then declined.

View larger version (14K): [in this window] [in a new window]   Fig. 1.   Effects of PGE2 and forskolin on intracellular cAMP level of human T cell line. Cells (107 cells/ml) were incubated with PGE2 (1 µM; ) or forskolin (10 µM; ) for 10 to 360 min. Intracellular cAMP levels were measured by EIA. Data are presented as mean ± S.E. (n = 3). Baseline value of cAMP was 11.0 ± 1.9 fmol/106 cells. *P < .05, **P < .01; compared with baseline.

Effect of intracellular cAMP on TCR-stimulated IL-5 production. We next examined the effects of PGE₂, forskolin and db-cAMP on IL-5 production by a T cell line stimulated through TCR by immobilized OKT3. The human T cell line did not produce detectable amount of IL-5 (<0.02 ng/ml) without stimulation and produced a significant amount of IL-5 (2.08 ± 0.21 ng/ml) with TCR-stimulation. Preliminary experiments indicated that well-immobilized OKT3 (10 µg/ml) produced half-maximum IL-5 production (data not shown). Each test compound was added at the start of culture, and IL-5 concentration in the resulting supernatants was measured by EIA. As shown in figure 2, PGE₂, forskolin and db-cAMP inhibited IL-5 production in a dose-dependent manner.

View larger version (11K): [in this window] [in a new window]   Fig. 2.   Effects of PGE2, forskolin and db-cAMP on TCR-stimulated IL-5 production by human T cell line. Cells (105 cells/ml) were incubated with immobilized OKT3 and PGE2 (), forskolin () or db-cAMP () for 24 h. Concentration of IL-5 in the supernatant was measured by EIA. Percent of control IL-5 production (2.08 ± 2.1 ng/ml) is shown (n = 3).

Role of IL-2 in IL-5 production by human T cell line. We have previously demonstrated that IL-2 induces IL-5 production by human T cells (Mori et al., 1996). Therefore, the effect of anti-IL-2 neutralizing antibody on IL-5 production by a T cell line was examined. As shown in figure 3, anti-IL-2 antibody suppressed TCR-stimulated IL-5 production in a dose-dependent manner, and complete suppression of IL-5 production was produced at 100 µg/ml. The results clearly indicated that the production of IL-5 by human T cell line was dependent on IL-2.

View larger version (25K): [in this window] [in a new window]   Fig. 3.   Effect of anti-IL-2 antibody on TCR-stimulated IL-5 production by human T cell line. Cells were stimulated with immobilized OKT3 for 24 hr. Each test compound was added from the start of culture (n = 3). *P < .05, **P < .01; compared with OKT3-stimulated control.

Effect of intracellular cAMP on IL-2-induced IL-5 production. The effects of PGE₂, forskolin and db-cAMP on IL-5 production by a T cell line stimulated with rhIL-2 was next examined (fig. 4). The human T cell line produced a significant amount of IL-5 (56 ± 5.6 pg/ml) upon stimulation with rhIL-2 (100 U/ml). Preliminary experiments indicated that the concentration of 100 U/ml rhIL-2 produced half-maximum IL-5 production (data not shown). PGE₂, forskolin and db-cAMP concentration-dependently enhanced IL-2-induced IL-5 production (fig. 4). H-89 (100 nM) suppressed IL-5 production by 27 ± 2.7% (n = 3). The effective doses of these compounds on rhIL-2-induced IL-5 production were approximately the same as those on OKT3-induced IL-5 production (fig. 2).

View larger version (11K): [in this window] [in a new window]   Fig. 4.   Effects of PGE2, forskolin and db-cAMP on rhIL-2-induced IL-5 production by human T cell line. Cells were incubated with rhIL-2 (100 U/ml) and PGE2 (), forskolin () or db-cAMP () for 24 hr. Percent of control IL-5 production (56 ± 5.6 pg/ml) is shown (n = 3).

Effects of cAMP on IL-5 gene expression. To examine IL-2 and IL-5 gene expression in a human T cell line, cells was stimulated with immobilized OKT3 or rhIL-2 for 6 hr. Total cytoplasmic RNA was extracted, reverse transcribed and amplified by 25 cycles of PCR using specific primer sets. A representative result of the effect of PGE₂ on mRNA expression in a human T cell line is shown in figure 5. Comparable amounts of mRNA of IL-2 and IL-5 were detected after 6 hr of stimulation. PGE₂ (1 µM) suppressed both IL-2 mRNA and IL-5 mRNA expression induced by TCR-stimulation. IL-5 mRNA expression was also induced by rhIL-2-stimulation and was enhanced by PGE₂. The expression of -actin mRNA did not change among the experimental groups (fig. 5). Essentially the same results were obtained when PCR was performed for 22 and 28 cycles (data not shown), indicating that the amounts of PCR products at 25 cycles reflect well the relative quantity of mRNA in the original RNA preparations. The results indicated that the effects of cAMP-modulating agents were exerted at the level of gene expression.

View larger version (31K): [in this window] [in a new window]   Fig. 5.   Effects of PGE2 on IL-2 and IL-5 mRNA expression of human T cell line. Cells (4 × 106 cells/ml) were incubated with PGE2 (1 µM) and immobilized OKT3 or rhIL-2 (100 U/ml). Total RNA was then extracted, reverse transcribed and amplified by PCR. The expected size of PCR amplification products was 305, 294 and 838 bp for IL-2, IL-5 and -actin, respectively. Lane 1, No stimulation, 2, immobilized OKT3, 3, immobilized OKT3 + PGE2, 4, rhIL-2, 5, rhIL-2 + PGE2.

	Discussion

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Our findings clearly demonstrated that cAMP has two differential effects on IL-5 production by human T cells. cAMP-elevating agents suppressed IL-5 production induced by TCR-stimulation, whereas they potentiated IL-5 production induced by IL-2-stimulation. Both effects were confirmed at the level of gene expression.

PGE₂ and forskolin clearly elevated intracellular cAMP levels and inhibited IL-5 production by TCR-stimulated T cells. db-cAMP also inhibited but PKA inhibitor enhanced TCR-stimulated IL-5 production. These findings suggest that intracellular cAMP down-regulates IL-5-producing activity of T cells stimulated through TCR. As the native activation signals for T cells are introduced through TCR, we might well expect these findings to reflect the responses occurring in vivo.

However, IL-2-induced IL-5 production by the T cell line was clearly enhanced by PGE₂, forskolin and db-cAMP. We (Mori et al., 1996) and other investigators (Watanabe et al., 1994) previously reported that IL-5 production by human T cells was affected by a T cell growth factor, IL-2. We, therefore, examined the role of IL-2 in TCR-stimulated IL-5 production. Anti-IL-2 neutralizing antibody completely suppressed IL-5 production (fig. 3), suggesting that IL-2 produced by T cells plays an essential role in the IL-5 production when stimulated through TCR. Moreover, rhIL-2 itself induced IL-5 production. Although IL-2 could not be detected in the culture supernatants upon TCR-stimulation (data not shown), IL-2 mRNA expression was definitely detected (fig. 5). The apparent discrepancy may be explained by the possibility that T cell-derived IL-2 was captured by its relevant receptors on the T cell surface and accordingly no detectable amount of IL-2 was then released into the culture supernatants. The amount of IL-5 produced by IL-2-stimulation was approximately 1/20 of that produced by TCR-stimulation, suggesting that both signals through TCR and IL-2 receptor may be required for full IL-5-producing capacity.

It has been reported that cAMP-elevating agents inhibited IL-2 production by human T cells (Bastin et al., 1990; Snijdewint et al., 1993). In this study, we confirmed these findings at the gene expression level. TCR-stimulated IL-2 mRNA expression was suppressed by PGE₂, suggesting that the suppression of IL-2 production may lead to the inhibition of IL-5 production. It has been reported that cAMP inhibits IL-2 receptor expression in human T cells (Anastassiou et al., 1992; Krause and Deutsch, 1991). Therefore, down-regulation of the IL-2 signaling pathway at the receptor expression level may also participate in the suppression of IL-5 production by cAMP.

The molecular basis of the IL-2 signal leading to IL-5 production in human T cells is not fully clear yet. It was reported that IL-2 induced DNA binding and transactivating effect of AP-1 (Guizani et al., 1996). Increase in AP-1 binding by PKA was also reported (De Groot and Sassone-Corsi, 1992), indicating the possibility that cAMP enhances IL-2-induced IL-5 production via the AP-1 pathway. Although the role of AP-1 in human IL-5 gene expression has not been fully clarified, we previously showed that dexamethasone, which reduced AP-1 binding activity, suppressed IL-5 production of human T cells (Mori et al., 1995a, 1997). A DNA sequence homologous to the AP-1 binding element identified in human IL-2 gene was identified in human IL-5 gene promoter region (Okudaira et al., 1994). These findings suggest that the regulation of AP-1 binding activity may be involved in the enhancement of IL-2-induced IL-5 production by cAMP. Electrophoresis mobility shift assay and reporter assay using the cis-regulatory elements of human IL-5 gene promoter may be effective ways for further examination and these studies are currently underway.

In conclusion, IL-5 synthesis of human T cells is differentially modulated by intracellular cAMP depending on the nature of the activation signals. cAMP inhibits IL-5 production stimulated through TCR, the native activation signal. A cAMP-elevating agent may show efficacy in the treatment of allergic diseases associated with eosinophilic inflammation via suppression of IL-5 production.