International Immunology

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International Immunology, Vol. 11, No. 6, 1001-1004, June 1999
© 1999 Japanese Society for Immunology

T_h1-derived cytokine IFN- is a potent inhibitor of eotaxin synthesis in vitro

Misato Miyamasu, Masao Yamaguchi, Toshiharu Nakajima¹, Yoshikata Misaki, Yutaka Morita, Kouji Matsushima², Kazuhiko Yamamoto and Koichi Hirai¹

Department of Allergy and Rheumatology,
¹ Department of Bioregulatory Function, and
² Department of Molecular Preventive Medicine and CREST, University of Tokyo Graduate School of Medicine, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan

Correspondence to: K. Hirai

	Abstract

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Eotaxin potentially plays an integral role in tissue eosinophilia. Inasmuch as T_h2-derived cytokine IL-4 has been shown to stimulate eotaxin generation, we investigated here the effect of T_h1-derived cytokine IFN- on human eotaxin production. IFN- but not - or -ß potently inhibited tumor necrosis factor (TNF)--induced eotaxin generation by dermal fibroblasts. The inhibitory effect was unique to eotaxin, because production of IL-8 or monocyte chemoattractant protein (MCP)-1 protein was not affected by the treatment with IFN-. Furthermore, the suppressive effect of IFN- was not cell-type or stimulus specific. The level of eotaxin mRNA increased within 2 h after activation with TNF- and continued to increase up to 72 h. IFN- did not inhibit, but rather augmented the TNF--induced accumulation of mRNA in the early phase (~6 h). However, in the later phase, IFN- completely prevented the subsequent elevation of eotaxin mRNA and sustained it at low levels. Although the protective effect of IFN- against allergic inflammation has been assumed to result from its sole regulation of the proliferation of T_h2-type T lymphocytes, these results imply that IFN- can also directly act on stromal cells to inhibit eotaxin production and consequently intervene in eosinophil recruitment.

Keywords: allergy, ELISA, eosinophil, fibroblast, IL-4

	Introduction

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Immigration of eosinophils is a hallmark of the inflammation associated with allergic diseases and a CC chemokine, eotaxin, potentially plays a pivotal role in tissue-specific eosinophil recruitment in humans (1) as well as in animals (2). A specific receptor for eotaxin, designated CC chemokine receptor (CCR) 3 (3), is selectively expressed in eosinophils (4), basophils (5,6) and T_h2 cells (7), indicating a crucial role for eotaxin in recruiting these cells at sites of allergic inflammation. The importance of eotaxin in allergic inflammation has become evident, but the mechanisms regulating the synthesis of eotaxin are less well elucidated.

During the development of an immune response, naive resting CD4⁺ T cells are polarized to either a T_h1 or T_h2 phenotype (8). Cytokines liberated by allergen-reactive T_h2 cells control the process leading to allergic inflammation and several lines of evidence indicate the involvement of T_h2-derived cytokine IL-4 in the expression of eotaxin. Murine eotaxin mRNA is induced in vivo in response to the transplantation of IL-4-secreting tumor cells (9). In vitro studies demonstrated that IL-4 induces eotaxin secretion and markedly enhances tumor necrosis factor (TNF)--induced eotaxin generation by human dermal fibroblasts (10). On the other hand, the T_h1-derived cytokine IFN- suppresses the development of eosinophilic inflammation in vivo. IFN- inhibits, while IFN- antibodies enhance, the recruitment of eosinophils to inflamed sites following either parasitic infection or antigen challenge (11–13). Given the potential importance of the T_h1/T_h2 paradigm in the development of allergic inflammation, we explored here the effect of IFN- on human eotaxin production.

When dermal fibroblasts were stimulated with TNF-, large amounts of eotaxin, IL-8 and monocyte chemoattractant protein (MCP)-1 were liberated in the supernatants (13.0 ± 4.7, 94.0 ± 18.4 and 71.6 ± 5.3 ng/ml respectively). TNF--induced eotaxin generation was inhibited by IFN- in a dose-dependent fashion: half-maximal inhibition was observed at 10 U/ml and almost complete abolishment was achieved at 100 U/ml (Fig. 1). The exquisite sensitivity to IFN- indicated that the inhibitory action was exerted via interaction with specific receptors. On the other hand, type I IFN, i.e. IFN- and -ß, exerted no significant inhibitory effects (Fig. 1). The inhibitory effect of IFN- was specific to eotaxin: the generation of IL-8 and MCP-1 was not significantly affected by IFN- (Fig. 1).

View larger version (18K): [in this window] [in a new window]   Fig. 1. Effects of IFN on TNF--induced chemokine production by dermal fibroblasts. Cultured human neonatal dermal fibroblasts (Cell Systems, Kirkland, WA) were grown to confluence in 96-well culture plates and stimulated with TNF- (50 ng/ml; Dainippon Pharmaceutical, Osaka, Japan) in combination with graded doses of IFN- (; Pepro Tech, Rocky Hill, NJ), -ß (; Pepro Tech) or - (•; Shionogi Pharmaceutical, Osaka, Japan). After 72 h, immunoreactive eotaxin in the supernatants was assayed by ELISA (16). Immunoreactive IL-8 () and MCP-1 () in the cultures of IFN- were assayed as previously described (17,18). The values are expressed as the percentage of the control TNF--induced production in the absence of IFN. Control production of eotaxin, IL-8 and MCP-1 was 13.0 ± 4.7, 94.0 ± 18.4 and 71.6 ± 5.3 ng/ml respectively. Spontaneous production of eotaxin, MCP-1 and IL-8 in the absence of any exogenous factors was <0.1 ng/ml. Bars represent the SD (n = 3). **P < 0.01 versus control TNF--induced production in the absence of IFN . When the viability was assessed by the Trypan blue dye exclusion test, we found no significant differences between cells before and after incubating with TNF- and IFN- (data not shown).

 
The inhibitory effect of IFN- on eotaxin generation was not stimulus specific. Stimulation of fibroblasts with IL-1 or IL-4 resulted in the secretion of substantial amounts of eotaxin (2.8 ± 0.5 and 2.3 ± 0.6 ng/ml respectively) and IL-4 markedly enhanced TNF--induced eotaxin generation (11.7 ± 0.4 versus 132.6 ± 28.1 ng/ml). Eotaxin production initiated by each of these stimuli was inhibited by IFN- in a dose-dependent fashion, albeit its inhibitory effect was less pronounced in the cultures stimulated with IL-4 alone (Fig. 2). Furthermore, the inhibitory effect of IFN- on eotaxin generation was not cell-type specific and not restricted to dermal fibroblasts. We investigated the effect of IFN- on eotaxin production by several types of cells. Although a recent report revealed that endothelial cells represent a major cellular source of eotaxin (14), both endothelial cells and A549 cells stimulated with TNF- resulted in liberation of marginal levels of eotaxin (<20 pg/ml). On the other hand, MRC5 lung fibroblast cell line cells and T98G glioblastoma cell line cells liberated considerable amounts of eotaxin in response to TNF- (2.8 ± 0.7 and 2.2 ± 0.9 ng/ml respectively). IFN- also effectively inhibited eotaxin generation by MRC5 and T98G in a dose-dependent fashion (Fig. 3).

View larger version (23K): [in this window] [in a new window]   Fig. 2. Effects of IFN- on eotaxin production induced in dermal fibroblasts by IL-1, IL-4 and TNF- plus IL-4. Cultured dermal fibroblasts were grown to confluence in 96-well culture plates and stimulated with IL-1 (, 10 ng/ml; Pepro Tech), IL-4 (•, 10 ng/ml; Pepro Tech), TNF- (, 50 ng/ml) and TNF- plus IL-4 () in the presence or absence of graded doses of IFN-. After 72 h, immunoreactive eotaxin in the supernatants was assayed by ELISA. The values are expressed as the percentage of the control production of eotaxin in the absence of IFN-. Control production with IL-1, IL-4, TNF- and TNF- plus IL-4 was 2.8 ± 0.5, 2.3 ± 0.6, 11.7 ± 0.4 and 132.6 ± 28.1 ng/ml respectively. Bars represent the SD (n = 3). *P < 0.05, **P < 0.01 versus control production in the absence of IFN.

 

View larger version (18K): [in this window] [in a new window]   Fig. 3. Effects of IFN- on TNF--induced eotaxin production by lung fibroblasts (MRC5) and glioblastoma cells (T98G). MRC5 cells (•, n = 3) and T98G cells (, n = 4) were grown to confluence in 96-well culture plates and then stimulated with TNF- (50 ng/ml) in combination with graded doses of IFN-. After 72 h (MRC5) or 96 h (T98G), immunoreactive eotaxin in the supernatants was assayed by ELISA. The values are expressed as the percentage of the control TNF--induced eotaxin production in the absence of IFN-. Control production by MRC5 and T98G was 2.8 ± 0.7 and 2.2 ± 0.9 ng/ml respectively. Spontaneous production of eotaxin in the absence of any exogenous factors by MRC5 and T98G was 0.1 ± 0.03 and 0.1 ± 0.02 ng/ml respectively. Bars represent the SD. *P < 0.05, ***P < 0.005 versus control TNF--induced production in the absence of IFN.

 
When dermal fibroblasts were stimulated with TNF-, the level of eotaxin mRNA increased within 2 h, followed by a linear increase which continued for 72 h of incubation. Until 6 h after stimulation, the addition of IFN- did not inhibit but rather augmented the accumulation of eotaxin mRNA induced by TNF-. After 24 h post-stimulation, however, the inhibitory effect of IFN- became evident: IFN- completely prevented the subsequent elevation of eotaxin mRNA and sustained it at low levels up to 72 h post-stimulation (Fig. 4). The time kinetic pattern of eotaxin protein expression paralleled that of mRNA: stimulation with TNF- resulted in the liberation of low, but consistent and significant, levels of eotaxin at 6 h post-stimulation, which continued to increase geometrically up to 72 h. IFN- showed no inhibitory effect on eotaxin secretion until 12 h, but it completely attenuated the elevation of eotaxin production at 24 h after stimulation (Fig. 5). Although we cannot completely deny the possibility that IFN- also inhibits eotaxin protein generation at post-transcriptional levels, these results strongly suggest that IFN- at least regulates eotaxin generation at the pre-translational level.

View larger version (75K): [in this window] [in a new window]   Fig. 4. Time kinetics of eotaxin mRNA induction in dermal fibroblasts. Cultured dermal fibroblasts were stimulated with TNF- (, 50 ng/ml), IFN- (, 300 U/ml), IFN- plus TNF- (•) or control medium () for various time periods. At the end of the indicated time periods, the levels of eotaxin mRNA were determined by PCR (A) and quantitated by competitive PCR-ELISA (B). Total RNA was extracted with a SNAP total RNA isolation kit (Invitrogen, Leek, Netherlands) according to the manufacturer's instructions. After precipitation with ethanol, the first-strand cDNA was reverse transcribed as described previously (19). The second-strand DNA synthesis and hot-start amplification were performed using a Takara thermal cycler MP (Takara, Ohtsu, Japan) under oil-free conditions. Amplification of cDNA was performed as previously described (19). Direct and reverse oligo primers for eotaxin (5'-CCCAACCACCTGCTGCTTTAACCTG-3' for sense and 5'-AAAAATGGTGATTATTTATGGC-3' for anti-sense) and ß-actin (5'-GGTCAGAAGGATTCCTATGTG-3' for sense and 5'-ATTGCCAATGGTGATGACCTG-3' for anti-sense) were constructed based on the published sequences of each mRNA. To activate DNA polymerase, preheating (9 min at 95°C) was performed. Then amplification was performed for 30 cycles of denaturation (0.5 min at 94°C), annealing (0.5 min at 56°C), elongation (0.5 min at 72°C) and final elongation (10 min at 72°C). (A) PCR products were electrophoresed through a 2% agarose gel and visualized with ethidium bromide. (B) The procedures for competitive PCR of eotaxin mRNA were basically the same as those described previously (19). Briefly, cDNA and varying amounts of competitor cDNA were used as templates, and quantification of the amplified PCR products was performed by ELISA. The amplified products were immobilized on carboxylated-surface plates. The covalently bound single-strand DNAs were hybridized with digoxigenin-labeled oligonucleotide probes (5'-TACCCCTTCAGCGACTAGAGA-3' for eotaxin and 5'-AAGTACCGTCGACGTCGGA-3' for competitor). The plates were developed with peroxidase-conjugated anti-digoxigenin antibodies and signals were visualized with tetramethyl benzidine. Because the ratio of the target and competitor templates remained constant during amplification, the quantity of competitor DNA in the PCR templates which yields an equal amount of the two PCR products shows the initial amount of the target gene. A representative out of two different experiments is shown.

 

View larger version (20K): [in this window] [in a new window]   Fig. 5. Time kinetics of eotaxin protein induction in dermal fibroblasts. Cultured dermal fibroblasts were stimulated with TNF- (, 50 ng/ml), IFN- (, 300 U/ml), IFN- plus TNF- (•) or control medium () for various time periods. At the end of the indicated time periods, the levels of eotaxin protein were assayed by ELISA. Bars represent SD (n = 4).

 
The eotaxin promoter contains consensus recognition sequences for several IFN- response elements (15). The detailed mechanisms underlying IFN--mediated inhibition of eotaxin synthesis such as the half-life of eotaxin mRNA and the effect of de novo protein synthesis are now being investigated. Irrespective of the underlying mechanisms, reduction of eotaxin generation by IFN- could be one of the major factors responsible for the inhibition of tissue eosinophilia. The contrasting responses elicited by T_h2-derived cytokine IL-4 and T_h1-derived cytokine IFN- in eotaxin generation might indicate that this spectrum of action and dichotomy is important in tissue eosinophilia: the regulation of the balance in the local production of these paracrine factors could be a therapeutic target for the management of allergic diseases such as bronchial asthma.

	Acknowledgments

 
We thank Drs H. Nakamura and N. Ida (Toray Industries, Shiga, Japan) for providing the mAb against MCP-1, Dr O. Yoshie (Kinki University, Osaka, Japan) for providing a cDNA for human eotaxin, and Dr S. Izumi (University of Tokyo) for measuring IL-8 and MCP-1. We also gratefully acknowledge the technical expertise of Ms M. Imanishi, S. Jibiki and Y. Asada. Sincere thanks are also extended to Ms A. Kikutake for her excellent secretarial help. This work was supported by a grant from the Manabe Medical Foundation, grants-in-aid from the Ministry of Education, Science, Sports and Culture of Japan (to M. M. and T. N.), and grants-in-aid from the Ministry of Health and Welfare of Japan (to K. H. and M. Y.). M. M. is a Research Fellow of the Japan Society for the Promotion of Science.

	Abbreviations

 

CCR	CC chemokine receptor 3
MCP	monocyte chemoattractant protein
TNF	tumor necrosis factor

	Notes

 
Transmitting editor: K. Sugamura

Received 28 December 1998, accepted 18 February 1999.

	References

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