Effect of IL-12 Encoding Plasmid Administration on Tight-Skin Mouse

Biochemical and Biophysical Research Communications 280, 707–712 (2001) doi:10.1006/bbrc.2000.4171, available online at http://www.idealibrary.com on

Effect of IL-12 Encoding Plasmid Administration on Tight-Skin Mouse Junko Tsuji-Yamada, Masatoshi Nakazawa,* Kazuo Takahashi, Katsumasa Iijima,† Shunji Hattori,† Kenji Okuda,‡ Mutsuhiko Minami,* Zenro Ikezawa, and Tetsuo Sasaki Department of Dermatology, *Department of Immunology and Parasitology, ‡Department of Bacteriology, Yokohama City University, School of Medicine, Yokohama, Japan; and †Nippi Research Institute of Biomatrix, Tokyo, Japan

Received November 17, 2000

The tight-skin (Tsk/ⴙ) mutant mice, a putative murine model of scleroderma, are characterized by the excessive deposition of collagen and the presence of antinuclear antibodies. Type 2 cytokines, such as IL-4 and IL-6, are capable of regulating the synthesis of various matrix molecules, including type I collagen, by fibroblasts. IL-12 is well known to induce type 1 cytokine production and to reduce type 2 activity. Here, we examined the effect of IL-12 encoding plasmid (pCAGGSIL-12) on the disease progression of Tsk/ⴙ mice. pCAGGSIL-12 plasmid or pCAGGS parental vector was injected intramuscularly 7 times at 3 week intervals into Tsk/ⴙ mice. One week after the last injection, pCAGGSIL-12 administered Tsk/ⴙ mice exhibited a marked decrease in the skin thickness compared with the mice treated with pCAGGS vector. The serum levels of antinuclear antibodies were diminished in pCAGGSIL-12 treated mice. IL-4 production by spleen cells from pCAGGSIL-12 plasmid treated mice was significantly lower than that from vector treated mice. These results indicate that pCAGGSIL-12 administration into Tsk/ⴙ mice had beneficial effects in preventing the collagen accumulation in the skin and suppressing the autoimmunity via improvement of Th1/Th2 balance. The present study suggests that the IL-12 encoding plasmid administration might have a therapeutic effect on systemic sclerosis. © 2001 Academic Press Key Words: IL-12; plasmid; tight-skin mouse; antinuclear antibody; systemic sclerosis; IL-4; collagen; gene therapy; skin; Th1/Th2.

Systemic sclerosis (SSc) is a systemic disease of unknown etiology which usually takes a chronic course and can be fatal within a few years. It affects the connective tissue containing blood vessels and a diffuse Abbreviations used: Tsk/⫹, tight-skin; SSc, systemic sclerosis; ANA, antinuclear antibody.

sclerosis of the skin and the internal organs occur. Excessive accumulation of collagen in skin and various internal organs is the hallmark of SSc (1). It has been hypothesized that SSc is an autoimmune disease with Th2 dominant response, and that IL-4 has an important role in its pathogenesis. Previous studies showed that IL-4 was expressed in the dermis of a large majority of patients with SSc (2) and that IL-4 stimulated collagen synthesis by fibroblasts from SSc skin in a dose-dependent manner (3, 4). Several investigators have suggested that IL-4 is a cytokine implicated in the early steps of the fibrotic process (2, 5, 6). The tight-skin (Tsk/⫹) mouse has been proposed and used as a putative model for SSc (7). Tsk/⫹ is a semidominant mutation in fibrillin-1 (Fbn-1), the major component of 10 nm microfibrils. Tsk/⫹ mice display connective tissue abnormalities characterized by excessive accumulation of collagen in skin, subcutaneous tissues, and multiple internal organs. In the skin of Tsk/⫹ mice, there is extensive replacement of subcutaneous adipose tissue by thick fibrous tracts above and below the panniculus carnosus, increasing the total thickness of the skin. The pathologic feature of the skin is characterized by an increase in the expression of type I and III procollagen genes (8), excessive accumulation of collagen, and increases in prolylase enzyme activity and hydroxyproline content (9). Immunologically the presence of autoantibodies such as antinuclear and anti-topoisomerase I antibodies has been found in Tsk/⫹ mice (10). Recently, serum anti-Fbn-1 autoantibodies have been reported to occur in Tsk/⫹ mice (11) and in patients with SSc (12) and localized scleroderma (13). Tsk/⫹ mice have some different changes from SSc. For example, lung is an internal organ that is often involved in SSc, but Tsk/⫹ mice show emphysema, not lung fibrosis. Further, emphysema was shown to be unaffected by both the CD4 or CD8 mutation, but skin fibrosis to be reduced by CD4 mutation and anti-topoisomerase activity to be reduced by CD8 mutation (14).

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Human IL-12 is a cytokine which consists of two disulfide-linked chains of 40 kDa and 35 kDa (15, 16). IL-12 polarizes undifferentiated T helper (Th) cells to commit to the Th1, and reduces Th2 activity. IL-12 produced by B-lymphoblastoid cell lines was first identified by its ability to activate NK cells and to induce the synthesis of IFN-␥ by NK cells (17, 18). IL-12 has been tested as an immunotherapeutic drug for cancer (19 –21). Further, the ability of IL-12 as an immunotherapeutic agent for a variety of Th2 mediated diseases or disease models has been assessed (22–24). Th1 or Th2 bias has been found in many diseases such as infections, autoimmune diseases and allergenic diseases (25). Although the pathogenesis of SSc is still unknown, SSc can be considered a Th2 mediated disease, since its collagen synthesis has been shown to be activated by Th2 cytokines such as IL-4 (3, 4) and IL-6 (26). While previous studies demonstrated that IFN-␥ has been effective for SSc (27), its practical usage is limited because of its side effects (28). IL-12 has a strong effect on induction of IFN-␥ production in vivo. Therefore, we hypothesized that a continuous production of IL-12 in vivo might have a therapeutical benefit in SSc. To verify the validity of this hypothesis, we investigated the effects of IL-12 encoding DNA plasmid administration on Tsk/⫹ mice. MATERIALS AND METHODS Mice. Tsk/⫹ and ⫹pa/⫹pa mutant mice on C57BL/6 background were obtained from Jackson Laboratories (Bar Harbor, ME) and maintained in the specific pathogen free conditions at the Yokohama City University animal facility. Tsk/⫹ mice were produced by mating male Tsk/⫹ mice and female ⫹pa/⫹pa mice. Three to five week old male Tsk/⫹ mice were used for experiments. Plasmids. A plasmid encoding murine IL-12 (designated pCAGGSIL-12) was used in these studies. Briefly, murine IL-12 p35 and p40 cDNA were inserted into the EcoRI site of pCAGGS expression vector. We previously reported that administered pCAGGSIL-12 plasmid induced the production of IL-12 mRNA by muscle cells for three weeks in vivo as detected by RT-PCR and that the bioactivity of the culture supernatant of L-cells transfected with pCAGGSIL-12 was 200 U/ml in vitro (29). The control (non-cytokine encoding) vector was constructed of pCAGGS alone. Plasmid administration and experimental design. Mice were injected in the gastrocnemius with 100 ␮g of either pCAGGSIL-12 or control plasmid pCAGGS in 50 ␮l of PBS. Plasmid injection was repeated every three weeks. The muscle bed was pretreated with 100 ␮l of 25% sucrose in phosphate buffered saline (PBS) approximately 30 minutes before plasmid administration. Blood samples were obtained from ophthalmic venous plexus before plasmid DNA administration, and one week after the 3rd and the 7th administration. Skin samples (approximately 3 ⫻ 5 mm) were obtained from the dorsal region one week after the 3rd and the 7th plasmid DNA administration. Cell culture. Spleen was obtained one week after the 7th plasmid DNA administration. Spleen cells (5 ⫻ 10 6 cells/ml) were cultured in the RPMI1640 medium supplemented with 10% FCS containing 2 mM glutamine, 100 ␮g/ml streptomycin, and 100 U/ml penicillin. These cells were stimulated with 50 nM PMA and 500 nM calcium

ionophore for 24 or 48 h to determine amounts of IL-4 and IFN-␥, respectively, produced in culture supernatant. ELISA. IL-4, IFN-␥ and IL-12 heterodimers (p70) in the serum or culture supernatants were measured using murine cytokine ELISA kit (Genzyme, Cambridge, MA). ELISA was performed according to the manufacture’s protocol. Briefly, 96 microwell plates were coated overnight with capture antibodies at 4°C. After several washing with PBS-0.02% tween-20, the plates were blocked with 4% BSA in PBS for 2 h at 37°C. After washing with PBS-0.02% tween-20, the standards and the samples were added and incubated at 37°C for 1 h. After washing with PBS-0.02% tween-20, the second antibodies were added and incubated at 37°C for 1 h. After washing with PBS-0.02% tween-20, horse-radish peroxidase (HRP)-streptavidin solution diluted at 1:300-1:500 was added to wells and incubated at 37°C for 30 minutes. After washing, the working TMB Microwell Peroxidase Substrate (Kiekegaard & Perry Laboratories, Inc. Gaithersburg, MD) solution was added and incubated at room temperature for 10 minutes. The reaction was stopped by stopping solution (1N H 2SO 4), and absorbance was read at 450 nm using a Model 3550 microplate reader (BIO-RAD Laboratories). The cytokine values were expressed in pg/ml, relative to a set of standards supplied with the test kit. The sensitivity of this assay was ⬍5 pg/ml for IL-4 and IL-12, and ⬍15 pg/ml for IFN-␥, respectively. Immunofluorescence assay for anti-nuclear antibody (ANA). HEp-2 cells were used as the substrate in evaluating the presence of ANA in the serum from the Tsk/⫹ mice. A FITC-conjugated antimouse immunoglobulin at a 1:50 dilution was used as a second antibody. The serum diluted serially from 1:10 to 1:640 or greater were added and incubated at 37°C for 30 min. After washing with PBS, the second antibody was added and incubated on the same conditions. After washing with PBS, slides were examined under the fluorescence microscope (BH2-RFCA Olympus, Tokyo, Japan) equipped with an excitation filter at 490 nm. Specific fluorescence at a serum dilution of 1:20 or greater was recorded as a positive response. Controls tested with the second antibody only gave consistently negative responses. Preparation of skin sections and histopathology. Skin samples obtained from the dorsal region of each mouse were collected into PBS and fixed 10% formaldehyde in PBS. The samples were processed for light microscopy by routine methods. After fixation, samples were sectioned and stained with hematoxylin-eosin (H-E) dye. The skin thickness was measured by dermis and epidermis under a light microscope. The skin thickness was measured for 10 regions of a skin section from each mouse, and the average skin thickness was calculated. Determination of hydroxyproline content. To measure the collagen content of skin, skin samples were obtained from the dorsal region of each mouse. For hydroxyproline determination, the dried samples were weighted and hydrolyzed in 6N HCl in sealed glass tubes at 110°C for 48 h and aliquots were then assayed as previously described (30). Evaporated samples were redissolved in 200 ␮l water, and filtered. Twenty ␮l of samples were used for assay of amino acid composition in the amino acid analyzer. The quantity of hydroxyproline was determined using a standard sample of hydroxyproline and chromatography software and expressed as nmol per mg (dry weight). Statistical studies. Student’s t-test was used to evaluate the significance of differences between groups.

RESULTS Effect of the IL-12 Encoding Plasmid Administration on the Thickness of Skin in Tsk/⫹ Mice To investigate the effect of IL-12 on the Tsk/⫹ mice, young Tsk/⫹ mice were treated with IL-12 encoding

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Effect of IL-12 Encoding Plasmid Administration on Cytokines in Vivo

FIG. 1. Effect of pCAGGSIL-12 plasmid administration on Tsk/⫹ mouse skin. The dorsal skin was taken one week after the 3rd (A, C) and the 7th (B, D) plasmid administration. H-E stained typical examples are shown. The pCAGGSIL-12 plasmid treated Tsk/⫹ mice (A, B) showed suppression of skin fibrosis compared with pCAGGS control vector treated Tsk/⫹ mice (C, D).

DNA plasmid and the development of dermal fibrosis and skin thickness was assessed. By palpation the improvement/prevention of skin fibrosis by the treatment appeared to be general. Therefore, dorsal skin was biopsied and skin sections were stained with hematoxylin-eosin. After the 7th plasmid administration, thick and closely packed collagen bundles were observed in the pCAGGS control plasmid treated Tsk/⫹ mice compared with the pCAGGSIL-12 plasmid treated Tsk/⫹ mice, whereas no difference was observed between two groups after the 3rd plasmid administration (Fig. 1). In other words, significant sclerotic changes were observed in pCAGGS treated Tsk/⫹ mice after the 7th treatment, while pCAGGSIL-12 treated Tsk/⫹ mice showed diminished disease progression after the 7th plasmid administration. The skin (dermis plus epidermis) thickness was measured for 10 regions of a skin section from each mouse under light microscope, and the average skin thickness was calculated. After the 3rd administration of plasmid, there was no difference in the skin thickness between pCAGGSIL-12 plasmid treated Tsk/⫹ mice (221.6 ⫾ 41.3 ␮m, n ⫽ 11) and pCAGGS vector plasmid treated Tsk/⫹ mice (190.2 ⫾ 28.1 ␮m, n ⫽ 8). After the 7th administration, the dorsal skin thickness of pCAGGSIL-12 plasmid treated Tsk/⫹ mice (235.2 ⫾ 43.0 ␮m, n ⫽ 11) was significantly thinner (p ⬍ 0.05) compared with that of pCAGGS vector plasmid treated Tsk/⫹ mice (290.5 ⫾ 60.5 ␮m, n ⫽ 8) (Fig. 2). The quantity of hydroxyproline in the biopsied skin was also assessed. The pCAGGSIL-12 plasmid treated Tsk/⫹mice showed significantly lower (p ⬍ 0.0005) amount of hydroxyproline (136.9 ⫾ 5.8 nmol/mg, n ⫽ 5) compared to pCAGGS vector treated Tsk/⫹ mice (195.0 ⫾ 18.1 nmol/mg, n ⫽ 5) after last plasmid administration, while they were comparable after 3rd administration.

To evaluate the effect of IL-12 encoding plasmid administration on type 1 or type 2 cytokine production, serum samples were obtained one week after the 3rd and the 7th plasmid treatment and serum IFN-␥, IL-4 and IL-12 were measured. Serum IL-4 and IL-12 levels of the both groups were under detection limits, and serum IFN-␥ level was comparable between the two groups one week after the last plasmid administration. However, serum IFN-␥ of pCAGGSIL-12 treated mice was slightly higher than that of pCAGGS control plasmid treated mice 24 h after the last plasmid administration (statistically not significant, data not shown). Next, we determined the ability of cytokine production by the spleen cells from each group. Spleen cells were obtained one week after the last plasmid administration, and were cultured for 24 or 48 h with PMA and calcium ionophore for analysis of IL-4 or IFN-␥, respectively. Spleen cells from pCAGGSIL-12 plasmid treated Tsk/⫹ mice produced significantly lower (p ⫽ 0.001) amounts of IL-4 (5.4 ⫾ 3.5 pg/ml, n ⫽ 10) compared with those from pCAGGS vector treated Tsk/⫹ mice (29.7 ⫾ 15.0 pg/ml, n ⫽ 9) (Fig. 3A). The amounts of IFN-␥ produced by the spleen cells from pCAGGSIL-12 plasmid treated Tsk/⫹ mice (597.7 ⫾ 295.7 pg/ml, n ⫽ 9) were higher than those from pCAGGS vector treated Tsk/⫹ mice (523.7 ⫾ 155.1 pg/ml), but they were not statistically significant (Fig. 3B).

FIG. 2. Effect of pCAGGSIL-12 plasmid administration on Tsk/⫹ mouse skin thickness. The dorsal skin thickness was determined one week after the 3rd and the 7th plasmid administration. The pCAGGSIL-12 plasmid treated Tsk/⫹ mice showed significant suppression of skin thickening compared with pCAGGS control vector treated Tsk/⫹ mice one week after the 7th treatment (*p ⬍ 0.05), while they showed no significant suppression one week after the 3rd treatment. Results are expressed as mean ⫹ SD.

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titer (1:80) showed thickened dorsal skin (262 ⫾ 30.8 ␮m) than the other ten mice (225 ⫾ 44.1 ␮m), but the difference was not statistically significant. DISCUSSION

FIG. 3. Effect of pCAGGSIL-12 plasmid administration on IL-4 (A) and IFN-␥ (B) production by cultured spleen cells. Spleen cells were taken and cultured after one week of the last plasmid administration. The amount of IL-4 produced by cultured spleen cells from pCAGGSIL-12 plasmid treated Tsk/⫹ mice was significantly lower (*p ⬍ 0.005) than that from pCAGGS control vector treated Tsk/⫹ mice (A). No significant differences in IFN-␥ production were detected (B). Results are expressed as mean ⫹ SD.

In this study, we examined the effects of IL-12 encoding DNA plasmid on the disease progression of Tsk/⫹ mutant mice. We found that 100 ␮g of pCAGGSIL-12, which can keep its function longer than 3 weeks in vivo (29, 31), prevented the development of both clinical and immunological manifestations of Tsk/⫹ mutant mice. Namely, treatment of young Tsk/⫹ mice with pCAGGSIL-12 diminished skin thickness, suppressed serum ANA production, and altered the balance between Th2 and Th1 cytokine production. Recently, IL-12 was shown to be the most useful regulator of a cytokine balance produced by helper T cell subsets in vivo (15). It was also reported that its half-life in vitro was about 5 or 6 h longer than other cytokines and that only one injection of IL-12 into mice activated IFN-␥ production in the cytokine network and led to upregulation of cellular immunity (19, 20). IL-12 suppresses IL-4 production through activating IFN-␥ secretion. It has been reported that IFN-␥ reduces excessive collagen synthesis by scleroderma fibroblasts (32, 33) and IL-4 stimulates collagen synthesis by scleroderma fibroblasts (3, 4). Treatment of SSc patients with IFN-␥ has been reported (23, 34), and anti IL-4 antibody treatment has been shown to be effective in preventing dermal collagen deposition in Tsk/⫹ mouse (35). However, the effects of exogenous cytokines on diseases have faced difficulty in maintain-

Effect of IL-12 Encoding Plasmid Administration on Antinuclear Antibody Production in TSK Mice Serum antinuclear antibody (ANA) was measured using HEp-2 cells after the last treatment with pCAGGSIL-12 plasmid or control pCAGGS vector. ANA (⭌1:20) was detected in 6 of 13 Tsk/⫹ mice treated with pCAGGSIL-12 plasmid and in 9 of 11 Tsk/⫹ mice treated with control pCAGGS vector. Comparing the ANA ratios indicated in Fig. 4, those of pCAGGSIL-12 treated Tsk/⫹ mice (0.9 ⫾ 1.2) were significantly lower (p ⬍ 0.001) than those of pCAGGS vector treated Tsk/⫹ mice (3.5 ⫾ 1.9) (Fig. 4). There was no positive correlation between the ANA titer and dorsal skin thickness of pCAGGSIL-12 plasmid treated mice (data not shown). However, in the pCAGGSIL-12 treated mice, the three mice with relatively high ANA

FIG. 4. Effect of pCAGGSIL-12 plasmid administration on serum antinuclear antibody (ANA). Serum ANA titers were measured using HEp-2 cells one week after the last plasmid administration. pCAGGSIL-12 plasmid treatment reduced the frequency of ANA positivity and its titer in Tsk/⫹ mice. Each circle indicates the ANA titer from the individual mouse. Bars show mean ⫾ SD, which were calculated as ⬍10 ⫽ 0, 20 ⫽ 1, 40 ⫽ 2, 80 ⫽ 3, 160 ⫽ 4, and 320 ⫽ 5).

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ing a steady state of protein level in vivo. A high dose of cytokine needs to be administered continuously, and it may induce side effects (34). In the case of anticytokine antibody treatment, a high protein concentration and continuous administration are also required (35). Thus, we used IL-12 encoding DNA plasmid against disease progression in Tsk/⫹ mice. In the Tsk/⫹ mice treated with pCAGGSIL-12 plasmid, significantly reduced IL-4 production from spleen cells was observed (Fig. 3A). Unfortunately, serum level of IL-4 and IL-12 was below detectable limit by ELISA. Ong et al. also could not detect IL-4 in the serum of Tsk/⫹ mice, but anti-IL-4 mAb treatment has prevented dermal collagen deposition in those mice (35). Their results indicate that even undetectable level of serum IL-4 might affect on dermal fibroblasts to stimulate collagen synthesis. As suggested from the results of cultured spleen cells, decreased IL-4 level appears to have prevented the collagen deposition in our Tsk/⫹ mice as well. One week after the last plasmid administration, serum level of IFN-␥ was comparable between the pCAGGSIL-12 treated and pCAGGS treated group. This result in the serum corresponds to that in the cultured spleen cells. However, 24 h after the last plasmid administration, serum level of IFN-␥ in the pCAGGSIL-12 treated Tsk/⫹ mice was relatively, but not significantly, higher than that in the pCAGGS treated mice. This slightly increased serum IFN-␥ also might have reduced collagen deposition in our Tsk/⫹ mice. Failure to detect serum IL-12 (p70), which was the case in our previous studies (29, 31), may relate to its relatively short t 1/2 in vivo. As an immunological parameter, ANAs are positive in over 90% of patients with SSc. ANA titers, however, show great variation and little correlation to clinical activity (36). ANA production was significantly reduced in the Tsk/⫹ mice treated with pCAGGSIL-12 plasmid compared with the Tsk/⫹ mice treated with pCAGGS control plasmid (Fig. 4). There was no significant correlation between the ANA titer and skin thickness in each group treated with pCAGGSIL-12 or pCAGGS plasmid. However, three mice having higher ANA titer (1:80) in the pCAGGSIL-12 treated group showed relatively thickened dorsal skin compared to the other mice having lower ANA titer (average: 1:10). This result indicates that pCAGGSIL-12 plasmid treatment diminished both ANA production and skin thickness, and suggests that improved balance of Th1/ Th2 suppressed both ANA production by B cells and collagen synthesis by dermal fibroblasts. The present study indicates that pCAGGSIL-12 administration to Tsk/⫹ mice has beneficial effects in presenting the collagen accumulation in the skin and suppressing the autoimmunity via improvement of Th1/Th2 balance. The previous treatments that regulate the Th1/Th2 balance, such as anti IL-4 antibody and IFN-␥, have many limitations to the clinical usage

because of their side effects and prescriptive difficulty. Administration of IL-12 could have more potential in regulating immune system than the application of anti IL-4 antibody and IFN-␥. IL-12 encoding plasmid has some advantages in supplying IL-12 in more stable manner (29). We injected the plasmid into the hind muscle and examined the dorsal skin, serum and spleen cells, and indicated that the effects were systemic. Although further studies are necessary before clinical use, our study suggests that this plasmid administration might have a therapeutic effect on SSc. ACKNOWLEDGMENTS This work was supported by a grant from the Ministry of Education, Science and Culture of Japan 09670891 to T.S.

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