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NDT Advance Access originally published online on July 31, 2007
Nephrology Dialysis Transplantation 2007 22(11):3131-3138; doi:10.1093/ndt/gfm465
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© The Author [2007]. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org


Systemic administration of interleukin-4 expressing plasmid DNA delays the development of glomerulonephritis and prolongs survival in lupus-prone female NZB x NZW F1 mice

Toshiharu Hayashi1, Keiko Hasegawa1, Yuji Sasaki1, Takashi Mori1, Cie Adachi1 and Ken Maeda2

1Laboratories of Veterinary Pathology and 2Veterinary Microbiology, Faculty of Agriculture, Yamaguchi University, 1677-1 Yoshida, Yamaguchi 753-8515, Japan.

Correspondence and offprint requests to: T. Hayashi, Laboratory of Veterinary Pathology, Faculty of Agriculture, Yamaguchi University, 1677-1, Yoshida, Yamaguchi, 753-8515, Japan Email: hayasi{at}yamaguchi-u.ac.jp



   Abstract
Top
 Abstract
Introduction
 Subjects and methods
 Results
 Discussion
 Acknowledgements
 References
 
Background. T helper (Th)1/Th2 balance determines the direction of some kinds of autoimmune diseases. Th1 cytokines, especially interferon (IFN)-{gamma} has been proven important in the pathogenesis in lupus. The present study examined the effects of administration of interleukin (IL)-4 (Th2 cytokine) expressing plasmid DNA (IL-4pDNA) on the development of glomerulonephritis and survival in lupus-prone female NZB x NZW (B/W)F1 mice.

Methods. B/WF1 mice were administrated intraperitoneally either with IL-4pDNA (100 µg/mouse), plasmid (100 µg/mouse) or saline at 4 and 6 weeks of age and at 4 week intervals from 8 to 32 weeks of age.

Results. Compared to the saline and plasmid groups (controls), the IL-4pDNA-treatment drastically delayed the development of glomerulonephritis with deposits of IgG2a and C3 leading to excretion of urine protein, and prolonged survival. Clinical improvement was associated with the reduction in productions of IgG anti-dsDNA autoantibody. Also, compared to the other two controls the IL-4pDNA-treatment reduced production of IFN-{gamma} and increased IL-4 production from splenic cells.

Conclusions. The present study suggests that systemic IL-4pDNA administration may delay lupus onset by suppressed IFN-{gamma} production due to shifting from Th1 to Th2 responses.

Keywords: B/WF1 mice; IFN-{gamma}; IL-4; IL-4 expressing plasmid; lupus nephritis; survival; Th1/Th2 balance



   Introduction
Top
 Abstract
 Introduction
Subjects and methods
 Results
 Discussion
 Acknowledgements
 References
 
Female NZB x NZW F1 (B/W)F1 mice spontaneously develop lupus resembling human systemic lupus erythematosus (SLE) with polyclonal B cell activation leading to productions of autoantibodies (anti-nuclear and anti-double-strand DNA antibodies, etc.) depending on complex interaction of endogenous and exogenous environmental factors, sex hormones and genetic susceptibility, and the development of immune complexes-mediated glomerulonephritis leads to death [1–4].

The dominance of helper T (Th)1 or Th2 cytokines in the pathogenesis of SLE patients and lupus model mice has been debated in reports over time until now. Earlier reports proposed a Th2 response (or a mixed Th1 and Th2 response) [5–9], whereas recent evidence demonstrated the importance of the Th1 response in lupus nephritis [2–4,10–12]. On the other hand, the notion of mutual inhibitory mechanisms between Th1 and Th2 cytokines [13] suggests that IL-4 may apply to lupus as one of effective therapies. For instance, the constitutive expression of IL-4 transgene by B cells prevents the development of lethal lupus-like glomerulonephritis in the (NZW x C57BL/6. Yaa)F1 murine model of SLE [14]. Also suppressive synthetic oligodeoxynucleotides (ODN) containing TTAGGG motifs in the telomeric region of mammalian chromosomes delay the onset of glomerulonephritis and prolong survival in lupus-prone B/WF1 mice with a significant reduction in productions of anti-dsDNA autoantibody, IFN-{gamma} and IL-12 [15]. In addition, production of proinflammatory cytokines, especially IL-6 which promotes the terminal differentiation of activated B cells and contributes to the development of glomerulonephritis, was blocked by those treatments [14,15]. On the other hand, Th2 cytokines such as IL-4 [16,17] or IL-10 [18] expressing plasmid DNA, including adenovirus vector expressing IL-4 gene [19] led to long-term systemic production of those cytokines with remarkable beneficial therapeutic effects on Th1-dependent autoimmune diseases, such as type I autoimmune insulin-dependent diabetes (IDDM) in animal models. However, the effects of IL-4-expressing plasmid DNA (IL-4pDNA) on lupus development remain unclear.

The present study examined whether IL-4pDNA could delay the onset of glomerulonephritis and disease progress in lupus-prone female B/WF1 mice.



   Subjects and methods
Top
 Abstract
 Introduction
 Subjects and methods
Results
 Discussion
 Acknowledgements
 References
 
Mice
Female B/WF1 mice (total numbers of mice used; n = 28) were obtained from SLC Co. (Shizuoka, Japan). They were kept in metal cages sterilized by heating and given autoclaved pellets (CA-1, Clea Japan, Inc., Tokyo, Japan) and water. They were kept at 25 ± 2°C room temperature, 55 ± 10% humidity and 12 h light-dark cycle (lighting time 08:00 to 20:00). The number of mice used in each set of experiments was shown in each section. The animal experiments were approved by Animal Research Ethics Board of Faculty of Agriculture, Yamaguchi University.

IL-4-expressing plasmid DNA (IL-4pDNA)
IL-4-expressing plasmid DNA was made in the method described previously [20]. In brief, plasmid pCAGGS (plasmid), which possesses a human cytomegalovirus immediate early enhancer and a modified chicken β-actin promoter (CAG promoter) [21], was kindly provided by Dr J. Miyazaki (Osaka University, Japan). For construction of cDNA library, pooled splenocytes from three BALB/c mice (Kyudo, Saga, Japan) were stimulated with either concanavalin A (Con A; 5 µg/ml, Sigma, St Louis, MO, USA) or pokeweed mitogen (5 µg/ml, Sigma) for 24 h [20]. Then, mRNA was extracted from a mixture of the stimulated splenocytes. The gene encoding mouse IL-4 was amplified from the cDNA library by polymerase chain reaction with primers 5'-TTCTCGGAGATGGGTCTCAACCCCCAG-3' and 5'-TTCTCGAGCTACGAGTAATCCATTTGCAT-3', and cloned into pCR2.1. The nucleotide sequences of these cloned DNA were analysed before sub-cloning into the XhoI site of pCAGGS, and plasmids were purified with EndFree Qiagen Plasmid Purification Kits (Qiagen, Hilden, GmbH, Germany).

Confirmation of IL-4 production by cells transfected with IL-4pDNA
Crandell's feline kidney cell line (CRFK; ATCC CCL-94, MA, USA) was transfected either with plasmid pCAGGS (n = 2) or IL-4pDNA (n = 2) using LipofectamineTM2000 (Invitrogen, CA, USA) according to the manufacture's protocol. Approximately 1 x 106 cells were plated in 2 ml of Dulbecco's modified Eagle's medium (DMEM; KC Biological, Lenexa, Kans, USA) containing 10% fetal bovine serum and then added 500 µl Opti-MEM I-reduced serum medium (Gibco, Grand Island, NY, USA) with 4 µg of plasmid DNA and 10 µl of LipofectamineTM2000. The culture supernatants were collected at 3 or 8 days post-transfection (dpt). In this system, transfected cells with IL-4pDNA produced IL-4 (183 pg/ml at 3 dpt and 204 pg/ml at 8 dpt, respectively) and did not produce IFN-{gamma} (0 pg/ml at 3 or 8 dpt, respectively), whereas plasmid pCAGGS did not produce both cytokines (0 pg/ml) at 3 or 8 dpt, respectively.

Administration of IL-4pDNA
Dose and time of administration of the IL-4pDNA were determined depending on our previous report [20] with minor modification. Mice were treated intraperitoneally either with 100 µg of the IL-4pDNA (IL-4pDNA group; n = 9), 100 µg of plasmid only (plasmid group; n = 10) or saline only (saline group; n = 9) at the age of 4, 6, 8, 12, 16, 20, 24, 28 and 32 weeks.

Sampling of urine, blood, spleen and kidney for disease evaluation
During experimental periods from the age of one to 32 weeks in each group (n = 7 at random) in each time point indicated, plasma and/or urine were obtained as described previously [22]. At 32 weeks of age, survived mice in each group (n = 4) were euthanatized by inhalation of over a dose of chloroform, since at this age some mice in the plasmid group began to die. Then, blood, spleens and kidneys were obtained, and they were provided for histopathology (left kidney) and immunohistochemistry (right kidney). For a further evaluation of the IL-4pDNA effects, the resident mice (n = 6; the plasmid group, n = 5; the saline and IL-4pDNA-treated groups, respectively) were examined for the survival rate until the age of 36 weeks.

Urine protein and leukocytes were assessed semiquantitatively using dip sticks (Multi-sticks SG-L, Bayer, Tokyo, Japan). The values of BUN and creatinine were determined using Fuji dry-chem 3500s and Fuji dry-chem slides (Fuji film Co., Tokyo, Japan).

Isolation of splenic cells for cytokine assay
Spleens from each group of mice were aseptically removed and placed in phosphate buffered-balanced salt solution (PBBS; pH 7.4) as described previously [23]. Total number of cells was determined using a haemocytomater after single cell suspensions and cells were diluted in DMEM to a density of 5 x 106 cells/ml. There was a more than 95% viability of cells by trypan blue dye exclusion test. Splenocytes (1 x 106 cells in 200 µl DMEM with 5% FCS) with or without Con A (5 µg/ml) were cultured for 48 h at 37°C in a 5% atmosphere and supernatants were stored at –80°C until use.

Measurements of cytokines in blood and cultured spleen cell fluid by ELISA
IFN-{gamma} and IL-4 mouse ELISA kits were purchased from Techre Co. (Minneapolis, MN, USA). The minimal detectable concentration was 1.2 pg/ml for IFN-{gamma} and 2 pg/ml for IL-4. Supernatants from cultured splenic cells (n = 4 in each group) at the age of 32 weeks were assayed for those cytokines.

In the case that cytokines could not be detected, their concentration was estimated as 0.

Concentration of IgG anti-dsDNA
Concentration of mouse IgG anti-dsDNA was measured by commercial ELISA kit according to manufacture's protocol (Alpha Diagnostic International, TX, USA).

Kidney histopathology and immunohistochemistry
Left kidneys were fixed in 10% neutral buffered formalin (pH 7.0) and embedded in paraffin, and sections (4 µm) were stained with haematoxylin and eosin (HE) and periodic acid-methenamine-silver (PAM).

On the other hand, for detection of IgG2a, IgG1 or C3 deposits in glomeruli, right kidneys were frozen in chilled n-hexane, and frozen sections were made by cryostat and fixed in acetone.

Evaluation of histopathology and IgG1, IgG2a and C3 deposits in glomeruli
The degree of glomerular damage was estimated semi-quantitatively as a method described previously [11] with a 0–4 scale based on the severity and extent of histological changes as follows: the index of glomerular lesions (IGL); no or minimal mesangial change (0), mild mesangial proliferation (1), marked mesangial proliferation (2), 2 plus capillary wall thickening (3), 3 plus sclerosis or ‘wire loop’ lesions (4).

The score of immunofluorescence (SIF) was based on the intensity and distribution of the deposits of C3 (FITC-labeled goat anti-mouse C3; Cappel, Durham, NC, USA), IgG1 (fluorescein isothiocyanate; FITC-labelled goat anti-mouse IgG1; Santa Cruz Biotechnology, CA, USA) and IgG2a (FITC-labelled goat anti-mouse IgG2a) in glomeruli from 0 to 4; no deposit (0), slight (1) and moderate (2) deposits in mesangial areas, moderate (3) and marked (4) deposits in both mesangial areas and capillaries or in capillary walls diffusely without deposition of mesangial areas. IGL and SIF were calculated using the following formula; (n0 x 0) + (n1 x 1) + (n2 x 2) + (n3 x 3) + (n4 x 4)/{sum}n. Twenty glomeruli from each kidney were examined by two different observers.

Statistical analysis
The data were expressed as the mean of samples examined ± SE. Unpaired Student's t-test was used to evaluate the significance of differences. Also, the survival rate was subjected to chi-square analysis. A P-value less than 0.05 was considered significant.



   Results
Top
 Abstract
 Introduction
 Subjects and methods
 Results
Discussion
 Acknowledgements
 References
 
IL-4pDNA administration reduces excretion of urine protein and leucocyte in B/WF1 mice
Compared to the two control (the saline and plasmid) groups, the IL-4pDNA group reduced excretion of urine protein statistically at the age of 25 and 32 weeks (Figure 1A: P < 0.05). Excretion of urine leucocytes (over plus one) was detected in the two controls (from 25 to 32 weeks of age in the saline group and from 30 to 32 weeks of age in the plasmid group), but not the IL-4pDNA group (Figure 1B). In the plasmid group, there was an increase in BUN (Figure 1C; P < 0.05, comparison with the saline and IL-4pDNA groups) and creatinine values (Figure 1D) compared to the saline and IL-4pDNA groups at 32 weeks of age.


Figure 1
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  		Fig. 1. Appearance of excretion of urine protein (A) and leucocytes (B), and increase in BUN (C) and creatinine (D). Each arrow indicates the time of administration (4, 6, 8, 12, 16, 20, 24, 28 and 32 weeks of age) of the plasmid (100 µg), IL-4pDNA (100 µg) or saline. The mean ± SE. *P < 0.05 (comparison between the IL-4pDNA group and the two controls). **P < 0.05 (comparison between the IL-4pDNA group and the plasmid groups or the saline group and the plasmid group).

 
IL-4pDNA administration reduces mouse IgG dsDNA concentration in B/WF1 mice
IgG dsDNA concentration increased from 16 to 32 weeks of age in the two controls, whereas that increased from 24 to 32 weeks in the IL-4pDNA group (Figure 2). In comparison with the plasmid (1.85 ± 0.09) and saline (1.79 ± 0.19) groups, the IL-4pDNA-treatment (1.14 ± 0.08) reduced IgG anti-dsDNA concentration significantly at 32 weeks of age (P < 0.05: comparison with the two controls).


Figure 2
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  		Fig. 2. Development of mouse IgG anti-dsDNA concentrations. The mean ± SE. *P < 0.05 (comparison between the IL-4pDNA group and the two controls).

 
IL-4pDNA administration reduces glomerular damages (IGL) and IgG2a and C3 deposits in glomeruli (SIF) in B/WF1 mice
At 32 weeks of age, in comparison with the plasmid (1.9 ± 0.3) and saline (1.6 ± 0.2) groups, respectively, the IL-4pDNA-treatment reduced IGL (0.9 ± 0.1, Figure 3A; P < 0.05). Also, compared to deposits of IgG2a in each control group (the plasmid; 1.2 ± 0.3 and saline; 0.9 ± 0.2) their deposits in the IL-4pDNA (0.3 ± 0.1) were statistically reduced, respectively (Figure 3B; P < 0.01). Moreover, C3 deposit in the IL-4pDNA group (0.4 ± 0.4) compared to the two controls (the plasmid; 2.4 ± 0.3 and saline; 1.8 ± 0.2) was reduced, respectively (Figure 3B; P < 0.01). Although IgG1 deposit increased slightly in the IL-4pDNA group compared to the two controls, there was no statistical difference of IgG1 deposit in glomeluri among three groups.


Figure 3
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  		Fig. 3. IGL (A) and SIF (B) at the age of 32 weeks. The mean ± SE. *P < 0.05 (comparison between the IL-4pDNA group and the two controls).

 
In the plasmid group, sizes of glomeruli were increased slightly to severely, and there were mild to severe mesangial expansion, proliferation of mesangial cells, diffuse thickening of capillary walls, swelling of endothelial and mesangial cells with infiltration of some neutrophils and monocytes (Figure 4A). On the other hand, in the saline group glomerular changes (Figure 4B) were less severe, compared to the plasmid group. There were no or slight glomerular changes (Figure 4C) in the IL-4pDNA group. In addition, there was infiltration of leucocytes (mainly lymphocytes and plasma cells) in pelvic areas in the plasmid (four of four cases), saline (two of four cases) or IL-4pDNA (two of four cases) groups, and there were differences in the degree of infiltration among three groups (severe in the plasmid; Figure 4D, mild in the saline; Figure 4E, and slight; Figure 4F in the IL-4pDNA groups). Also focal infiltration around blood vessel in the cortical interstitium in the plasmid group was found (one of four case).


Figure 4
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  		Fig. 4. Representative glomerular lesions in the plasmid (A), saline (B) and IL-4pDNA (C) groups at the age of 32 weeks. Also, representative pelvic interstitial lesions in the plasmid (D), saline (E) and IL-4pDNA (F) groups: x100 (A, B and C), x200 (insert; high magnification of arrows), x50 (D, E and F) HE.

 
IgG2a (Figure 5A) and C3 (Figure 5B) in glomeruli were moderately deposited in the IL-4pDNA group, whereas those were mild (Figure 5C and D) in the saline group. In the IL-4 group no or slight IgG2a (Figure 5E) and C3 (F) deposits were seen in the IL-4pDNA group.


Figure 5
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  		Fig. 5. Representative glomerular IgG2a (A, C and E) and C3 (B, D and F) deposits in the plasmid, saline and IL-4pDNA groups at the age of 32 weeks; x200, immunofluorescence. Plasmid (A and B), saline (C and D) and IL-4pDNA (E and F) groups. Immunofluorescence. x200.

 
IL-4pDNA administration increases spontaneous IL-4 production from splenic cells in B/WF1 mice
In the IL-4pDNA group, IL-4 production (2.2 ± 0.9 pg/ml) from splenic cells without Con A stimulation was higher than that in two controls (0.2 ± 0.6 pg/ml or 0 pg/ml in the plasmid or saline group, respectively) (Figure 6A; P < 0.05 compared to the plasmid group), whereas IFN-{gamma} production (0.2 ± 0.1 pg/ml) compared to two controls (8.1 ± 2.2 pg/ml or 0.7 ± 0.3 pg/ml in plasmid- or saline-treated group, respectively) (Figure 6B; P < 0.05) was reduced at the age of 32 weeks. On the other hand, both cytokines from splenic cells stimulated with Con A were increased and there were no differences of their value among three groups (Figure 5C and D).


Figure 6
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  		Fig. 6. Production of IL-4 (A and C) and IFN-{gamma} (B and D) from splenic cells without (A and B) or with ConA (C and D) at the age of 32 weeks. The mean ± SE. *P < 0.05 (comparison between the IL-4pDNA and the two controls).

 
IL-4pDNA administration prolongs the survival in B/WF1 mice
As shown in Figure 7, mice treated with the plasmid began to die at the age of 32 weeks (33%), and 70% of the mice had died at the age of 36 weeks, whereas the saline-treated mice began to die at age of 34 weeks (33%), and 40% had died at age 36 weeks. The IL-4pDNA-treated mice were all alive at 36 weeks of age, and survival rate in this group was statistically higher than that in two controls (P < 0.001). In addition, survival rate in the plasmid group was lower than that in the saline group (P < 0.05).


Figure 7
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  		Fig. 7. Survival rate among three groups. *P < 0.001(comparison between the IL-4pDNA group and two controls). **P < 0.05 (comparison between the two controls).

 
Clinically, the two controls dying showed dull movement, severe emaciation, loss of hair smoothness, subcutaneous oedema and ascites or hydrothorax, or both, whereas all the mice treated with IL-4pDNA showed no such changes.



   Discussion
Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
Acknowledgements
 References
 
The present study clearly demonstrated that the IL-4pDNA-treatment reduced the development of glomerulonephritis with excretion of urine protein and prolonged survival in lupus-prone B/WF1 mice, compared to the plasmid and saline controls. This is accompanied by reduced productions of IgG anti-mouse dsDNA antibodies with decreased deposition of IgG2a and C3 in glomeruli. Also, IL-4pDNA-treatment decreased production of IFN-{gamma}, which class-switches from IgM to nephritogenic IgG2a and may contribute to the development of glomerulonephritis by up-regulating the expression of major histocompatibility complex gene products and adhesion molecules as well as by activating macrophages [1,3,4,15,24]. In addition, interstitial infiltration of mononuclear cells was reduced in the IL-4pDNA group compared to the two controls, suggesting less severe kidney damage by the IL-4pDNA-treatment. It seems likely, since it has been reported that (i) recruitment of CD4+, CD8+ and double negative T cells into the kidney, (ii) kidney-infiltrating T cell activation resulting in IFN-{gamma} production and (iii) expansion of CD4+ T cells proliferating within the kidney resulted in kidney injury in lupus model (MRL-FAslpr) mice [25]. Moreover, the present result may be compatible with our previous observations that the IL-4pDNA group compared to the plasmid [20] and saline (unpublished observation) groups delayed the disease progress. Taken together, the present result suggests that kidney disease may be reduced by the IL-4pDNA-treatment.

Compared to the two controls, the IL-4pDNA-treatment increased IL-4 and decreased IFN-{gamma} productions by splenic cells, suggesting that IL-4 secretion may be shifted from Th1 to Th2 subsets in splenic cell population, IFN-{gamma} production may be suppressed by mutual Th1/Th2 inhibitory effects [13]. On the other hand, splenocytes stimulated with Con A produced a large amount of IL-4 and IFN-{gamma} compared to those stimulated without Con A, and there was no difference in their values among three groups. This suggests that function and/or ratio of Th1/Th2 subsets of splenocytes by the treatment of the IL-4pDNA and plasmid may be reversible. If so, continuous IL-4pDNA-treatment may be required for the prevention of the development of lupus.

A large dose and frequent administrations of recombinant IL-4 (rIL-4) are necessary to maintain the effective concentration of IL-4 on prevention of the Th1-type autoimmune diseases such as type I IDDM in murine models [17,26], since its half life is very short (t1/2 = 19 ± 2 min in the circulation in mice administrated intravenously by murine rIL-4 [27]). Moreover, a large dose of rIL-4 may evoke unexpected side effects in recipients, such as an increased susceptibility to Th2 allergic diseases (e.g., asthma and rhinitis) other than the preventive effects of lupus. On the other hand, in the present study mice treated with the IL-4pDNA at relatively long intervals (2–4-week intervals), since it has been shown that CRFK cell line transfected with the IL-4pDNA produced enough concentration of IL-4 more than 8 days in vitro as described in section ‘Subjects and methods’) were effective to prevent disease development. And the IL-4pDNA-treated mice did not show any clinical signs. Thus, compared to the administration of the rIL-4, IL-4pDNA may be advantageous and useful for the reduction of a Th1-dependent autoimmune disease, including lupus.

The IL-4pDNA injected intraperitoneally may be taken up by peritoneal immune cells (e.g., macrophages, dendritic cells and lymphocytes), since it is known that DNA from bacteria containing many CpG motifs, a 6-base DNA motif consisting of unmethylated CpG dinucleotide flanked by two 5' purines and 3' pyrimidines, interacts with Toll-like receptors, which are expressed on a variety of immune cells described above [28]. Further study is needed to clarify cell types, including efficiency of transfection, which take up the IL-4pDNA and how long the expression of the IL-4pDNA lasts, including how secreted IL-4 protein affects, IL-4 receptor, since the IL-4 receptor {alpha}-chain is a component of both the IL-4 and the IL-13 receptor [29].

The present study demonstrated that the plasmid accelerated the lupus development compared to not only the IL-4pDNA group but also the saline group. It seems likely, since B/WF1 mice administrated with plasmid containing many CpG motifs [30] or synthetic CpG oligodeoxynucleotides [11], may interact and stimulate a variety of immune cells [28] as discussed earlier, and then those cells may secrete mainly Th1(IFN-{gamma}) and Th1-related (IL-12 and IL-18) cytokines including IL-6 [31]. As a result, the plasmid-treatment compared to the saline- and IL-4pDNA-treatment may accelerate kidney disease. Thus, although there were contradictory issues in the role of Th1 and Th2 cytokines, the present study may be another example that Th1 cytokines are responsible for lupus pathogenesis. Although, one might expect that plasmid, a component of IL-4pDNA, may have a capacity to induce productions of Th1 and Th1-related cytokines, IL-4 production may overcome and inhibit productions of Th1 cytokines by mutual inhibitory effects of Th1/Th2 cells [13].

In conclusion, the present study suggests that the administration of IL-4pDNA during the pre-active and active phases may delay the development of lupus. The systemic administration of IL-4pDNA may be advantageous for cytokine therapy for lupus. This is the first report that the L-4pDNA delayed lupus development in lupus-prone female NZB x NZWF1 mice to our knowledge.



   Acknowledgements
Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 Acknowledgements
References
 
We thank Dr J. Miyazaki (Osaka University, Japan) for kindly providing the plasmid pCAGGS. This study was supported in part by a grant-in-aid of the Ministry of Education, Science, Sports and Culture of Japan (No. 15380210).

Conflict of interest statement. None declared.



   Notes
 
The authors wish it to be known that, in their opinion, the first two authors contributed equally to this work.



   References
Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 Acknowledgements
 References
 

  1. Hass CB, Ryffel B, Hir ML. IFN-{gamma} receptor depletion prevents autoantibody production and glomerulonephritis in (NZBxNZW)F1 mice. J Immunol (1998) 160:3713–3718.[Abstract/Free Full Text]
  2. Theofilopoulos AN, Koundoris S, Kono DH, Lawson BR. The role of IFN-gamma in systemic lupus erythematosus:a challenge to the Th-1/Th-2 paradigm in autoimmunity. Arthritis Res (2001) 3:136–144.[CrossRef][Web of Science][Medline]
  3. Richards HB, Satoh M, Jennete JC, et al. Interferon-{gamma} is required for lupus nephritis in mice treated with the hydrocarbon oil pristine. Kidney Int (2001) 60:2173–2180.[CrossRef][Web of Science][Medline]
  4. Pollard KM, Hultman P, Kono DH. Using single-gene deletion to identify checkpoints in the progression of systemic autoimmunity. Ann NY Acad Sci (2003) 987:336–339.
  5. Ishida H, Muchamuel T, Sakaguchi S, et al. Continuous administration of anti-interleukin 10 antibodies delays onset of autoimmunity in NZB/WF1 mice. J Exp Med (1994) 179:300–310.
  6. Klinman DM, Steinberg AD. Inquiry into murine and human lupus. Immunol Rev (1995) 144:157–193.[CrossRef][Web of Science][Medline]
  7. Kaneko Y, Hirose S, Abe M, et al. CD40-mediated stimulation of B1 and B2 cells;implication in autoantibody in murine lupus. Eur J Immunol (1996) 26:3061–3065.[Web of Science][Medline]
  8. Nakajima A, Hirose S, Yagita H, et al. Role of IL-4 and IL-12 in the development of lupus in NZB/WF1 mice. J Immunol (1997) 158:1466–1472.[Abstract]
  9. Peng SL, Moslehi J, Craft J. Roles of interferon-{gamma} and interleukin-4 in murine lupus. J Clin Invest (1997) 99:1936–1946.[Web of Science][Medline]
  10. Takahashi S, Fossati L, Iwamoto M, et al. Imbalance towards Th1 predominance is associated with acceleration of lupus-like autoimmune syndrome in MRL mouse. J Clin Invest (1996) 97:1597–1604.[Web of Science][Medline]
  11. Hasegawa K, Hayashi T. Synthetic CpG oligodeoxynucleotides accelerate the development of lupus nephritis during preactive phase in NZB x NZW F1 mice. Lupus (2003) 12:1–8.[Free Full Text]
  12. Sugiyama M, Funauchi M, Yamagata T, et al. Predominant inhibition of Th1 cytokines in New Zealand black/white F1 mice treated with FK 506. Scand J Rhematol (2004) 33:108–114.[CrossRef]
  13. Mosmann TR, Coffman RL. Th1 and TH2 cells; different patterns of lymphokine secretion lead to different functional properties. Ann Rev Immunol (1989) 7:145–173.[CrossRef][Web of Science][Medline]
  14. Santiago ML, Fossati L, Jacquet C, et al. Interleukin-4 protects against a genetically linked lupus-like autoimmune syndrome. J Exp Med (1997) 185:65–70.[Abstract/Free Full Text]
  15. Dong L, Ito S, Ishii KJ, et al. Suppressive oligodeoxynucleotides delay the onset of glomerulonephritis and prolong survival in lupus-prone NZB x NZW mice. Arth Rheum (2005) 52:651–658.[CrossRef][Web of Science][Medline]
  16. Lee M, Koh JJ, Han S, et al. Prevention of autoimmune insulitis by delivery of interleukin-4 plasmid using a soluble and biodegraded polymeric carrier. Pharm Res (2002) 19:246–249.[CrossRef][Web of Science][Medline]
  17. Hayashi T, Yasutomi Y, Hasegawa K, et al. Interleukin-4-expressing plasmid DNA inhibits reovirus type-2-triggered autoimmune insulitis in DBA/1J suckling mice. Int J Exp Pathol (2003) 284:101–106.
  18. Nitta Y, Tashiro F, Tokui M, et al. Systemic delivery of interleukin 10 by intramuscular injection of expression plasmid DNA prevents autoimmune diabetes in nonobese diabetic mice. Human Gene Ther (1998) 9:1701–1707.[Web of Science][Medline]
  19. Cameron MJ, Arreaza GA, Waledhauser L, et al. Immunotherapy of spontaneous type I diabetes in nonobese diabetic mice by systemic interleukin-4 treatment employing adenovirus vector-mediated gene transfer. Gene Ther (2000) 1:111–123.
  20. Hasegawa K, Hayashi T, Maeda K. Promotion of lupus in NZBxNZWF1 mice by plasmids encoding interferon(IFN)-{gamma} but not by those encoding interleukin(IL)-4. J Comp Path (2002) 127:1–6.[CrossRef][Web of Science][Medline]
  21. Niwa H, Yamamura K, Miyazaki J. Efficient selection for high-expression transfectants with a novel eukaryotic vector. Gene (1991) 108:193–200.[CrossRef][Web of Science][Medline]
  22. Hayashi T, Hasegawa K, Adachi C. Elimination of CD4+CD25+ T cell accelerates the development of glomerulonephritis during the preactive phase in autoimmune-prone female NZBxNZWF1 mice. Int J Exp Path (2005) 86:289–296.[CrossRef][Web of Science][Medline]
  23. Hayashi T, Hasegawa K, Sasaki Y, et al. Elimination of CD4+CD25+ regulatory T cells break down reovirus type 2-triggered and CpG ODN-induced prolonged mild autoimmune insulitis in DBA/1 mice. Scand J Immunol (2006) 63:116–124.[CrossRef][Web of Science][Medline]
  24. Kameyama Y, Hayashi T. Suppression of development of glomerulonephritis in NZBxNZWF1 mice by persistent infection with lactic dehydrogenase virus: relations between intercellular adhesion molecule-1 expression on endothelial cells and leucocyte accumulation in glomeruli. Int J Exp Path (1994) 75:295–304.[Web of Science][Medline]
  25. Schwarting A, Tesch G, Kinoshita K, et al. IL-12 drives IFN-{gamma}-dependent autoimmune kidney disease in MRL-Faslpr mice. J Immunol (1999) 163:6884–6891.[Abstract/Free Full Text]
  26. Tominaga Y, Nagata M, Yasuda H, et al. Administration of IL-4 prevents autoimmune diabetes but enhance pancreatic insulitis in NOD mice. Clin Immunol Immunopathol (1998) 86:209–218.[CrossRef][Web of Science][Medline]
  27. Conlon PJ, Tyler S, Grabstein KH, et al. Interleukin-4 (B-cell stimulatory factor-1) auguments the in vivo generation of cytotxic cells in immunosuppressed animals. Biotechnol Tec (1990) 1:31–34.
  28. Sano K, Shirota H, Terui T, et al. ODN without CpG motifs work as adjuvant for the induction of Th2 differentiation in a sequence-independent manner. J Immunol (2003) 170:2367–2373.[Abstract/Free Full Text]
  29. Mohrs M, Ledermann B, Kohler G, et al. Differences between IL-4- and IL-4 receptor alpha-deficient mice in chronic leishmaniasis reveal a protective role for IL-13 receptor signaling. J Immunol (1999) 162:7302–7308.[Abstract/Free Full Text]
  30. Pisetsky DS. Immune activation by bacterial DNA:a new genetic code. A review. Immunity (1996) 5:303–310.[CrossRef][Web of Science][Medline]
  31. Klinman DM, Yi AK, Beaucage SL, et al. CpG motifs present in bacterial DNA rapidly induce lymphocytes to secrete interleukin 6, interleukin 12, and interferon {gamma}. Proc Natl Acad Sci USA (1996) 93:2879–2883.[Abstract/Free Full Text]
Received for publication: 20. 3.07
Accepted in revised form: 19. 6.07


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