IK cytokine ameliorates the progression of lupus nephritis in MRL/lpr mice

Authors


Abstract

Objective

IK cytokine has been isolated as a factor that inhibits interferon-γ (IFNγ)–induced expression of class II major histocompatibility complex (MHC) antigens. Aberrant expression of class II MHC antigens has reportedly been recognized in the target organs of autoimmune diseases and been associated with disease activity. In this study, we investigated whether IK cytokine can ameliorate the progression of lupus nephritis in MRL/lpr mice.

Methods

A truncated IK analog was prepared and transfected into a nonmetastatic fibroblastoid cell line, and then injected subcutaneously into MRL/lpr mice at ages 8 weeks (before the onset of lupus nephritis) and 12 weeks (at the early stage of the disease).

Results

An IK cytokine, when it was translated from methionine at position 316, acted as a secretory protein. This truncated IK cytokine (tIK) reduced IFNγ-induced class II MHC expression in various cells through decreased expression of class II MHC transcription activator. Treatment of MRL/lpr mice with tIK significantly reduced renal damage as compared with control mice. A significant decrease in macrophage and T cell infiltration was found in the kidneys of tIK-treated mice, resulting in decreased production of IFNγ and interleukin-2. Mice treated with tIK also showed significant reduction of anti-DNA antibodies and circulating immune complexes. A specific reduction of class II MHC expression was observed on B cells and monocytes as well as in the kidney.

Conclusion

We prepared a potent IK analog and demonstrated its ability to ameliorate the progression of lupus nephritis. This agent may therefore provide a new therapeutic approach for lupus nephritis.

Systemic lupus erythematosus (SLE) is an autoimmune disease that involves increased production of autoantibodies, immune complex (IC) deposition in the microvasculature of various organs, complement activation, leukocyte infiltration, and tissue damage. The IC glomerulonephritis of SLE is both a major cause of morbidity and a determinant of disease outcome (1). Studies of the pathogenesis of SLE have been facilitated by the availability of murine lupus models. Autoimmune diseases in MRL/MpJ-lpr/lpr (MRL/lpr) mice resemble human SLE and are characterized by dysregulation of both cellular and humoral immunity (2, 3). MRL/lpr mice spontaneously develop fatal glomerular disease in association with an increase in circulating ICs, autoantibody production, and cytokine abnormalities. The main histopathologic characteristics of the glomerular lesions in MRL/lpr mice are endocapillary proliferative, crescentic, and/or wire loop–like features, closely resembling those seen in human lupus nephritis, especially class IV.

Class II major histocompatibility complex (MHC) antigens play a critical role in the initiation, development, and regulation of the immune response (4, 5). These surface molecules are constitutively expressed on antigen-presenting cells (APCs), including B cells, macrophages, and dendritic cells. In addition, class II MHC expression can be induced on most cell types by exposure to various cytokines, the most potent of which is interferon-γ (IFNγ) (6). Aberrant expression of class II MHC antigens has been reported to be recognized in the target organs of autoimmune diseases and to be associated with disease activity (4, 7). Expression of class II MHC antigens on glomerular endothelial cells and tubular epithelial cells of the kidney has been reported in humans with SLE and in animal models of SLE (7–10). Since alteration of class II MHC expression on APCs and target cells can affect both the type and the magnitude of immune responses, immune regulation through class II MHC antigens may become a therapeutic target in the treatment of lupus nephritis.

IK cytokine was originally isolated from the conditioned culture medium of the K562 erythroleukemic cell line as a factor that inhibited the IFNγ-induced expression of class II MHC antigens (11). Moreover, IK cytokine also reportedly inhibits class II MHC constitutive expression as a regulatory protein (12, 13). We previously established a system that allows for long-term observation of the effect of secretory agents such as chemokine antagonists on autoimmune diseases in MRL/lpr mice using the nonmetastatic fibroblastoid cell line MRL/N-1, which is derived from the MRL mouse strain (14, 15). In the present study, we prepared a potent secretory form of IK cytokine and demonstrated its therapeutic effect on lupus nephritis in MRL/lpr mice using this system.

MATERIALS AND METHODS

Mice and cells.

MRL/lpr mice were purchased from The Jackson Laboratory (Bar Harbor, ME). The fibroblastoid cell line MRL/N-1, which does not metastasize after injection into the skin, was established from the spleen of an MRL/MpTn-gld/gld mouse (16). RAW264.7, a murine macrophage cell line, was obtained from Riken Cell Bank (Tsukuba, Japan) (17, 18). The MRL/N-1 and RAW264.7 cells were maintained in RPMI 1640 medium supplemented with 10% heat-inactivated fetal calf serum (Life Technologies, Gaithersburg, MD).

Antibodies and reagents.

Fluorescein isothiocyanate (FITC)–conjugated anti-mouse macrophage (MOMA-2) monoclonal antibody (mAb) and FITC-conjugated anti-mouse CD45R (B220) mAb were obtained from Beckman Coulter (Roissy, France). Phycoerythrin (PE)–conjugated anti-mouse I-Ak mAb and biotin-conjugated anti-mouse I-Ak mAb were purchased from BD Biosciences (San Diego, CA).

Construction of expression vector encoding the truncated IK analog (tIK).

To amplify the fragment of tIK translated from methionine at position 316, the following primer set for murine IK cytokine (19) was used: 5′-AGGCTGACATGAACATTTTTG-3′ (forward) and 5′-TGTTTCTAGAAAGATTAGTACTTTGGTCTTTTCAC-3′ (reverse). Total cellular RNA was extracted from murine spleen cells as previously described (20). The required fragment was amplified by a reverse transcriptase–polymerase chain reaction (RT-PCR) technique using an RNA PCR kit (Takara Shuzo, Kyoto, Japan), as described previously (20). After confirming the entire nucleotide sequence, the fragment was cloned into the Eco RI site of the pCXN2 vector (21) after the site had been end-filled to form blunt ends.

Cell transfection and selection of tIK-transfected cell line.

MRL/N-1 cells were transfected with 10 μg of pCXN2 containing the tIK gene using Lipofectamine (Life Technologies), as described previously (22). The transfectants were selected for resistance to G418 (Life Technologies) at a concentration of 500 μg/ml for MRL/N-1 cells. The stable transfectants were isolated and examined for expression of the tIK gene by Northern blot analysis. The clone (MRL/N-1/tIK) showing the strongest expression of tIK was then selected for use in the study.

Purification of tIK protein.

Purification of the tIK protein from the culture supernatant of the transfectant was performed as previously described (11). The culture supernatant was concentrated with an Amicon Ultra centrifugal filter (Millipore, Bedford, MA) and subjected to anion exchange chromatography on a Mono Q column (Amersham Biosciences, Piscataway, NJ). The fractions containing bioactivity were pooled and further purified using an S12 gel filtration column (Amersham Biosciences). The protein concentration was determined using a bicinchoninic acid assay kit (Pierce, Rockford, IL), and the purity was analyzed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and silver staining. The purity of the tIK protein was >90%.

RT-PCR detection of class II MHC, cytokines in tissues and cells, and class II MHC transcription activator (CIITA).

Total RNA was extracted from snap-frozen kidneys and spleens using RNAzol B (Tel-Test, Friendswood, TX). Messenger RNA (mRNA) was prepared using a TaKaRa Bio mRNA purification kit (Takara Shuzo). The following specific primers for murine class II MHC, CIITA, and cytokines were used: I-Ak (23) 5′-AAGAAGGAGACTGTCTGGATGC-3′ (forward), 5′-TGAATGATGAAGATGGTGCCC-3′ (reverse); CIITA (24) 5′-TCCTCTGGAAAGACTCAGTCCA-3′ (forward), 5′-ATATCCACCACGTGCTTTCTCA-3′ (reverse); interleukin-2 (IL-2) (25) 5′-GACACTTGTGCTCCTTGTCAACAG-3′ (forward), 5′-TGATGAAATTCTCAGCATCTTCCA-3′ (reverse); IFNγ (14) 5′-CACGGCACAGTCATTGAAAGCC-3′ (forward), 5′-CTTATTGGGACAATCTCTTCCC-3′ (reverse); GAPDH (14) 5′-GGTGGAGGTCGGAGTCAACG-3′ (forward), 5′-CAAAGTTGTCATGGATGACC-3′ (reverse). The required fragments were amplified by RT-PCR using an RNA PCR kit (Takara Shuzo). First-strand complementary DNA (cDNA) was synthesized from 2 μg total RNA and subjected to 28 PCR cycles (94°C for 1 minute, 58°C for 30 seconds, and 72°C for 1 minute) to detect I-Ak, IFNγ, and IL-2. First-strand cDNA was synthesized from 2 μg mRNA and subjected to 20 PCR cycles (94°C for 1 minute, 55°C for 1 minute, and 72°C for 1 minute) to detect CIITA.

Flow cytometric analysis.

RAW264.7 cells (1 × 105/ml) were incubated for 48 hours with 2 ng/ml murine IFNγ (Diaclone, Besançon, France) in the presence or absence of 50 ng/ml tIK protein. The spleens of control and tIK-treated MRL/lpr mice were harvested. Single-cell suspensions of splenocytes were prepared after the erythrocytes had been removed with hemolytic buffer. Flow cytometric analysis of these cells was performed as previously described (26). Briefly, RAW264.7 cells and splenocytes were blocked by Fc receptor blocking reagent (Miltenyi Biotec, Auburn, CA). Class II MHC expression of these cells was analyzed using PE-conjugated anti-mouse I-Ak mAb. To further identify the splenocyte populations, anti-MOMA as well as anti-B220 conjugated to FITC were used.

Immunohistochemistry.

To detect class II MHC expression, formalin-fixed sections were deparaffinized and analyzed using avidin–biotin–peroxidase complex with biotin-labeled anti-mouse I-Ak mAb, as described previously (14). Preimmune biotin-labeled goat serum served as a negative control. The phenotypes of the infiltrating cells were also analyzed using a rabbit anti-human CD3 polyclonal antibody (Dako, Carpinteria, CA) for T cells, and a rat anti-mouse macrophage mAb (Mac-2; Boehringer Mannheim, Mannheim, Germany).

Evaluation of nephritis, vasculitis, and sialadenitis.

Kidneys were fixed in 10% formalin for 24 hours at 4°C. Paraffinized sections of kidneys were stained with hematoxylin and periodic acid–methenamine–silver reagent. Histopathologic findings in glomerular lesions were graded on a scale of 0–3, where 0 = normal, 1 = mild (cell proliferation and/or cell infiltration), 2 = moderate (cell proliferation and/or cell infiltration with membrane proliferation), and 3 = severe (cell proliferation and/or cell infiltration, membrane proliferation, and crescent formation and/or hyalinosis). The glomerular lesion index was calculated from the sum of the scores for 40 random glomeruli per kidney.

Histopathologic findings in renal vascular lesions were graded on a scale of 0–3, where 0 = normal, 1 = mild (perivascular cell infiltration), 2 = moderate (destruction of arterial wall), and 3 = severe (myointimal thickening). The vascular lesion index was calculated from the sum of the scores for all vessels per section.

The degree of sialadenitis in the submandibular glands was graded on a scale of 0–3, where 0 = normal, 1 = mild (cell infiltration localized in the perivascular and/or periductal regions), 2 = moderate (cell infiltration localized in the perivascular and/or periductal regions with extension to the parenchyma), and 3 = severe (cell infiltration localized in the perivascular and/or periductal regions with extension to the parenchyma, with fibrosis and/or granuloma). The sialadenitis index was calculated from the sum of the scores for all ducts and all vessels per section.

Lymphadenopathy and splenomegaly.

When the mice were killed, all swollen lymph nodes (cervical, bronchial, inguinal, and mesenteric) and the spleens were removed and weighed to evaluate lymphadenopathy and splenomegaly.

Measurement of IFNγ and IL-2 in mouse serum.

The serum levels of IFNγ and IL-2 in the mice were measured using a mouse Th1/Th2 cytokine kit (BD cytometric bead array; BD Biosciences), following the manufacturer's instructions.

Evaluation of circulating ICs and anti-DNA antibody.

The levels of circulating ICs and anti-DNA antibodies were measured by enzyme-linked immunosorbent assay (ELISA), as described previously (14). Briefly, ELISA plates were coated with 10 μg/ml human C1q (Sigma, St. Louis, MO) to measure circulating ICs or with 5 μg/ml double-stranded calf thymus DNA (Sigma) for measurement of anti-DNA antibody. Sera were added in serial dilutions starting at 1:40 and incubated for 30 minutes at room temperature. After washing, horseradish peroxidase–conjugated goat anti-mouse IgG antibody was added. A mouse anti–double-stranded DNA mAb (Chemicon International, Temecula, CA) served as a positive control, allowing a reference standard curve to be drawn.

Colony formation assay.

The colony formation assay was performed as described by Han et al (27). Briefly, murine bone marrow cells were isolated from the femurs of 8-week-old MRL/lpr mice, and stem cells were separated from the murine bone marrow cells using anti–Sca-1 MicroBeads with a MACS column (Miltenyi Biotec). Cells (2 × 103 cells/ml) were plated in MethoCult GF M3434 (StemCell Technologies, Vancouver, British Columbia, Canada) with purified tIK at a concentration of 50 ng/ml. The numbers of burst-forming unit–erythrocyte (BFU-E), colony-forming unit–granulocyte–macrophage (CFU-GM), and CFU–granulocyte–erythrocyte–macrophage–megakaryocyte (CFU-GEMM) colonies were counted individually after 11 days of incubation. Three plates were examined per experiment, and each experimental set was performed in triplicate.

Statistical analysis.

Values are expressed as the mean ± SD. Statistical analysis was performed using Student's t-test. P values less than 0.05 were considered significant.

RESULTS

Preparation of tIK-transfected cell lines and purification of tIK protein.

In the preliminary experiment, we prepared stable full-length IK cytokine–expressing cells (MRL/N-1/IK), in which the 1.7-kb IK cytokine fragment containing the full coding region was cloned into the pCXN2 vector and transfected into MRL/N-1 cells. However, hardly any IK cytokine was present in the culture supernatant from MRL/N-1/IK cells, even though IK mRNA was highly expressed in the cells themselves (data not shown). Next, we prepared tIK-expressing cells (MRL/N-1/tIK), in which the 0.74-kb amplified fragment translated from methionine at position 316 was cloned into the pCXN2 vector and transfected into MRL/N-1 cells. The tIK protein was secreted sufficiently into the culture supernatant from the MRL/N-1/tIK cells. The tIK protein was purified from this culture supernatant by anion-exchange chromatography on a Mono Q column and, subsequently, on an S12 gel filtration column. The molecular weight of the purified murine tIK protein was ∼28 kd.

Reduction by tIK protein of IFNγ-induced class II MHC expression by RAW264.7 cells.

We examined whether purified tIK protein reduced constitutive or IFNγ-induced class II MHC expression by the RAW264.7 murine macrophage cell line. The tIK protein did not suppress constitutive class II MHC expression by RAW264.7 cells, even at the high concentration of 1 μg/ml (data not shown). The expression of class II MHC by RAW264.7 cells (1 × 105/ml) was up-regulated by IFNγ at a concentration of >0.2 ng/ml. The purified tIK protein began to suppress IFNγ-induced class II MHC expression by RAW264.7 cells at a concentration of 5 ng/ml, and its activity reached a plateau at a concentration of 50 ng/ml, when IFNγ was added at a concentration of 2 ng/ml (Figure 1A). Next, using RT-PCR of mRNA, we analyzed the expression of the CIITA promoter by IFNγ-treated RAW264.7 cells in the presence or absence of tIK. As shown in Figure 1B, the level of CIITA mRNA was decreased by 62% in tIK-treated RAW264.7 cells in comparison with tIK-untreated RAW264.7 cells. This finding suggests that the reduction of IFNγ-induced class II MHC expression by RAW264.7 cells by tIK occurs through decreased expression of CIITA.

Figure 1.

A, Class II major histocompatibility complex (MHC) expression by RAW264.7 cells treated with interferon-γ (IFNγ) in the presence or absence of truncated IK cytokine (tIK). RAW264.7 cells (1 × 105/ml) were incubated for 48 hours with 2 ng/ml murine IFNγ in the presence or absence of 50 ng/ml tIK.B, Class II MHC transcription activator (CIITA) mRNA expression by RAW264.7 cells treated with IFNγ in the presence or absence of tIK. The expression of CIITA mRNA in RAW264.7 cells was examined by reverse transcriptase–polymerase chain reaction as described in Materials and Methods.

Evaluation of lupus nephritis and renal vasculitis in tIK-treated MRL/ lpr mice.

Control MRL/N-1/pCXN2 or MRL/N-1/tIK cells (5 × 106) were injected subcutaneously into MRL/lpr mice at ages 8 weeks (before the onset of lupus nephritis) and 12 weeks (at the early stage of the disease), since MRL/lpr mice began to show a significant increase in renal mRNA for I-Ak at 8 weeks of age (data not shown). Two weeks after injection, tIK was present in the serum of mice, as confirmed by reduction of which represent concentration with respect to the IFNγ (0.2 ng/ml)–induced class II MHC expression by RAW264.7 cells (mean ± SD 15 ± 10% reduction; P < 0.05). Furthermore, the serum of MRL/N-1/tIK–bearing MRL/lpr mice 6 weeks after injection also reduced IFNγ-induced class II MHC expression by RAW264.7 cells (72 ± 10% reduction; P < 0.01). The mean ± SD volume of MRL/N-1/pCXN2 and MRL/N-1/tIK tumors in MRL/lpr mice was 780 ± 240 mm3 and 840 ± 320 mm3, respectively, at 6 weeks after injection.

We then compared the extent of lupus nephritis between control and tIK-treated MRL/lpr mice 6 weeks after injection into 8-week-old animals (before the onset of the disease). As shown in Figure 2A, control MRL/N-1/pCXN2–bearing mice demonstrated progressive development of renal damage, such as the presence of enlarged hypercellular glomeruli with increased numbers of both resident cells and infiltrating leukocytes, glomerulosclerosis, crescent formation, hyalinosis, and prominent interstitial mononuclear cell infiltrates. The tIK-treated mice showed significantly diminished glomerular hypercellularity, crescent formation, glomerulosclerosis, and interstitial mononuclear cell infiltrates in comparison with control mice (mean ± SD index of glomerular lesions score 1.19 ± 0.56 versus 2.04 ± 0.58; P < 0.05) (Figure 2B).

Figure 2.

A, Comparison of lupus nephritis and renal vasculitis in control and truncated IK cytokine (tIK)–treated MRL/lpr mice. Eight-week-old mice were injected, and the extent of lupus nephritis (periodic acid–methenamine–silver stain; top) and renal vasculitis (hematoxylin and eosin stain; bottom) in control and tIK-treated MRL/lpr mice was examined 6 weeks after injection. MRL/N-1/pCXN2–bearing MRL/lpr mice were used as controls (see Materials and Methods) (original magnification × 200).B, Evaluation of glomerulonephritis and vasculitis in control and tIK-treated MRL/lpr mice. Eight-week-old (before the onset of lupus nephritis) and 12-week-old (at the early stage of lupus nephritis) mice were injected, and the extent of tissue damage was compared between control and tIK-treated MRL/lpr mice 6 weeks after injection. MRL/N-1/pCXN2–bearing MRL/lpr mice were used as controls. The degree of tissue damage was graded as described in Materials and Methods. Values are the mean and SD index of glomerular lesions (IGL; 40 random glomeruli per kidney) and index of vascular lesions (IVL; all vessels in the kidney) (n = 6 mice per group). Similar results were obtained in 2 independent experiments.

Moreover, in renal vasculitis, tIK-treated mice also demonstrated significantly reduced destruction of the vessel wall associated with the formation of granulomatous lesions in the perivascular regions compared with control mice (mean ± SD index of vascular lesions score 0.67 ± 0.40 versus 1.20 ± 0.39; P < 0.05) (Figure 2B). As shown in Figure 3, infiltrating macrophages and T cells in the glomerulus, tubulointerstitial, and perivascular regions were significantly diminished in tIK-treated MRL/lpr mice compared with control mice. In addition, tIK-treated mice also had significantly less severe proteinuria than control mice (mean ± SD 220 ± 80 μg protein/mg creatinine versus 480 ± 180 μg protein/mg creatinine; P < 0.01).

Figure 3.

Quantitative analysis of mononuclear cells in glomerular, tubulointerstitial, and perivascular areas in kidneys from control and truncated IK cytokine (tIK)–treated MRL/lpr mice. Formalin-fixed sections were deparaffinized and then analyzed by immunohistochemistry as described in Materials and Methods. The numbers of CD3+ T cells and macrophages (Mϕ) were determined. Values are the mean and SD number of infiltrating and parenchymal cells in glomerular (cell counts in 20 random glomeruli) interstitial (10 random fields at 400× magnification), and perivascular (5 random perivascular areas) regions (n = 6 mice per group).

Next, we compared renal damage between control and tIK-treated MRL/lpr mice 6 weeks after inoculation at 12 weeks of age (at the early stage of the disease). As shown in Figure 2B, tIK-treated mice also had significantly reduced (P < 0.05) glomerular damage and vasculitis compared with control mice (mean ± SD index of glomerular lesions score 1.80 ± 0.60 versus 2.55 ± 0.44; index of vascular lesions score 1.01 ± 0.62 versus 1.88 ± 0.51). Mice treated with tIK also had significantly reduced proteinuria compared with control mice (320 ± 120 μg protein/mg creatinine versus 540 ± 200 μg protein/mg creatinine; P < 0.05). These findings indicate that tIK ameliorated the progression of lupus nephritis and renal vasculitis in MRL/lpr mice.

In contrast to the above observations, no significant differences in sialadenitis, lymphadenopathy, or splenomegaly were observed between the 2 groups (data not shown).

Measurement of IFNγ and IL-2 in the kidneys and serum of tIK-treated MRL/ lpr mice.

It has been reported that Th1-associated cytokines, especially IFNγ, play an important role in the progression of renal injury in MRL/lpr mice (28–31). Using RT-PCR, we examined the expression of IFNγ and IL-2 mRNA in total kidney RNA prepared from control and tIK-treated MRL/lpr mice at 6 weeks after injection into 8-week-old animals (Figures 4A and B). In the kidneys of tIK-treated mice, IFNγ and IL-2 transcripts were reduced by 67% and 60%, respectively, as compared with those in the kidneys of control mice. Moreover, the serum levels of IFNγ and IL-2 in tIK-treated mice were significantly lower than those in control mice (mean ± SD 14.03 ± 4.01 pg/ml and 7.73 ± 3.04 pg/ml versus 41.15 ± 17.61 pg/ml and 21.05 ± 10.29 pg/ml, respectively; P < 0.05). These findings indicated that tIK suppressed the production of IFNγ and IL-2 systemically and locally, and that amelioration of lupus nephritis by tIK was associated with a reduction of IFNγ and IL-2 production.

Figure 4.

Expression of interferon-γ (IFNγ) and interleukin-2 (IL-2) mRNA in kidneys from control and truncated IK (tIK) cytokine–treated MRL/lpr mice. A, Results of reverse transcriptase–polymerase chain reaction (RT-PCR). Eight-week-old mice were injected, and total kidney RNA was prepared from control and tIK-treated MRL/lpr mice 6 weeks after injection. MRL/N-1/pCXN2–bearing MRL/lpr mice were used as controls. B, Results of quantitative analysis of IFNγ and IL-2 mRNA expression in kidneys of control and tIK-treated MRL/lpr mice. Values are the mean and SD ratio of IFNγ mRNA or IL-2 mRNA to each respective GAPDH mRNA, as detected by RT-PCR (n = 6 mice per group).

Anti-DNA antibodies and circulating ICs in tIK-treated MRL/ lpr mice.

To determine whether tIK altered the production of anti-DNA antibodies and circulating ICs in MRL/lpr mice, we evaluated serum levels of anti-DNA antibodies and circulating ICs in control and tIK-treated mice. As shown in Figure 5, serum levels of both anti-DNA antibodies and circulating ICs in tIK-treated mice were significantly lower than those in control mice, indicating that tIK suppressed the production of anti-DNA antibodies and circulating ICs in MRL/lpr mice.

Figure 5.

Production anti-DNA antibodies (Ab) and circulating immune complexes in the serum of control and truncated IK (tIK) cytokine–treated MRL/lpr mice. A, Eight-week-old (before the onset of lupus nephritis) and B, 12-week-old (at the early stage of lupus nephritis) mice were injected, and anti-DNA antibodies and circulating immune complexes were measured according to standard procedures. Values are the mean ± SD (n = 6 mice per group). Similar results were obtained in 2 independent experiments.

Reduction of class II MHC expression in the kidneys and spleens of tIK-treated MRL/lpr mice.

We analyzed the expression levels of class II MHC and costimulatory molecules in target organs such as kidneys and spleens in tIK-treated MRL/lpr mice, since expression of these molecules is critical to the activation and expansion of autoreactive T cells as well as autoantibody production (4). As shown in Figure 6A, class II MHC expression was recognized by immunohistochemistry mainly on proximal tubules, interstitium, mesangial cells, and infiltrating macrophages in the kidneys of control mice at 14 weeks of age. In tIK-treated mice, the expression level of class II MHC on these cells was reduced significantly in comparison with control mice. This finding was also confirmed by RT-PCR, which showed decreased expression of I-Ak mRNA in total kidney RNA (Figure 6B). In addition, treatment with tIK significantly reduced class II MHC expression on splenic B cells as well as monocytes compared with the levels in control mice (Figures 6A and B).

Figure 6.

A, Class II major histocompatibility complex (MHC) expression in kidneys and splenocytes from control and truncated IK cytokine (tIK)–treated MRL/lpr mice. Top, Representative immunohistochemistry results. Formalin-fixed sections were deparaffinized, incubated with biotin-labeled rabbit anti-murine I-Ak monoclonal antibody, and the expression of class II MHC was detected using avidin–biotin–peroxidase complex. Sections were counterstained with hematoxylin (original magnification × 400). Bottom, Representative flow cytometry results. Splenocytes were blocked with Fc receptor blocking reagent. Class II MHC expression was analyzed using phycoerythrin-conjugated anti-mouse I-Ak monoclonal antibody. To further identify the splenocyte populations, fluorescein isothiocyanate–conjugated anti–MOMA-2 or anti-B220 monoclonal antibody was used. Data were collected with a FACScan flow cytometer and analyzed using CellQuest software. B, Results of reverse transcriptase–polymerase chain reaction of kidneys and spleens from control and tIK-treated MRL/lpr mice. Total RNA from kidneys and spleens was prepared 6 weeks after injection, and class II MHC mRNA expression was examined as described in Materials and Methods.

However, soluble tIK protein did not reduce constitutive class II MHC expression on splenic B cells and monocytes isolated from 8-week-old MRL/lpr mice whose serum level of IFNγ was not elevated (data not shown). In addition, the expression of costimulatory molecules such as CD80, CD86, and CD40 on B cells also remained unaffected in tIK-treated mice (data not shown). These findings demonstrate that tIK reduced IFNγ-induced class II MHC expression rather than constitutive class II MHC expression, and that tIK ameliorated the progression of lupus nephritis in MRL/lpr mice through a specific reduction of IFNγ-induced class II MHC expression on B cells and monocytes as well as in target organs such as the kidney.

Effect of tIK on colony formation by bone marrow cells in MRL/lpr mice.

To examine the effect of tIK on the proliferation and differentiation of pluripotent stem cells, we performed a colony formation assay using stem cells isolated from bone marrow cells derived from MRL/lpr mice. After 11 days of incubation, there were no significant differences in the numbers of BFU-E, CFU-GM, and CFU-GEMM colonies between control and tIK-treated groups (mean ± SD BFU-E 11.98 ± 1.95 versus 11.09 ± 1.83; CFU-GM 48.09 ± 2.90 versus 48.61 ± 3.74; and CFU-GEMM 2.78 ± 0.77 versus 2.58 ± 0.68, respectively).

DISCUSSION

This is the first study to show that IK cytokine can ameliorate the progression of lupus nephritis in MRL/lpr mice. Our main findings are as follows: IK cytokine acted as a secretory protein when translated from methionine at position 316; the tIK reduced IFNγ-induced class II MHC expression in various cells through decreased expression of CIITA; treatment of MRL/lpr mice with tIK significantly reduced renal damage compared with control mice; a significant decrease in the infiltration of macrophages and T cells was found in the kidneys of tIK-treated mice, resulting in decreased IFNγ and IL-2 production; a significant reduction of anti-DNA antibodies and circulating ICs was also observed in tIK-treated mice; these findings were due to a specific reduction of class II MHC expression on B cells and monocytes as well as in the kidneys, leading to a reduction in the activation and expansion of autoreactive T cells, autoantibody production, and renal injury.

IK cytokine was shown to inhibit IFNγ-induced expression of class II MHC antigen (11). In addition, IK cytokine also inhibited constitutive expression by class II MHC through repression of CIITA transcription in the IK-transfected Raji B cell line (12). The human and murine forms of IK cytokine share 98% sequence identity at the amino acid level. The murine IK cytokine consists of 557 amino acid residues, which contain a trimeric coiled-coil motif, a nuclear localization signal (NLS), and a region rich in tandem repeats of arginine (R)–aspartic acid (D) or R–glutamic acid (E), which are commonly found in RNA nuclear-binding proteins (19). These findings suggest that IK cytokine acts as a regulatory protein and is possibly involved in CIITA transcription.

On the other hand, IK cytokine was originally isolated and purified from the conditioned culture medium of the K562 erythroleukemic cell line (11). This isolated IK cytokine was a 19-kd protein encoded by a truncated gene that consisted of 162 amino acid residues, from methionine at position 316 to proline at position 477. Isolated IK showed deletion of the trimeric coiled-coil motif and NLS sequence at the C-terminal end, but retained the RD/RE tandem repeat region. Moreover, tIK in which 315 amino acid residues are deleted from the N-terminal end has been reported to be incapable of localizing to the nucleolus, although the full-length IK cytokine shows efficient sorting and nucleolar localization (19).

In the present study, we found that tIK was secreted sufficiently from transfected cells and did not suppress constitutive class II MHC expression, but reduced IFNγ-induced expression of class II MHC antigen through CIITA repression. Although the receptors and mechanism involved are well understood, tIK is likely to induce a cascade of downstream transduction signals affecting IFNγ-mediated CIITA mRNA accumulation through its extracellular receptor. From these findings and our present results, it appears that IK cytokine acts not only as a nuclear-binding protein, but also as a secreting protein when translated from methionine at position 316.

Class II MHC antigens play a critical role in the immune response by presenting antigenic peptides to CD4+ T cells. Although the normal pattern of expression by class II MHC antigen is restricted to APCs, a wide range of cells can express class II MHC antigens on their surface when stimulated with cytokines such as IFNγ. Th1-associated cytokines, especially IFNγ, have been reported to play an important role in the progression of renal injury in MRL/lpr mice (28–31). In MRL/lpr mice, it has been previously postulated that class II MHC expression is enhanced on tubular epithelial cells, glomerular endothelium, and mesangial cells, preceding overt glomerulonephritis and proteinuria (9), and that an increase in renal class II MHC expression might accompany the renal lesions in parallel with an increase in T cell infiltrates, since most of the infiltrating cells within the kidneys of diseased MRL/lpr mice are CD4+ T cells (32, 33).

These findings suggest that the increased production of IFNγ and up-regulation of class II MHC expression are likely to contribute to disease pathogenesis in MRL/lpr mice. The B cell–deficient MRL/lpr mice not only had an obvious lack of autoantibodies, but also lacked T cell–mediated manifestations of the disease, such as proliferation and activation of autoreactive T cells and T cell infiltrates in the target organs, suggesting that B cells play a key immunoregulatory role as APCs in autoimmune diseases besides their established pathogenic role as autoantibody-secreting cells (34, 35). In addition, the self peptides bound by class II MHC molecules, which are required for the generation of autoantibodies, have also been isolated from MRL/lpr mice (36, 37). From these findings, it is suggested that immune regulation through class II MHC antigens plays a critical role not only in the production of autoantibodies but also in the activation of autoreactive T cells in target organs such as the kidneys in MRL/lpr mice.

On the basis of these findings, we attempted to ameliorate lupus nephritis in MRL/lpr mice by using tIK protein to reduce class II MHC expression on APCs and in target organs. In kidney specimens from patients with lupus nephritis, expression by class II MHC antigens has been observed mainly on glomerular endothelial cells, tubular epithelial cells, mesangial cells, and macrophages (7). Moreover, it has been reported that the aberrant expression by class II MHC antigens in glomeruli and serum IFNγ concentrations is related to proliferative and active lesions, including leukocyte infiltration, in human lupus nephritis (10). Therefore, since the development of renal damage through the increased production of IFNγ and the up-regulation of class II MHC expression in patients with lupus nephritis closely resembles that in MRL/lpr mice, our findings in MRL/lpr mice will be applicable to the treatment of lupus nephritis in humans.

In the present study, inoculation of tIK into MRL/lpr mice before the onset and at the early stage of lupus nephritis significantly reduced glomerular damage, interstitial mononuclear cell infiltration, and renal vasculitis as compared with control MRL/lpr mice. The tIK-treated MRL/lpr mice showed a significant reduction of IFNγ and IL-2 both systemically and in the kidneys, as well as a significant reduction of circulating ICs and anti-DNA antibodies compared with control mice. In addition, intrarenal deposition of IgG and C3 was also significantly milder in tIK-treated MRL/lpr mice than in control mice (Hasegawa H: unpublished observations). It was considered that down-modulation of class II MHC expression on B cells and monocytes, as well as in the kidneys, suppressed the activation and expansion of autoreactive T cells, leading to decreased production of IFNγ, IL-2, and autoantibodies, resulting in amelioration of renal injury. In contrast, there was no difference in lymphadenopathy and splenomegaly between control and tIK-treated MRL/lpr mice. This may be because a massive expansion of CD4−,CD8−,B220+ T cells is observed in the lymph nodes and spleen of MRL/lpr mice with increasing age, and these cells are not restricted to class II MHC antigens (38, 39).

The up-regulation of class II MHC expression on salivary epithelial cells is reportedly related to the promotion of autoimmune reactions between epithelial cells and lymphocytes in the salivary glands of MRL/lpr mice (40). However, in the present study, there was no difference in sialadenitis between control and tIK-treated MRL/lpr mice. This indicated that other factors, such as cytokines, chemokines, and adhesion molecules, rather than class II MHC antigens, might play an important role in the development of autoimmune sialadenitis in MRL/lpr mice. Cao et al (13) have shown that IK expression increased and was inversely correlated with gradual loss of HLA–DR during growth factor–induced CD34+ cell proliferation and differentiation, and that IK antisense oligonucleotide inhibited colony formation by multilineage early erythroid and granulomonocytic CD34+ progenitors. Full-length IK acts as a nuclear-binding protein and inhibits constitutive class II MHC expression, whereas tIK acts as a secreting protein and reduces IFNγ-induced class II MHC expression. Therefore, tIK did not affect the proliferation and differentiation of pluripotent stem cells in MRL/lpr mice because it did not inhibit constitutive class II MHC expression.

It has been reported that class II MHC–deficient MRL/lpr mice fail to produce significant levels of autoantibodies or to develop lupus nephritis, despite the development of lymphadenopathy and CD4−,CD8−,B220+ T cell expansion (41). Recently, statins have been reported to reduce autoantibody production and delay the development of lupus nephritis in (NZB × NZW)F1 mice through down-modulation of class II MHC expression (24, 42). These studies support our present results. The secretory form of IK cytokine may therefore provide a potentially useful approach to the treatment of lupus nephritis in humans.

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