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The Journal of Immunology, 2006, 177: 5676-5686.
Copyright © 2006 by The American Association of Immunologists, Inc.

Development of Autoimmunity in IL-14{alpha}-Transgenic Mice1

Long Shen*, Chongjie Zhang*,{dagger}, Tao Wang*, Stephen Brooks{ddagger}, Richard J. Ford§, Yen Chiu Lin-Lee§, Amy Kasianowicz, Vijay Kumar, Lisa Martin||, Ping Liang#, John Cowell# and Julian L. Ambrus, Jr2,*

* Division of Allergy, Immunology and Rheumatology, Department of Medicine, School of Medicine and Biomedical Sciences, State University of New York, Buffalo, NY 14203 and Kaleida Health, Buffalo, NY; {dagger} Department of Immunology, Sichuan University, Sichuan, China; {ddagger} Department of Surgery, Kaleida Health, Buffalo, NY 14203; § Department of Hemato-Pathology, M. D. Anderson Cancer Center, Houston, TX 77030; Immco, Buffalo, NY 14228; || Department of Comparative Medicine, State University of New York, Buffalo, NY 14260; and # Department of Cancer Genetics, Roswell Park Cancer Institute, Buffalo, NY 14263


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Multiple genetic loci contribute to the development of systemic lupus erythematosus (SLE). In murine models for SLE, various genes on chromosome four have been implicated. IL-14 is a cytokine originally identified as a B cell growth factor. The il14 gene is located on chromosome 4. IL-14{alpha} is a cytokine encoded by the plus strand of the IL-14 gene using exons 3–10. The expression of IL-14{alpha} is increased in (NZB x NZW)F1 mice. In this study, we produced IL-14{alpha}-transgenic mice to study the role of IL-14{alpha} in the development of autoimmunity. At age 3–9 mo, IL-14{alpha}-transgenic mice demonstrate increased numbers of B1 cells in the peritoneum, increased serum IgM, IgG, and IgG 2a and show enhanced responses to T-dependent and T-independent Ags compared with littermate controls. At age 9–17 mo, IL-14{alpha}-transgenic mice develop autoantibodies, sialadenitis, as in Sjögren’s syndrome, and immune complex-mediated nephritis, as in World Health Organization class II SLE nephritis. Between the ages 14–18 mo, 95% of IL-14{alpha}-transgenic mice developed CD5+ B cell lymphomas, consistent with the lymphomas seen in elderly patients with Sjögren’s syndrome and SLE. These data support a role for IL-14{alpha} in the development of both autoimmunity and lymphomagenesis. These studies may provide a genetic link between these often related disorders.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The development of autoimmunity involves the complex interactions among various genetic and environmental factors (1, 2, 3, 4, 5). Different murine models have demonstrated that modification of single genes involving lymphocyte activation, apoptosis, degradation of nucleosomes generated during apoptosis, or clearance of immune complexes can induce an systemic lupus erythematosus (SLE)3-like phenotype (6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19). Abnormalities in some of these genes, such as C1q, DNase1, and Fas, have been identified in rare patients with SLE or lymphoproliferative disorders (20, 21, 22, 23). Murine studies have also emphasized the importance of background genes in determining the phenotypes resulting from single gene alterations (7, 24, 25). The (NZB x NZW)F1 mouse has been carefully analyzed genetically because it represents a polygenic model for SLE (1, 26, 27). Genes contributed from both New Zealand Black (NZB) and New Zealand White (NZW) influence the ultimate phenotype. One such example is the sle2 gene locus on murine chromosome 4 that encompasses at least three intervals designated sle2a, sle2b, and sle2c. The sle2a and sle2b loci are contributed by NZW and sle2c by NZB (28). Within this 27 cM region marked on the 5' end by D4MIT6 are found several genes including cd72, tlr4, ifn, and lck. The most robust phenotype resulting from this locus is an increase in peritoneal CD5+ B-1a cells. However, the sle2 locus has also been shown to be responsible for B cell hyperreactivity resulting in elevated serum IgM and IgM Abs to various autoantigens but not IgG anti-nuclear Abs (ANA) (28, 29, 30). Mice congenic for sle2 on a C57BL/6 (B6) background demonstrated enhanced IgM responses to the T-dependent Ag dinitrophenyl-keyhole limpet hemocyanin and the T-independent Ag trinitrophenyl-LPS (29).

IL-14 is a cytokine that was originally designated as high molecular weight B cell growth factor (31). It was identified and cloned from a Burkitt lymphoma cell line (31, 32) and shown to enhance B cell proliferation, especially of germinal center B cells and surface Ig (sIg)Dlow human tonsillar B cells, which includes B1 cells and activated B2 cells (31, 33). The murine il14 gene was mapped to chromosome 4 near lck by M. Seldin (University of California at Davis, CA), within the locus designated sle2. The human IL14 gene was mapped to 1p34-6, the syntenic region for mouse chromosome 4 (H. Donath-Keller, unpublished data). Interestingly, two studies have identified 1p36 as a lupus susceptibility locus in humans (34, 35). The NCBI has designated the IL14 gene Txln for taxilin, a designation given to this gene by a publication more recent than any publications on IL-14 (36). To investigate the hypothesis that the il14 gene may play a role in the development of autoimmunity through regulating B cell function, transgenic mice were established expressing IL-14 in the B cell compartment.

Two transcripts are produced from opposite strands of the IL14 gene that we have designated IL-14{alpha} and IL-14beta. The current study focuses on IL-14{alpha}. The IL-14{alpha} transcript is produced from the plus strand of the IL-14 gene using exons 3–10. We first examined the expression of IL-14{alpha} in a murine model of SLE, (NZB x NZW)F1 mice. We then examined the phenotype of transgenic mice expressing IL-14{alpha} and demonstrated that they develop hypergammaglobulinemia and IgM anti-cardiolipin Abs but only rarely IgG ANA and anti-DNA Abs. IL-14{alpha}-transgenic mice also show enhanced responses to T-dependent and T-independent Ags, sialadenitis, and immune complex-mediated nephritis with deposition of IgM in their glomeruli. The IL-14{alpha} transgene induced a phenotype that is very similar to SLE and Sjögren’s syndrome. In addition, most aged IL-14{alpha}-transgenic mice develop a CD5+ B cell lymphoma similar to the tumors seen in elderly patients with SLE and Sjögren’s syndrome (37, 38, 39).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

B6 mice were obtained from The Jackson Laboratory and housed in the Laboratory Animal Facility at State University of New York (SUNY; Buffalo, NY) in accordance with institutional guidelines. IL-14{alpha}-transgenic mice were made by the Gene Targeting and Transgenic Facility at Roswell Park Cancer Institute (Buffalo, NY) and maintained in the Laboratory Animal Facility at SUNY Buffalo.

Production of transgenic mice

To produce the transgenic mice, the pEµSR vector expressing IL-14{alpha} was constructed as previously described (40). In brief, the cDNA for human IL-14{alpha} (amplified by RT-PCR from total RNA purified from PHA stimulated-Namalva cells with primers that also included the necessary polylinker sites and a hemagglutinin tag) was cloned into pBluescript using the endonuclease sites NcoI and BamHI. The cDNA for hemagglutinin-IL-14 was then inserted into the pEuSR polylinker sites by using the endonuclease sites HindIII and SST1. The completed vectors were transfected into DH5{alpha} cells that were expanded in Luria-Bertani medium. The transgenic vectors were then isolated using the Lysozyme-Triton plasmid prep kit (Qiagen) and further purified by CsCl centrifugation. The purified vectors were cut with NotI and the vector DNA containing the human IL-14{alpha} cDNA was purified using the QIAquick gel extraction kit (Qiagen).

Purified vectors were then injected into C3H oocytes fertilized with B6 sperm and placed into B6 pseudopregnant females. The presence of the transgenic vector in the offspring was determined by PCR of tail DNA purified by the DNeasy Tissue kit (Qiagen) using the vector-specific primers: forward-AGGCCTGTACGGAAGTGTTACTTC and reverse-CAGCCTGACCCTGAGGAGTGAATT. Positive offspring were then crossed with B6 partners for a minimum of five generations before studies were performed. The majority of the studies in this manuscript were performed on IL-14{alpha}-transgenic mice after >10 backcrosses with B6 partners. Initial studies (serum Igs, responses to vaccination with T-dependent and T-independent Ags) were performed with three independent founder lines, all of which gave similar results. Thereafter, the studies were performed with a single line, which is the topic of this manuscript.

Fluorescence in situ hybridization (FISH)

For the analysis of the chromosomal localization of the IL-14{alpha} transgene, metaphase chromosome spreads were prepared from cultured fibroblasts derived from the spleens of IL-14{alpha}-transgenic mice using standard hypotonic and air-drying procedures (41). The probe was derived from a 5.3-kb plasmid containing the 1.2-kb IL-14 cDNA insert. The DNA probe was labeled with digoxigenin-tagged dUTP using a standard nick-translation protocol (Roche). Detection of the hybridized probe was conducted using a FITC-tagged anti-digoxigenin Fab. The mouse chromosomes were stained with 4',6'-diamidino-2-phenylindole. Interpretation of the banded structure of the mouse karyotype was performed as described by Cowell (42). Metaphase chromosomes were prepared from three different mice and the localization of the IL-14 transgene was on the distal region of the long arm of chromosome 18 in each case.

Mouse cell separation

Dynabeads mouse pan B (B220; Invitrogen Life Technologies) was used to isolate mouse B220-positive and -negative cells from spleen following the manufacturer’s instruction.

Total RNA purification

Dependent on the source of samples, two different methods were used to collect total RNA. TRIzol purified total RNA from the whole spleen of mice, according to the manufacturer’s instructions (Invitrogen Life Technologies). Total RNA from cells harvested from the spleens of mice was purified with the QIAamp RNA Blood mini kit according to the manufacturer’s instructions (Qiagen).

Semiquantitative RT-PCR

cDNA was first produced from total RNA using the Superscript first-strand synthesis system for RT-PCR according to the manufacturer’s instructions (Invitrogen Life Technologies), then amplified with specific primers under the following conditions for PCR: 3 min of denaturation at 94°C, followed by 35 cycles of annealing at 58oC for 1 min, extension at 72°C for 1 min and denaturation at 94°C for 1 min. At the end of the 35 cycles, the reactions were maintained at 72°C for 5 min and then stored at 4°C. The primers used for the actin control were: forward-5'-GTGGGGCGCCCCAGGCACCA and reverse-5'-CTCCTTAATGTCACGCACGATTTC. For IL-14{alpha} mRNA, the primers were forward-5'-GCCACAGGAGAAGAAGAAAGCCAA and reverse-5'-GACTTTCAACCCCTTGTTCCTTGG.

RT-PCR products were separated on 1% agarose gels and visualized by UV light after incorporation of ethidium bromide, as described previously (32).

Northern blot analysis for murine IL-14{alpha}

The RNA probes for the Northern blots for murine IL-14{alpha} were prepared as follows: RT-PCR using the forward primers-5'-ATGGACGACCCAGACAGACA and reverse primers-5'-AGTGATCTCCTTCCCTAGAC for murine IL-14{alpha} were used to generate cDNA. The T7 promoter adapter was then added to these PCR products using the No-cloning promoter addition kit (Ambion). Biotin-labeled RNA probes were generated from this template by in vitro transcription using T7 phage RNA polymerase with biotin label UTP according to the manufacturer’s instructions (MAXI Script In Vitro Transcription kit; Ambion). The probes were purified using 6% Tris-borate/disodium/EDTA urea gels according to the manufacturer’s instructions (Invitrogen Life Technologies).

Total RNA was purified from murine spleen cells before and after stimulation with 10 µg/ml PHA for 8 h at 37°C in RPMI 1640/5% FBS as previously described (32). RNA was first separated for 2 h on denaturing agarose gels containing formaldehyde and then transferred to positively charged nylon membranes (Ambion) as described (32).

Hybridization of filters with biotin-labeled RNA probes was performed according to the Northern Max system for Northern blots (Ambion). The signal from the probes was detected using the Bio Detect kit according to the manufacturer’s instructions (Ambion).

Immunization of mice

For the study of responses to T-dependent and T-independent Ags, mice were immunized with 100 µg of either nitrophenyl (NP)-OVA (Biosearch Technologies) or NP-Ficoll (Biosearch Technologies), respectively, in IFA on days 0 and 14 i.p. Blood was drawn on days 0, 14, 28, and 70 for analysis by ELISA.

ELISA

The ELISA for IgG, IgM, and anti-NP were performed in 96-well plates coated with anti-murine IgG (Sigma-Aldrich), anti-murine IgM (Sigma-Aldrich), or NP-BSA (Biosearch Technologies), respectively, as previously described (33). For the IgM ELISA, sera were diluted 1/2,000 and for the IgG ELISA, sera were diluted 1/25,000 in PBS. Sera from the IL-14{alpha}-transgenic and control mice were incubated with plates, washed three times with PBS/0.1% Tween 20, and then alkaline phosphatase-conjugated anti-murine IgG or anti-murine IgM was added as indicated. Plates were again washed with PBS/0.1% Tween 20 and pNP phosphate disodium salt substrate (Pierce) added. Plates were then analyzed with a spectrophotometer reading at 405 nm as described previously (33).

The ELISA for IFN-{alpha}, IFN-beta, and IFN-{gamma} in the sera of mice were performed by using mouse IFN-{alpha} ELISA kit, mouse IFN-beta ELISA kit, and mouse IFN-{gamma} ELISA kit from BioSource International according to the manufacturer’s instructions.

The ELISA for autoantibodies in the sera of mice were performed as previously described (43, 44). ANA and anti-dsDNA Abs were performed by both the Kotzin laboratory (43) and by Immco. Anti-chromatin and anti-histone Abs were performed only by the Kotzin laboratory (43). Anti-Ro, anti-La, anti-Sm, anti-nRNP, and anti-cardiolipin Abs were performed by Immco.

Histological analysis of kidneys and parotid glands

Kidneys and parotid glands were harvested from IL-14{alpha}-transgenic and littermate control mice and placed in either 10% formalin (Baxter Diagnostics) or Zeus tissue fixative (Zeus Scientific). Kidneys and parotid glands in 10% formalin were sectioned and stained with H&E or Giemsa and evaluated by standard light microscopy, as described (45). Kidneys in Zeus tissue fixative were sectioned and stained with FITC-conjugated goat anti-murine IgM (Sigma-Aldrich). Analysis was then performed using fluorescence microscopy, as described (45).

Flow cytometry

Cells collected from peritoneal cavity of the mice, spleens, or tumor tissue of the mice were stained with panels composite with anti-mouse Ab CD19-allophycocyanin, CD138-PE, CD21-FITC, CD5-PE-Cy5, CD23-PE, IgD-FITC (BD Pharmingen), CD38-PE-Cy5 (eBioscience), and IgM-PE (Abcam) and analyzed on a BD Biosciences FACSCalibur machine and Winlist software.

Immunohistochemistry for CD5 and B220

Tumor tissues from IL-14{alpha}-transgenic mice were collected and fixed in 10% formalin then embedded in paraffin. Thin sections were prepared on slides and the slides stained with the Histomouse-Max kit (Invitrogen Life Technologies) as instructed by the manufacturer. Anti-mouse CD5 and B220 Abs were purchased from BD Pharmingen.

Ig gene rearrangements

Clonal B cell determination of mouse tumor was performed by high-fidelity PCR using 0.1 mg of DNA extracted from normal B6J mouse liver, spleen, and transgenic mouse tumors using the DNeasy Tissue kit (Qiagen). The primers for Ig gene rearrangement in the lymphomas from the IL-14{alpha}-transgenic mice were: DHL, 5'-GGAATTCGMTTTTTGTSAAGGGATCTACTACTGTG J3; 5'-GTCTAGATTCTCACAAGAGTCCGATAGACCCTGG; and DHR, 5'-TTTTGYTGMTGGATATAKCACTGAG; J3, 5'-GTCTAGATTCTCACAAGAGTCCGATAGACCCTGG. PCR was performed as described (46) with the following protocol: incubation 95°C for 5 min then 95°C 1 min, 55°C 1 min, and 72°C 2 min for 35 cycles, followed by extension at 72°C for 10 min. The PCR products were analyzed on 1.2% agarose gel electrophoresis.

Oversight

The animal studies were all approved by the Institutional Animal Care and Use Committee (SUNY Buffalo School of Medicine and Biomedical Sciences).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
The transcript we have designated IL-14{alpha} is generated from the plus strand of the il14 gene using exons 3–10. The predicted amino acid sequence encoded by this transcript predicts a 47.4-kDa protein with >90% amino acid sequence identity between human and mouse (Fig. 1). This transcript is expressed normally in activated T lymphocytes, a subpopulation of activated B lymphocytes (see Fig. 3C) and in follicular dendritic cells (our unpublished data). Because the il14 gene was mapped to a potential lupus susceptibility locus in both human and mouse (34, 47), we first examined the expression of IL-14{alpha} in the splenic lymphocytes of NZB, NZW, and (NZB x NZW)F1 mice, a well-established animal model for SLE. As demonstrated in Fig. 2 by Northern blot analysis, the IL-14{alpha} mRNA is expressed in 8-mo-old female NZW and (NZB x NZW)F1 splenic mRNA but not in mRNA from unstimulated 8-mo-old female NZB or B6 spleen. Previous studies demonstrated that expression of IL-14 is up-regulated in cells after stimulation with PHA (31, 32). Splenic cells from B6 mice were activated with PHA for 18 h in vitro and used as a positive control to verify the detection of the IL-14{alpha} mRNA by the Northern blot assay. The expression of IL-14{alpha} in murine SLE encouraged the evaluation of the ability of IL-14{alpha} to induce autoimmunity in transgenic mice.


Figure 1
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  		FIGURE 1. The protein sequences were aligned with Clustal X and then box-shaded via the BOXSHADE server at <www.ch.embnet.org/software/BOX_form.html>. Dark shaded residues are identical; light shaded residues are similar; unshaded residues differ.

 

Figure 3
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  		FIGURE 3. A, FISH was performed on chromosomal preparations from the fibroblasts of the spleens of IL-14{alpha}-transgenic mice using the pEµSR-IL-14{alpha} vector as a probe, as described in Materials and Methods. Positive immunofluorescence (green) is noted on chromosome 18. B, Splenic lymphocytes from IL-14{alpha}-transgenic mice were isolated and separated with anti-B220 magnetic beads into B220-positive and -negative subpopulations. The RNA was isolated from each of these subpopulations and evaluated by RT-PCR for expression of IL-14{alpha} and actin, as described in Materials and Methods. Lane 1, Female IL-14{alpha}-transgenic, B220– cells; lane 2, female IL-14{alpha}-transgenic, B220+ cells; lane 3, male IL-14{alpha}-transgenic, B220– cells; lane 4, male IL-14{alpha}-transgenic, B220+ cells; lane 5, nontemplate control. C, Splenic lymphocytes from B6 mice were first isolated and separated with anti-B220 magnetic beads into B220-positive and -negative subpopulations. Then B220-positive cells were placed in culture for 18 h either in medium alone (RPMI 1640/5% FBS) or medium plus LPS (100 µg/ml). B220-negative cells were placed in culture for 18 h with medium alone or medium plus PHA (10 µg/ml). Cells were then harvested, RNA was prepared, and RT-PCR was performed for IL-14 and actin as described in Materials and Methods. Lane 1, C57; lane 2, C57, B220+ cells; lane 3, C57, B220+ cells stimulated with LPS; lane 4, C57, B220– cells; lane 5, C57, B220– cells stimulated with PHA; lane 6, NZBW F1; lane 7, IL-14{alpha}-transgenic; lane 8, nontemplate control.

 

Figure 2
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  		FIGURE 2. Splenic lymphocytes were obtained from 8-mo-old female mice and total RNA was prepared as described in Materials and Methods. Northern blots were performed using a cDNA probe based on exons 2 and 3 of IL-14, as described in Materials and Methods. RNA was made from the spleens of individual mice. The mouse strains are B6, NZB, NZW, and (NZB x NZW)F1 (F1). The RNA bands were visualized with the BioDetect kit (Ambion). The IL-14{alpha} signal was identified as a 4.1-kb band whereas actin was identified as a 2.1-kb band.

 
Generation of IL-14{alpha}-transgenic mice

To understand physiological roles for IL-14{alpha} in vivo, we generated transgenic mice using pEµSR (40). pEµSR vector leads to gene expression predominantly in the B cell compartment. Three lines of transgenic mice were initially derived and studied for various properties including serum Igs and responses to vaccinations with T-dependent and T-independent Ags. All three lines behaved similarly in these assays, so one line was selected for further backcrosses to enhance the purity of the B6 background and for further study. This transgenic line of mice contained the pEuSR-IL-14{alpha} transgene predominantly on murine chromosome 18 (Fig. 3A), and expressed IL-14{alpha} mRNA predominantly in the B lymphocyte compartment (Fig. 3B). The level of expression of the transgene in these mice was similar to the level of IL-14{alpha} expression seen in T cells activated with PHA or B cells activated with LPS from normal B6 mice or from splenic lymphocytes of 8-mo-old female (NZB x NZW)F1 splenocytes (Fig. 3C). Chromosome 18 does not contain genes that are known to influence either autoimmunity or lymphomagenesis. The IL-14{alpha} protein was detectable in the sera of these mice using rabbit polyclonal antisera against human IL-14{alpha} (data not shown). The IL-14{alpha}-transgenic mice used in this study were backcrossed for eight generations with B6 mice to minimize the presence of non-B6 genes. Both male and female mice were used in all the studies. No qualitative differences were found between males and females, although females tended to develop autoimmunity somewhat earlier and more severely than males.

Serum studies in IL-14{alpha}-transgenic mice

Serum was collected from IL-14{alpha}-transgenic mice at different ages to evaluate production of Igs and autoantibodies. The IL-14{alpha}-transgenic mice develop hypergammaglobulinemia by 6 mo of age involving both IgG and IgM that becomes more exaggerated as they get older (Fig. 4A). At 9 mo of age, the IL-14{alpha}-transgenic mice had a statistically significant increase in IgA compared with the littermate controls (p < 0.0001). They had a more prominent elevation in IgG2a (p < 0.0001), but not in the other IgG subclasses (Fig. 4B). Because IgG2a is usually induced by IFN, we also examined levels of IFN-{alpha}, IFN-beta, and IFN-{gamma} in the sera of these mice. No IFN-beta or IFN-{gamma} was detected. A significant amount of IFN-{alpha} was noted in the sera of the IL-14{alpha}-transgenic mice, but not in the littermate controls at 10–12 mo of age (Fig. 4C). We next examined in the sera of these mice the presence of autoantibodies often associated with SLE and Sjögren’s syndrome, IgG ANA, anti-dsDNA, anti-chromatin, anti-Ro, anti-La, anti-Sm, and anti-nRNP (48, 49). Forty mice were included in these studies. Although some of the mice had high titers of one or more of these Abs, many of the IL-14{alpha}-transgenic mice did not express any of them. The only autoantibody identified in the sera of all of the IL-14{alpha}-transgenic mice was IgM anti-cardiolipin. The titer of this autoantibody increased as the mice aged and was statistically significantly different from the normal controls at all the ages tested (a representative study with six mice in each group: 6 mo, p = 0.0034; 9 mo, p = 0.0084; 12 mo, p < 0.0001) (Fig. 4D).


Figure 4
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  		FIGURE 4. A, Sera were obtained from IL-14{alpha}-transgenic mice and littermate controls at 3, 6, and 12 mo of age. ELISA was used to determine the levels of IgM and IgG as outlined in Materials and Methods. Data shown are the mean and SEM for the four mice in each group at each time point. The differences between the levels of IgM in the sera of the IL-14{alpha}-transgenic mice and littermate controls were significant at all the time points studied (3 mo, p = 0.0022; 6 mo, p = 0.015, and 12 mo, p = 0.0021) as were the levels of IgG (3 mo, p = 0.04, 6 mo, p = 0.0031, and 12 mo, p < 0.0001). B, Sera were obtained from IL-14{alpha}-transgenic mice and littermate controls at 9 mo of age. ELISAs were used to determine the levels of IgA, IgE, and the IgG subclasses. Data shown are the individual values for the six mice studied in each group. The only statistically significant differences between the IL-14a-transgenic mice and littermate controls were IgA (p < 0.0001) and IgG 2a (p < 0.0001). C, ELISA was used to determine the levels of IFN-{alpha} in the sera of IL-14{alpha}-transgenic mice and littermate controls at the times indicated. Data shown are the mean and SEM for four mice studied in each group. (Figure legend continues) The difference between the values for the IL-14{alpha}-transgenic mice and littermate controls were significant at 6 mo (p = 0.014) and 10 mo (p = 0.0007) but not at 4 mo. D, ELISA was used to determine the levels of IgM anti-cardiolipin Abs in the sera of IL-14{alpha}-transgenic mice and littermate controls. Data shown are the mean and SEM for six mice studied in each group at the times indicated. The differences in the levels of IgM anti-cardiolipin Abs between IL-14{alpha}-transgenic mice and littermate controls was statistically significant at all of the time points tested (6 mo, p = 0.034; 9 mo, p = 0.084; and 12 mo, p < 0.001).

 
Vaccination studies and B cell subpopulations in IL-14{alpha}-transgenic mice

Because previous studies had suggested that IL-14 may influence memory B cell function (33), we vaccinated the IL-14{alpha}-transgenic mice with both T-independent and T-dependent Ags and measured their Ag-specific Ab responses. As demonstrated in Fig. 5, IL-14{alpha}-transgenic mice demonstrated increased IgG anti-NP responses to the T-dependent Ag NP-OVA compared with littermate controls. When the T-independent Ag NP-Ficoll was used IL-14{alpha}-transgenic mice demonstrated increased IgM anti-NP responses (Fig. 5). These studies suggested that several B cell subpopulations might be increased either in number or function in the IL-14{alpha}-transgenic mice. Therefore, we examined subpopulations of B lymphocytes in the spleen and peritoneal cavities of IL-14{alpha}-transgenic mice compared with littermate controls at 12 mo of age. Flow cytometry studies were performed using groups of six IL-14{alpha}-transgenic mice and littermate control mice and Abs to CD5, CD19, CD21, CD38, CD138, IgM, and IgD. Several interesting findings are summarized in Table I. In the peritoneal washes, there was a statistically significant increase in the percentage of CD5+, CD19+, IgM+, sIgDlow B1 cells in the IL-14{alpha}-transgenic mice compared with the littermate controls (14.91 ± 3.0 vs 4.9 ± 0.65; p < 0.0001). In the spleen, the IL-14{alpha}-transgenic mice compared with the littermate controls had an increased percentage of total CD19+ B cells (33.69 ± 4.27 vs 18.42 ± 1.6; p < 0.0001), CD19+, CD21+, IgM+ marginal zone B cells (11.78 ± 3.35 vs 1.76 ± 0.49; p < 0.0001), and CD19+, CD38+, sIgDlow germinal center B cells (9.51 ± 1.65 vs 5.30 ± 1.15; p = 0.0004). There was a statistically significant increase in CD19–, CD138+ plasma cells in the IL-14{alpha}-transgenic mice, although the numbers were small (1.45 ± 0.43 vs 0.48 ± 0.1; p = 0.0003). The absolute numbers of lymphocytes in the spleens of IL-14{alpha}-transgenic mice were not significantly different from those in littermate controls (for six mice in each group: IL-14{alpha}-transgenic –3.23 ± 0.49 x 107; littermate controls –2.25 ± 0.53 x 107; p = 0.2).


Figure 5
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  		FIGURE 5. A, Groups of four littermate control and four IL-14{alpha}-transgenic mice were immunized with 100 µg of NP-Ficoll, as indicated, i.p. in IFA on days 0 and 14. NP-specific IgM and IgG were determined by ELISA on days 0, 14, 28, and 70 as outlined in Materials and Methods. Data shown for each point are the mean values of triplicate wells for each mouse, and four mice in each group. The differences between the responses of the two groups of mice were significant for IgM (day 14, p = 0.001; day 28, p < 0.001; and day 70, p = 0.003) but not IgG. This experiment is representative of four similar experiments done with these mice. B, Groups of four littermate control and four IL-14{alpha}-transgenic mice were immunized with 100 µg of NP-OVA in IFA i.p. on days 0 and 14. NP-specific IgM and IgG was determined by ELISA on days 0, 14, 28, and 70 as outlined in Materials and Methods. Data shown for each point are the mean values of triplicate wells for each mouse, and four mice in each group. The differences between the responses of the two groups of mice were significant only for IgG on days 28 (p = 0.002) and 70 (p = 0.001). This experiment is representative of four similar experiments done with these mice.

 

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  		Table I. Comparison of B cell subpopulations in IL-14{alpha}-transgenic mice and littermate controlsa

 
Histological demonstration of autoimmunity

The IL-14{alpha}-transgenic mice live a normal lifespan. To study the development of organ injury, mice were autopsied at different ages. By 8 mo of age, most of the IL-14{alpha} transgenic mice (21 of 25 studied) have mild proteinuria, but never develop renal dysfunction. Further studies were performed on 18 mice at 10 mo of age. Histological evaluation of their kidneys revealed only a mild increase in mesangial cells (Fig. 6A). Immunohistochemistry revealed deposition of IgM in the glomeruli (Fig. 6B). Weak deposition of IgG and complement was also noted (data not shown). Interestingly, IgM deposition was also noted in blood vessels. The majority of IL-14{alpha}-transgenic mice (39 of 40 mice studied), but not the littermate controls, also developed lymphocytic infiltration of their salivary glands that was age dependent (Fig. 6C). These findings are consistent with SLE and secondary Sjögren’s syndrome.


Figure 6
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  		FIGURE 6. A, Kidneys were harvested from IL-14{alpha}-transgenic mice and littermate controls mice at 12 mo of age. Staining with Giemsa and IgM Ab was performed as described in Materials and Methods and visualization by standard microscopy and fluorescence microscopy. Increase in mesangial cells was observed in transgenic mice but not in littermate control. Deposition of IgM in the glomeruli and blood vesicle was also observed in transgenic but not in littermate control. Photos were taken at x400. B, Parotid glands were harvested from IL-14{alpha}-transgenic mice at 8, 12, and 17 mo of age. Parotid glands were harvested from littermate control mice at 17 mo of age. Tissues were sectioned, stained with H&E, and visualized by standard microscopy, as outlined in Materials and Methods. Photographs were taken at x400.

 
Lymphoma in IL-14{alpha}-transgenic mice

When aged, the IL-14{alpha}-transgenic mice all develop mild splenomegaly and lymphoid hyperplasia but not clinically detectable lymph node swelling. Autopsies done on IL-14{alpha}-transgenic mice between the ages of 12–20 mo revealed lymphoma in 96% of 40 mice studied. A few mice had lymphoma restricted to the spleen, but the majority had lymphoma in the liver and gastrointestinal tract or lung (Fig. 7). The lymphoma had the histological features of a large B cell lymphoma, expressed CD5 and CD19 but not CD21 and variably CD23. It contained Ig gene rearrangements suggesting a B cell tumor of monoclonal origin (Fig. 7). This development of B cell lymphomas with this pattern of organ involvement is consistent with what is seen in patients with SLE and Sjögren’s disease (38, 39, 50).


Figure 7
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  		FIGURE 7. A, Micrograph is shown of the tumors in the gastrointestinal tract of an IL-14{alpha}-transgenic mouse autopsied at the age of 17 mo. B, Staining of the tumor from an IL-14{alpha}-transgenic mouse is shown with H&E, anti-B220, and anti-CD5. C, Flow cytometry evaluation of a tumor from an IL-14{alpha}-transgenic mouse is shown with anti-CD5, anti-CD19, and anti-IgM. D, Evaluation of Ig gene rearrangements in the tumor from an IL-14{alpha}-transgenic mouse (tumor) compared with normal spleen (control) by PCR, as outlined in Materials and Methods. Arrow, The unique band in the tumor suggesting a monoclonal tumor.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
This study demonstrates that IL-14{alpha} is a highly conserved protein in humans and mice. Its expression is increased in an animal model for SLE, the (NZB x NZW) F1 mouse. With enhanced expression of IL-14{alpha}, transgenic mice developed many physiological changes similar to those observed in patients with SLE and Sjögren’s syndrome. These include hypergammaglobulinemia, involving IgG, IgA, and IgM autoantibodies, deposition of Igs in the kidney and infiltration of the salivary glands with lymphocytes. In addition, the IL-14{alpha}-transgenic mice demonstrate increased numbers of B1 cells in their peritoneal cavities and enhanced responses to vaccinations with both T-dependent and T-independent Ags, features that have been ascribed to the sle2 locus of the NZM mouse (28, 51). Aged IL-14{alpha}-transgenic mice develop B cell malignancies that are similar to those seen in patients with SLE and Sjögren’s syndrome (37, 39, 48, 52, 53).

The il14 gene is located near lck, a region that has been associated with various lupus susceptibility as well as lupus suppressor genes (28). A syntenic region on human chromosome 1 was also identified as a lupus susceptibility locus in certain human studies (34, 35). The sle2 locus in on chromosome 4 (28). The dominant phenotype ascribed to this locus is increased B1 cells in the peritoneal cavity (28, 51, 54). Other features ascribed to this locus that are also identified in the IL-14{alpha}-transgenic mice are increased serum IgM, enhanced responses to T-dependent and T-independent Ags, and production of anti-cardiolipin Abs (29, 30, 55). Various studies have demonstrated, however, that the effects of sle2 in generating these phenotypic changes are contributed from NZB, rather than NZW (56). The expression of IL-14{alpha} is noted in NZW but not NZB mice (Fig. 2). NZW contributes genes that suppress rather than induce this phenotype (56). The IL-14{alpha}-transgenic mice have features not ascribed to the sle2 locus, such as increase in serum IgG and IgG2a, mesangial expansion in the glomeruli with deposition of immunoglobulins, infiltration of the salivary glands with lymphocytes, and the development of lymphoma. We observed lymphomas in the majority of IL-14{alpha}-transgenic mice by 18 mo of age. This is consistent with the fact that patients with SLE and Sjögren’s syndrome also tend to develop lymphomas late in life (37, 50, 57, 58, 59). Further studies will be necessary to determine how il14 relates to the SLE susceptibility genes that have been mapped in (NZB x NZW)F1, various NZM strains, and other SLE models.

The il14 gene is on chromosome 4 as are the majority of type 1-IFN genes. Previous studies demonstrated that IL-14 lacked antiviral effects of IFN-{alpha} (31). In the present studies, we demonstrated that IL-14{alpha}-induced IgG2a, an IFN-inducible gene (60, 61, 62). IL-14{alpha} induced the production of IFN-{alpha} (Fig. 4C), but not IFN-{gamma}, that may have been responsible for this change (60). At the same time, however, other cytokines, such as IL-27, have recently been implicated in inducing the switch to IgG2a (63). Whether IL-14 directly induces the switch to IgG2a or expands IgG2a-producing cells will have to be determined in future studies. Type 1 IFNs have been implicated in the induction of autoimmunity in (NZB x NZW)F1 mice as well as in patients with SLE and Sjögren’s syndrome (64, 65, 66). Features of SLE are severely abrogated in (NZB x NZW)F1 mice lacking the type 1 IFN receptor (67). The generation of IL-14{alpha}-transgenic mice lacking the type 1 IFN receptor will be necessary to determine whether the activities of IL-14{alpha} require the induction of IFN-{alpha}.

The observation that IL 14{alpha}-transgenic mice developed elevated serum levels of IgM is consistent with the observation that they contain increased numbers of B1 cells and marginal zone B cells. The majority of IgM is derived from B1 B lymphocytes and marginal zone B lymphocytes, especially in mice (68, 69, 70). Previous data derived in vitro with native high molecular weight B cell growth factor/IL-14 suggested that IL-14 might act selectively on sIgD– tonsillar B lymphocytes, which includes B1 lymphocytes and memory B2 lymphocytes (33). Our current data also suggest a role for IL-14 in IgM Ab responses to T-independent Ags, which come from B1 and marginal zone B lymphocytes.

The IL-14{alpha}-transgenic mice also spontaneously developed a significant increase in serum IgG and IgG2a. The IL-14{alpha}-transgenic mice have increased numbers of splenic B2 cells and germinal center B lymphocytes compared with littermate controls that would contribute to this response. They demonstrate an increased IgG response to a T-dependent Ag. The production of IgG Ab involves additional cell interactions not required for the production of IgM. The switch from IgM to IgG production requires stimulation of CD40 on B lymphocytes by CD154 on T lymphocytes or less commonly B lymphocytes, NK cells, or mast cells (71, 72, 73, 74, 75, 76, 77). IFN-{gamma} was first described to direct H chain class switching to IgG2a (62, 78), however, the same can be accomplished with type1 IFNs (60). IL-14{alpha} may be working directly in the induction of the IgG/IgG2a response or through the stimulation of IFN-{alpha} production. IL-14{alpha} is expressed in follicular dendritic cells and this suggests that it may be somehow involved with the generation or selection of memory B lymphocytes (33). Further work will be needed to examine this issue.

The development of deposits of IgM in the kidneys and the infiltration of the parotid glands with lymphocytes in IL-14-transgenic mice suggests an SLE-like phenotype. Autoimmunity in diseases such as SLE and Sjögren’s syndrome involves excessive activation of B lymphocytes and the production of autoantibodies (79). The causes of this abnormal activation have not been fully elucidated and are likely to vary in different patients and in different animal models of these diseases. Animal models have established that alterations of individual genes that regulate lymphocyte proliferation (8, 9), inhibit apoptosis (10, 11), regulate signaling through the BCR (12, 13, 14), determine degradation or clearance of autoantigens (15, 80) or clearance of immune complexes (6, 7) can result in serum autoantibodies and immune complex-mediated glomerulonephritis. In all cases, the genetic background upon which the defined gene is added determines the severity of the disease phenotype (7, 24). The IL-14{alpha}-transgenic mice are on a B6 background that is permissive to the expression of autoimmune features. A minority of the genetically altered mice that develop a SLE-like disease also develop Sjögren’s syndrome. Sjögren’s syndrome is seen in NZW, (NZB x NZW)F1, MRL/lpr, C3H/lpr, NFS/sld, aromatase-deficient mice and IQI/jic mice (81, 82, 83, 84, 85). All of these mice, like patients with Sjögren’s disease, develop elevated serum IgM levels (81, 84, 85, 86). The exact mechanism(s) by which IL-14 induces hypergammaglobulinemia is unclear. It is also unclear which autoantibodies produced in IL-14{alpha}-transgenic mice are responsible for deposition of Igs in the kidney, as high titers of anti-DNA Abs, anti-chromatin Abs, and anti-histone Abs were produced in only a minority of mice on a B6 background. Glomerulonephritis in the absence of these autoantibodies has also been observed in female NZM.C57Lc4 mice (87). Furthermore, a minority of IL-14{alpha}-transgenic mice develop Abs to Ro and La, autoantibodies that have been associated with the development of Sjögren’s syndrome (88, 89, 90). Murine models have suggested several other autoantigens may be important for the development Sjögren’s syndrome (84, 85, 91). Which autoantigens are relevant to Sjögren’s disease in IL-14{alpha}-transgenic mice is an area of current investigation.

The development of malignancies is felt to involve the dysregulation of genes involved with cell cycle regulation, growth, and/or apoptosis (92, 93). Pre-B cell lymphomas have been observed in mice overexpressing myc, especially in association with ras, raf, or bcl-1/cyclin D1 (94, 95, 96). Transgenic mice overexpressing the t (14:18) translocation including the bcl-2 gene develop large B cell lymphomas often involving dysregulated myc expression (97). The only cytokine whose overexpression alone has resulted in a B cell malignancy is IL-6, which produces plasmacytomas in transgenic mice (98). The development of lymphoma in aged IL-14{alpha}-transgenic mice was not surprising for several reasons. First, IL-14 was originally identified in a Burkitt lymphoma cell line and subsequently shown to be constitutively expressed in high-grade germinal center-derived human B cell lymphomas (31, 99). Second, IL-14 induces B cell growth and proliferation. Genes regulating B lymphocyte growth and survival are often identified to be dysregulated in B cell lymphomas (100, 101, 102). Finally, patients in their 60s and 70s with autoimmune diseases such as SLE and Sjögren’s disease often develop B cell lymphomas (48, 103, 104). The lymphomas seen in the aged IL-14{alpha}-transgenic mice most closely resembled large B cell lymphomas, which are generally derived from germinal cell B lymphocytes. It is surprising that more malignancies have not been described in the various other animal models for SLE and Sjögren’s syndrome (39, 48, 52, 57, 105).

Transgenic mice were chosen to evaluate physiological roles of IL-14{alpha} for several reasons. IL-14{alpha} is very unstable in vitro and many in vitro assays are likely affected by this property. The in vivo system allows the determination of the influence of IL-14{alpha} on intact immune systems, rather than isolated cell populations in vitro. The data resulting from these transgenic mice must, however, be viewed with several caveats. The levels of IL-14{alpha} achieved in the transgenic mice are much higher than would be seen under normal physiological conditions (Fig. 3C). The expression of IL-14{alpha} in the B lymphocyte compartment in the transgenic mice may mimic pathological more than normal physiological conditions (99, 106).

In summary, IL-14{alpha} is a highly conserved protein across species, suggesting its fundamental importance to mammalian physiology. The fact that it enhances Ab responses to vaccinations, induces autoimmunity, and contributes to the formation of B cell lymphomas suggests that it may have fundamental roles in these processes. Further work will be necessary to elucidate the mechanisms by which IL-14 participates in these disorders and to establish its roles during normal physiology.


   Acknowledgments
 
We thank various individuals for assistance with these studies. Advice regarding the construction of the IL-14-expressing pEµSR vectors was provided by Katherine Siminovich and Jinyi Zhang, University of Toronto (Toronto, Canada). Paul Soloway helped generate IL-14{alpha}-transgenic mice. Philip Simonian and Brian Kotzin, Division of Clinical Immunology, University of Colorado (Denver, CO) provided assistance and advice with various aspects of these studies. Judith Horn did the immunofluorescence studies for Ig deposition in the kidneys. Hua Zeng, Shangguo Tang, and Donald Sykes helped with various technical aspects of the work. We are grateful to Sei-Ichi Matsui and Jeff LaDuca of the Roswell Park Cancer Institute SKY/FISH facility for assistance in FISH mapping. We also thank Linda Ludwig and Oleh Pankewycz for helpful suggestions and comments regarding this work. Tracey Roth provided editorial assistance in the preparation of this manuscript.


   Disclosures
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by grants from the Wendt Foundation (to J.L.A.), John R. Oishei Foundation (to J.L.A.), Troup Foundation (to J.L.A.), and Kaleida Health Foundations (to J.L.A.). Back

2 Address correspondence and reprint requests to Dr. Julian L. Ambrus, Jr., Division of Allergy, Immunology and Rheumatology, School of Medicine and Biomedical Sciences, State University of New York, Room 308 Multiple Lab Building, Buffalo General Hospital, 100 High Street, Buffalo, NY 14203. E-mail address: jambrus{at}buffalo.edu Back

3 Abbreviations used in this paper: SLE, systemic lupus erythematosus; NZB, New Zealand Black; NZW, New Zealand White; ANA, anti-nuclear Ab; sIg, surface Ig; FISH, fluorescence in situ hybridization; NP, nitrophenyl. Back

Received for publication April 4, 2006. Accepted for publication July 20, 2006.


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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
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