Sle1ab Mediates the Aberrant Activation of STAT3 and Ras-ERK Signaling Pathways in B Lymphocytes
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免疫学杂志 2005年第3期
Abstract
The Sle1ab genomic interval on murine chromosome 1 mediates the loss of immune tolerance to chromatin resulting in antinuclear Abs (ANA) production in the lupus-prone NZM2410 mouse. Global gene expression analysis was used to identify the molecular pathways that are dysregulated at the initiation of B lymphocyte autoimmunity in B6.Sle1ab mice. This analysis identified that STAT3 and ras-ERK signaling pathways are aberrantly activated in Sle1ab B lymphocytes, consistent with increased production of IL-6 by splenic B lymphocytes and monocytes in B6.Sle1ab mice. In vitro treatment of splenic mononuclear cells isolated from ANA-positive Sle1ab mice with anti-IL-6 Ab or AG490, an inhibitor of STAT3 signaling pathway, suppressed ANA production in short-term culture, indicating that this pathway was essential to the production of autoantibodies. In vivo treatment of ANA-positive B6.Sle1ab mice with the ras pathway inhibitor, perillyl alcohol, suppressed the increase of ANA. These findings identify IL-6 as a early key cytokine in Sle1ab-mediated disease development and indicate that the STAT3 and ras-ERK signaling pathways are potential therapeutic targets for treating systemic lupus erythematosus.
Introduction
Susceptibility to systemic lupus erythematosus is mediated by a complex interaction of genetic and environmental elements (1, 2). Our laboratory has used congenic dissection in the NZM2410 lupus-prone mouse to model the genetic mechanisms responsible for disease pathogenesis. Congenic dissection is a strategy in which individual genes that contribute to a polygenic disease (such as lupus) are segregated into unique substrains of a specific inbred strain, thus allowing the component phenotype contributed by each gene to be analyzed separately. Phenotypic analyses of the collection of B6-congenic strains that we have produced from the NZM2410 model have allowed the characterization of component phenotypes associated with three pathways that contribute to lupus pathogenesis (1, 3, 4). These studies have identified the Sle1 gene cluster, which mediates a breach in immune tolerance and the production of antinuclear Abs (ANA),3 as a key pathway in the initiation of the disease cascade.
B6.Sle1 mice develop hypergammaglobulinemia, splenomegaly, and expanded populations of activated CD4 T cells and B cells in their spleens, leading to a progressive loss in immune tolerance and the production of high-titered IgG antinuclear Abs beginning at 4 mo of age (5). Our ongoing genetic analyses of this interval indicate that a cluster of four loci, denoted Sle1a through Sle1d, mediate these Sle1 phenotypes (6). Detailed analyses of truncated congenic intervals containing the individual genes in the Sle1 cluster have identified Sle1a and Sle1b as the most potent loci, each capable of mediating fatal lupus when combined with additional autoimmune genes, such as lpr or yaa. These studies have demonstrated that Sle1ab is expressed in a cell intrinsic fashion in lymphocytes and that they cause progressive functional changes in both T and B lymphocytes, leading to chronic immune activation and autoantibody production.
Phenotypic analyses have indicated that B lymphocytes are strongly dysregulated by Sle1ab (6, 7). Although previous analyses have identified a variety of cell surface phenotypic changes, little is currently known about transcriptional changes in B lymphocytes from B6.Sle1ab. In this study, we describe a global gene expression analysis of B lymphocytes from B6.Sle1ab mice as they transition to an ANA-positive phenotype. Our results implicate IL-6 as a key early cytokine in disease development and identify several molecular pathways as candidates for therapeutic intervention.
Materials and Methods
Mice
C57BL/6 (B6) mice were obtained from The Jackson Laboratory and subsequently bred in the University of Texas Southwestern Medical Center specific pathogen-free animal colony. B6.Sle1ab mice are B6 mice congenic for a 3-cM interval of NZM2410 origin on chromosome 1, with termini at D1Mit400 and D1Mit206 (6). The derivation of B6-congenic mice bearing NZM2410-lupus susceptibility intervals has been detailed previously (3, 6). The immunological phenotypes of this strain have been reported previously (3, 5, 6). All experiments were conducted with the approval of the Institutional Animal Care and Use Committee of the University of Texas Southwestern Medical Center.
Cell isolation and RNA extraction
B lymphocytes were isolated from mouse spleens by using DynaBeads coated with anti-B220 mAb according to the manufacturer’s instruction (Dynal). Briefly, splenocytes were depleted of RBC by incubation with ACK lysing buffer (containing 0.15 M NH4Cl, 10 mM KHCO3, and 0.1 mM EDTA, pH 7.4) for 5 min. After three washes at 4°C, the cells were incubated at 4°C for 20 min with Dynabeads Mouse pan B (B220) at a target:bead ratio of 1:4 in PBS containing 1% FCS. The rosetted cells were then washed three times. The purity of the isolated B lymphocytes was >97%. Total cellular RNA was prepared using an Atlas Total RNA Isolation kit (BD Clontech) per the manufacturer’s instruction.
cDNA microarray hybridization and analysis
For Atlas Arrays, the complex [-32P]dATP-labeled first-strand cDNA probes were synthesized and purified according to the protocol provided in the Atlas Mouse 1.2 Array User’s Manual (BD Clontech). Ten micrograms of total RNA was used as template in a 10-μl reverse transcription reaction. A gene-specific primer mixture (BD Clontech) was used to prime the reverse transcription. The Atlas Mouse 1.2 cDNA array membranes were prehybridized in ExpressHyb (BD Clontech) containing 100 mg/5 ml of sheared salmon testes DNA at 71°C for 30 min. Then these membranes were hybridized with 32P-labeled first-strand cDNA probes (10 x 106 cpm) overnight at 71°C. After washing, the arrays were exposed to a phosphorimaging screen for 24 h at room temperature. The screen was scanned on a PhosphorImager (Molecular Dynamics) and the images were analyzed by using AtlasImage software (BD Clontech). The scatter plots were generated using GeneSpring Software (Silicon Genetics). Mouse 12k glass arrays containing 12,000 cDNAs were constructed in the University of Texas Southwestern Medical Center Microarray Core Facility (see http://microarraycore.swmed.edu for gene list). Each array contains 5,600 sequence-verified cDNA clones from Research Genetics, Inc., and 6,200 IMAGE Consortium mouse cDNA clones (1,536 clones from B cell line, 1,920 clones from T cell line, 768 clones from thymus, 768 clones from macrophage cell line, and 1152 clones from spleen) from Incyte Genomics. Generation of cDNA probes and array hybridization were performed with a Submicro Expression kit (Genisphere) (8) according to the manufacturer’s protocol. Briefly, 1 μg of total RNA was reverse-transcribed using reverse transcription primers tagged with either Cy3- or Cy5-specific 3DNA capture sequence. The synthesized tagged cDNAs were then labeled by Cy3–3DNA or Cy5–3DNA based on the complementary of capture sequence with 3DNA capture reagents. The hybridizations were performed at 50°C, with three subsequent washes of the slides in 2x SSC, 0.2% SDS, 2x SSC, and 0.2x SSC buffers. The arrays were scanned using the GenePix 4000A scanner (Axon Instruments). The results were analyzed using GenePix 4.0 (Axon Instruments) and GeneSpring (Silicon Genetics).
Relative quantitative RT-PCR
Relative quantitative RT-PCR was performed using reverse transcription and coamplification of 18S ribosomal RNA as internal control following the protocol provided with a QuantumRNA 18S internal standard kit (Ambion) according to the manufacturer’s instructions. Briefly, 0.5 μg of RNA was mixed with 2 μl of random primers (50 μM; Ambion) and RNase-free water up to 10 μl of volume, denatured at 80°C for 10 min, and rapidly cooled on ice for 5 min. Then 1 μl of dNTP mix (10 mM), 0.5 μl of RNasin RNase inhibitor (Roche Applied Science), 1 μl of 0.1 M DTT, 4 μl of 5x first-strand buffer (Invitrogen Life Technologies), and 1 μl of Moloney murine leukemia virus-reverse transcriptase (Invitrogen Life Technologies) were added to each sample. The mixtures were incubated for 1 h at 42°C. PCR was performed using 1 μl of the reverse transcription product in a 50-μl reaction mixture containing 5 μl of 10x PCR buffer (Roche Applied Science), 1 μl of dNTPs (10 mM), 2 μl of gene-specific primer pair (10 μM each), 4 μl of 18 S Classic Primer and Competimer pair (Ambion), and 2.5 U of TaqDNA polymerase (Roche Applied Science). After incubation at 94°C for 5 min, the PCR was performed for 29 cycles followed by a 5-min extension at 72°C. Each cycle consisted of 94°C for 30 s, 60°C for 30 s, and 72°C for 45 s. Both suppressor of cytokine signaling (SOCS) 3 mRNA and 18S rRNA coamplified within a linear range. Because of the low abundance of IL-6 mRNA in spleen, RT-PCR of IL-6 mRNA was performed on equal amounts of RNA without using 18S RNA internal control. The PCR cycle number for IL-6 is 35. Amplified PCR products were visualized by ethidium bromide staining and relatively quantified by EagleEye software (Stratagene). The nucleotide sequences of the primers for SOCS3 are 5'-GGA GAC TCC TGA GTT AAC ACT GGG-3' (sense) and 5'-GAC CAG TTC CAG GTA ATT GCA TGG C-3' (antisense). The nucleotide sequences of the primers for IL-6 are 5'-ATA GTC AAT TCC AGA AAC CGC TAT GAA G-3' (sense) and 5'-GAT TAT ATC CAG TTT GGT AGC ATC CAT C-3' (antisense).
SDS-PAGE and immunoblotting
Isolated B lymphocytes were lysed in a buffer containing 300 mM NaCl, 50 mM Tris-Cl (pH 7.6), 0.5% Triton X-100, 10 μg/ml leupeptin, 10 μg/ml aprotinin, 1 mM PMSF, 1 mM sodium orthovanadate, 1 mM sodium molybdate, and 1 mM NaF. Cellular extracts were incubated on ice for 30 min and then centrifuged at 12,000 x g for 20 min. The supernatants were collected, and the protein concentration was determined using a Bio-Rad protein assay kit. Equivalent amounts of total cellular protein extract (4 μg) were fractionated on 10% SDS-PAGE and electrotransferred to a polyvinylidene difluoride membrane using a Bio-Rad transblot apparatus. The membrane was blocked overnight with TBST buffer (10 mM Tris-Cl (pH 8.0), 0.9% NaCl, and 0.1% Tween 20) plus 4% BSA (for phosphorylated proteins) or 5% nonfat dry milk and incubated for 1 h with primary mAb in TBST buffer plus 4% BSA (for phosphorylated proteins) or 5% nonfat dry milk. Following three washes in TBST, the membranes were incubated with a secondary Ab for 1 h. The blots were again washed three times in TBST and then developed using an ECL Plus kit (Amersham) according to the manufacturer’s instructions. Protein bands were quantified by densitometric analysis using a computerized densitometer (Molecular Dynamics) and ImageQuant software (Molecular Dynamics). Rabbit polyclonal Abs specific for P44/42 MAP kinase, STAT3, or phospho-STAT3 were purchased from Cell Signaling. Rabbit anti-phospho-ERK1/2 MAP kinase polyclonal Abs were purchased from Promega. Rabbit anti-SOCS3 polyclonal Abs and the secondary Abs (goat anti-rabbit IgG) were purchased from Santa Cruz Biotechnology.
ELISA
Sera collected from the mice studied were assayed for the presence of anti-histone-DNA Abs by ELISA as previously described (4, 5, 9). Comparisons of Ab levels between different strains and genders in sera were conducted by testing simultaneously all of the samples from different mice on the same ELISA plate. Briefly, Immulon II plates (Dynatech Laboratories), precoated with methylated BSA, were coated overnight with 50 μg/ml dsDNA (Sigma-Aldrich) and 10 μg/ml total histones (Roche Applied Science) at 4°C. The concentrations of Ags used in these ELISAs have been shown to saturate all available binding sites (4, 9). After blocking with PBS/3% BSA/0.1% gelatin/3 mM EDTA, 1/100 dilutions of the test sera were incubated in duplicate for 2 h at room temperature. Bound IgG was detected with alkaline phosphatase-conjugated anti-mouse IgG (Jackson ImmunoResearch Laboratories) using pNPP as a substrate. Raw OD was converted to units per milliliter using a positive control serum from an NZM2410 mouse. The reactivity of a 1/100 dilution of this serum was arbitrarily set to 100 U/ml. For quantitation of serum IL-6, a Quantikine M IL-6 kit (R&D Systems) was used according to the manufacturer’s instruction.
In vitro culture of splenic mononuclear cells
Splenic mononuclear cells were isolated using Histopaque-1083 (Sigma-Aldrich) and cultured in RPMI 1640 containing 10% FCS for 2 days with or without drugs. Supernatant was collected and the levels of anti-histone/dsDNA Abs were determined using ELISA at 1/2 dilutions. The levels of total IgG were determined using ELISA at 1/5 dilutions. The drugs used were AG490 (Alexis Biochemicals), manumycin A (Sigma-Aldrich), and perillyl alcohol (POH) (Sigma-Aldrich). Monoclonal anti-IL-6 Ab and the isotype control were purchased from BD Pharmingen.
Flow cytometry and intracellular staining
Intracellular IL-6 staining was conducted using a Cytofix/Cytoperm Plus kit (BD Pharmingen) according to the manufacturer’s instruction. Briefly, freshly isolated splenic mononuclear cells were stained with FITC conjugate of anti-B220, anti-CD4, or anti-CD11b (BD Pharmingen) at 4°C for 20 min after incubation with Fc Block (BD Pharmingen) for 15 min. After washing, the cells were permeabilized with Cytofix/Cytoperm, washed twice with Perm/Wash buffer, and stained with PE conjugate of anti-IL-6 mAb or isotype control (BD Pharmingen) for 30 min at 4°C. The cells were washed twice with Perm/Wash buffer and resuspended in PBS containing 1% FCS and 0.1% sodium azide before acquisition of the data on a FACScan (BD Biosciences) with CellQuest software (BD Biosciences). The data were analyzed with FlowJo software (Tree Star).
Treatment of mice with POH
B6.Sle1ab mice were treated with i.p. administration of 75 mg of POH/kg of body weight or vehicle control daily for 14 days. The vehicle for POH was Tricaprylin (Sigma-Aldrich). Sera were collected before and after treatment. Levels of anti-histone-DNA Abs in the sera were measured by ELISA as described above.
Statistics
Results were expressed as the mean ± SE and compared using unpaired Student’s t test. Differences were considered significant at p < 0.05.
Results
Comparisons of global gene expression patterns in B cells of B6 vs B6.Sle1ab mice
Variations in global gene expression were sought by comparing B220+ splenic B lymphocytes isolated from age- and gender-matched B6 and B6.Sle1ab mice early in the initiation of autoimmunity to nuclear Ags (3- to 4-mo-old female mice recently converted to ANA). Our initial analyses used Clontech’s Atlas Mouse 1.2 Array, which detects 1,176 genes with a variety of roles in biological processes such as oncogenesis, cell signaling, cell cycle, and apoptosis. Subsequently, we expanded the analysis by the addition of custom-fabricated cDNA slide arrays containing 12,000 genes (see http://microarraycore.swmed.edu/website for gene list). Representative results of some of these microarray analyses are presented in Fig. 1. As expected, B lymphocytes from separate B6 mice express these genes at similar levels (Fig. 1A). In contrast, comparisons of B lymphocytes of age- and gender-matched B6 and B6.Sle1ab revealed a distinct subset of genes with changes in gene expression.
FIGURE 1. Analysis of gene expression by using Atlas Mouse 1.2 Arrays has identified the up-regulated expression of the SOCS3 gene in splenic B lymphocytes of B6.Sle1ab mice. A and B, Scatter plots of hybridization intensities for probes synthesized from total RNA of splenic B lymphocytes isolated from B6 and B6.Sle1ab mice. For each plot, the horizontal axis represents the hybridization intensity for genes expressed in B6 B lymphocytes, and the vertical axis represents the hybridization intensity for these genes in either B6 or B6.Sle1ab B lymphocytes. Each dot in the plots represents the hybridization intensity for a single gene in each sample. The parallel lines flanking the diagonal indicate 2- and 3-fold changes in gene expression. A, Gene expression in B lymphocytes from two separate B6 mice is compared. B, Up-regulated expression of the SOCS3 gene in B6.Sle1ab B lymphocytes is indicated by a circle. C, Array hybridization patterns of SOCS1 and SOCS3 for B6 and B6.Sle1ab are shown. SOCS1 is indicated by a white arrow and SOCS3 is indicated by a black arrow. Of the SOCS family genes, both SOCS1 and SOCS3 are expressed in B lymphocytes, but only the SOCS3 gene shows up-regulated expression in B6.Sle1ab B lymphocytes. D, Relative quantitative RT-PCR assessment of expression of SOCS3 in splenic B lymphocytes of B6, B6.Sle1, and B6.Sle1ab mice, confirming that expression of the SOCS3 gene is up-regulated in Sle1-congenic mice. 18S rRNA is used as the internal control.
Table I lists the genes that exhibited >2-fold expression variations in three or more replicate comparisons of B220+ splenic B cells from B6 vs B6.Sle1ab mice. None of these genes are located within the congenic interval, indicating that they reflect variations in molecular pathways downstream from Sle1ab. There is no significant difference in the distribution of B cells within B cell subsets as determined by CD5, CD21, and CD23 staining. However, there is a higher percentage of activated B cells in B6.Sle1ab mice as measured by CD69 staining. Many of the up-regulated genes observed in this analysis are consistent with the presence of an expanded population of proliferating splenic B lymphocytes in B6.Sle1ab mice. Thus, Ig genes, c-myc, p55CDC, G2-M-specific cyclin B2, and a variety of other cell proliferation-related genes are all significantly up-regulated in B6.Sle1ab B lymphocytes early in the initiation of detectable humoral autoimmunity. A small number of genes were also down-regulated, although most of these were expressed sequence tags (ESTs). Interestingly, similar profiles were detected in 4-mo-old female B6.Sle1ab mice regardless of their ANA phenotype, suggesting that chronic immune activation is a consistent feature of these mice and precedes ANA production.
Table I. Genes differentially regulated in splenic B lymphocytes of B6. Sle1ab mice
Dysregulation of SOCS3 in Sle1ab B lymphocytes
SOCS3 expression is consistently up-regulated >3-fold in comparisons of age- and gender-matched B6 and B6.Sle1ab splenic B lymphocytes (Fig. 1, B and C), while SOCS1 expression is unchanged. This up-regulation was confirmed by quantitative RT-PCR of splenic B cell mRNAs isolated from cohorts of both B6.Sle1 and B6.Sle1ab mice (Fig. 1D). The SOCS gene family encodes proteins that are induced in cells responding to stimulation by cytokines (10). Since SOCS family transcription has been shown to be up-regulated in response to STAT phosphorylation (11, 12), we compared the levels of phosphorylated STATs in B lymphocytes freshly isolated from B6 and B6.Sle1ab mice. As shown in Fig. 2, phosphorylated STAT3 is clearly increased in Sle1ab B lymphocytes (Fig. 2A), although no difference was observed in the levels of phosphorylated STAT1, STAT5, or STAT6 (data not shown). These results are consistent with previous reports indicating that SOCS3 is induced in response to STAT3 phosphorylation (13, 14, 15, 16).
FIGURE 2. Sle1ab mediates aberrant activation of the STAT3 signaling pathway in B lymphocytes. A, Representative immunoblots of phospho-STAT3, STAT3, and SOCS3 proteins in lysates of splenic B lymphocytes isolated from B6 and B6.Sle1ab mice. Four micrograms of cell lysate from each mouse was resolved by SDS-PAGE and immunoblotted using phospho-STAT3-, STAT3-, or SOCS3-specific Abs. Results of densitometric analysis for these blots are shown in the bar graph below the blots. The mean of pSTAT3/STAT3 or SOCS3 in the B6 group is designated as 1, and each measurement is compared with this mean. ANA (B) and spleen weights (C) are shown for the mice analyzed in A. B, The cutoff level for autoantibody production is indicated by the dashed line which represents mean + 3 SD of the ANA levels in B6 control mice.
IL-6 plays a key role in ANA production by B6.Sle1ab mice
STAT3 phosphorylation is induced by IL-6 and potentially several other cytokines (17, 18, 19, 20). Consequently, we assayed serum levels of IL-2, IL-4, IL-5, IL-6, IL-10, TNF-, and IFN- levels in B6 and B6.Sle1ab mice at 4 mo of age. As shown in Fig. 3A, this analysis revealed statistically significant increases in the IL-6 levels in the serum of B6.Sle1ab mice. Levels of TNF- and IL-5 were only slightly elevated in some B6.Sle1ab mice, whereas no change in the level of IL-2, IL-4, IFN-, or IL-10 was detected (data not shown).
FIGURE 3. Sle1ab mediates an elevated serum level of IL-6. A, Sera from 4-mo-old B6 and B6.Sle1ab mice were measured for IL-6 levels by ELISA. Mean value is indicated in the graph as a short dash. *, p < 0.05. B, Amplification of IL-6 mRNA from B6.Sle1ab mice by RT-PCR. C–F, IL-6 is secreted by some B220+ cells and CD11b+ cells in spleens of B6.Sle1ab mice. Freshly isolated splenocytes were stained for surface markers and intracellularly with PE-labeled anti-IL-6 mAb or isotype control and then analyzed using flow cytometry. The numbers shown in the gates represent the percentage (mean ± SE of three experiments and corrected with isotype control staining) of cells positive for both IL-6 and B220 or IL-6 and CD11b staining. *, p < 0.05. Spleens of B6.Sle1ab mice have more CD11b+ mononuclear cells than those of B6 mice (1.5-fold in percentage), whereas there is no significant difference in the percentage of CD11b+ mononuclear cells secreting IL-6 (17% in B6.Sle1ab vs 14% in B6).
To investigate whether more IL-6 was produced in the spleen of B6.Sle1ab mice, we performed quantitative RT-PCR on mRNA isolated from age- and gender-matched B6 and B6.Sle1ab spleens. As shown in Fig. 3B, B6.Sle1ab spleens contain higher levels of IL-6 mRNA. Flow cytometry analysis with intracellular staining for IL-6 indicated that B6.Sle1ab mice differ from B6 mice via an expansion of splenic B lymphocytes and monocytes that express this cytokine (Fig. 3C).
Although these analyses of ex vivo splenic B cells from B6.Sle1ab mice establish that variations in STAT3 activation and IL-6 are correlated with ANA production, they do not establish that B cells producing ANA are dependent upon these phenotypes. To address this issue, we assessed the effect of inhibiting these functions on the production of ANA by short-term cultures of splenocytes isolated from ANA+ B6.Sle1ab mice. As shown in Fig. 4A, neutralizing anti-IL-6 Ab significantly inhibited ANA production in 48-h in vitro cultures of B6.Sle1ab splenocytes. These results establish that IL-6 plays a key role in driving the production of ANA by autoimmune B lymphocytes in B6.Sle1ab mice. Similarly, the STAT3 inhibitor AG490 greatly suppressed ANA production in a similar experimental system (Fig. 4B). These results indicate that STAT3 activation via IL-6 stimulation is key in the generation of ANA by splenic B cells of B6.Sle1ab mice.
FIGURE 4. Inhibition of ANA production in splenic mononuclear cells by anti-IL-6 Ab and AG490. Splenic mononuclear cells isolated from ANA-positive B6.Sle1ab mice were cultured in vitro with different concentrations of isotype control or neutralizing anti-IL-6 Ab (A) or AG490 (B) for 48 h. ANA levels were determined by ELISA. T0, ANA level for control group; T, ANA level in control and treatment group. Each data point represents the mean ± SE of six experiments. *, p < 0.05. A, Anti-IL-6 group is compared with the isotype control group at each dose, and the AG490 group is compared at each dose to the control group in B. At the concentration of 50 μM, AG490 slightly decreased the viability of cells (by 10%) at the end of the 48-h culture period, mainly by inducing apoptosis as measured by annexin V and 7-aminoactinomycin D staining. It dramatically decreased the cell viability at 100 μM (by 50%).
Sle1ab mediates dysregulation of the Ras-ERK pathway
As shown in Fig. 5A, gene expression profiling of B6.Sle1ab splenic B cells revealed a significant up-regulation in the expression of the gene encoding the -chain of farnesyltransferase. Since farnesyltransferase catalyzes the posttranslational farnesylation of ras (21, 22, 23), a key step in the activation of the ras pathway, this result suggests that the ras signaling pathway in B lymphocytes of B6.Sle1ab mice is activated. As shown in Fig. 5B, immunoblot analysis of ERK1/ERK2 indicated that the ERK2 pathway is more active in B6.Sle1 B lymphocytes, consistent with increased Ras-mediated signaling in bulk splenic B lymphocytes of B6.Sle1ab mice. However, no significant difference in JNK and P38 MAP kinase pathways was detected (data not shown).
FIGURE 5. Ras-ERK MAP kinase pathway is dysregulated in splenic B lymphocytes isolated from B6.Sle1ab mice. A, Microarray analyses indicate that more mRNAs for farnesyltransferase -chain are present in B lymphocytes isolated from B6.Sle1ab mice. For each line, the left end represents the microarray hybridization intensity of probes generated from the farnesyltransferase -chain mRNA in B6 B lymphocytes, and the right end of the line represents that for the same mRNA in B6 or B6.Sle1ab B lymphocytes. The numbers at the top of each graph represent the ratios of their hybridization intensities to that for B6 B lymphocytes. B, Representative immunoblots of phospho-ERK1/2 and ERK1/2 proteins in lysates of splenic B lymphocytes isolated from B6 and B6.Sle1ab mice. Lysates of splenic B lymphocytes were diluted in 2x SDS sample buffer, resolved on SDS-PAGE (4 μg protein/lane), and immunoblotted using Abs specific for phospho-ERK1/2 MAP kinases or total ERK1/2 MAP kinases. Results of densitometric analysis for these blots are shown in the bar graph below the blots. The mean of pERK1/ERK1 or pERK2/ERK2 in the B6 group is designated as 1, and each measurement is compared with this mean. ANA level (C) and spleen weights (D) are shown for the mice analyzed in B. C, The cutoff level for ANA production is indicated by the dashed line which represents the mean + 3 SD of the ANA levels in B6 control mice.
Although elevated ERK phosphorylation is correlated with splenomegaly and ANA development in most B6.Sle1ab mice examined, the occurrence of aberrant ERK activation may precede the development of splenomegaly and ANA (Fig. 5B). This further indicates that the dysregulation of the Ras-ERK signaling pathway may not be a consequence of B cell activation, but instead one element in the mechanism that leads to B cell activation. Consistent with this interpretation, we observed elevated Ras-ERK activation in Sle1ab B cells before the detection of increased levels of the activated CD4+ T cell population (measured by CD69 staining) in B6.Sle1ab mice (data not shown). Furthermore, an increase of ERK2 phosphorylation in B lymphocytes of B6.Sle1ab mice can appear as early as 2 mo of age, when ANA is still negative and STAT3 phosphorylation is not seen increased.
The impact of farnesyltransferase inhibitors manumycin A and POH on ANA production by short-term cultures of B6.Sle1ab splenocytes was assessed to determine whether the detected Ras-ERK activation was directly involved with ANA production by splenic B cells. Both of these compounds inhibit ras signaling via inhibition of ras protein farnesylation (24, 25, 26). Splenocytes from ANA-positive B6.Sle1ab mice were cultured with different concentrations of manumycin A or POH for 2 days, and the levels of anti-histone/dsDNA Abs in the supernatants were determined. As shown in Fig. 6, A and B, both agents inhibit ANA production and total IgG production in these short-term cultures. These results establish that the ras pathway plays a key role in ANA production in B6.Sle1ab mice.
FIGURE 6. Inhibition of ANA production by farnesyltransferase inhibitors. Inhibition of ANA production in splenic mononuclear cells cultured in vitro by manumycin A (A) and POH (B) is shown. One million splenic mononuclear cells isolated from ANA-positive B6.Sle1ab mice were cultured in vitro with or without manumycin A or POH at 37°C in 5% CO2 for 48 h. The supernatant was collected and assayed for ANA level by ELISA. T0, The ANA concentration in supernatant of the control group (without drug); T, the ANA concentration in supernatant of the drug treatment group. Each point represents the mean ± SE of three experiments. *, p < 0.05. At the end of the 48-h culture period, these two drugs decreased the cell viability by 60% at the highest concentrations, mainly by inducing apoptosis as measured by annexin V and 7-aminoactinomycin D staining. C, POH inhibits ANA production in B6.Sle1ab mice. ANA-positive B6.Sle1ab mice were treated with i.p. administration of 75 mg of POH/kg of body weight daily for 2 wk. Sera were collected before and after treatment. ANA levels were measured by ELISA. T0, The ANA level before treatment; T, the ANA level after treatment.
Treatment with POH has been shown to be effective in preventing in vivo tumor development in mice and farnesyltransferase inhibitors are being evaluated in clinical trials for cancer treatment in humans (24, 25). To assess the potential of POH as a therapeutic modality for systemic autoimmunity, we selected B6.Sle1ab mice with high levels of ANA and treated them with daily i.p. injections of POH at a dose of 75 mg/kg body weight for 2 wk. This dose was found to be safe for mice by other investigators (25). Sera were collected before and after treatment and the levels of ANA were determined by ELISA. As shown in Fig. 6C, POH will prevent the increase in ANA production that normally occurs subsequent to seroconversion in B6.Sle1ab mice. These results indicate that the ras pathway plays an important role in the in vivo production of ANA by B6.Sle1ab mice and suggests that ras inhibitors may be of value as therapeutics for systemic autoimmunity.
Discussion
Our analysis of global gene expression profiles in B lymphocytes from B6.Sle1ab mice identified a variety of differentially expressed genes early in the initiation of autoantibody production. Interestingly, these activation phenotypes were present in mice already actively producing ANA as well as those that had not yet initiated ANA production. This suggests that the Sle1ab susceptibility alleles mediate a chronic immune activation and that ANA production is a subsequent stochastic event that has a high probability of occurring by 9 mo of age, at which time ANA penetrance is 90% in females. Since the anti-chromatin Abs produced by B6.Sle1ab mice are not pathogenic and do not preferentially utilize the same VH genes as pathogenic autoantibodies that are produced in polycongenic strains such as B6.Sle1Sle3 (9), these results suggest that the crucial role that Sle1ab plays in the initiation of severe autoimmunity is to potentiate the development of a chronic activation phenotype in lymphocytic lineages, rather than the specific production of anti-chromatin Abs.
Although the majority of the genes that are differentially expressed in B6.Sle1ab B lymphocytes are predictable molecular players in immune activation, our analysis identified an aberrant activation in three interrelated molecular pathways with relevance to lupus susceptibility: IL-6 secretion, SOCS3/STAT3 activation, and ras/MAPK activation. The importance of each of these pathways in the production of autoantibodies by B lymphocytes was validated via analysis of protein expression and/or phosphorylation status in freshly isolated ex vivo B lymphocytes. In addition, inhibitors of the pathways could be shown to suppress ANA production by B lymphocytes in short-term culture and in vivo administration of farnesyltransferase inhibitors significantly decreased ANA production in B6.Sle1ab mice. Taken together, these results validate this approach for the identification of dysregulated molecular pathways in autoimmunity and identify three strong candidate pathways for therapeutic intervention in autoantibody production.
Previous studies have implicated IL-6 as a key cytokine in systemic lupus erythematosus pathogenesis in humans as well as lupus-prone murine models (26, 27, 28, 29). Our results indicate that this pathway is an important early element in the production of autoantibodies by B6.Sle1ab mice. Based on the patterns of activation detected in our analysis, increased proportions of monocytes and B lymphocytes secreting IL-6 are detectable early in the development of ANA production. It is reasonable to predict that increased levels of IL-6 drive the activation of the STAT3 and SOCS3 pathways as well as ras activation and ERK/MAPK phosphorylation. However, since Ras-ERK activation is downstream of many signaling pathways, it is also possible that the aberrant activation of the Ras-ERK pathway is independent of the IL-6 signaling pathway. As aforementioned, we did see the increase of ERK2 phosphorylation preceding the increase of STAT3 phosphorylation when ANA was still negative. It has been documented that B cell activation can lead to the production of IL-6 (30, 31). Therefore, one possible mechanism for increased IL-6 production in B6.Sle1ab mice is that intrinsic B cell signaling defects involving aberrant activation of ERK2 lead to the dysregulated B cell activation, which in turn results in the elevated production of IL-6. IL-6 can then act on B cells to enhance the activation (through STAT3 as well as Ras-ERK pathways) and Ab production. In this case, altered CD4+ T cell activation and an increase in the size of CD11b+ mononuclear cell population may only be the downstream effects of B cell activation.
It is interesting to note that we detected activation of SOCS3 and STAT3 simultaneously in ex vivo B lymphocytes of B6.Sle1ab mice. Since SOCS3 is a suppressor of IL-6 signaling, its elevated expression under normal circumstances would be predicted to down-regulate IL-6 signaling in B lymphocytes and subsequently extinguish STAT3 activation (11, 16, 19, 32, 33). Thus, there are at least three possible explanations for the coexistence of elevated levels of activated STAT3 and up-regulated expression of SOCS3 in B6.Sle1ab. First, chronic elevation of IL-6 levels may mediate activation in some subsets of B lymphocytes so strongly that an elevated expression of SOCS3 is insufficient to suppress the activated STAT3 signaling. Alternatively, STAT3 activation may be mediated by an IL-6-independent pathway in Sle1ab B lymphocytes which cannot be extinguished by SOCS3. Finally, elevated SOCS3 and active STAT3 may be expressed in distinct subsets of splenic B lymphocytes in B6.Sle1ab mice. In this scenario, B lymphocytes that actively produce ANA have silenced SOCS3 and are being driven by the activated IL-6/STAT3 pathway, whereas B lymphocytes that are not responding have suppressed STAT3-mediated activation by up-regulating SOCS3. This possibility is consistent with the potent effect of inhibitors of both IL-6 and STAT3 on ex vivo secretion of ANAs by B lymphocytes from B6.Sle1ab mice. We are in the process of developing an in vivo system to assess the potential impact of modulating IL-6 secretion and SOCS3 expression by B lymphocytes to evaluate in more detail the role of this pathway in the activation of B lymphocytes in B6.Sle1ab mice.
Finally, our in vivo analysis of inhibition of farnesyltransferase with POH validates the potential efficacy of inhibiting this pathway as a therapeutic intervention in lupus. Farnesyltransferase has been identified as a target for cancer therapy and pharmaceutical companies are currently testing specific inhibitors in human trials. Our results indicate that these inhibitors may also be of value in the treatment of systemic autoimmunity. We are in the process of assessing the efficacy of farnesyltransferase inhibitors in the treatment of other murine models of systemic autoimmunity, to assess the importance of the ras pathway in murine models with more severe pathology.
Acknowledgments
We thank Drs. Chandra Mohan and Nicolai van Oers for helpful discussions. We also thank Xiang-Hong Tian for technical assistance and Dr. Jose Casco for the management of the mouse colony.
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 National Institutes of Health (PO1 AI 39824 to E.K.W.) and Alliance for Lupus Research (to E.K.W.). K.L. is a recipient of National Institutes of Health National Research Service Award (5 F32 AR48058).
2 Address correspondence and reprint requests to Drs. Kui Liu and Edward Wakeland, Center for Immunology, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75390-9093. E-mail addresses, respectively: kui.liu@utsouthwestern.edu and edward.wakeland@utsouthwestern.edu
3 Abbreviations used in this paper: ANA, antinuclear antibody; POH, perillyl alcohol; SOCS, suppressor of cytokine signaling; EST, expressed sequence tag.
Received for publication August 25, 2004. Accepted for publication November 2, 2004.
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The Sle1ab genomic interval on murine chromosome 1 mediates the loss of immune tolerance to chromatin resulting in antinuclear Abs (ANA) production in the lupus-prone NZM2410 mouse. Global gene expression analysis was used to identify the molecular pathways that are dysregulated at the initiation of B lymphocyte autoimmunity in B6.Sle1ab mice. This analysis identified that STAT3 and ras-ERK signaling pathways are aberrantly activated in Sle1ab B lymphocytes, consistent with increased production of IL-6 by splenic B lymphocytes and monocytes in B6.Sle1ab mice. In vitro treatment of splenic mononuclear cells isolated from ANA-positive Sle1ab mice with anti-IL-6 Ab or AG490, an inhibitor of STAT3 signaling pathway, suppressed ANA production in short-term culture, indicating that this pathway was essential to the production of autoantibodies. In vivo treatment of ANA-positive B6.Sle1ab mice with the ras pathway inhibitor, perillyl alcohol, suppressed the increase of ANA. These findings identify IL-6 as a early key cytokine in Sle1ab-mediated disease development and indicate that the STAT3 and ras-ERK signaling pathways are potential therapeutic targets for treating systemic lupus erythematosus.
Introduction
Susceptibility to systemic lupus erythematosus is mediated by a complex interaction of genetic and environmental elements (1, 2). Our laboratory has used congenic dissection in the NZM2410 lupus-prone mouse to model the genetic mechanisms responsible for disease pathogenesis. Congenic dissection is a strategy in which individual genes that contribute to a polygenic disease (such as lupus) are segregated into unique substrains of a specific inbred strain, thus allowing the component phenotype contributed by each gene to be analyzed separately. Phenotypic analyses of the collection of B6-congenic strains that we have produced from the NZM2410 model have allowed the characterization of component phenotypes associated with three pathways that contribute to lupus pathogenesis (1, 3, 4). These studies have identified the Sle1 gene cluster, which mediates a breach in immune tolerance and the production of antinuclear Abs (ANA),3 as a key pathway in the initiation of the disease cascade.
B6.Sle1 mice develop hypergammaglobulinemia, splenomegaly, and expanded populations of activated CD4 T cells and B cells in their spleens, leading to a progressive loss in immune tolerance and the production of high-titered IgG antinuclear Abs beginning at 4 mo of age (5). Our ongoing genetic analyses of this interval indicate that a cluster of four loci, denoted Sle1a through Sle1d, mediate these Sle1 phenotypes (6). Detailed analyses of truncated congenic intervals containing the individual genes in the Sle1 cluster have identified Sle1a and Sle1b as the most potent loci, each capable of mediating fatal lupus when combined with additional autoimmune genes, such as lpr or yaa. These studies have demonstrated that Sle1ab is expressed in a cell intrinsic fashion in lymphocytes and that they cause progressive functional changes in both T and B lymphocytes, leading to chronic immune activation and autoantibody production.
Phenotypic analyses have indicated that B lymphocytes are strongly dysregulated by Sle1ab (6, 7). Although previous analyses have identified a variety of cell surface phenotypic changes, little is currently known about transcriptional changes in B lymphocytes from B6.Sle1ab. In this study, we describe a global gene expression analysis of B lymphocytes from B6.Sle1ab mice as they transition to an ANA-positive phenotype. Our results implicate IL-6 as a key early cytokine in disease development and identify several molecular pathways as candidates for therapeutic intervention.
Materials and Methods
Mice
C57BL/6 (B6) mice were obtained from The Jackson Laboratory and subsequently bred in the University of Texas Southwestern Medical Center specific pathogen-free animal colony. B6.Sle1ab mice are B6 mice congenic for a 3-cM interval of NZM2410 origin on chromosome 1, with termini at D1Mit400 and D1Mit206 (6). The derivation of B6-congenic mice bearing NZM2410-lupus susceptibility intervals has been detailed previously (3, 6). The immunological phenotypes of this strain have been reported previously (3, 5, 6). All experiments were conducted with the approval of the Institutional Animal Care and Use Committee of the University of Texas Southwestern Medical Center.
Cell isolation and RNA extraction
B lymphocytes were isolated from mouse spleens by using DynaBeads coated with anti-B220 mAb according to the manufacturer’s instruction (Dynal). Briefly, splenocytes were depleted of RBC by incubation with ACK lysing buffer (containing 0.15 M NH4Cl, 10 mM KHCO3, and 0.1 mM EDTA, pH 7.4) for 5 min. After three washes at 4°C, the cells were incubated at 4°C for 20 min with Dynabeads Mouse pan B (B220) at a target:bead ratio of 1:4 in PBS containing 1% FCS. The rosetted cells were then washed three times. The purity of the isolated B lymphocytes was >97%. Total cellular RNA was prepared using an Atlas Total RNA Isolation kit (BD Clontech) per the manufacturer’s instruction.
cDNA microarray hybridization and analysis
For Atlas Arrays, the complex [-32P]dATP-labeled first-strand cDNA probes were synthesized and purified according to the protocol provided in the Atlas Mouse 1.2 Array User’s Manual (BD Clontech). Ten micrograms of total RNA was used as template in a 10-μl reverse transcription reaction. A gene-specific primer mixture (BD Clontech) was used to prime the reverse transcription. The Atlas Mouse 1.2 cDNA array membranes were prehybridized in ExpressHyb (BD Clontech) containing 100 mg/5 ml of sheared salmon testes DNA at 71°C for 30 min. Then these membranes were hybridized with 32P-labeled first-strand cDNA probes (10 x 106 cpm) overnight at 71°C. After washing, the arrays were exposed to a phosphorimaging screen for 24 h at room temperature. The screen was scanned on a PhosphorImager (Molecular Dynamics) and the images were analyzed by using AtlasImage software (BD Clontech). The scatter plots were generated using GeneSpring Software (Silicon Genetics). Mouse 12k glass arrays containing 12,000 cDNAs were constructed in the University of Texas Southwestern Medical Center Microarray Core Facility (see http://microarraycore.swmed.edu for gene list). Each array contains 5,600 sequence-verified cDNA clones from Research Genetics, Inc., and 6,200 IMAGE Consortium mouse cDNA clones (1,536 clones from B cell line, 1,920 clones from T cell line, 768 clones from thymus, 768 clones from macrophage cell line, and 1152 clones from spleen) from Incyte Genomics. Generation of cDNA probes and array hybridization were performed with a Submicro Expression kit (Genisphere) (8) according to the manufacturer’s protocol. Briefly, 1 μg of total RNA was reverse-transcribed using reverse transcription primers tagged with either Cy3- or Cy5-specific 3DNA capture sequence. The synthesized tagged cDNAs were then labeled by Cy3–3DNA or Cy5–3DNA based on the complementary of capture sequence with 3DNA capture reagents. The hybridizations were performed at 50°C, with three subsequent washes of the slides in 2x SSC, 0.2% SDS, 2x SSC, and 0.2x SSC buffers. The arrays were scanned using the GenePix 4000A scanner (Axon Instruments). The results were analyzed using GenePix 4.0 (Axon Instruments) and GeneSpring (Silicon Genetics).
Relative quantitative RT-PCR
Relative quantitative RT-PCR was performed using reverse transcription and coamplification of 18S ribosomal RNA as internal control following the protocol provided with a QuantumRNA 18S internal standard kit (Ambion) according to the manufacturer’s instructions. Briefly, 0.5 μg of RNA was mixed with 2 μl of random primers (50 μM; Ambion) and RNase-free water up to 10 μl of volume, denatured at 80°C for 10 min, and rapidly cooled on ice for 5 min. Then 1 μl of dNTP mix (10 mM), 0.5 μl of RNasin RNase inhibitor (Roche Applied Science), 1 μl of 0.1 M DTT, 4 μl of 5x first-strand buffer (Invitrogen Life Technologies), and 1 μl of Moloney murine leukemia virus-reverse transcriptase (Invitrogen Life Technologies) were added to each sample. The mixtures were incubated for 1 h at 42°C. PCR was performed using 1 μl of the reverse transcription product in a 50-μl reaction mixture containing 5 μl of 10x PCR buffer (Roche Applied Science), 1 μl of dNTPs (10 mM), 2 μl of gene-specific primer pair (10 μM each), 4 μl of 18 S Classic Primer and Competimer pair (Ambion), and 2.5 U of TaqDNA polymerase (Roche Applied Science). After incubation at 94°C for 5 min, the PCR was performed for 29 cycles followed by a 5-min extension at 72°C. Each cycle consisted of 94°C for 30 s, 60°C for 30 s, and 72°C for 45 s. Both suppressor of cytokine signaling (SOCS) 3 mRNA and 18S rRNA coamplified within a linear range. Because of the low abundance of IL-6 mRNA in spleen, RT-PCR of IL-6 mRNA was performed on equal amounts of RNA without using 18S RNA internal control. The PCR cycle number for IL-6 is 35. Amplified PCR products were visualized by ethidium bromide staining and relatively quantified by EagleEye software (Stratagene). The nucleotide sequences of the primers for SOCS3 are 5'-GGA GAC TCC TGA GTT AAC ACT GGG-3' (sense) and 5'-GAC CAG TTC CAG GTA ATT GCA TGG C-3' (antisense). The nucleotide sequences of the primers for IL-6 are 5'-ATA GTC AAT TCC AGA AAC CGC TAT GAA G-3' (sense) and 5'-GAT TAT ATC CAG TTT GGT AGC ATC CAT C-3' (antisense).
SDS-PAGE and immunoblotting
Isolated B lymphocytes were lysed in a buffer containing 300 mM NaCl, 50 mM Tris-Cl (pH 7.6), 0.5% Triton X-100, 10 μg/ml leupeptin, 10 μg/ml aprotinin, 1 mM PMSF, 1 mM sodium orthovanadate, 1 mM sodium molybdate, and 1 mM NaF. Cellular extracts were incubated on ice for 30 min and then centrifuged at 12,000 x g for 20 min. The supernatants were collected, and the protein concentration was determined using a Bio-Rad protein assay kit. Equivalent amounts of total cellular protein extract (4 μg) were fractionated on 10% SDS-PAGE and electrotransferred to a polyvinylidene difluoride membrane using a Bio-Rad transblot apparatus. The membrane was blocked overnight with TBST buffer (10 mM Tris-Cl (pH 8.0), 0.9% NaCl, and 0.1% Tween 20) plus 4% BSA (for phosphorylated proteins) or 5% nonfat dry milk and incubated for 1 h with primary mAb in TBST buffer plus 4% BSA (for phosphorylated proteins) or 5% nonfat dry milk. Following three washes in TBST, the membranes were incubated with a secondary Ab for 1 h. The blots were again washed three times in TBST and then developed using an ECL Plus kit (Amersham) according to the manufacturer’s instructions. Protein bands were quantified by densitometric analysis using a computerized densitometer (Molecular Dynamics) and ImageQuant software (Molecular Dynamics). Rabbit polyclonal Abs specific for P44/42 MAP kinase, STAT3, or phospho-STAT3 were purchased from Cell Signaling. Rabbit anti-phospho-ERK1/2 MAP kinase polyclonal Abs were purchased from Promega. Rabbit anti-SOCS3 polyclonal Abs and the secondary Abs (goat anti-rabbit IgG) were purchased from Santa Cruz Biotechnology.
ELISA
Sera collected from the mice studied were assayed for the presence of anti-histone-DNA Abs by ELISA as previously described (4, 5, 9). Comparisons of Ab levels between different strains and genders in sera were conducted by testing simultaneously all of the samples from different mice on the same ELISA plate. Briefly, Immulon II plates (Dynatech Laboratories), precoated with methylated BSA, were coated overnight with 50 μg/ml dsDNA (Sigma-Aldrich) and 10 μg/ml total histones (Roche Applied Science) at 4°C. The concentrations of Ags used in these ELISAs have been shown to saturate all available binding sites (4, 9). After blocking with PBS/3% BSA/0.1% gelatin/3 mM EDTA, 1/100 dilutions of the test sera were incubated in duplicate for 2 h at room temperature. Bound IgG was detected with alkaline phosphatase-conjugated anti-mouse IgG (Jackson ImmunoResearch Laboratories) using pNPP as a substrate. Raw OD was converted to units per milliliter using a positive control serum from an NZM2410 mouse. The reactivity of a 1/100 dilution of this serum was arbitrarily set to 100 U/ml. For quantitation of serum IL-6, a Quantikine M IL-6 kit (R&D Systems) was used according to the manufacturer’s instruction.
In vitro culture of splenic mononuclear cells
Splenic mononuclear cells were isolated using Histopaque-1083 (Sigma-Aldrich) and cultured in RPMI 1640 containing 10% FCS for 2 days with or without drugs. Supernatant was collected and the levels of anti-histone/dsDNA Abs were determined using ELISA at 1/2 dilutions. The levels of total IgG were determined using ELISA at 1/5 dilutions. The drugs used were AG490 (Alexis Biochemicals), manumycin A (Sigma-Aldrich), and perillyl alcohol (POH) (Sigma-Aldrich). Monoclonal anti-IL-6 Ab and the isotype control were purchased from BD Pharmingen.
Flow cytometry and intracellular staining
Intracellular IL-6 staining was conducted using a Cytofix/Cytoperm Plus kit (BD Pharmingen) according to the manufacturer’s instruction. Briefly, freshly isolated splenic mononuclear cells were stained with FITC conjugate of anti-B220, anti-CD4, or anti-CD11b (BD Pharmingen) at 4°C for 20 min after incubation with Fc Block (BD Pharmingen) for 15 min. After washing, the cells were permeabilized with Cytofix/Cytoperm, washed twice with Perm/Wash buffer, and stained with PE conjugate of anti-IL-6 mAb or isotype control (BD Pharmingen) for 30 min at 4°C. The cells were washed twice with Perm/Wash buffer and resuspended in PBS containing 1% FCS and 0.1% sodium azide before acquisition of the data on a FACScan (BD Biosciences) with CellQuest software (BD Biosciences). The data were analyzed with FlowJo software (Tree Star).
Treatment of mice with POH
B6.Sle1ab mice were treated with i.p. administration of 75 mg of POH/kg of body weight or vehicle control daily for 14 days. The vehicle for POH was Tricaprylin (Sigma-Aldrich). Sera were collected before and after treatment. Levels of anti-histone-DNA Abs in the sera were measured by ELISA as described above.
Statistics
Results were expressed as the mean ± SE and compared using unpaired Student’s t test. Differences were considered significant at p < 0.05.
Results
Comparisons of global gene expression patterns in B cells of B6 vs B6.Sle1ab mice
Variations in global gene expression were sought by comparing B220+ splenic B lymphocytes isolated from age- and gender-matched B6 and B6.Sle1ab mice early in the initiation of autoimmunity to nuclear Ags (3- to 4-mo-old female mice recently converted to ANA). Our initial analyses used Clontech’s Atlas Mouse 1.2 Array, which detects 1,176 genes with a variety of roles in biological processes such as oncogenesis, cell signaling, cell cycle, and apoptosis. Subsequently, we expanded the analysis by the addition of custom-fabricated cDNA slide arrays containing 12,000 genes (see http://microarraycore.swmed.edu/website for gene list). Representative results of some of these microarray analyses are presented in Fig. 1. As expected, B lymphocytes from separate B6 mice express these genes at similar levels (Fig. 1A). In contrast, comparisons of B lymphocytes of age- and gender-matched B6 and B6.Sle1ab revealed a distinct subset of genes with changes in gene expression.
FIGURE 1. Analysis of gene expression by using Atlas Mouse 1.2 Arrays has identified the up-regulated expression of the SOCS3 gene in splenic B lymphocytes of B6.Sle1ab mice. A and B, Scatter plots of hybridization intensities for probes synthesized from total RNA of splenic B lymphocytes isolated from B6 and B6.Sle1ab mice. For each plot, the horizontal axis represents the hybridization intensity for genes expressed in B6 B lymphocytes, and the vertical axis represents the hybridization intensity for these genes in either B6 or B6.Sle1ab B lymphocytes. Each dot in the plots represents the hybridization intensity for a single gene in each sample. The parallel lines flanking the diagonal indicate 2- and 3-fold changes in gene expression. A, Gene expression in B lymphocytes from two separate B6 mice is compared. B, Up-regulated expression of the SOCS3 gene in B6.Sle1ab B lymphocytes is indicated by a circle. C, Array hybridization patterns of SOCS1 and SOCS3 for B6 and B6.Sle1ab are shown. SOCS1 is indicated by a white arrow and SOCS3 is indicated by a black arrow. Of the SOCS family genes, both SOCS1 and SOCS3 are expressed in B lymphocytes, but only the SOCS3 gene shows up-regulated expression in B6.Sle1ab B lymphocytes. D, Relative quantitative RT-PCR assessment of expression of SOCS3 in splenic B lymphocytes of B6, B6.Sle1, and B6.Sle1ab mice, confirming that expression of the SOCS3 gene is up-regulated in Sle1-congenic mice. 18S rRNA is used as the internal control.
Table I lists the genes that exhibited >2-fold expression variations in three or more replicate comparisons of B220+ splenic B cells from B6 vs B6.Sle1ab mice. None of these genes are located within the congenic interval, indicating that they reflect variations in molecular pathways downstream from Sle1ab. There is no significant difference in the distribution of B cells within B cell subsets as determined by CD5, CD21, and CD23 staining. However, there is a higher percentage of activated B cells in B6.Sle1ab mice as measured by CD69 staining. Many of the up-regulated genes observed in this analysis are consistent with the presence of an expanded population of proliferating splenic B lymphocytes in B6.Sle1ab mice. Thus, Ig genes, c-myc, p55CDC, G2-M-specific cyclin B2, and a variety of other cell proliferation-related genes are all significantly up-regulated in B6.Sle1ab B lymphocytes early in the initiation of detectable humoral autoimmunity. A small number of genes were also down-regulated, although most of these were expressed sequence tags (ESTs). Interestingly, similar profiles were detected in 4-mo-old female B6.Sle1ab mice regardless of their ANA phenotype, suggesting that chronic immune activation is a consistent feature of these mice and precedes ANA production.
Table I. Genes differentially regulated in splenic B lymphocytes of B6. Sle1ab mice
Dysregulation of SOCS3 in Sle1ab B lymphocytes
SOCS3 expression is consistently up-regulated >3-fold in comparisons of age- and gender-matched B6 and B6.Sle1ab splenic B lymphocytes (Fig. 1, B and C), while SOCS1 expression is unchanged. This up-regulation was confirmed by quantitative RT-PCR of splenic B cell mRNAs isolated from cohorts of both B6.Sle1 and B6.Sle1ab mice (Fig. 1D). The SOCS gene family encodes proteins that are induced in cells responding to stimulation by cytokines (10). Since SOCS family transcription has been shown to be up-regulated in response to STAT phosphorylation (11, 12), we compared the levels of phosphorylated STATs in B lymphocytes freshly isolated from B6 and B6.Sle1ab mice. As shown in Fig. 2, phosphorylated STAT3 is clearly increased in Sle1ab B lymphocytes (Fig. 2A), although no difference was observed in the levels of phosphorylated STAT1, STAT5, or STAT6 (data not shown). These results are consistent with previous reports indicating that SOCS3 is induced in response to STAT3 phosphorylation (13, 14, 15, 16).
FIGURE 2. Sle1ab mediates aberrant activation of the STAT3 signaling pathway in B lymphocytes. A, Representative immunoblots of phospho-STAT3, STAT3, and SOCS3 proteins in lysates of splenic B lymphocytes isolated from B6 and B6.Sle1ab mice. Four micrograms of cell lysate from each mouse was resolved by SDS-PAGE and immunoblotted using phospho-STAT3-, STAT3-, or SOCS3-specific Abs. Results of densitometric analysis for these blots are shown in the bar graph below the blots. The mean of pSTAT3/STAT3 or SOCS3 in the B6 group is designated as 1, and each measurement is compared with this mean. ANA (B) and spleen weights (C) are shown for the mice analyzed in A. B, The cutoff level for autoantibody production is indicated by the dashed line which represents mean + 3 SD of the ANA levels in B6 control mice.
IL-6 plays a key role in ANA production by B6.Sle1ab mice
STAT3 phosphorylation is induced by IL-6 and potentially several other cytokines (17, 18, 19, 20). Consequently, we assayed serum levels of IL-2, IL-4, IL-5, IL-6, IL-10, TNF-, and IFN- levels in B6 and B6.Sle1ab mice at 4 mo of age. As shown in Fig. 3A, this analysis revealed statistically significant increases in the IL-6 levels in the serum of B6.Sle1ab mice. Levels of TNF- and IL-5 were only slightly elevated in some B6.Sle1ab mice, whereas no change in the level of IL-2, IL-4, IFN-, or IL-10 was detected (data not shown).
FIGURE 3. Sle1ab mediates an elevated serum level of IL-6. A, Sera from 4-mo-old B6 and B6.Sle1ab mice were measured for IL-6 levels by ELISA. Mean value is indicated in the graph as a short dash. *, p < 0.05. B, Amplification of IL-6 mRNA from B6.Sle1ab mice by RT-PCR. C–F, IL-6 is secreted by some B220+ cells and CD11b+ cells in spleens of B6.Sle1ab mice. Freshly isolated splenocytes were stained for surface markers and intracellularly with PE-labeled anti-IL-6 mAb or isotype control and then analyzed using flow cytometry. The numbers shown in the gates represent the percentage (mean ± SE of three experiments and corrected with isotype control staining) of cells positive for both IL-6 and B220 or IL-6 and CD11b staining. *, p < 0.05. Spleens of B6.Sle1ab mice have more CD11b+ mononuclear cells than those of B6 mice (1.5-fold in percentage), whereas there is no significant difference in the percentage of CD11b+ mononuclear cells secreting IL-6 (17% in B6.Sle1ab vs 14% in B6).
To investigate whether more IL-6 was produced in the spleen of B6.Sle1ab mice, we performed quantitative RT-PCR on mRNA isolated from age- and gender-matched B6 and B6.Sle1ab spleens. As shown in Fig. 3B, B6.Sle1ab spleens contain higher levels of IL-6 mRNA. Flow cytometry analysis with intracellular staining for IL-6 indicated that B6.Sle1ab mice differ from B6 mice via an expansion of splenic B lymphocytes and monocytes that express this cytokine (Fig. 3C).
Although these analyses of ex vivo splenic B cells from B6.Sle1ab mice establish that variations in STAT3 activation and IL-6 are correlated with ANA production, they do not establish that B cells producing ANA are dependent upon these phenotypes. To address this issue, we assessed the effect of inhibiting these functions on the production of ANA by short-term cultures of splenocytes isolated from ANA+ B6.Sle1ab mice. As shown in Fig. 4A, neutralizing anti-IL-6 Ab significantly inhibited ANA production in 48-h in vitro cultures of B6.Sle1ab splenocytes. These results establish that IL-6 plays a key role in driving the production of ANA by autoimmune B lymphocytes in B6.Sle1ab mice. Similarly, the STAT3 inhibitor AG490 greatly suppressed ANA production in a similar experimental system (Fig. 4B). These results indicate that STAT3 activation via IL-6 stimulation is key in the generation of ANA by splenic B cells of B6.Sle1ab mice.
FIGURE 4. Inhibition of ANA production in splenic mononuclear cells by anti-IL-6 Ab and AG490. Splenic mononuclear cells isolated from ANA-positive B6.Sle1ab mice were cultured in vitro with different concentrations of isotype control or neutralizing anti-IL-6 Ab (A) or AG490 (B) for 48 h. ANA levels were determined by ELISA. T0, ANA level for control group; T, ANA level in control and treatment group. Each data point represents the mean ± SE of six experiments. *, p < 0.05. A, Anti-IL-6 group is compared with the isotype control group at each dose, and the AG490 group is compared at each dose to the control group in B. At the concentration of 50 μM, AG490 slightly decreased the viability of cells (by 10%) at the end of the 48-h culture period, mainly by inducing apoptosis as measured by annexin V and 7-aminoactinomycin D staining. It dramatically decreased the cell viability at 100 μM (by 50%).
Sle1ab mediates dysregulation of the Ras-ERK pathway
As shown in Fig. 5A, gene expression profiling of B6.Sle1ab splenic B cells revealed a significant up-regulation in the expression of the gene encoding the -chain of farnesyltransferase. Since farnesyltransferase catalyzes the posttranslational farnesylation of ras (21, 22, 23), a key step in the activation of the ras pathway, this result suggests that the ras signaling pathway in B lymphocytes of B6.Sle1ab mice is activated. As shown in Fig. 5B, immunoblot analysis of ERK1/ERK2 indicated that the ERK2 pathway is more active in B6.Sle1 B lymphocytes, consistent with increased Ras-mediated signaling in bulk splenic B lymphocytes of B6.Sle1ab mice. However, no significant difference in JNK and P38 MAP kinase pathways was detected (data not shown).
FIGURE 5. Ras-ERK MAP kinase pathway is dysregulated in splenic B lymphocytes isolated from B6.Sle1ab mice. A, Microarray analyses indicate that more mRNAs for farnesyltransferase -chain are present in B lymphocytes isolated from B6.Sle1ab mice. For each line, the left end represents the microarray hybridization intensity of probes generated from the farnesyltransferase -chain mRNA in B6 B lymphocytes, and the right end of the line represents that for the same mRNA in B6 or B6.Sle1ab B lymphocytes. The numbers at the top of each graph represent the ratios of their hybridization intensities to that for B6 B lymphocytes. B, Representative immunoblots of phospho-ERK1/2 and ERK1/2 proteins in lysates of splenic B lymphocytes isolated from B6 and B6.Sle1ab mice. Lysates of splenic B lymphocytes were diluted in 2x SDS sample buffer, resolved on SDS-PAGE (4 μg protein/lane), and immunoblotted using Abs specific for phospho-ERK1/2 MAP kinases or total ERK1/2 MAP kinases. Results of densitometric analysis for these blots are shown in the bar graph below the blots. The mean of pERK1/ERK1 or pERK2/ERK2 in the B6 group is designated as 1, and each measurement is compared with this mean. ANA level (C) and spleen weights (D) are shown for the mice analyzed in B. C, The cutoff level for ANA production is indicated by the dashed line which represents the mean + 3 SD of the ANA levels in B6 control mice.
Although elevated ERK phosphorylation is correlated with splenomegaly and ANA development in most B6.Sle1ab mice examined, the occurrence of aberrant ERK activation may precede the development of splenomegaly and ANA (Fig. 5B). This further indicates that the dysregulation of the Ras-ERK signaling pathway may not be a consequence of B cell activation, but instead one element in the mechanism that leads to B cell activation. Consistent with this interpretation, we observed elevated Ras-ERK activation in Sle1ab B cells before the detection of increased levels of the activated CD4+ T cell population (measured by CD69 staining) in B6.Sle1ab mice (data not shown). Furthermore, an increase of ERK2 phosphorylation in B lymphocytes of B6.Sle1ab mice can appear as early as 2 mo of age, when ANA is still negative and STAT3 phosphorylation is not seen increased.
The impact of farnesyltransferase inhibitors manumycin A and POH on ANA production by short-term cultures of B6.Sle1ab splenocytes was assessed to determine whether the detected Ras-ERK activation was directly involved with ANA production by splenic B cells. Both of these compounds inhibit ras signaling via inhibition of ras protein farnesylation (24, 25, 26). Splenocytes from ANA-positive B6.Sle1ab mice were cultured with different concentrations of manumycin A or POH for 2 days, and the levels of anti-histone/dsDNA Abs in the supernatants were determined. As shown in Fig. 6, A and B, both agents inhibit ANA production and total IgG production in these short-term cultures. These results establish that the ras pathway plays a key role in ANA production in B6.Sle1ab mice.
FIGURE 6. Inhibition of ANA production by farnesyltransferase inhibitors. Inhibition of ANA production in splenic mononuclear cells cultured in vitro by manumycin A (A) and POH (B) is shown. One million splenic mononuclear cells isolated from ANA-positive B6.Sle1ab mice were cultured in vitro with or without manumycin A or POH at 37°C in 5% CO2 for 48 h. The supernatant was collected and assayed for ANA level by ELISA. T0, The ANA concentration in supernatant of the control group (without drug); T, the ANA concentration in supernatant of the drug treatment group. Each point represents the mean ± SE of three experiments. *, p < 0.05. At the end of the 48-h culture period, these two drugs decreased the cell viability by 60% at the highest concentrations, mainly by inducing apoptosis as measured by annexin V and 7-aminoactinomycin D staining. C, POH inhibits ANA production in B6.Sle1ab mice. ANA-positive B6.Sle1ab mice were treated with i.p. administration of 75 mg of POH/kg of body weight daily for 2 wk. Sera were collected before and after treatment. ANA levels were measured by ELISA. T0, The ANA level before treatment; T, the ANA level after treatment.
Treatment with POH has been shown to be effective in preventing in vivo tumor development in mice and farnesyltransferase inhibitors are being evaluated in clinical trials for cancer treatment in humans (24, 25). To assess the potential of POH as a therapeutic modality for systemic autoimmunity, we selected B6.Sle1ab mice with high levels of ANA and treated them with daily i.p. injections of POH at a dose of 75 mg/kg body weight for 2 wk. This dose was found to be safe for mice by other investigators (25). Sera were collected before and after treatment and the levels of ANA were determined by ELISA. As shown in Fig. 6C, POH will prevent the increase in ANA production that normally occurs subsequent to seroconversion in B6.Sle1ab mice. These results indicate that the ras pathway plays an important role in the in vivo production of ANA by B6.Sle1ab mice and suggests that ras inhibitors may be of value as therapeutics for systemic autoimmunity.
Discussion
Our analysis of global gene expression profiles in B lymphocytes from B6.Sle1ab mice identified a variety of differentially expressed genes early in the initiation of autoantibody production. Interestingly, these activation phenotypes were present in mice already actively producing ANA as well as those that had not yet initiated ANA production. This suggests that the Sle1ab susceptibility alleles mediate a chronic immune activation and that ANA production is a subsequent stochastic event that has a high probability of occurring by 9 mo of age, at which time ANA penetrance is 90% in females. Since the anti-chromatin Abs produced by B6.Sle1ab mice are not pathogenic and do not preferentially utilize the same VH genes as pathogenic autoantibodies that are produced in polycongenic strains such as B6.Sle1Sle3 (9), these results suggest that the crucial role that Sle1ab plays in the initiation of severe autoimmunity is to potentiate the development of a chronic activation phenotype in lymphocytic lineages, rather than the specific production of anti-chromatin Abs.
Although the majority of the genes that are differentially expressed in B6.Sle1ab B lymphocytes are predictable molecular players in immune activation, our analysis identified an aberrant activation in three interrelated molecular pathways with relevance to lupus susceptibility: IL-6 secretion, SOCS3/STAT3 activation, and ras/MAPK activation. The importance of each of these pathways in the production of autoantibodies by B lymphocytes was validated via analysis of protein expression and/or phosphorylation status in freshly isolated ex vivo B lymphocytes. In addition, inhibitors of the pathways could be shown to suppress ANA production by B lymphocytes in short-term culture and in vivo administration of farnesyltransferase inhibitors significantly decreased ANA production in B6.Sle1ab mice. Taken together, these results validate this approach for the identification of dysregulated molecular pathways in autoimmunity and identify three strong candidate pathways for therapeutic intervention in autoantibody production.
Previous studies have implicated IL-6 as a key cytokine in systemic lupus erythematosus pathogenesis in humans as well as lupus-prone murine models (26, 27, 28, 29). Our results indicate that this pathway is an important early element in the production of autoantibodies by B6.Sle1ab mice. Based on the patterns of activation detected in our analysis, increased proportions of monocytes and B lymphocytes secreting IL-6 are detectable early in the development of ANA production. It is reasonable to predict that increased levels of IL-6 drive the activation of the STAT3 and SOCS3 pathways as well as ras activation and ERK/MAPK phosphorylation. However, since Ras-ERK activation is downstream of many signaling pathways, it is also possible that the aberrant activation of the Ras-ERK pathway is independent of the IL-6 signaling pathway. As aforementioned, we did see the increase of ERK2 phosphorylation preceding the increase of STAT3 phosphorylation when ANA was still negative. It has been documented that B cell activation can lead to the production of IL-6 (30, 31). Therefore, one possible mechanism for increased IL-6 production in B6.Sle1ab mice is that intrinsic B cell signaling defects involving aberrant activation of ERK2 lead to the dysregulated B cell activation, which in turn results in the elevated production of IL-6. IL-6 can then act on B cells to enhance the activation (through STAT3 as well as Ras-ERK pathways) and Ab production. In this case, altered CD4+ T cell activation and an increase in the size of CD11b+ mononuclear cell population may only be the downstream effects of B cell activation.
It is interesting to note that we detected activation of SOCS3 and STAT3 simultaneously in ex vivo B lymphocytes of B6.Sle1ab mice. Since SOCS3 is a suppressor of IL-6 signaling, its elevated expression under normal circumstances would be predicted to down-regulate IL-6 signaling in B lymphocytes and subsequently extinguish STAT3 activation (11, 16, 19, 32, 33). Thus, there are at least three possible explanations for the coexistence of elevated levels of activated STAT3 and up-regulated expression of SOCS3 in B6.Sle1ab. First, chronic elevation of IL-6 levels may mediate activation in some subsets of B lymphocytes so strongly that an elevated expression of SOCS3 is insufficient to suppress the activated STAT3 signaling. Alternatively, STAT3 activation may be mediated by an IL-6-independent pathway in Sle1ab B lymphocytes which cannot be extinguished by SOCS3. Finally, elevated SOCS3 and active STAT3 may be expressed in distinct subsets of splenic B lymphocytes in B6.Sle1ab mice. In this scenario, B lymphocytes that actively produce ANA have silenced SOCS3 and are being driven by the activated IL-6/STAT3 pathway, whereas B lymphocytes that are not responding have suppressed STAT3-mediated activation by up-regulating SOCS3. This possibility is consistent with the potent effect of inhibitors of both IL-6 and STAT3 on ex vivo secretion of ANAs by B lymphocytes from B6.Sle1ab mice. We are in the process of developing an in vivo system to assess the potential impact of modulating IL-6 secretion and SOCS3 expression by B lymphocytes to evaluate in more detail the role of this pathway in the activation of B lymphocytes in B6.Sle1ab mice.
Finally, our in vivo analysis of inhibition of farnesyltransferase with POH validates the potential efficacy of inhibiting this pathway as a therapeutic intervention in lupus. Farnesyltransferase has been identified as a target for cancer therapy and pharmaceutical companies are currently testing specific inhibitors in human trials. Our results indicate that these inhibitors may also be of value in the treatment of systemic autoimmunity. We are in the process of assessing the efficacy of farnesyltransferase inhibitors in the treatment of other murine models of systemic autoimmunity, to assess the importance of the ras pathway in murine models with more severe pathology.
Acknowledgments
We thank Drs. Chandra Mohan and Nicolai van Oers for helpful discussions. We also thank Xiang-Hong Tian for technical assistance and Dr. Jose Casco for the management of the mouse colony.
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 National Institutes of Health (PO1 AI 39824 to E.K.W.) and Alliance for Lupus Research (to E.K.W.). K.L. is a recipient of National Institutes of Health National Research Service Award (5 F32 AR48058).
2 Address correspondence and reprint requests to Drs. Kui Liu and Edward Wakeland, Center for Immunology, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75390-9093. E-mail addresses, respectively: kui.liu@utsouthwestern.edu and edward.wakeland@utsouthwestern.edu
3 Abbreviations used in this paper: ANA, antinuclear antibody; POH, perillyl alcohol; SOCS, suppressor of cytokine signaling; EST, expressed sequence tag.
Received for publication August 25, 2004. Accepted for publication November 2, 2004.
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