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Recent studies in mice have demonstrated that the protein tyrosine phosphatase SHP-1 is a crucial negative regulator of cytokine signaling, inflammatory gene expression, and demyelination in central nervous system. The present study investigates a possible similar role for SHP-1 in the human disease multiple sclerosis (MS). The levels of SHP-1 protein and mRNA in PBMCs of MS patients were significantly lower compared to normal subjects. Moreover, promoter II transcripts, expressed from one of two known promoters, were selectively deficient in MS patients. To examine functional consequences of the lower SHP-1 in PBMCs of MS patients, we measured the intracellular levels of phosphorylated STAT6 (pSTAT6). As expected, MS patients had significantly higher levels of pSTAT6. Accordingly, siRNA to SHP-1 effectively increased the levels of pSTAT6 in PBMCs of controls to levels equal to MS patients. Additionally, transduction of PBMCs with a lentiviral vector expressing SHP-1 lowered pSTAT6 levels. Finally, multiple STAT6-responsive inflammatory genes were increased in PBMCs of MS patients relative to PBMCs of normal subjects. Thus, PBMCs of MS patients display a stable deficiency of SHP-1 expression, heightened STAT6 phosphorylation, and an enhanced state of activation relevant to the mechanisms of inflammatory demyelination.
Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system (CNS) that remains a major cause of disability.1,2 Several studies demonstrate that MS lesions and PBMCs from MS patients contain activated transcription factors like NF-κB, STAT1, STAT3, and STAT6,3-5 which can lead to enhanced signaling of inflammatory stimuli. These findings suggest that regulation of inflammatory signaling may be altered in MS and may be responsible for inflammatory demyelination.
SHP-1 is a protein tyrosine phosphatase with two SH2 domains and acts as a negative regulator of cytokine signaling via STAT1, STAT3, and STAT6.6-9 We have shown that mice genetically lacking in SHP-1 (motheaten mice) display myelin deficiency, which may be mediated by increased inflammatory mediators in the CNS.10,11 Furthermore, when motheaten mice are infected with Theiler’s murine encephalomyelitis virus (TMEV), the CNS displays substantially more virus-induced inflammatory demyelination than wild-type mice.12 In agreement with these studies, lower levels of SHP-1 have also been implicated in autoimmune-mediated demyelination in rodents12,13 and in lymphoproliferative diseases in humans.14-17 Moreover, low SHP-1 levels have been associated with excessive tyrosine phosphorylation of STAT1 following IFN-β treatment in MS patients.18 These studies suggest that SHP-1 plays multiple roles in leukocytes, including controlling activation state relevant to mechanisms of inflammatory demyelination.
Two distinct promoters are responsible for expression of each of the two known SHP-1 transcripts produced from the SHP-1 gene.19 These distinct transcripts in turn encode two slightly different SHP-1 isoforms, which have the same catalytic activity.20 It was previously shown that the two SHP-1 transcripts are differentially expressed in human tissues and cell lines.21,22 Promoter I transcripts are highly expressed in epithelial cells, while promoter II transcripts are more abundant in hematopoietic cells.22,23 Several reports show differential regulation of the SHP-1 gene promoters by distinct transcription factors.19,24,25 Importantly, promoter-specific regulation of SHP-1 expression has been associated with human disease.14,23,26
In this study, we show that the levels of SHP-1 are lower in PBMCs from MS patients compared to normal subjects. Corresponding to this deficiency, we have shown that STAT6 phosphorylation and STAT6-responsive genes are constitutively higher in PBMCs of MS patients compared to those of normal subjects. Additionally, we delineate the contribution of two promoter-specific transcripts in SHP-1 deficiency, which points to a specific decrease in promoter II activity in PBMCs of MS patients. Taken together, we propose the potential involvement of SHP-1 promoter II dysregulation in the pathogenesis of MS.
Patients were clinically diagnosed as either having active relapsing–remitting (RR) MS, active secondary progressive (SP) MS, or inactive RR (Inc) MS.27 Active RR MS was defined as a moderate to severe exacerbation within 6 months prior to entry and SP MS was defined as a continuous progression over the preceding 6 months of the study. Patients who had not received any disease-modifying treatment like IFN-β, glatiramir acetate, or other immunosuppressive agents at least two months prior to donating blood were selected. In this study, PBMCs were isolated from 23 MS patients, and 18 control subjects. The Institutional Review Board of SUNY Upstate University approved all studies, and both patients and controls granted informed consent before providing blood.
Patients and normal subjects donated 50 ml of blood collected in heparinized tubes. Blood was diluted 1:1 with HBSS and overlaid onto lymphocyte separation medium (Cellgro, Herndon, VA, USA). After centrifugation, 10 ml of the interface containing the PBMCs was collected and diluted to 50 ml with HBSS. After another two washes with HBSS, a sample of the freshly isolated cells was resuspended in STAT-60 (Tel-Test, Friendswood, TX, USA) for RNA analysis. The rest of the cells were cultured for a week in RPMI media with 20 U/ml IL-2 (R&D Systems, Minneapolis, MN, USA) and 10% fetal bovine serum. After 1 week, cells were either resuspended in STAT-60 or in RIPA buffer (50 mM Tris-HCl, pH 7.4, 1% NP-40, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM EDTA, 1 mM PMSF, 1 μg/ml aprotinin, 1 μg/ml leupeptin, 1 μg/ml pepstatin, and 1 mM activated Na3VO4) for protein analysis or fixed for intracellular flow analysis. In all experiments, PBMCs, either freshly isolated or cultured for a week in the presence of IL-2, were used with exception to lentiviral vector transductions. In the lentiviral vector transduction experiments, cells were incubated for 25 days to allow the vector to integrate into the host DNA and expand the number of transduced cells.
PBMCs of MS patients and normal subjects were cultured for 1 week and were then treated with either 10 ng/ml of IL-4 for 24 h or received medium alone (R&D Systems). A portion of PBMCs were transfected with siRNA against human SHP-1 or scramble siRNA (Dharmacon, Chicago, IL, USA) at a concentration of 1 μg/106 cells. The transfection reagent (Dharmafect 4; Dharmacon) was used as specified by the manufacturer. Cells were incubated in the transfection medium for 24 h, after which the medium was replaced with complete growth medium for another 48 h before IL-4 treatment. The effectiveness of the SHP-1 siRNA to lower SHP-1 expression was evaluated by both real-time RT-PCR and flow cytometry.
Total RNA was isolated using RNA STAT-60. RNA was quantified spectrophotometrically and 0.5 μg of total RNA was converted into cDNA. Briefly, total RNA and random primers (Invitrogen, Carlsbad, CA, USA) were incubated at 72°C for 10 min. Reverse transcription was performed using the Superscript II RT enzyme (Invitrogen) following the specification of the manufacturer. cDNA was diluted to 200 μl with water and 4 μl was used for quantitative real-time PCR using SYBR Green kit (Abgene, Epson, UK). The PCR parameters were 15 min for 95°C and 35 cycles of 95°C for 15 s and 60°C for 1 min in ABI prism 700 (Applied Biosystems, Foster city, CA, USA). The primers were used at 10 nM. Serial dilutions of cDNA containing a known copy number of each gene were used in each quantitative PCR run to generate a standard curve relating copy number with threshold amplification cycle.28 Gene expression levels were calculated during the logarithmic amplification phase by determining the initial mRNA copy number using the standard curve. Amplification of each gene-specific fragment was confirmed both by examination of melting peaks and by agarose gel electrophoresis. The following primer pairs were used in this study: SHP-1 (BC002523) forward-TGGCGTGGCAGGAGAACAG and reverse-GCAGTTGGTCACAGAGTAGGGC, SHP-1 (I) (NM080548) forward-TGGCTTCCCCCTCCCTACAG and reverse-CCCTGGTTCTTGCGACTGG, SHP-1 (II) (NM002831) forward-ATCTGAGGCTTAGTCCCTGAGC and reverse-CTGAGGTCTCGGTGAAACCAC, IL-4 (NM000589) forward-TCCCAACTGCTTCCCCC and reverse-TCTTCTGCTCTGTGAGGCTG, IL-4rα (IL-4rα) forward-CGTGTATCCCTGAGAACAACG and reverse-CGTGTATCCCTGAGAACAACG, CCL17 (NM002987) forward-CGAGGGACCAATGTGGGC and reverse-GGGTGAGGAGGCTTCAAGACC, CCL11 (NM002986) forward-CACTTCTGTGGCTGCTGCTC and reverse-GCTTTCTGGGGACATTTGC, CCR4 (NM005508) forward-GGTCCTGTTCAAATACAAGCG and reverse-AAACCCACTGGTCTGCTGC, CCR8 (NM005201) forward-CAGTGTGACAACAGTGACCGAC and reverse-GCAATAAAAGACAGCAAGGAGC, ADAM8 (NM001109) forward-ATCCCGAGAGACCCGCTAC and reverse-TGATTCACCACCTCCAGCAC, lymphotoxin-α (LTα) (NM000595) forward-CACCTCATTGGAGACCCCAG and reverse-TGTTGCTCAAGGAGAAACCATC, Arginase I (NM000045) forward-GACCTGCCCTTTGCTGACATC and reverse-TTGACTTCTGCCACCTTGCC, IFN-γ (NM000619) forward-TGCAGGTCATTCAGATGTAG and reverse-AGCCATCACTTGGATGAGTT, MCP-1 (NM002982) forward-GCTCATAGCAGCCACCTTC and reverse-GCTTCTTTGGGACACTTGC, GAPDH (NM002046) forward-ACCACCATGGAGAAGGC and reverse-GGCATGGACTGTGGTCATGA.
Whole-cell extracts were prepared as previously described.9,29 Briefly, PBMCs were rinsed with PBS, and then lysed with RIPA buffer. Protein (100 μg) per lane was electrophoresed through a 7.5% polyacrylamide-resolving gel and electroblotted to a polyvinylidene difluoride membrane (Millipore Corporation, Burlington, MA, USA). Membranes were blocked with 5% non-fat dry milk for 1 h, and then incubated with anti-SHP-1 (Upstate, Lake Placid, NY) antibodies followed by horseradish peroxidase-conjugated rabbit IgG antibody (Dako Corporation, Carpinteria, CA, USA). Enhanced chemiluminescence (Amersham Life Sciences Inc., Cleveland, OH, USA) was used to visualize reactive protein bands on X-ray film.
PBMCs were sorted into the different cell populations (T cells, B cells, and monocytes) to examine the expression of SHP-1 in individual populations. Cells (4 × 106/200 μl) were incubated on ice for 30 min with 20 μl of CD3-FITC (349201) to stain T cells, 20 μl of CD19-PE (340720) to stain B cells, and 10 μl of CD14-APC (340684) to stain for monocytes (Becton Dickinson Immunocytometry Systems, Mountain View, CA, USA). Cells were washed and resuspended in 1mL of Hank’s solution and were analyzed by fluorescence-activated cell sorter (FACS Vantage S/E; Becton Dickinson). The percentage of each cell population was determined and four aliquots labeled as T cells, B cells, monocytes, and negative cells were collected. The cells of each aliquot were lysed in the RNA isolation reagent.
PBMCs were treated for 15 min with 10ng/ml IL-4. Cells (106/1 ml) received 100 μl of 16% stock formaldehyde for fixation and then incubated in 90% methanol at 4°C for half an hour to permeabilize cells for intracellular staining. Cells were washed twice with the staining media containing 0.5% BSA and 0.02% sodium azide in PBS. Cells were resuspended in a 100 μl of staining media and incubated with 20 μl either phosphoSTAT6 (PY-641) with Alexa-488 (612600), CD3-FITC (349201), or mouse IgG with Allexa-488 isotype control (557721) (Becton Dickinson).
Also, the levels of SHP-1 were concurrently analyzed with pSTAT6. Fixed and permeabilized cells were incubated overnight at 4°C with either 1μg of rabbit anti-SHP-1 (Upstate), rabbit anti-total STAT6 (phosphorylated plus unphosphorylated) (Santa Cruz Biotech., Inc., Santa Cruz, CA, USA) antibodies, or rabbit polyclonal IgGs for isotype control. The cells were incubated for 3 h in 1 μg of goat anti-rabbit secondary antibody conjugated to PE (Invitrogen). Cells were analyzed on an LSRII analyzer (Becton Dickinson) and the mean florescence intensity (MFI) was recorded. To quantify the levels of STAT6-responsive genes by flow cytometry, anti-human ADAM8-PE IgG (R&D Systems; Cat no. 143338) was used to stain ADAM8 and anti-human LTα (R&D Systems; Cat no. 5808) was used to stain LTα (R&D Systems). Fixed and permeabilized cells were stained for 45 min at room temperature and analyzed. To quantify the levels of CCL17/TARC, cells were treated with 5 mg/ml of Brefeldin-A prior to fixation to block chemokine secretion and after 12 h cells were fixed and stained with anti-human CCL17/TARC (R&D Systems; Cat no. 54015) for 45 min and then analyzed.
The lentiviral vector prepared as described30 was used to stably infect PBMCs of MS patients. The vector carried the human SHP-1 coding sequence (NCBI no. BC002523), allowing the bicistronic expression of green fluorescent protein (GFP) and SHP-1 in the transduced cells. PBMCs were incubated in RPMI medium and infected at an approximate MOI of 0.1 for 16 h. The virus was then removed and the cells were cultured for 25 days both to allow stable integration of the lentiviral DNA in the host DNA and expand the number of transduced cells. Transduced PBMCs of MS patients and control PBMCs of normal subjects’ cells were incubated in IL-2-containing medium to maintain cell variability for the culture period. Cell viability was assessed by cell morphology and Trypan blue exclusion. GFP expression was analyzed with flow cytometry and GFP-expressing cells were sorted using FACS (Becton Dickinson). The GFP-positive and -negative cells were analyzed for the expression of SHP-1 and pSTAT6 using flow cytometry and the expression of several STAT6-responsive genes using real-time RT-PCR.
Arginase enzymatic activity was measured as previously described.31 Briefly, PBMCs were lysed in RIPA buffer and incubated in 10 mM MnCl2 and 0.5 M l-arginine at 37°C for 75 min. After the assay was stopped by addition of H3PO4, 1-phenyl-1,2-propanedione-2-oxime (Sigma, St Louis, MO, USA) was added and the samples were incubated at 100°C for 60 min. Urea production by arginase was measured by optical density at 540 nm.
Histograms or tables contain statistical means with the standard error values. The number of samples used in each assay is indicated in the figure legends. The P-values were generated using the unpaired Student’s t-test and a P-value of less than 0.05 was chosen to indicate statistical significance between two sample means.
To examine SHP-1 expression in MS patients, isolated PBMCs were prepared from whole heparinized human blood. The levels of SHP-1 mRNA were quantified using primers flanking the coding region of the gene common to both transcripts. SHP-1 mRNA levels in freshly isolated PBMCs were significantly lower in MS patients compared to normal subjects (Figure 1a). To examine whether the SHP-1 deficiency in PBMCs of MS patients was stable, PBMCs were expanded for 1 week in vitro. Similarly, SHP-1 mRNA was significantly lower in cultured PBMCs of MS patients compared to those of normal subjects (Figure 3a). Moreover, the levels of SHP-1 mRNA did not differ between PBMCs of MS patients who were clinically diagnosed with either RR MS or SP MS, indicating that depressed SHP-1 was a general marker across these clinical subclassifications (Figure 4). In accordance with mRNA levels, PBMCs of MS patients had significantly lower levels of SHP-1 protein than those of normal subjects measured by western immunoblotting (Figures 2a and b). To corroborate these findings, the levels of SHP-1 protein in PBMCs were measured by intracellular flow cytometry (Figures 2c and d). Constitutive levels of SHP-1 in PBMCs of MS patients were half the levels of SHP-1 in cells of normal subjects. Analysis of protein (Figure 2a) and mRNA (Figure 3b) of housekeeping genes showed no differences between MS and normal subject PBMCs.
To determine the individual contribution of each of two known transcripts on the expression levels of SHP-1 in PBMCs, promoter I and II transcript copy numbers were measured using promoter-specific RT-PCR primers. Constitutive levels of SHP-1 promoter I and GAPDH transcripts were not significantly lower in PBMCs of MS patients compared to controls (Figures (Figures1c,1c, 3b and 3c). On the other hand, constitutive levels of SHP-1 promoter II transcripts were two-fold lower in PBMCs of MS patients compared to controls (Figures (Figures1d1d and and3d).3d). Additionally, the levels of SHP-1 transcripts I and II did not differ between PBMCs of MS patients with different clinical subclassifications (Figure 4). Taken together, we concluded that PBMCs of MS patients have constitutively lower levels of SHP-1 protein and mRNA compared to normal subjects and furthermore that a lack of promoter II transcripts is primarily responsible for this deficiency.
To determine whether the lower levels of SHP-1 seen in PBMCs of MS patients could be attributed to differences in the proportions of cell types between subjects, we analyzed SHP-1 expression in sorted PBMCs. Antibodies against CD3, CD19, and CD14 were used to separate PBMCs into T cells, B cells, and monocytes, respectively. The percentage of each cell population was measured in PBMCs and RNA was isolated from the sorted fractions to measure the levels of SHP-1. The proportion of cell types making up the PBMCs of MS patients and controls were the same (80% T cells, 5% B cells, 3% monocytes, and 12% of negative cells). A majority of negative cells probably represent natural killer cells based on their scatter properties.32
The levels of SHP-1 mRNA in the sorted populations were quantified and T cells had similar levels of SHP-1 as in unsorted PBMCs. Importantly, T-cell populations of MS patients had significantly lower levels of SHP-1 compared to normal subjects (Figure 5a), similar to that seen in the unsorted population. Additionally, B cells and unstained cells of MS patients had significantly lower levels of SHP-1 mRNA compared to the same cell types of normal subjects. As expected, SHP-1 promoter II transcripts were primarily responsible for the lower levels of SHP-1 in MS patients in the sorted populations with more dramatic differences seen in B cells and negative cells (six-fold lower in MS compared to normal subjects). T cells had a three-fold lower level of SHP-1 promoter II transcripts in MS patients compared to normal subjects (Figure 5d). In contrast, promoter I and GAPDH transcripts were not significantly different in either unsorted PBMCs or sorted cell populations of MS patients compared to normal subjects (Figures 5b and c). These data indicate that MS patients and normal subjects contain similar proportions of leukocytes; however, the levels of SHP-1 expression in specific mononuclear cell populations are significantly lower in MS patients compared to normal subjects.
SHP-1 regulates phosphorylation of several STATs by removal of the phosphate group from tyrosine either on activated cytokine receptors or downstream signaling molecules. Thus, SHP-1 modulates STAT activation, translocation into the nucleus, and transcriptional activity on responsive genes. Because several studies showed that SHP-1 profoundly modulates IL-4R/STAT6 signaling molecules,33-36 STAT6 was chosen for further analysis of possible altered function of SHP-1 in MS PBMC. For analysis, we measured the levels of total STAT6 (unphosphorylated plus phosphorylated, tSTAT6) and tyrosine phosphorylated STAT6 (pSTAT6) in PBMCs using intracellular flow cytometry. As expected, PBMCs of MS patients had significantly elevated constitutive phosphorylation of STAT6 compared to PBMCs of normal subjects (Figure 6c). In contrast, there were no differences in the expression levels of total STAT6 protein (Figures 6a and b), verifying that the differences observed in pSTAT6 levels are attributed to the relative phosphorylation state of STAT6. Staining the same cells for SHP-1 revealed a reciprocal expression pattern of SHP-1 and pSTAT6, such that lower constitutive levels of SHP-1 in MS patients correlated with higher pSTAT6 levels compared to PBMCs of normal subjects (Figure 7). Furthermore, PBMCs were treated with IL-4 to determine possible differences in STAT6 activation following engagement of the IL-4 receptor. Treatment with IL-4 caused higher phosphorylation of STAT6 in PBMCs of MS patients compared to normal subjects (Figure 6d).
To directly determine whether a specific deficiency in SHP-1 could mediate increased activation of STAT6 in human PBMCs, PBMCs of patients and controls were treated with siRNA against SHP-1 and levels of pSTAT6 were measured. The efficiency of the siRNA against SHP-1 was verified by both real-time RT-PCR and intracellular flow cytometry (Figure 7a and Table 1). SHP-1 siRNA treatment effectively increased the levels of pSTAT6 in PBMCs of normal subjects to levels equal that of MS patients. Also, SHP-1 siRNA significantly increased inducible pSTAT6 levels following IL-4 treatment (Figure 7d). These results indicate that lower SHP-1 in PBMCs of MS patients could be directly responsible for higher constitutive levels of pSTAT6 and heightened IL-4-induced signaling to STAT6.
As a reciprocal approach, PBMCs of MS patients were transduced with a lentiviral expression vector carrying the human SHP-1 coding sequence, which allowed the bicistronic expression of GFP and SHP-1. PBMCs were then sorted according to GFP expression and were stained for SHP-1 and pSTAT6. Cells expressing GFP expressed heightened SHP-1 and lower pSTAT6 levels compared to cells in the same culture that did not express GFP (Figure 9), further substantiating that lower SHP-1 as seen in MS patients directly leads to increased pSTAT6 levels.
To determine whether constitutively higher pSTAT6 in PBMCs of MS patients relative to PBMCs of normal subjects corresponded to heightened expression of STAT6-responsive genes, the mRNA levels of several genes that are STAT6 responsive were measured in PBMCs of MS patients, normal subjects, and normal subjects treated with siRNA against SHP-1. The expression levels of certain STAT6-responsive chemokines and their receptors were of particular interest because these mediate trafficking, maturation, and attraction of T cells and macrophages to areas of CNS inflammation.37 CCL17/TARC38,39 was highly expressed in PBMCs and was more than five-fold higher in PBMCs of MS patients compared to normal subjects measured by both real-time RT-PCR and flow cytometry (Table 1 and Figure 8a). Treatment with siRNA to SHP-1 tripled the constitutive amount of CCL17/TARC in PBMCs of normal subjects, indicating that SHP-1 regulated CCL17/TARC expression. Treatment with IL-4 for 24 h induced significantly higher levels of CCL17/TARC in PBMCs of MS patients compared to PBMCs of normal subjects. Moreover, SHP-1 siRNA-treated PBMCs of normal subjects had heightened expression of CCL17/TARC in response to IL-4. Furthermore, transduction of the SHP-1-expressing vector in PBMCs of MS patients (Figure 9) decreased CCL17 expression by 15-fold (Table 2).
Another STAT6-responsive chemokine, CCL11/eotaxin,40 was significantly higher in MS patient PBMCs and normal subject PBMCs treated with SHP-1 siRNA than in PBMCs of normal subjects (Table 1). When the mRNA transcript levels of two of the chemokine receptors CCR4 and CCR8 that bind the CCL17/TARC and CCL11/eotaxin ligands were measured,14 CCR8 mRNA was not different between the PBMCs of MS patients and normal subjects and its levels did not change with either siRNA or IL-4 treatment. However, CCR4 mRNA transcripts were higher in PBMCs of MS patients compared to PBMCs of normal subjects, yet its expression levels did not change with either siRNA or IL-4 treatment.
Other STAT6-responsive genes, including ADAM8, LTα, and arginase I were analyzed and both mRNA and protein expression were elevated in PBMCs of MS patients compared to normal subjects (Table 1 and Figure 8). ADAM8 is a STAT6-responsive41,42 disintegrin matrix metalloproteinase that may be involved in demyelination.43 LTα is also STAT6-inducible in human leukocytes44 and is a member of the TNF superfamily that has been shown to mediate inflammation and demyelination in mouse models of MS.45,46 Arginase I is expressed both in the CNS and the immune system and is highly inducible by IL-4 through STAT6.47 Apart from participating in the urea cycle, arginase I also plays an important role in both virus-induced35 and autoimmune-mediated demyelination.48 Interestingly, ADAM8, LTα, and arginase I, showed higher inducible mRNA and protein levels in PBMCs of MS patients compared to normal subjects following IL-4 treatment consistent with the fact that they are STAT6 inducible (Table 1 and Figure 8). Furthermore, treatment of normal PBMCs with siRNA against SHP-1 significantly raised the levels of ADAM8, LTα, and arginase I (Table 1). Additionally, transduction of the SHP-1-expressing lentiviral vector resulted in a decrease of ADAM8, LTα, and arginase I expression in PBMCs of MS patients (Table 2).
Other genes not associated with STAT6 activation were also measured (Tables (Tables11 and and2).2). We did not observe any differences in the expression of the cytokine IFN-γ,49 or the chemokine receptor CXCR350,51 between PBMCs of MS patients and normal subjects. In contrast, the expression levels of the STAT1-responsive chemokine IP-10 were elevated in PBMCs of MS patients compared to normal subjects in accord with several studies.52-56 Furthermore, IP-10 mRNA showed a modest but significant increase in PBMCs of normal subjects following treatment with SHP-1 siRNA, but was not inducible by IL-4 treatment. To determine whether effects on the above genes were specific, neither the STAT-independent chemokine MCP-157 nor the housekeeping gene GAPDH showed differences in expression levels between normal subjects and MS patients (Table 1).
The present study demonstrates that PBMCs of MS patients express lower levels of the protein tyrosine phosphatase SHP-1. Furthermore, this SHP-1 deficiency corresponds to a diminished expression of promoter II transcripts. Accordingly, PBMCs of MS patients have constitutively higher activated STAT6, a transcription factor that is stringently modulated by SHP-1. In turn, several STAT6-inducible genes that may play a role in the mechanism of inflammatory demyelination are shown to be both higher in MS patients and regulated by SHP-1.
The relevance of the present findings to the pathogenetic mechanisms of MS is suggested by previous reports on signaling molecules in the CNS of MS subjects that are regulated by SHP-1. For instance, pSTAT6 was found to be highly expressed in oligodendrocytes and reactive microglia in active MS lesions compared to cells in white matter of normal subjects or patients with other neurological diseases.5 In animal models of MS, mice that lack SHP-1 show increased constitutive activation of STAT6 and heightened levels of STAT6-inducible genes both in the CNS35 and in peripheral immune cells (personal unpublished observations). Thus, the present study indicates that expression of several genes with confirmed STAT6-responsive elements may have functional significance to the pathophysiology of MS.
The chemokine CCL17/TARC is highly expressed by both T cells and monocytes and binds to the CCR4 and CCR8 chemokine receptors to attract T cells58 and monocytes59 to inflammatory sites. Here, we show that CCL17/TARC is elevated in the PBMCs of MS patients, suggesting that CCL17/TARC may act to further accentuate the infiltration of T cells and monocytes into areas of demyelination. Also, CCL11/eotaxin, which is a potent chemoattractant of monocytes to areas of inflammation,60 was elevated in PBMCs of MS patients and its expression was controlled by SHP-1.
Analysis of other STAT6-responsive genes showed that the matrix metalloproteinase ADAM8 is elevated in PBMCs of MS patients and SHP-1 controls its expression. Proteinases may be involved in the pathogenesis of MS, since these can mediate the proteolysis of extracellular matrix molecules, disrupt the blood–brain barrier, and promote lymphocyte entry into the brain parenchyma. Additionally, ADAM8 was shown to hydrolyze myelin basic protein43 and can therefore directly contribute to demyelination.
Previously, it was shown that LTα mediates inflammation and demyelination both in EAE and the cuprizone models of MS.46,61 In particular, function-blocking antibodies against LTα were shown to prevent EAE.62 Here, we show that LTα, a STAT6-inducible gene,44 is elevated in the PBMCs of MS patients compared to normal subjects. The fact that SHP-1 controls the expression of inflammatory genes like LTα illustrates the potential impact of SHP-1 deficiency in MS patients.
Arginase I is a STAT6-responsive gene that has be shown to be constitutively elevated in the CNS of SHP-1-deficient mice and plays an important role in virus-induced demyelinating disease.35 Additionally, arginase I inhibition results in attenuation of EAE onset and progression.48 Although the mechanism by which arginase I activity in either the CNS or immune systems may promote inflammatory demyelination still remains to be elucidated, its role in MS in light of the present findings should be further investigated.
Apart from STAT6, there are several other transcription factors that are elevated in MS patients and are controlled by SHP-1. In particular, tyrosine-phosphorylated STAT1 (pY-STAT1) that mediates interferon signaling was found to be elevated in MS patients.3,4 We have verified with flow cytometry that pY-STAT1 was increased in PBMCs of MS patients compared to normal subjects (personal unpublished data) and verified that STAT1-inducible genes like IP-10 are elevated in PBMCs of MS patients (Table 1) as previously shown.52-56 SHP-1 has been shown to control STAT1 activation.3,9,63 Thus, lower levels of SHP-1, as seen in PBMCs of MS patients, can provide a potential explanation for the heightened STAT1 activation seen in MS.
Furthermore, the transcription factor NF-κB mediates inflammatory cytokine signaling and is elevated in MS lesions.64 Thus, it may be important that mice genetically lacking SHP-1 show elevated NF-κB activity and increased NF-κB-inducible genes both in CNS glia and in lymphocytes.29,65 Further investigation is needed to determine whether several of the abnormalities seen in MS, including overactive inflammatory transcription factors, can be attributed to the stable deficiency of SHP-1 seen in MS patients.
SHP-1 deficiency has been associated with several immune abnormalities.16 Importantly, several signaling events of antigen receptors on lymphocytes involve tyrosine phosphorylation. Lack of the protein tyrosine phosphatase SHP-1 was shown to effectively increase the activity of the protein tyrosine kinases Src and Lyn in B cells and Lck and Fyn in T cells.66 Absence of SHP-1 in B cells results in increased antigen receptor signaling, polyclonal B cell activation, and autoimmunity.67-69 In addition, several studies have demonstrated that lack of SHP-1 in T cells leads to lower activation threshold and prolonged proliferation.70 In the light that much of the CNS damage seen in MS may be mediated through reactive T cells and increased antibody production, the SHP-1 deficiency seen in MS patients could play a role both in initiating or exacerbating immune responses that cause demyelination.
Importantly, in both the TMEV and the EAE mouse models of MS, lack of SHP-1 leads to more severe disease progression and demyelination.12,13 Furthermore, SHP-1 is highly expressed in oligodendrocytes and astrocytes, and the lack of SHP-1 leads to pronounced Jak/STAT signaling within CNS glia that might be important in oligodendrocyte pathology.10 In support for this role, motheaten mice have reduced myelin basic protein expression and dysmyelination compared to wild-type mice, suggesting that SHP-1 is important in myelin formation and/or maintenance.11 The latter may relate to the preferential expression of STAT6 in oligodendrocytes5 and to important regulatory functions for SHP-1 in these cells, especially during inflammatory events in the white matter. Therefore, it will be important to investigate whether the lower SHP-1 levels seen in the PBMCs of MS patients are also seen in CNS glia within MS lesions.
In this study, the expression levels of SHP-1 both in freshly isolated PBMCs and PBMCs cultured for 1 week in vitro were analyzed. We found that in either freshly isolated or cultured PBMCs, MS patients had a deficient expression of SHP-1 compared to normal subjects. This observation both allowed a determination of the stability of this difference and further subsequent experimental manipulation of considerably increased numbers of cultured cells than was possible using freshly isolated cells. Further, we verified that culturing PBMCs did not differentially affect the proportions of cell types between MS patients and PBMCs of normal subjects, which may have otherwise confounded our analysis. Finally, we document that pSTAT6 levels and STAT6-responsive genes are elevated in cultured PBMCs of MS patients. In keeping with stable SHP-1 differences, freshly isolated PBMCs had similar gene expression patterns of STAT6- and STAT1-responsive genes as cultured PBMCs in that genes like CCL17 and IP-10 were significantly higher in MS patients compared to normal subjects (personal unpublished data).
Two distinct promoters drive the expression of two different SHP-1 transcripts from the SHP-1 gene. Here, we demonstrated that promoter II transcripts, which are more abundantly expressed in hematopoietic cells, are selectively lower in the PBMCs of MS patients compared to normal subjects. Several studies have examined the contribution of genetics in MS71-74 and none have shown any linkage associated with the SHP-1 locus, chromosome 12p12.21 In contrast, numerous reports document the downregulation of the promoter II transcripts in leukemia/lymphoma cell lines and attribute SHP-1 deficiency to epigenetic modifications and in particular CpG methylation of SHP-1 promoter II.14,15 Interestingly, in the context of MS, either viral infections75-79 or inflammation80-83 can cause de novo methylation or hypermethylate CpG promoter sequences resulting in decreased gene expression. In accordance with these reports, preliminary data indicate that treatment of PBMCs of MS patients with the demethylating agent 2′-deoxy-5-azacytidine increase SHP-1 promoter II transcripts to normal levels (personal unpublished observations). Furthermore, a recent study reports that the HTLV-1 Tax protein profoundly suppresses the activity of the SHP-1 promoter II through recruitment of histone deacetylase-1.84 Interestingly, HTLV-1 is a lymphotropic virus that has been associated with CNS-demyelinating disease known as HAM/TSP (HTLV-1-associated myelopathy/tropical spastic paraparesis).85 The observation that lymphotropic viruses may selectively decrease SHP-1 expression lends support to the hypothesis that viral infection, possibly through epigenetic downmodulation of genes, can trigger immune-mediated demyelination.86,87 Whether CpG methylation and/or histone deacetylation lead to stable epigenetic modification of promoter II in MS patients, is responsible for stable deficiency of SHP-1, and is responsible for increased CNS inflammation in MS lesions requires further investigation.
This study was supported in part by research grants from the National Multiple Sclerosis Society (RG2569C5) to Paul T Massa, NIH grant (NS041593) to Paul T Massa, and Serono Inc. to Burk Jubelt, Cornelia Mihai, and Paul T Massa. We also thank Dr Nick J Gonchoroff for his expertise in flow cytometry/cell sorting performed in this study and Dr Gerold Feuer for assistance in design and production of lentivirus expression vectors.