Laboratory Investigation (2008) 88, 243–255 & 2008 USCAP, Inc All rights reserved 0023-6837/08 $30.00

SHP-1 deficiency and increased inflammatory expression in PBMCs of multiple sclerosis patients George P Christophi1,2, Chad A Hudson1,2, Ross C Gruber1, Christoforos P Christophi1, Cornelia Mihai1, Luis J Mejico1, Burk Jubelt1,2 and Paul T Massa1,2

Recent studies in mice have demonstrated that the protein tyrosine phosphatase SHP-1 is a crucial negative regulator of 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 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. Laboratory Investigation (2008) 88, 243–255; doi:10.1038/labinvest.3700720; published online 21 January 2008

KEYWORDS: autoimmunity; demyelination; inflammation; STAT6

Multiple sclerosis (MS) is an inflammatory demyelinating levels of SHP-1 have also been implicated in autoimmune- disease of the central nervous system (CNS) that remains a mediated demyelination in rodents12,13 and in lymphopro- major cause of disability.1,2 Several studies demonstrate that liferative diseases in humans.14–17 Moreover, low SHP-1 levels MS lesions and PBMCs from MS patients contain activated have been associated with excessive tyrosine phosphorylation transcription factors like NF-kB, STAT1, STAT3, and of STAT1 following IFN-b treatment in MS patients.18 These STAT6,3–5 which can lead to enhanced signaling of studies suggest that SHP-1 plays multiple roles in leukocytes, inflammatory stimuli. These findings suggest that regulation including controlling activation state relevant to mechanisms of inflammatory signaling may be altered in MS and may be of inflammatory demyelination. responsible for inflammatory demyelination. Two distinct promoters are responsible for expression of SHP-1 is a protein tyrosine phosphatase with two SH2 each of the two known SHP-1 transcripts produced from the domains and acts as a negative regulator of cytokine signaling SHP-1 gene.19 These distinct transcripts in turn encode two via STAT1, STAT3, and STAT6.6–9 We have shown that slightly different SHP-1 isoforms, which have the same mice genetically lacking in SHP-1 (motheaten mice) display catalytic activity.20 It was previously shown that the two SHP- myelin deficiency, which may be mediated by increased 1 transcripts are differentially expressed in human tissues inflammatory mediators in the CNS.10,11 Furthermore, when and cell lines.21,22 Promoter I transcripts are highly expressed motheaten mice are infected with Theiler’s murine in epithelial cells, while promoter II transcripts are encephalomyelitis virus (TMEV), the CNS displays sub- more abundant in hematopoietic cells.22,23 Several reports stantially more virus-induced inflammatory demyelination show differential regulation of the SHP-1 gene promo- than wild-type mice.12 In agreement with these studies, lower ters by distinct transcription factors.19,24,25 Importantly,

1Department of Neurology, SUNY Upstate Medical University, Syracuse, NY, USA and 2Department of Microbiology & Immunology, SUNY Upstate Medical University, Syracuse, NY, USA Correspondence: Dr PT Massa, PhD, Department of Neurology, SUNY Upstate Medical University, State University of New York, 750 East Adams Street, Syracuse, NY 13210, USA. E-mail: [email protected] Received 12 October 2007; revised 2 December 2007; accepted 5 December 2007 www.laboratoryinvestigation.org | Laboratory Investigation | Volume 88 March 2008 243 SHP-1 deficiency in PBMCs of MS patients GP Christophi et al

promoter-specific regulation of SHP-1 expression has been Cytokine and siRNA Treatment associated with human disease.14,23,26 PBMCs of MS patients and normal subjects were cultured for In this study, we show that the levels of SHP-1 are lower in 1 week and were then treated with either 10 ng/ml of IL-4 for PBMCs from MS patients compared to normal subjects. 24 h or received medium alone (R&D Systems). A portion of Corresponding to this deficiency, we have shown that STAT6 PBMCs were transfected with siRNA against human SHP-1 phosphorylation and STAT6-responsive genes are con- or scramble siRNA (Dharmacon, Chicago, IL, USA) at a stitutively higher in PBMCs of MS patients compared to concentration of 1 mg/106 cells. The transfection reagent those of normal subjects. Additionally, we delineate the (Dharmafect 4; Dharmacon) was used as specified by the contribution of two promoter-specific transcripts in SHP-1 manufacturer. Cells were incubated in the transfection deficiency, which points to a specific decrease in promoter II medium for 24 h, after which the medium was replaced with activity in PBMCs of MS patients. Taken together, we complete growth medium for another 48 h before IL-4 propose the potential involvement of SHP-1 promoter II treatment. The effectiveness of the SHP-1 siRNA to lower dysregulation in the pathogenesis of MS. SHP-1 expression was evaluated by both real-time RT-PCR and flow cytometry. MATERIALS AND METHODS Real-Time RT-PCR Patient Selection Total RNA was isolated using RNA STAT-60. RNA was Patients were clinically diagnosed as either having active re- quantified spectrophotometrically and 0.5 mg of total RNA lapsing–remitting (RR) MS, active secondary progressive was converted into cDNA. Briefly, total RNA and random (SP) MS, or inactive RR (Inc) MS.27 Active RR MS was de- primers (Invitrogen, Carlsbad, CA, USA) were incubated at fined as a moderate to severe exacerbation within 6 months 721C for 10 min. Reverse transcription was performed using prior to entry and SP MS was defined as a continuous pro- the Superscript II RT enzyme (Invitrogen) following the gression over the preceding 6 months of the study. Patients specification of the manufacturer. cDNA was diluted to 200 ml who had not received any disease-modifying treatment like with water and 4 ml was used for quantitative real-time PCR IFN-b, glatiramir acetate, or other immunosuppressive using SYBR Green (Abgene, Epson, UK). The PCR agents at least two months prior to donating blood were parameters were 15 min for 951C and 35 cycles of 951C for selected. In this study, PBMCs were isolated from 23 MS 15 s and 601C for 1 min in ABI prism 700 (Applied Biosys- patients, and 18 control subjects. The Institutional Review tems, Foster city, CA, USA). The primers were used at 10 nM. Board of SUNY Upstate University approved all studies, and Serial dilutions of cDNA containing a known copy number of both patients and controls granted informed consent before each gene were used in each quantitative PCR run to generate providing blood. a standard curve relating copy number with threshold am- plification cycle.28 Gene expression levels were calculated PBMC Isolation and Expansion during the logarithmic amplification phase by determining Patients and normal subjects donated 50 ml of blood col- the initial mRNA copy number using the standard curve. lected in heparinized tubes. Blood was diluted 1:1 with HBSS Amplification of each gene-specific fragment was confirmed and overlaid onto lymphocyte separation medium (Cellgro, both by examination of melting peaks and by agarose gel Herndon, VA, USA). After centrifugation, 10 ml of the in- electrophoresis. The following primer pairs were used in this terface containing the PBMCs was collected and diluted to study: SHP-1 (BC002523) forward-TGGCGTGGCAGGA 50 ml with HBSS. After another two washes with HBSS, a GAACAG and reverse-GCAGTTGGTCACAGAGTAGGGC, sample of the freshly isolated cells was resuspended in STAT- SHP-1 (I) (NM080548) forward-TGGCTTCCCCCTCCCTA 60 (Tel-Test, Friendswood, TX, USA) for RNA analysis. The CAG and reverse-CCCTGGTTCTTGCGACTGG, SHP-1 (II) rest of the cells were cultured for a week in RPMI media with (NM002831) forward-ATCTGAGGCTTAGTCCCTGAGC and 20 U/ml IL-2 (R&D Systems, Minneapolis, MN, USA) and reverse-CTGAGGTCTCGGTGAAACCAC, IL-4 (NM000589) 10% fetal bovine serum. After 1 week, cells were either forward-TCCCAACTGCTTCCCCC and reverse-TCTTCTGC resuspended in STAT-60 or in RIPA buffer (50 mM Tris-HCl, TCTGTGAGGCTG, IL-4ra (IL-4ra) forward-CGTGTATCCCT pH 7.4, 1% NP-40, 0.25% sodium deoxycholate, 150 mM GAGAACAACG and reverse-CGTGTATCCCTGAGAACAA NaCl, 1 mM EDTA, 1 mM PMSF, 1 mg/ml aprotinin, 1 mg/ml CG, CCL17 (NM002987) forward-CGAGGGACCAATGTG leupeptin, 1 mg/ml pepstatin, and 1 mM activated Na3VO4) GGC and reverse-GGGTGAGGAGGCTTCAAGACC, CCL11 for protein analysis or fixed for intracellular flow analysis. In (NM002986) forward-CACTTCTGTGGCTGCTGCTC and all experiments, PBMCs, either freshly isolated or cultured reverse-GCTTTCTGGGGACATTTGC, CCR4 (NM005508) for a week in the presence of IL-2, were used with exception forward-GGTCCTGTTCAAATACAAGCG and reverse-AAAC to lentiviral vector transductions. In the lentiviral vector CCACTGGTCTGCTGC, CCR8 (NM005201) forward-CAG transduction experiments, cells were incubated for 25 days to TGTGACAACAGTGACCGAC and reverse-GCAATAAAAGA allow the vector to integrate into the host DNA and expand CAGCAAGGAGC, ADAM8 (NM001109) forward-ATCCC the number of transduced cells. GAGAGACCCGCTAC and reverse-TGATTCACCACCTCCA

244 Laboratory Investigation | Volume 88 March 2008 | www.laboratoryinvestigation.org SHP-1 deficiency in PBMCs of MS patients GP Christophi et al

GCAC, lymphotoxin-a (LTa) (NM000595) forward-CACCT Intracellular SHP-1 flow cytometric analysis CATTGGAGACCCCAG and reverse-TGTTGCTCAAGGAGA Also, the levels of SHP-1 were concurrently analyzed with AACCATC, Arginase I (NM000045) forward-GACCTGCC pSTAT6. Fixed and permeabilized cells were incubated CTTTGCTGACATC and reverse-TTGACTTCTGCCACCTTG overnight at 41C with either 1mg of rabbit anti-SHP-1 CC, IFN-g (NM000619) forward-TGCAGGTCATTCAGATG (Upstate), rabbit anti-total STAT6 (phosphorylated plus TAG and reverse-AGCCATCACTTGGATGAGTT, MCP-1 unphosphorylated) (Santa Cruz Biotech., Inc., Santa Cruz, (NM002982) forward-GCTCATAGCAGCCACCTTC and re- CA, USA) antibodies, or rabbit polyclonal IgGs for isotype verse-GCTTCTTTGGGACACTTGC, GAPDH (NM002046) control. The cells were incubated for 3 h in 1 mg of goat anti- forward-ACCACCATGGAGAAGGC and reverse-GGCATG rabbit secondary antibody conjugated to PE (Invitrogen). GACTGTGGTCATGA. Cells were analyzed on an LSRII analyzer (Becton Dickinson) and the mean florescence intensity (MFI) was recorded. To Western Blotting quantify the levels of STAT6-responsive genes by flow Whole-cell extracts were prepared as previously described.9,29 cytometry, anti-human ADAM8-PE IgG (R&D Systems; Cat a Briefly, PBMCs were rinsed with PBS, and then lysed with no. 143338) was used to stain ADAM8 and anti-human LT a RIPA buffer. Protein (100 mg) per lane was electrophoresed (R&D Systems; Cat no. 5808) was used to stain LT (R&D through a 7.5% polyacrylamide-resolving gel and electro- Systems). Fixed and permeabilized cells were stained for blotted to a polyvinylidene difluoride membrane (Millipore 45 min at room temperature and analyzed. To quantify the Corporation, Burlington, MA, USA). Membranes were levels of CCL17/TARC, cells were treated with 5 mg/ml of blocked with 5% non-fat dry milk for 1 h, and then Brefeldin-A prior to fixation to block secretion incubated with anti-SHP-1 (Upstate, Lake Placid, NY) anti- and after 12 h cells were fixed and stained with anti-human bodies followed by horseradish peroxidase-conjugated rabbit CCL17/TARC (R&D Systems; Cat no. 54015) for 45 min and IgG antibody (Dako Corporation, Carpinteria, CA, USA). then analyzed. Enhanced chemiluminescence (Amersham Life Sciences Inc., Cleveland, OH, USA) was used to visualize reactive protein bands on X-ray film. Transduction of SHP-1-Expresssing Lentiviral Vector The lentiviral vector prepared as described30 was used to stably infect PBMCs of MS patients. The vector carried the Sorting and Flow Cytometry human SHP-1 coding sequence (NCBI no. BC002523), Percentage analysis of PBMC cell types by flow cytometry allowing the bicistronic expression of green fluorescent pro- PBMCs were sorted into the different cell populations tein (GFP) and SHP-1 in the transduced cells. PBMCs were (T cells, B cells, and ) to examine the expression of incubated in RPMI medium and infected at an approximate 6 SHP-1 in individual populations. Cells (4 Â 10 /200 ml) were MOI of 0.1 for 16 h. The virus was then removed and the cells incubated on ice for 30 min with 20 ml of CD3-FITC (349201) were cultured for 25 days both to allow stable integration of to stain T cells, 20 ml of CD19-PE (340720) to stain B cells, the lentiviral DNA in the host DNA and expand the number and 10 ml of CD14-APC (340684) to stain for monocytes of transduced cells. Transduced PBMCs of MS patients and (Becton Dickinson Immunocytometry Systems, Mountain control PBMCs of normal subjects’ cells were incubated in View, CA, USA). Cells were washed and resuspended in 1mL IL-2-containing medium to maintain cell variability for the of Hank’s solution and were analyzed by fluorescence- culture period. Cell viability was assessed by cell morphology activated cell sorter (FACS Vantage S/E; Becton Dickinson). and Trypan blue exclusion. GFP expression was analyzed with The percentage of each cell population was determined and flow cytometry and GFP-expressing cells were sorted using four aliquots labeled as T cells, B cells, monocytes, and FACS (Becton Dickinson). The GFP-positive and -negative negative cells were collected. The cells of each aliquot were cells were analyzed for the expression of SHP-1 and pSTAT6 lysed in the RNA isolation reagent. using flow cytometry and the expression of several STAT6- responsive genes using real-time RT-PCR. Intracellular pSTAT6 flow cytometric analysis PBMCs were treated for 15 min with 10ng/ml IL-4. Cells (106/1 ml) received 100 ml of 16% stock formaldehyde for Arginase Activity Assay fixation and then incubated in 90% methanol at 41C for half Arginase enzymatic activity was measured as previously an hour to permeabilize cells for intracellular staining. Cells described.31 Briefly, PBMCs were lysed in RIPA buffer and were washed twice with the staining media containing 0.5% incubated in 10 mM MnCl2 and 0.5 M L-arginine at 371C for BSA and 0.02% sodium azide in PBS. Cells were resuspended 75 min. After the assay was stopped by addition of H3PO4, in a 100 ml of staining media and incubated with 20 ml either 1-phenyl-1,2-propanedione-2-oxime (Sigma, St Louis, MO, phosphoSTAT6 (PY-641) with Alexa-488 (612600), CD3- USA) was added and the samples were incubated at 1001C for FITC (349201), or mouse IgG with Allexa-488 isotype 60 min. Urea production by arginase was measured by optical control (557721) (Becton Dickinson). density at 540 nm.

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Statistical Analysis the levels of SHP-1 mRNA did not differ between PBMCs of Histograms or tables contain statistical means with the MS patients who were clinically diagnosed with either RR MS standard error values. The number of samples used in each or SP MS, indicating that depressed SHP-1 was a general assay is indicated in the figure legends. The P-values were marker across these clinical subclassifications (Figure 4). In generated using the unpaired Student’s t-test and a P-value of accordance with mRNA levels, PBMCs of MS patients had less than 0.05 was chosen to indicate statistical significance significantly lower levels of SHP-1 protein than those of between two sample means. 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 RESULTS SHP-1 Protein and mRNA are lower in MS Patients cytometry (Figures 2c and d). Constitutive levels of SHP-1 in To examine SHP-1 expression in MS patients, isolated PBMCs of MS patients were half the levels of SHP-1 in cells PBMCs were prepared from whole heparinized human blood. of normal subjects. Analysis of protein (Figure 2a) and The levels of SHP-1 mRNA were quantified using primers mRNA (Figure 3b) of housekeeping genes showed no flanking the coding region of the gene common to both differences between MS and normal subject PBMCs. transcripts. SHP-1 mRNA levels in freshly isolated PBMCs were significantly lower in MS patients compared to normal SHP-1 Promoter II Transcripts are Selectively Lower in MS Patients subjects (Figure 1a). To examine whether the SHP-1 deficiency in PBMCs of MS patients was stable, PBMCs were To determine the individual contribution of each of two expanded for 1 week in vitro. Similarly, SHP-1 mRNA was known transcripts on the expression levels of SHP-1 in significantly lower in cultured PBMCs of MS patients PBMCs, promoter I and II transcript copy numbers compared to those of normal subjects (Figure 3a). Moreover, 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

Figure 1 SHP-1 mRNA levels in freshly isolated PBMCs of MS patients and Figure 2 SHP-1 protein expression in cultured PBMCs of MS patients normal subjects. The absolute mRNA transcript copy numbers per 10 ng of compared to normal subjects. (a) Western blot analysis of SHP-1 and actin total RNA were quantified using real-time RT-PCR in MS patients (n ¼ 23) in PBMCs of MS patients compared to normal subjects. (b) The Western and normal subjects (n ¼ 18). (a) Using primers in the mid-region of the immunoblot protein bands of MS patients (n ¼ 7) and control subjects SHP-1 gene that would amplify both SHP-1 transcripts, the levels of the (n ¼ 7) were analyzed with Scion Image software program and the average common SHP-1 transcripts were measured (*significance of Po0.05). levels of SHP-1 relative to actin are shown (*significance of Po0.05). (b) The mRNA levels of the housekeeping gene GAPDH were quantified (c) SHP-1 protein levels of MS patients (n ¼ 12) and normal subjects (n ¼ 12) between MS patients and normal subjects. (c) Sequence-specific primers were measured with intracellular flow cytometry. The histograms are to the SHP-1 gene were used to quantify the expression of the SHP-1 labeled as follows: filled histogram for isotype control, solid line for normal promoter I transcripts. (d) Similarly, sequence-specific primers allowed the subject, and dashed line for MS patient. (d) SHP-1 expression measured by quantification of the SHP-1 promoter II transcripts. flow cytometry was quantified using the mean florescence intensity (MFI).

246 Laboratory Investigation | Volume 88 March 2008 | www.laboratoryinvestigation.org SHP-1 deficiency in PBMCs of MS patients GP Christophi et al

patients compared to controls (Figures 1c, 3b and 3c). On the negative cells probably represent natural killer cells based on other hand, constitutive levels of SHP-1 promoter II tran- their scatter properties.32 scripts were two-fold lower in PBMCs of MS patients The levels of SHP-1 mRNA in the sorted populations were compared to controls (Figures 1d and 3d). Additionally, the quantified and T cells had similar levels of SHP-1 as in levels of SHP-1 transcripts I and II did not differ between unsorted PBMCs. Importantly, T-cell populations of MS PBMCs of MS patients with different clinical subclassifica- patients had significantly lower levels of SHP-1 compared to tions (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.

Expression of SHP-1 in Specific PBMC Types 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

Figure 4 SHP-1 mRNA levels in freshly isolated (a–d) or cultured (e–h) PBMCs of patients diagnosed with different clinical subclassifications of MS. The absolute specific mRNA transcript numbers per 10 ng of total RNA were quantified with real-time RT-PCR in patients with active Figure 3 SHP-1 mRNA levels in cultured PBMCs of MS patients compared relapsing–remitting (RR) MS (n ¼ 10), active secondary progressive (SP) MS to normal subjects. The absolute mRNA transcript copy numbers per 10 ng (n ¼ 6), or inactive RR (Inc) MS (n ¼ 7). The mRNA levels of the (a) common of total RNA were quantified using real-time RT-PCR in MS patients (n ¼ 23) SHP-1 transcript, (b) housekeeping gene GAPDH, (c) SHP-1 promoter I and normal subjects (n ¼ 18). (a) The levels of the common SHP-1 transcripts, and (d) SHP-1 promoter II transcripts were quantified in freshly transcript, (b) the housekeeping gene GAPDH, (c) the SHP-1 promoter I isolated PBMCs of MS patients. Similarly, the mRNA levels of (e) the transcripts, and (d) the SHP-1 promoter II transcripts were quantified with common SHP-1 transcript, (f) the housekeeping gene GAPDH, (g) SHP-1 real-time RT-PCR in cultured PBMCs of MS patients and normal subjects promoter I transcripts, and (h) SHP-1 promoter II transcripts were quantified (*significance of Po0.05). in PBMCs of MS patients that were cultured for 1 week.

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normal subjects (Figure 5a), similar to that seen in the SHP-1 modulates STAT activation, translocation into the unsorted population. Additionally, B cells and unstained cells nucleus, and transcriptional activity on responsive genes. of MS patients had significantly lower levels of SHP-1 mRNA Because several studies showed that SHP-1 profoundly compared to the same cell types of normal subjects. As modulates IL-4R/STAT6 signaling molecules,33–36 STAT6 was expected, SHP-1 promoter II transcripts were primarily chosen for further analysis of possible altered function of responsible for the lower levels of SHP-1 in MS patients in SHP-1 in MS PBMC. For analysis, we measured the levels the sorted populations with more dramatic differences seen of total STAT6 (unphosphorylated plus phosphorylated, in B cells and negative cells (six-fold lower in MS compared tSTAT6) and tyrosine phosphorylated STAT6 (pSTAT6) in to normal subjects). T cells had a three-fold lower level of PBMCs using intracellular flow cytometry. As expected, SHP-1 promoter II transcripts in MS patients compared to PBMCs of MS patients had significantly elevated constitutive normal subjects (Figure 5d). In contrast, promoter I and phosphorylation of STAT6 compared to PBMCs of normal GAPDH transcripts were not significantly different in either subjects (Figure 6c). In contrast, there were no differences in unsorted PBMCs or sorted cell populations of MS patients the expression levels of total STAT6 protein (Figures 6a and b), compared to normal subjects (Figures 5b and c). These data verifying that the differences observed in pSTAT6 levels indicate that MS patients and normal subjects contain similar are attributed to the relative phosphorylation state of STAT6. proportions of leukocytes; however, the levels of SHP-1 Staining the same cells for SHP-1 revealed a reciprocal expression in specific mononuclear cell populations are signi- expression pattern of SHP-1 and pSTAT6, such that lower ficantly lower in MS patients compared to normal subjects. constitutive levels of SHP-1 in MS patients correlated with

Analysis of STAT6 Activation in MS PBMCs 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,

Figure 6 Total STAT6 and phosphorylated STAT6 (pSTAT6) levels in cultured PBMCs of MS patients compared to normal subjects. PBMCs from MS patients and normal controls were fixed, permeabilized, and stained with antibodies against either total STAT6 (unphosphorylated plus phosphorylated STAT6) or pSTAT6. The histogram overlays are labeled as follows: filled histogram for isotype control, solid line for normal subject, Figure 5 Quantification of the SHP-1 mRNA in different cell types of MS and dashed line for MS patient. (a) The representative levels of total STAT6 patients and normal subjects. The absolute mRNA transcript copy numbers in MS patients and normal subjects are shown after quantification with per 10 ng of total RNA were quantified with real-time RT-PCR in MS patients intracellular flow cytometry. (b) The levels of tSTAT6 were quantified based (n ¼ 23) and normal subjects (n ¼ 18). Cultured PBMCs were sorted into on the relative MFI in PBMCs of MS patients (n ¼ 9) and normal subjects T cells, B cells, monocytes, and negatively stained cells. Using sequence- (n ¼ 8) before and after treatment with IL-4. (c) The representative levels of specific primers the levels of (a) the common SHP-1 transcripts, (b) GAPDH pSTAT6 in MS patients and normal subjects are shown. (d) The levels of mRNA levels, (c) SHP-1 promoter I transcripts, and (d) SHP-1 promoter II pSTAT6 were quantified based on the relative MFI in PBMCs of MS patients transcripts were quantified using real-time RT-PCR in the different cell types (n ¼ 16) and normal subjects (n ¼ 13) before and after treatment with IL-4 of both MS patients and normal subjects (*significance of Po0.05). (*significance of Po0.05).

248 Laboratory Investigation | Volume 88 March 2008 | www.laboratoryinvestigation.org SHP-1 deficiency in PBMCs of MS patients GP Christophi et al

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 heigh- tened 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.

Heightened Expression of STAT6-Inducible Genes in MS 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 and their receptors were of particular interest because these mediate trafficking, maturation, and attraction of T cells and to areas of CNS inflammation.37 Figure 7 pSTAT6 levels in PBMCs after siRNA treatment against SHP-1. 38,39 Cultured PBMCs from MS patients and normal subjects were stained with CCL17/TARC was highly expressed in PBMCs and was antibodies against the phosphorylated form of STAT6 (pSTAT6) or against more than five-fold higher in PBMCs of MS patients SHP-1. The histogram overlays are labeled as follows: filled histogram for compared to normal subjects measured by both real-time isotype control, solid line for normal subject, dashed line for MS patient, RT-PCR and flow cytometry (Table 1 and Figure 8a). Treat- and dotted line for PBMCs treated with siRNA against SHP-1. (a) SHP-1 and ment with siRNA to SHP-1 tripled the constitutive amount of (b) pSTAT6 expression in PBMCs of normal subjects, MS patients, and PBMCs treated with siRNA against SHP-1. (c) PBMCs were pretreated with CCL17/TARC in PBMCs of normal subjects, indicating that scramble (nonspecific) siRNA and the levels of pSTAT6 were measured in SHP-1 regulated CCL17/TARC expression. Treatment with MS patients (n ¼ 6) and normal subjects (n ¼ 6) before and after IL-4 IL-4 for 24 h induced significantly higher levels of CCL17/ treatment. (d) PBMCs were treated with siRNA against SHP-1 and the levels TARC in PBMCs of MS patients compared to PBMCs of of pSTAT6 were measured both in MS patients (n ¼ 6) and normal subjects normal subjects. Moreover, SHP-1 siRNA-treated PBMCs of (n ¼ 6) before and after IL-4 treatment (*significance of Po0.05). 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) higher pSTAT6 levels compared to PBMCs of normal subjects decreased CCL17 expression by 15-fold (Table 2). (Figure 7). Furthermore, PBMCs were treated with IL-4 to Another STAT6-responsive chemokine, CCL11/eotaxin,40 determine possible differences in STAT6 activation following was significantly higher in MS patient PBMCs and normal engagement of the IL-4 receptor. Treatment with IL-4 caused subject PBMCs treated with SHP-1 siRNA than in PBMCs of higher phosphorylation of STAT6 in PBMCs of MS patients normal subjects (Table 1). When the mRNA transcript levels compared to normal subjects (Figure 6d). of two of the chemokine receptors CCR4 and CCR8 that To directly determine whether a specific deficiency in bind the CCL17/TARC and CCL11/eotaxin ligands were SHP-1 could mediate increased activation of STAT6 in measured,14 CCR8 mRNA was not different between the human PBMCs, PBMCs of patients and controls were treated PBMCs of MS patients and normal subjects and its levels did with siRNA against SHP-1 and levels of pSTAT6 were not change with either siRNA or IL-4 treatment. However, measured. The efficiency of the siRNA against SHP-1 was CCR4 mRNA transcripts were higher in PBMCs of MS verified by both real-time RT-PCR and intracellular flow patients compared to PBMCs of normal subjects, yet its cytometry (Figure 7a and Table 1). SHP-1 siRNA treatment expression levels did not change with either siRNA or IL-4 effectively increased the levels of pSTAT6 in PBMCs of treatment. normal subjects to levels equal that of MS patients. Also, Other STAT6-responsive genes, including ADAM8, LTa, SHP-1 siRNA significantly increased inducible pSTAT6 levels and arginase I were analyzed and both mRNA and protein following IL-4 treatment (Figure 7d). These results indicate expression were elevated in PBMCs of MS patients compared that lower SHP-1 in PBMCs of MS patients could be directly to normal subjects (Table 1 and Figure 8). ADAM8 is a responsible for higher constitutive levels of pSTAT6 and STAT6-responsive41,42 disintegrin matrix metalloproteinase heightened IL-4-induced signaling to STAT6. that may be involved in demyelination.43 LTa is also

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Table 1 Gene profile of PBMCs of MS patients and normal subjects

Gene No treatment IL-4 treatment

Normal subjects MS patients SHP-1 siRNA Normal subjects MS patients SHP-1 SiRNA

w w SHP-1 1.0±0.1 0.4±0.10* 0.1±0.1* 1.1±0.1 0.3±0.2 0.1±0.1 w w IL-4 1.0±0.1 3.2±0.8* 2.8±0.6* 1.8±0.2 4.1±0.5 3.9±1.0 IL-4-ra 1.0±0.1 0.8±0.2 1.3±0.3 1.2±0.1 1.01±0.2 1.1±0.4 w w CCL17 1.0±0.2 5.9±1.4* 2.8±0.5* 2.8±0.1 21.5±6.3 6.9±1.3 w w CCL11 1.0±0.3 2.2±0.4* 7.2±2.7* 2.9±0.7 6.4±2.1 16.8±7.2 w CCR4 1.0±0.2 2.4±0.6* 1.3±0.4 1.6±0.2 2.8±0.5 1.4±0.3 CCR8 1.0±0.2 1.5±0.3 1.3±0.5 1.2±0.1 1.8±0.2 1.5±0.2 w w ADAM8 1.0±0.3 3.4±0.4* 2.4±1.2* 2.6±0.6 4.8±0.6 6.4±3.0 w LTa 1.0±0.2 3.7±1.2* 2.8±1.0* 1.8±0.2 6.2±1.6 3.1±1.0 w w Arginase I 1.0±0.2 2.4±0.6* 5.1±2.1* 2.8±0.3 5.2±1.6 10.1±5.7 IFN-g 1.0±0.2 1.2±0.4 1.3±0.4 0.8±0.2 0.9±0.2 1.1±0.2 w IP-10 1.0±0.3 3.0±0.6* 1.9±0.4 0.7±0.4 2.9±0.5 1.8±0.5 CXCR3 1.0±0.2 0.7±0.2 0.8+0.4 1.1+0.3 0.9+0.4 0.7+0.5 MCP-1 1.0±0.3 0.8±0.2 0.7±0.3 0.7±0.3 1.0±0.5 0.6±0.2 GAPDH 1.0±0.2 0.9±0.2 0.8±0.2 0.8±0.2 0.9±0.1 0.9±0.1

*Significance of Po0.05 compared to untreated normal subjects. w Significance of Po0.05 compared to IL-4-treated normal subjects. The average is shown followed by the standard error after the ± sign. A number of genes, including several STAT6 inducible, were quantified in PBMCs of normal subjects (n ¼ 18), PBMCs of MS patients (n ¼ 23), or PBMCs of normal subjects treated with siRNA against SHP-1 (n ¼ 6) before and after 24 h treatment with IL-4. Real-time RT-PCR was used to measure the mRNA levels of SHP-1 common transcript, IL-4, IL-4 receptor a, Arginase I, the protease ADAM8, the chemokines CCL17/TARC and CCL11/eotaxin, the chemokine receptors CCR4 and CCR8, lymphotoxin-a, interferon gamma, the chemokine IP-10, the CXCR3, the chemokine CCL2/MCP-1, and the housekeeping GAPDH. The actual transcript copy number of each gene is reported in relation to the untreated normal subjects, which is designated as 1.

STAT6-inducible in human leukocytes44 and is a member of levels of the STAT1-responsive chemokine IP-10 were ele- the TNF superfamily that has been shown to mediate in- vated in PBMCs of MS patients compared to normal subjects flammation and demyelination in mouse models of MS.45,46 in accord with several studies.52–56 Furthermore, IP-10 Arginase I is expressed both in the CNS and the immune mRNA showed a modest but significant increase in PBMCs system and is highly inducible by IL-4 through STAT6.47 of normal subjects following treatment with SHP-1 siRNA, Apart from participating in the urea cycle, arginase I also but was not inducible by IL-4 treatment. To determine plays an important role in both virus-induced35 and auto- whether effects on the above genes were specific, neither the immune-mediated demyelination.48 Interestingly, ADAM8, STAT-independent chemokine MCP-157 nor the house- LTa, and arginase I, showed higher inducible mRNA and keeping gene GAPDH showed differences in expression levels protein levels in PBMCs of MS patients compared to normal between normal subjects and MS patients (Table 1). subjects following IL-4 treatment consistent with the fact that they are STAT6 inducible (Table 1 and Figure 8). Further- DISCUSSION more, treatment of normal PBMCs with siRNA against SHP- The present study demonstrates that PBMCs of MS patients 1 significantly raised the levels of ADAM8, LTa, and arginase I express lower levels of the protein tyrosine phosphatase (Table 1). Additionally, transduction of the SHP-1-expressing SHP-1. Furthermore, this SHP-1 deficiency corresponds to a lentiviral vector resulted in a decrease of ADAM8, LTa, and diminished expression of promoter II transcripts. Accord- arginase I expression in PBMCs of MS patients (Table 2). ingly, PBMCs of MS patients have constitutively higher Other genes not associated with STAT6 activation were activated STAT6, a transcription factor that is stringently also measured (Tables 1 and 2). We did not observe any modulated by SHP-1. In turn, several STAT6-inducible genes differences in the expression of the cytokine IFN-g,49 or the that may play a role in the mechanism of inflammatory chemokine receptor CXCR350,51 between PBMCs of MS demyelination are shown to be both higher in MS patients patients and normal subjects. In contrast, the expression and regulated by SHP-1.

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Figure 9 PBMCs of MS patients were transduced with a lentiviral vector co-expressing GFP and SHP-1. Cells were sorted into two populations, GFP negative (solid line) or GFP positive (dashed line). The sorted aliquots were then analyzed for the expression of (a) GFP, (b) SHP-1 and (c) pSTAT6 using Figure 8 Protein levels of STAT6-inducible genes in MS patients were flow cytometry. verified using flow cytometry and enzymatic assays. (a) CCL17/TARC, (b) ADAM8 and (c) lymphotoxin-a protein levels were measured using flow cytometry in cultured PBMCs of MS patients (n ¼ 7) and normal subjects (n ¼ 6) before and after treatment with IL-4. Protein expression was CCL11/eotaxin, which is a potent chemoattractant of quantified using the mean florescence intensity (MFI) and are reported 60 relative to the untreated control samples. (d) Arginase activity was monocytes to areas of inflammation, was elevated in measured in PBMCs of MS patients and normal subjects and is shown PBMCs of MS patients and its expression was controlled by relative to the untreated control samples (*significance of Po0.05). SHP-1. Analysis of other STAT6-responsive genes showed that the matrix metalloproteinase ADAM8 is elevated in PBMCs of The relevance of the present findings to the pathogenetic MS patients and SHP-1 controls its expression. Proteinases mechanisms of MS is suggested by previous reports on may be involved in the pathogenesis of MS, since these can signaling molecules in the CNS of MS subjects that are mediate the proteolysis of extracellular matrix molecules, regulated by SHP-1. For instance, pSTAT6 was found to be disrupt the blood–brain barrier, and promote lymphocyte highly expressed in oligodendrocytes and reactive microglia entry into the brain parenchyma. Additionally, ADAM8 was in active MS lesions compared to cells in white matter of shown to hydrolyze myelin basic protein43 and can therefore normal subjects or patients with other neurological diseases.5 directly contribute to demyelination. In animal models of MS, mice that lack SHP-1 show Previously, it was shown that LTa mediates inflammation increased constitutive activation of STAT6 and heightened and demyelination both in EAE and the cuprizone models of levels of STAT6-inducible genes both in the CNS35 and in MS.46,61 In particular, function-blocking antibodies against peripheral immune cells (personal unpublished observa- LTa were shown to prevent EAE.62 Here, we show that LTa,a tions). Thus, the present study indicates that expression STAT6-inducible gene,44 is elevated in the PBMCs of MS of several genes with confirmed STAT6-responsive patients compared to normal subjects. The fact that SHP-1 elements may have functional significance to the patho- controls the expression of inflammatory genes like LTa physiology of MS. illustrates the potential impact of SHP-1 deficiency in MS The chemokine CCL17/TARC is highly expressed by both patients. T cells and monocytes and binds to the CCR4 and CCR8 Arginase I is a STAT6-responsive gene that has be shown to chemokine receptors to attract T cells58 and monocytes59 to be constitutively elevated in the CNS of SHP-1-deficient mice inflammatory sites. Here, we show that CCL17/TARC is and plays an important role in virus-induced demyelinating elevated in the PBMCs of MS patients, suggesting that disease.35 Additionally, arginase I inhibition results in CCL17/TARC may act to further accentuate the infiltration of attenuation of EAE onset and progression.48 Although the T cells and monocytes into areas of demyelination. Also, mechanism by which arginase I activity in either the CNS or

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Table 2 Regulation of STAT6-inducible genes in PBMCs of MS autoimmunity.67–69 In addition, several studies have patients after overexpressing SHP-1 demonstrated that lack of SHP-1 in T cells leads to lower 70 Gene Normal MS patients GFP MS patients activation threshold and prolonged proliferation. In the subjects negative GFP positive light that much of the CNS damage seen in MS may be mediated through reactive T cells and increased antibody CCL17 1.0±0.2 4.5±1.4* 0.3±0.1 production, the SHP-1 deficiency seen in MS patients could ADAM8 1.0±0.3 3.0±0.9* 1.2±0.5 play a role both in initiating or exacerbating immune responses that cause demyelination. LTa 1.0±0.2 4.3±1.2* 1.4±0.4 Importantly, in both the TMEV and the EAE mouse ± ± ± Arginase I 1.0 0.2 2.7 0.7* 0.5 0.2 models of MS, lack of SHP-1 leads to more severe disease IFN-g 1.0±0.2 1.1±0.3 1.3±0.4 progression and demyelination.12,13 Furthermore, SHP-1 is GAPDH 1.0±0.2 0.9+0.4 1.1+0.3 highly expressed in oligodendrocytes and astrocytes, and the lack of SHP-1 leads to pronounced Jak/STAT signaling within *Significance of Po0.05 compared to untreated normal subjects. CNS glia that might be important in oligodendrocyte The average is shown followed by the standard error after the ± sign. pathology.10 In support for this role, motheaten mice have PBMCs of MS patients were transduced with a lentiviral vector co-expressing reduced myelin basic protein expression and dysmyelination GFP and SHP-1 and cells were sorted into two populations. Then, mRNA was isolated from the GFP-negative and -positive cell population and the expres- compared to wild-type mice, suggesting that SHP-1 is 11 sion of several STAT6-inducible genes and IFN-g and GAPDH was analyzed important in myelin formation and/or maintenance. The using real-time RT-PCR. The relative gene expression levels of GFP-positive and latter may relate to the preferential expression of STAT6 in -negative transduced PBMCs of MS patients (n ¼ 4) were compared to PBMCs 5 of normal subjects (n ¼ 7). oligodendrocytes 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 immune systems may promote inflammatory demyelination investigate whether the lower SHP-1 levels seen in the PBMCs still remains to be elucidated, its role in MS in light of the of MS patients are also seen in CNS glia within MS lesions. present findings should be further investigated. In this study, the expression levels of SHP-1 both in freshly Apart from STAT6, there are several other transcription isolated PBMCs and PBMCs cultured for 1 week in vitro were factors that are elevated in MS patients and are controlled by analyzed. We found that in either freshly isolated or cultured SHP-1. In particular, tyrosine-phosphorylated STAT1 PBMCs, MS patients had a deficient expression of SHP-1 (pY-STAT1) that mediates interferon signaling was found to compared to normal subjects. This observation both allowed be elevated in MS patients.3,4 We have verified with flow a determination of the stability of this difference and further cytometry that pY-STAT1 was increased in PBMCs of MS subsequent experimental manipulation of considerably patients compared to normal subjects (personal unpublished increased numbers of cultured cells than was possible using data) and verified that STAT1-inducible genes like IP-10 are freshly isolated cells. Further, we verified that culturing elevated in PBMCs of MS patients (Table 1) as previously PBMCs did not differentially affect the proportions of cell shown.52–56 SHP-1 has been shown to control STAT1 acti- types between MS patients and PBMCs of normal subjects, vation.3,9,63 Thus, lower levels of SHP-1, as seen in PBMCs of which may have otherwise confounded our analysis. Finally, MS patients, can provide a potential explanation for the we document that pSTAT6 levels and STAT6-responsive genes heightened STAT1 activation seen in MS. are elevated in cultured PBMCs of MS patients. In keeping Furthermore, the transcription factor NF-kB mediates with stable SHP-1 differences, freshly isolated PBMCs had inflammatory cytokine signaling and is elevated in MS similar gene expression patterns of STAT6- and STAT1-res- lesions.64 Thus, it may be important that mice genetically ponsive genes as cultured PBMCs in that genes like CCL17 lacking SHP-1 show elevated NF-kB activity and increased and IP-10 were significantly higher in MS patients compared NF-kB-inducible genes both in CNS glia and in lympho- to normal subjects (personal unpublished data). cytes.29,65 Further investigation is needed to determine Two distinct promoters drive the expression of two whether several of the abnormalities seen in MS, including different SHP-1 transcripts from the SHP-1 gene. Here, we overactive inflammatory transcription factors, can be attri- demonstrated that promoter II transcripts, which are more buted to the stable deficiency of SHP-1 seen in MS patients. abundantly expressed in hematopoietic cells, are selectively SHP-1 deficiency has been associated with several immune lower in the PBMCs of MS patients compared to normal abnormalities.16 Importantly, several signaling events of subjects. Several studies have examined the contribution antigen receptors on lymphocytes involve tyrosine phos- of genetics in MS71–74 and none have shown any linkage phorylation. Lack of the protein tyrosine phosphatase SHP-1 associated with the SHP-1 locus, chromosome 12p12.21 In was shown to effectively increase the activity of the protein contrast, numerous reports document the downregulation of tyrosine kinases Src and Lyn in B cells and Lck and Fyn in the promoter II transcripts in leukemia/lymphoma cell lines T cells.66 Absence of SHP-1 in B cells results in increased and attribute SHP-1 deficiency to epigenetic modifications antigen receptor signaling, polyclonal activation, and and in particular CpG methylation of SHP-1 promoter II.14,15

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Interestingly, in the context of MS, either viral infections75–79 12. Massa PT, Ropka SL, Saha S, et al. Critical role for protein tyrosine or inflammation80–83 can cause de novo methylation or phosphatase SHP-1 in controlling infection of central nervous system glia and demyelination by Theiler’s murine encephalomyelitis virus. hypermethylate CpG promoter sequences resulting in J Virol 2002;76:8335–8346. decreased gene expression. In accordance with these reports, 13. Deng C, Minguela A, Hussain RZ, et al. Expression of the tyrosine preliminary data indicate that treatment of PBMCs of MS phosphatase SRC homology 2 domain-containing protein tyrosine 0 phosphatase 1 determines activation threshold and severity of patients with the demethylating agent 2 -deoxy-5-azacytidine experimental autoimmune encephalomyelitis. J Immunol increase SHP-1 promoter II transcripts to normal levels 2002;168:4511–4518. (personal unpublished observations). Furthermore, a recent 14. Zhang Q, Raghunath PN, Vonderheid E, et al. Lack of phosphotyrosine phosphatase SHP-1 expression in malignant T-cell lymphoma cells study reports that the HTLV-1 Tax protein profoundly results from methylation of the SHP-1 promoter. Am J Pathol suppresses the activity of the SHP-1 promoter II through 2000;157:1137–1146. recruitment of histone deacetylase-1.84 Interestingly, HTLV-1 15. Oka T, Yoshino T, Hayashi K, et al. Reduction of hematopoietic cell-specific tyrosine phosphatase SHP-1 gene expression in natural is a lymphotropic virus that has been associated with killer cell lymphoma and various types of lymphomas/leukemias: CNS-demyelinating disease known as HAM/TSP (HTLV-1- combination analysis with cDNA expression array and tissue associated myelopathy/tropical spastic paraparesis).85 The microarray. Am J Pathol 2001;159:1495–1505. 16. Shultz LD, Rajan TV, Greiner DL. 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