Christelle Le Dantec

Marie-Michèle Varin

Wesley H Brooks

Jacques-Olivier Pers

Pierre Youinou

Yves Renaudineau
email: yves.renaudineau@univ-brest.fr

Christelle Le Dantec

Epigenetics and Sjögren's Syndrome

Keywords: Sjögren's syndrome, epigenetics, DNA methylation, microRNAs, post-translational modifications, human endogenous retrovirus

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INTRODUCTION

Sjögren's syndrome (SS) is a chronic autoimmune disease affecting exocrine glands, mainly the salivary and lacrimal glands, leading to its designation as autoimmune exocrinopathy or autoimmune epithelitis [START_REF] Mavragani | The geoepidemiology of Sjögren's syndrome[END_REF]. Systemic features may be present, and patients with SS have a 20-40 fold increased risk of developing lymphoma [START_REF] Voulgarelis | Malignant lymphoma in primary Sjögren's syndrome: a multicen-ter, retrospective, clinical study by the European Concerted Action on Sjögren's Syndrome[END_REF][START_REF] Baimpa | Hematologic manifestations and predictors of lymphoma development in primary Sjögren syndrome: clinical and pathophysiologic aspects[END_REF][START_REF] Tobón | The FMS-like tyrosine kinase3-ligand, a mediator for B cell survival is also a marker for lymphoma in primary Sjögren's syndrome[END_REF], mostly salivary gland lymphoma. In the different SS manifestations, the epithelium is affected and injury is associated with dense lymphocytic infiltrates composed of activated T and B cells [START_REF] Christodoulou | Characteristics of the minor salivary gland infiltrates in Sjögren's syndrome[END_REF]. Epithelial cells play a central role in the disease through the recruitment and activation of lymphocytes, and the release of apoptotic blebs containing autoantigens (52 kDa and 60 kDa Ro/SSA, La/SSB, Sm) [START_REF] Salomonsson | Cellular basis of ectopic germinal center formation and autoantibody production in the target organ of patients with Sjögren's syndrome[END_REF].

The etiology of SS is multifactorial and arises from an interplay of genetic predispositions, in particular HLA-DR allele subtypes, and specific gene polymorphisms including the ancestral haplotype 8.1, IL-10, SSA1, FCGR3B, CCL3L1, IRF5 and STAT4 [START_REF] Cobb | Genes and Sjögren's syndrome[END_REF][START_REF] Mohammed | Association of severe inflammatory polyarthritis in primary Sjögren's syndrome: clinical, serologic, and HLA analysis[END_REF][START_REF] Delgado-Vega | Genetic associations in type I interferon related pathways with autoimmunity[END_REF]; immunological deregulation such as autoantibody (Ab) production; and environmental factors like UV light, nutrition, exposure to infectious agents, and drugs [START_REF] Pers | Conference Scene: Updating the highlights of Sjögren's Syndrome[END_REF]. Interestingly, exposure to environmental factors, geographical particularities, radiation exposure, and sex have been advanced recently to explain the broad prevalence, 0.1% to 4.8%, reported in SS [START_REF] Mavragani | The geoepidemiology of Sjögren's syndrome[END_REF].

Initially, epigenetics was introduced to describe how environmental factors may influence a cell heritably or reversibly without affecting the nucleotide sequence of the DNA. Now, a more precise definition of epigenetics relates it to specific cellular processes including the control of transcription by DNA methylation and histone modifications, the control of transcripts by microRNAs (miRNA), and the influence on cellular regulatory processes by protein post-translational modifications (PTM). As a consequence, epigenetic processes control cellular differentiation and development, cellular life span, progression of carcinogenesis, and last but not least, the immune system.

DNA METHYLATION AND CHROMATIN REMO-DELLING

DNA Methylation and Histone Modifications

The density of chromatin varies along each chromosome with dense regions referred to as heterochromatin and less dense regions referred to as euchromatin. Heterochromatin participates in the inactivation of the genes, in the compaction of centromeres and telomeres, and in the silencing of transposons, such as human endogenous retroviruses (HERV) that are present in the "junk DNA". Regulatory gene sequences present in the heterochromatin are characterized by increased DNA methylation of cytosines in the CpG motifs, histone deacetylation, and recruitment of non-histone DNA-binding proteins, like the heterochromatin protein 1 that blocks transcription factor access to promoters. In contrast, euchromatin is found in areas of transcriptionally active genes and shows decreased DNA methylation, and increased histones modifications such as acetylation.

Throughout the genome, methylated sites are distributed on about 70% of CpGs. The remaining demethylated motifs are concentrated in short repeated CpG sequence domains, called CpG islands, that control gene expression when associated with gene regulatory regions. In cancer cells, DNA methylation has been shown to contribute to gene silencing of tumor suppressor genes, and to DNA mutations and chromosomal instability when DNA methylation concerns CpG islands outside of regulatory areas. DNA methylation occurs by covalent addition of a methyl group from the methyl donor S-adenosyl-L-methionine (SAM) to the 5 carbon of the cytosine ring in CpG pairs. At least five DNA methyltransferases have been characterized (DNMT1, DNMT3a, DNMT3b, DNMT3L and DNMT2). DNMT1 preferentially methylates hemi-methylated substrates, such as appear during cell division, and thereby DNMT1 contributes to the maintenance of DNA methylation patterns. DNMT3a and DNMT3b methylate unmethylated DNA and thus contribute to de novo methylation. DNMT2 displays only weak DNA methyl transferase activity. Its main function is to methylate aspartyl-tRNA. DNMT3L binds DNMT3a and DNMT3b to regulate their functions. DNA methylation causes transcriptional repression of associated genes directly by reducing the affinity between transcription factors and their target DNA, or indirectly via methyl-CpGbinding proteins (MBD), a group that includes MECP2, MBD1-MBD4 and Kaiso. The main function of MBD is to recruit proteins that do not bind directly to DNA, such as the histone deacetylases (HDACs) and HP1. Genetics and epigenetics are linked since it has been demonstrated that polymorphisms in the X-linked MECP2 gene confer a risk for development of systemic lupus erythematosus (SLE) [START_REF] Webb | Variants within MECP2, a key transcription regulator, are associated with increased susceptibility to lupus and differential gene expression in patients with systemic lupus erythematosus[END_REF]. However, association with SS is currently unknown.

Histones are small well-conserved proteins associated with DNA in a fundamental unit called the nucleosome that controls chromatin condensation and decondensation. Noncondensed nucleosomes resemble "beads on a string", and each bead or nucleosome consists of two copies of the four histones (H2A, H2B, H3 and H4) surrounded by 146 bp of DNA coiling twice around the histone core. The number of nucleosomes per cell has been estimated at around 10 million, and they are separated by 10-80 bp of linker DNA. In condensed DNA, the linker histone H1 binds to the linker DNA between nucleosomes to facilitate compaction. The histone amino terminal tails protruding from the nuclesomes are subject to many PTM including acetylation, methylation, ubiquitination, phosphorylation, sumoylation, deimination/citrullinisation, ADP ribosylation, and proline isomerization [START_REF] Dieker | Epigenetic Histone Code and Autoimmunity[END_REF]. Histone PTM are recognized by specific proteins in order to control DNA folding via DNA-histone, histonehistone and nucleosome-nucleosome interactions. The most intensively studied modifications are acetylation and methylation. Indeed, the activity of histone acetyl transferases (HATs) is counterbalanced by HDACs that remove the acetyl groups. HDACs are divided into four classes: class I consists of HDAC 1-3 and 8 (nuclear localization), class II consists of HDAC 4-7 and 9-10 (cytoplasmic and nuclear localization), class III consists of sirtuins (SIRT 1-7) and class IV consists of HDAC 11 which exhibits features of classes I-II. Similarly, the impact of arginine or lysine methylation by histone methyl transferases (HMTs) is reversed by demethylating enzymes. It should be kept in mind that one histone can carry different modifications at any given time and that histone modifications are dynamic and can change rapidly in response to stimuli. In addition, PTM are not restricted to histones since, for example, HDAC2 can modify up to 200 non-histone targets including the U1 small nuclear (sn)-ribonucleoprotein (RNP) and the small cytoplasmic (sc)-RNP proteins 60 kDa Ro/SSA and La/SSB.

Twin Studies

Studies involving monozygotic twins (MT) that are genetically identical constitute the ideal model to study epigenetic modifications [START_REF] Ballestar | Epigenetics Lessons from Twins: Prospects for Autoimmune Disease[END_REF]. Fraga et al. have demonstrated that DNA methylation patterns and histone acetylation profiles diverge in the peripheral blood between younger and older healthy MT [START_REF] Fraga | Epigenetic differences arise during the lifetime of monozygotic twins[END_REF]. Furthermore, it was also noted that MT with the greatest differences were those who have spent less of their lives together, thus providing evidence that changes may be related to differences in environmental exposures and/or diet. Using peripheral blood mononuclear cells (PBMC) from pairs of siblings discordant for SLE, rheumatoid arthritis (RA) and dermatomyositis (DM), the same group has reported that only MT discordant for SLE, but not for RA or DM, exhibited global loss in their DNA methylation status when 807 promoters were analyzed [START_REF] Javierre | Changes in the pattern of DNA methylation associate with twin discordance in systemic lupus erythematosus[END_REF], and this was associated with a reduction in DNMT1 and DNMT3b transcripts. Methyl cytosine modifications among 5 SLE twins and their corresponding healthy MT were scrutinized in more detail revealing a common set of 49 demethylated genes. Among the differentially methylated genes, some of them are involved in the immune response including IL-10, CD9, CD82, PECAM, the interferon gamma receptor 2 (IFNRG2), and the colony stimulating factor 3 receptor (CSFR3).

Epigenetics in Aging

The combined influence of aging and environmental factors in the epigenome may provide an explanation for agerelated AID development. Indeed, when analysing DNA methylation status, a reduction with age is observed in the elderly and it appears to be proportional to the life expectancy [START_REF] Grolleau-Julius | The Role of Epigenetics in Aging and Autoimmunity[END_REF]. Similarly, chromatin reorganization is observed in senescent cells in association with histone modifications [START_REF] Mccord | Telomeric chromatin: roles in aging, cancer and hereditary disease[END_REF]. However, studies of histone modifications in the elderly have concentrated mostly on the association between cellular life span and the NAD+ dependent HDAC class III sirtuin family. Indeed, manipulation of sirtuin activity influences cellular longevity positively when over-expressed and negatively when under-expressed [START_REF] Finkel | Recent progress in the biology and physiology of sirtuins[END_REF]. Interestingly, young SIRT1 -/-mice develop an SLE-like disease with antinuclear Abs (ANA) and glomerulonephritis. When they survive up to two years of age, a diabetes insipidus appears [START_REF] Sequeira | sirt1-null mice develop an autoimmune-like condition[END_REF]. Sirtuin expression is controlled by environmental factors. In fact, smoking is associated with SIRT1-2 downregulation and pro-inflammatory cytokine production, an effect prevented with the utilization of the SIRT1 activator resveratrol isolated from red wine. The beneficial effects of sirtuin inhibitors and HDAC class I-II inhibitors have also been demonstrated in different AID mouse models showing that the effect was related to reduction of cytokine production while autoAb synthesis remained unaffected [START_REF] Grabiec | Histone Deacetylase Inhibitors Suppress Inflammatory Activation of Rheumatoid Arthritis Patient Synovial Macrophages and Tissue[END_REF][START_REF] Lee | Overexpression of SIRT1 protects pancreatic beta-cells against cytokine toxicity by suppressing the nuclear factor-kappaB signaling pathway[END_REF][START_REF] Trenkmann | Epigenetics in Rheumatoid Arthritis[END_REF]. The relation between life span and HDAC class I-II is still being debated.

CHROMATIN MODIFICATIONS IN SS

DNA Methylation in SS

Hydralazine and procainamide, drugs known to induce SS with immunological features of an SLE like disease [START_REF] Darwaza | Hydrallazine-induced Sjögren's syndrome[END_REF][START_REF] Taylor | Procainamide toxicity[END_REF], possess the capacity to remove the methyl group from cytosines present in CpG islands and, therefore, are referred to as demethylating agents. Both drugs, when given orally to mice for several weeks, are responsible for the development of an SLE-like disease with ANA [START_REF] Cannat | Induction by isoniazid and hydrallazine of antinuclear factors in mice[END_REF]. Variations were observed depending on the animal strain, age, and sex and the effect disappeared after discontinuation of the drug. Richardson's group has demonstrated that hypomethylated CD4 + T cells became autoreactive. Indeed, passive transfer of CD4 + T cells pre-treated with either of two distinct DNA methyltransferase inhibitors, 5-azacytidine or procainamide, into mice induced anti-dsDNA Ab production with the characteristics of a severe immune complex glomerulonephritis [START_REF] Quddus | Treating activated CD4+ T cells with either of two distinct DNA methyltransferase inhibitors, 5-azacytidine or procainamide, is sufficient to cause a lupus-like disease in syngeneic mice[END_REF]. The influence of DNMT inhibitors was also addressed in B cells using bone marrow B cells pretreated with hydralazine and re-injected by passive transfer into syngenic mice leading to the detection of anti-nucleosome Abs in the recipients [START_REF] Mazari | Subversion of B lymphocyte tolerance by hydralazine, a potential mechanism for drug-induced lupus[END_REF]. Similar to what was observed in SLE, scleroderma and DM [START_REF] Lei | Abnormal DNA methylation in CD4+ T cells from patients with systemic lupus erythematosus, systemic sclerosis, and dermatomyositis[END_REF], Yin et al. have recently reported that DNA methylation is impaired in CD4+ T cells isolated from patients with SS [START_REF] Yin | Hypomethylation and overexpression of CD70 (TNSF7) in CD4+ T cells of primary Sjögren's syndrome[END_REF]. As a consequence, several methylation sensitive autoreactive related genes are overexpressed. Among these genes, the promoter region flanking the CD70 gene, a B cell co-stimulatory molecule, is demethylated and CD70 is over-expressed in SS CD4+ T cells.

Different T cell and B cell subpopulations are characterized by abnormal DNA methylation patterns. First, the senescent CD4+ CD28 CD70+ Kir+ subpopulation, which is increased in RA and spondyloarthritis [START_REF] Chen | Decreased ERK and JNK signaling contribute to gene overexpression in "senescent" CD4+CD28-T cells through epigenetic mechanisms[END_REF], second the defective pre-Treg CD4+ CD25 subpopulation that overexpress FoxP3 but lack regulatory functions [START_REF] Lal | Epigenetic mechanisms of regulation of Foxp3 expression[END_REF], and third the CD5 + B cell [START_REF] Garaud | Selection of the alternative exon 1 from the cd5 gene down-regulates membrane level of the protein in B lymphocytes[END_REF]. In SS, whereas the CD4+ CD28-subpopulation is not altered, FoxP3+ T cells and CD5+ B cells are overexpressed in the blood and salivary glands from SS patients [START_REF] Christodoulou | Foxp3+ T-regulatory cells in Sjögren's syndrome: correlation with the grade of the autoimmune lesion and certain adverse prognostic factors[END_REF][START_REF] Sarigul | The numbers of Foxp3 + Treg cells are positively correlated with higher grade of infiltration at the salivary glands in primary Sjogren's syndrome[END_REF][START_REF] Youinou | CD5-expressing B lymphocytes in the blood and salivary glands of patients with primary Sjögren's syndrome[END_REF][START_REF] Vaudo | Precocious intima-media thickening in patients with primary Sjögren's syndrome[END_REF]. Like CD4+ CD25-FoxP3+ isolated from SLE patients, FoxP3+ T cells isolated from SS patients are unable to suppress the immune response suggesting that in both diseases FoxP3+ T cells are in fact demethylated and defective pre-Treg cells, an hypothesis that needs further confirmation [START_REF] Katsifis | T lymphocytes in Sjögren's syndrome: contributors to and regulators of pathophysiology[END_REF]. Of note is the observation that the Treg/FoxP3 -/-Scurfy mice develop a rapid salivary gland inflammation and impaired salivation function after oral application of lipopolysaccharide (LPS) confirming that functional Treg are important to prevent SS [START_REF] Sharma | X-linked Foxp3 (Scurfy) mutation dominantly inhibits submandibular gland development and inflammation respectively through adaptive and innate immune mechanisms[END_REF].

SS and HERV Elements

Throughout the genome, HERV are particularly sensitive to DNA methylation changes and several reports have identified such elements in salivary glands from SS patients [START_REF] Brookes | The immune response to and expression of cross-reactive retroviral gag sequences in autoimmune disease[END_REF][START_REF] Yamano | Retrovirus in salivary glands from patients with Sjögren's syndrome[END_REF]. In an attempt to confirm the importance of HERV expression in salivary glands from SS, we have tested by RT-PCR three HERV-E elements Fig. [START_REF] Mavragani | The geoepidemiology of Sjögren's syndrome[END_REF]. Interestingly, all patients tested, except one, were positive for at least one HERV-E element. HERV are sequences derived from the integration of retroviral elements that account for at least 8% of the genome in comparison to only 3% of the genome being essential for life. Integration of most HERV into the genome occurred millions of years ago and, after mutations and deletions that render infection impossible, HERV are transmitted like other stable Mendelian elements. Analysis of the HERV mutation rate, 0.15-0.21% mutations per million years, between the 5' long terminal repeat (LTR) and 3' LTR, may be helpful for dating chromosomal integration [START_REF] Renaudineau | Characterization of the human CD5 endogenous retrovirus-E in B lymphocytes[END_REF]. HERV reactivation has been associated with exogenous viruses, mitogens, pro-inflammatory cytokines, steroid hormones, UVB irradiation, and X-ray exposure. When overexpressed, HERV may interfere with the immune response by different mechanisms [START_REF] Renaudineau | Autoreactive B Cells and Epigenetics[END_REF]. First, is the possibility of molecular mimicry between a retroviral protein and an autoantigen. For example, HRES-1 p30gag that crossreacts with the SLE autoantigen U1-snRNP is recognized as an autoantigen in up to 30% of SS patients and 50% of SLE patients compared to less than 5% in a healthy control group [START_REF] Brookes | The immune response to and expression of cross-reactive retroviral gag sequences in autoimmune disease[END_REF]. Similarly, another unknown HERV, which may be Erv-3, cross-reacts with the p24 Gag of HIV-1 leading to the detection of anti-p24 Abs in the blood of patients with SS (33%) and p24 related antigen in the salivary glands (47%) in the absence of HIV and HTLV-1 infection [START_REF] Yamano | Retrovirus in salivary glands from patients with Sjögren's syndrome[END_REF]. Second, HERV gene products may possess immunomodulatory functions leading to repression or expansion of T cell populations as demonstrated in multiple sclerosis [START_REF] Perron | The human endogenous retrovirus link between genes and environment in multiple sclerosis and in multifactorial diseases associating neuroinflammation[END_REF]. Third, HERV gene products may be homologous to a cellular protein and its over-expression modifies the cellular physiology [START_REF] Fernandez | Activation of mammalian target of rapamycin controls the loss of TCRzeta in lupus T cells through HRES-1/Rab4-regulated lysosomal degradation[END_REF]. Fourth, HERV may have integrated into or adjacent to immune-related genes and provide a new promoter that generates truncated proteins, or isoforms by alternative splicing. An example is the HERV-CD5 endogenous retrovirus, located 8 kb upstream CD5 gene, which generates an alternative transcript with CD5 [START_REF] Renaudineau | An alternative exon 1 of the CD5 gene regulates CD5 expression in human B lymphocytes[END_REF][START_REF] Garaud | IL-6 modulates CD5 expression in B cells from patients with lupus by regulating DNA methylation[END_REF][START_REF] Hippen | CD5 maintains tolerance in anergic B cells[END_REF]. Fifth, HERV can also influence neighbouring genes in an abnormal way. Presence of HERV-K(C4) in the 9 th intron of the C4 complement gene, which occurs in 70% of the population, resulted in a reduction of C4B protein [START_REF] Wouters | High-throughput analysis of the C4 polymorphism by a combination of MLPA and isotype-specific ELISA's[END_REF]. Sixth, HERV integration may be considered as a genetic risk factor for AID, as reported for HERV-K113 and patients with SS and MS [START_REF] Moyes | The distribution of the endogenous retroviruses HERV-K113 and HERV-K115 in health and disease[END_REF].

SS and Lymphoma

The methylation status of CpG islands of the proapoptotic death associated protein kinase (DAPK) was studied by Tosos et al. in samples from primary salivary gland lymphoma, the main SS associated lymphoma [START_REF] Toso | Clinical and molecular features of mucosa-associated lymphoid tissue (MALT) lymphomas of salivary glands[END_REF]. Surprisingly, the authors reported that demethylation of the DAPK gene characterized the SS sub-group when compared to the non-SS sub-group that are aberrantly hypermethylated. As a consequence, the tumor processes may be different between both sub-groups.

CHROMOSOME X AND SS

In humans, females inactivate one of their two X chromosomes in each cell in order to express X-linked genes at similar levels as males that have only one X chromosome since most X-linked genes are not sex-specific [START_REF] Brooks | X Chromosome Inactivation and Autoimmunity[END_REF]. The active chromosome is referred to as Xa, whereas the inactive one is referred to as Xi. The choice of which X chromosome to inactivate, the maternally-derived or the paternallyderived, is a random choice made in somatic cells early in development. As a consequence, in newborn females we observe a mosaic of groups of cells with the maternallyderived X inactivated next to groups of cells with the paternally-derived X inactivated. This pattern will be conserved throughout life since the daughter cells and all subsequent generations will keep the same parentally-derived X chromosome inactive as was chosen by its original ancestor cell.

X chromosome inactivation (XCI) originates on the long arm of the X at position Xq13.3 in an area of DNA referred to as the X-Inactivation Center (XIC) that contains the 35kb human X-Inactivation Specific Transcript (XIST) gene that yields a long processed RNA transcript of approximately 19 kb. The XIST RNA does not appear to code for protein and remains in the nucleus where it can repress the Xi by coating the contiguous chromosome and recruiting proteins that further establish an inactive heterochromatic state. Furthermore, stable Xi repression involves continuous activity of DNMT1, DNA methylation, a series of histone modifications (hypoacetylation, methylation, and ubiquitination), plus the accumulation of the repressor histone variant, macroH2A. All these modifications contribute to formation of the heterochromatic Xi structure, also referred to as the Barr body, which is generally located at the periphery of the nucleus. The Xa chromosome (i.e. euchromatin) assumes a more central location in the nucleus, similar to autosomes, and remains active.

Several arguments suggest a role for the X chromosome in the development of SS. The first, and indirect one, relates to the female predominance in SS with a sex ratio of 9:1 between females and males. The second one is related to the association between numerical abnormalities of the X chromosome and SS since several reports of SS women with trisomy X (47,XXX) or a super female phenotype (mosaic of XXXXX/XXXX/XXX/XX/XO) have been reported. On the other hand, the skewed XCI, (i.e. a significant preferential inactivation of one parentally-derived X over the other) reported in scleroderma was not observed in SS [START_REF] Ozcelik | X chromosome inactivation and female predisposition to autoimmunity[END_REF]. The recent observation that increased demethylation of the Xi is associated with over-expression of the B-cell co-stimulator CD40-ligand in CD4 + T cells from women with SLE suggests that this should be tested in SS [START_REF] Lu | Demethylation of CD40LG on the inactive X in T cells from women with lupus[END_REF]. Cumulatively, the possible involvement of the X chromosome and related epigenetic defects in SS needs further investigation.

RNA TRANSCRIPTS

miRNA

MicroRNAs (miRNA) are genome-encoded 18-to 23base-pair (bp) RNAs that regulate gene expression at the post-transcriptional level. MiRNAs are transcribed in the nucleus by RNA polymerase II or III forming long preliminary transcripts which are cleaved by Drosha in the nucleus, exported to the cytoplasm by exportin 5, processed by Dicer into mature miRNA duplexes, and separated into single strands at the core of the multiprotein RNA-induced silencing complex (RISC) by argonaut proteins to generate miR-NAs. It is generally accepted that miRNAs, by base pairing, bind the 3 untranslated region (UTR) of specific messenger RNAs (mRNA) for degradation or translational repression Fig. (2A). One third of the transcriptome is suspected to be regulated by the 800-1,000 human miRNAs since one miRNA can alter the expression of hundreds of mRNAs. MiRNAs regulate different cellular processes such as embryonic development, cell differentiation, cell cycle, apoptosis, immune cell development, and immune responses. One of the characteristics of miRNAs is that they are highly resistant to degradation by RNases and can be isolated from biopsies and all types of body fluids including plasma, saliva, urine, and exosomes thus making miRNAs excellent biomarkers.

miRNA in SS

To date, few studies on miRNAs have been conducted in SS patients. According to Pauley et al., miR-146a and miR-155, two miRNAs up-regulated in response to the adaptative immune response in multiple cell types, are increased prior to disease onset in the salivary glands and in the peripheral blood mononuclear cells (PBMC) from both SS patients and from the SS prone mouse model C57Bl/6.NOD-Aec1 Aec2 [START_REF] Pauley | Altered miR-146a expression in Sjögren's syndrome and its functional role in innate immunity[END_REF]. MiR-146a is a master gene regulator, activated by NF-kappaB, that controls the TLR/IFN pathway through the TNF receptor-associated factor 6 (TRAF6), the IL-1 receptor associated kinase (IRAK1), the signal transducer and activator of transcription 1 (STAT1), and the interferon regulatory factor 5 (IRF5) [START_REF] Tsitsiou | microRNAs and the immune response[END_REF]. Examination of miR-155 targets reveals an effect on the response of toll-like receptors and interleukin-1 receptors (TIRs) that are suspected to affect the immune response. Interestingly, the FoxP3 transcription factor, which is over-expressed in T cells infiltrating SS salivary glands (see above), has been shown to induce miR-155 expression. Another source of miR-155 may be salivary gland epithelial cells since we have observed that cultured salivary epithelial cells from SS express two fold more miR-155 than controls Fig. (2B). Two other miRNA, were also tested revealing that miR-181a but not miR-150 was overexpressed in cultured salivary glands from SS patients. MiR-181a participates in differentiation and proliferation by targeting, respectively, an homeobox protein (Hox-A11) and oncogenes (Tcl-1). Testing salivary gland biopsies from SS patients, Illei's group has observed that three miRNAs within the miR-17-92 cluster, miR-18a, hsa-miR-19a, and hsa-miR-19b, were upregulated [START_REF] Alevizos | MicroRNAs in Sjogren's syndrome as a prototypic autoimmune disease[END_REF]. The miR-17-92 cluster is crucial for T and B cell development and activation, and its overexpression is associated with a lymphoproliferative disease and an SLE-like disease with antinuclear Abs and glomerulonephritis. Two targets have been characterized for miR-17-92, the pro-apoptotic protein Bim and the tumor suppressor (PTEN).

miRNA in RA and SLE

Similar to SS, the expression of miR-146a and miR-155 is increased in PBMC, and in fibroblast-like synoviocytes isolated from RA patients when compared to healthy controls [START_REF] Chan | Contrast in aberrant mi-croRNA expression in systemic lupus erythematosus and rheumatoid arthritis: is microRNA-146 all we need?[END_REF]. A correlation with RA disease activity was observed for miR-146a and miR-16. In contrast, miR-146a is underexpressed in PBMC from SLE patients and this underexpression negatively correlates with disease activity, thus providing an explanation for the link observed between interferon over-expression and miR-146a downregulation in SLE [START_REF] Brooks | Epigenetics and autoimmunity[END_REF]. MiRNA analysis in SLE should not be restricted to miR-146a since the analysis of PBMC from SLE patients has revealed nine up-regulated miRNAs (miR-21, miR-61, miR-78, miR-142-3p, miR-189, miR-198, miR-298, miR-299-pp, and miR-342) and seven down-regulated miRNAs (miR-17-5p, miR-112, miR-141, miR-184, miR-196a, miR-383, and miR-409-3p). In kidney biopsies from SLE patients, there are 66 miRNAs that are differentially expressed.

POST-TRANSLATIONAL MODIFICATIONS

A dysregulation of apoptosis is suspected to be involved in the early stage of SS and autoAb production. Supporting this possibility is the presence of histones, 52 kDa and 60 kDa Ro/SSA, La/SSB, and proteins relocated from the nucleus to apoptotic blebs on the cell surface of early apoptotic cells. Muller's group was the first to postulate that specific PTM histone modifications during cell death may generate neo-epitopes [START_REF] Dieker | Epigenetic Histone Code and Autoimmunity[END_REF]. Indeed, apoptosis-associated acetylation of histones H2B and H4 provides important epitopes for pathogenic autoAbs in SLE, and administration of acetylated peptides in lupus prone mice accelerated the disease, while non-acetylated peptides are ineffective and phosphorylated peptides are protective. Similarly, apoptosis and PTM have been proposed to influence 60 kDa Ro/SSA and La/SSB autoimmunity [START_REF] Reed | B cell apotopes of the 60-kDa Ro/SSA and La/SSB autoantigens[END_REF]. In fact, when using sera from SS patients, the Ab binding and the relative avidity is increased when La/SSB peptides phosphorylated at Serine 367 are used instead of crude peptides [START_REF] Terzoglou | Preferential recognition of the phosphorylated major linear B-cell epitope of La/SSB 349-368 aa by anti-La/SSB autoantibodies from patients with systemic autoimmune diseases[END_REF]. Modifications such as phosphorylation, citrullinisation or oxidation influence 60 kDa Ro/SSA peptide antigenicity, thus explaining why immunization with lipid oxidized 60 kDa Ro/SSA peptides increases the antigenicity and favors epitope spreading to La/SSB, U1-snRNP and Sm [START_REF] Terzoglou | Post-translational modifications of the major linear epitope 169-190aa of Ro60 kDa autoantigen alter the autoantibody binding[END_REF][START_REF] Scofield | Modification of lupus-associated 60-kDa Ro protein with the lipid oxidation product 4-hydroxy-2-nonenal increases antigenicity and facilitates epitope spreading[END_REF]. The preferential recognition of the post-transcriptional forms of La/SSB by anti-La/SSB Abs in SS was confirmed when using parotid glands from patients [START_REF] Stea | Analysis of parotid glands of primary Sjögren's syndrome patients using proteomic technology reveals altered autoantigen composition and novel antigenic targets[END_REF]. Then, as demonstrated with citrullinisation in RA [START_REF] Klareskog | Immunity to citrullinated proteins in rheumatoid arthritis[END_REF], it appears that apoptotic specific PTM could influence autoimmunity at different levels: initiation of autoimmunity, autoAb production with epitope spreading, and production of pathogenic autoAb. Such a hypothesis warrants further investigation.

CONCLUSION

While our current knowledge is limited of how epigenetic modifications may influence the course of SS, epigenetic studies will undoubtedly help to understand SS features: age-related development, female predominance, geolocalization, cycles of flare and remission, association with lymphoma, and so forth. In addition, the reversible nature of epigenetic modifications offers the opportunity to identify target sites with potential impact on the prevention or progression of AID (Table 1). However, the revolution of epigenetics in AID and in SS [START_REF] Renaudineau | The Revolution of Epigenetics in the Field of Autoimmunity[END_REF] needs new collaborations between researchers from different research areas and the creation of the Brest Epigenetic task force during the Xth International Symposium on Sjögren's syndrome, 1-3 October 2009, Brest, France, will serve this purpose [START_REF] Lu | Epigenetics in autoimmune disorders: Highlights of the 10th Sjögren's Syndrome Symposium[END_REF].

CD4+ T cells

Yes [START_REF] Lee | Overexpression of SIRT1 protects pancreatic beta-cells against cytokine toxicity by suppressing the nuclear factor-kappaB signaling pathway[END_REF] Yes [START_REF] Grabiec | Histone Deacetylase Inhibitors Suppress Inflammatory Activation of Rheumatoid Arthritis Patient Synovial Macrophages and Tissue[END_REF] Yes [START_REF] Fraga | Epigenetic differences arise during the lifetime of monozygotic twins[END_REF] Senescent CD4+ CD28-No [START_REF] Lei | Abnormal DNA methylation in CD4+ T cells from patients with systemic lupus erythematosus, systemic sclerosis, and dermatomyositis[END_REF] No [START_REF] Trenkmann | Epigenetics in Rheumatoid Arthritis[END_REF] Yes [START_REF] Trenkmann | Epigenetics in Rheumatoid Arthritis[END_REF] Pre-Treg CD4+ CD25-Foxp3+ (Yes ?) Yes [START_REF] Darwaza | Hydrallazine-induced Sjögren's syndrome[END_REF] No B cells ? Yes [START_REF] Brookes | The immune response to and expression of cross-reactive retroviral gag sequences in autoimmune disease[END_REF] ? miRNA miR-146a Upregulated [START_REF] Hippen | CD5 maintains tolerance in anergic B cells[END_REF] Downregulated [START_REF] Toso | Clinical and molecular features of mucosa-associated lymphoid tissue (MALT) lymphomas of salivary glands[END_REF] Upregulated [START_REF] Toso | Clinical and molecular features of mucosa-associated lymphoid tissue (MALT) lymphomas of salivary glands[END_REF] miR-155 Upregulated [START_REF] Hippen | CD5 maintains tolerance in anergic B cells[END_REF] Unaffected Upregulated [START_REF] Toso | Clinical and molecular features of mucosa-associated lymphoid tissue (MALT) lymphomas of salivary glands[END_REF] miR-17-92 Upregulated [START_REF] Moyes | The distribution of the endogenous retroviruses HERV-K113 and HERV-K115 in health and disease[END_REF] Unaffected ?

miR-181a Upregulated [Fig 2]

Upregulated [START_REF] Reed | B cell apotopes of the 60-kDa Ro/SSA and La/SSB autoantigens[END_REF] ?

X chromosome

Skewed XCI No [START_REF] Renaudineau | An alternative exon 1 of the CD5 gene regulates CD5 expression in human B lymphocytes[END_REF] No [START_REF] Renaudineau | An alternative exon 1 of the CD5 gene regulates CD5 expression in human B lymphocytes[END_REF] Yes [START_REF] Renaudineau | An alternative exon 1 of the CD5 gene regulates CD5 expression in human B lymphocytes[END_REF] CD40L expression on the inactive X ? Yes [START_REF] Garaud | IL-6 modulates CD5 expression in B cells from patients with lupus by regulating DNA methylation[END_REF] ?

MECP2 polymorphism ? Yes [START_REF] Tobón | The FMS-like tyrosine kinase3-ligand, a mediator for B cell survival is also a marker for lymphoma in primary Sjögren's syndrome[END_REF] No

Fig. ( 1 )

 1 Fig. (1). Detection of human endogenous retrovirus transcripts in salivary glands from primary Sjögren's syndrome (SS) patients. A-Using the online basic local alignment search tool (BLAST) provided by ensemble (http://www.ensembl.org/Multi/ blastview), the HERV-E 4.1 sequence yielded several elements over the whole genome including a copy from 19p13.1 showing 100% identity to the 4.1 clone. B-Detection of HERV-E transcripts in salivary glands from SS patients by RT-PCR using HERV 4.1, HERV-Mid1and HERV-LTR7 specific primers. Details have been described previously[START_REF] Renaudineau | Characterization of the human CD5 endogenous retrovirus-E in B lymphocytes[END_REF].

Fig. ( 2

 2 Fig. (2).MiRNA in epithelial cells isolated from primary Sjögren's syndrome (SS) salivary glands. A-A schematic diagram illustrating how miRNAs can potentially modulate mRNA degradation and translation through interactions with the 3 UTR. B-A quantitative PCR expression analysis of three miRNAs (miR-150, miR-155 and miR-181a) in epithelial cells cultured from six SS patients and six controls reveals that the expression levels of miR-155 and miR-181a were significantly higher in SS. Results, normalized to the house keeping miR-RNU48, are expressed relative to controls and there is no significant difference in the expression of miR-150.

Table 1 . Epigenetic Abnormalities Reported in Three Autoimmune Diseases: Sjögren's Syndrome (SS), Systemic Lupus Erythema- tosus (SLE), and Rheumatoid Arthritis (RA)

 1

		Autoimmune Diseases
	SS	SLE	RA

ACKNOWLEDGEMENTS

We are grateful to the "Association Française du Gougerot-Sjögren et des Syndromes Secs" for their support.

CONFLICT OF INTEREST

None declared.


Jean-Franc ¸ois Se ´ïte ´

Divi Cornec

Yves Renaudineau

Pierre Youinou

Rizgar A Mageed

Sophie Hillion

IVIg modulates BCR signaling through CD22 and promotes apoptosis in mature human B lymphocytes

Among various mechanisms for interactions with B cells, intravenous immunoglobulin (IVIg) may operate through the insertion of its Fc part into the Fc-␥ receptor, or the binding of its sialic acid (SA)bearing glycans to the negatively regulating CD22 lectin. It appeared that IVIg reduces B lymphocyte viability in a dose-and time-dependent manner. Furthermore, we show by confocal microscopy that SApositive IgG, but not SA-negative IgG bind to CD22. This interaction reduces the strength of B-cell receptor-mediated signaling trough down-regulating tyrosine phosphorylation of Lyn and the B-cell linker proteins, and up-regulating phospholipase C␥2 activation. This cascade resulted in a sustained activation of Erk 1/2 and arrest of the cell cycle at the G 1 phase. These changes may be accounted for the efficacy of IVIg in autoimmune diseases. (Blood.

Introduction

Although intravenous immunoglobulin (IVIg) is widely used to treat idiopathic thrombocytopenic purpura, Kawasaki disease, systemic lupus erythrematosus and Guillain-Barre ´syndrome, [START_REF] Nimmerjahn | Anti-inflammatory actions of IVIg[END_REF] the mechanisms by which immune functions are influenced by such preparations remain to be delineated. [START_REF] Seite | What is the contents of the magic draft IVIg? Autoimmun[END_REF] Because B lymphocytes play a central role in the immunopathologic processes that cause these diseases, several studies have suggested that B cells are the target cells for the beneficial effect of IVIg. For this hypothesis to apply, IVIg would modulate a broad range of B lymphocyte functions including activation, proliferation, [START_REF] Amran | Suppression of cytokine-dependent human T-cell proliferation by IVIg[END_REF] and survival. [START_REF] Prasad | Therapeutic preparations of normal polyspecific IgG (IVIg) induce apoptosis in human lymphocytes and monocytes: a novel mechanism of action of IVIg involving the Fas apoptotic pathway[END_REF] In other words, IVIg would interfere with the expression of genes and, potentially, the functions of proteins involved in cell growth and death.

The interaction between IVIg and B lymphocytes could thus modulate intracellular signaling resulting from the engagement of the B-cell antigen (Ag) receptor (BCR). The earliest signaling events of the ensuing cascade involves activation of the Src-family protein-tyrosine kinase (PTK) Lyn. [START_REF] Goodnow | Balancing immunity and tolerance: deleting and tuning lymphocyte repertoires[END_REF] Activated Lyn phosphorylates immunoreceptor tyrosine-based activation motifs (ITAMs) on the cytoplasmic domain of the membrane ␣ and ␤ proteins (CD79␣ and CD79␤). Once phosphorylated, CD79␣ and CD79␤ recruit another PTK protein, Syk. Syk associates with phospho-ITAMs via its tandem Src-homology 2 (SH2) domains, is phosphorylated, and in turn, phosphorylates the adaptor protein B-cell linker protein (BLNK). The adaptor thus acquires the capacity to bind to and activate Bruton's tyrosine kinase (Btk). This sequence of signaling events leads to the activation of phospholipase C␥2 (PLC␥2), hydrolysis of phosphatidylinisitol-4,5-bisphosphate and the production of inositol-1,4,5-trisphosphate.

As a result, released intracellular calcium synergizes with other signals to induce gene transcription. [START_REF] Defranco | Signal transduction by the BCR[END_REF] This promotion implies the recruitment of several transcription factors (TFs) that influence B-cell proliferation, differentiation, cytokine production, and apoptosis. [START_REF] Rathmell | B-cell homeostasis: digital survival or analog growth?[END_REF][START_REF] Tobo ´n | Are autoantibodies triggering endothelial cell apoptosis really pathogenic?[END_REF] The outcome of BCR engagement is variable, depending on the status of the B cell and commitment of coreceptors. The latter dictate the ultimate response and associate positive regulators (such as CD19 and CD21) and negative regulators (such as CD22 and CD32) and these ultimately determine the outcome of B-cell responses. Under normal conditions, both groups of proteins are expressed at varying levels on the surface of all B lymphocytes and their engagement contributes to setting the threshold of B-cell activation. This is often achieved by increasing or decreasing tyrosine phosphorylation, thereby enhancing or reducing signal transduction. [START_REF] Dal Porto | BCR signaling 101[END_REF] CD22 conveys the SH2 domain-containing phosphatase 1 (SHP-1) over to the BCR through the immunoreceptor tyrosinebased inhibition motifs (ITIMs) located in its cytoplasmic domain. [START_REF] Schulte | Tyrosine phosphorylation of CD22 during B cell activation[END_REF] This membrane protein belongs to the sialic acid (SA)binding Ig-like lectin (Siglec) superfamily, with 7 Ig-like extracellular domains and an amino-terminal Ig domain. CD22 is unique in that its amino terminal domain recognizes the galactoselinked SA␣2-6 sequence that is typically found on N-linked glycans of glycoproteins widely expressed on the surface of various cell types. Known ligands for CD22 include polysaccharides attached to antibodies (Abs), [START_REF] O'reilly | Bifunctional CD22 ligands use multimeric immunoglobulins as protein scaffolds in assembly of immune complexes on B cells[END_REF] but the intimate nature of the binding of CD22 to endogenous glycoproteins in a cis manner remains to be determined. In contrast, there is good evidence that CD22 binding by exogenous cross-linkers affects B-cell responses. [START_REF] Poe | CD22 regulates B lymphocyte function in vivo through both ligand-dependent and ligand-independent mechanisms[END_REF] Whether different ligands produce different effects on B-cell responses is unclear, yet the ligand-binding domain of CD22 most notably its ␣2-6-linked SA-binding domain is needed for CD22 to inhibit BCR signaling. [START_REF] Kelm | The ligand-binding domain of CD22 is needed for inhibition of the BCR signal, as demonstrated by a novel human CD22-specific inhibitor compound[END_REF][START_REF] Jin | Sialic acid binding domains of CD22 are required for negative regulation of BCR signaling[END_REF] To gain insights into how IVIg modulates immune responses, we explored the capacity of IVIg to modulate BCR signaling and thus influence the fate of B cells. Our results suggest that SAcontaining IgG within IVIg peparations bind to CD22 on B cells. This binding modulates B-cell functions, and could therefore contribute to the therapeutic efficacy of IVIg.

Methods

B-cell isolation and culture

Blood samples were collected from healthy volunteers, and tonsils from children undergoing routine tonsillectomy. All volunteers and parents of children gave informed consent in accordance with the Declaration of Helsinki, and the study was approved by the Institutional Review Board at the Brest University Medical School Hospital. Peripheral blood mononuclear cells were separated on Ficoll-Hypaque, T lymphocytes rosetted out, and B-cell-enriched preparations collected. All monoclonal Abs (mAbs) were from Beckman-Coulter unless otherwise specified. Fluorescein isothiocyanate (FITC)-conjugated anti-CD19 and phycoerythrin (PE)conjugated anti-CD5 mAbs were combined to ensure the sample purity by fluorescence-activated cell sorting (FACS) analyses. There were more than 98% B lymphocytes in all preparations.

Human Ramos lymphoma line B cells were purchased from ATCC. B cells were cultured in RPMI-1640 medium containing 10% fetal calf serum and supplemented with 2mM L-glutamine, 200 U/mL penicillin and 1 g/mL streptomycin.

Assessment of apoptosis

To assess their apoptosis, 2 ϫ 10 5 B cells in RPMI-1640 were incubated for 24 hours with increasing amount of IVIg (LFB). Cells were then stained with FITC-annexin V (AV) and propidium iodide (PI) according to Beckman Coulter's instructions. Briefly, B cells were suspended in 100 L phosphate-buffered saline (PBS) buffer and incubated for 10 minutes at 4°C with 4 L PI, and 1 L FITC-AV. In the FACS analyses, cells in the early stage of apoptosis were positive for FITC-AV and negative for PI. They were enumerated, and their mean fluorescence intensities (MFIs) recorded.

IVIg preparation

IVIg was dissolved in serum-free RPMI-1640 medium to a concentration of 100 mg/mL, and prepared for conjugation with FITC by dialysis of IVIg in carbonate-bicarbonate buffer, pH 9.5. Fluorescein-5-thiosemicarbazide (Molecular Probes) was then added with 100 g in dimethylsulfoxyde for each 1 mg of IVIg for 1 hour at 37°C. Excess FITC was removed by extensive dialysis against PBS. This preparation was assessed in an UV2 spectrophotometer (Unicam) by reading the absorbance at 280 nm and 495 nm, and by expressing the fluorescence as [(absorbance 280 nm) Ϫ 0.31(absorbance 495 nm)]/1.4. An IVIg-devoid preparation was examined in parallel to guarantee that the fluorescence came from conjugated IVIg, and not from dissociated FITC. SA-IgG was purified from the bulk of IVIg preparations by lectinaffinity chromatography with a Sambucus nigra agglutinin (SNA) agarose column (Vector Laboratories). Enrichment of the eluate was verified by Western blotting (WB) with biotinylated SNA at 4 g/mL and horseradish peroxidase (HRP)-conjugated Streptavidin.

For preparing Fab and Fc fragment, IVIg preparations were digested with 2 mg/mL papain in 0.04M cystein-PBS for 6 hours at 37°C. The resulting Fab and Fc fragments were separated from the undigested IVIg using a Sepharose S300 column and the Fc fragments were further separated from the Fab fragments on a protein G column. All purification steps were carried out using a fast protein liquid chromatography system (Pharmacia). Purity of the fragments was assessed by WB using Abs specific either for Fab of for Fc.

Analysis of phosphorylation by FACS

Phosphorylation assays were carried out using PE-conjugated mouse anti-phospho-BLNK (Phosphotyrosine PY 84; BD Biosciences), PEconjugated mouse anti-phospho-PLC␥2 (PY759) mAbs (BD Biosciences), rabbit anti-phospho-CD19 (PY531) Ab, rabbit anti-phospho-Lyn (PY396) and rabbit anti-phospho-CD22 (PY822) Abs (all from Abcam) developed by FITC donkey anti-rabbit Ab (Jackson ImmunoResearch Laboratories). For FACS analysis of phosphorylation, the cells were either unstimulated or stimulated with 20 g/mL anti-IgM-coated onto beads in the presence, or absence of 20 mg/mL IVIg for 20 minutes at 4°C, and then for 3 minutes at 37°C. In some experiments, 1 mg/mL sialylated IVIg (IVIg SAϩ), or 20 mg/mL nonsialylated IgG (IVIg SAϪ) was added. Activated cells were fixed and permeabilized by incubation for 30 minutes in 70% methanol before staining. Data were analyzed using EXPO32 program. Changes in phosphorylation expressed in folds increase and decrease were calculated in cells stimulated in the presence or absence of IVIg.

Confocal microscopy

To evaluate its relationships with CD22, 20 mg/mL of IVIg was associated with 5 ϫ 10 5 B lymphocytes. Interactions were revealed using goat FITC-conjugated FabЈ 2 anti-human IgG Ab (Jackson ImmunoResearch Laboratories), and rabbit anti-CD22 (Abcam), wich was revealed with tetrarhodamine isothiocyanate (TRITC)-conjugated donkey anti-rabbit Ig Ab (Jackson ImmunoResearch Laboratories). In some experiments, 1 mg/ mL of purified IVIg SAϩ or 20 mg/mL of the IVIg SAϪ fraction was added to 5 ϫ 10 5 tonsil B cells, and binding revealed as before. After a 1-hour incubation at 4°C, B lymphocytes were fixed with 4% filtered p-formaldehyde solution, cytospined and visualized on a Leica TCS laser scanning confocal microscope at 100ϫ magnification. Slides were mounted with Vectashield from Vector Laboratories. Acquisition was made with TCS NT Leica software.

Immunoprecipitation

B cells were incubated with 20 g/mL anti-IgM Ab-coated beads and/or 20 mg/mL IVIg for 20 minutes at 4°C before activation at 37°C for 3 minutes. The cells were then lysed in 50mM Tris (hydroxymethyl) aminomethane, 150mM NaCl, 5mM sodium fluoride, 2mM ethylenediaminetetraacetic acid, 40 M sodium orthovanadate, protease inhibitor cocktail and 0.2% Brij-96 (all from Sigma-Aldrich) for 1 hour at 4°C. After centrifugation, supernatants were collected and pre-cleared with 50 L protein G-coated Sepharose beads for 30 minutes at 4°C. The lysates were incubated with 10 g/mL rabbit anti-CD22 Ab, or rabbit anti-PY476-SHP-1 Ab and 100 L protein G-coated Sepharose beads overnight at 4°C. The beads were washed 5 times with lysis buffer and bound proteins in the immunoprecipitates eluted with 80 L of warm reducing loading buffer containing 0.125mM Tris, 10% glycerol, 4% SDS and 1.8% ␤-mercaptoethanol, and separated on 7% polyacrylamide gel electrophoresis (PAGE). WB was carried out with the same anti-CD22 and anti-SHP-1 Abs. Bindings of the Abs was revealed with HRP-conjugated anti-rabbit Ab (Amersham) and enhanced chemiluminescence (ECL) detection kit.

Western blotting

Lysates were prepared as described above and analyzed on 7% to 13% SDS-PAGE. Separated proteins were transferred to a polyvinylidene difluoride membrane, and quenched with 5% nonfat milk in PBS or with 5% of BSA for 1 hour at room temperature and then incubated with primary Ab for 1 to 6 hours, depending on the Ab. Rabbit anti-caspase 9 and anti-caspase 3 Abs were from Cell Signaling Technology. Rabbit anti-CDK2, mouse anti-E2F, anti-phosphoP38 (PT180ϩPY182), anti-phospho-Erk1/Erk2, anti-p27, and anti-p85 of phosphoinositide 3-kinase Abs were from Abcam. The anti-poly ADP-ribose polymerase (PARP) was Ab from Roche Applied Science and HRP-conjugated sheep anti-mouse as well as HRP-conjugated donkey anti-rabbit Abs were from GE Healthcare. These Abs were used at 1 in 40 000 dilution in 1% nonfat milk protein containing 0.05% Tween in PBS. Bound Abs were revealed using the ECL kit.

Statistical analyses

Analysis of variance and 2-tailed paired Student t test were used to determine the significance of difference between sample means. Significance refers to difference from the controls unless otherwise indicated.

Results

IVIg induces B-cell apoptosis through a selective pathway

We first examined the influence of IVIg on B-cell survival. FITC-AV/IP staining of blood and tonsillar primary B cells and lymphoma B-cell line cells revealed that these cells underwent apoptosis in a dose-dependent and time-related manner in the presence of IVIg (Figure 1A and supplemental Figure 1, available on the Blood Web site; see the Supplemental Materials link at the top of the online article). IVIg at 20 mg/mL significantly reduced cell viability at 16 to 24 hours (P Ͻ .005). Thus, the basal rate of apoptosis at 16 and 24 hours were significantly enhanced by the presence of IVIg. Intriguingly, B lymphocytes from blood and Ramos cells were somewhat resistant to spontaneous and IVIginduced death, compared with tonsillar B cells.

To ensure that IVIg was the genuine killing agent, this preparation was dialyzed against RPMI 1640 to exclude that stabilizers were responsible for cell death. We repeated the experiments using FabЈ 2 fragments of IVIg instead of the whole molecules. Culturing dialyzed IVIg preparation with tonsillar B cells showed similar kinetics, thus excluding a role for stabilizers. In addition, significant differences in the uptake of AV conjugate by the B cells was seen when the cells were cultured in the presence of the complete Ig comparing with their F(abЈ) 2 fractions at 8 hours (Figure 1B). Next, the induction of apoptosis was assessed with high protein concentration by comparing the rate of cell death in the presence of IVIg with an equimolar concentration of bovine serum albumin (BSA). The apoptosis rate in the presence of BSA was identical to spontaneous apoptosis and significantly less than with IVIg (P Ͻ .01).

The binding of IVIg to B cells is mediated by interaction with CD22

Given that no direct interaction between IVIg and B cells has hitherto been proven, we labeled IVIg with FITC to obtain direct evidence for its binding to B cells. The IVIg bound Ramos cells in a dose-dependent manner (Figure 2A), until the cells were saturated, thus, confirming specificity of the binding. To identify the potential molecule through which IVIg bind B cells, we analyzed the interactions between IVIg and B cells obtained from different sources. This experiment was carried out based on the hypothesis that different B-cell populations would have different expression levels of the molecule(s) to which the IVIg binds. This experiment revealed that, indeed, the IVIg bound more to Ramos cells than to cells from tonsils and blood (Figure 2B). IVIg could thus have a selective effect on a subpopulation of B cells.

To gain insight into how IVIg modulate B-cell functions, we examined the capacity of IVIg to bind potential membrane proteins on the B cells. B cells express several molecules that have potential to modulate B-cell functions upon interaction with their specific ligands and receptors. CD22, a member of the SA binding Ig Siglec family, is one such key molecule. It is a B cell-specific glycoprotein that associates with the BCR. The Fc parts of IVIg have a Siglec N-linked glycan covalently linked with a core containing N-acetylglucosamine and mannose. Further modification of this core is observed in the IgG pool of which 5% terminate with SA.

Confocal microscopy analyses served to address the issue of CD22 as a ligand for IVIg. The use of FITC-conjugated IVIg confirmed that within the IVIg preparation, some IgG bind to the surface of B cells (Figure 3A). This was contrasted with the binding of aggregated IgG, used as positive control and goat anti-human IgG as a negative control. The pattern of IVIg binding to the B cells membrane was somewhat similar to that obtained for the binding of aggregated IgG. However, the binding of aggregated IgG show larger green fluorescence lumps than seen for the IVIg, which appears to spread thinner on the surface. Overlay of FITC-conjugated IVIg and TRITC-conjugated CD22 shown as yellow clearly indicated that IVIg and CD22 colocalized on the B-cell surface (Figure 3B). To directly show that the interaction between CD22 and IVIg involved SA on IgG, IVIg SAϩ and IVIg SAϪ fraction were obtained and their purity verified by WB (Figure 3D). IVIg and IVIgSAϩ fraction, but not IVIg SAϪ bound SNA. Thus, only the IVIg SAϩ fraction colocalized with CD22 (Figure 3C upper panel), although some in the IVIg SAϪ fraction also bound to the B-cell membrane (Figure 3C lower panel).

Binding of the IVIg fraction to CD22 was further confirmed by immunoprecipitation. B cells were incubated with the IVIg preparations for 20 minutes at 4°C and for 5 minutes at 37°C, lysed and bound IgG-CD22 immunoprecipitated. Within the IVIg, IgG was associated with CD22 on the WB (Figure 3E). Interestingly, these experiments showed that the optimal binding of the IVIg preparation with CD22 occurred when the cell-IVIg mixtures were maintained at 37°C. These experiments provide evidence that the binding of IgG to CD22 is optimal at a physiologic temperature.

Effect of IVIg on BCR-mediated signaling involves CD22

To assess the likely effects of IVIg binding to B cells on BCRmediated signaling, we examined the strength of anti-IgMgenerated signaling. Although the exact dynamics of signaling events that translate BCR engagement to cell responses are not fully established, modulation of the level of intracellular calcium level is considered a reliable measure.

In addition, we assessed the phosphorylation status of several key molecules associated with BCR-mediated signaling by FACS. This technique allows the quantification of PY in individual subpopulations of cells, and reduces the effects of manipulation on measurement of B cells. Analyses were performed on gated viable cells. After 3 minutes of stimulation with anti-IgM, 22% of tonsillar B cells showed elevated levels of phosphorylated CD22 at its ITIMs in the absence of IVIg (Figure 4A). The percentage phosphorylated of CD22 in B-cells increased from 22 in cells cultured without IVIg to 58 of total CD22 in the presence of IVIg. In contrast, tyrosine phosporylation of CD19 diminished. The analysis also showed decreased phosphorylation of Lyn, and of BLNK at Y84, which is one of the 3 tyrosine residues involved in recruiting PLC␥2. [START_REF] Chiu | BLNK: molecular scaffolding through 'cis'mediated organization of signaling proteins[END_REF] Indeed, there was a significant reduction (P Ͻ .001) in PLC␥2 phosphorylation at Y759 in the presence of IVIg. No changes in the phosphorylation level of the signaling proteins were seen in unstimulated B cells in the absence, or presence of either total IVIg or its fractions (Figure 4A). However, when B cells were stimulated with anti-IgM in the presence of either 20mg/ML total IVIg or with 1mg/mL IVIg SAϩ there was a significant elevation in the level of CD22 phosphorylation and reduction in CD19, Lyn, BLNK and PLC␥2 phosphorylation (Figure 4B). The IVIg SAϪ fraction, in contrast, did not alter the level of phosphorylation of these proteins even at 20 mg/mL. All the noted down-regulation of phosphorylation strongly indicated an effective modulation of the calcium pathway.

To confirm that the reduction in the calcium signaling pathway was due to CD22, we immunoprecipitated CD22 and measured associated SHP-1. As a result of hypo-phosphorylation of CD19, Lyn, BLNK and PLC␥2, SHP-1 was more recruited by adherence of IVIg to CD22 (Figure 5A). The constitutive association between CD22 and SHP-1 was slightly increased upon BCR cross-linking but significantly so in the presence of IVIg (Figure 5B). The ability of CD22 to modulate B-cell responses by interacting in trans with glycosylated Ags has previously been demonstrated. [START_REF] Courtney | Sialylated multivalent antigens engage CD22 in trans and inhibit B cell activation[END_REF] Here, we show that high concentrations of IVIg could act in a similar manner and reduce the strength of BCR-mediated signaling. This occurs through the CD22-SHP-1 inhibitory pathway and by downregulating calcium pathway activation, that determine the fate of B cells. (B) Tonsillar B cells were stained with rabbit anti-CD22 followed by TRITC conjugated anti-rabbit Ab. After several washes, the cells were incubated with IVIg and and followed by FITC-conjugated anti-human IgG F(abЈ)2. (C) Sialylated IVIg (IVIg SAϩ) and asialylated (IVIg SAϪ) fractions were used to stain B cells followed by FITC-conjugated F(abЈ) 2 anti-human IgG antibody. B cells were also stained with anti-CD22. (D) IVIg was separated according to their SA content using SNA affinity chromatography. Purity of the fractions was assessed by lectin blotting. Staining for CD22 was followed by secondary HRP-conjugated Ab, and the blots developed using the ECL system. (E) Tonsillar B cells were incubated with IVIg before warming the cells up at 37°C (middle lane) or examined without warming (left lane). Control for warming up at 37°C without IVIg is also shown (right lane) is shown. After the incubation, cell lysates were prepared and immunoprecipates with protein G Sepharose beads and analyzed by WB using anti-human CD22 Ab followed by secondary HRP conjugated Ab. The blots were developed using the ECL system.

IVIg promotes apoptosis through sustained activation of Erk 1/2 and cell-cycle arrest

Erk1/2 phosphorylation results from BCR signaling, and influences the behavior of B-cells. [START_REF] Irish | Kinetics of BCR signaling in human B cell subsets mapped by phosphospecific flow cytometry[END_REF] As IVIg binding influences BCR signaling, we next assessed the effect of this interaction on Erk1/2 phosphorylation. For this purpose, we studied tonsillar B cells in presence of IVIg, with and without BCR engagement. Treatment with IVIg for 30 seconds induced Erk1/2 phosphorylation sustained after 2 minutes (Figure 6A). Further study of the impact of IVIg binding on other MAPK kinases showed that IVIg decreased p85 PI3K level but increased p38 phosphorylation (Figure 6B). Erk1/2 thus has a dual role in regulating cell proliferation and cell cycle arrest, so that the outcome of altered phosphorylation of these proteins depends on several factors including duration of the modulating stimulus.

To determine the consequences of these changes in phosphorylation on the fate of B cells, we studied the level of several proteins involved in the cell cycle. Because IVIg promoted apoptosis, we examined the effect of IVIg binding on proteins regulating the G 1 phase of the cell cycle. Previous studies have highlighted a critical role for p27/Kip1 in cell cycle progression though the G 0 -G 1 phase, [START_REF] Nevins | Role of the Rb/E2F pathway in cell growth control[END_REF] as we also confirmed in another model. [START_REF] Hillion | IL-6 contributes to the expression of RAGs in human mature B cells[END_REF] Treatment of B cells with IVIg resulted in a significant increase in the level of p27/Kip1 with and without BCR engagement (Figure 7A). Semiquantitative measurements, based on band density showed a 2-fold increase in the level of p27/Kip1 expression. In contrast, a 24-hour treatment of B cells with IVIg abrogated cyclin-dependent kinase (CDK)2 expression. This reduction was exaggerated by BCR engagement, in line with up-regulation of its inhibitor p27/Kip1.

Because down-regulation of CDK2 inhibits the E2F-1 growth promoting agent, [START_REF] Krek | Negative regulation of the growth-promoting transcription factor E2F-1 by a stably bound cyclin A-dependent protein kinase[END_REF] we therefore evaluated the expression of the latter TF. A 24-hour BCR engagement in the presence of IVIg resulted in a striking reduction in the E2F-1 expression (Figure 7A). Consistent with this data, IVIg induced a G 1 cell cycle arrest and led to apoptosis. Both processes are controlled by p53, which governs expression of key pro-and anti-apoptotic molecules such as Bcl2, which, in turn, regulate activation of caspases. In this respect, treatment of B cells with IVIg and BCR engagement decreased the levels of pro-caspases 3 and 9, and increased that of cleaved PARP (Figure 7B).

All in all, the data generated in this study indicate that IVIg modulates B-cell responses promotes cell cycle arrest and apoptosis by mediating Erk phosphorylation and p38 activation while reducing PI3K activation.

Discussion

An ever-growing array of autoimmune conditions has successfully been treated with IVIg. Although the exact mode of action remains elusive, IVIg modulates the immune system through binding to membrane receptors. B lymphocytes possess some of these receptors. In this study, we provide evidence that CD22 is a key receptor in the beneficial effects of IVIg. Further, we report for the first time that IVIg binds primary human B cells and modulate BCRmediated signaling. Numerous investigators have previously demonstrated that IVIg influences B cells, [START_REF] Prasad | Therapeutic preparations of normal polyspecific IgG (IVIg) induce apoptosis in human lymphocytes and monocytes: a novel mechanism of action of IVIg involving the Fas apoptotic pathway[END_REF][START_REF] Dussault | Immunomodulation of human B cells following treatment with intravenous immunoglobulins involves increased phosphorylation of extracellular signalregulated kinases 1 and 2[END_REF][START_REF] De Grandmont | IVIg induces the in vitro differentiation of human B lymphocytes and the secretion of IgG[END_REF][START_REF] Proulx | Spontaneous internalization of IVIg in activated B cells[END_REF] however, none have really identified the pathways through which B-cell fate would be influenced by IVIg. Modulation of Erk activation in B cells by IVIg has been described before [START_REF] Dussault | Immunomodulation of human B cells following treatment with intravenous immunoglobulins involves increased phosphorylation of extracellular signalregulated kinases 1 and 2[END_REF] in association with growth inhibition. In this study, we report that IVIg influences the cell cycle and leads to apoptosis. A previous report has proposed that IVIg induces apoptosis though the Fas pathway. Altough our study has identified a different mechanism, we cannot totally exclude apoptosis through the-Fas pathway, because different IVIg preparations can vary as they are from different donors. [START_REF] Prasad | Therapeutic preparations of normal polyspecific IgG (IVIg) induce apoptosis in human lymphocytes and monocytes: a novel mechanism of action of IVIg involving the Fas apoptotic pathway[END_REF] Although IVIg has been shown not to affect B-cell viability when stimulated with CD40L, [START_REF] De Grandmont | IVIg induces the in vitro differentiation of human B lymphocytes and the secretion of IgG[END_REF] we observed that B-cell activation through the BCR was critically important, underlying the role of B-cell activation state in IVIginduced apoptosis.

There is good evidence that CD22 interacts in a cis fashion with ligands to control BCR-mediated signaling. However, controversy persists as to the requirement for Ag glycosylation. In this case, IVIg could bind CD22 in trans to influence signaling. In fact, the latter ligands are so ubiquitous that it renders impossible to discriminate the contribution of cis and trans modes of interactions. We reasoned that IVIg SAϩ could bind CD22 and interfere with BCR-mediated signaling. Modulation of B-cell activation by IVIg, especially through the calcium pathway, is due to binding to CD22 and recruitment of SHP-1. Together, our results indicate that trans interaction by CD22 suppresses BCR-mediated B-cell activation, as previously proposed. [START_REF] Courtney | Sialylated multivalent antigens engage CD22 in trans and inhibit B cell activation[END_REF][START_REF] Gross | Developmental acquisition of the Lyn-CD22-SHP-1 inhibitory pathway promotes B cell tolerance[END_REF] Incubation of IVIg with anti-Ig-activated B cells enhances CD22 phosphorylation, which is necessary for SHP-1 recruitment. [START_REF] Jin | Sialic acid binding domains of CD22 are required for negative regulation of BCR signaling[END_REF] Similar results were obtained using synthetic sialosides to abrogate ligand binding in in vitro and in vivo. [START_REF] Durandy | Intravenous immunoglobulins-understanding properties and mechanisms[END_REF] Thus, ligand binding cis and trans modes are required for the inhibitory role of CD22. [START_REF] Kelm | The ligand-binding domain of CD22 is needed for inhibition of the BCR signal, as demonstrated by a novel human CD22-specific inhibitor compound[END_REF] In this regard, a recent study has provided evidence for the effect of sialylated antigens on B cells. [START_REF] Duong | Decoration of T-independent antigen with ligands for CD22 and Siglec-G can suppress immunity and induce B cell tolerance in vivo[END_REF] These investigators showed that B-cell Ag activation could be modulated by interaction of glycosylated antigen with CD22 leading to the induction of tolerance. The avidity of IVIg-CD22 interaction is a challenge to measure, but probably weaker than the maximum CD22 cross-linking. There are, however, some suggestions that direct interaction between CD22 and Ag could influence the fate of B cells by modulating secondary signals such as Erk. [START_REF] Tuscano | CD22 cross-linking generates BCR-independent signals that activate the JNK/SAPK signaling cascade[END_REF] Furthermore, a link between CD22 engagement and apoptosis has been suggested for B cells in patients with chronic lymphocytic leukemia. [START_REF] Qu | Bispecific anti-CD20/22 antibodies inhibit B-cell lymphoma proliferation by a unique mechanism of action[END_REF] Immunotoxins that bind CD22 would favor cytotoxicity through activating caspase 3 and cleaving PARP. [START_REF] Decker | Induction of caspase-dependent programmed cell death in B-cell CLL by anti-CD22 immunotoxins[END_REF] Our data indicated that a similar outcome of cell death is achieved by IVIg in B cells. However, it remains to be formally demonstrated that cytotoxicity is mediated by IVIg-CD22 interaction in this setting.

Analysis of the potential pathways through which IVIg-CD22 could influences B cells revealed that IVIg enhances Erk1/2 phosphorylation as suggested by others investigators. [START_REF] Dussault | Immunomodulation of human B cells following treatment with intravenous immunoglobulins involves increased phosphorylation of extracellular signalregulated kinases 1 and 2[END_REF][START_REF] Bayry | DC-SIGN and alpha2,6-sialylated IgG Fc interaction is dispensable for the anti-inflammatory activity of IVIg on human dendritic cells[END_REF] IVIg also enhanced p38-phosphorylation but down-regulated p85-activation. All these signaling molecules are involved in regulating apoptosis in B cells. [START_REF] Richards | Regulation of B-cell entry into the cell cycle[END_REF][START_REF] Opferman | Apoptosis in the development of the immune system[END_REF] The p38 MAPK plays a role in cell cycle progression of B cells that are exposed to oxidative stress. [START_REF] He | Syk is required for p38 activation and G2/M arrest in B cells exposed to oxidative stress[END_REF] In addition, p38 could induce G 1 cycle arrest and Bcl2 phosphorylation leading to its degradation in the proteasome. [START_REF] Rosini | NGF withdrawal induces apoptosis in CESS B cell line through p38 MAPK activation and Bcl-2 phosphorylation[END_REF] p38 and JNK are stressassociated PTKs that regulate apoptosis depending on the cell type and stimulus. [START_REF] Raingeaud | Proinflammatory cytokines and environmental stress cause p38 MAP kinase activation by dual phosphorylation on tyrosine and threonine[END_REF][START_REF] Kim | Involvement of p38 MAP kinase during iron chelator-mediated apoptotic cell death[END_REF] Although p38 has been implicated in apoptosis in several systems, the mechanism by which this is brought about is unknown. Hsu and colleagues reported that p38 mediates apoptosis through increased expression of Fas ligand. [START_REF] Hsu | p38 mitogen-activated protein kinase is involved in Fas ligand expression[END_REF] Our results showed that IVIg-CD22 interaction enhanced p38 phosphorylation and led to the activation caspase-3 cleavage, which is consistent with a recent report. [START_REF] O'donnell | Phosphatase inhibition augments anti-CD22-mediated signaling and cytotoxicity in non-hodgkin's lymphoma cells[END_REF] Analysis of potential pathways influenced by IVIg-CD22 interaction indicate that IVIg interferes with BCR signaling and B-cell responses through sustained Erk1/2 phosphorylation, and down-regulation of PI3K activation. The PI3K/Akt pathway (which is essential for survival) constitutes a common response pathway of cells to growth-factor stimulation. Erk1/2 has a dual role in regulating B-cell responses, and is thus required for cell proliferation as well as cell cycle arrest. [START_REF] Lloyd | Distinct functions for Erks?[END_REF][START_REF] Vantaggiato | Erk1/2 MPA kinases affect Ras-dependent cell signaling differentially[END_REF] However, our study could not exclude the possibility that CD22 engagement by IVIg disrupts the MAPK pathway, cell cycle arrest and apoptosis as has been reported for CD22 cross-linking. [START_REF] Decker | Induction of caspase-dependent programmed cell death in B-cell CLL by anti-CD22 immunotoxins[END_REF][START_REF] O'donnell | Phosphatase inhibition augments anti-CD22-mediated signaling and cytotoxicity in non-hodgkin's lymphoma cells[END_REF][START_REF] Herrera | Immunotoxins against CD19 and CD22 are effective in killing precursor-B acute lymphoblastic leukemia cells in vitro[END_REF] This results in cell cycle arrest at G 1 phase, down-regulation of CDK2 and up-regulation of its inhibitor p27, which activates caspase-3. This findings leads to the prediction that other inducible CDKs, such as CDK4 and CDK6, could be down-regulated by IVIg-CD22 interaction. For example, down-regulation of CDK2/6 causes inactivation of pRb, which down-regulates the E2F family of TFs, known as proteins required for S phase. [START_REF] Deshpande | Cyclins and CDKs in development and cancer: a perspective[END_REF] Indeed, IVIg reduced E2F-1 protein expression, and thereby related target genes.

In summary, we believe that IVIg binds B cells by interacting with CD22. This ligation interferes with several BCR-mediated signaling pathways. These include inhibition of the PLC␥2 cascade, sustained activation of Erk1/2, enhanced p38 activation and down-regulation of PI3K. These changes are associated with the induction of CDK inhibitor p27/Kip1, which inhibits cell cycle progression at the G 1 phase and results in with apoptosis. Modulation of the viability of primary B cells and the growth of cancer line B cell provides for new interest in IVIg therapy in the control of B-cell malignancies.

Figure 2 .

 2 Figure 2. IVIg binds to B-cell surface in a dose-dependent manner. (A) Ramos B cells were incubated with a range of FITC-labeled IVIg for 1 hour at 4°C. Binding of IVIg was determined by FACS and MFIs measured at each point of FITC-IVIg binding. Unconjugated FITC was used as the negative control. (B) Blood B cells (open bars), tonsillar B cells (gray bars) and Ramos B cells (closed bars) were incubated with increasing concentrations of IVIg-FITC and binding measured by FACS.

Figure 1 .

 1 Figure 1. Reduced B-cell viability in the presence of IVIg. (A) Blood and tonsillar B cells and lymphoma cell line B cells (2 ϫ 10 5 ) were incubated for 24 hours with increasing concentrations of IVIg. Cell viability was assessed by FITC-AV uptake. BSA was used at 20 mg/mL as a control for the effect of high protein content. (B) Tonsillar B cells were cultured in the presence of 20 mg of IVIg (f), IgG F(abЈ)2 (ࡗ), BSA (OE), or in medium alone (F) for the indicated periods. The figure depicts the mean Ϯ SD values from 5 independent experiments. Statistical analysis was performed using the 2-tailed paired Student t test. *P Ͻ .05 vs cells cultured in medium alone.

Figure 3 .

 3 Figure 3. IVIg binds to CD22 on the surface of B cells. (A) Tonsillar B cells were incubated with IVIg (left panel), or with aggregate IgG (middle panel). The binding of IgG was revealed using FITC-conjugated goat FabЈ2 anti-human IgG. The right panel shows binding of the secondary Ab alone with the TRITC-conjugated donkey anti-rabbit IgAb, used as negative control.(B) Tonsillar B cells were stained with rabbit anti-CD22 followed by TRITC conjugated anti-rabbit Ab. After several washes, the cells were incubated with IVIg and and followed by FITC-conjugated anti-human IgG F(abЈ)2. (C) Sialylated IVIg (IVIg SAϩ) and asialylated (IVIg SAϪ) fractions were used to stain B cells followed by FITC-conjugated F(abЈ) 2 anti-human IgG antibody. B cells were also stained with anti-CD22. (D) IVIg was separated according to their SA content using SNA affinity chromatography. Purity of the fractions was assessed by lectin blotting. Staining for CD22 was followed by secondary HRP-conjugated Ab, and the blots developed using the ECL system. (E) Tonsillar B cells were incubated with IVIg before warming the cells up at 37°C (middle lane) or examined without warming (left lane). Control for warming up at 37°C without IVIg is also shown (right lane) is shown. After the incubation, cell lysates were prepared and immunoprecipates with protein G Sepharose beads and analyzed by WB using anti-human CD22 Ab followed by secondary HRP conjugated Ab. The blots were developed using the ECL system.

Figure 4 .

 4 Figure 4. IVIg modulate BCR-associated signaling pathways. Phosphorylation levels of CD22, CD19, Lyn, BLNK and PLC␥2 were determined by FACS. (A) Tonsillar B-cell subpopulations were incubated with or without 20 mg/mL IVIg or 1 mg/mL IVIg SAϩ or 20 mg/mL IVIg SAϪ for 20 minutes at 4°C and warmed up for 3 minutes at 37°C without any stimulation. B cells were subsequently fixed with p-formaldehyde, permeabilized and then stained with rabbit anti-phospho-CD22, anti-phosphoCD19, and anti-phospho-Lyn and developed by FITC-donkey anti-rabbit Ig Ab, and with PE-conjugated anti-phospho-BLNK and anti-phospho-PLC␥2. (B) Tonsillar B cells were stimulated with anti-IgM Ab-coated beads for 3 minutes at 37°C in the presence or absence of 20 mg/mL IVIG, 1 mg/mL IVIg SAϩ or 20 mg/mL IVIg SAϪ for 20 minutes at 4°C. Activated B cells were stained with anti-phospho-CD22, antiphospho-CD19, anti-phospho-Lyn, anti-phospho-BLNK and anti-phospho-PLC␥2 with the same protocol as before. The results represent mean percentages Ϯ SD of 3 independent experiments. Statistical analyses were performed using the 2-tailed paired Student t test. *P Ͻ .001 vs cells incubated with no IVIg.

Figure 5 .

 5 Figure 5. IVIg binding to CD22 enhances SHP-1 recruitment. (A) Tonsillar B cells were incubated with anti-IgM-coated beads in the presence or absence of IVIg for 20 minutes at 4°C and 3 minutes at 37°C before lysing the cells. Lysates were incubated with anti-CD22 and protein G-coated Sepharose beads and immunoprecipitates analyzed by WB using anti-CD22 or anti-SHP-1 Ab. Nonstimulated B cells (Unst.) are shown as control. (B) The degree of coprecipitation is presented as a ratio of intensity of SHP-1 band/intensity of CD22 band.

Figure 6 .

 6 Figure 6. IVIg modulates the MAPK pathway. Tonsillar B cells were incubated with anti-IgM-coated beads in the presence or absence of IVIg for 30 seconds or 2 minutes before lysis. Total cell lysates were prepared for WB using anti-phospho ERK1 and anti-phospho ERK2 (A) or anti-phospho p38 and anti-PI3K (B) Abs. Values indicate relative density of the WB bands normalized to that obtained for ␤-actin. Data are representative of 3 separate experiments.

Figure 7 .

 7 Figure 7. Effect of IVIg on cell cycle protein expression and apoptosis. Tonsillar B cells were incubated with anti-IgM-coated beads in the presence or absence of IVIg for 24 hours before lysis. Total cell lysates were separated and stained in WB with (A) anti-p27, anti-CDK2 and anti-E2F Abs, and (B) anti-caspase 9, anti-caspase 3, and anti-PARP Abs. Values indicate relative density of the bands normalized to that obtained for ␤-actin. Data are representative of 3 independent experiments.

Acknowledgments

We thank Ms Genevie `ve Michel and Ms Simone Forest for expert secretarial assistance.

Conflict-of-interest disclosure: The authors declare no competing financial interests.

Correspondence: Professor Pierre Youinou, Laboratory of Immunology, Brest University Medical School Hospital, BP 824, F29609, Brest, France; e-mail: youinou@univ-brest.fr.