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Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 2007 Jul 17;104(30):12451–12456. doi: 10.1073/pnas.0703451104

Human endometriosis is associated with plasma cells and overexpression of B lymphocyte stimulator

Aniko Hever *, Richard B Roth *, Peter Hevezi *, Maria E Marin †, Jose A Acosta †, Hector Acosta †, Jose Rojas †, Rosa Herrera †, Dimitri Grigoriadis *, Evan White *, Paul J Conlon *, Richard A Maki *, Albert Zlotnik *,‡
PMCID: PMC1941489  PMID: 17640886

Abstract

Endometriosis affects 10–20% of women of reproductive age and is associated with pelvic pain and infertility, and its pathogenesis is not well understood. We used genomewide transcriptional profiling to characterize endometriosis and found that it exhibits a gene expression signature consistent with an underlying autoimmune mechanism. Endometriosis lesions are characterized by the presence of abundant plasma cells, many of which produce IgM, and macrophages that produce BLyS/BAFF/TNFSF13B, a member of the TNF superfamily implicated in other autoimmune diseases. B lymphocyte stimulator (BLyS) protein was found elevated in the serum of endometriosis patients. These observations suggest a model for the pathology of endometriosis where BLyS-responsive plasma cells interact with retrograde menstrual tissues to give rise to endometriosis lesions.

Keywords: autoimmunity, cytokines, microarrays

Endometriosis, adenomyosis, and uterine fibroids (leiomyomata) represent the most common benign gynecological diseases of women of reproductive age and are collectively responsible for significant morbidity. Affecting an estimated 10–20% of women in this age group (1), endometriosis is associated with pain and other discomforts and may play a role in infertility (2, 3). Despite its high prevalence, the underlying mechanism of endometriosis is still poorly understood. Diagnosis of endometriosis is also difficult, relying primarily on symptomatology (i.e., dysmenorrhea or painful menstruation), with laparoscopic examination being the only definitive confirmation. Because the latter is a surgical procedure, the diagnosis and subsequent treatment of endometriosis are often delayed (4). Treatment options for endometriosis include surgery and/or suppression of ovarian steroids (5). However, these strategies may offer only temporary relief because the disease usually recurs (6). Endometriosis may be confused with other gynecological disorders, such as adenomyosis or uterine fibroids. In endometriosis, endometrial tissue is found outside the uterus. In adenomyosis, the endometrium grows abnormally into the myometrium, while uterine fibroids are pathological outgrowths of the myometrium. The interrelationship of these diseases, if any, has not been well established (7). We have profiled endometriosis at the molecular level by using genomewide genearrays. Our results indicate that endometriosis, in contrast to adenomyosis or leiomyomas, exhibits a unique molecular signature that reveals underlying immune system-associated pathology.

Results

Endometriosis Exhibits a Unique Gene Expression Pattern Characterized by Underlying Immunological Pathways.

We sought to understand endometriosis at the molecular level. Toward this end, we performed microarray analyses of these three diseases as well as control tissues (endometrium, myometrium, and ovary) by using the Affymetrix U133 Plus 2.0 genomewide gene array (Affymetrix, Santa Clara, CA). We compared ovarian endometriosis lesions with control endometrium obtained from the same patient at the same time to avoid detecting gene expression differences due to the menstrual cycle (8). Genes significantly up-regulated in endometriosis versus control endometrium were analyzed by using ingenuity pathway analysis (IPA) (9). This analysis determined that genes associated with immune responses represent the most significant functional class in endometriosis, including 53 genes with altered expression that form two networks shown in Fig. 1. These genes are shown in supporting information (SI) Table 1. Network 1 identified genes associated with immune responses, cell-to-cell signaling/interaction, and hematological system development/function; whereas Network 2 included genes associated with inflammatory diseases, immune responses, and cellular movement. These results suggest that endometriosis has an immunological basis.

Fig. 1.

Fig. 1.

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IPA was used to identify pathways altered in endometriosis compared with normal endometrium, and the two most significant networks associated with immune responses are shown (merged). The significance values of each were Network 1, P = 10−38, and Network 2, P = 10−26. For explanation of significance values, see Materials and Methods.

SI Fig. 4 is a heat map indicating many genes up-regulated in endometriosis versus control endometrium. SI Table 2 lists the top 50 genes up-regulated in endometriosis versus normal endometrium. All these genes had false discovery rates of P < 0.05 (Benjamini–Hochberg multiple testing correction), indicating that their induction in endometriosis was highly significant. Because all the endometriosis samples were ovarian, we also filtered out genes highly expressed in the ovary by using our database of gene expression that includes 93 female human tissues as described (10). We also compared endometriosis with uterine fibroids and adenomyosis (one-way ANOVA; P < 0.00001) and observed that each of these diseases exhibits a discrete molecular signature of gene expression (SI Fig. 5). When adenomyosis or uterine fibroid-associated genes were analyzed by IPA, no immune response-associated pathways were detected (11).

Endometriosis Gene Expression Pattern Indicates Presence of Plasma Cells and Activated Macrophages.

The endometriosis-associated genes included several immunoglobulins, complement components, and cytokines such as PBEF (pre-B cell colony-enhancing factor) (12) and BlyS/BAFF/TNFSF13B (13). However, B cell markers like CD19, CD20, or the transcription factor PAX-5 were not detected in endometriosis lesions. The Ig switching mechanism involves the expression of activation-induced cytidine deaminase (14), which was not detected, indicating that there are few or no B cells undergoing Ig class switching in endometriosis lesions. Similarly, T cell-specific genes (T cell antigen receptor chains α and β, as well as CD3ε) were not up-regulated in endometriosis. This paucity of mature B and T cells was confirmed by immunohistochemistry, which revealed no CD20-positive cells and few CD3ε-positive cells in the lesions (data not shown). Conversely, many genes associated with macrophage activation were strongly up-regulated in endometrial lesions such as CD163 (a macrophage scavenger receptor for hemoglobin) (15), CD14 (lipopolysaccharide receptor) class II major histocompatibility complex molecules, and the intracellular adhesion molecule (ICAM-1) (SI Table 2). Last, no neutrophils were observed in the lesions.

Histopathology of Endometriosis Lesions Confirmed Presence of Plasma Cells and Macrophages.

Tissue sections stained with H&E revealed abundant plasma cells and macrophages (Fig. 2a) in the endometriosis lesions. Additional endometrial sections were stained with antibodies against IgM (Fig. 2b) (or λ/κ light chains, data not shown), revealing that ≤30% of the plasma cells were producing IgM. The rest of the plasma cells were likely producing IgG because we observed strong up-regulation of IgG mRNA (the gene array IgG data were unreliable, so we confirmed this by PCR; data not shown), whereas no up-regulation of IgA or IgE mRNAs was detected. The presence of hemosiderin-containing macrophages was also evident in endometriosis lesions (Fig. 2c). Staining for the macrophage-specific antigen CD68 (macrosialin/gp110) confirmed the presence of abundant macrophages in endometriosis lesions (Fig. 2d). Histological examination of control endometrium revealed no plasma cells with few macrophages present. Thus, immunopathological examination of endometriosis lesions confirmed the molecular results, pointing to a strong presence of plasma cells (some producing IgM) and activated macrophages.

Fig. 2.

Fig. 2.

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Endometriosis lesions contain macrophages and plasma cells. (a) Staining with H&E showing plasma cells in endometriosis lesions (arrows). (Magnification: ×10.) (b) Immunohistochemical analysis shows that some of the plasma cells express IgM (arrows). (Magnification: ×40.) (c) H&E staining revealed many macrophages with hemosiderin in the lesions of ovarian endometriosis. (Magnification: ×10.) (d) Staining with anti-CD68 confirmed the presence of macrophages in these lesions. (Magnification: ×40.)

Role for B Lymphocyte Stimulator (BlyS) in Endometriosis.

Several of the cytokines up-regulated in endometriosis lesions are known to specifically activate B cells (16). BLyS was of particular interest because it was highly up-regulated, is a cytokine necessary for normal B cell development, and also induces differentiation into plasma cells (17). We confirmed that BLyS mRNA levels were elevated in endometriosis lesions by real-time PCR (Fig. 3a). BLyS is capable of binding to three different receptors of the TNF superfamily (BAFF-R, TACI, and BCMA) (18). We analyzed the expression of these receptors by real-time PCR. Only BCMA was strongly up-regulated in endometriosis lesions (Fig. 3b). Interestingly, BCMA is expressed by plasma cells (19) and supports their survival (18). These observations strongly suggest that the BLyS/BCMA interaction supports the plasma cells observed in endometriosis (Fig. 2 a and b). Immunohistochemical staining revealed that the cells producing this cytokine were macrophages present in the endometriosis lesions (Fig. 3c). We then measured BLyS protein in the sera of 31 endometriosis patients and found that its levels were significantly elevated compared to 21 healthy controls (Fig. 3d). Patients with adenomyosis and uterine fibroids did not have elevated BLyS serum levels compared to controls (data not shown). More important, BLyS levels are elevated in other autoimmune diseases, including rheumatoid arthritis, systemic lupus erythematosus (SLE), and Sjögren's syndrome (20–23).

Fig. 3.

Fig. 3.

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BLyS and its receptor BCMA are significantly altered in endometriosis. (a) Qualitative RT-PCR (TaqMan) confirmed the overexpression of BLyS mRNA in endometriosis vs. control endometrium, as originally identified through microarray analysis. Relative expression values are expressed as mean of 2(40−Ct) ± SEM: endometrium, 100 ± 15 (n = 16); endometriosis, 886 ± 263 (n = 13). (b) BCMA mRNA was also found to be overexpressed in endometriosis vs. control endometrium by quantitative RT-PCR (TaqMan). Relative expression values are expressed as mean of 2(40−Ct) ± SEM: endometrium, 43 ± 13 (n = 16); endometriosis, 290 ± 89 (n = 13). (c) Immunohistochemical staining revealed that BlyS is produced by macrophages in endometriotic lesions. (Magnification: ×100.) (d) Individual serum BlyS protein levels in 21 healthy control females and 31 patients with endometriosis were measured by ELISA. The data show the mean pg/ml values of each group ± SEM: control, 523 ± 74; endometriosis, 1,695 ± 188.

Complement System Is Altered in Endometriosis.

Complement components were also strongly up-regulated in endometriosis lesions (Fig. 1 and SI Tables 1 and 2). The expression of some complement components fluctuates during the menstrual cycle, but the levels observed in endometriosis are much higher than those induced during the menstrual cycle (8). Several complement components are produced by endometrial cells (24, 25), and changes in complement levels have previously been reported to be associated with endometriosis (26, 27). We measured the serum levels of C3, C4, C6, and C7 complement components in patients with endometriosis, adenomyosis, and uterine fibroids compared to normal controls. Although no changes in serum C4, C6, and C7 were observed between endometriosis patients and controls, serum levels of C3 were slightly reduced [endometriosis patients, 939 ± 47 μg/ml; healthy controls, 1,352 ± 23 μg/ml (n = 26 and 11, respectively; P < 0.0001)]. Patients with adenomyosis or uterine fibroids had no significant changes in C3 levels. Lower C3 levels are consistent with findings from other autoimmune diseases, including SLE (28) as well as Sjögren's syndrome (20).

Are There Atypical Plasma Cells in Endometriotic Lesions?

Our results indicate that endometriosis lesions contain many activated macrophages and plasma cells with few or no mature B or T cells. Because typical B cell responses (those represented by B-2 cells) (29) require T cell help, these observations suggest that either the development of these immune responses occurs elsewhere (and the plasma cells then migrate to the endometriotic lesions) or the plasma cells in endometriosis represent atypical B cell responses similar to the B-1 or marginal zone cells that have been characterized in the mouse (29, 30). To further investigate this, we cloned and sequenced Ig μ clones from the endometriosis lesions and compared their sequences to Ig μ clones amplified from tonsils (representing normal B-2 responses). As shown in SI Fig. 6 A and B, the IgM clones produced in endometriosis lesions exhibit sharply reduced diversity when compared to tonsil-derived IgMs (SI Tables 3 and 4), suggesting that the IgM-producing plasma cells in endometriosis lesions produce a restricted Ig repertoire.

Discussion

Endometriosis is a common gynecological disease affecting a large number of reproductive age women. Treatment options include surgery (laparoscopy) as well as hormonal treatment targeting the GnRH receptor (1). Despite its high prevalence, its pathogenesis remains obscure. Although the exact origins of endometriosis are unclear, a widely accepted model postulates that endometriosis originates when some endometrial tissue, which is normally eliminated with the menstrual flow, instead undergoes retrograde flow (via the Fallopian tubes) to the ovaries and peritoneum (5, 31). The endometrial tissues then develop into inflammatory foci that respond to hormonal cycles and cause the symptoms associated with endometriosis. Previous endometriosis genomic studies focused on endometriosis-associated infertility (32), whereas another study identified Cyr61 as an endometriosis-associated gene (33). The present study represents the most comprehensive, genomewide analysis of ovarian endometriosis lesions to date. Given that endometriosis is difficult to diagnose, we also compared endometriosis with adenomyosis and uterine fibroids, two other common gynecological diseases that share symptoms with endometriosis. Our results indicate that these diseases can be readily differentiated at the molecular level (SI Fig. 5), and that only endometriosis exhibits a gene expression signature reminiscent of other autoimmune disorders (Fig. 1 and SI Tables 1 and 2). It has been postulated that endometriosis has an autoimmune etiology (34), but the nature of this disorder has not been characterized. Patients with endometriosis have a higher incidence of autoantibodies of both IgM and IgG isotypes directed against phospholipids, histones, or DNA (35). Our molecular and pathological analyses indicate that endometriosis lesions are characterized by the presence of abundant plasma cells and activated macrophages. One of the most up-regulated cytokines in these lesions is BLyS, a cytokine known to have important effects on B cells. Notably, BLyS has been shown to be critical for normal B-2 cell development (36), while high levels of BLyS overstimulate various B cell responses, leading to the initiation and exacerbation of autoimmune responses. Indeed, BLyS overexpression (using transgenic mice) has been shown to result in an autoimmune condition similar to SLE through the expansion of a population of B-1-related lymphocytes (29, 30). We should note that the activation of macrophages present in endometriosis lesions likely involves specific signals. For example, although macrophages were found to produce BLyS, other genes like IL1 or TNFα (present in diseases like SLE) (37), were not up-regulated, suggesting that endometriosis lesions represent a unique microenvironment.

B cell responses have been subdivided into thymus-independent (B1-like and related subsets) and thymus-dependent (B2) responses. B1 and related cells may not require T cell help to make antibodies, producing primarily IgM with some IgG3 (29, 30). B1 cells typically recognize antigens that induce multivalent cross-linking of the B cell receptor. Their repertoire is likely selected by self-antigens, making them potentially autoreactive. In the mouse, B1 cells primarily reside in the peritoneum, where they have been shown to be essential for resistance to certain pathogens and play an important role in mucosal immunity (38). B-1 cells are also known to exist in the human body and to produce autoreactive antibodies (38). For example, in arthritis, B1 cells make antibodies to self-antigens (i.e., DNA), which are involved in the disease process (39). The peritoneal location of B1-like cells is ideal for them to interact with retrograde menstrual tissues and to be involved in the pathogenesis of endometriosis (29, 30). The production of limited repertoire IgM (SI Fig. 6 A and B) by endometriosis-associated plasma cells suggests that some of these may derive from atypical B cells. This hypothesis is also consistent with other clinical findings of endometriosis, including the presence of autoantibodies against endometrial antigens (29, 30).

Other characteristics of endometriosis, like its recurring nature, are suggestive of autoimmunity. Furthermore, epidemiological studies have already documented a higher incidence of other autoimmune diseases among endometriosis patients (40).

Taken together, our results suggest the following model for the pathogenesis of endometriosis: The disease commences when menstrual tissue fails to exit the body and instead travels back through the fallopian tubes to the ovaries and/or peritoneal cavity (retrograde menstruation). These tissues then interact with B cells found in the peritoneal cavity (41). These cells likely include the precursors of the plasma cells we have detected in endometriosis lesions. BlyS production may be of particular importance for the plasma cells to develop and for the disease to become established. The fact that only BCMA mRNA is up-regulated in endometriosis lesions, along with the known biology of this receptor (18), strongly suggests that BLyS mediates the survival and activation of plasma cells through this receptor (35). This model predicts that neutralization of BLyS may have therapeutic effects in endometriosis.

Our results represent a significant advance in our understanding of the pathogenesis of endometriosis, but questions remain. First, it is unclear why only some women develop endometriosis, although retrograde menstrual flow likely occurs in most women of reproductive age. Second, what is the connection between dysmenorrhea and the plasma cells found in endometriosis lesions? We should note that CD5+ mouse B cells have been reported to respond to estrogen by increased antibody production (42). Thus, it is important to know what happens to the plasma cells present in endometriosis lesions after hormonal treatment (with GnRH agonists or antagonists). Finally, the plasma cells present in endometriosis lesions may represent lineages related to B1, marginal zone, or other atypical B cell subsets (29). The present study identified new directions that can now be approached experimentally, and future studies should investigate the nature and role that the plasma cells and activated macrophages play in endometriosis.

Materials and Methods

Human Samples.

Ten ovarian endometriosis and 10 matched control endometrium from the same patients (follicular phase, n = 2; luteal phase, n = 8), 10 adenomyosis (follicular phase, n = 2; luteal phase, n = 6; unknown phase, n = 2), and 10 uterine fibroid (leiomyoma) (follicular phase, n = 5; luteal phase, n = 3; unknown phase, n = 1; postmenopausal, n = 1) samples were obtained from Zoion Diagnostics (Hawthorne, NY). All donors were Caucasian, were not taking medications, and had not received hormone therapy before surgery. All of the diseased tissue samples were from discarded organs from scheduled surgeries, following Institutional Review Board approvals and informed consent. After removal, the samples were snap-frozen in liquid N2 and stored at −80°C. In addition to control endometrium and ovary samples, our database of gene expression (10) contains 93 tissues from five female donors. Flash-frozen tissue samples were obtained between 3 and 5 h postmortem (Zoion Diagnostics).

RNA Extraction and Microarray Analysis.

Total RNA was prepared from frozen samples by using TRIzol (43). Tissues were ground under liquid nitrogen in a mortar and pestle, and the resulting powder was solubilized in 1 ml of TRIzol (Invitrogen, Carlsbad, CA) in a FastPrep microfuge tube containing Lysing Matrix D ceramic beads. The RNA was subsequently isolated from the resulting TRIzol solution and further purified with an additional RNeasy step (QIAGEN, Chatsworth, CA). Five micrograms of total RNA from each sample was used to direct cDNA synthesis by using a T7 oligo(dT)24 primer and PowerScript RT (BD Clontech, Palo Alto, CA). After second-strand synthesis, double-stranded cDNA was used in a MEGAscript T7 RNA polymerase in vitro transcription reaction (Ambion, Austin, TX) containing biotin-labeled ribonucleotides, CTP (Enzo Diagnostics, Farmingdale, NJ) and UTP (Roche, Indianapolis, IN). The biotinylated cRNA was fragmented and hybridized to the Affymetrix Human Genome U133 Plus 2.0 gene array, stained with streptavidin phycoerythrin conjugate, and scanned in a GeneChip Scanner 3000 as described in the Affymetrix technical manual. The data sets presented in this study have been deposited in the GEO database (accession no. GSE7305).

Statistical Analysis.

All statistical analyses were performed by using Genedata Expressionist Pro 2.0 software (Genedata, Basel, Switzerland). Raw data from Affymetrix U133 Plus 2.0 cel files were uploaded to Expressionist Pro 2.0 and processed by using robust multiarray analysis (44) and subsequently log-transformed. Ovarian endometriosis and control endometrium samples were compared by using a two-sample paired t test with a significance threshold P value of 0.05. A Benjamini–Hochberg multiple testing correction was applied with a false discovery rate threshold of P < 0.05. Additionally, a fold-change filter was applied, with a minimum fold-change threshold of 2.0. Normal endometrium and ovary samples were also compared, with identical thresholds for significance as described above. To identify probe sets significantly up-regulated in ovarian endometriosis compared to control endometrium, an additional set of filters was applied because many probe sets were the result of ovary tissue contamination in ovarian endometriosis samples. If a probe set was identified as differentially expressed between normal ovary and normal endometrium, indicating a nondisease-related difference in expression, it was removed from the ovarian endometriosis vs. control endometrium differentially regulated list of probe sets. In some instances, probe sets were not removed if (i) the fold-change ratios of endometriosis/control endometrium and normal ovary/normal endometrium were of opposite signs, or (ii) the fold-change ratios of endometriosis/control endometrium and normal ovary/normal endometrium were at least 3-fold different from one another. Endometriosis, adenomyosis, and uterine fibroid samples were analyzed by using a one-way ANOVA (P < 0.00001) to identify significantly differentially regulated genes.

Ingenuity Pathway Analysis.

The genes that were identified as significantly up- or down-regulated in endometriosis vs. endometrium (two-sample paired t test, P < 0.01, filtered for ovary) were used for network and gene ontology analysis. Data sets containing the Affymetrix probe set identifiers, fold changes, and P values were uploaded into IPA software (www.ingenuity.com). The IPA program searches the Ingenuity Pathway Knowledge Base for interactions (known from the literature) between the uploaded genes and all other genes contained in the Ingenuity Pathway Knowledge Base and generates a series of networks. In addition, the program calculates a statistical score for each network. The significance reflects the number of modulated genes represented in a given network and is calculated by using Fisher's exact test. A P value <0.01 is considered significant.

Hierarchical Clustering.

The robust multiarray analysis data set, including all ovarian endometriosis vs. control endometrium, or endometriosis vs. adenomyosis and uterine fibroids, was compared by using a paired t test (P < 0.05 plus 2-fold change) or one-way ANOVA (P < 0.00001), respectively. Clustering was performed by using GeneSpring GX 7.3 (Agilent Technologies, Palo Alto, CA) software by applying the standard correlation with average linkage after per gene normalization.

Quantitative Real-Time PCR.

Total RNAs from diseased or normal samples were converted into single-stranded cDNAs by using the high-capacity cDNA Archive Kit (Applied Biosystems, Foster City, CA) according to the manufacturer's instructions. PCR was performed on the ABI PRISM 7900HT Sequence Detection System in 384-well plates. TaqMan Universal PCR MasterMix and Assays-on-Demand Gene Expression probes (Applied Biosystems) were used for the PCR step according to the manufacturer's instructions. Expression values obtained were corrected for loading by measuring 18S RNA relative expression levels and quantified by converting the cycle threshold (Ct) value into a numerical value by using the following formula: expression value = 2(40−Ct).

IgM PCR and Sequence Analysis.

Variable regions of IgM transcripts were cloned from endometriosis and control tissues (tonsil) by using SMART RACE cDNA amplification (Clontech, Mountain View, CA). 5′RACE-ready cDNA was prepared in a template-switching reaction using 500 ng of total RNA and the protocol supplied by the manufacturer. Reverse primers specific for IgM were: IGHM_1R 5′-TTCGTCTGTGCCCTGCATGACGTC and IGHM_2R 5′-AAGGCAGCAGCACCTGTGAGGTG.

Next, 100 ng of RACE-ready cDNA was used in an initial 30-cycle PCR using the manufacturer's universal primer mix and the 1R (outlying) gene-specific primers. The second 30-cycle PCR contained one-tenth of the products from the first reaction and the nested primers (nested universal and gene-specific; 2R). Advantage 2 polymerase was used in both reactions. We have found this proofreading polymerase to have a very low (<1 mutation in 104 bases) error frequency. RACE products were resolved by 1% low-melt agarose gel electrophoresis directly cloned into pCR2.1 TOPO (Invitrogen) and sequenced on both strands by using T3 and T7 primers.

Serum Samples from Endometriosis Patients.

Serum samples were collected from 31 patients confirmed to have endometriosis by laparoscopy and before receiving any hormonal treatment (Lupron, etc.). Control samples were obtained from 21 age-matched women who tested negative for routine clinical chemistry screenings and did not exhibit any symptoms of gynecological diseases.

Measurement of Serum Complement Factors.

Individual serum complement components were measured in sera from patients by ELISA (Abbott Laboratories, Chicago, IL) performed as per the manufacturer's instructions.

Measurement of Serum BLyS.

BLyS was measured by ELISA (R&D Systems, Minneapolis, MN) according to the manufacturer's specifications. The plates were read at an OD of 450 nm (with a correction wavelength set at 570 nm), and a standard curve was generated by using recombinant human BLyS.

Immunohistochemistry.

Paraffin-embedded sections were processed for epitope retrieval (heating under pressure). The sections were stained with appropriate dilutions of antibodies against human CD68 (clone 514H12; Serotec, Oxford, U.K.), polyclonal rabbit anti-human IgM (Dako, Glostrup, Denmark), or rat anti-human BLyS (ab16107; Abcam, Cambridge, MA). These antibodies were followed by appropriate anti-mouse, anti-rat, or anti-rabbit antibodies conjugated with biotin (CalTag, Burlingame, CA), followed by a streptavidin-biotin-peroxidase complex (Dako). The peroxidase was developed with diaminobenzidine, and the stained slides were counterstained with hematoxylin and examined by microscopy.

Supplementary Material

Supporting Information
pnas_0703451104_index.html (3.4KB, html)

Acknowledgments

We thank Dr. Leopoldo Santos for suggestions and critical review of the manuscript; Jerry Lee, Dorian Willhite, Anil Pahuta, and Xin-Jun Liu for technical support; and Drs. Hector Herrera, Wayne Muller, and Juan Pablo Flores for help with the immunohistochemistry experiments.

Abbreviations

BlyS

B lymphocyte stimulator

IPA

ingenuity pathway analysis

SLE

systemic lupus erythematosus.

Footnotes

Conflict of interest statement: A.H., R.B.R., P.H., D.G., E.W., P.J.C., R.A.M., and A.Z. were employed by Neurocrine Biosciences and owned common stock of this company at the time this study was performed.

This article contains supporting information online at www.pnas.org/cgi/content/full/0703451104/DC1.

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