Fatty Acid Amide Hydrolase Regulates Peripheral B Cell Receptor Revision, Polyreactivity, and B1 Cells in Lupus Free

The genetic basis of self-reactivity and B cell autoimmunity is poorly understood. This work focuses on a murine genetic interval, Sle2^z, which is associated with autoantibody, polyreactivity, and B1a cell expansion. Importantly, diverse autoantibody specificities, such as antierythrocyte, anticardiolipin, and anti-gp70 Abs, are associated with this interval in different strains (1–4). When the NZM2410-derived Sle2^z locus was introgressed onto the relatively nonautoimmune C57BL/6 (B6) background to yield B6.Sle2^z/z congenics, these mice displayed abnormal B cell development with an accumulation of B-1a cells in the peritoneal cavity and spleen, intrinsic B cell hyperactivity, and polyclonal B cell activation with increased serum IgM (5). The epistatic interaction of Sle2^z with Sle1^z and Sle3^z led to early-onset full-blown lupus with fully penetrant nephritis (6). More recently, the Sle2^z locus was shown to encompass a cluster of at least three subintervals: Sle2a^z, Sle2b^z, and Sle2c^z (7). The mechanism by which Sle2^z impacts B cell tolerance and development is unknown.

B cell tolerance is a complex process, and our understanding of this process has been shaped by studies using BCR-transgenic (Tg) models (8). One such model that has been useful in uncovering key checkpoints and molecules in B cell tolerance is the antihen egg lysozyme (HEL) BCR-Tg (HEL^Ig) model. In this model, the nature of the cognate self-antigen, soluble HEL (sHEL) or membrane HEL, dictates the type of tolerance mechanism elicited. Although in HEL^Ig.mHEL double-Tg mice, self-reactive B cells are deleted in the bone marrow (BM), in HEL^Ig.sHEL double-Tg mice, self-reactive B cells are allowed to escape to the periphery but are functionally tolerized (9, 10). Key tolerance mechanisms exercised in the latter model include anergy and receptor editing/revision, both of which ensure the absence of reactivity to self (11). In this study, we bred the antiHEL BCR-Tg model to B6.Sle2^z mice to explore whether and how Sle2^z impacts B cell tolerance.

We found that the Sle2^z lupus-susceptibility interval has a significant impact on mature BCR revision. Progressive narrowing of the Sle2^z interval that still maintains the impact on BCR revision led to a critical interval harboring fatty acid amide hydrolase (FAAH). This critical Sle2^z interval was associated with FAAH overexpression, and this biochemical alteration was mechanistically linked to heightened BCR revision, RAG expression, and autoimmunity.

FAAH, a member of the serine hydrolase family, is one of the primary hydrolytic enzymes that cause degradation of endogenous lipid ligands, such as fatty acid amides and fatty acid esters (12, 13). These ligands bind to the receptors of the cannabinoid signaling pathway, CB1 and CB2 (14, 15), and to the nuclear receptors of the peroxisome proliferator–activated receptor (PPAR) family (16). Therefore, FAAH negatively regulates cannabinoid (17) and PPAR (18) signaling by degradation of their endogenous ligands. Cannabinoid (19) and PPAR (20) signaling were shown to have potent immunosuppressive and anti-inflammatory effects (21). Recent studies also demonstrated an important role for the cannabinoid signaling pathway in the development of B cells in the BM (22) and periphery (23). Therefore, FAAH stands at an important crossroad, with the potential to redirect the immune system toward self-reactivity by altering the level of signaling through key signaling pathways.

B6.Sle2^{NZM2410/NZM2410} (B6.Sle2^z/z) mice were described previously (5). B6.HEL^Ig mice [B6-Tg(IghelMD4)4Ccg/J] and B6.sHEL [B6-Tg(ML5sHEL)5Ccg/J] mice (11, 12) were obtained from The Jackson Laboratory (Bar Harbor, ME). B6.HEL^Ig and B6.HEL^Ig.sHEL double-Tg mice (obtained by cross-breeding the respective single-Tg mice) were interbred for two generations with B6.Sle2^z/z mice to obtain mice that were homozygous for the Sle2^z interval and hemizygous for the HEL^Ig BCR Tg, with or without the sHEL Ag Tg. These are referred to as B6.Sle2^z/z.HEL^Ig and B6.Sle2^z/z.HEL^Ig.sHEL mice, respectively. The Sle2^z locus was progressively narrowed by breeding B6.Sle2^z/z.HEL^Ig mice with B6 mice, to derive B6.Sle2^b/z.HEL^Ig mice, which were subsequently bred with B6 mice to obtain various subcongenic recombinant mice bearing various subintervals of the parental Sle2^z interval. All mice used for this study were bred and housed in a specific pathogen–free colony at the Department of Animal Resources, University of Texas Southwestern Medical Center. Comparable numbers of male and female mice were pooled for all experiments, because no significant sex differences were noted in the phenotypes studied.

For flow cytometric analysis, cells were first blocked with staining buffer (PBS, 5% horse serum, 0.05% azide) containing 10% normal rabbit serum. Cells were then stained on ice with the following dye- or biotin-coupled Abs (Pharmingen, San Diego, CA) for 30 min on ice: IgM^a (DS-1), IgM^b (AF6-7A), I-A^b (AF6-120.1), Early B cell Ag (AA4.1), λ₁₊₂₊₃ (R26-46), CD4 (RM4-5), CD5 (53-7.3), CD8 (Ly-2), CD19 (1D-3), CD21 (7G6), CD23 (B3B4), CD45R/B220 (RA3-6B2), CD69 (H1.2F3), CD86/B7-2 (GL1), CD138 (281-2), and CD43 (S7). This was followed by the addition of streptavidin-allophycocyanin to reveal biotin binding. Dead cells were excluded on the basis of scatter characteristics, and 10,000–50,000 events were acquired per sample. Analysis was performed using FlowJo (TreeStar, San Carlos, CA) and BD CellQuest (BD Biosciences, San Jose, CA).

Antihistone/DNA and antissDNA, IgM^a antiHEL Abs were assayed using ELISA, as described (5, 6). Raw OD was converted to U/ml, using a positive-control serum derived from a B6.HEL^Ig mouse for the IgM^a antiHEL ELISA or from a B6.Sle1^z/z.FAS^lpr mouse for the antihistone/DNA and antissDNA ELISA, arbitrarily setting the reactivity of a 1:200 dilution of the positive control serum to 100 U/ml. Autoantigen array slides were prepared by University of Texas Southwestern Microarray Core Facility and used as described (24).

Total genomic DNA was extracted from whole splenocytes using the phenol-chloroform precipitation method. The band intensities between various samples after PCR were analyzed using ImageJ software, and the ratio of V-Jκ1/V-Jκ2 band intensities was calculated.

Total mRNA was isolated from splenic B220⁺ selected B cells using an RNeasy mini kit (QIAGEN) and quality checked using an Agilent 2100 Bioanalyzer. Microarray analysis was performed by the University of Texas Southwestern Microarray Core Facility using MouseWG-6 V2 BeadChip (Illumina), according to the manufacturer’s instructions. Microarray data were extracted using BeadStudio v3.1, background subtracted, and normalized using a cubic spline algorithm. Genes that were differentially expressed between groups were identified using the Illumina custom error model implemented in BeadStudio. Genes were considered significantly differentially expressed at p < 0.001 and if the change was >2-fold. The microarray data presented in this article have been submitted to the National Center for Biotechnology Information Gene Expression Omnibus under accession number GSE73112 (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE73112).

Single-cell suspensions of whole splenocytes were prepared and cocultured for 18, 24, or 42 h, with or without various stimuli, including polyclonal anti-IgM F(ab)′₂ (10 μg/ml; Jackson ImmunoResearch, West Grove, PA), LPS (1 μg/ml; Sigma), and HEL (50–1000 ng/ml; Sigma). Live cells were enumerated using trypan blue exclusion, resuspended in FACS buffer, and stained with Abs to CD45R/B220 and CD86/B7-2 to assess the activation status. Alternately, the cells were used to extract RNA using an RNeasy kit (Sigma).

Spleens were collected from three B6 and three B6.Sle2^z mice. The tissue samples were weighed, homogenized (100 mg tissue/ml homogenization buffer), and centrifuged at 1000 × g for 10 min at 4°C. Clarified tissue homogenates were incubated with 100 μM [¹⁴C]–N-acylethanolamine (20,000 dpm) in 50 mM Bis-Tris buffer (pH 9), in a final volume of 450 μl, for 30 min at 37°C with shaking (120 rpm). Enzyme activity was tested for two substrates: [1-14C]–N-palmitoylethanolamine (PEA) and [1-14C]-anandamide (AEA). Enzyme assays were terminated by the addition of 2 ml hot isopropyl alcohol (70°C), and lipids were extracted into chloroform. Lipid products were separated by thin-layer chromatography, and the distribution of radioactivity was evaluated by radiometric scanning with a Bioscan AR 2000. FAAH activity was calculated using the percentage of radiolabeled N-acylethanolamine converted into free fatty acid.

Group differences were analyzed using the Student t test. These tests were executed using GraphPad Prism (San Diego, CA).

A key marker of effective tolerance induction in the HEL^Ig.sHEL model is the absence of anti-HEL Abs in the serum (9). Sle2^z did not appear to breach B cell tolerance, as underscored by the similar or even lower levels of anti-HEL IgM^a Abs in B6.Sle2^z/z.HEL^Ig.sHEL sera compared with B6.HEL^Ig.sHEL sera at all ages examined (Supplemental Fig. 1A, 1B). Interestingly, old B6.Sle2^z/z.HEL^Ig mice produced significantly lower titers of anti-HEL Ab compared with age-matched B6.HEL^Ig controls (Supplemental Fig. 1B).

Naive B6.HEL^Ig and B6.Sle2^z/z.HEL^Ig B cells (which have not been exposed to HEL) resembled each other with regard to CD86 upregulation and proliferative responses to BCR stimulation, either with anti-IgM or sHEL (data not shown). Also, in concordance with their serology, B6.Sle2^z/z.HEL^Ig.sHEL B cells behaved very similarly to B6.HEL^Ig.sHEL anergic B cells, in that B cells from both strains barely upregulated the activation marker CD86 following sHEL stimulation (Supplemental Fig. 1C). Anti-IgM stimulation induced CD86 expression to a similar extent in both strains, albeit to a lesser degree than that seen in naive B6.HEL^Ig B cells (Supplemental Fig. 1C). Similar results were obtained when proliferation in response to anti-IgM–mediated BCR cross-linking was measured using CFDA-SE dilution (Supplemental Fig. 1D). IL-4 or a combination of PMA and ionomycin rescued this BCR-induced unresponsiveness to a similar degree in B cells from both strains (Supplemental Fig. 1D).

Sle2^z was implicated in polyclonal or polyreactive hypergammaglobulinemia (5) Hence, we next explored whether this phenotype was still apparent in the presence of the BCR transgene. Although the strains did not differ significantly with regard to the serum levels of Tg-encoded anti-HEL Abs, B6.Sle2^z/z.HEL^Ig mice exhibited significantly elevated levels of IgM anti-ssDNA Abs (Fig. 1A), non-Tg total IgM^b Abs (Fig. 1B), and class-switched (total) IgG Abs (Fig. 1C) compared with controls. Interestingly, these sera also exhibited elevated levels of total Igλ (Fig. 1D), also suggestive of increased BCR revision.

Given these findings, we used an autoantigen array to examine whether the elevated serum Abs in Sle2^z/z BCR-Tg mice might bind additional autoantigen specificities (25). Commensurate with the high titers of total IgG in B6.Sle2^z/z.HEL^Ig mice, the sera from these mice exhibited a high degree of polyreactivity with specificities for a variety of autoantigens, including nuclear Ags such as chromatin, dsDNA, total histone, histone H2A, La/SS-B, Ro/SS-A, and Scl-70, compared with control B6.HEL^Ig sera (Fig. 1E).

However, the increased level of autoantibodies against different autoantigens in the sera of B6.Sle2^z/z.HEL^Ig mice might simply be the result of an overall increase in the total IgM and IgG Ab levels in these mice. To overcome this issue, we performed ELISA to assay total IgG and IgM Ab in the different strains and calculated the average fold change in total IgG and IgM levels in Sle2^z/z.HEL^Ig.sHEL serum compared with B6.HEL^Ig.sHEL controls. We then diluted the Sle2^z/z.HEL^Ig.sHEL and control serum samples differentially to normalize for total Ig levels and performed ELISA for ssDNA IgM, anti-histone IgG, and dsDNA IgG autoantibodies. We found that all of these specificities were still significantly elevated in Sle2^z/z.HEL^Ig.sHEL mice relative to controls (data not shown).

Examination of Ig H chain (HC) allele expression in splenic B cells revealed an increased proportion of B cells that bore IgM^b in >3-mo-old B6.Sle2^z/z.HEL^Ig mice compared with control B6.HEL^Ig mice (Fig. 2A, 2B, Supplemental Tables I–III). Thus, although only ∼7% of splenic B cells in 1–3-mo-old B6.Sle2^z/z.HEL^Ig mice bore surface IgM^b, comparable to age-matched B6.HEL^Ig mice, this number increased over time to 21% at >3–6 mo of age and to 35% by 8–14 mo of age, with these percentages being significantly higher than the corresponding values in B6.HEL^Ig controls (Fig. 2A, 2B, Supplemental Tables I–III). However, no differences in surface IgM^a/IgM^b expression were noted on BM B cells (data not shown), suggesting that the impact of Sle2^z on allelic exclusion may be exercised primarily in the periphery.

Interestingly, the presence of the surrogate self-antigen sHEL accelerated this age-associated increase in IgM^b expression, such that the percentage of B cells that solely expressed endogenous IgM^b increased from 10% in the 1–3-mo age group up to ∼45% in the 8–14-mo age group in B6.HEL^Ig.sHEL spleens, with a complementary decrease in Tg-encoded IgM^a expression (Fig. 2C, 2D, Supplemental Tables I–III). The presence of Sle2^z drastically accentuated the usage or expression of the endogenous IgM^b allele; the percentage of B cells in B6.Sle2^z/z.HEL^Ig.sHEL spleens that were exclusively IgM^b+ progressively increased from ∼18% (1–3 mo) to 38% (>3–6 mo) to 70% (8–14 mo) (Fig. 2C, 2D, Supplemental Tables I–III). Such a robust differential in the usage of Tg versus endogenous Ig HC in the periphery is suggestive of either a selective expansion of IgM^b+ B cells or a process of vigorous receptor revision in Tg-encoded B cells that are chronically exposed to the HEL self-antigen, in the presence of Sle2^z.

Although the different Tg strains, with or without Sle2^z, did not differ with regard to the absolute numbers of splenic B or T cells (data not shown), the presence of Sle2^z led to a profound enlargement in splenic marginal zone (MZ)-type B cells, with a concomitant decrease in CD21^intCD23^hi follicular-type B cells in B6.Sle2^z/z BCR-Tg spleens at all ages (Supplemental Tables I–III). We also noticed an expansion of splenic B1a cells, particularly in older B6.Sle2^z/z BCR-Tg mice (Supplemental Tables I–III).

Skewing in HC allele expression was also observed among splenic B cell subsets, especially in the B6.Sle2^z/z.HEL^Ig strain (Table I). Although the B cells that solely expressed IgM^a were predominantly follicular-type B cells, splenic B cells that bore IgM^b encompassed large numbers of MZ and B1a cells. Interestingly, among the IgM^a−IgM^b+ HC-edited B cells, B1a cells exhibited the largest fold increase in BCR-Tg and double-Tg mice with Sle2^z (Table I). Notably, a higher proportion of IgM^a+IgM^b+ double-positive splenic B cells in B6.Sle2^z/z BCR-Tg mice seem to be of the MZ and B1a type (Table I), similar to what was reported for Igλ and Igκ dual-expressing B cells in the B6.56R anti-DNA BCR-Tg model (26).

Interestingly, the degree of HEL binding by IgM^a+ cells, as gauged by the percentage of HEL⁺ cells and the absolute mean fluorescence intensity of HEL binding, was significantly lower in B6.Sle2^z/z.HEL^Ig.sHEL B cells compared with B6.HEL^Ig.sHEL B cells at all ages examined, with a progressive accentuation of this phenotype with age (Supplemental Tables I–III). In fact, about half of the IgM^a+ B cells from 10-mo-old B6.Sle2^z/z.HEL^Ig.sHEL mice had lost HEL binding. This reduced HEL binding was not due to greater downregulation of sIgM itself, because B6.Sle2^z/z.HEL^Ig.sHEL and B6.HEL^Ig.sHEL B cells had comparable levels of IgM^a at all ages examined (Supplemental Tables I–III). Similar patterns were noted when B6.Sle2^z/z.HEL^Ig B cells were compared with B6.HEL^Ig B cells, but only with age (Supplemental Table III). These findings raised the possibility that an increased fraction of IgM^a-Tg HC on Sle2^z-bearing B cells may have been paired with non-Tg L chain (LC; Igκ or Igλ), thus abrogating HEL reactivity. The Tg LC is composed of a rearrangement to Jκ2. Hence, we compared the relative degree of Ig LC rearrangements to Jκ2 (i.e., reflective of the Tg) versus Jκ1 (reflective of a non-Tg endogenous LC). Indeed, B6.Sle2^z/z Tg B cells exhibited significantly elevated usage of non-Tg LC, based on increased rearrangements to Jκ1 (Fig. 2E, 2F). B6.Sle2^z/z.HEL^Ig B cells also exhibited increased usage of endogenous Ig λ LC compared with B6.HEL^Ig B cells, within the IgM^a+ and IgM^b+ subsets (Table I). Similarly, B6.Sle2^z/z.HEL^Ig.sHEL B cells exhibited increased usage of endogenous Ig λ LC compared with B6.HEL^Ig.sHEL B cells, but only within the IgM^b+ subset.

To ascertain whether Sle2^z/z actively impacted BCR revision in mature B cells or simply promoted the selection and expansion of B cells bearing endogenous (presumably self-reactive) BCR, B cells were stimulated in vitro and monitored for RAG expression. Compared with B6.HEL^Ig B cells, B6.Sle2^z/z.HEL^Ig B cells exhibited significantly higher RAG re-expression upon BCR stimulation (Fig. 2G). These results suggest that Sle2^z-bearing B cells may be constantly editing or revising their BCR away from HEL specificity in a RAG-dependent manner, both in the presence and absence of HEL, with self-antigen exposure simply accelerating this process.

Next, we bred a series of subcongenic recombinant lines harboring different subintervals of the Sle2^z locus together with the HEL^Ig transgene (Fig. 3A) and tested them for increased Ig HC editing in splenic B cells. As shown in Fig. 3B, congenic mice bearing the D4MIT331-D4MIT16 telomeric subinterval exhibited similar levels of HC revision as mice with the full-length Sle2^z interval. This telomeric subinterval was inherited from the New Zealand Black (NZB) strain. However, the D4MIT205-D4MIT12 and D4MIT12-D4MIT16 subcongenics also exhibited increased HC editing, albeit to a lesser degree than the D4MIT331-D4MIT16 subcongenics. Because we cannot exclude the possibility that the D4MIT12-D4MIT16 subcongenic mice harbor chromosomal regions beyond the D4MIT12 marker (up to any length between the D4MIT331 and D4MIT12 markers), the observed increased editing in this subcongenic mouse could result from the candidate gene being located within this chromosomal fragment in close proximity to the D4MIT12 marker. In contrast, the D4MIT205-D4MIT331 subcongenic mice also exhibited increased HC editing, thereby excluding the possibility of the candidate gene being present within the D4MIT12-D4MIT16 interval. Overall, these data suggested that the candidate gene responsible for the Sle2^z-associated BCR-revision phenotype resided in the D4MIT331-D4MIT12 interval.

We next performed microarray analysis of total mRNA isolated from splenic B cells from B6 and B6.Sle2^z mice and examined which genes within the D4MIT331-D4MIT12 interval were significantly differentially expressed (≥2-fold) in Sle2^z versus B6 mature splenic B cells. Nine genes met these criteria, all located within this interval (Fig. 4A). Both in terms of its known function and expression profile in different B cell subsets, FAAH, a gene that encodes a membrane-bound hydrolase, emerged as the most attractive candidate. This gene resides approximately eight megabases from D4MIT12 within the D4MIT331-D4MIT12 interval and was found to be upregulated ∼5-fold in Sle2^z splenic B cells in the microarray study (Fig. 4A).

We next surveyed the mRNA transcript levels of this enzyme in different B cell subsets (Fig. 4B), as documented in the immunological genome project (ImmGen) database. FAAH transcripts were documented to be higher in the later stages of B cell development, with the highest levels in splenic and peritoneal cavity B1a cells. This is especially important given that Sle2^z mice exhibit an expansion of B1a cells (5), a major source of serum IgM (27). Moreover, we noticed an increased percentage of B1a cells in the IgM^b+ edited B cells in Sle2^z BCR-Tg mice (Table I). Similar to the microarray results, quantitative real-time PCR and Western blotting indicated significant upregulation of FAAH expression in Sle2^z splenic B cells (Fig. 4C, 4D). Concordant with these findings, we also noted a significant increase in FAAH enzyme activity in Sle2^z spleens, as indicated by the increased breakdown of two of the primary substrates of this enzyme: AEA and PEA (Fig. 4E).

All of the above findings are not simply due to the increased numbers of B1 and MZ B cells in B6.Sle2^z mice, because these studies were carried out at the age of 2–3 mo, a time when B6.Sle2^z and B6 mice have similar B cell subset profiles (5).

URB597 (cyclohexylcarbamic acid 3′-carbamoylbiphenyl-3-yl ester) is a widely used, potent, and selective inhibitor of FAAH (28). Intraperitoneal injection of B6.Sle2^z.HEL^Ig.sHEL mice with URB597 for 8 wk resulted in a dramatic decrease in the percentage of IgMb⁺ HC-revised B cells in the spleen (Fig. 5A, 5B). We also noticed a significant decrease in the percentage of splenic B cells that had revised their Ig LC (Fig. 5C) and cells that had revised both Ig HC and LC (Fig. 5D). Likewise, there was a significant decrease in the level of B220⁺IgMb⁺ peritoneal cavity B cells following inhibition of FAAH in Sle2^z.HEL^Ig.sHEL mice (Fig. 5E). We also noticed a decreased level of RAG expression in Sle2^z.HEL^Ig.sHEL splenic B cells following FAAH inhibition (Fig. 5F), suggesting that FAAH may accentuate BCR revision by regulating the expression of RAG in Sle2^z.HEL^Ig.sHEL peripheral B cells. However, we could not study the effect of FAAH overexpression on the B1a cell compartment because B6.Sle2^z mice only exhibit an expansion of this cell population when they are significantly older.

The NZB strain, the strain of origin of the Sle2^z interval where FAAH resides, exhibits expanded B1a cells in the spleen and peritoneal cavity earlier in life. A striking observation was a dramatic decrease in B1a cells in NZB mice following FAAH inhibition (Fig. 6A, 6B). NZB peritoneal cavity B cells also exhibited a significant reduction in B220⁺λ⁺ cells, indicating reduced levels of edited B cells following FAAH inhibition (Fig. 6C). FAAH inhibition led to a significant decrease in total IgM and anti-ssDNA Ab levels, hallmark features of this strain (29) (Fig. 6D, 6E), suggesting that decreased peripheral BCR revision following FAAH inhibition ultimately leads to a decrease in polyclonal IgM autoantibody levels.

To test whether FAAH leads to increased Rag2 expression following BCR ligation, we treated peritoneal cavity B1a cells from B6.Sle2^z/z mice with anti-IgM F(ab)′₂ and the FAAH inhibitor URB597 in vitro. As expected, activation of BCR by anti-IgM F(ab)′₂ led to dramatic upregulation of FAAH and Rag2 (Fig. 6F, 6G). Interestingly, Rag2 upregulation was abrogated when cells were treated concomitantly with the FAAH inhibitor (Fig. 6G). This indicates an important role for FAAH in upregulating Rag following BCR ligation.

Previous congenic dissection studies revealed that B6.Sle2^z/z mice did not exhibit anti-nuclear Abs (ANAs) but did display hypergammaglobulinemia (5). In contrast, the interaction of Sle2^z with Sle1^z and Sle3^z in B6.Sle1^z/z.Sle2^z/z.Sle3^z/z triple-congenic mice led to pathogenic ANAs and fatal autoimmune disease (6). These studies suggested that Sle2^z had a very significant impact on B cell function in lupus. Subsequent BM adoptive-transfer studies revealed these influences to be B cell intrinsic (30). Our present results suggest that FAAH, a gene within Sle2^z, contributes to humoral autoimmunity through a novel and unique mechanism: enhanced BCR revision in mature B cells.

The presence of a considerable proportion of splenic B cells bearing endogenous IgM^b HC and reduced HEL binding, even among B cells bearing Tg IgM^a HC, as well as the increased rearrangement and/or usage of endogenous Igκ/Igλ LC alleles, suggests an unequivocal role for Sle2^z in BCR revision and/or repertoire selection. In contrast, a similar increase in the shift from Tg-encoded IgM^a to endogenous IgM^b BCR usage was not seen in BM B cells, indicating that the receptor editing in BM B cells is still intact. However, this finding may not be exclusive to Sle2^z. Even splenic B cells from B6.HEL^Ig.sHEL mice display a progressive increase in endogenous IgM^b HC usage with age. Similar findings were observed in another BCR-Tg model, 3-83/H-2k^b/d, wherein a considerable proportion of splenic B cells, and not BM B cells, from aged mice displayed endogenous IgM^b HC usage, with an increase in serum ANA titers (31). One important finding in the present study is that even naive splenic B cells from B6.Sle2^z/z.HEL^Ig mice that had never been exposed to the surrogate self-antigen sHEL displayed this phenomenon, with the presence of the surrogate self-antigen accentuating and hastening this process in B6.Sle2^z/z.HEL^Ig.sHEL mice. Hence, this process seems to be an intrinsic abnormality of Sle2^z-bearing mature B cells that is accentuated by BCR ligation by self-antigen. This increased drive to revise the primary BCR in B6.Sle2^z/z mice appears to be associated with very interesting cellular and serological phenotypes.

One consequence of exaggerated BCR revision in Sle2^z B cells is the emergence of serum autoantibodies in mice that express the endogenous HC and LC genes (Fig. 1B, 1D). When the antigenic specificity of their non-Tg Abs was examined, sera from Sle2^z/z-Tg mice were found to be remarkably polyreactive and autoreactive, including Abs to ssDNA, chromatin, dsDNA, histones, La/SSB, Scl-70, and U1-snRNP. Hence, one of the consequences of the exaggerated peripheral BCR revision in this model appears to be heightened polyreactivity and autoreactivity. Parallel with these findings, Weigert and colleagues (27) reported that, when chronic graft-versus-host disease was induced in anti-DNA BCR-Tg mice, incomplete LC editing as a component of receptor revision led to polyreactivity toward a variety of self-antigens in addition to DNA.

Another phenotype that we observed was the increase in splenic MZ and B1a cells among the IgM^a−IgM^b+ edited B cells and IgM^a+IgM^b+ double expressor cells in B6.Sle2^z/z.HEL^Ig and B6.Sle2^z/z.HEL^Ig.sHEL mice. This is especially interesting given that B1a cells express the highest level of FAAH among all of the B cell subsets (Fig. 4B). Conversely, we noticed a decrease in B1a cells following inhibition of FAAH (Fig. 6A, 6B). The additional findings that FAAH inhibition reverses the exaggerated receptor revision seen in B6.Sle2^z/z.HEL^Ig.sHEL mice (Fig. 5), dampens λ- LC usage in another autoimmune strain examined (Fig. 6C), and shuts off RAG2 upregulation following BCR activation in B1a cells (Fig. 6F, 6G) strongly suggest that FAAH may play an important role in B1a cell differentiation, as well as in promoting receptor revision in these cells. The decrease in peritoneal B1a cells after FAAH inhibition could be a result of decreased survival of these cells following FAAH inhibition. However, we did not notice increased B1a cell death following FAAH inhibition in vitro for 24 h (data not shown). Hence, the reduced B1a cells in the peritoneal cavity could be a result of altered differentiation or homing of these cells when FAAH is inhibited.

Earlier studies showed that the specificity of the BCR, notably polyreactivity, can skew B cells toward becoming B1a cells (32). Hence, the elevated B1a cell numbers elicited by Sle2^z, both in BCR-Tg and non-Tg models, may simply be the readout of elevated peripheral BCR revision. Thus, the altered BCR repertoire in Sle2^z BCR-Tg mice (toward polyreactivity and self-reactivity) may be responsible for the increased skewing toward B1a cells, as well as MZ B cells, in these mice.

An important question is whether Sle2^z actively regulates the degree of BCR revision or simply facilitates the selective expansion of polyreactive/autoreactive B cells arising as a consequence of BCR revision. The observations that BCR cross-linking augments RAG re-expression in mature Sle2^z B cells and inhibition of FAAH dampens RAG expression in B6.Sle2^z/z.HEL^Ig.sHEL splenic B cells suggest that Sle2^z, through FAAH, may play a direct role in amplifying RAG-mediated BCR revision in mature B cells. The notion that exaggerated peripheral BCR revision may be a common theme in systemic autoimmunity is supported by studies that reported elevated frequencies of VH replacement products in human autoimmune diseases, including systemic lupus erythematosus and rheumatoid arthritis (33, 34).

The molecular mechanism(s) through which overexpression of FAAH leads to increased RAG expression following BCR activation of peripheral B cells is not known. Two attractive candidates are cannabinoid signaling and PPAR signaling. Further studies are warranted to examine whether overexpression of FAAH contributes to RAG upregulation through negative regulation of one or both of these signaling pathways.

Interestingly, a study by Lugar et al. (35) showed that plasma cells from lupus patients overexpress FAAH compared with naive and memory B cells. This suggests an important correlation between upregulation of FAAH and the increased production of Abs in human lupus. Finally, inhibition of FAAH or modulation of related pathways using pharmacological inhibitors to treat various autoimmune and inflammatory conditions, such as rheumatoid arthritis, multiple sclerosis, and inflammatory bowel disease, gained significant attention recently (24, 36). This study identified FAAH as a novel player in the pathogenesis of lupus, and it will be important to examine whether inhibition of FAAH has therapeutic benefit in human lupus.

We thank Anne Satterthwaite for critical reading of the manuscript.

The authors have no financial conflicts of interest.