Contributions of B cells to lupus pathogenesis

Abstract

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the production of autoantibodies. This review summarizes first the results obtained in the mouse that have revealed how B cell tolerance is breached in SLE. We then review the B cell subsets, in addition to the autoAb producing cells, which contribute to SLE pathogenesis, focusing on marginal zone B cells, B-1 cells and regulatory B cells. Finally, we review the interactions between B cells and other immune cells that have been implicated in SLE, such as dendritic cells, macrophages, neutrophils and T cells.

1. Introduction

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the production of autoantibodies (autoAbs) (Ceppellini et al., 1957; Robbins et al., 1957). These autoAbs are produced by both long-lived plasma cells (PCs) and short-lived plasmasblasts (PBs) (Hoyer et al., 2004; Liu et al., 2011), some of which are generated through germinal centers (GCs) (Vinuesa et al., 2010) while others bypass GCs and differentiate into PBs in extrafollicular foci (Shlomchik, 2008). This review summarizes first the results obtained in the mouse that have revealed how B cell tolerance is breached in SLE. We will then review which B cell subsets, in addition to the autoAb producing cells, contribute to SLE pathogenesis. Finally, we will review the interactions between B cells and other immune cells that have implicated in SLE. This review will refer to several spontaneous mouse models of SLE which have distinct genetic backgrounds, and have provided different insights to the mechanism of lupus pathogenesis in general, including the role of B cells (Table 1).

Table 1.

Spontaneous Mouse Models of Lupus

Strain	Parental strains	Lupus-like phenotype specific to the model	References
(NZB x NZW) F1	NZB and NZW	Lymphadenopathy, splenomegaly, high level anti-dsDNA IgG, lupus nephritis (GN). Strong female bias	(Helyer and Howie, 1963)
NZM2410 NZM2328	NZM2410 and NZM2328 are 2 of 27 recombinant inbred strains between NZB and NZW	Similar to (NZB x NZW) F1 with less pronounced female bias	(Rudofsky et al., 1993)
B6.NZM2410.Sle1.Sle2.Sle3	NZM2410 derived genetic loci, Sle1 - 3, are introduced to a B6 non-autoimmune background	Milder phenotypes than the parental NZM2410	(Morel et al., 2000)
MRL/lpr	MRL strain is generated from inbreeding between several strains of mice. The lpr, lymphoproliferation, mutation is a loss of function in the pro-apoptotic Fas gene.	High level of autoAbs: anti-DNA, anti-Sm, rheumatoid factors, GN. Lymphadenopathy contributed mainly by accumulation of CD4− CD8− T cells.	(Cohen and Eisenberg, 1991)
MRL/gld	The gld, generalized lymphoproliferative disease, mutation is a loss of function mutation in the FasL gene.	Lymphadenopathy, autoAbs, GN.
BXD2	C57BL/6J x DBA/2J recombinant inbred strain	High level of IL - 17, autoAbs (anti-DNA, anti-histone, and rheumatoid factor), GN and arthritis.	(Hsu et al., 2008)
BXSB/Yaa	(B6 x SB/Le) F1 x SB/Le --> Inbreeding. Yaa, Y-linked autoimmune accelerator, refers to the translocation of 16 genes from the X chromosome, including TLR7 onto the Y chromosome	Only males are affected. AutoAbs skewed toward RNA-specificities, monocytosis.	(Murphy and Roths, 1979; Santiago-Raber et al., 2008)

2. B cell Tolerance

Maintenance of B cell tolerance is essential for preventing the secretion of autoAbs with potential pathogenic specificities. In SLE, failure in B cell tolerance sits at the core of the disease process. Indeed, it is largely accepted that tissue injury results from the production of autoAbs which combine with self-antigens (self-Ags) to form immune complexes (ICs) that deposit into organs leading to inflammation and cellular damage. The mechanisms by which normal B cells from healthy subjects maintain tolerance against lupus-associated antigens follow the same general basic principles that have been described for generic antigens, which will be briefly reviewed below. In addition, more specific mechanisms are involved to prevent the production of lupus-associated autoAbs, due to the nature of the prevalent lupus autoAgs. Indeed, lupus-associated autoAgs are largely confined to nucleoprotein complexes that are released during cell death and that activate TLR7 and TLR9 (Marshak-Rothstein and Rifkin, 2007). These specific mechanisms will be reviewed in sections 2.1 and 2.2.

Given that 55–75% of B cell receptors (BCR) on human immature B cells are self-reactive, strict tolerance mechanisms are required to eliminate them from the B cell repertoire (Wardemann et al., 2003). Classic studies using BCR transgenic (Tg) mouse models have identified several tolerance checkpoints at which autoreactive B cells are regulated (Pillai et al., 2011). Central tolerance in the bone marrow (BM) eliminates self-reactive immature B cells primarily by receptor editing (Gay et al., 1993; Murphy and Roths, 1979; Tiegs et al., 1993). Failure in receptor editing results in the autoreactive B cells becoming either anergized or deleted depending on receptor affinity (Cambier et al., 2007). Immature B cells that pass the central tolerance checkpoint migrate to the spleen where they develop into mature B cells. At this stage, self-reactive B cells are regulated by peripheral checkpoints, such as deletion, anergy, follicular exclusion, and clonal ignorance (Shlomchik, 2008). In addition, recent work has shown that self-reactive B cells that arise from a GC reaction are tolerized if the self-Ag is expressed in large amounts and in close proximity to the GC (Chan et al., 2012). Elimination of autoreactive B cells has been a major therapeutic goal in SLE. This cannot be achieved without a thorough understanding of how these multiple tolerance mechanisms are affected in SLE. The knowledge gained in this field from mouse models will be reviewed in this section.

2.1 Breakdown of B cell tolerance in BCR tg mouse models of lupus

Studies crossing the classic BCR Tg tolerance models, such as HEL x anti-sHEL (Rathmell and Goodnow, 1994) or anti-MHCI (Rubio et al., 1996), to the MRL/lpr lupus-prone background did not reveal significant tolerance defects, which has been attributed to the lack of specificity of these models towards a lupus relevant self-Ag (Shlomchik, 2008). However, Tg mouse models targeting lupus-associated self-Ags such as DNA, RNA-containing particle such as Sm, and IgG have shown dysregulated B cell tolerance when crossed to an autoimmune background. A summary of the findings from these models is given in Table 2.

Table 2.

Self-Ag specific BCR transgenic models in a non-autoimmune versus lupus-prone background

Specificity	Models	Non-autoimmune background	Lupus-prone background
Anti-dsDNA	3H9, 3H9/Vκ8, 3H9/56R	anergized (Nguyen et al., 1997) or developmentally arrested after Ag encounter (Mandik-Nayak et al., 1997)	anti-dsDNA Ab secretion (Mandik-Nayak et al., 1999) or differentiation into MZB (Liu et al., 2007)
RF (anti-IgG)	AM14, AM14/Vκ8	clonal ignorance (Hannum et al., 1996)	RF secretion and SHM at EF zones (Wang and Shlomchik, 1999; William et al., 2005)
Anti-Sm	2-12, 2-12/Vκ8	developmentally arrested or anergized (Santulli-Marotto et al., 1998)	accelerated anti-Sm response (Santulli-Marotto et al., 2001)

2.1.1 Anti-DNA

Anti-dsDNA IgG is an important disease marker, given that it was detected in 55% of patient sera prior to disease diagnosis and in the great majority of them after diagnosis (Arbuckle et al., 2003). The 3H9 heavy chain (HC) Tg, derived from an MRL/lpr anti-DNA Ab, combines with several endogenous light chains (LC) to produce a BCR reactive to either ssDNA or dsDNA (Erikson et al., 1991). The HC 3H9 Tg model has been used extensively to study the mechanism of tolerance to DNA. Its main advantage is that it maintains a physiological polyclonal B cell repertoire, while nearly all the B cells in HC/LC double Tg mice are specific to a single Ag.

In the non-autoimmune BALB/c background, both the 3H9 HC tg and the 3H9/Vκ8 double Tg mice show high levels of B cells carrying DNA-specific BCRs, but anti-DNA autoAbs could not be detected. Therefore, in a healthy background tolerance mechanisms prevent the differentiation of self-reactive B cells into Ab forming cells (AFC). Further studies showed that the 3H9 B cells were Ag-experienced, yet developmentally arrested at the T-B cell interface of the splenic follicle (Mandik-Nayak et al., 1997). Meanwhile, the 3H9/Vκ8 B cells, which are primarily anti-ssDNA, had an anergic phenotype characterized by reduced proliferation in response to stimulation despite being long-lived (Nguyen et al., 1997).

In contrast, 3H9 B cells in the MRL/lpr autoimmune background were no longer developmentally arrested and entered the follicles (Mandik-Nayak et al., 1999). Furthermore, site-directed 3H9/Vκ8 B cells in the MRL/lpr background were activated, class-switched, and underwent somatic hypermutation (SHM) which led them to acquire specificity to other autoAbs (Brard et al., 1999). The effect of anti-dsDNA reactivity on receptor editing was studied in a 3H9 HC mouse with a site-directed mutation from aspartate to arginine at position 56 in the CDR2 region (3H9/56R) which resulted in a BCR with higher affinity for dsDNA when combined with most LCs (Li et al., 2001). In the non-autoimmune BALB/c background, the 3H9/56R B cells successfully underwent LC receptor editing to produce a BCR that did not bind to dsDNA. In contrast, this mechanism was defective in the MRL/lpr background as most B cells were still specific for dsDNA following receptor editing (Li et al., 2002). In addition, the NZM2410-derived Sle2 lupus susceptibility locus also breach tolerance of the 3H9/56R B cells by preferentially inducing their differentiation into marginal zone B cells (MZB) (Liu et al., 2007).

The 3H9 model has also provided insights into the dysregulation of DNA-specific B cells by comparing two non-autoimmune strains. While the BALB/c background prevents the secretion of anti-DNA autoAb, anti-DNA Abs are found in B6.3H9/56R mice (Tsao et al., 2008) due to defect in a post-GC checkpoint (Fukuyama et al., 2005). Therefore, some non-autoimmune genetic backgrounds already possess a predisposition to autoimmunity that is only apparent in the presence of high numbers of self-reactive B cells.

2.1.2 RF Specificity

Rheumatoid factors (RF) are autoAbs directed against self-IgG and the presence of serum RF has been associated with active SLE (Kessel et al., 2009). The AM14 HC, derived from an MRL/lpr hybridoma, was used to generate a RF specific Tg model in which the AM14 HC combines with endogenous Vκ8 LC to form a BCR specific for IgG2a of the “a” allotype (Shlomchik et al., 1993). Therefore, this system enables the study of B cell tolerance with a SLE-relevant specificity (RF) in the presence or absence of the IgG2a^a autoAg. In the non-autoimmune BALB/c background, the moderate affinity of the AM14/Vκ8 BCR rendered the autoreactive B cells clonally ignorant rather than deleted or anergized in the presence of the autoAg (Hannum et al., 1996). However, in the MRL/lpr background, the AM14 B cells were spontaneously activated and differentiated into AFCs when IgG2a^a was expressed (Wang and Shlomchik, 1999).

Further studies showed that MRL/lpr RF B cells underwent SHM in the extra-follicular (EF) zones bypassing GC reactions, and developed into short-lived plasmablasts (William et al., 2002; William et al., 2005). These results were validated in a site-directed AM14 HC model where the spleen and BM of MRL/lpr, but not BALB/c mice, contained activated and class-switched RF B cells located in EF clusters (Sweet et al., 2010). The presence of IgG2a^a ICs found in MRL/lpr but not in BALB/c mice, led to the activation of the RF B cells (Rifkin et al., 2000) and in vitro studies have shown activation of AM14 B cells depended on dual ligation of the BCR and TLR7/TLR9 (Lau et al., 2005; Leadbetter et al., 2002). Finally, administration of anti-chromatin IgG2a^a to either Tg MRL/lpr or BALB/c mice was sufficient to activate their AM14 B cells (Herlands et al., 2007). Therefore, the excess DNA/RNA ICs generated by lupus-prone mice leads to the activation of clonally ignorant RF B cells.

T cells are not required for differentiation of AM14 AFCs, class switching, and SHM when BCR and TLR7/TLR9 ligation was provided in vivo (Herlands et al., 2008; Sweet et al., 2011). However, CD40L and IL-21 signaling provides by CD4⁺ T cells increased the number of RF plasmablasts and the frequency of SHM. In addition, AM14 B cells can differentiate into memory B cells and provide secondary responses only with T-cell help (Sweet et al., 2013). Therefore, the activation of AM14 B cells is complex with other immune cells enhancing their pathogenic potential.

The AM14 model has been extensively studied in the MRL/lpr genetic background. A potential confounding factor arises with this model since the autoimmune background is primarily dependent on the Fas mutation (lpr). However, Fas deficiency in humans (Autoimmune Lymphoproliferative Syndrome) only shares some clinical manifestations with SLE (Teachey et al., 2010). We have crossed the AM14 HC with the Fas-sufficient C57BL/6-based B6.NZM2410.Sle1.Sle2.Sle3 triple congenic (B6.TC) mouse model of lupus (Morel et al., 2000). In this model, the B6.TC lupus background induced spontaneous activation of RF B cells in the presence of the autoAg in a TLR7/9 dependent manner. Just like in the MRL/lpr model, the activated AM14 B cells followed an EF response with high levels of SHM hypermutation (Sang et al., in preparation). Further studies will reveal the mechanisms by which RF B cell tolerance is broken in this autoimmune background.

2.1.3 Anti-Sm

AutoAbs against snRNPs, known as anti-Sm, are found in a large subset of SLE patients. To study Sm-specific B cells, a 2-12 HC Tg mouse was generated from an anti-Sm MRL/lpr hybridoma (Santulli-Marotto et al., 1998). The B cell repertoire of these mice is reactive towards Sm, ssRNA, as well as non-self Ags. Anti-Sm secretion could not be detected in 2-12 HC Tg B6 mice with anti-Sm B cells arrested at an immature stage with a shortened half-life. However, some anti-Sm B cells matured in an anergized state as immunization with murine snRNPs induced their activation and autoAb secretion. Meanwhile, crossing the 2-12 HC to the MRL/lpr background accelerated the anti-Sm response when compared to non-Tg MRL/lpr mice (Santulli-Marotto et al., 2001). A more thorough analysis of 2-12 HC Tg MRL/lpr mice revealed a defect in the differentiation of anti-Sm B cells to the B-1 lineage where they are tolerized (Santulli-Marotto et al., 2001). In non-autoimmune mice, 2-12 B cells preferentially differentiated into peritoneal B-1 cells that remained tolerant towards the self-Ag (Qian et al., 2001). This B-1 cell differentiation was dependent on a strong signaling threshold as lowering BCR signaling through CD19 deficiency resulted in differentiation to the B-2 compartment, and a breach of tolerance. In addition, 2-12/Vκ8 double Tg B cells have a low affinity for Sm Ags and only differentiated to the B-2 lineage and displayed an anergic phenotype (Borrero and Clarke, 2002). Furthermore, 2-12 B-1 as well as MZ B cells were clonally ignorant by being sequestered from self-Ag, as a deficiency in the clearance of apoptotic cells led to anti-Sm secretion from both B cell populations (Qian et al., 2004). Therefore, the 2-12 model illustrates how the signaling threshold as well as the availability of self-Ag to the different B cell lineages play a role in the maintenance of tolerance and how this complex mechanism is dysregulated in a lupus-prone background resulting in autoAb production.

2.2 Dysregulation of tolerance checkpoints are found in SLE patients

SLE patients show an abnormal distribution of B cell populations in comparison to healthy controls, which indicates defects in tolerance checkpoints. Studies using a dsDNA mimetope tetramer (DWEYS) showed that SLE patients had both naive and Ag-experienced B cells that were reactive against dsDNA. This observation was independent of disease activity suggesting a failure in both early and late selection checkpoints (Jacobi et al., 2009). Broader studies looking at multiple disease-associated autoAbs showed a defect in early tolerance mechanisms due to the increased presence of autoreactive mature naive B cells in SLE patients (Yurasov et al., 2005). Furthermore, patients in remission maintained elevated numbers of autoreactive mature naive B cells suggesting that the accumulation of self-reactive B cells can predispose individuals to disease (Yurasov et al., 2006).

In addition to early and peripheral tolerance checkpoints, a third checkpoint has been identified for Ag experienced B cells as self-reactive IgM⁺ CD27⁺ memory B cells are excluded from the circulation whereas B cells specific for common bacterial pathogens are expanded (Dunn-Walters et al., 1995; Tangye et al., 1998; Tsuiji et al., 2006). Surprisingly, IgG⁺ memory B cells produce self-reactive Abs, including anti-nuclear specificities, in the sera of healthy individuals (Tiller et al., 2007). The majority of these autoAbs are derived de novo through SHM during the differentiation of self-Ag activated B cells. These results point towards a breach in tolerance at the GC level. Autoreactive B cells were excluded from the GCs in the tonsils of healthy controls but not in SLE patients (Cappione et al., 2005). Therefore, defects regulating GC reactions could lead to the production of de novo autoreactive B cells which would explain the high levels of memory and plasma cells characteristically seen in SLE.

3. Antigen-independent mechanisms of B cell tolerance

3.1 Endosomal Toll-like Receptors (TLRs) play an important role in the activation of pathogenic B cells

Recent studies have linked the endosomally localized TLR7 and TLR9 to the regulation of B cell tolerance. The dual ligation of BCR and TLR7 or TLR9 is necessary for the activation of AM14/Vk8 Tg B cells into RF secreting cells (Lau et al., 2005; Leadbetter et al., 2002), demonstrating that BCR-mediated internalization of the ICs delivers the TLR7/9 ligands into the endosomal compartment. In support of this hypothesis, lupus-prone MRL/lpr or MRL/gld mice lacking the TLR adaptor molecule MyD88 had reduced levels of autoAb. Furthermore, blocking endosomal TLR signaling decreased ANAs and improved survival in the B6.lpr and BXSB lupus-prone mice (Kono et al., 2009). Finally, the expression of TLR7 and TLR9 induces ANAs and RF production in a B-cell intrinsic manner (Koh et al., 2013; Teichmann et al., 2013). These nucleic acid sensing TLRs are also required for the production of pathogenic autoantibodies with non-nucleic acid specificities, most likely through dendritic cell activation (Koh et al., 2013).

TLR9 is essential for the development of anti-dsDNA and anti-chromatin Abs as TLR9 deficient MRL/lpr mice lacked autoAbs with these specificities but, unexpectedly, suffered from exacerbated lupus (Christensen et al., 2005). Meanwhile, TLR7 deficiency prevented the production of autoAbs against RNA-containing Ags and ameliorated disease in MRL/lpr mice. These opposing roles for TLR7 and TLR9 were replicated in the lupus-prone congenic strain B6.Nba2 (Santiago-Raber et al., 2010). Consistent with these results, the duplication of X-linked TLR7 gene results in a lupus-like phenotype in mice carrying the Y-linked autoimmune accelerating locus (yaa) or a Tg (Deane et al., 2007; Subramanian et al., 2006). Finally, genetic polymorphisms regulating TRL7 expression have been associated to SLE susceptibility in males (Deng et al., 2013; Shen et al., 2010).

The comparison of MRL/lpr mice deficient in TLR7, TLR9, and/or Myd88 revealed that TLR9 regulates TLR7 and suppresses the production of TLR7-dependent anti-RNA autoAbs (Nickerson et al., 2010). Furthermore, ANA production by MRL/lpr mice was solely attributed to TLR7/TLR9 signaling. Mechanistic studies have indicated that TLR9 restricts the survival of anergic anti-DNA B cells, while TLR7 requires type I IFN signaling to exacerbate disease symptoms (Nickerson et al., 2013a; Nickerson et al., 2013b). This suggests that TLR7 and TLR9 represent ideal therapeutic targets for SLE with TLR9 agonists used to eliminate anti-DNA Ab producing B cells and TLR7 antagonists used to dampen disease.

3.2 Dendritic Cells (DCs) modulate B cell responses via cytokine secretion

Alterations in cytokine levels are seen in SLE patients (Davis et al., 2011) and thus may play an important role in B cell mediated pathogenesis. Recent work showed that monocyte-derived DCs generated in the presence of serum from SLE patients promoted either naive and memory B cells to differentiate into IgG-secreting plasmablasts in a BAFF and IL-10 dependent manner (Joo et al., 2012). These results correlated with the elevated BAFF expression observed in blood DCs from SLE patients (Gerl et al., 2010).

In vitro studies showed that activated BM-derived DCs (BMDC) from the lupus prone B6.TC mice induced a greater B cell proliferation, Ab production, and PC differentiation than B6 BMDCs (Wan et al., 2008). The enhanced B cell response was mediated by soluble factors, including IL-6 and IFN-γ (Wan et al., 2008) and Sang et al. in preparation). In addition, DC deletion decreased autoAb titers and plasmablast numbers, which correlated with disease amelioration in MRL/lpr mice (Teichman et al., 2010). MyD88/TLR signaling in DCs contributes to the autoimmune pathology of MRL/lpr mice as deficient DCs secreted lower amounts of inflammatory cytokines. This, however, did not affect the production of pathogenic autoAbs (Teichman et al., 2013).

Finally, DC subsets regulate B cells differently. Immature BMDCs (iBMDC) as well as BM resident DCs (BM-RDC) inhibited TLR-induced B cell proliferation and differentiation whereas splenic resident DCs had no effect (Sindhava et al., 2012). Meanwhile, it is well accepted that IL-6, a cytokine produced at high levels by activated DCs, promotes B cell terminal differentiation into PCs. However, IL-6 secretion by DCs repressed LPS-induced Ab secretion in autoreactive B cells chronically exposed to self-Ag such as in the 2-12 anti-Sm or HEL-Ig X sHEL models (Kilmon et al., 2005). Therefore, cytokines secreted by DCs can play dual roles in promoting or repressing autoimmune responses.

4. Role of specific B cell subsets in lupus

Largely based on murine models of SLE, it has been proposed that marginal zone B (MZB) cells and B-1a cells contribute to the production of pathogenic autoAbs while B regulatory cells (Breg or B10) suppress these responses.

4.1 Marginal zone B cells expand and migrate to the follicle where they engage CD4⁺ T cells to promote autoantibody production

The vast majority of studies on MZBs have been conducted in the mouse, but there are important differences between the two species. In particular, human MZB cells are present in the spleen and circulation, but murine MZB cells are restricted to the MZ in the spleen (Steiniger et al., 2006). Because blood flow into the spleen initially passes through the MZ sinus, MZB cells are the first of B cells to encounter blood-borne Ag (Mebius and Kraal, 2005). Ag-activated MZB cells migrate toward the follicle (FO) where they can either receive CD4⁺ T cell help then become PCs or they can activate CD4⁺ T cells, which in turn activate cognant follicular B (FOB) cells (Förster et al., 1996; Lu and Cyster, 2002; MacLennan and Liu, 1991; Phan et al., 2005; Zhou et al., 2011). A weak affinity for self Ag suggests that MZB cells can become pathogenic in the context of lupus. The number of MZB cells expand with progression of disease in several lupus models, including NZB/W F1 mice (Wither et al., 2000), in which they generate more anti-dsDNA IgM than FOB cells (Zeng et al., 2000). NZB/W F1 MZB cells express CD80 at a high level equivalent to that of CD40-activated B cells (Wither et al., 2000). Because NZB/W F1 T cells express normal level of CD40L, this indicates that the expanded MZB cell population is intrinsically ‘active’, and is capable of activating autoreactive CD4⁺ T cells (Wither et al., 2000). Estrogen treatment of BALB/C mice carrying a dsDNA specific Tg BCR resulted in the expansion of the Tg MZB cells, which correlated with an increase in anti-dsDNA Ab titers (Grimaldi et al., 2001). The number of MZB cells also greatly expands in BAFF Tg mice (Enzler et al., 2006; Mackay et al., 1999). BAFF promotes B cell survival via the alternative NFκB pathway and induces class-switch and the production of autoAbs via the classical NFκB pathway. Disruption of either pathway reduces the MZB cell population, and disruption of the alternative NFκB pathway impairs the production of anti-dsDNA IgM (Enzler et al., 2006).

The expansion of MZB cells was also observed in the spontaneous triple congenic (TC) B6.Sle1.Sle2.Sle3 model (Duan et al., 2008). In addition, TC MZB cells breach follicular exclusion by migrating to the FO instead of staying in the MZ, and this is associated with high level of anti-dsDNA IgG (Duan et al., 2008). The breach of follicular exclusion occurs before autoAbs are detected in TC mice, suggesting that TC MZBs contribute to the production of pathogenic autoAbs. CD86-deficiency normalized both MZB cells location and anti-dsDNA IgG titers in TC mice (Duan et al., 2008), which suggested that T cells and MZB cells interact. Indeed, TC MZB cells were found co-localized with TC CD4⁺ T cells in the FO (Zhou et al., 2011). Furthermore, TC MZB cells proliferated more and secreted more anti-DNA IgM than B6 MZB cells in response to anti-CD40 stimulation (Zhou et al., 2011). TC MZB cells also induced B6 CD4⁺ T cells to proliferate more than did B6 MZB cells (Zhou et al., 2011). This suggests that autoreactive TC MZB cells contribute to disease by interacting with autoreactive CD4⁺ T cells in the follicles.

Expansion of MZB cells does not always correlate with disease in mouse models of lupus. NZM TAN mice only manifest a mild lupus-like phenotype although their MZB cell population is enlarged (Duan et al., 2007). NZM TAN MZB cells express high levels of CD5, a negative regulator of BCR signaling, which may be the reason why they do not respond to T cell-independent Ag stimulation and do not migrate to the FO (Duan et al., 2007; Duan et al., 2008). BXSB.Yaa mice represent a model which lupus develops in spite of a drastically reduced number of MZB cells in the spleen (Amano et al., 2003).

4.2 Autoreactive B1a cells contribute to glomerulonephritis and T cell activation

B-1 cells represent a separate lineage of B lymphocytes found mostly in the pleural and peritoneal cavities, and in lower numbers in the spleen. They have been subdivided into CD5⁺ B-1a and CD5⁻ B-1b cells (Baumgarth, 2011; Godin et al., 1993; Hayakawa et al., 1985; Kantor, 1991). CD5 is a negative regulator of BCR signaling, which explains why CD5⁻ B-1b, but not CD5⁺ B-1a cells undergo clonal expansion in response to Ag challenge (Alugupalli et al., 2003; Alugupalli et al., 2004; Bikah et al., 1996). As an alternative to BCR induced activation, B1a cells are activated by TLR signaling, which induce their migration from the peritoneum to the spleen or to sites of inflammation where they can class-switch and differentiate into PCs (Yang et al., 2007). B-1 cells are the main source of natural IgM, which are Abs generated in the absence of Ag exposure (Baumgarth, 2011; Baumgarth et al., 2005; Bouvet and Dighiero, 1998; Stewart, 1992), that have a low affinity polyreactivity and autoreactivity (Avrameas, 1991; Baumgarth et al., 1999; Casali and Schettino, 1996; Choi and Baumgarth, 2008; Coutinho et al., 1995). The autoreactivity of natural Abs has suggested that B-1 cells contribute to autoimmune pathogenesis (Duan and Morel, 2006). Accordingly, the removal of peritoneal B-1 cells from NZB/W F1 mice correlated with disease attenuation (Mihara et al., 1988), and osteopontin-induced B-1 cell expansion paralleled an increased anti-dsDNA Ab titers (Iizuka et al., 1998). On the other hand, B-1a cells are not responsible for autoAb production in Fas-deficient mice (Reap et al., 1993), and IL-5 induced expansion of B-1a cells in NZB/W F1 was associated with disease protection (Wen et al., 2004).

B-1a cells have been shown to contribute to the development of GN in several murine models of lupus. In NZB/W F1 mice, class switched B-1a cells travel to the spleen (Enghard et al., 2010) or kidneys where they secrete anti-dsDNA IgG (Ishikawa et al., 2002; Ishikawa et al., 2001; Ito et al., 2004). A correlation between the expansion of B-1a cells and development of GN has been linked to the NZM2410-derived Sle2 lupus susceptibility locus (Xu et al., 2005), in which Sle2c1 provides the most significant contribution (Xu et al., 2011). Sle2c1 contains a SNP in the promoter of the Cdkn2c gene that encodes for the cyclin-dependent kinase inhibitor p18^INK4c. This SNP creates a second binding site for YY-1 that represses p18 transcription. Contrary to conventional B cells, B-1 cells are maintained by self-renewal in which p18 is critical to regulate cell cycle. The decreased expression of p18 promotes cell division hence the observed B-1a cell expansion in mice carrying Sle2c1 (Potula et al.; Xu et al.). B-1a cells can also exacerbate lupus in mice by engaging CD4⁺ T cells and promoting Th17 differentiation (Zhong et al., 2007). IL-17 has been implicated in lupus in mice and humans (Crispín and Tsokos, 2010).

Human B1a cells have been recently been described (Griffin et al., 2011), SLE patients have an enlarged population of B1 cells that are activated and induced the expansion of CD4⁺ T cells (Griffin and Rothstein, 2011). Even though B-1a cells do not contribute to pathology in all mouse models of lupus, the fact that B1 cells are expanded in SLE patients warrants further investigation of the mechanisms by which B-1a cells expand and contribute to systemic autoimmunity.

4.3 Regulatory B cells can suppress lupus before disease onset

A subset of B cells that share surface markers, including CD5 and CD1d, with MZB and B-1a cells possesses regulatory function by their production of IL-10 (Blair et al., 2010). In the Palmerston North mouse model of lupus, TLR9 activated MZB cells secrete high level of IL-10, which is associated with a reduction of the pro-inflammatory cytokine subunit IL-12p40 (Lenert et al., 2005). B cell depletion before disease onset accelerated the development of proteinuria in NZB/W F1 mice, indicating that Bregs have a protective role early in the disease process (Haas et al., 2010). However, depletion of B cells from NZB/W F1 mice during disease onset reduced disease severity (Haas et al., 2010), indicating that when pathogenic autoAbs are produced, Bregs have a very limited, if any, protective role. Adoptive transfers of CD1d^hi CD5⁺ B cells into CD19⁻ deficient NZB/W F1 mice significantly prolonged their survival, possibly through the expansion of regulatory T cells (Watanabe et al., 2010). Tim-1 deficient mice lacked IL-10 expression in B cells, and this resulted in systemic autoimmunity that was enhanced by Fas-deficiency (Xiao et al., 2012). On the other hand, B cell-specific depletion of IL-10 had no protective effect on disease progression in the MRL/lpr mice, indicating that B10 cells were not involved, at least in this model (Teichmann et al., 2012).

A very small numbers of B cells that secrete IL-10 in vitro in response to CpG have been found in the blood of some SLE patients in a higher amount than in healthy controls (Iwata et al., 2011). CD40-stimulated CD19⁺ CD24^hi CD38^hi B cells have also been found to suppress human Th1 differentiation, partly via IL-10. Furthermore, the suppressive capacity of CD19⁺ CD24^hi CD38^hi is defective in SLE patients (Blair et al., 2010). Overall, these studies suggest that regulatory B cells may play a protective role in the early stages of lupus, but the mechanisms may differ between mice and humans (Fujio et al., 2013).

4. Interactions of B cells with other immune cells that contribute to lupus

5.1 Neutrophils can activate autoreactive B cells

Neutrophils (PMNs) offer protection from pathogens by secreting neutrophil extracellular traps (NETs) that contain antimicrobial peptides and chromatin. NETs are not quickly cleared in SLE patients, leading to the formation of autoAbs-DNA ICs (Knight and Kaplan, 2012). In addition to leading to organ damage, ICs stimulate plasmacytoid dendritic cells (pDCs) to secrete IFNα, which re-stimulate PMNs to produce more NETs, thereby creating a positive feedback loop which exacerbates disease (Knight and Kaplan, 2012; Lande et al., 2011). Furthermore, IFNα in combination with IL-6 induce B cell differentiation into PCs (Jego et al., 2003). IFNα also stimulate myeloid DCs (mDCs) that activate and induce class-switching in autoreactive B cells either directly or indirectly through autoreactive T helper cells (Banchereau and Pascual, 2006; Caux et al., 1997). A recent study has unveiled a subset of splenic B cell helper neutrophils (N_BH), that, when activated by microbial products, secrete BAFF, APRIL and IL-21 to stimulate Ab production and class-switch by MZB cells (Puga et al., 2012). Although the N_BH subset has been found to be defective in immune deficiencies (Puga et al., 2012), its role in lupus has not yet been explored. These findings have been summarized in Figure 1.

Figure 1.

Activated neutrophils produce neutrophil extracellular traps (NETs). This mechanism of immune protection exposes large amounts of autoantigens that form immune complexes (IC) with autoAbs. The IC can induce plasmacytoid dendritic cells (pDCs) to secrete IFNα, which in turn stimulates neutrophils to generate more NETs. Furthermore, pDC-derived IFNα and IL6 induce B cell differentiation into plasma cells. In the spleen, TLR activation triggers neutrophils to differentiate into B cell helper neutrophils (N_BH), which are located in the perifollicular region and secrete APRIL, IL–21, and BAFF to induce MZB cells to secrete antibodies. In addition, N_BH can induce MZB cells to express Activation Induced Cytidine Deaminase (AID) and undergo class-switch.

5.2 Marginal zone macrophages are necessary for the clearance of apoptotic cell debris and arrest of autoreactive marginal zone B cells in the marginal zone

MZ macrophages (MZMφ) retain MZB cells within the MZ by processing apoptotic cell (AP) debris, preventing them to activate autoreactive B cells which migrate to the FO (Karlsson, 2003; Wermeling et al., 2007). Another way to retain MZB cells in the MZ is via direct contact via MARCO receptor on MZMφ and unknown receptor on MZB cells (Chen et al., 2005; Yokota et al., 1998). Both mechanisms work together to prevent release of Ag-activated autoreactive MZB cells into the FO where they can initiate the process that leads to autoAbs production.

In BXD2 mice, the MZMφ population gradually decreases with progression of disease, and they are inherently unable to clear AP debris (Li et al., 2013). This exposes autoreactive MZB and MZB precursor (MZP) cells to autoAgs. BXD2 MZP cells upload more AP debris than MZB cells, and only MZP cells migrate to the FO (Li et al., 2013). Mice deficient in scavenger receptors SR–A and MARCO, which are used by MZMφ to bind apoptotic debris (Kraal and Mebius, 2006; Peiser and Gordon, 2001; Platt et al., 1996; Wermeling et al., 2007) produce high titer of DNA specific Abs in response to APs (Wermeling et al., 2007) (see Figure 2). Furthermore, blocking scavenger receptor mediated signaling increased the anti-DNA Ab titer in FcγRIIB^−/− and NZB/W F1 mice (Wermeling et al., 2007). Finally, MARCO has been proposed as a lupus susceptibility gene in the BXSB.Yaa model (Rogers et al., 2009).

Figure 2.

Marginal zone macrophages (MZMϕ) retain MZB cells in the marginal zone and clear apoptotic cell debris. In their absence, such as in the BXD2 mouse, exposes autoreactive MZB cells to autoantigens from apoptotic cells. Such antigen-activated autoreactive MZB cells can either migrate to the red pulp and become short-lived plasmablasts or migrate into the follicle where they engage cognate CD4⁺ T cells from the T cell zone. Those activated CD4⁺ T cells can activate cognate follicular B cell, which proliferate in the follicle to form a germinal center (GC). Proliferating GC B cells undergo affinity maturation in the dark zone, then enter the light zone where it encounters follicular helper T (T_FH) cells. T_FH help induce the engaged B cell to undergo class switch and become either long lived antibody secreting plasma cells or memory B cells. Mountz’s group have shown in the BXD2 model that IL – 17 signaling arrests both T_FH cells and GC B cells in the GC, and thereby prolongs GC reaction and promote antibody production.

Functional MZMφ can also contribute to murine lupus by supporting the expansion of MZB cells via direct contact. Splenic Mφ and most likely MZMφ express delta-like 1 (DL1) ligand, which when engaged with Notch2 receptor (Notch2R) on transitional B cells or MZPs to promote MZB cell differentiation (Moriyama et al., 2008). While anti-DL1 monoclonal Ab (mAb) treatment of young non-autoimmune B6 and NZB/W F1 mice successfully depleted MZB cells, the same treatment of diseased NZB/W F1 mice was not able to reduce their MZB cell population (Moriyama et al., 2008). This suggests that the expansion of MZB cells in old NZB/W F1 mice may result from an enhanced Notch2R signaling, and possibly an increased expression of DL1.

5.3 T cells activate autoreactive B cells

Several T cell subsets contribute to lupus by activating autoreactive B cells. Follicular helper T (T_FH) cells can be divided into two subsets: CXCR5⁺ T_FH cells are attracted to CXCL13 in the GC where they induce B cells to undergo class switch and produce Abs, meanwhile, CXCR4⁺ extrafollicular T cells (T_HEF) are attracted by CXCL12 to extrafollicular sites in lymphoid organs where they induce differentiation of cognate B cells into short-lived plasmablasts (Breitfeld et al., 2000; Chan and Brink, 2012; Craft, 2012; Goodnow et al., 2010; Kim et al., 2001; Kim et al., 2005; Schaerli et al., 2000). Some SLE patients show elevated numbers of circulating T_FH cells that positively correlate with levels of autoAbs, circulating GC B cells and disease severity (Feng et al., 2012; Simpson et al., 2010; Terrier et al., 2012). The Sanroque mutation results in an increased ICOS expression, which results in expansion of IL-21-secreting T_FH cells, the spontaneous formation of GCs and lupus-like phenotypes (Luzina et al., 2001; Vinuesa et al., 2005). IL-21, CD40L, and ICOSL are the key mediators of the interactions between T_FH cells and GC B cells in the differentiation of long-lived PCs producing high affinity class-switched Abs. Blockade of each of these three pathways reduced anti-dsDNA IgG titers and alleviated renal pathology in lupus-prone mice (Daikh et al., 1997; Herber et al., 2007; Iwai et al., 2003; Ma et al., 1996). Anti-CD40L treatment has been shown to be effective in SLE patients (Grammer et al., 2003), but was not further pursued due to thromboembolic side effects caused by the aggregation of activated platelets expressing CD40L (Peters et al., 2009). In the MRL/lpr lupus-prone mice, T_HEF cells secrete IL-21 and induce B cells outside the FO to undergo SHM, class-switch, and differentiate into short-lived autoAb producing PCs (Odegard et al.; Rankin et al.). The same phenomenon was reported for the AM14Tg BCR producing rheumatoid factor (William et al., 2002), although in this model T cell help is not necessary to, but enhances autoAb production (Sweet et al., 2011). Nevertheless, both T_FH and T_HEF produced IL-21 stimulates B cells to differentiate into autoAb-secreting cells (Odegard et al.; Vinuesa et al.), and polymorphisms in the IL21 and IL21R genes have been associated with human SLE disease (Sawalha et al., 2008; Webb et al., 2009).

T_H17 cells represent another B cell activating T cell subset. Increased serum IL-17 in SLE patients is correlated with disease severity (Doreau et al., 2009; Garrett-Sinha et al., 2008). IL-17 in combination with BAFF promotes autoreactive human B cell survival and differentiation into PCs (Doreau et al., 2009). BXD2 mice have large populations of IL17 receptor expressing (IL17-R⁺) B cells and Th17 cells (Hsu et al., 2008). IL-17 induces BXD2 B cells to up-regulate the expression of regulator of G signaling proteins 16, which leads to a decreased sensitivity of G protein coupled chemokine receptors on B cells to chemokine gradients and thereby maintain GC stability (Xie et al., 2010). This explains how IL-17 activated B cells are retained in GCs where they receive prolonged T_FH help in BXD2 mice (Hsu et al., 2008) (see Figure 2).

Finally, in vitro assays have shown that NK T cells engage MZB and B-1 cells with CD1d and CD40L (Takahashi and Strober, 2008). This interaction induced MZB and B-1 cells from 12 weeks old NZB/W F1 to secrete anti-dsDNA IgM, and B-1 cells, to a lesser extent than MZB cells, from older NZB/W F1 to class witch to anti-dsDNA IgG (Takahashi and Strober, 2008).

• B cells producing pathogenic autoantibodies are the primary effector cells in systemic lupus erythematosus

• B cell tolerance to self antigens is breached through multiple mechanisms

• B cell subsets such as marginal zone B cells, B1-a cells and regulatory B cells, modulate autoimmune pathogenesis

• Interactions between B cells and other immune cell types such as T cells, dendritic cells, macrophages and neutrophils, are important to sustain autoantibody production.

Abbreviations

Ab

antibody

AFC

antibody forming cells

BCR

B cell receptor

DC

dendritic cell

GC

germinal center

IC

immune complex

ICOS

inducible T cell co-stimulator

PC

plasma cell

SLE

systemic lupus erythematosus

Tg

transgenic

Ag

antigen

MZ

marginal zone

FO

follicle

MZB cells

marginal zone B cells

FOB cells

follicular B cells

TC

triple congenic

GN

Glomerulonephritis

SNP

single nucleotide polymorphism

PMN

polymorphonuclear neutrophil

NET

neutrophil extracellular traps

pDCs

plasmacytoid dendritic cells

mDC

myeloid DC

AID

activation induced (cytidine) deaminase

MZMφ

marginal zone macrophage

AP

apoptotic cell

DL1

delta-like 1

mAb

monoclonal antibody

T_FH

follicular helper T cells

T_HEF

extrafollicular T cells

SHM

somatic hypermutation