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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Eur J Immunol. Author manuscript; available in PMC Jul 1, 2011.
Published in final edited form as:
PMCID: PMC3057185
NIHMSID: NIHMS268905

Separate Checkpoints Regulate Splenic Plasma Cell Accumulation and IgG Autoantibody Production in Lyn-deficient Mice

Summary

Accumulation of plasma cells and autoantibodies against nuclear antigens characterize both human and murine lupus. Understanding how these processes are controlled may reveal novel therapeutic targets for this disease. Mice deficient in Lyn, a negative regulator of B and myeloid cell activity, develop lupus-like autoimmune disease. Here, we show that lyn−/− mice exhibit increased splenic plasmablasts and plasma cells and produce IgM against a wide range of self-antigens. Both events require Btk, a target of Lyn-dependent inhibitory pathways. A Btk-dependent increase in the expression of the plasma cell survival factor IL-6 by lyn−/− splenic myeloid cells was also observed. Surprisingly, IL-6 was not required for plasma cell accumulation or polyclonal IgM autoreactivity in lyn−/− mice. IL-6 was, however, necessary for the production of IgG autoantibodies, which we show are focused in lyn−/− mice towards a limited set of nucleic acid-containing and glomerular autoantigens. A similar uncoupling of plasma cell accumulation from IgG autoantibodies was seen in lyn+/− mice. Plasma cell accumulation and polyclonal IgM autoreactivity are therefore controlled separately from, and are insufficient for, the production of IgG against lupus-associated autoantigens. Regulators of either of these two checkpoints may be attractive therapeutic targets for lupus.

Keywords: autoimmunity, Lyn, Btk, IL-6, plasma cell

Introduction

The autoimmune disease systemic lupus erythematosus (SLE) is characterized by loss of tolerance to nuclear antigens resulting in autoantibody production, immune complex deposition, inflammation, and end organ damage. Aberrant accumulation of antibody secreting cells, which include among them autoreactive specificities, is a common feature of human SLE [1] and several genetically distinct models of murine lupus, including NZB x NZW and Lyn-deficient mice [29]. This has been attributed to an unusually favorable splenic plasma cell survival niche and impaired plasma cell homing to the bone marrow in the NZB x NZW model [5,6]. However, events that promote increased plasma cells in lyn−/− mice are poorly defined. An understanding of the range of cellular and molecular mechanisms that result in plasma cell accumulation and autoantibody production may lead to novel therapeutic approaches for SLE.

Mice lacking the Src family kinase Lyn develop an autoimmune disease with several features characteristic of SLE, including anti-dsDNA IgM and IgG, plasma cell accumulation, and glomerulonephritis [24]. Decreased Lyn expression has been observed in SLE patients[10] and polymorphisms in the lyn gene are associated with human lupus [11]. Thus, lyn−/− mice are a useful model for understanding SLE.

Lyn inhibits responses to BCR crosslinking by phosphorylating the ITIMs of several inhibitory receptors [12]. B cells from lyn−/− mice are thus hyperresponsive to BCR stimulation [4,12]. lyn−/− mice also demonstrate numerous defects in myeloid cells, such as increased myelopoiesis [13], increased integrin signaling [14] and increased production of cytokines by activated macrophages, mast cells, and, in some circumstances, dendritic cells[1517]. These cytokines include IL-6, a plasma cell survival factor [1820], the expression of which has been shown to be elevated in both murine and human lupus [2123]. It plays a role in the production of anti-DNA antibodies in murine lupus models both in vitro and in vivo [23,24].

A major target of Lyn-dependent inhibitory pathways in B cells is the BCR signaling component Btk. [2527]. Btk and Lyn also have opposing effects in macrophages [15,28], dendritic cells [29,30], and mast cells [16]. Mice deficient in both Lyn and Btk have a severe block in B-cell development. Residual B cells fail to respond to BCR crosslinking or produce autoantibodies [31,32]. A transgene (Btklo) that expresses a low level of Btk in B cells, but no Btk in myeloid cells, restores both B cell development and BCR signaling in lyn−/−Btk−/− mice. B cells from lyn−/− and lyn−/−Btklo mice are present in similar numbers and are equally hyperresponsive to BCR stimulation [31,33]. Despite this, lyn−/−Btklo mice do not develop autoantibodies [33]. Thus, Btk-dependent events subsequent to or separate from the initial hyperactivation of B cells by antigen are required for autoimmunity in lyn−/− mice.

Here, we demonstrate that Btk mediates both the accumulation of splenic plasmablasts and plasma cells and increased expression of IL-6 by splenic myeloid cells in lyn−/− mice. Surprisingly, increased plasmablasts and plasma cells as well as elevated IgM reactive to a wide range of autoantigens were still observed in lyn−/− mice in the absence of IL-6. However, IL-6 was required for the production of IgG autoantibodies, which in lyn−/− mice are focused towards a limited number of lupus associated autoantigens such as DNA-containing, RNA-containing, and glomerular antigens. Uncoupling of plasma cell expansion and IgM autoreactivity from the production of IgG autoantibodies was also observed in lyn+/− mice. Thus, plasma cell accumulation in lyn−/− mice is dependent on Btk, independent of IL-6, and insufficient for pathogenic autoimmunity. Additional events requiring IL-6 and complete deficiency of Lyn are required for the production of autoreactive IgG focused towards nucleic acids and glomeruli.

Results

lyn−/− mice demonstrate a Btk-dependent increase in splenic plasmablasts and plasma cells

lyn−/−Btklo mice are lyn−/−Btk−/− mice carrying a transgene expressing a low level of Btk in B cells and no Btk in myeloid cells. We have previously shown that B cells from these mice retain hypersensitivity to BCR crosslinking, but the animals fail to develop autoantibodies [31,33]. Thus, Btk contributes to autoimmunity in lyn−/− mice by regulating events subsequent to or separate from the initial hyperactivation of B cells by antigen. We therefore asked whether Btk regulates plasma cell accumulation in lyn−/− mice.

Consistent with previous reports of increased antibody secreting cells and total IgM in the absence of Lyn [24,3133], our studies reveal an increase in splenic plasmablasts and plasma cells in lyn−/− mice by flow cytometry (Figure 1). lyn−/− spleens had a significantly higher frequency and total number of CD138hiB220+ plasmablasts and CD138hiB220−/lo plasma cells than did their wild type counterparts. These cells expressed high levels of intracellular IgK, confirming their identity as plasmablasts and plasma cells (Supplemental Figure 1). Taking into account the reduced frequency and number of B cells (Figure 1), lyn−/− spleens had a striking 10 fold increase in the number of plasmablasts and plasma cells per B cell. The frequency and total number of plasmablasts and plasma cells as well as the ratio of plasmablasts and plasma cells to B cells were normalized in lyn−/−Btklo mice (Figure 1). This was due to reduced Btk levels and not to the integration site of the transgene or unappreciated ectopic expression of Btk, as splenic plasmablasts and plasma cells remained elevated in lyn−/− mice expressing both the endogenous Btk gene and the transgene (lyn−/−Btkhi) (Figure 1). This effect of reduced Btk dosage was specific to lyn−/− mice, as splenic plasmablast and plasma cell numbers were not significantly different between wild type and Btklo mice (data not shown).

Figure 1
lyn−/− mice have a Btk-dependent increase in splenic plasmablasts and plasma cells

At 7–9 months of age, the frequency of splenic plasmablasts and plasma cells remained elevated in lyn−/−, but not lyn−/−Btklo mice (wt 1.3 +/− 0.13%, n = 8; lyn−/− 4.2 +/− 3.1%, n = 8; lyn−/−Btklo 1.2 +/− 0.58%, n = 5; p = 0.03 for lyn−/− vs. both wt and lyn−/−Btklo). Thus, reduced Btk dosage does not simply delay the appearance of these cells. There was no significant difference in the frequency of CD138hiB220−/lo plasma cells in the bone marrow of 7–9 month old wt, lyn−/−, and lyn−/−Btklo mice (wt 0.75 +/− 0.23%, n = 8; lyn−/− 1.18 +/− 0.60%, n = 8; lyn−/−Btklo 0.7 +/− 0.21%, n = 5), indicating that plasma cell accumulation in lyn−/− mice occurs primarily in the periphery.

Thus, lyn−/− mice demonstrate a Btk-dependent increase in splenic plasmablasts and plasma cells. These data also indicate that heightened sensitivity to BCR crosslinking is insufficient for the increase in plasma cells observed in the absence of Lyn.

lyn−/− mice exhibit a Btk-dependent increase in IL-6

The plasma cell survival factor IL-6 has been implicated in the production of anti-DNA antibodies in murine models of lupus [1824]. We therefore asked whether lyn−/− mice expressed increased levels of IL-6, and, if so, whether this depended on Btk. There was a trend towards increased serum IL-6 levels in 5–7 month old lyn−/− mice, but not lyn−/−Btklo mice (Figure 2a). This was not observed in younger mice (Figure 2a), although it is possible that local increases in IL-6 occur earlier, becoming systemic with time.

Figure 2
Increased levels of serum IL-6 and increased expression of IL-6 by splenic myeloid cells is observed in lyn−/− mice and is dependent on Btk

lyn−/− macrophages and dendritic cells have previously been shown to secrete increased amounts of IL-6 under certain conditions [15,17]. We therefore asked whether more cells express IL-6 in the lyn−/− splenic environment, where the increased plasmablasts and plasma cells are observed, and whether this requires Btk. Splenocytes were incubated for 4 hours with brefeldin A plus or minus LPS. IL-6 expression was subsequently measured by flow cytometry. Minimal IL-6 staining was observed in unstimulated cells (Figure 2c). However, an increased frequency of IL-6 expressing cells was observed in LPS-stimulated splenocytes from lyn−/− mice compared to their wild type or lyn−/−Btklo counterparts (Figure 2b,c). The majority of these cells were B220- and expressed the myeloid cell marker CD11b. A subset were also positive for the dendritic cell marker CD11c (Figure 2b).

IL-6 is dispensable for plasma cell accumulation and polyclonal IgM autoreactivity but mediates the production of lupus-associated IgG autoantibodies

Given a) the known roles of IL-6 in plasma cell survival and the production of anti-DNA antibodies in other lupus models [1824] and b) the requirement for Btk in both the plasma cell accumulation and the increased IL-6 expression observed in the absence of Lyn, we hypothesized that IL-6 mediates plasma cell accumulation and autoantibody production in lyn−/− mice. To address this issue we generated mice deficient in both Lyn and IL-6. Surprisingly, there was no difference in the frequency of splenic plasmablasts and plasma cells between lyn−/− and lyn−/−IL-6−/− mice in young (Figure 3a, Supplemental Figure 2) or aged mice (Figure 3b,c). In addition, the ratio of plasmablasts and plasma cells per B cell was similarly elevated in lyn−/− and lyn−/−IL-6−/− mice. Thus, IL-6 is dispensable for both the initial increase in splenic plasmablasts and plasma cells in lyn−/− mice and the maintenance of this phenotype. Nor did IL-6 deficiency reduce the frequency of bone marrow plasma cells in either young or aged lyn−/− mice (Figure 3d,e, Supplemental Figure 2). If anything, there was a trend towards increased plasma cells in the bone marrow of aged lyn−/−IL-6−/− mice, although this was not significant (Figure 3e, Supplemental Figure 2).

Figure 3
Splenic plasmablast and plasma cell accumulation in lyn−/− mice is independent of IL-6

To gain a broad perspective on the role of IL-6 in autoantibody production in lyn−/− mice, we employed an autoantigen array containing approximately 70 antigens associated with a range of autoimmune diseases [34]. The antigens include nucleic acids, histones, extracellular matrix components, and various other protein antigens. Sera from 18 week old wild type, lyn−/−, and lyn−/−IL-6−/− mice were hybridized to the array, and reactivity detected with anti-IgM or anti-IgG secondary antibodies. lyn−/− mice demonstrated a broad spectrum of autoreactivity in the IgM compartment, which was also observed in lyn−/−IL-6−/− mice (Figure 4a). This result was not due simply to the increased total IgM levels in lyn−/− and lyn−/−IL-6−/− serum (wt 0.61 +/− 0.27 mg/ml; lyn−/− 4.02 +/− 0.49* mg/ml; lyn−/−IL-6−/− 3.68 +/− 1.54* mg/ml; n = 4, * p < 0.05 vs. wt), as only a subset of the total antigens interrogated were recognized. Thus, IL-6 is also dispensable for polyclonal IgM autoreactivity. Quite a different scenario was observed in the IgG compartment, however. Autoreactive IgG in lyn−/− mice was much more focused towards a limited number of antigens which are characteristic of SLE (Figure 4a). These include DNA- and RNA- containing antigens and glomerular extracts. Intriguingly, almost no autoreactive IgG was observed in the absence of IL-6. This was despite the fact that there was no significant difference in total IgG levels between wt (2.65 +/− 2.30 mg/ml, n = 4), lyn−/− (5.50 +/− 1.58 mg/ml, n = 4) and lyn−/−IL-6−/− (4.55 +/− 2.48 mg/ml, n = 4) mice. To determine whether the lack of IL-6 merely delays the appearance of autoreactive IgG, we repeated the array experiments on aged mice. Similar results were obtained with respect to both IgM (data not shown) and IgG autoantibody profiles (Figure 4b).

Figure 4
lyn−/− mice demonstrate IL-6 independent IgM autoreactivity against a wide range of autoantigens and IL-6 dependent, focused IgG autoreactivity against lupus associated autoantigens

lyn+/− mice have an intermediate degree of plasma cell accumulation and polyclonal IgM autoreactivity but do not produce lupus-associated IgG autoantibodies

The increased signaling that occurs in lyn−/− mice as a result of impaired inhibitory receptor activity is likely dampened by reduced dosage of Btk. To determine whether altering the balance of activating and inhibitory signals in general affects plasma cell accumulation and/or IgG autoantibody levels, we examined lyn+/− mice, which have a partial impairment of lyn-mediated inhibitory signaling [35]. lyn+/− mice had plasmablast and plasma cell frequencies between those of wt and lyn−/− mice (Figure 5). This was reflected in an intermediate level of total IgM (wt 0.61 +/− 0.27 mg/ml, n = 4; lyn+/− 1.03 +/− 0.39 mg/ml, n = 5; lyn−/− 4.02 +/− 0.49 mg/ml, n = 4).

Figure 5
lyn+/− mice exhibit an intermediate degree of splenic plasmablast and plasma cell accumulation

To assess the effect of altered Lyn and/or Btk dosage on autoantibody profiles, we compared the reactivity of sera from wild type, lyn−/−, lyn+/−, and lyn−/−Btklo mice with the autoantigen array described above (Figure 4c). Neither IgM nor IgG autoantibodies were present in lyn−/−Btklo mice [33], consistent with the requirement for Btk in the accumulation of plasma cells. Intriguingly, several lyn+/− mice demonstrated the IgM autoantibody signature characteristic of lyn−/− mice, but did not produce IgG against the lupus associated autoantigens (Figure 4c). Total IgG levels were not significantly different between lyn−/− and lyn+/− mice, however (lyn−/− 5.5 +/− 1.58 mg/ml, n = 4; lyn+/− 3.78 +/− 1.22 mg/ml, n = 5). These observations reinforce the results from lyn−/−IL-6−/− mice demonstrating that increased plasma cells and polyclonal IgM autoreactivity are insufficient for the production of IgG against lupus related autoantigens.

Discussion

We demonstrate here that lyn−/− mice have an increase in splenic plasmablasts and plasma cells that is associated with the production of autoreactive IgM against a wide range of self antigens (checkpoint 1, Figure 6). Both events are mediated by Btk, a target of Lyn-dependent inhibitory pathways, and are observed to some extent even when Lyn dosage is only partially reduced. Surprisingly, both accumulation and polyclonal IgM autoreactivity are independent of the plasma cell survival factor IL-6. This is despite the observation that lyn−/− myeloid cells overexpress IL-6 in a Btk-dependent manner. Ongoing studies are aimed at defining whether Lyn-deficiency affects the differentiation, survival, or migration of plasma cells or alters the plasma cell niche in the spleen. The role of Btk in each of these processes is also being examined.

Figure 6
Multiple checkpoints focus IgG autoreactivity towards lupus-associated antigens in lyn−/− mice

The current studies do not address whether plasma cell accumulation is necessary for autoimmune disease. However, the fact that this is a common feature of human SLE and many distinct murine lupus models [19] suggests that it plays an important role in disease pathogenesis. Our results do demonstrate that neither increased plasmablasts and plasma cells nor polyclonal IgM autoreactivity are sufficient for the production of pathogenic IgG autoantibodies.

In contrast to the wide range of self antigens recognized by IgM in lyn−/− mice, autoreactive IgG is much more focused towards DNA- and RNA- containing and glomerular antigens, all of which are characteristic of SLE (checkpoint 2, Figure 6). The production of IgG against these lupus associated autoantigens is not observed in lyn−/−IL-6−/− or lyn+/− mice, both of which demonstrate increased splenic plasma cells and widespread autoreactivity in the IgM compartment. This suggests that while many types of autoreactive B cells escape tolerance mechanisms and differentiate into IgM secreting plasma cells in the absence of Lyn, only a subset receive the appropriate signals to induce class switching to pathogenic IgG isotypes. The latter process requires IL-6 and complete deficiency of Lyn.

There are several possible mechanisms by which IL-6 may control the production of autoreactive IgG. Some of the effects of IL-6 on B cells have recently been shown to be indirect, via CD4+ T cells [36]. IL-6 promotes the development of TFH cells and Th17 cells [37,38], both of which have been implicated in the pathogenesis of lupus [39,40]. Inhibition of Treg function as a result of increased IL-6 expression has also been demonstrated in the Sle1.Sle2.Sle3 lupus model [41]. Anergic anti-DNA B cells in Balb/c mice can be activated in the presence of CD4+ T cell help, a process which is suppressed by Tregs [42]. It will be interesting to determine whether autoimmunity in lyn−/− mice is T-cell dependent. IL-6 has also been shown to promote myelopoiesis in the Sle1.Yaa mice [43]. Myelopoiesis [13] and IL-6 expression are both enhanced in lyn−/− mice. This could lead to an increase in T cell activation due to greater numbers of antigen presenting cells, or facilitate T-cell independent class switching of anti-DNA B cells[44] in response to myeloid derived factors such as BAFF [45]. In non-autoimmune mice, IL-6 expressed by myeloid cells can inhibit the differentiation of autoreactive, anergic B cells [46]. This tolerance mechanism is clearly ineffective in the absence of Lyn, as IL-6 has no effect on IgM autoantibodies and promotes IgG autoreactivity in lyn−/− mice.

CD11b+ splenocytes are likely an important source of IL-6 in lyn−/− mice. Increased expression of IL-6 in these cells in response to LPS was dependent on Btk. The role of Btk in IL-6 production by myeloid cells is controversial based on previous results analyzing macrophages and dendritic cells from Btk−/− mice or X-linked agammaglobulinemia patients, who have loss of function mutations in Btk. Some studies indicate that Btk has no effect on LPS-induced IL-6 production [30,47], while others show that IL-6 expression is enhanced in the absence of Btk [48]. In contrast to these reports but consistent with our results, a Tec kinase inhibitor blocks LPS-induced IL-6 production in human neutrophils [49]. Taken together, these observations suggest two possibilities. The requirement for Btk in IL-6 production may differ depending on the context, such as the microenvironment or whether or not Lyn is expressed. Alternatively, various myeloid cell subsets may have different requirements for Btk in IL-6 production.

These results presented here indicate that therapeutic approaches that target the production of IgG autoantibodies (checkpoint 2, Figure 6) may have efficacy for SLE even if they do not prevent B cell hyperactivation, plasma cell accumulation, or IgM autoreactivity (checkpoint 1, Figure 6). Since they affect steps that occur late in the process of pathogenic autoantibody production, such approaches should be significantly less immunosuppressive than either currently approved therapies or B cell depletion. Of further clinical interest, the phenotype of lyn−/−IL-6−/− mice bears some resemblance to incomplete lupus in humans. These individuals have some autoimmune features but have insufficient criteria for a diagnosis of SLE. Autoantibody array studies similar to those performed here recently revealed that IgM autoreactivity predominates in incomplete lupus patients [34]. SLE patients, in contrast, have a much higher ratio of autoreactive IgG to IgM for most autoantigens. This suggests that the switch to pathogenic IgG isotypes may underlie the transition from incomplete lupus to full blown SLE. It would be intriguing to determine whether IL-6 levels vary between incomplete lupus and SLE patients.

Materials and Methods

Mice

All mice were backcrossed onto the C57BL/6 background. lyn−/− [4] and lyn−/−Btklo [31, 33] have been described previously. IL-6−/− mice were purchased from Jackson Laboratories. Animals were housed in an SPF barrier facility. All procedures were approved by the UT Southwestern Institutional Animal Care and Use Committee.

Flow cytometry

Single-cell suspensions were prepared from whole spleen by mechanical dissociation or flushed from bone marrow and treated with RBC-lysis buffer. Cells were Fc-blocked with anti-mouse CD16/CD32 (BD Biosciences) prior to incubation with some combination of monoclonal antibodies to B220-PerCP, CD138-biotin, CD11b-FITC, CD11c-biotin, IgK-PE (Southern Biotech), or IL-6-PE for four-color flow cytometry. Biotinylated antibodies were detected with strepavidin-allophycocyanin. All antibodies were from BD Biosciences unless otherwise indicated. After staining for extracellular markers, cells were permeabilized and fixed according to the manufacturer’s protocol using BD Cytofix/Cytoperm kit (BD Biosciences) followed by staining with anti-IgK or anti-IL-6. Prior to intracellular staining for IL-6, cells were incubated with media alone or 10 ug/ml LPS for 4 hrs in the presence of GolgiPlug (brefeldin A) (BD Biosciences), which was used according to the manufacturer’s instructions. Samples were acquired on a FACSCalibur cytometer and analyzed using CellQuest software (both from BD Biosciences).

ELISA

Blood was collected from a saphenous vein puncture using clot-activating microvettes (Sarstedt) and serum was isolated. IL-6 was detected using an OptEIA anti-mouse IL-6 ELISA kit (BD Biosciences) according to the manufacturer’s instructions; Immulon II plates (Dynatech Laboratories) were used. Total IgM and IgG ELISAs were performed as described previously [33].

Autoantigen arrays

Autoantibodies were measured on an autoantigen proteomic array that has been described previously [34]. The array includes 70 autoantigens and 4 control proteins. 1ul of serum samples were diluted 1:100 and added to the arrays in duplicate. Detection was with Cy3 labeled anti-mouse IgM and Cy5 labeled anti-mouse IgG (Jackson ImmunoResearch). A Genepix 4000B scanner with laser wavelengths 532 (for Cy3) and 635 (for Cy5) was used to generate images for analysis. Images were analyzed using Genepix Pro 6.0 software to generate a GPR file. Net fluorescence intensities (nfi) were normalized using anti-mouse IgG or IgM spotted onto each array. Values obtained from duplicate spots were averaged. Hierarchical clustering analysis of autoantibodies were performed using Cluster and Treeview software (http://rana.lbl.gov/EisenSoftware.htm).

Supplementary Material

Supplementary

Acknowledgments

We thank Sandirai Musuka and Arturo Menchaca for excellent technical support and Dr. Rochelle Hinman for critical reading of the manuscript. This work was funded by NIH grants P01 AI039824 (A.B.S.) and 1 F31 GM076982 (T.G.). A.B.S. is a Southwestern Medical Foundation Scholar in Biomedical Research. A.J.C. was supported in part by the UT Southwestern Summer Undergraduate Fellowship Program.

Abbreviations

SLE
systemic lupus erythematosus

Footnotes

Conflicts of Interest

None

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