hen egg-white lysozyme
New Zealand Black
(New Zealand Black × New Zealand White)F1
systemic lupus erythematosus
Polyclonal B cell activation is a hallmark of the immune dysregulation in New Zealand Black (NZB) mice. We have previously shown that the splenic B cell activation is associated with increased CD80 expression. Here we show that abnormal expansions of CD80-expressing GC, CD5+, marginal zone (MZ) precursor and MZ B cells produce this increase. To investigate the role of BCR engagement in the generation and activation of these populations, a non-self-reactive Ig Tg was introduced onto the NZB background. NZB Ig-Tg mice lacked Tg CD5+ and peanut agglutinin+ B cells, confirming the role of endogenous Ag in their selection. Although the increased proportion of MZ B cells was retained in NZB Ig-Tg mice, CD80 expression on these cells was reduced as compared to non-Tg NZB mice, suggesting a role for BCR engagement with endogenous Ag in their activation. Examination of CD40L-knockout NZB mice showed no difference in the abnormal activation or selection of the B cell populations, with the exception of GC cells, as compared to wild-type NZB mice. Thus, polyclonal B cell activation in NZB mice does not require CD40 engagement, but results, in part, from dysregulated BCR-specific mechanisms.
Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by production of pathogenic autoantibodies directed predominantly towards nuclear Ag. The New Zealand Black (NZB) mouse and its F1 hybrid with the New Zealand White mouse (NZB/W) both spontaneously develop a lupus-like autoimmune disease and are considered to be excellent models of human SLE. In NZB mice autoimmune disease is characterized by the production of IgG anti-RBC and anti-ssDNA autoantibodies leading to hemolytic anemia and mild glomerulonephritis, respectively, late in life. NZB/W mice produce in addition high-affinity IgG anti-dsDNA antibodies resulting in a severe immune complex-mediated glomerulonephritis beginning at approximately 5 months of age (reviewed in 1).
Polyclonal B cell activation is found in a large proportion of SLE patients and a number of lupus-prone mouse strains 2, 3. In NZB and NZB/W mice, polyclonal B cell activation is observed early in life (approximately 4 wk of age) and is characterized by increased serum levels of IgM, increased numbers of IgM-secreting B cells with specificity for both auto-Ag and conventional Ag, enhanced spontaneous proliferation of B cells in vitro, and increased numbers of activated B cells isolated on discontinuous Percoll gradients 1, 2, 4–7. Our laboratory has shown that young NZB and NZB/W mice have an expanded splenic B cell population expressing high levels of costimulatory and adhesion molecules, such as CD80 and ICAM-1 8. Sorted CD80+ NZB splenic B cells, isolated from the low-buoyant density fraction on discontinuous Percoll gradients, were enriched for polyclonally activated IgM-secreting B cells 8.
Consistent with this in vitro data, we found that the increased expression of CD80 on splenic B cells co-segregated with increased numbers of IgM-secreting spleen cells in a genetic mapping study, and was linked to a region located at ∼37 cM on NZB chromosome 13 9. This NZB chromosomal region was previously shown to contain lupus susceptibility genes, suggesting that the immune mechanisms leading to polyclonal B cell activation and expansion of CD80+ splenic B cells play an important role in the development of autoimmunity in lupus-prone mice 10, 11. In further support of this concept, we recently showed that C57BL/6 (B6) congenic mice with an introgressed homozygous NZB chromosome 13 interval extending from ∼24 to 73 cM develop polyclonal B cell activation characterized by increased IgM production and an abnormal expansion of CD80+ splenic B cells together with high titer of IgG anti-chromatin autoantibodies and mild glomerulonephritis 12.
Although splenic polyclonal B cell activation has been shown to precede and predict the onset of disease in lupus-prone NZB and NZB/W mice, the origin of this B cell activation remains unclear 13. Merino et al.14 showed, by depleting CD4+ T cells from birth using an anti-CD4 mAb, that CD4+ T cells do not participate in the induction of IgM hypergammaglobulinemia in NZB/W mice. Consistent with a T cell-independent defect, young NZB/W nu/nu mice had similar numbers of IgM anti-TNP plaque-forming cells to NZB/W nu/+ autoimmune control mice 15. Furthermore, transfer of pre-B cell lines from NZB and NZB/W mice into SCID mice was sufficient to produce IgM hypergammaglobulinemia 16, 17. In contrast, Chen et al.18 found reduced numbers of IgM-secreting B cells in NZB.CD4–/– or NZB.CD8–/– mice compared with wild-type NZB mice. Thus, the role of T cell signals in the generation of polyclonal B cell activation remains controversial. The role of other signals, such as those delivered through the BCR, on the generation of the activation phenotype has not been examined.
In this study, we have further investigated the origin of the polyclonally activated CD80+ splenic B cell population in NZB mice. We show that this population is comprised of peanut agglutinin (PNA)+ GC, CD5+, marginal zone (MZ) precursor and MZ B cells, all of which are expanded in NZB mice. As these populations are all proposed to be positively selected by endogenous Ag, such as ubiquitous environmental, bacterial, or self Ag, we investigated the impact of BCR specificity on the selection and activation of these populations 19–24. This was achieved by generating NZB mice in which the endogenous BCR repertoire was removed by the introduction of an anti-hen egg-white lysozyme (HEL) Ig Tg. We show that NZB Ig-Tg mice lack PNA+ GC and CD5+ B cells, confirming the role of BCR engagement with endogenous Ag in the generation of these populations. Although the expansion of MZ B cells was retained in NZB Ig Tg mice as compared to their B6 counterparts, we found decreased MZ B cell activation in NZB Ig-Tg mice when compared with NZB non-Tg (NTg) littermates, suggesting that engagement of MZ B cells with endogenous Ag is required to maintain their abnormal state of activation in vivo.
Nevertheless, MZ B cells and their precursors remained activated in NZB Ig-Tg mice as compared to their B6 counterparts, also implicating BCR specificity-independent mechanisms in the generation of this phenotype. These specificity-independent mechanisms do not involve CD40 signaling since the abnormal selection and activation of non-GC B cell subsets in NZB CD40L-knockout mice is identical to wild-type NZB mice.
Abnormal expansion of several activated B cell populations in young NZB mice
We have previously shown that approximately 50% of CD80+ splenic B cells in NZB mice have a MZ B cell phenotype, suggesting that abnormal CD80 expression was not restricted to this B cell population 8. In this study, we have conducted a detailed characterization of the different splenic B cell populations expressing CD80 in young lupus-prone mice. We first assessed CD80 expression in B cell populations defined by CD21 expression. CD21 is expressed at low levels on transitional T1 and PNA+ GC B cells, low-intermediate levels on CD5+ B cells, intermediate levels on mature follicular B cells, and high levels on MZ B cell precursors and MZ B cells 12, 25, 26. As shown in Fig. 1A, B, increased proportions of CD80+ B cells were seen in the CD21low, CD21int and CD21high B cell subsets of both NZB and NZB/W mice when compared with BALB/c mice. Similar results were obtained for ICAM-1 expression (data not shown). These findings confirm that the abnormal expansion of CD80+ splenic B cells in NZB and NZB/W mice cannot be attributed solely to the expanded MZ B cell population, but also results from an increased proportion of CD80+ cells in each of the CD21-defined B cell subsets.
Although the majority of CD21low B cells have a transitional T1 B cell phenotype in normal mice, we recently showed that the CD21low B cell subset in 4-month-old NZB mice contains increased proportions of PNA+ GC and CD5+ B cells compared with B6 mice 12. We therefore examined whether the increased proportion of CD80+ cells within the CD21low B cell subset in 4- to 6-wk-old NZB mice reflects expansion of PNA+ GC and/or CD5+ B cells expressing CD80 or aberrant activation of transitional T1 B cells. As shown in Fig. 2A, B, the CD21low B cell subset in young NZB mice contains an increased proportion of PNA+ GC B cells when compared with controls. The proportion of PNA+ GC B cells expressing CD80 is similar in NZB and B6 mice (Y.-H. Cheung and J. Wither, unpublished observations). A small proportion of the CD5+CD21low/int B cell population is also present within the CD21low B cell subset of NZB mice (Fig. 2A, B), but in contrast to GC B cells, the proportion of CD80+ B cells within the CD5+ B cell population was increased in NZB mice when compared with non-autoimmune mice (% CD80+CD5+ B cells ± SD: BALB/c, 67.08 ± 9.32; B6, 51.33 ± 1.32; NZB, 81.69 ± 1.47; n=6–7, p<0.005).
Transitional T1 and CD5+, but not GC, B cells have high levels of CD24 expression 12, 25. In NZB mice the 12% increase in the proportion of CD5+ B cells within the CD21lowCD24high B cell subset, as compared to the non-autoimmune mouse strains, approximates the 12–14% increase in CD21low B cells that are CD80+ (Fig. 1B, data not shown). Since most CD5+ B cells in NZB mice are CD80+, this suggests that the majority of CD80+CD21low B cells are CD5+ and not T1 B cells. This concept is supported by the observation that the percentage of CD80+ B cells within the bone marrow B220lowCD24highIgM+ immature B cell compartment, the immediate precursors of the T1 B cell population in the spleen, was similar between NZB and B6 mice (% CD80+CD24highIgM+B220low cells ± SD: NZB, 7.82 ± 2.77; B6, 7.61 ± 2.50; n=9–10, p=0.86). Unfortunately, the AA4.1 mAb that stains transitional B cells could not be used to confirm the lack of aberrant CD80 expression on NZB T1 B cells because NZB splenic B cells do not stain with this reagent (data not shown). Nevertheless, the data suggest that T1 B cells do not contribute to the increased proportion of CD80+CD21low B cells observed in NZB mice.
As shown in Fig. 2A, the CD21int B cell subset in NZB mice also contains an increased proportion of CD5+ B cells which are almost entirely CD80+ and therefore, CD5+ B cells also contribute to the increased proportion of CD80+CD21int B cells found in these mice (% CD5+CD21int B cells ± SD: BALB/c, 2.6 ± 0.8; B6, 2.9 ± 0.7; NZB, 6.4 ± 1.7; n=9–10, p<0.0001).
To investigate whether CD21int follicular B cells abnormally express CD80 in NZB mice, we examined CD80 expression following staining with anti-CD21 and anti-CD23. Mature follicular B cells are CD21intCD23+ and therefore can be distinguished from CD5+CD21low/intCD23– B cells using CD23 expression. Although NZB mice have a CD23 polymorphism that leads to reduced CD23 cell surface expression 27, necessitating adjustment of the regions used to gate the CD23+ and CD23– populations, these populations can still be readily discriminated in NZB mice (Fig. 3A). The percentage of CD21intCD23+ B cells is similar between NZB and BALB/c mice, but reduced in NZB mice when compared with B6 controls (Fig. 3A, B). As shown in Fig. 3C, D, we found an increased proportion of CD80+ B cells within the CD21intCD23+ B cell population of NZB mice when compared with non-autoimmune mice. However, further staining of the CD80+CD21int B cell population indicated that these cells express high levels of CD24 and therefore, CD80+CD21intCD23+ B cells do not have a mature follicular B cell phenotype in NZB mice (data not shown).
The CD21high B cell subset can also be divided into two populations based upon CD23 staining; CD21highCD23– MZ B cells and CD21highCD23+ MZ B cell precursors 26, 28. As shown in Fig. 3A, B, there is an increased proportion of MZ B cell precursors and MZ B cells in NZB mice when compared with non-autoimmune mice, suggesting that in NZB mice there is altered B cell selection leading to enhanced recruitment into the MZ precursor and MZ populations. Furthermore, the proportion of CD80+ B cells was significantly increased in these B cell populations in NZB mice when compared with controls and therefore, both MZ B cell precursors and MZ B cells contribute to the increased proportion of CD80+CD21high B cells observed in NZB mice (Fig. 3C, D). Thus, the increased CD80+ splenic B cell population in NZB mice is derived from abnormal expansions of activated PNA+ GC, CD5+, MZ, and MZ precursor B cells. Interestingly, all of these B cell populations can be generated following encounter with Ag in vivo.
BCR engagement contributes to the generation of activated NZB B cell subsets
Given the possible role of Ag encounter in the abnormal selection and activation of splenic B cell populations in NZB mice, it was of interest to determine the effect of removal of the endogenous B cell repertoire on this phenotype. To remove the endogenous BCR repertoire, an Ig Tg recognizing a foreign protein, HEL, was backcrossed onto the NZB background. As in non-autoimmune B6 mice, expression of the anti-HEL Ig Tg blocks endogenous Ig gene rearrangement and therefore almost all peripheral B cells (∼96%) express the Tg-derived a-allotype IgM heavy chain and have HEL specificity in NZB Ig-Tg mice (Fig. 4A, 29). Consistent with a lack of overt binding to endogenous Ag, IgMa antibodies produced in both B6 and NZB Ig-Tg mice do not cross-react with lupus-specific auto-Ag such as ssDNA (data not shown). Furthermore, the abnormal Bcl-2 expression in NZB T1 B cells that we have previously shown resulting from BCR engagement with endogenous Ag in vivo is blocked by the introduction of the anti-HEL Ig Tg on the NZB background 30. Thus, investigation of anti-HEL Ig-Tg mice permits assessment of the role of BCR engagement with endogenous Ag in the generation of the B cell activation phenotype in NZB mice.
As shown in Fig. 4B, production of PNA+CD21low GC B cells was abrogated within the IgMa+ population of NZB Ig-Tg mice, suggesting that PNA+ GC B cells in young NZB mice are derived from Ag-engaged B cells in vivo. Similarly, generation of CD5+ B cells was prevented by introduction of the anti-HEL Ig Tg onto the NZB background (Fig. 4B). This finding is consistent with observations in anti-HEL Ig-Tg B6 mice (Fig. 4B, 31) and indicates that NZB immune defects are insufficient to overcome the requirement for BCR engagement with endogenous Ag for selection into this compartment.
To determine whether the abnormal distribution of CD21intCD23+, CD21highCD23+, or CD21highCD23– B cells in NZB mice is also affected by introduction of the anti-HEL Ig Tg, we examined the proportion of cells within these populations following staining with anti-IgMa mAb. As was seen in NTg mice, the percentage of CD21intCD23+ cells was reduced, and CD21highCD23– and CD21highCD23+ cells were increased within the IgMa+ B cell population of NZB Ig-Tg mice as compared to B6 Ig-Tg mice (Fig. 4C, D). The expansion of MZ precursors and MZ B cells in NZB Ig-Tg mice was not due to an accumulation of cells expressing endogenous receptors, because the proportion of light-chain-edited cells (represented by the HELlo/–IgMa+ cells in Fig. 4A) and IgMa+ cells co-expressing IgMb was very low and similar between B6 and NZB Ig-Tg mice (% HELlo/–IgMa+ cells ± SD: NZB, 0.69 ± 0.23; B6, 0.86 ± 0.32; n=3–5, p>0.05; % IgMa+IgMb+ ± SD: NZB, 4.74 ± 2.31; B6, 3.75 ± 2.64; n=3–5, p>0.05). Furthermore, the distribution of these cells within the CD21int and CD21high B cell subsets did not differ between the two strains (data not shown). Thus, factors other than BCR specificity appear to contribute to the abnormal expansion of the MZ B cells and their precursors in NZB mice.
Finally, we assessed whether the introduction of the anti-HEL Ig Tg prevented the abnormal activation of CD21intCD23+, CD21highCD23+, or CD21highCD23– B cells in NZB Ig-Tg mice. Although the percentage of CD80+ B cells within the CD21intCD23+, CD21highCD23+, and CD21highCD23– B cell populations was decreased in B6 Ig-Tg mice compared with B6 NTg littermates, the abnormal CD80 expression within the CD21intCD23+ and the CD21highCD23+ B cell populations was not significantly affected by the introduction of the anti-HEL Ig Tg in NZB mice (Fig. 5A, B). In contrast, the percentage of CD80+ B cells within the CD21highCD23– MZ B cell population of NZB Ig-Tg mice was significantly reduced when compared with NZB NTg littermates.
Nevertheless, CD80 expression remained elevated on the MZ B cell population of NZB Ig-Tg mice when compared with B6 Ig-Tg mice (% CD80+CD21highCD23– B cells ± SD: NZB Ig-Tg, 57.6 ± 12.5; B6 Ig-Tg, 20.0 ± 4.5; n=4–7, p=0.0005). It is unlikely that this increase is due to contamination of the CD21highCD23– population with B cells that express specificities other than HEL that have engaged endogenous Ag, because as outlined above the proportion of these cells was small and examination of IgMa+CD21hi cells (which encompass both MZ B cells and their precursors) in NZB Ig-Tg mice demonstrated a similar increase relative to B6 Ig-Tg mice (Fig. 5C, data not shown). Furthermore, if the increased CD80 expression in the NZB Ig-Tg MZ B cell population was due solely to contamination with cells expressing endogenous receptors, we would have seen two peaks of CD80 expression, one with the same expression as the majority of B6 Ig-Tg MZ B cells representing HEL-specific cells that lack binding to endogenous Ag and the other with increased expression relative to B6 Ig-Tg MZ cells representing B cells with other specificities. However, this is not what was observed. As shown in Fig. 5A, the CD80 expression in the CD21highCD23– population of NZB Ig-Tg mice demonstrated a single peak which was shifted to the right relative to that seen in B6 Ig-Tg mice (MFI CD80 expression ± SD: NZB Ig-Tg, 62.7 ± 20.5; B6 Ig-Tg, 20.8 ± 6.7; n=4–7, p=0.0016). Thus, BCR engagement with endogenous Ag in vivo appears to contribute to the abnormal activation of NZB MZ B cells, but is clearly not the sole factor driving this activation.
In summary, replacement of the endogenous B cell repertoire in NZB mice had variable effects on the generation and activation of the abnormally expanded and activated B cell populations in NZB mice. While generation of PNA+ and CD5+ B cells was abrogated and MZ B cell activation was reduced, altered selection into the MZ B cell lineage was unaffected. Furthermore, MZ B cells and their precursors still retained a significant component of their abnormal activation phenotype. Consistent with this observation, serum IgM levels remain elevated in NZB Ig-Tg mice as compared to NTg littermate controls (IgM mg/mL ± SD: NZB NTg, 2.92 ± 1.06; NZB Ig-Tg, 2.34 ± 0.76; B6 NTg, 0.54 ± 0.08; B6 Ig-Tg, 0.55 ± 0.08; n = 11–12, 6–10-wk-old mice; both p>0.05 for Ig-Tg vs. NTg, both p<0.0001 for B6 vs. corresponding NZB strain) and IgMa anti-HEL levels (OD ± SD: NZB Ig-Tg, 2.23 ± 0.29; B6 Ig-Tg, 1.54 ± 0.24; n=15–18, 8–10-wk-old mice, p<0.0001) are increased relative to their B6 Ig-Tg counterparts, indicating that polyclonal B cell activation is, at least in part, driven by mechanisms that are independent of BCR specificity.
The abnormal splenic B cell activation in NZB mice is CD40L-independent
Signals through the BCR integrate with those delivered by other cell surface receptors, such as cytokine receptors and CD40, to induce B cell proliferation and activation 32, 33. We have previously shown that resting B cells from NZB mice abnormally up-regulate CD80 following CD40 cross-linking in vitro, raising the possibility that the abnormal expression of CD80 on NZB B cells is CD40-dependent 34. Therefore to investigate whether CD40 signals contribute to the abnormal generation of CD80+ splenic B cells in NZB mice in vivo, a CD40L gene deletion was backcrossed onto the NZB genetic background using the speed congenic technique (see Materials and methods, 35). The CD40L gene is X-linked and therefore is not located within any of the chromosomal regions previously linked to lupus susceptibility in NZB mice 36.
As in non-autoimmune B6.CD40L–/– mice, abrogation of the CD40/CD40L pathway prevents GC formation in NZB.CD40L–/– mice and therefore a study of PNA+ B cells was not performed (35; E. Pau et al., manuscript in preparation). At 8 wk of age, there was no difference in the proportion of CD5+, CD21intCD23+, CD21highCD23+, or CD21highCD23– B cells between NZB.CD40L+/– and NZB.CD40L–/– mice (data not shown). Similarly, abnormal CD80 expression in each of these splenic B cell populations was not affected by the CD40L gene deletion (Fig. 6A, B).
Consistent with these findings, serum total IgM remained elevated in NZB.CD40L–/– mice (IgM mg/mL ± SD: NZB.CD40L+/–, 5.81 ± 0.40; NZB CD40L–/–, 5.52 ± 0.84; B6.CD40L+/–, 1.58 ± 0.79; B6.CD40L–/–, 0.89 ± 0.47; n = 9–19, 4-month-old mice, p = 0.18 and p = 0.012, respectively, for CD40L+/– as compared to CD40L–/–). The levels of IgM anti-ssDNA and anti-dsDNA antibodies were also unaffected by CD40L gene deletion (OD IgM anti-ssDNA ± SD: NZB.CD40L+/–, 1.78 ± 0.30; NZB CD40L–/–, 1.82 ± 0.26; B6.CD40L+/–, 0.33 ± 0.20; B6.CD40L–/–, 0.28 ± 0.08; OD IgM anti-dsDNA ± SD: NZB.CD40L+/–, 1.12 ± 0.29; NZB CD40L–/–, 1.16 ± 0.25; B6.CD40L+/–, 0.14 ± 0.04; B6.CD40L–/–, 0.13 ± 0.05; n = 9–22, 4-month-old mice, all p>0.05 for CD40L+/– as compared to CD40L–/– within each strain). Thus, the signaling defects leading to abnormal B cell selection and activation of splenic B cells in NZB mice are CD40L-independent.
In this study we have performed a detailed characterization of the CD80+ splenic B cell population in young NZB mice. We demonstrate that the generation of this B cell population results from abnormal expansions of activated PNA+ GC, CD5+, MZ precursor and MZ B cells. Several lines of evidence indicate that BCR signaling and/or engagement plays a crucial role in the generation of CD5+ and MZ B cells 19, 21–23. Both populations contain poly-specific B cells with specificity for endogenous bacterial and/or self Ag, are positively selected, and are susceptible to changes in BCR signaling thresholds. In addition, GC are formed by recirculating B cells following encounter with Ag in the T cell zone of secondary lymphoid organs, or MZ B cells following contact with blood-born Ag in the MZ 20, 24.
As all of the cell populations that are expanded in NZB mice are Ag-selected, we examined the role of endogenous environmental and/or self Ag in their generation and activation by replacing the B cell repertoire with a non-self-reactive anti-HEL Ig Tg. Our results show that although BCR engagement with endogenous Ag in vivo plays a crucial role in the generation or activation of several splenic B cell populations in young NZB mice, BCR specificity-independent mechanisms are also involved.
We also explored the role of the CD40/CD40L pathway in the abnormal generation of CD80+ splenic B cells in NZB mice and showed that, with the exception of the GC population, this phenotype is retained in NZB.CD40L–/– mice. Consistent with this data, NZB.CD40L–/– mice had similar elevated serum total IgM and IgM autoantibody levels to wild-type NZB mice, confirming that polyclonal B cell activation in these mice is CD40L-independent.
Our study extends the results of previous reports suggesting that this B cell activation is T cell-independent 14–17, by demonstrating that not only IgM hyper-gammaglobulinemina but also other manifestations of the abnormal activation phenotype are CD40L-independent. Furthermore, we rule out a significant role for expression of CD40L on other cell populations, such as B cells, in the generation of the abnormal B cell activation phenotype 37. Although the findings reported herein do not exclude the possibility that other activating molecules expressed on or secreted by αβ T cells play a role in the generation of B cell activation phenotype in NZB mice, we think that this is unlikely since NZB.TCRα–/– mice have the same proportion of CD80+ splenic B cells within all CD21-defined B cell subsets as NZB.TCRα+/+ mice (data not shown). Taken together these findings indicate that the polyclonal B cell activation in NZB mice is both CD40L- and T cell-independent.
Replacement of the endogenous BCR repertoire with an anti-HEL Ig Tg abrogated the production of GC and CD5+ B cells. These findings indicate that, similar to non-autoimmune mice, generation of the GC and CD5+ B cell subsets in NZB mice requires engagement with endogenous Ag, which is prevented by the introduction of a high-affinity anti-HEL Ig Tg that lacks overt reactivity with these Ag. In contrast, introduction of the anti-HEL Ig Tg had variable effects on MZ B cells and their precursors in NZB mice. While the expansion of these subsets was unaffected by the introduction of the anti-HEL Ig Tg, activation of MZ B cells was reduced. This later observation suggests that BCR specificity plays an important role in dictating the activation of NZB MZ B cells, probably through engagement with endogenous Ag.
Consistent with the previously proposed activation of normal MZ B cells by endogenous Ag in non-autoimmune mice 38, CD80 expression on B6 MZ B cells was also reduced by introduction of the anti-HEL Ig Tg. Whether the reduced expression of CD80 on anti-HEL Ig-Tg MZ B cells reflects a decreased signal through the BCR, due to a lack of cross-reactivity with endogenous Ag, or whether anti-HEL Ig Tg MZ-B cells fail to recruit additional signals that must act in tandem with BCR signaling to drive CD80 expression is currently unknown. Resting murine B cells require additional signals such as LPS, IL-4, or cross-linking of CD21 to up-regulate CD80 in response to BCR engagement in vitro39–41. It is therefore possible that the reduced activation of anti-HEL Ig-Tg MZ B cells reflects their lack of cross-reactivity with Ag that recruit these additional signals in vivo. Such signals could include those mediated by TLR for BCR that recognize DNA, RNA, and bacterial Ag 42, 43, or CD21 for Ag or immune complexes that activate complement 39, 44, 45.
Although introduction of the anti-HEL Ig Tg reduced expression of CD80 on MZ B cells of both NZB and control mice, CD80 expression on NZB anti-HEL Ig-Tg MZ B cells remained elevated as compared to B6 anti-HEL Ig-Tg MZ B cells. There are several potential explanations for this difference. First, it is possible that NZB B cells have an intrinsic signaling abnormality that leads to increased expression of CD80 in MZ B cells. We have previously shown that NZB resting B cells demonstrate enhanced up-regulation of CD80 in response to CD40 engagement 34. While the CD40-CD40L pathway does not play a role in the increased CD80 expression on MZ B cells observed in vivo, other BCR-independent signals that act through the NF-κB signaling pathway might be responsible for the generation of this B cell phenotype in NZB mice.
Second, the immune dysregulation in NZB mice could lead to increased generation of these BCR-independent activating signals. As mentioned above, CD80 up-regulation on B cells can be induced in vitro by LPS or IL-4 alone; however, it is unlikely that increases in these molecules lead to the abnormal expression of CD80 on NZB MZ B cells, because the activation of NZB MZ B cells occurs in the absence of T cells and the mice are housed in specific pathogen-free conditions.
MZ B cells express high levels of CD21 and it has been shown that C3d-tagged immune complexes can bind to CD21 non-specifically on B cells, leading to their activation 45. It is possible that residual immune complexes in NZB anti-HEL Ig Tg contribute to the increased activation of the MZ B cells because, despite introduction of the Tg, NZB anti-HEL Ig-Tg mice still produce small amounts of endogenous IgMb and IgG anti-ssDNA autoantibodies (data not shown). These residual immune complexes might also serve to activate plasmacytoid dendritic cells leading to increased generation of interferon-α, which has been shown to increase B cell activation 43, 46. Ongoing experiments in the laboratory are seeking to discriminate between these possibilities.
Due to their anatomic location, increased basal state of activation, and close proximity with recirculating Ag, MZ B cells respond rapidly to antigenic stimulation, migrating to B cell follicles to form GC 24, 47, 48. We have previously shown that MZ B cells isolated from NZB Ig-Tg mice are abnormally recruited into GC following transfer into NZB soluble HEL recipient mice where they engage self Ag in vivo49. Consistent with this previous study, we show here that expansion of abnormally activated MZ B cells and PNA+ GC B cells occurs early in life and almost simultaneously in NZB mice. Furthermore, introduction of the anti-HEL Ig-Tg mice onto the NZB background reduced MZ B cell activation and prevented generation of PNA+ B cells. Taken together these findings suggest that MZ B cells are contacting self Ag and being spontaneously recruited into GC in NZB mice.
The increased expression of multiple costimulatory and adhesion molecules on B cells in NZB mice, as compared with non-autoimmune controls, may be relevant to this abnormality. Young NZB mice have an increased proportion of CD86high and ICAM-1high cells within their CD21high B cell compartment, suggesting that these molecules are co-expressed at increased levels on CD80+ MZ B cells (8 and data not shown). Furthermore, we have previously reported that the levels of CD80 expression on NZB MZ B cells are similar to those induced in vitro by an anti-CD40 mAb or a CD8a-CD40L fusion protein, suggesting that they are sufficient to costimulate naive T cells 8. Thus, NZB CD80+ MZ B cells that engage self Ag may be better able to activate naive autoreactive T cells following migration into the B cell follicles leading to abnormal initiation of self-reactive GC reactions.
In contrast to expression of CD80, the expansion of MZ B cells in NZB mice was unaffected by the introduction of the anti-HEL Ig Tg. This finding suggests that, at least in part, the immune mechanisms leading to this expansion in NZB mice are independent of BCR specificity. One of these mechanisms may involve BAFF, as we have recently found that NZB anti-HEL Ig-Tg and non-Tg mice have elevated levels of BAFF as compared to their B6 counterparts (N. Chang et al., manuscript in preparation). These elevated levels of BAFF may not only affect the MZ population quantitatively, but also qualitatively. We have previously demonstrated that NZB transitional T1 B cells have an IgM-mediated apoptosis defect. NZB transitional T1 B cells express Rag-2, suggesting that they have already contacted Ag in vivo and are enriched for self-reactive cells 30. BAFF has previously been shown to promote selection of low-affinity self-reactive B cells into the MZ compartment 50. Thus, increases in BAFF and an apoptosis defect may act in concert to promote selection of self-reactive cells into the MZ B cell compartment of NZB mice. Consistent with this hypothesis, we and others have previously demonstrated that the polyclonally activated B cell population in NZB mice contains a large proportion of DNA-specific B cells, which suggests that MZ B cells and their precursors are enriched with self-reactive clones 2, 6, 8.
In this study, we show that the generation of the CD80+ splenic B cell population in NZB mice results from the expansion of abnormally activated Ag-selected populations. We provide evidence that polyclonal B cell activation results from both BCR specificity-dependent and -independent mechanisms but not from aberrant CD40 B cell signaling. Since polyclonal B cell activation is a hallmark of the immunologic profile in human SLE 3 and we have recently shown that the activated B cell populations in human SLE are also Ag-engaged (N. Chang et al., submitted), it is likely that the findings outlined herein are relevant to human disease. Indeed, further characterization of the signaling pathway(s) leading to the generation of these activated self-reactive B cell populations will be crucial to our understanding of the events leading to pathogenic autoantibody production.
Materials and methods
BALB/c and B6 mice were purchased from Taconic. NZB mice were purchased from Harlan-Sprague-Dawley (Blackthorn, UK). B6 mice expressing Tg encoding IgM/IgD H and L chains specific for HEL (MD4) and B6.CD40L-knockout mice were purchased from The Jackson Laboratories and subsequently bred in our animal facility. Offspring were genotyped by PCR, using primers specific for the V region of IgH, neo, or CD40L gene 29, 35. NZB Ig-Tg and NZB.CD40L–/– mice were produced using the speed congenic technique 51. Mice were genotyped using a panel of polymorphic microsatellite primers spaced on average 14 cM apart (range 1–29 cM). Fully backcrossed NZB Ig-Tg and NZB.CD40L–/– mice were obtained in seven and eight generations, respectively. All mice used for the experiments were female, 4–12 wk old, and housed in microisolators in a specific pathogen-free facility at the Toronto Western Hospital.
Flow cytometric analysis
RBC-depleted splenocytes (5×105) were incubated with 10 μg/mL mouse IgG (Sigma-Aldrich) for 15 min to block FcR and stained with various combinations of directly conjugated mAb. After washing, allophycocyanin-conjugated streptavidin (BD Pharmingen) was used to reveal biotin-conjugated mAb staining. Dead cells were excluded by staining with 0.6 μg/mL PI (Sigma-Aldrich). Flow cytometry of the stained splenocytes was performed using a dual laser FACScalibur (BD Biosciences) and analyzed using Cell Quest software (BD Biosciences).
The following directly conjugated mAb were purchased from BD Pharmingen: biotin-anti-B220 (RA3-6B2), biotin-anti-CD80 (16-10A1), biotin-anti-IgMa (DS-1), PE-anti-CD80 (16-10A1), PE-anti-CD5 (53-7.3), PE-anti-B220 (RA3-6B2), PE-anti-CD23 (B3B4), PE-anti-IgMa (DS-1), PE-hamster IgG1 anti-TNP isotype control (G235-2356), FITC-anti-CD21 (7G6), and FITC anti-B220 (RA3-6B2). Biotinylated HEL was prepared using an EZ-Link Sulfo-NHS-LC Biotinylation kit (Pierce). Biotinylated PNA was purchased from Sigma-Aldrich.
Measurement of antibody production
Levels of total serum IgM, IgM anti-ssDNA and anti-dsDNA, and IgMa anti-HEL antibodies were measured by ELISA. ELISA plates (Immunolon II) were coated overnight at 4° C with nuclear Ag (dsDNA, 20 μg/mL; ssDNA, 10 μg/mL) or goat anti-mouse IgM (for total serum IgM) diluted in PBS, or HEL (50 μg/mL) in 50 mM sodium bicarbonate pH 9.8 binding buffer. The plates were washed with 0.05% Tween-20/PBS, and blocked with 2% BSA/PBS for 1 h at room temperature. After further washing, serum samples diluted in PBS/BSA/Tween-20 at a concentration of 1/100 for measurement of anti-DNA and anti-HEL antibodies or 1/3000 for measurement of IgM, were added to the ELISA plates, and incubated for 1 h at room temperature. The presence of bound antibodies was detected by adding alkaline phosphatase-conjugated anti-IgM (Caltag, Burlingame, CA) or biotinylated anti-IgMa (BD Pharmingen) followed by alkaline phosphatase-conjugated streptavidin (BD Pharmingen). Serum concentrations of total IgM were calculated by reference to a standard curve using a log-log plot.
Comparisons of differences between groups of mice for continuous data were performed using a two-tailed unpaired Student's t-test. A p value of <0.05 was considered to be significant.
This work was supported by grants from the Canadian Institute of Health Research and the Arthritis Society of Canada (J.E.W). J.E.W. is the recipient of an Arthritis Society/Canadian Institutes of Health Research investigator award. We would like to thank Dr. Michael Ratcliffe and Dr. J. C. Zúñiga-Pflücker for critical reading of the manuscript.