B lymphocyte stimulator (BLyS; trademark of Human Genome Sciences, Rockville, MD) protein (also known as BAFF, TALL-1, THANK, TNFSF13B, and zTNF4) is a 285–amino acid member of the tumor necrosis factor (TNF) ligand superfamily (1–6). It is expressed as a type II transmembrane protein, which is cleaved from the cell surface by a furin protease to release a biologically active soluble 17-kd protein (1–5).
BLyS is a potent B cell survival factor (7–10). The numbers of mature B cells in secondary lymphoid organs, as well as baseline serum Ig levels and Ig responses to T cell–dependent and T cell–independent antigens, are markedly reduced in mice rendered genetically deficient in BLyS (11, 12). Conversely, in vivo administration of exogenous BLyS to mice induces B cell expansion and polyclonal hypergammaglobulinemia (1). Although 3 distinct BLyS receptors are known (BCMA, TACI, and BAFF-R) (6, 7, 13–18), the agonist effects of BLyS on B cells are mediated predominantly (if not solely) via BAFF-R (17–20).
A clear relationship between BLyS overexpression and systemic lupus erythematosus (SLE) has been established through 3 sets of seminal in vivo observations in mice. First, constitutive overproduction of BLyS in some (albeit not all) mice transgenic for the human or murine blys gene leads not just to polyclonal hypergammaglobulinemia, but to elevated titers of multiple autoantibodies (including anti–double-stranded DNA [anti-dsDNA]), circulating immune complexes, and renal Ig deposits as well (6, 21, 22). Second, mice genetically prone to spontaneous development of SLE ([NZB × NZW]F1 and MRL-lpr/lpr mice) harbor elevated circulating levels of BLyS by the time of disease onset (6). Third, treatment of these SLE mice with a BLyS antagonist (a soluble fusion protein between one of the BLyS receptors and IgG Fc) ameliorates the progression of disease and improves survival (6, 19).
BLyS overexpression is a feature of human SLE as well. Cross-sectional studies have documented elevated circulating levels of BLyS in ∼30% of SLE patients (23, 24). A weak (but significant) correlation was observed between circulating levels of BLyS and total IgG, and a stronger correlation was observed between circulating levels of BLyS and anti-dsDNA autoantibodies. This is consistent with observations in blys-transgenic mice that elevations in autoantibody titers are out of proportion to elevations in total Ig levels (6, 21, 22).
Based on the likely contributory role of BLyS to SLE, there is considerable interest in the development and testing of BLyS antagonists as therapeutic agents. A fully human anti-BLyS monoclonal antibody (mAb) (25) has undergone phase I testing in SLE patients and has been shown to be both safe and biologically active (26). It remains to be determined, however, whether all SLE patients would be candidates for BLyS-antagonist therapy or whether only certain patients might be suitable. Suitability may depend upon the duration and persistence of BLyS dysregulation in a given patient. To better delineate such dysregulation in SLE, we conducted a longitudinal observational study of SLE patients in which we assessed BLyS expression over time.
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- MATERIALS AND METHODS
Cross-sectional studies have documented elevated circulating levels of BLyS in variable percentages of patients with SLE, rheumatoid arthritis, Sjögren's syndrome, or human immunodeficiency virus infection (23, 24, 33–35). The entry of at least 1 BLyS antagonist (anti-BLyS mAb) into clinical trials in SLE (26) underscores the need to fully delineate the “normal” fluctuations in BLyS expression among both healthy individuals and patients with SLE. Previous cross-sectional studies indicated that ∼30% of SLE patients have elevated circulating levels of BLyS at a single point in time (23, 24). Since cross-sectional studies are silent with regard to the duration of an abnormality, it remained unknown whether the ∼30% of SLE patients have abnormally elevated circulating BLyS levels 100% of the time, whether 100% of SLE patients have abnormally elevated circulating BLyS levels ∼30% of the time, or whether some intermediate percentage of SLE patients have abnormally elevated circulating BLyS levels some intermediate percentage of time. To address this unresolved issue, we longitudinally monitored serum BLyS protein levels, blood BLyS mRNA ratios, and PBMC surface expression of BLyS along with clinical laboratory and disease features in a cohort of SLE patients.
Our findings permit us to draw several conclusions. First, there is marked heterogeneity in the serum BLyS phenotype among SLE patients that is in dramatic contrast to the almost monolithic response observed among normal controls (Figure 1 and Table 2). Approximately 50% of SLE patients displayed abnormal serum BLyS phenotypes (intermittently elevated or persistently elevated). The patients we studied may not be completely representative of the entire SLE population, since the presence of active disease was a criterion for study enrollment. The broader SLE population may therefore manifest a somewhat lower percentage of abnormal serum BLyS phenotypes. On the other hand, our empirically determined percentage is based on a median observation period of 369 days. The true frequency of abnormal serum BLyS phenotypes in SLE may actually be greater than ∼50% and may only become apparent with longer followup.
In any case, it is likely that the elevated serum BLyS levels reflect dysregulated (increased) BLyS production in vivo. Indeed, blood BLyS mRNA levels and PBMC surface expression of BLyS, each of which likely reflects in vivo BLyS production, were also substantially increased in SLE (Figures 2 and 3 and Table 2). It is noteworthy that a subset of CD14– cells from several SLE patients expressed surface BLyS, an observation not made for corresponding cells from control subjects (Figure 3). Whether these CD14–,BLyS+ cells are monocytes that have down-regulated their surface CD14 consequent to differentiation into dendritic cells (36), other myeloid lineage cells that are inherently CD14–, or CD14– non–myeloid-lineage cells that “aberrantly” express surface BLyS in SLE patients remains to be established.
Discordance between serum BLyS levels and blood BLyS mRNA levels or PBMC surface BLyS expression was not uncommon (data not shown), pointing to complex regulation of serum BLyS levels. For example, surface expression of BLyS by monocytes in synovial fluid from patients with rheumatoid arthritis is decreased despite the presence of increased levels of soluble BLyS in these same synovial fluid samples (31). This raises the possibility that the rate of cleavage of membrane BLyS to soluble BLyS may be an important regulatory factor in the joints of rheumatoid arthritis patients, and it may play a similar important regulatory role in SLE patients. In addition, elaboration of BLyS by activated neutrophils can occur in the absence of surface BLyS expression (37). Either of these two phenomena may contribute to a dissociation between circulating BLyS levels and cell surface BLyS expression.
Furthermore, surface expression of BLyS receptors on B cells varies with the activation/differentiation state of the B cells (38). Since the vast majority of B cells reside in extravascular tissues (e.g., spleen, lymph nodes, bone marrow), unmeasured variations in BLyS receptor expression by these B cells may affect consumption of soluble BLyS and lead to a selective decrease in circulating BLyS levels without concomitant changes in blood BLyS mRNA levels or in cell surface BLyS expression. Moreover, the bulk of endogenous BLyS production likely occurs extravascularly (e.g., in spleen and lymph nodes that are rich in myeloid-lineage cells), so BLyS mRNA levels of circulating BLyS-producing cells may not faithfully reflect BLyS mRNA levels of tissue BLyS-producing cells. Indeed, elevated levels of BLyS mRNA are present in the kidneys of MRL-lpr/lpr mice but not in their PBMCs (Baker KP, Wu Y: unpublished observations). This disparity between BLyS expression in tissues and BLyS expression in the circulation notwithstanding, it is clear that chronic persistent or intermittent overexpression of BLyS is highly prevalent among SLE patients.
A second conclusion from our findings is that in patients with elevated serum BLyS levels, intense courses of high-dose corticosteroids profoundly reduce these levels, oftentimes to normal (Table 3). It may be that corticosteroids can “mask” BLyS dysregulation, leading to an underestimation of the true percentage of SLE patients with persistently elevated or intermittently elevated serum BLyS phenotypes. Although this might represent a direct effect of corticosteroids on blys gene transcription and/or on BLyS protein translation, it may actually be an indirect consequence of corticosteroid-mediated inhibition of interferon (IFN). IFNγ and IFNα each up-regulate BLyS production (30, 39), and PBMCs from many (but not all) SLE patients bear an “IFN signature” indicative of IFN up-regulation (40, 41). High-dose corticosteroid treatment extinguishes the IFN signature (41), raising the possibility that IFN is a major inducer of BLyS overproduction in SLE patients. Experiments are currently in progress to address this issue.
In any case, it is tempting to speculate that part of the salutary clinical effect of high-dose corticosteroids is related to the decrease in serum BLyS levels. Indeed, serum BLyS levels often inversely correlated with the corticosteroid dosage (Figure 4). It may be that the requirement for corticosteroids in many patients can be reduced (eliminated) by neutralization of BLyS. Clinical trials will be necessary to address this issue.
A third conclusion is that the serum BLyS phenotype does not define a specific constellation of affected organ systems in SLE and, for any given SLE patient, serum BLyS levels do not correlate with disease activity (Table 4 and Figure 1). This was predictable, given that BLyS has no known direct or immediate proinflammatory properties. Changes in serum BLyS levels at any point in time should not be expected to acutely promote changes in systemic or organ-specific inflammation (which would be reflected in the SLEDAI score). Even when we focused on the subset of patients with “substantial” proteinuria (≥2.0 gm/day) who presumably had increased renal excretion of BLyS (32), we failed to find a correlation between the degree of proteinuria and serum BLyS levels in most patients. In only 3 of the 17 informative patients was there any suggestion of an inverse relationship between these 2 parameters (Figure 5). It may be that our previous finding of an inverse correlation between serum BLyS levels and the presence of nephrotic-range proteinuria (24) was not solely a consequence of the proteinuria per se but was also influenced by the high doses of corticosteroids that these patients were taking.
The failure to document a correlation between serum BLyS levels and disease activity for any individual patient should not be interpreted to mean that elevated serum BLyS levels have no effect on disease activity. Of note, the positive correlation between serum BLyS levels and anti-dsDNA titers previously documented in cross-sectional studies (23, 24) was confirmed and extended by the current longitudinal study (Figure 6). It may be that increases in BLyS levels, by virtue of the effects on autoantibody production, do increase the likelihood of aggravating and/or exacerbating disease. Such an association might be appreciated in a large SLE population when analyzed in aggregate. Indeed, preliminary findings in a larger cohort of SLE patients suggest that plasma BLyS levels correlate significantly with SLEDAI scores across all patient visits in aggregate over time (42). However, analysis of single individual patients would fail to demonstrate such association because of the numerous factors other than BLyS that contribute to fluctuations in disease activity.
Based on our results, then, are all SLE patients candidates for BLyS-antagonist therapy? The answer to this question depends upon how one views the role of BLyS in SLE. One model has BLyS functioning as a contributor to development of SLE. BLyS per se does not cause loss of tolerance to self antigens, but once such tolerance is broken, the ever-present nature of the autoantigen permits it to repetitively stimulate the host immune system. In the presence of increasing amounts of BLyS, the autoimmune response is exaggerated. In a susceptible host, this exaggerated autoimmune response can trigger/maintain frank clinical disease (SLE).
According to this model, reducing SLE-contributory BLyS levels to normal should ameliorate disease by suppressing the BLyS-driven acceleration or exaggeration of the autoimmune response. Thus, SLE patients with the most elevated circulating BLyS levels should be the ones who are most responsive to BLyS-antagonist therapy. Those patients with persistently normal circulating BLyS levels might be relatively resistant to BLyS-antagonist therapy, since “excess” BLyS is not driving the clinical autoimmunity in these patients.
An alternative model has BLyS functioning as a passive facilitator in the development of SLE. In this model, development of the pathogenic anti-self response is inherently BLyS-independent. Regardless of whether BLyS levels are normal or elevated, the pathogenic component of the autoimmune response is similar. That is, the trigger of autoimmunity elicits a response so robust that its pathogenicity is not further amplified by elevated levels of BLyS. Accordingly, the critical genetic and/or environmental factors that lead to the autoimmune response do not directly require BLyS. Indeed, the fact that many SLE patients have normal circulating BLyS levels strongly suggests that BLyS overexpression is not absolutely essential to development of SLE. Nevertheless, given the indispensable role of BLyS in B cell development (11, 12), a certain threshold level of BLyS is required to permit any antibody responses (including autoantibody responses). When BLyS levels are reduced below this critical threshold level, the ability to fully mount the pathogenic autoimmune response (along with other B cell and humoral responses) is impaired. According to this second model, the SLE patients who should be most responsive to BLyS-antagonist therapy are those with normal, rather than elevated, circulating levels of BLyS, since in such patients, less neutralization of BLyS would be required to reach the critical threshold level.
These two models are not necessarily mutually exclusive. Within the population of humans with SLE, there may be individuals in whom BLyS plays more of a contributor role, and there may be others in whom BLyS plays more of a facilitator role. Indeed, from a therapeutic perspective, the models may operationally be viewed as a continuum, with some patients requiring more neutralization of BLyS than is required by others before salutary clinical effects can be appreciated. Thus, all SLE patients may benefit from BLyS-antagonist therapy. Appropriate clinical trials should be able to resolve these issues.