Biomarkers in Systemic Lupus Erythematosus

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1 ARTHRITIS & RHEUMATISM Vol. 50, No. 7, July 2004, pp DOI /art , American College of Rheumatology REVIEW Biomarkers in Systemic Lupus Erythematosus II. Markers of Disease Activity Gabor G. Illei, Edward Tackey, Larissa Lapteva, and Peter E. Lipsky Introduction Systemic lupus erythematosus (SLE) is the most heterogeneous chronic autoimmune disease. Biologic markers that reliably describe various aspects of SLE could be used to predict prognosis, characterize disease activity, and guide therapy. In order to become a validated biomarker, a candidate biomarker must be evaluated in a scientifically rigorous manner. Only a subset of candidate biomarkers will fulfill criteria for surrogate end points after a vigorous qualification process. This process was reviewed in detail in the first part of this review (1) and is summarized in Figure 1. Potential genetic markers of susceptibility were also addressed in the first part of this review (1). Herein we present a review of the literature on biomarkers of disease activity, focusing on more recent publications about novel candidate biomarkers. It is important to note that no candidate has yet been validated as a true biomarker. We have summarized our assessment of the potential of various candidate biomarkers by grouping them as promising, probable, or unlikely, based on the available data (Table 1). Classic markers of lupus activity The value of commonly available immunologic studies, such as tests to determine levels of anti doublestranded DNA (anti-dsdna), measures of complement activation, and immune complexes, remains uncertain. Gabor G. Illei, MD, Edward Tackey, MD, Larissa Lapteva, MD, Peter E. Lipsky, MD: National Institute of Arthritis and Musculoskeletal and Skin Diseases, NIH, DHHS, Bethesda, Maryland. Address correspondence and reprint requests to Gabor G. Illei, MD, Office of the Clinical Director, National Institute of Arthritis and Musculoskeletal and Skin Diseases, NIH, 10 Center Drive, Room 9S205, Bethesda, MD illeig@mail. nih.gov. Submitted for publication July 30, 2003; accepted in revised form March 16, There is a large body of literature about the associations of various autoantibodies with disease activity and/or specific clinical manifestations. Despite extensive evaluation, discussion of which is beyond the scope of this review, there is remarkably little consensus on the value of these examinations in specific situations in individual patients. Anti-dsDNA and complement have been found to be good markers of disease activity and predictors of outcome in lupus nephritis in some, but not all, studies. The results of some studies have suggested that an increase in anti-dsdna antibody levels is such a reliable predictor of disease flare that preemptive treatment is warranted (2,3). Other studies, however, have indicated that such changes have no value in predicting flares (4,5). Previously, high levels of anti-dsdna antibodies were widely accepted as markers of active renal disease; however, a decrease, and not an increase, in anti-dsdna levels was found at the time of renal flares in a recent longitudinal study of 53 patients (6). Although this controversy will not be discussed in detail here since several recent publications and reviews have addressed it (2,4,5,7 10), it is clear that methodologic differences, such as the frequency of testing, the tools used to assess activity, the definition of flares, and the statistical methods used all contributed to the conflicting results. Candidate biomarkers of lupus activity Complement activation products. Complement has an important role in the pathogenesis of SLE. Traditionally, CH50, C3, and C4 measurements have been used to monitor disease activity. However, they have low sensitivity and specificity because plasma levels reflect the result of the dynamic state of complement synthesis and complement consumption, both of which are increased during inflammation. Activation of the complement system is characterized by the generation of 2048

2 BIOMARKERS IN SLE 2049 Figure 1. Development of biomarkers and surrogate end points for systemic lupus erythematosus (SLE). Candidate biomarkers must be validated to be used as true biomarkers. A subset of these will fulfill criteria for surrogate end points after a vigorous qualification process. The intended purpose of biomarkers may be different at various stages of the disease and must be specified at the earliest point in development. Some biomarkers may be appropriate to be used as markers for various specific features, such as disease activity and response to therapy. Different biomarkers may be needed to describe the same attribute, such as disease activity, at different stages of SLE. The circles represent potential markers that describe specific aspects of SLE. Different patterns indicate that different biomarkers may describe the same aspect of the disease, such as activity, at different stages of pathogenesis. The vertically decreasing size of the circles demonstrates that the number of candidate biomarkers will decrease with each step in the validation/qualification process. activated breakdown products of precursor molecules. Complement activation products, such as C1r, C1s, C1 inhibitor, and C4a d (specific for activation of the classical pathway of the complement cascade), Bb (specific for the alternative pathway), and C3a d, C5a, and C5b 9 (terminal complement complex [TCC]) (specific for the common terminal pathway), may be more specific for complement activation than C3 or C4 and have been evaluated as markers of disease activity in several studies. In earlier studies, increased levels of C3a, but not C5a, were seen in patients with more active disease (11,12), and rises in C3a levels preceded clinical flares in 40% of patients. In a subsequent larger study of the same group of patients (380 plasma samples obtained from 86 SLE patients who were followed up prospectively for 15 months), levels of Ba were significantly elevated in patients with more active disease, whereas mean levels of Bb, C5b 9, and C4d did not differ significantly between patients with active and those with inactive disease. However, in patients with inactive/ stable disease, elevated C4d was the most sensitive marker of subsequent flare, and elevated Bb had the highest specificity and greatest predictive value for flare (13). Similarly, in a prospective study of 31 SLE patients in which disease activity was determined using the SLE Disease Activity Index (SLEDAI) (14), Systemic Lupus Activity Measure (SLAM) (15), and physician global assessment at 3 consecutive visits 4 months apart, C4d and Bb were found to be more sensitive indicators of current moderate-to-severe lupus disease activity than were C5b 9, C3, and C4; serum C4d and Bb were also sensitive in predicting flare at the subsequent visit, 4 months later (16). C5b 9 levels did not correlate with disease activity in this cohort, in contrast to the findings in other studies (17,18).

3 2050 ILLEI ET AL Table 1. Serologic Classic Potential biomarkers of disease activity in SLE* Category Promising Possible Unlikely Cytokines and cytokine receptors Anti-dsDNA Immune complexes Complement activation products IFN IL-1Ra IL-4 sil-2r (scd25) IL-6 IFN stnfr IL-10 IL-12 IL-12 p40 IL-15 IL-13 IL-16 IL-18 TNF Endothelial activation svcam-1 slcam-1 Soluble E-selectin Soluble thrombomodulin Measures of NO production Soluble cell surface molecules Miscellaneous scd27 scd40l (CD154) Soluble HLA class I BLyS Kynurenine/tryptophan quotient Leucine aminopeptidase Urinary IL-6 Glycosaminoglycans von Willebrand factor svcam-1 Immunoglobulin free light chains Cellular CD27 high plasma cells CD8,DR T cells / T cells CD19 B cells CD4,CD80 T cells CD40L lymphocytes sfas Double-negative T cells * No assessment has been validated as a biomarker in systemic lupus erythematosus (SLE). The likelihood of development into a biomarker has been categorized into promising, possible, or unlikely. To be categorized as promising, an assessment must have been shown to associate/correlate with SLE or some aspect of SLE in the majority of studies with at least 3 studies conducted or show a strong association if fewer studies were done. To be categorized as possible, the data about the association/correlation of the assessment with SLE or some aspect of SLE must be controversial or must have been shown to associate/correlate only weakly or moderately. To be classified as unlikely, the assessment must have been shown not to associate/correlate with SLE in the majority of the studies. Classification may change as new data become available. Anti-dsDNA anti double-stranded DNA; IFN interferon- ; sil-2r soluble interleukin-2 receptor; TNFR tumor necrosis factor receptor; IL-1Ra IL-1R antagonist; svcam-1 soluble vascular cell adhesion molecule 1; sicam-1 soluble intercellular adhesion molecule 1; NO nitric oxide; scd4ol soluble CD40 ligand; BLyS B lymphocyte stimulator. Porcel et al studied serum levels of C3, C4, factor B, C3a desarg (stable inactive form of C3a), ic3b, C4a, and TCC over a 1-year period in 61 patients with lupus and found that C5b 9 showed the strongest correlation with disease activity scores (17). Similar observations were made in a study of 41 Brazilian lupus patients (18). All parameters, with the exception of C3 and C3b(Bb)P, showed a statistically significant correlation with disease activity. Although none of the variables was able to discriminate between patients whose disease was in remission and those with mild disease activity, C5b 9, C3a desarg, and C4 levels differed significantly between patients with mild and those with moderate or severe disease. Among all the variables, the most highly significant results were found for levels of C5b 9, and these levels correlated well with the severity of the disease (P ). Nagy et al also found a moderate correlation (r 0.3) between SLEDAI scores and C5b 9 levels, but in their study of 65 patients, plasma C3b(Bb)P, a marker of activation of the alternative pathway, correlated best (r 0.41) with disease activity (19). Most of these studies included patients with various organ manifestations including nephritis. Any association between complement activation products and disease activity was either restricted to or stronger for patients with moderate-to-high disease activity. Whether complement tests are of value in routine examination of patients with mild SLE without renal involvement was addressed by Mollnes et al (20). They

4 BIOMARKERS IN SLE 2051 prospectively studied 21 SLE patients without active renal disease, with monthly evaluations for 1 year. They measured C1q, C1 inhibitor, C3, C4, Clrs C1 inhibitor complexes, C4bc, Bb, C3a, C3bc, C5a, and TCC. Of all the complement activation products measured, only TCC correlated significantly with SLEDAI scores (r 0.72), although levels were at the upper limit of normal. No variable was able to predict flares. They concluded that complement testing in general is of limited value in routine examination of patients with mild SLE and that TCC seems to be one of the most sensitive laboratory markers of disease activity in SLE patients even in the absence of nephritis (20). Membrane cofactor protein (CD46) acts as a cofactor for the cleavage of C3b/C4b. Kawano et al studied 21 SLE patients and found higher serum levels of soluble CD46 (scd46) in patients with active lupus; these levels decreased with treatment and were negatively correlated with CH50 levels (21). In summary, there is good rationale to use complement activation products as markers of disease activity. However, available studies have conflicting results, with markers of the classical, alternative, or common pathways showing correlation with activity in some but not in other studies. Some of this may result from methodologic differences, such as the use of plasma versus serum and differences in the definition of disease activity. Further work and large-scale trials in this area are needed to help in further defining appropriate complement split products for assessing lupus disease activity and determining whether any of these can be used as a reliable biomarker. Cytokines in SLE. Cytokines play an important and diverse role in the pathogenesis of lupus. There are a variety of hypothetical explanations for cytokine imbalances in this disease. Considerable attention has been given to the Th1/Th2 model as well as to combinations of pro- and antiinflammatory mediators, possibly profiling certain phenotypes and stages of the disease, with the goal of identifying patterns of cytokine production that may be of importance with regard to further prognostic and treatment decisions. Interleukin-1 and interleukin-1 receptor antagonist. With its potent antiinflammatory properties, interleukin-1 (IL-1) is an important mediator of tissue damage in several chronic autoimmune diseases. IL-1 receptor antagonist (IL-1Ra) blocks IL-1 in a dosedependent manner, and its production is regulated at least partially through Fc receptor (Fc R) dependent pathways. Lipopolysaccharide-stimulated IL-1 release by monocytes from patients with SLE was found to be decreased regardless of disease activity, whereas monocytes from patients with active lupus had deficient Fc R-mediated production of IL-1Ra (22) but produced increased amounts of IL-1Ra in response to adherent IgG (23). Serum IL-1Ra levels correlated with C-reactive protein levels (r 0.70) in 20 patients with newly diagnosed, untreated lupus (24). High levels of IL-1Ra were observed during disease flares not involving the kidney, whereas relatively low levels characterized renal flares in a longitudinal study of 20 patients (25). Strong correlation (r 0.75) was observed between serum IL-1Ra levels and SLEDAI scores in a cohort of 31 patients (21 with active disease and 10 with inactive disease) (23), but not in another study of 15 patients who had not yet received treatment (26). IL-2 and sil-2 receptor. IL-2 is a growth factor for both T cells and B cells. An important role for IL-2 in autoimmunity was demonstrated in IL-2 knockout mice, which developed lymphoid hyperplasia and inflammatory bowel disease. SLE T cells have been found to produce decreased amounts of IL-2 following antigenic stimulation in vitro, an abnormality that is more pronounced in cells from patients with active disease (27). Baseline or mitogen-induced production of IL-2 in whole blood cultures was not different between SLE patients (n 24) and controls, but a correlation (r 0.5) between SLAM scores and mitogen-induced IL-2 production was found in patients (28). Although the correlation was statistically significant, the potential clinical utility is limited because of the variance of the data. In another study, in vitro IL-2 production was measured following various forms of stimulation of peripheral blood mononuclear cells (PBMCs) from SLE patients (n 150) with various degrees of disease activity. Three different patterns were observed, and diminished T cell function correlated with higher disease activity (29). Soluble IL-2 receptor (scd25) is released by activated lymphocytes and may be a measure of lymphocyte activation. Numerous studies have investigated serum sil-2r levels as a marker of disease activity, and most revealed elevated levels in patients with active disease (30 38). Several longitudinal studies demonstrated an increase in sil-2r levels during flares and a decrease with treatment and clinical improvement (36,39,40). In a longitudinal study of 62 patients (32 with lupus nephritis) (39), sil-2r levels paralleled lupus disease activity primarily in patients with nephritis, and in a retrospective study of 71 patients (40), rises in sil-2r levels preceded flares in 18 of 21 cases but did not correlate with disease activity scores. Levels of sil-2r were elevated during periods of active disease in

5 2052 ILLEI ET AL a cross-sectional study of 69 patients, and levels increased before and during flares in the 3 patients who were followed up longitudinally (36). Strong correlation between sil-2r levels and scores on the SLEDAI and on the European Consensus Lupus Activity Measure (ECLAM) (41) (r 0.75 and r 0.83, respectively) was observed in a cohort of 40 patients (42). This and some additional studies demonstrated that the increase in sil-2r levels was most pronounced in conjunction with major flares, especially in patients with nephritis (40,42 44); in 1 study of exacerbations in 16 consecutive patients, no change was observed during minor flares (44). These results suggest that sil-2r can be considered a promising candidate biomarker for disease activity in patients with SLE, especially among those with kidney involvement. IL-4. Increased in vitro production of IL-4 in lupus patients compared with healthy controls was reported by some investigators (45,46), whereas others found reduced IL-4 production in patients (47) or no difference between patients and controls (19,28,48). In a longitudinal study of 20 patients with SLE (49), serum IL-4 levels were elevated, but no correlation with SLEDAI scores was found. No difference in IL-4 levels was seen during inactive versus active disease in another study (37). IL-6. Data from several studies suggest that IL-6 plays a critical role in the B cell hyperactivity and immunopathology of human SLE, and may have a direct role in mediating tissue damage. Lymphoblastoid cells isolated from lupus patients produce increased levels of IL-6, and blocking of IL-6 inhibited anti-dsdna production by lymphoblastoid cells in vitro (50). Increased basal and stimulated IL-6 messenger RNA (mrna) levels in PBMCs have been demonstrated in several studies (47,51 54) and were shown to correlate with disease activity in the studies examining this correlation (47,53). The frequency of IL-6 secreting PBMCs was significantly higher in patients with SLE compared with healthy controls, independent of disease activity (55). Lupus patients have elevated serum levels of IL-6, and these levels have correlated with disease activity or anti-dna levels in some studies (41,56,57) but not in others (51,58,59). Boehme et al studied 124 serial serum samples from 30 patients with different levels of disease activity. IL-6 levels showed a moderate correlation with SLAM scores (r 0.43), and the strongest association was observed in the group with the most active disease (SLAM score 10) (56); a similar degree of correlation of serum IL-6 levels with scores on the SLEDAI (r 0.50) and ECLAM (r 0.48) was observed in a cohort of 40 patients (41). Several other studies, however, have failed to show any association (51,58,59). Although IL-6 levels were elevated, no correlation with SLEDAI or SLAM scores was noted in a study of 52 patients (51), and there was also no correlation with ECLAM scores in serial samples from 9 patients followed up for 2 years (59). In another study (58), although only 7 of 56 patients had elevated serum IL-6 levels and serum concentrations of IL-6 did not correlate with SLAM scores, 16 of 50 had increased urinary IL-6 levels, and urinary levels of this cytokine correlated with overall disease activity and the presence of active urinary sediment. The data on the correlation of serum IL-6 levels and disease activity are not sufficiently strong to make IL-6 a reliable marker of activity. Given that IL-6 has potent inflammatory effects locally, further studies on the correlation between urinary IL-6 and nephritic activity are warranted. IL-10. IL-10 is a pleiotropic cytokine with strong antiinflammatory effects. It also contributes to B cell stimulation and autoantibody production. It is not clear, however, whether the increased IL-10 levels observed in murine and human lupus are causative or rather represent an attempt to regulate a strong Th1 immune response, as suggested in a recent study using the IL-10 knockout mouse lupus model. In that investigation, animals lacking the IL-10 gene had more severe lupus and higher mortality compared with wild-type controls (60). Both spontaneous and mitogen-induced IL-10 production have been found to be increased in PBMCs of patients with SLE (47,48,51,54,61,62). However, in only 1 of these studies (47) did in vitro IL-10 production correlate with disease activity (47). Several studies have assessed the correlation between serum (or plasma) levels of IL-10 and disease activity. Houssiau et al detected IL-10 in sera from 27 of 72 SLE patients and 1 of 30 controls. IL-10 levels correlated weakly with SLEDAI scores (r 0.36) and anti-dna antibody levels (r 0.35) and were negatively correlated with C3 levels (r 0.39) (63). Park and colleagues studied sera from 41 SLE patients before and after the institution of treatment with immunosuppressive therapy and showed that levels of IL-10 correlated with global changes in SLEDAI scores (r 0.45), but not with specific laboratory parameters (64). A moderate correlation between IL-10 levels and SLAM scores (r 0.51) was observed by Boehme et al (56). Miret and colleagues found no difference in IL-10 levels between SLE patients and healthy controls, but did note a

6 BIOMARKERS IN SLE 2053 significant correlation (r 0.40) between levels of this cytokine and SLEDAI scores (65). Grondal et al found increased serum levels of IL-10 in SLE patients; however, these did not correlate with SLEDAI or SLAM scores (51). No difference in IL-10 levels between patients with inactive disease (SLEDAI score 8) and those with active disease (SLEDAI score 8) was found in a cross-sectional study of 20 patients (49). In yet another study of 52 patients followed up at 3 consecutive time points over a 6-month period, no correlation was found between IL-10 levels and ECLAM scores (66). Overall, the findings of studies of IL-10 as a biomarker of lupus activity cover the full range from strong to no association; therefore, it is unlikely to be a valid biomarker. IL-12, IL-18, and interferon-. Interferon- (IFN ) is the classic Th1 cytokine. Its expression is regulated by a number of IFN-inducing monokines, such as IL-12, IL-18, and IL-15. Elevated serum levels of IL-12, IL-18, and IFN have been observed in patients with active lupus (19,45,49,67 69). Wong et al described elevated levels of IL-18 in a cohort of 36 Chinese SLE patients (70). They analyzed the relationship between disease activity and the ratio of selected Th1 cytokines and IL-4 and found moderate correlation between the serum IL-18:IL-4 ratio and SLEDAI scores (r 0.43). The same authors compared patients with (n 35) and patients without (n 37) renal disease and found that plasma IL-18 levels correlated with SLEDAI scores much more strongly in patients with renal disease (r 0.62 and r 0.25 in patients with and those without renal disease, respectively) (70). Amerio and colleagues (49) also observed a strong correlation between IL-18 levels and SLEDAI scores (r 0.72) in a cohort of 20 patients with active lupus (SLEDAI score 5). In contrast, in a study of 60 patients (68), Robak and colleagues found no correlation between IL-18 concentrations and disease activity. There are conflicting data in the literature with regard to production of IL-12 and its interaction with other cytokines (IFN, IL-18). Defective IL-12 production in lupus has been described by some investigators (71) but not by others (72). Serum levels of IL-12 did not correlate with disease activity in several studies (49,68,69); however, serum levels of the p40 subunit of IL-12 correlated with SLEDAI scores (r 0.56) and declined significantly following immunosuppressive therapy (73). Min et al attempted to demonstrate an association between IL-12, IFN, and IL-4 levels in lupus nephritis and found significantly lower levels of IL-12 and IFN in 17 patients with lupus nephritis compared with 23 patients with nonrenal lupus; a significant but weak correlation (r 0.32) was seen between SLEDAI scores and serum IL-4:IFN ratios (74). Although no statistically significant difference in basal or mitogen-stimulated IFN production was found between lupus patients and controls, a strong, significant correlation between SLAM scores and IFN production (r 0.63) was observed in one study (28), whereas in another study, phytohemagglutinin-induced IFN production was decreased and there was no correlation with disease activity (48). These data do not support the use of IL-12 or IFN as biomarkers of disease activity, whereas IL-18 warrants further investigation. Kynurenine and tryptophan. IFN stimulates the enzyme indoleamine dioxygenase (IDO), which converts tryptophan to kynurenine, which is further metabolized to end products such as quinolinic acid and nicotinamides. Widner et al studied serum tryptophan and kynurenine levels and the kynurenine/tryptophan quotient (K/T) in 55 SLE patients, to assess IDO enzyme activity. Lower tryptophan levels and higher kynurenine levels and K/T quotients were found in SLE patients compared with healthy donors. A weak but significant correlation between K/T quotients and SLEDAI scores was found (r 0.24, P 0.04) (75). IL-15. IL-15 induces T cell proliferation and proinflammatory cytokine production and reduces apoptosis. Two studies have shown elevated levels of IL-15 in SLE patients; in neither of those, however, was there any correlation with disease activity (76,77). Tumor necrosis factor and soluble tumor necrosis factor receptors. Controversial views on the role of tumor necrosis factor (TNF ) in the pathogenesis of SLE probably reflect the spectrum of its effects in different tissues during various stages of the disease. Increased levels of TNF have been observed in the serum of patients with SLE (41,49,65,78 80), but correlation with disease activity has varied among studies. Davas et al (42) found a strong correlation between serum TNF levels and SLEDAI (r 0.51) and ECLAM (r 0.54) scores in a cohort of 40 patients. A weaker correlation (r 0.35) with ECLAM scores was seen in a study of serially obtained serum samples from 9 patients (59). However, serum TNF levels did not correlate with SLEDAI scores in studies that included 51 patients (65) and 20 patients (49), respectively. Accelerated TNF expression was found in kidney biopsy specimens from 17 patients with type III or IV lupus nephritis, with 52% of the samples exhibiting TNF along the glomeruli and tubules (81).

7 2054 ILLEI ET AL Interestingly, titers of circulating stnfr p55 and p75 have been found in several studies to be significantly increased in lupus patients and to correlate with disease activity (42,79,82,83). Levels of stnfr p55 and p75 correlated more strongly (r 0.69 and r 0.72, respectively) than TNF levels (r 0.51) with disease activity indices in the study by Davas and colleagues (42). Gabay et al compared 73 lupus patients with patients with inflammatory arthritis and found that levels of stnfr were significantly higher in SLE patients than in patients with rheumatoid arthritis or spondylarthropathies, and p55 and p75 levels correlated with SLAM scores (r 0.5 for both) (79). A correlation of p55 and p75 levels with disease activity measured by the University College Hospital Middlesex score (r 0.54 and r 0.53, respectively) was found in a study of 46 consecutive SLE patients (82); the correlation with scores on the British Isles Lupus Assessment Group (BILAG) index (84) was similar in an additional 7 patients followed up longitudinally (82), whereas a weaker correlation with ECLAM scores was found in a study of serial samples from 9 patients (r 0.35 for both p55 and p75) (59). Soluble TNFR, in elevated levels, has been shown to be biologically active (80,82). A recent study by Aringer et al demonstrated that, despite the increased amount of circulating stnfr, sera from patients with SLE contained biologically active TNF, possibly interacting with functional receptors expressed by peripheral blood lymphocytes. Moreover, serum levels of TNFRII (p75) correlated with disease activity and with the percentage of lymphocytes undergoing apoptosis (83). Elevated concentrations of stnfr have been found to correlate with lupus activity in most studies. Soluble TNFR is a promising candidate as a biomarker of lupus activity. IL-16. IL-16 is produced by CD8 T lymphocytes. It induces chemotaxis of CD4 T cells and monocytes and expression of CD25 and HLA class II molecules on T cells. It can also cause CD4 T cell anergy. IL-16 levels have been found to be elevated in Japanese lupus patients (n 49) compared with healthy controls and correlated strongly (r 0.75) with SLEDAI scores in a subset of these patients (n 29) (85). A similar positive correlation with SLEDAI scores (r 0.62) was found in a European cohort of 34 patients (86). Although there is relatively little information on IL-16, the strong correlation between IL-16 levels and disease activity observed in 2 ethnically different cohorts makes it a possible biomarker candidate. B lymphocyte stimulator. B lymphocyte stimulator (BLyS; trademark of Human Genome Sciences, Rockville, MD) (also known as BAFF, THANK, TALL-1, ztnf-4), a member of the TNF ligand family, has recently been found to play a major role in B cell immunity. BLyS is expressed by macrophages, dendritic cells, and B cells and is up-regulated by IFN and IL-10. BLyS may exist as a type II membrane protein and as a biologically active soluble product. It promotes B cell survival and enhances T cell independent antibody responses (for review, see ref. 87). Transgenic expression of BLyS in mice leads to polyclonal hypergammaglobulinemia, elevated levels of autoantibodies, and renal Ig deposits (88 90). Moreover, elevated BLyS levels have been found in murine models of SLE, and blocking BLyS with a soluble receptor ameliorated disease and improved survival in these models (88). Circulating plasma levels of BLyS are elevated in several autoimmune diseases, including SLE (91,92). In a cross-sectional study of 150 lupus patients, elevated BLyS levels were associated with hypergammaglobulinemia and anti-dsdna antibodies. No association of BLyS levels with SLAM scores was seen in a subset of 110 patients (92). In another study, 19 of 95 patients with SLE had elevated BLyS levels compared with healthy controls; BLyS levels did not correlate with IgG levels and correlated only weakly with anti dsdna levels (r 0.31), and patients with nephrotic-range proteinuria had lower plasma BLyS levels (91). In summary, measurement of BLyS levels appears to reflect B cell activation that parallels autoantibody production, rather than clinical manifestations of lupus activity. Type I interferon system. The role of antigenpresenting cells and the type I IFN system in response to possible environmental triggers appears to be one of the important components in the development and maintenance of abnormal autoimmune responses. Several studies have demonstrated elevated levels of IFN in patients with SLE (93 98). Serum IFN levels correlated with SLEDAI scores (r 0.47) in serial samples from 30 patients (5 samples per patient) (93). IFN levels showed a strong correlation with disease activity (r 0.6) in a study of untreated patients (95). In another study, the allostimulatory capacity of lupus sera, which was attributed mainly to IFN, showed a moderate correlation with disease activity (r 0.47) in pediatric lupus patients receiving various types of treatment (94). Genome-wide gene expression profiling using microarrays is a powerful technology that allows the simultaneous measurement of thousands of mrna

8 BIOMARKERS IN SLE 2055 transcripts. In a recent study, the gene expression profile of PBMCs from lupus patients with relatively quiescent disease was compared with that of PBMCs from healthy volunteers (99). The analysis identified 161 unique genes that were differentially expressed between the 2 groups. Using hierarchical clustering, 2 distinct patterns that distinguished SLE patients from controls were identified, although considerable overlap was described. One of the most striking mrna clusters contained several IFN-regulated genes. An IFN score was generated for each patient based on the levels of expression of genes in the IFN cluster. Approximately half of the SLE patients exhibited an elevated IFN score. Further analysis showed that the IFN score was moderately but significantly correlated (r 0.5) with the number of major SLE features exhibited and that major organ involvement (previous or concurrent) was more frequent in patients with high levels of expression of IFN-induced genes (99). The overexpression of IFN-regulated genes was confirmed in another study that included 30 pediatric lupus patients with various levels of disease activity (100). Using the most stringent statistical analysis (Bonferroni correction), gene expression profiling of PBMCs identified 15 genes that were highly up-regulated in SLE patients; 14 of these are targets of IFN. Analysis using a more liberal correction (Benjamini and Hochberg correction) yielded 18 additional genes, 12 of which are IFN regulated. A surprising finding was the overexpression of genes of immature granulocytes, especially defensin. Indeed, immature neutrophils were identified in the white blood cells of a large proportion of SLE patients. Treatment with high-dose glucocorticoids inhibited the appearance of the IFN signature, but not the expression of immature granulocyte-associated genes. The expression of 10 genes correlated with disease activity according to the SLEDAI. The strongest correlation (r 0.55) was found with the formyl peptide receptor like 1 protein, which mediates chemotactic activities of defensins that are products of immature neutrophils (100). These results need confirmation in larger populations, but suggest that distinct gene expression profiles may be associated with lupus and with specific organ involvements, and may change with disease activity. Moreover, the IFN signature was found in many patients who did not have detectable serum levels of IFN, suggesting that microarray analysis can be used to detect the activation of distinct pathways, which can lead to better understanding of disease pathogenesis and can suggest therapeutic targets. The utility of using microarray expression profiles to monitor disease activity remains to be determined. Markers of endothelial activation and lupus disease activity. Adhesion molecules mediate specific cell cell and cell extracellular matrix interactions. They are expressed on vascular endothelium and on circulating leukocytes and play an important role in the inflammatory response by regulating adhesion of leukocytes to the endothelial cells, transmigration through the endothelium, and cell interactions in the immune response. Proinflammatory cytokines up-regulate the expression of selectins and adhesion molecules. Increased expression of vascular cell adhesion molecule 1 (VCAM-1), intercellular adhesion molecule 1 (ICAM-1), and E-selectin has been demonstrated in the skin of SLE patients, and this was most pronounced in patients with active disease and laboratory markers of complement activation (101). Soluble adhesion molecules can be found in the plasma and may serve as useful and practical markers of inflammation. ICAM-1 (CD54). Increased sicam-1 levels have been found in patients with active lupus in some studies ( ) but not in others ( ). The literature is more controversial on the issue of whether sicam-1 levels correlate with disease activity or major organ involvement. Some studies show no correlation with disease activity (105,112). Tulek et al studied 41 patients with lupus and 16 healthy controls and found that, although mean sicam-1 levels were slightly elevated in patients with active disease, there was no statistically significant difference between levels in those with active and those with inactive SLE. No correlation with SLEDAI scores or any organ involvement was observed (105). Other studies of similar size demonstrated significant correlation with disease activity (110,113,114); however, these did not include a cohort with inactive lupus, making comparison difficult. In a study of 24 lupus patients, a strong correlation between serum sicam-1 levels and SLEDAI scores was found (r 0.739) (113). Soluble ICAM-1 levels were elevated in pediatric lupus patients with active disease (n 18), and they correlated with SLEDAI scores (r 0.79) (110). Sfikakis et al found increased sicam-1 levels in SLE patients (n 61), with a strong correlation between these levels and both SLEDAI (r 0.46) and ECLAM (r 0.63) scores in a representative subgroup of 30 patients (103). Tesar et al observed high sicam-1 levels, which changed with disease activity, in 6 patients with lupus nephritis (115). Data from longitudinal studies are also conflicting. Spronk et al found no correlation between disease

9 2056 ILLEI ET AL activity and sicam-1 levels over a 6-month period (102) in their study of sera obtained preceding 22 exacerbations of SLE, whereas Egerer et al found that in 9 patients followed up for 2 3 years, persistently elevated levels of sicam-1 were associated with persistently active disease that was poorly responsive to therapy (116). Soluble VCAM-1 (CD106). Soluble VCAM-1 levels are elevated in patients with active lupus and correlate with global measures of disease activity (66,102,106,112,115,117); the correlation with serologic markers of disease activity has been more variable. Janssen and colleagues found a good correlation (r 0.68) with disease activity measured by the BILAG index in 17 patients with various manifestations of SLE (112). Ho et al (117) described good correlation between SLEDAI scores and plasma svcam-1 levels in Chinese SLE patients; the correlation was stronger among those who had nephritis (r 0.69) (n 35) than among those who only had extrarenal disease (r 0.54) (n 38). Ikeda and colleagues, in a study of 72 SLE patients (27 with lupus nephritis), also found an association between svcam-1 levels and disease activity; higher levels of svcam-1 were found at the time of biopsy in patients with an activity index of 4 on kidney biopsy (106). In longitudinal studies, good correlation between svcam-1 levels and disease activity was found. In a study of serially obtained sera (obtained every 6 months), samples obtained before or during 22 consecutive exacerbations of SLE exhibited increased svcam-1 levels, and there was good association between svcam-1 levels and SLEDAI scores (102). Kaplanski et al measured adhesion molecules, including serum svcam-1, in patients with primary antiphospholipid syndrome (APS; n 24), patients with SLE and APS (n 15), and patients with SLE without APS (n 22). Soluble VCAM-1 levels were high in all 3 of these groups. Patients with SLE-associated APS had the highest svcam-1 levels, and these levels correlated positively with the severity of thrombosis. Those authors suggested that svcam-1 may have a role in the pathogenesis of APS and may also serve as a disease activity marker (118). Soluble E-selectin (CD62E). Soluble E-selectin levels are elevated in lupus patients. However, no correlation with disease activity has been found in most studies (102,106,112), although one study demonstrated a correlation of soluble E-selectin levels with active cutaneous disease without systemic symptoms in a mixed population of patients with discoid LE (n 19), subacute cutaneous LE (n 8), and SLE (n 24) (119). In summary, the controversy surrounding adhesion molecules and lupus disease activity is still ongoing as a result of the conflicting findings in several studies. The most consistent data showing an association of disease activity with soluble adhesion molecules are for svcam-1, for which the association appears most strong in lupus nephritis; the correlation of sicam-1 levels with disease activity is controversial. Most studies have not demonstrated any correlation between soluble E-selectin and disease activity. Serum thrombomodulin. Thrombomodulin (TM) is expressed on the luminal surface of the vascular endothelium. Its soluble form can be detected in the plasma and urine after endothelial cell injury. All studies have shown elevated levels of stm in patients with active SLE, especially those with nephritis (66,117, ). In patients with renal disease, Ho et al found stm levels to be moderately correlated (r 0.41) with SLEDAI scores (117), whereas in other studies a strong correlation with ECLAM scores (r 0.69) or SLAM scores (r 0.89) was detected in SLE patients overall (66). The consistent results observed throughout the relevant studies make stm a good candidate as a biomarker of disease activity, especially in patients with lupus nephritis. Measures of nitric oxide synthesis. Nitric oxide (NO) plays a vital role in immunity by modulating inflammatory responses. NO is synthesized from L-arginine by constitutive NO synthase and inducible NO synthase. It is readily transformed into nitrate and nitrite, which are excreted into the urine. Several studies have shown elevated levels of NO and/or nitrate/nitrite in the blood (117, ) and urine (125) of lupus patients, with higher levels in patients with active disease. The correlation of serum NO or nitrite levels with disease activity is more variable among studies. Ho and colleagues studied plasma NO concentrations in 73 SLE patients (35 with and 38 without renal disease) and found that these concentrations correlated with disease activity (117). Serum nitrite levels correlated with disease activity (r 0.47) and with levels of antibodies to dsdna (r 0.35) in the sera of 46 SLE patients in another study (123), and a significant correlation between serum nitrate/nitrite levels and SLEDAI scores was found in a study of 26 patients followed up for 1 3 years; kidney-related parameters within the SLEDAI had the highest correlation with serum nitrate/nitrite levels (124). Other studies, however, have not confirmed these observations. Although serum nitrate concentrations showed moderate correlation (r 0.35) with SLEDAI scores in a univariate cross-sectional analysis of 50 SLE

10 BIOMARKERS IN SLE 2057 patients, there was no correlation (r 0.13) between SLEDAI scores and serum nitrate levels when serial measurements were analyzed (125). No statistically significant correlation between serum nitrite levels and SLEDAI scores was found in a cohort of Chinese patients with (n 35) or without (n 37) kidney disease (117). Some of these conflicting results may be explained by methodologic differences. For example, nitrate and nitrite levels can be falsely elevated by exogenous sources, such as foods and medications. Therefore, some studies have investigated other by-products of NO synthesis. In a prospective investigation, levels of 3-nitrotyrosine, an indicator of long-term NO-mediated protein modification, correlated with SLEDAI scores in African American patients (r 0.591) but not in white patients, whereas nitrate/nitrite levels correlated with SLEDAI scores in white patients (r 0.653) (126). Further studies are needed to assess the various measures of NO production as potential markers of disease activity. Immune complexes and SLE disease activity. Circulating immune complexes (CICs) exist in the serum of patients with SLE ( ), and correlation with disease activity was described in several reports published in the late 1970s and the 1980s ( ). Abrass et al conducted a prospective study of 48 patients to assess the potential value of serial measurements of CICs in SLE patients not selected for particular organ involvement. CICs were measured by the fluid-phase and solid-phase C1q binding assays. Elevated results on the solid-phase C1q binding assay were associated with the presence of manifestations of SLE, as well as with changes in degree of disease activity that prompted physician action. The predictive accuracy of an elevated result on the C1q binding assay in relation to disease manifestations was found to be This assay also predicted, 82% of the time, physician action to change therapy (133). Levels of IgG-, IgM-, IgA-, and IgE-containing ICs were measured in sera of 44 patients with SLE in another study (134). Disease activity was determined retrospectively according to criteria described by Morimoto et al (135). ICs of all immunoglobulin isotypes correlated with disease activity (134). Other investigators have studied the relationship between molecular sizes of DNA fragments from DNA anti-dna antibody ICs and disease activity. Morimoto and colleagues examined sera from 28 patients with SLE. Three molecular sizes of DNA were identified, and the patients were divided into 4 groups based on these molecular sizes. The amount of DNA from ICs showed strong correlation with disease activity (r 0.86), but the size of the DNA fragment did not (135). In a more recent study of serial serum samples from 28 patients, severe flares were associated with lower C1q levels and lower amounts of DNA in ICs, and levels of DNA in ICs correlated inversely with SLEDAI scores (r 0.47) and with C1q at flares (r 0.62) (136). Further studies are warranted to define these changes more completely and to establish standardized methods that could be used to assess their clinical value as markers of disease activity. T cells and disease activity. T lymphocytes play a major role in the immune dysregulation in SLE. Therefore, assessing the number of activated or abnormal T cell subsets is an obvious avenue for monitoring of disease activity. Lymphocyte activation is associated with changes in the expression of selected surface markers, such as CD69, HLA DR, and CD27. Su et al described a moderate correlation (r 0.5) between the ratio of CD69 and CD3 PBMCs and SLEDAI scores in 42 lupus patients (137). Viallard and colleagues studied HLA DR expression by subsets of peripheral T lymphocytes in relation to disease activity in 60 SLE patients over a 3-year period, using flow cytometric analysis. Thirty-four patients had quiescent disease (SLEDAI scores 1 6 throughout the study) and 26 had active disease (at least a 3-point increase in the SLEDAI score at some point). In univariate analyses, all serologic parameters (anti-dsdna antibody levels, CH50 levels, erythrocyte sedimentation rate) and the percentage of DR T cells were significantly higher in the active disease group, with the increase in the percentage of CD8,DR cells being the most significant (P 10 7 ). Multivariate logistic regression analysis identified CH50 levels and CD4,DR and CD8,DR cell percentages as independent variables predicting a flare. CD8,DR cells were most strongly associated with flares (P 0.001). In a longitudinal analysis of patients who had flares, an increase in the percentage of CD8,DR lymphocytes was seen before the flare was diagnosed clinically (138). CD3,CD4,CD8 double-negative T cells constitute 1 15% of all peripheral blood lymphocytes in humans. Double-negative T cells can express either the / or the / T cell receptor. Compared with agematched normal controls, a significantly lower frequency of / T cells was found by flow cytometry in peripheral blood lymphocytes of 35 SLE patients (3.2% versus 5.9%). No correlation with either clinical or laboratory measures of disease activity was found (139). Similarly,

11 2058 ILLEI ET AL in another study (140), a decreased number of / T cells was found in 29 SLE patients (mean SD / l) compared with a control group ( / l), and no statistically significant correlation between disease activity and the number of / T cells was demonstrated. However, the percentage of / T lymphocytes in clinically normal skin of SLE patients was twice that found in the skin of healthy subjects (mean SD % versus %), and it correlated with disease activity (r 0.594) (140). Oishi et al found that double-negative V 24 T cells were significantly increased in patients with active SLE compared with healthy subjects. During active disease, lupus patients had a selective reduction and disappearance of double-negative T cells with the invariant V 24J Q chain (the human counterpart of murine natural killer 1 [NK1 ] T cells) and oligoclonal expansion of some other subpopulations; all of these changes reverted with corticosteroid therapy (141). Anand et al found a significantly higher percentage of / double-negative T cells in 22 patients with SLE compared with controls, although there was no significant increase in the total percentage of double-negative T cells that included / double-negative T cells (142). The expression of the activation markers HLA DR and CD69 on the surface of double-negative T cells did not correlate with disease activity (measured by the BILAG index) (142). CD80 is a costimulatory molecule that is expressed on activated T cells. The percentage of CD4,CD80 cells correlated (r 0.522) with disease activity in one study of 28 Japanese SLE patients (111); however, no such association was found in a study of 23 European patients (13 with active and 10 with inactive disease) (143). The observation that the percentage of CD8,DR T cells is increased before flares is intriguing but has to be confirmed in additional investigations. Studies with more detailed analysis of peripheral T cell subsets are necessary to determine whether any of these subsets are suitable markers of activity. Leucine aminopeptidase (LAP) is a ubiquitous exopeptidase, and increased levels of LAP in the serum may reflect release from damaged cells or increased secretion by activated cells. Inokuma et al studied 46 consecutive hospitalized patients with active SLE (mean SLEDAI score 15) who were being treated with steroids at stable doses and found that 70% of patients had an elevated LAP level on at least 1 occasion; LAP levels correlated with leukopenia, lymphocytopenia, and SLEDAI scores (144). Those authors postulated that the increased LAP levels during active lupus might be related to increased T cell apoptosis or T cell activation since high levels of LAP have been observed following stimulation of T lymphocytes but not B lymphocytes in patients with infections that directly affect T cells, such as measles and rubella (145). LAP levels were within normal limits in patients with rheumatoid arthritis, suggesting that increased lymphocyte apoptosis, rather than T cell activation, is the source of LAP. B cells and disease activity. The central role of B cells in the pathogenesis of lupus has been increasingly recognized (146). These cells produce pathogenic autoantibodies, serve as antigen-presenting cells, produce cytokines, and influence T cell activation. In mice, B cells expressing the CD5 marker are a major source of autoantibody production. In humans, however, CD5 behaves as an activation marker rather than as a marker for a distinct lineage of cells (147), and CD5 cells do not correlate with disease activity in SLE ( ). Other cellular markers of B cell activation have also been investigated. In a study of 13 patients with active and 10 patients with inactive SLE, a panel of activation markers of B and T lymphocytes was evaluated, and only CD19,CD86 B cells correlated with disease activity (r 0.57) (143). Recently, a more detailed analysis of the distribution of B cell subsets was performed by flow cytometry and the results were compared with laboratory and clinical measures of disease activity (152). In that study, the number and frequency of plasma cells strongly expressing CD27 (CD27 high ) was significantly correlated with scores on 2 SLE disease activity indices (SLEDAI [r 0.49] and ECLAM [r 0.42]) and with the titer of anti-dsdna antibodies. Patients with highly active disease (SLEDAI score 8) were also found to have an increased frequency of CD19 cells. Using a nonparametric data-sieving algorithm, these B cell abnormalities were found to exhibit predictive values of 78% and 78.9%, respectively, for inactive and active disease. The predictive value of the B cell abnormalities was greater than that of the humoral/clinical data pattern, making this B cell subpopulation a promising candidate biomarker of disease activity (152). Several studies have shown that soluble CD27 levels were higher in patients with SLE compared with healthy controls and that patients with active disease had higher levels than patients with inactive disease (36,153). In a study of 70 patients (25 with active disease, as judged by an independent physician), those with active disease had higher levels of scd27, but classifying patients as having high or low scd27 levels had low