Systemic lupus erythematosus (SLE) is a chronic autoimmune disease with diverse presentations. Its pathogenesis remains elusive; however, multifactorial interactions among genetic and environmental factors may be involved 12. SLE is characterized by dysregulation of the immune system that involves hyperactivity of T cells and B cells, production of pathogenic autoantibodies, and the formation of immune complexes, which can lead to multiorgan damage.

Certain nuclear and cytoplasmic autoantigens become clustered in the surface blebs of apoptotic cells 3. Under normal circumstances, apoptotic cells are engulfed by macrophages in the early phase of cell death without inducing inflammation or the immune response. In SLE, however, the clearance of apoptotic cells by macrophages is impaired, which may allow apoptotic cells to serve as immunogens for the induction of autoreactive T and B cells and drive the production of autoantibodies 4.

The reasons for the defective clearance of apoptotic cells in SLE are not clear. The past decade has provided significant evidence that complement deficiencies, immunoglobulin (Ig) M deficiency, pentraxin deficiency and defects in macrophage handling may each contribute to defective clearance of apoptotic bodies 567. Macrophages recognize apoptotic cells through an array of surface receptors. Among them, the tyro 3, axl, mer (TAM) kinases, especially the c-mer receptor tyrosine kinase, play an especially important role in the clearance of apoptotic cells 89. Mice lacking c-mer have impaired clearance of apoptotic cells and develop progressive lupus-like autoimmunity 10. The two ligands that bind to and activate c-mer are growth arrest-specific 6 (Gas6) and protein S, which in turn bind to phosphatidylserine residues exposed early in apoptosis on the surface of the apoptotic cell 11121314.

Gas6, a 75 kDa multimodular vitamin K-dependent protein that has 46 to 48% amino acid identity to protein S, was discovered in the early 1990 s 15. It contains an N-terminal γ-carboxyglutamic acid (Gla) domain, interacting with phosphatidylserine containing membranes, followed by four epidermal growth factor-like domains and a large C-terminal region homologous to the sex hormone binding globulin, can ligate TAM receptor tyrosine kinases 16. Gas6 is expressed in many tissues, including capillary endothelial cells, vascular smooth muscle cells, and bone marrow cells. Unlike protein S, Gas6 is not expressed in the liver, and its concentration in plasma is 1,000-fold lower than that of protein S 17.

Protein S has a critical function in regulating coagulation by serving as a cofactor for activated protein C-dependent proteolytic inactivation of factor Va and factor-VIIIa. Protein S circulates as approximately 40% free protein S and 60% as a complex with C4-binding protein; only free protein S is active as a cofactor for activated protein C and a ligand for the TAM receptor kinases. In the absence of free protein S, there is increased risk of thromboembolism 18.

It is reasonable to hypothesize that Gas6 and protein S might have important roles in the pathogenesis of SLE. Recently, plasma Gas6 was reported to be elevated in patients with severe sepsis, septic shock, and severe acute pancreatitis 192021. However, there are no reports about Gas6 levels in SLE. Low levels of protein S are reported in SLE, and could be contributing to the thrombotic propensity in certain SLE patients 222324. We have therefore compared Gas6 and free protein S concentrations in patients with SLE, examining their possible use as biomarkers of clinical phenotype and/or disease activity.

Abnormal clearance of apoptotic cells may be important in the development of autoantibodies in SLE. As the TAM kinases may be important in the disposition of apoptotic cells, we evaluated plasma concentrations of their ligands Gas6 and free protein S. Although the levels of Gas6 and free protein S were not different overall between patients with SLE and matched healthy controls, free protein S was decreased in subsets of SLE patients with a history of serositis, neurologic, hematologic, and immunologic disorder. It was especially noteworthy that the concentrations of free protein S were correlated with C3 and C4. Protein S was decreased in SLE patients with active hematologic disease as defined by the BILAG index. In contrast to the findings for protein S, reduced levels of Gas6 were not associated with more active disease, with the possible exception of neurologic disorder, although this analysis was limited by a small number of patients. Surprisingly, active hematologic disease as defined by BILAG revealed an unexpected association with elevated, not reduced Gas6 levels.

Protein S is a vitamin K-dependent plasma anticoagulant protein and its deficiency leads to hypercoagulability syndromes with increased risk for venous thrombosis. However, there have been limited reports about functional effects of protein S independent of its anticoagulant function. After identification of TAM kinases as receptors for protein S, this protein was shown to be required for the efficient uptake of apoptotic cells by macrophages in vitro 34, suggesting an important role in immune clearance. Protein S may play a particularly significant role in the removal of apoptotic cells because of its high plasma concentration, despite its apparent lower affinity for the receptor than Gas6. In the study of c-mer-mediated phagocytosis of apoptotic cells, protein S stimulated phagocytosis as well as or better than Gas6 3536. Therefore, it is possible that insufficient levels of protein S may lead to inefficient clearance of apoptotic cells, resulting in exposure of cellular contents to immune cells and promoting an autoimmune response.

Several reports have suggested that the levels of free protein S may be lower in patients with SLE 222332. In the present study, there was no significant difference overall in circulating free protein S between patients with SLE and matched healthy controls. However, the concentrations of free protein S did appear to be decreased in subsets of those patients with a history of certain clinical manifestations, and low protein S correlated with acute evidence of hematologic disease activity and complement consumption. These findings support the possibility of a novel functional link between the coagulation system and distinct inflammatory responses in SLE. It is well known that there is increased cardiovascular mortality and morbidity among SLE patients, which is not fully explained by traditional risk factors 3738. Our results raise the possibility that, in a definable subset of patients with SLE, disease activity may lead to a decrease in the level of free protein S, which then may increase thrombogenicity. It should be considered that the protein that regulates levels of free protein S is the C4b-binding protein, which is a critical complement regulator as well 39. The failure in our series to find decreased levels of protein S in patients with previous thrombosis could reflect the very small number of patients in that category, along with the multiple risk factors that are probably involved in the pathologic hypercoagulability of SLE. Additionally, this was a cross-sectional analysis, whereas at least one report has suggested that decreased free protein S may be more likely to be observed closer in time to a thrombotic event in patients with SLE 40. Although decreased protein S levels may be secondary to SLE activity, we favor the hypothesis that a decrease in protein S may actually contribute to SLE pathogenesis, as discussed above and suggested by Rothlin and colleagues 41.

Previous reports have observed an association between reduced levels of free protein S and antiphospholipid antibody in SLE 2330. It has been suggested that acquired protein S deficiency could contribute to increased risk of thrombosis in patients with antiphospholipid antibody. However, other investigations have not confirmed an association 22313233. These reports evaluated free protein S in a relatively small number of SLE patients (30 to 50 patients). In the present study assessing 107 SLE patients, free protein S levels were significantly lower only in those patients with ACA, but not in those with LAC and anti-β2 glycoprotein I. Autoantibodies directed against protein S have been associated with thrombosis in patients with APS and SLE 32424344. However, the presence of anti-protein S antibodies in patients have not been found to reduce the concentrations of free protein S 3242. Our findings were consistent with these results although the prevalence of anti-protein S was lower in our patients (5%) than previous reports (26 to 31%).

Gas6 is a cell survival, proliferation and chemotactic factor and also a recognition bridge between phagocytes and apoptotic cells. Gas6 is present at a low concentration in plasma; however, it can be released by endothelial cells and leukocytes during serum starvation or under inflammatory conditions 1921454647. The receptors that bind Gas6 (Tyro3, Axl, and c-mer) have an immunoregulatory role, modulating macrophage activation following an initial immune stimulus 948. Gas6 may thus be supposed to participate in inflammation by interfering with macrophage-lymphocyte crosstalk. Furthermore, Gas6 might be involved in other chronic systemic autoimmune diseases, such as rheumatoid arthritis and chronic inflammatory demyelinating polyneuropathy 4950. It has been suggested that Gas6 is involved in macrophage activation in chronic autoimmunity as an autocrine or paracrine regulatory molecule for monocytes 51.

In the present study, plasma Gas6 levels in patients with SLE were the same as in matched HC and levels were unrelated to age and gender. The concentration of Gas6 was increased in the patients with a history of neurologic disorder and acute activity in the BILAG hematology system. The latter results may reflect the inducible nature of Gas6. Basal levels of Gas6 were low, yet it is known to be upregulated in certain states of intense inflammation such as septic shock and severe acute pancreatitis 192021. A recent report finding that almost all Gas6 present in healthy subjects is bound by soluble Axl may explain why there is actually little free Gas6 present in either normal or SLE serum, although the extent to which our ELISA can detect axl-bound Gas6 has not been tested 52.

In SLE, free protein S was decreased in patients characterized by a history of serositis, neurologic, hematologic, and immunologic disorder. Protein S was also decreased in patients with low C3 and C4 and active hematologic activity. Thus, free protein S may be useful as a biomarker of clinical phenotype and disease activity.

Furthermore, the decrease of protein S and increase of Gas6 in patients with acute activity in the BILAG hematologic system suggests the possibility of a unique link between inflammation and thrombotic risk that could be explored mechanistically.

The TAM ligands are important apoptotic debris receptors and regulators of innate immunity. Our study shows that low levels of one TAM ligand, protein S, correlate with C3 and C4 levels and with clinical manifestations of SLE. In contrast, circulating levels of Gas6, the other principal TAM ligand, have little apparent relation to SLE laboratory or clinical manifestations. These data support the view that ligation of the TAM ligands through protein S but not Gas6 is important in clearance of debris and regulation of the innate immune system in patients with SLE.

ACA: anticardiolipin; ACR: American College of Rheumatology; APS: antiphospholipid syndrome; BILAG: British Isles Lupus Assessment Group Instrument; BSA: bovine serum albumin; CV: coefficient of variation; ELISA: enzyme linked immunosorbent assay; Gas6: growth arrest-specific 6; HRP: horseradish peroxidase; Ig: immunoglobulin; NC: normal controls; SLE: systemic lupus erythematosus; SLEDAI: systemic lupus erythematosus disease activity index; TAM kinases: tyro 3, axl, mer.

The authors declare that they have no competing interests.

CHS designed and executed experiments, interpreted data, and wrote the manuscript. BH performed experiments and interpreted data. SL performed pilot experiments and interpreted data. JTM supplied samples and clinical data, interpreted results, and edited the manuscript. PLC designed experiments, interpreted data, and edited the manuscript.