Helena Enocsson

Interferon-alpha Mediates Suppression of C-Reactive Protein Explanation for Muted C-Reactive Protein Response in Lupus Flares?

OBJECTIVE C-reactive protein (CRP) is synthesized by hepatocytes in response to interleukin-6 (IL-6) during inflammation. Despite raised IL-6 levels and extensive systemic inflammation, serum CRP levels remain low during most viral infections and disease flares of systemic lupus erythematosus (SLE). Because both viral infections and SLE are characterized by high levels of interferon-alpha (IFNalpha), the aim of this study was to determine whether this cytokine can inhibit the induction of CRP. METHODS The interference of all 12 IFNalpha subtypes with CRP promoter activity induced by IL-6 and IL-1beta was studied in a CRP promoter- and luciferase reporter-transfected human hepatoma cell line, Hep-G2. CRP secretion by primary human hepatocytes was analyzed by enzyme-linked immunosorbent assay. RESULTS CRP promoter activity was inhibited by all single IFNalpha subtypes, as well as by 2 different mixtures of biologically relevant IFNalpha subtypes. The most prominent effect was seen using a leukocyte-produced mixture of IFNalpha (56% inhibition at 1,000 IU/ml). The inhibitory effect of IFNalpha was confirmed in primary human hepatocytes. CRP promoter inhibition was dose dependent and mediated via the type I IFN receptor. Transferrin production and Hep-G2 proliferation/viability were not affected by IFNalpha. CONCLUSION The current study demonstrates that IFNalpha is an inhibitor of CRP promoter activity and CRP secretion. This finding concords with previous observations of up-regulated IFNalpha and a muted CRP response during SLE disease flares. Given the fundamental role of both IFNalpha and CRP in the immune response, our results are of importance for understanding the pathogenesis of SLE and may also contribute to understanding the differences in the CRP response between viral and bacterial infections.

Soluble urokinase plasminogen activator receptor levels reflect organ damage in systemic lupus erythematosus

Assessments of disease activity and organ damage in systemic lupus erythematosus (SLE) remain challenging because of the lack of reliable biomarkers and disease heterogeneity. Ongoing inflammation can be difficult to distinguish from permanent organ damage caused by previous flare-ups or medication side effects. Circulating soluble urokinase plasminogen activator receptor (suPAR) has emerged as a potential marker of inflammation and disease severity, and an outcome predictor in several disparate conditions. This study was done to evaluate suPAR as a marker of disease activity and organ damage in SLE. Sera from 100 healthy donors and 198 patients with SLE fulfilling the 1982 American College of Rheumatology classification criteria and/or the Fries criteria were analyzed for suPAR by enzyme immunoassay. Eighteen patients with varying degree of disease activity were monitored longitudinally. Disease activity was assessed by the SLE disease activity index 2000 and the physicians global assessment. Organ damage was evaluated by the Systemic Lupus International Collaborating Clinics/American College of Rheumatology damage index (SDI). Compared with healthy control subjects, serum suPAR levels were elevated significantly in patients with SLE. No association was recorded regarding suPAR levels and SLE disease activity in cross-sectional or consecutive samples. However, a strong association was observed between suPAR and SDI (P < 0.0005). Considering distinct SDI domains, renal, neuropsychiatric, ocular, skin, and peripheral vascular damage had a significant effect on suPAR levels. This study is the first to demonstrate an association between serum suPAR and irreversible organ damage in SLE. Further studies are warranted to evaluate suPAR and other biomarkers as predictors of evolving organ damage.

Four anti-dsDNA antibody assays in relation to systemic lupus erythematosus disease specificity and activity

Objective. Analysis of antibodies against dsDNA is an important diagnostic tool for systemic lupus erythematosus (SLE), and changes in anti-dsDNA antibody levels are also used to assess disease activity. Herein, 4 assays were compared with regard to SLE specificity, sensitivity, and association with disease activity variables. Methods. Cross-sectional sera from 178 patients with SLE, of which 11 were followed consecutively, from a regional Swedish SLE register were analyzed for immunoglobulin G (IgG) anti-dsDNA by bead-based multiplex assay (FIDIS; Theradig), fluoroenzyme-immunoassay (EliA; Phadia/Thermo Fisher Scientific), Crithidia luciliae immunofluorescence test (CLIFT; ImmunoConcepts), and line blot (EUROLINE; Euroimmun). All patients with SLE fulfilled the 1982 American College of Rheumatology and/or the 2012 Systemic Lupus International Collaborating Clinics (SLICC-12) classification criteria. Healthy individuals (n = 100), patients with rheumatoid arthritis (n = 95), and patients with primary Sjögren syndrome (n = 54) served as controls. Results. CLIFT had the highest SLE specificity (98%) whereas EliA had the highest sensitivity (35%). When cutoff levels for FIDIS, EliA, and EUROLINE were adjusted according to SLICC-12 (i.e., double the reference limit when using ELISA), the specificity and sensitivity of FIDIS was comparable to CLIFT. FIDIS and CLIFT also showed the highest concordance (84%). FIDIS performed best regarding association with disease activity in cross-sectional and consecutive samples. Fisher’s exact test revealed striking differences between methods regarding associations with certain disease phenotypes. Conclusion. CLIFT remains a good choice for diagnostic purposes, but FIDIS performs equally well when the cutoff is adjusted according to SLICC-12. Based on results from cross-sectional and consecutive analyses, FIDIS can also be recommended to monitor disease activity.

Association of Serum C‐Reactive Protein Levels With Lupus Disease Activity in the Absence of Measurable Interferon‐α and a C‐Reactive Protein Gene Variant

The type I interferon (IFN) system is important in the pathogenesis of systemic lupus erythematosus (SLE). We previously demonstrated an inhibitory effect of IFNα on interleukin‐6 (IL‐6)–induced C‐reactive protein (CRP) in vitro, hypothetically explaining the poor correlation between disease activity and CRP levels in SLE. This study was undertaken to investigate disease activity, IL‐6 levels, and CRP levels in relation to a CRP gene polymorphism and IFNα.Objectives: The type I interferon (IFN) system is important in the pathogenesis of systemic lupus erythematosus (SLE). We previously demonstrated an inhibitory effect of IFNα on interleukin 6 (IL-6 ...

High prevalence of autoantibodies to C-reactive protein in patients with chronic hepatitis C infection: association with liver fibrosis and portal inflammation

The presence of autoantibodies against C-reactive protein (anti-CRP) has been reported in association with autoimmunity and histopathology in chronic hepatitis C virus (HCV) infection. Resistin could play a role in the pathogenesis of hepatitis, although results on HCV infection are ambiguous. Here we retrospectively analyzed anti-CRP and resistin levels in the sera of 38 untreated and well-characterized HCV patients at the time of their first liver biopsy. HCV activity and general health were assessed by a physician at least yearly until follow-up ended. Anti-CRP and resistin were also measured in patients with autoimmune hepatitis (AIH) and nonalcoholic fatty liver disease (NAFLD). Anti-CRP antibodies were registered in all HCV patients, whereas only a few AIH (11%) and NAFLD (12%) sera were positive. Anti-CRP levels were related to histopathological severity and were highest in patients with cirrhosis at baseline. Resistin levels were similar in HCV, AIH, and NAFLD patients, but high levels of resistin were associated with early mortality in HCV patients. Neither anti-CRP nor resistin predicted a response to interferon-based therapy or cirrhosis development or was associated with liver-related mortality. We conclude that anti-CRP antibodies are frequently observed in chronic HCV infection and could be a useful marker of advanced fibrosis and portal inflammation.

Soluble urokinase plasminogen activator receptor : A valuable biomarker in systemic lupus erythematosus?

Systemic lupus erythematosus (SLE) is a potentially severe autoimmune condition with an unpredictable disease course, often with fluctuations in disease activity over time. Long term inflammation and drug-related side-effects may subsequently lead to permanent organ damage, a consequence which is intimately connected to decreased quality of life and mortality. New lupus biomarkers that convey information regarding inflammation and/or organ damage are thus warranted. Today, there is no clinical biomarker that indicates the risk of damage accrual. Herein we highlight the urokinase plasminogen activator receptor (uPAR) and especially its soluble form (suPAR) that besides having biological functions in e.g. proteolysis, cell migration and tissue homeostasis, recently has emerged as a promising biomarker of inflammation and prognosis of several disorders. A strong association between suPAR and organ damage in SLE was recently demonstrated, and preliminary data (presented in this review) suggests the possibility of a predictive value of suPAR blood levels. The involvement of suPAR in the pathogenesis of SLE remains obscure, but its effects in leukocyte recruitment, phagocytic uptake of dying cells (efferocytosis) and complement regulation suggests that the central parts of the SLE pathogenesis could be regulated by suPAR, and vice versa.

Osteopontin is associated with disease severity and antiphospholipid syndrome in well characterised Swedish cases of SLE

Objective The variety of disease phenotypes among patients with SLE challenges the identification of new biomarkers reflecting disease activity and/or organ damage. Osteopontin (OPN) is an extracellular matrix protein with immunomodulating properties. Although raised levels have been reported, the pathogenic implications and clinical utility of OPN as a biomarker in SLE are far from clear. Thus, the aim of this study was to characterise OPN in SLE. Methods Sera from 240 well-characterised adult SLE cases classified according to the American College of Rheumatology (ACR) and/or the Systemic Lupus International Collaborating Clinics (SLICC) criteria, and 240 population-based controls were immunoassayed for OPN. The SLE Disease Activity Index 2000 (SLEDAI-2K) was used to evaluate disease activity and the SLICC/ACR Damage Index (SDI) to detect damage accrual. Results Serum OPN levels were in average raised fourfold in SLE cases compared with the controls (p<0.0001). OPN correlated with SLEDAI-2K, especially in patients with a disease duration of <12 months (r=0.666, p=0.028). OPN was highly associated with SDI (p<0.0001), especially in the renal (p<0.0001), cardiovascular (p<0.0001) and malignancy (p=0.012) domains. Finally, OPN associated with coherent antiphospholipid syndrome (APS; p=0.009), and both clinical and laboratory criteria of APS had significant positive impact on OPN levels. Conclusions In this cross-sectional study, circulating OPN correlates with disease activity in recent-onset SLE, reflects global organ damage and associates with APS. Longitudinal studies to dissect whether serum OPN also precedes and predicts future organ damage are most warranted.

Designed Surface with Tunable IgG Density as an in Vitro Model for Immune Complex Mediated Stimulation of Leukocytes

We present the design of an in vitro model for immune-complex-mediated stimulation of leukocytes and its functional characteristics with respect to monocyte adhesion. The model was based on the orientation-controlled immobilization of a humanized IgG1 monoclonal antibody (rituximab) via its interaction with a biotinylated peptide epitope derived from the CD20 marker. The peptide was linked to neutravidin covalently attached to a mixed self-assembled monolayer of carboxyl- and methoxy-terminated oligo(ethylene glycol) alkane thiolates on gold. The surface adhesion propensity of human monocytes (cell line U937) was highly dependent on the lateral IgG density and indicated that there exists a distance between IgG-Fc on the surface where interactions with Fc gamma receptors are optimal. This well-defined platform allows for a careful control of the size and orientation of artificial IgG immune complexes, it is easily made compatible with, for example, cellular imaging, and it will become useful for in vitro studies on the importance of Fc gamma receptor interactions in chronic immune-mediated diseases.

S2A:6 Soluble urokinase plasminogen activator receptor (supar) predicts the development of organ damage over 5 years in systemic lupus erythematosus: results from the slicc inception cohort

Background The urokinase plasminogen activator receptor (suPAR) participates in proteolysis, migration and adhesion. Receptor shedding yields a soluble form (suPAR) that has emerged as a promising severity biomarker in malignancies, inflammatory and infectious diseases. Previously, suPAR was shown to reflect accumulated organ damage in systemic lupus erythematosus (SLE). Here, we investigate suPAR as a potential predictor of future organ damage in patients with recent-onset SLE. Methods 345 SLE cases (at least 4 ACR criteria) from North America, Europe and Asia were included. All patients were from the SLICC inception cohort and were selected based on a minimum of 5 years follow-up and absence of organ damage (SLICC/ACR damage index; SDI>0) at inclusion. Patients were enrolled within 15 months of diagnosis. Estimated glomerular filtration rate (eGFR) was available for 180 patients. Serum suPAR levels were measured by ELISA at inclusion only, and levels were related to SDI after 5 years of follow-up. Age- and sex-matched controls (1:1) were from the Swedish population. Results Baseline suPAR levels were higher in patients who acquired damage (SDI>1) over a 5 year period (n=33) compared to patients without damage development (n=246; p<0.001) and controls (n=345; p=0.007) (figure 1). There were no significant differences in suPAR with regard to ethnicity (Caucasians vs non-Caucasians) or sex in patients/controls, but a weak correlation between age and suPAR among controls (p<0.001, r=0.23). No correlations (r>0.2) were found between suPAR and disease activity (SLEDAI-2K), corticosteroid dose or eGFR. Logistic regression revealed significant impact of baseline suPAR on future damage (SDI>1) (p=0.014; area under curve, AUC=0.64) and the predictive value became stronger after adjustment for age, sex, ethnicity and corticosteroid dose (p=0.008; AUC=0.74). Examining individual components of SDI revealed significant impact of suPAR on musculoskeletal damage (SDI>0) (p=0.018; AUC=0.66) lso when adjusting for covariates (p=0.020; AUC=0.68). Conclusion Prognostic biomarkers of disease severity in SLE could identify patients in need of tight control and improved treatment strategies. Here, suPAR is for the first time shown to have predictive potential of damage accrual in SLE. Continued follow-up of patients could elucidate the association between suPAR and damage in specific organ domains.Abstract S2A:6 Figure 1

Interferon-α coincides with suppressed levels of pentraxin-3 (PTX3) in systemic lupus erythematosus and regulates leucocyte PTX3 in vitro

Dysfunctional elimination of cell debris, and the role of opsonins such as pentraxins, is of interest regarding systemic lupus erythematosus (SLE) pathogenesis. Interferon (IFN)‐α is typically elevated during SLE flares, and inhibits hepatocyte production of the pentraxin ‘C‐reactive protein’ (CRP), partly explaining the poor correlation between CRP levels and SLE disease activity. The extrahepatically produced ‘pentraxin 3’ (PTX3) shares waste disposal functions with CRP, but has not been studied extensively in SLE. We analysed serum PTX3 in SLE, and assessed its interference with IFN‐α in vitro. Serum samples from 243 patients with SLE and 100 blood donors were analysed regarding PTX3. Patient sera were analysed for IFN‐α, and genotyped for three PTX3 single nucleotide polymorphisms reported previously to associate with PTX3 levels. Stimulated PTX3 release was assessed in the presence or absence of IFN‐α in blood donor neutrophils and peripheral blood mononuclear cells (PBMC). Serum PTX3 was 44% lower in patients with SLE compared to blood donors (P < 0·0001) and correlated with leucocyte variables. Patients with undetectable IFN‐α had 29% higher median PTX3 level than patients with detectable IFN‐α (P = 0·01). PTX3 production by PBMC was inhibited by IFN‐α, whereas neutrophil degranulation of PTX3 was increased. No differences in PTX3 levels were observed between the SNPs. In conclusion, median serum PTX3 is lower in SLE (especially when IFN‐α is detectable) compared to blood donors. In addition to its potential consumption during waste disposal, it is plausible that IFN‐α also attenuates PTX3 by inhibiting synthesis by PBMC and/or exhausting PTX3 storage in neutrophil granules.