Elevated Serum Levels of Zonulin Family Peptides in Anticitrullinated Protein Antibody-Positive At-Risk Individuals Without Arthritis.

OBJECTIVE: Recent advances imply that early events triggering rheumatoid arthritis (RA) occur at mucosal surfaces. We aimed to evaluate whether intestinal permeability is altered in patients at increased risk of RA, and/or predicts the development of clinical arthritis, by measuring serum zonulin family peptides (ZFP) levels, which are shown to reflect intestinal barrier integrity. METHODS: Two independent prospective observational cohorts were studied, including subjects with musculoskeletal symptoms and anticitrullinated protein antibodies (ACPA), but without clinical arthritis at baseline. In Sweden, 82 such at-risk patients were compared to 100 age-matched healthy blood donors. In the UK, 307 at-risk patients were compared to 100 ACPA-negative symptomatic controls. ZFP was measured in baseline sera by enzyme-linked immunoassays. RESULTS: In the Swedish at-risk cohort, ZFP levels were significantly increased in patients compared to controls (mean 41.4 vs 33.6 ng/mL, P < 0.001) and Cox regression analysis showed prognostic value of ZFP for arthritis development (hazard ratio [HZ] 1.04 per ng/mL ZFP increase, 95% CI 1.01-1.07, P = 0.02). Elevated ZFP levels among ACPA-positive at-risk patients compared to symptomatic ACPA-negative controls were confirmed in the UK at-risk cohort (mean 69.7 vs 36.0 ng/mL, P < 0.001), but baseline ZFP were not associated with arthritis development (HR 1.00 per ng/mL ZFP increase, 95% CI 1.00-1.01, P = 0.30). CONCLUSION: Serum ZFP levels are elevated in ACPA-positive at-risk patients when compared to both healthy blood donors and symptomatic ACPA-negative controls. Thus, gut barrier function may be of importance in RA-associated autoimmunity. A possible prognostic value of serum ZFP merits further investigation, preferably in larger prospective cohorts.

Variation of Complement Protein Levels in Maternal Plasma and Umbilical Cord Blood during Normal Pregnancy: An Observational Study.

The complement system constitutes a crucial part of the innate immunity, mediating opsonization, lysis, inflammation, and elimination of potential pathogens. In general, there is an increased activity of the complement system during pregnancy, which is essential for maintaining the host's defense and fetal survival. Unbalanced or excessive activation of the complement system in the placenta is associated with pregnancy complications, such as miscarriage, preeclampsia, and premature birth. Nonetheless, the actual clinical value of monitoring the activation of the complement system during pregnancy remains to be investigated. Unfortunately, normal reference values specifically for pregnant women are missing, and for umbilical cord blood (UCB), data on complement protein levels are scarce. Herein, complement protein analyses (C1q, C3, C4, C3d levels, and C3d/C3 ratio) were performed in plasma samples from 100 healthy, non-medicated and non-smoking pregnant women, collected during different trimesters and at the time of delivery. In addition, UCB was collected at all deliveries. Maternal plasma C1q and C3d/C3 ratio showed the highest mean values during the first trimester, whereas C3, C4, and C3d had rising values until delivery. We observed low levels of C1q and C4 as well as increased C3d and C3d/C3 ratio, particularly during the first trimester, as a sign of complement activation in some women. However, the reference limits of complement analyses applied for the general population appeared appropriate for the majority of the samples. As expected, the mean complement concentrations in UCB were much lower than in maternal plasma, due to the immature complement system in neonates.

The Complex Role of C-Reactive Protein in Systemic Lupus Erythematosus.

C-reactive protein (CRP) is well-known as a sensitive albeit unspecific biomarker of inflammation. In most rheumatic conditions, the level of this evolutionarily highly conserved pattern recognition molecule conveys reliable information regarding the degree of ongoing inflammation, driven mainly by interleukin-6. However, the underlying causes of increased CRP levels are numerous, including both infections and malignancies. In addition, low to moderate increases in CRP predict subsequent cardiovascular events, often occurring years later, in patients with angina and in healthy individuals. However, autoimmune diseases characterized by the Type I interferon gene signature (e.g., systemic lupus erythematosus, primary Sjögren's syndrome and inflammatory myopathies) represent exceptions to the general rule that the concentrations of CRP correlate with the extent and severity of inflammation. In fact, adequate levels of CRP can be beneficial in autoimmune conditions, in that they contribute to efficient clearance of cell remnants and immune complexes through complement activation/modulation, opsonization and phagocytosis. Furthermore, emerging data indicate that CRP constitutes an autoantigen in systemic lupus erythematosus. At the same time, the increased risks of cardiovascular and cerebrovascular diseases in patients diagnosed with systemic lupus erythematosus and rheumatoid arthritis are well-established, with significant impacts on quality of life, accrual of organ damage, and premature mortality. This review describes CRP-mediated biological effects and the regulation of CRP release in relation to aspects of cardiovascular disease and mechanisms of autoimmunity, with particular focus on systemic lupus erythematosus.

Comparison of Surrogate Markers of the Type I Interferon Response and Their Ability to Mirror Disease Activity in Systemic Lupus Erythematosus.

Objectives: Type I interferons (IFNs) are central and reflective of disease activity in systemic lupus erythematosus (SLE). However, IFN-α levels are notoriously difficult to measure and the type I IFN gene signature (IGS) is not yet available in clinical routine. This study evaluates galectin-9 and an array of chemokines/cytokines in their potential as surrogate markers of type I IFN and/or SLE disease activity. Methods: Healthy controls and well-characterized Swedish SLE patients from two cross-sectional cohorts (n=181; n=59) were included, and a subgroup (n=21) was longitudinally followed. Chemokine/cytokine responses in immune complex triggered IFN-α activity was studied in healthy donor peripheral blood mononuclear cells (PBMC). Levels of chemokines/cytokines and galectin-9 were measured by immunoassays. Gene expression was quantified by qPCR. Results: The IGS was significantly (p<0.01) correlated with galectin-9 (rho=0.54) and CXCL10 (rho=0.37) levels whereas serum IFN-α correlated with galectin-9 (rho=0.36), CXCL10 (rho=0.39), CCL19 (rho=0.26) and CCL2 (rho=0.19). The strongest correlation was observed between galectin-9 and TNF (rho=0.56). IFN-α and disease activity (SLEDAI-2K) were correlated (rho=0.20) at cross-sectional analysis, but no significant associations were found between SLEDAI-2K and galectin-9 or chemokines. Several inflammatory mediators increased at disease exacerbation although CCL19, CXCL11, CXCL10, IL-10 and IL-1 receptor antagonist were most pronounced. Immune complex-stimulation of PBMC increased the production of CCL2, CXCL8 and TNF. Conclusion: Galectin-9 and CXCL10 were associated with type I IFN in SLE but correlated stronger with TNF. None of the investigated biomarkers showed a convincing association with disease activity, although CXCL10 and CCL19 performed best in this regard.

Pentraxin-3 detected in human saliva shows limited correlation with biomarkers associated with systemic inflammation.

Pentraxin-3 (PTX3) is a conserved protein of the innate immune system which has been less studied than the pentraxin C-reactive protein (CRP), but it is of relevance in, for example, vascular pathology and pregnancy morbidities. Since the interest in salivary biomarkers in general is increasing, we asked whether PTX3 could be detected in saliva and if any substantial diurnal variation occurs. In addition, we evaluated association with biomarkers of systemic inflammation (interleukin (IL)-1ß, IL-6, and IL-8 and CRP), body mass index (BMI), smoking, and age. PTX3 in morning and evening saliva from 106 middle-aged participants of the general population was investigated by ELISA and total protein levels by spectrophotometry. PTX3 was detectable in saliva, and concentrations varied over the day with higher morning concentrations, but the PTX3 relative protein levels (percentage of total protein) were significantly higher in the evening. Sex and age did not impact salivary PTX3, but smoking was associated with lower PTX3 levels. BMI correlated positively with PTX3 in evening saliva. There was no general association with biomarkers of systemic inflammation, except for IL-6. Salivary PTX3 likely reflects the local inflammatory milieu, and adjustments for sampling time, smoking habits, and BMI are needed to adequately interpret PTX3 in saliva.

Presence of salivary IgA anti-citrullinated protein antibodies associate with higher disease activity in patients with rheumatoid arthritis.

BACKGROUND: Circulating IgA anti-citrullinated protein antibodies (ACPA) associate with more active disease, but a previous study implied that salivary IgA ACPA is related to a less severe disease. Therefore, we aimed to characterize the IgA ACPA response in the saliva and serum in relation to clinical picture and risk factors among patients with rheumatoid arthritis (RA). METHODS: RA patients (n = 196) and healthy blood donors (n = 101), included in the cross-sectional study "Secretory ACPA in Rheumatoid Arthritis" (SARA), were analyzed for ACPA of IgA isotype, and for subclasses IgA1 and IgA2 ACPA in paired saliva and serum samples using modified enzyme-linked immunosorbent assays (ELISA) targeting reactivity to a cyclic citrullinated peptide (anti-CCP). Cutoff levels for positive tests were set at the 99th percentile for blood donors. Antibody levels were related to clinical characteristics, radiographic damage, smoking habits, and carriage of HLA-DRB1/shared epitope (SE). RESULTS: IgA ACPA in the saliva was found in 12% of RA patients, IgA1 occurred in 10%, and IgA2 in 9%. In serum, IgA ACPA was found in 45% of the patients, IgA1 in 44%, and IgA2 in 39%. Levels of IgA ACPA in the saliva correlated significantly with serum levels of IgA (r = 0.455). The presence of salivary IgA ACPA was associated with a higher erythrocyte sedimentation rate (ESR), 28-joint disease activity score, tender joint count, and patient global assessment at the time of sampling. None of the antibodies was associated with smoking, SE, or radiographic damage. CONCLUSION: Salivary IgA ACPAs were detected in a subset of RA patients in association with higher disease activity. This suggests that mucosal ACPA responses in the oral cavity may contribute to disease-promoting processes in RA.

Elevated free secretory component in early rheumatoid arthritis and prior to arthritis development in patients at increased risk.

OBJECTIVES: Considering growing evidence of mucosal involvement in RA induction, this study investigated circulating free secretory component (SC) in patients with either recent-onset RA or with ACPA and musculoskeletal pain. METHODS: Two prospective cohorts were studied: TIRA-2 comprising 452 recent-onset RA patients with 3 years of clinical and radiological follow-up, and TIRx patients (n = 104) with ACPA IgG and musculoskeletal pain followed for 290 weeks (median). Blood donors and three different chronic inflammatory diseases served as controls. Free SC was analysed by sandwich ELISA. RESULTS: Serum levels of free SC were significantly higher in TIRA-2 patients compared with TIRx and all control groups (P < 0.01). Among TIRx patients who subsequently developed arthritis, free SC levels were higher compared with all control groups (P < 0.05) except ankylosing spondylitis (P = 0.74). In TIRA-2, patients with ACPA had higher baseline levels of free SC compared with ACPA negative patients (P < 0.001). Free SC status at baseline did not predict radiographic joint damage or disease activity over time. In TIRx, elevated free SC at baseline trendwise associated with arthritis development during follow-up (P = 0.066) but this disappeared when adjusting for confounders (P = 0.72). Cigarette smoking was associated with higher levels of free SC in both cohorts. CONCLUSION: Serum free SC levels are increased in recent-onset RA compared with other inflammatory diseases, and associate with ACPA and smoking. Free SC is elevated before arthritis development among ACPA positive patients with musculoskeletal pain, but does not predict arthritis development. These findings support mucosal engagement in RA development.

Pronounced Diurnal Pattern of Salivary C-Reactive Protein (CRP) With Modest Associations to Circulating CRP Levels.

C-reactive protein (CRP), a humoral component of the innate immune system with important functions in host-defense, is extensively used as a sensitive biomarker of systemic inflammation. During inflammation, hepatocyte-derived CRP rises dramatically in the blood due to increased interleukin-6 (IL-6) levels. Reliable detection of CRP in saliva, instead of blood, would offer advantages regarding sampling procedure and availability but using saliva as a diagnostic body fluid comes with challenges. The aims of this study were to evaluate associations between salivary CRP, total protein levels in saliva and serum CRP. Furthermore, we examined associations with plasma IL-6, body mass index (BMI), tobacco smoking and age. Salivary CRP was investigated by ELISA in 107 middle-aged participants from the general population. We employed spectrophotometric determination of total protein levels. Correlation analyses were used for associations of salivary CRP with serum CRP (turbidimetry), plasma IL-6 (Luminex®), BMI and smoking habits. Salivary median CRP was 68% higher (p=0.009), and total protein levels were 167% higher (p<0.0001), in morning compared to evening saliva. The correlation coefficients between serum and salivary CRP were low to moderate, but stronger for evening than morning saliva. Plasma IL-6 correlated significantly with serum CRP (rs =0.41, p<0.01), but not with morning or evening salivary CRP. Non-smokers showed 103% higher salivary CRP levels (p=0.015), whereas serum CRP was independent of smoking status. As opposed to CRP in serum, salivary CRP was not associated with BMI. Salivary CRP was 90% higher among the age interval 60-69 years compared to subjects aged 45-59 (p=0.02) while serum CRP levels did not differ between the age groups. In conclusion, CRP in saliva did not straightforwardly reflect serum concentrations. This raises questions regarding adequate reflection of biological events. The pronounced diurnal salivary CRP pattern accentuates the importance of standardizing the time-point of sampling.

C-Reactive Protein Levels in Systemic Lupus Erythematosus Are Modulated by the Interferon Gene Signature and CRP Gene Polymorphism rs1205.

Objectives: Patients with systemic lupus erythematosus (SLE) often display modest elevations of C-reactive protein (CRP) despite raised disease activity and increased interleukin (IL-) 6. We asked to what extent IL-6 levels, the CRP polymorphism rs1205, and the type I interferon (IFN) gene signature affects the basal CRP levels in patients with SLE during a quiescent phase of the disease. Methods: CRP and IL-6 were analyzed in plasma from 57 patients meeting established classification criteria for SLE. The CRP polymorphism rs1205 was assessed and gene expression analyzed including four type I IFN-regulated genes (IGS). Results: CRP was increased in patients with detectable IL-6 levels (p=0.001) and decreased among IGS-positive subjects (p=0.033). A multiple linear regression model revealed IL-6 to have a positive association with CRP levels, whereas both IGS-positivity and CRP genotype (rs1205) AA/GA were negatively associated with CRP-levels. Conclusion: Our data offer an explanation to the modest CRP levels seen in viral infections and IFN-α driven autoimmunity and corroborate prior observations showing an IFN-α dependent downregulation of CRP. The latter observation, together with the fact that the CRP-lowering polymorphism rs1205 is overrepresented in human SLE, could explain low basal CRP and inadequate CRP-responses among patients with active SLE.

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.

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.

OBJECTIVE: 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. METHODS: Sera from 155 SLE patients and 100 controls were analyzed for CRP. Patients were genotyped for a CRP single-nucleotide polymorphism (rs1205) associated with low CRP levels. Serum IFNα and IL-6 levels were quantified by immunoassays. Clinical disease activity was assessed using the SLE Disease Activity Index 2000 (SLEDAI-2K). RESULTS: CRP levels were increased in SLE patients compared to controls, but were not associated with SLEDAI-2K or IL-6 levels. However, exclusion of patients carrying at least one rs1205 minor allele revealed an association between disease activity and CRP levels (P = 0.005). We found a strong association between disease activity and CRP levels (P < 0.0005) when patients with measurable IFNα levels as well as the minor allele of rs1205 were excluded from the analysis. Similarly, when patients with elevated IFNα levels and/or the rs1205 polymorphism were excluded, IL-6 levels were associated with CRP levels. CONCLUSION: The present study demonstrates that the serum IFNα level as well as the CRP genotype affect the CRP response in SLE patients. Lack of correlation between serum levels of CRP and disease activity could therefore be explained by activation of the type I IFN system and polymorphisms in the CRP gene.

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 physician's 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.

C-reactive protein, immunoglobulin G and complement co-localize in renal immune deposits of proliferative lupus nephritis.

The pattern recognition molecules C-reactive protein (CRP) and C1q are of big interest in relation to the pathogenesis of systemic lupus erythematosus (SLE). Circulating autoantibodies against CRP and C1q are frequently found in SLE patients with active disease, particularly in lupus nephritis (LN), and rising levels reportedly relate to disease activity and outcome. If CRP-, or dsDNA- and/or C1q-containing immune complexes (ICs) are pathogenic in LN, glomerular IgG-deposits would be expected to co-localize with these antigens. In search for proof of this concept, renal biospsies from patients with active LN (n = 5) were examined with high-resolution immunogold electron microscopy. Renal biopsies from patients with Henoch-Schönlein purpura, pauci-immune nephritis and renal cancer served as controls. IgG antibodies against CRP, C1q and nucleosomes were analyzed in pre-post flare sera. We could demonstrate that CRP, C1q, C3c and dsDNA were co-localized with IgG in electron dense deposits in the glomerular basement membrane/subendothelial space in all of the 5 LN patients. Deposits of IgG, CRP, complement and dsDNA were 10-fold higher in LN compared to controls. All SLE patients had circulating anti-nucleosome antibodies; 4/5 had serum antibodies against CRP, dsDNA, and C1q at biopsy/flare. Despite a limited number of cases, the results support the notion of a pathogenic role not only for anti-dsDNA antibodies, but also for anti-CRP and anti-C1q in LN. The glomerular ICs may have been generated by deposition of circulating ICs or by in situ IC formation.

Beware of antibodies to dietary proteins in "antigen-specific" immunoassays! falsely positive anticytokine antibody tests due to reactivity with bovine serum albumin in rheumatoid arthritis (the Swedish TIRA project).

OBJECTIVE: To evaluate (1) to what extent sera from healthy subjects and patients with rheumatoid arthritis (RA) contain antibodies to bovine serum albumin (BSA); and (2) if anti-BSA antibodies interfere with results of enzyme-linked immunoassays (ELISA) containing BSA. METHODS: The ELISA used was a previously developed in-house assay of autoantibodies to tumor necrosis factor (TNF). Anti-TNF and anti-BSA antibodies were analyzed by ELISA in 189 patients with early RA and 186 healthy blood donors. TNF preparations containing either BSA or human serum albumin (HSA) as carrier proteins were used as antigens in the anti-TNF assay. The presence and levels of antibodies were analyzed in relation to disease course and to the presence/absence of rheumatoid factor (RF). RESULTS: In patients with RA, anti-TNF/BSA levels strongly correlated with anti-BSA levels (r = 0.81, p < 0.001), whereas anti-TNF/HSA did not (r = -0.09). Neither the presence nor the levels of anti-BSA in RA patients were associated with disease progression, and antibody levels were not significantly altered compared to controls (p = 0.11). IgG reactivity with TNF/HSA was neglible. In paired sera, preincubation with BSA abolished the anti-TNF/BSA reactivity. There were no indications of RF interference with anti-BSA or anti-TNF reactivity. CONCLUSION: Antibodies to BSA are common in patients with RA as well as in healthy individuals. Their presence does not seem to be associated with RA disease activity or disease course, but may severely interfere with ELISA containing BSA. The use of BSA as a "blocking agent" or carrier protein in immunoassays should therefore be avoided.

C1q induces a rapid up-regulation of P-selectin and modulates collagen- and collagen-related peptide-triggered activation in human platelets.

Blood platelets are emerging as important immunomodulatory cells, but complement interaction with platelets is not well understood. Several platelet structures have been described as complement protein 1q (C1q) binding receptors, such as C1qRp/CD93 and gC1qR. However, there are conflicting results whether these receptors are C1q binding structures, or even at all expressed on the cell surface. Recently, the collagen-binding integrin αIIßI was reported to bind C1q on mast cells, and this receptor is also present on platelets. The aim of this study was to further characterize the effects of C1q on platelets, by quantifying the platelet surface expression of P-selectin (CD62P) and monitoring the formation of platelet-neutrophil aggregates. Using flow cytometry, we found that C1q dose-dependently triggered a rapid but moderate and transient up-regulation of P-selectin already within 5s of C1q exposure. Pre-incubation with an antibody directed against gC1qR significantly inhibited (with 57% compared to control) the up-regulation, whereas an antibody towards the αIIßI-integrin showed no effect. Stimulation with C1q did not change the cytosolic calcium-levels, as measured with the fluorescent ratiometric probe Fura-2, however, a protein kinase C inhibitor (GF109203x) blocked the C1q-induced P-selectin expression. Furthermore, pre-incubation of platelets with C1q diminished both the collagen as well as the collagen-related peptide-induced up-regulation of P-selectin, most evident after 90s of stimulation. This indicates that C1q may regulate platelet activation via the GPVI receptor, which is a novel finding. Moreover, C1q antagonized the collagen-induced formation of platelet-neutrophil aggregates, indicating a reduced interaction between platelet P-selectin and neutrophil P-selectin glycoprotein ligand-1(PSGL-1/CD162). In summary, C1q induces a moderate rapid platelet P-selectin expression, modulates subsequent collagen and collagen-related peptide stimulation of platelets, and inhibits the formation of platelet-neutrophil aggregates. These immuno-regulatory effects of C1q may have a crucial role in innate immunity and inflammation.

Serum levels of autoantibodies against C-reactive protein correlate with renal disease activity and response to therapy in lupus nephritis.

INTRODUCTION: Serum levels of C-reactive protein (CRP) seldom reflect disease activity in systemic lupus erythematosus (SLE). We have previously shown that autoantibodies against neo-epitopes of CRP often occur in SLE, but that this does not explain the modest CRP response seen in flares. However, we have repeatedly found that anti-CRP levels parallel lupus disease activity, with highest levels in patients with renal involvement; thus, we aimed to study anti-CRP in a material of well-characterized lupus nephritis patients. METHODS: Thirty-eight patients with lupus nephritis were included. Treatment with corticosteroids combined with cyclophosphamide, mycophenolate mofetil or rituximab was started after baseline kidney biopsy. A second biopsy was taken after > or = 6 months. Serum creatinine, cystatin C, complement, anti-dsDNA, anti-CRP and urinalysis were done on both occasions. Biopsies were evaluated regarding World Health Organisation (WHO) class and indices of activity and chronicity. Renal disease activity was estimated using the British Isles Lupus Assessment Group (BILAG) index. RESULTS: At baseline, 34/38 patients had renal BILAG-A; 4/38 had BILAG-B. Baseline biopsies showed WHO class III (n = 8), IV (n = 19), III to IV/V (n = 3) or V (n = 8) nephritis. Seventeen out of 38 patients were anti-CRP-positive at baseline, and six at follow-up. Overall, anti-CRP levels had dropped at follow-up (P < 0.0001) and anti-CRP levels correlated with renal BILAG (r = 0.29, P = 0.012). A positive anti-CRP test at baseline was superior to anti-dsDNA and C1q in predicting poor response to therapy as judged by renal BILAG. Baseline anti-CRP levels correlated with renal biopsy activity (r = 0.33, P = 0.045), but not with chronicity index. Anti-CRP levels were positively correlated with anti-dsDNA (fluorescence-enhanced immunoassay: r = 0.63, P = 0.0003; Crithidia luciliae immunofluorescence microscopy test: r = 0.44, P < 0.0001), and inversely with C3 (r = 0.35, P = 0.007) and C4 (r = 0.29, P = 0.02), but not with C1q (r = 0.14, P = 0.24). No associations with urinary components, creatinine, cystatin C or the glomerular filtration rate were found. CONCLUSIONS: In the present study, we demonstrate a statistically significant correlation between anti-CRP levels and histopathological activity in lupus nephritis, whereas a baseline positive anti-CRP test predicted poor response to therapy. Our data also confirm previous findings of associations between anti-CRP and disease activity. This indicates that anti-CRP could be helpful to assess disease activity and response to therapy in SLE nephritis, and highlights the hypothesis of a pathogenetic role for anti-CRP antibodies in lupus nephritis.

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.

Reduced serum levels of autoantibodies against monomeric C-reactive protein (CRP) in patients with acute coronary syndrome.

INTRODUCTION: Inflammation is pivotal in atherosclerosis. Minor C-reactive protein (CRP) response reflects low-grade vascular inflammation and the high-sensitivity CRP test with levels > or = 3.0 mg/l predicts coronary vascular events and survival in angina pectoris as well as in healthy subjects. We and others recently reported autoantibodies against monomeric CRP (anti-CRP) in rheumatic conditions, e.g. systemic lupus erythematosus (SLE), and a connection between anti-CRP and cardiovascular disease in SLE has been suggested. PATIENTS AND METHODS: Anti-CRP serum levels were determined with ELISA in 140 individuals; 50 healthy controls and 90 patients with angiographically verified coronary artery disease of which 40 presented with acute coronary syndrome (ACS) and 50 with stable angina pectoris (SA). RESULTS: Significantly lower anti-CRP levels were observed in ACS compared to SA and controls (p=0.019). ACS patients, who had not been prescribed statins before their respective cardiovascular event, had lower anti-CRP (p=0.049). BMI correlated directly to anti-CRP levels in cross section analysis (p=0.043), but there was no association between anti-CRP and smoking or cholesterol. DISCUSSION: In ACS, it is plausible that ruptured plaques and inflamed tissue may be more prone to opsonization by monomeric CRP leading to consumption of anti-CRP. Hypothetically, surface-bound anti-CRP could thereby enhance the local inflammation in plaques.

Immobilized chemoattractant peptides mediate adhesion and distinct calcium-dependent cell signaling in human neutrophils.

Chemotaxis is the stimulated directional migration of cells in response to chemotactic factors, manifested for instance during leukocyte interaction with chemoattractants in inflammation. The N-formyl-Met-Leu-Phe (fMLF) bacterial peptide family is particularly potent in attracting and activating neutrophilic granulocytes. To accomplish defined circumstances for recruitment and activation of cells, we fabricated semitransparent gold-coated glass coverslips functionalized with chemoattractant fMLF receptor peptide agonist analogues. Peptides based on a common leading four-amino-acid sequence Gly-Gly-Gly-Cys were thus coupled to two potent fMLF receptor agonists, N-formyl-Tyr-Nle-Phe-Leu-Nle-Gly-Gly-Gly-Cys and N-formyl-Met-Leu-Phe-Gly-Gly-Gly-Cys, and a formylated control peptide, N-formyl-Gly-Gly-Gly-Cys. They were anchored via the SH group of Cys either directly to the gold surface or a mixed self-assembled monolayer composed of maleimide- and hydroxyl-terminated oligo(ethylene glycol) alkyldisulfides. The overall peptide immobilization procedure was characterized with ellipsometry, contact angle measurement, and infrared spectroscopy. When exposed to granulocytes, the agonist surface rapidly recruited neutrophils and the cells responded with extensive spreading and intracellular calcium transients within minutes. The reference peptide generated no such activation, and the cells maintained a more spherical morphology, suggesting that we have been able to immobilize chemoattractant receptor agonist peptides with retained bioactivity. This is a crucial step in designing surfaces with specific effects on cellular behavior.