Jonas Wetterö

On the binding of complement to solid artificial surfaces in vitro.

Since the realization of a complement activation capacity by artificial surfaces upon contact with blood, a common belief has evolved that charged nucleophilic surface groups such as amine (-NH2) and hydroxyl (-OH) react with and eventually bind to the internal thioester in complement factor 3 (C3). A covalent amide or ester linkage is thereby supposed to form between C3b and the surface itself. In this report, we present complement surface binding data by null-ellipsometry for two nucleophilic surfaces (-NH2 and -OH), for surfaces with immunoglobulin G (IgG) covalently bound, and for IgG spontaneously pre-adsorbed to hydrophobic silicon. The results reveal that the plasma proteins that were deposited during complement activation became eluted by sodium dodecyl sulfate. Hence the direct covalent binding between C3 and solid nucleophilic surfaces seems to be only of moderate importance, at least during shorter serum incubations. This strongly suggests that the prevalent covalent linkage model between solid artificial surfaces and C3b is not accurate. Instead we suggest a more pronounced role for C3 associations to other adsorbed proteins and or electrostatic and hydrophobic protein-surface interactions.

The influence of plasma proteins and platelets on oxygen radical production and F-actin distribution in neutrophils adhering to polymer surfaces

It is well known that blood cell interactions with artificial surfaces might have deleterious effects on host tissue, however, the mechanisms involved are far from understood. In this study, neutrophil-platelet interaction on uncoated or protein-coated polymer surfaces was investigated. Cell spreading, reorganization of actin filaments and release of oxygen metabolites (measured as luminol-amplified chemiluminescence) were used as criteria for cell activation on positively charged, hydrophilic 1,2-diaminocyclohexane, and negatively charged, hydrophobic hexamethylene-disiloxane. The model surfaces were made by radio frequency plasma discharge polymerization. Neutrophil contact with the uncoated polymers induced a prolonged generation of oxygen radicals. Precoating of the polymer surfaces with human serum albumin (HSA) or fibrinogen, markedly reduced neutrophil activation, whereas coating with human immunoglobulin G (IgG), a well-known opsonin, resulted in significantly higher levels of cell activation. Consequently, protein coating overruled the activating effects of the polymer surfaces. The presence of unstimulated or thrombin-stimulated platelets markedly increased the reactivity of neutrophils against fibrinogen- and IgG-coated surfaces. However, neutrophils remained relatively unreactive in the presence of platelets on HSA-treated surfaces. Comparison of the different types of surfaces used, reveals a correlation between the degree of cell spreading, reorganization of the actin cytoskeleton and the amount of oxygen radicals produced. Our results suggest that the acute inflammatory reaction on a biomaterial surface is highly dependent on the nature and composition of the first adsorbed protein layer and the extent of platelet activation.

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.

Role of the actin cytoskeleton during respiratory burst in chemoattractant-stimulated neutrophils

The aim of this study was to clarify the role of the actin cytoskeleton during chemotactic peptide fMet‐Leu‐Phe (fMLF)‐stimulated respiratory burst in human neutrophil granulocytes. Reactive oxygen species (ROS) was measured as luminol‐amplified chemiluminescence (CL) and F‐actin content as bodipy phallacidin fluorescence in neutrophils treated with latrunculin B or jasplakinolide, an inhibitor and activator of actin polymerization, respectively. Latrunculin B markedly decreased, whereas jasplakinolide increased, the F‐actin content in neutrophils, unstimulated or stimulated with fMLF. Latrunculin B enhanced the fMLF‐triggered ROS‐production more than tenfold. Jasplakinolide initially inhibited the fMLF‐induced CL‐response, however, caused a potent second sustained phase (>400% of control). Both actin drugs triggered a substantial CL‐response when added 5–25 min after fMLF. This was also valid for chemotactic doses of fMLF, where latrunculin B and jasplakinolide amplified the ROS‐production 5–10 times. By using specific signal transduction inhibitors, we found that the NADPH oxidase activation triggered by destabilization of the actin cytoskeleton occurs downstream of phospholipase C and protein kinase C but is mediated by Rho GTPases and tyrosine phosphorylation. In conclusion, rearrangements of the actin cytoskeleton are a prerequisite in connecting ligand/receptor activation, generation of second messengers and assembly of the NADPH oxidase in neutrophil granulocytes.

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

IntroductionSerum 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.MethodsThirty-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 ≥ 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.ResultsAt 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.ConclusionsIn 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.

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.

Human articular chondrocytes on macroporous gelatin microcarriers form structurally stable constructs with blood‐derived biological glues in vitro

Biodegradable macroporous gelatin microcarriers fixed with blood‐derived biodegradable glue are proposed as a delivery system for human autologous chondrocytes. Cell‐seeded microcarriers were embedded in four biological glues—recalcified citrated whole blood, recalcified citrated plasma with or without platelets, and a commercially available fibrin glue—and cultured in an in vitro model under static conditions for 16 weeks. No differences could be verified between the commercial fibrin glue and the blood‐derived alternatives. Five further experiments were conducted with recalcified citrated platelet‐rich plasma alone as microcarrier sealant, using two different in vitro culture models and chondrocytes from three additional donors. The microcarriers supported chondrocyte adhesion and expansion as well as extracellular matrix (ECM) synthesis. Matrix formation occurred predominantly at sample surfaces under the static conditions. The presence of microcarriers proved essential for the glues to support the structural takeover of ECM proteins produced by the embedded chondrocytes, as exclusion of the microcarriers resulted in unstable structures that dissolved before matrix formation could occur. Immunohistochemical analysis revealed the presence of SOX‐9‐ and S‐100‐positive chondrocytes as well as the production of aggrecan and collagen type I, but not of the cartilage‐specific collagen type II. These results imply that blood‐derived glues are indeed potentially applicable for encapsulation of chondrocyte‐seeded microcarriers. However, the static in vitro models used in this study proved incapable of supporting cartilage formation throughout the engineered constructs. Copyright

C1q-independent activation of neutrophils by immunoglobulin M-coated surfaces.

Neutrophil granulocytes are known to rapidly adhere and undergo frustrated phagocytosis upon contact with immunoglobulin and/or complement protein opsonized artificial surfaces. In this study, we examined the relation between serum protein deposition and human neutrophil activation on hydrophobic glass and silicon model surfaces that were coated with immunoglobulin G or M (IgG/IgM), both initiators of the classical complement pathway. Protein adsorption from normal human serum (NHS) was quantified with null-ellipsometry combined with antibody techniques. The neutrophil oxygen radical production was registered by luminol-amplified chemiluminescence (CL) and the morphology, as well as changes in the content of filamentous actin (F-actin), were documented by fluorescence microscopy. Complement factor 3 (C3) bound to both IgG- and IgM-coated surfaces, but surprisingly C1q was found only on IgG-coated surfaces. Both immunoglobulins triggered complement dependent neutrophil activation. However, CL and F-actin accumulation were found sensitive to the presence of C1q in the serum only at the IgG-coated surface. We suggest that spontaneously adsorbed IgM activates the complement system and interacts with neutrophils by C1q-independent mechanisms.

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.

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.