Irma Rensink

Complement Activation by Apoptotic Cells Occurs Predominantly via IgM and is Limited to Late Apoptotic (Secondary Necrotic) Cells

Apoptotic cells activate complement via various molecular mechanisms. It is not known which of these mechanisms predominate in a physiological environment. Using Jurkat cells as a model, we investigated complement deposition on vital, early and late apoptotic (secondary necrotic) cells in a physiological medium, human plasma, and established the main molecular mechanism involved in this activation. Upon incubation with recalcified plasma, binding of C3 and C4 to early apoptotic cells was similar to background binding on vital cells. In contrast, late apoptotic (secondary necrotic) cells consistently displayed substantial binding of C4 and C3 and low, but detectable, binding of C1q. Binding of C3 and C4 to the apoptotic cells was abolished by EDTA or Mg-EGTA, and also by C1-inhibitor or a monoclonal antibody that inhibits C1q binding, indicating that complement fixation by the apoptotic cells was mainly dependent on the classical pathway. Late apoptotic cells also consistently bound IgM, in which binding significantly correlated with that of C4 and C3. Depletion of plasma for IgM abolished most of the complement fixation by apoptotic cells, which was restored by supplementation with purified IgM. We conclude that complement binding by apoptotic cells in normal human plasma occurs mainly to late apoptotic, secondary necrotic cells, and that the dominant mechanism involves classical pathway activation by IgM.

Nucleosome-releasing factor: a new role for factor VII-activating protease (FSAP)

Plasma proteins such as early comple ment components and IgM are involved in the removal of late apoptotic or secondary necrotic (sn) cells. We have recently described how a plasma protease that could be inhibited by the protease inhibitor aprotinin was essential to remove nucleosomes from sn cells. An obvious candidate, plasmin, did indeed have nucleo some‐releasing factor (NRF) activity. However, recalci fied plasma (r‐plasma) retained its NRF activity after plasminogen depletion, which suggests the existence of another protease responsible for NRF activity in plasma. In this study we have used size‐exclusion and anion‐exchange chromatography to purify the protease responsible for NRF activity in plasma. SDS‐PAGE analysis of chromatography fractions containing NRF activity revealed a protein band corresponding with NRF activity. Sequence analysis showed this band to be factor VII‐activating protease (FSAP). We developed monoclonal antibodies to FSAP and were able to com pletely inhibit NRF activity in plasma with monoclonal antibodies to FSAP. Using affinity chromatography we were able to purify single‐chain (sc) FSAP from r‐ plasma. Purified scFSAP efficiently removes nucleo‐ somes from sn cells. We report that factor VII‐activat‐ ing protease may function in cellular homeostasis by catalyzing the release of nucleosomes from secondary necrotic cells.— Zeerleder, S., Zwart, B., te Velthuis, H., Stephan, F., Manoe, R., Rensink, I., Aarden, L. A. Nucleosome‐releasing factor: a new role for factor VII‐activating protease (FSAP). FASEB J. 22, 4077–4084 (2008)

A plasma nucleosome releasing factor (NRF) with serine protease activity is instrumental in removal of nucleosomes from secondary necrotic cells

We observed that interaction of secondary necrotic (sn) cells with human serum or plasma leads to loss of DNA staining. The decrease turned out to be a result of nucleosome release and was specific for apoptotic cells as necrotic cells did not show this phenomenon. We named this activity in plasma nucleosome releasing factor (NRF). NRF activity was completely inhibited by trypsin inhibitors suggesting that a serine protease is involved. Upon testing a number of plasma candidate serine proteases we found that plasmin did have NRF activity. However, plasminogen‐deficient plasma still had NRF activity indicating that NRF is not plasmin. We conclude that a yet unidentified plasma serine protease is involved in removal of nucleosomes from sn cells.

A New Principle for Rapid Immunoassay of Proteins Based on In Situ Precipitate-Enhanced Ellipsometry

A new technique is presented that allows measurement of protein concentrations in the picomolar range with an assay time of only 10-20 min. The method is an enzyme-linked immunosorbent assay (ELISA), but uses in-situ ellipsometric measurement of a precipitating enzyme product instead of the usual colorimetric detection of accumulating enzyme product in solution. Quantitative validation was obtained by use of annexin V, a protein with high binding affinity for phosphatidylserine-containing phospholipid membranes, resulting in a transport-limited adsorption rate. This property was exploited to obtain a range of low surface concentrations of annexin V by timed exposures of phospholipid bilayers to known concentrations of annexin V. Using polyvinylchloride (PVC)-coated and silanized silicon slides, various versions of this technique were used for the rapid assay of fatty acid-binding protein (FABP), a recently introduced early marker for acute myocardial infarction with a normal plasma concentration below 1 nmol/l, interleukin 6 (IL-6), a cytokine with normal plasma concentrations below 1 pmol/l, and again, annexin V. A possible future application of the method in the development of a one-step ELISA is discussed.

Cytokines and Intracellular Signals Involved in the Regulation of B-CLL Proliferation

Cytokines play an important role in the regulation of both normal and malignant B cells. In this paper we give a brief overview of the major cytokines involved in the regulation of B-CLL proliferation. In vitro experiments have indicated that there is an antagonistic interaction between TNF-alpha as a growth-enhancing factor and IL-4, which inhibits the growth of B-CLL. We have extended these findings with recent experiments on the intracellular signals which might be involved in these processes. We show that increased levels of intracellular cAMP dose-dependently inhibit the TNF-alpha-induced proliferation of B-CLL. On the basis of these results, we propose a model for the signals involved in the regulation of B-CLL proliferation. The implications for possible new ways of treatment are discussed.

ELISA to measure neutralizing capacity of anti-C1-inhibitor antibodies in plasma of angioedema patients.

BACKGROUND Neutralizing autoantibodies (NAbs) against plasma serpin C1-inhibitor (C1-inh) are implicated in the rare disorder, acquired angioedema (AAE). There is insufficient understanding of the process of antibody formation and its correlation with disease progression and severity. We have developed an ELISA for detecting neutralizing capacity of anti-C1-inh positive plasma samples that can be used to study changes in NAb repertoire in patient plasma over the course of disease. METHODS The ELISA is based on the specific interaction of active C1-inh with its target protease C1s. Decrease in the amount of C1s bound to immobilized C1-inh in the presence of test samples is proportional to the neutralizing capacity of the sample. Assay specificity, intra- and inter-assay variation and assay cut-off are determined using anti-C1-inh antibodies. Assay capability is demonstrated using plasma samples from AAE patients. RESULTS The assay is specific to a neutralizing anti-C1-inh antibody and shows no interference by a non-neutralizing anti-C1-inh antibody or by the plasma matrix. Intra-assay and inter-assay variations are determined as 17 and 18% respectively. Neutralizing capacity of antibody positive AAE patient plasma samples (n=16) with IgG or IgM type antibodies is readily determined. All samples show positive neutralizing capacity. CONCLUSION We have developed a robust, specific and semi-quantitative assay to detect the neutralizing capacity of plasma samples containing anti-C1-inh antibodies. This assay can be an important tool for the study of clinical implications of anti-C1-inh NAbs.

Comprehensive approach to study complement C4 in systemic lupus erythematosus: Gene polymorphisms, protein levels and functional activity

HighlightsLow gene copy number of C4A is associated with increased susceptibility to SLE.Serositis is associated with low gene copy number of C4A in SLE patients.A basepair insertion (A) after A1112 causes a silencing mutation in C4A.A novel and accurate substrate binding assay for C4A and C4B was developed.The binding capacity of available C4 to its substrates is unaffected in SLE. ABSTRACT Genetic variation of the genes encoding complement component C4 is strongly associated with systemic lupus erythematosus (SLE), a chronic multi‐organ auto‐immune disease. This study examined C4 and its isotypes on a genetic, protein, and functional level in 140 SLE patients and 104 healthy controls. Gene copy number (GCN) variation, silencing CT‐insertion, and the retroviral HERV‐K(C4) insertion) were analyzed with multiplex ligation‐dependent probe amplification. Increased susceptibility to SLE was found for low GCN (⟨ 2) of C4A. Serositis was the only clinical manifestation associated with low C4A GCN. One additional novel silencing mutation in the C4A gene was found by Sanger sequencing. This mutation causes a premature stop codon in exon 11. Protein concentrations of C4 isoforms C4A and C4B were determined with ELISA and were significantly lower in SLE patients compared to healthy controls. To study C4 isotypes on a functional level, a new C4 assay was developed, which distinguishes C4A from C4B by its binding capacity to amino or hydroxyl groups, respectively. This assay showed high correlation with ELISA and detected crossing over of Rodgers and Chido antigens in 3.2% (8/244) of individuals. The binding capacity of available C4 to its substrates was unaffected in SLE. Our study provides, for the first time, a complete overview of C4 in SLE from genetic variation to binding capacity using a novel test. As this test detects crossing over of Rodgers and Chido antigens, it will allow for more accurate measurement of C4 in future studies.