IgM antibodies against apoptotic cells and phosphorylcholine in patients with acute myocardial infarction in relation to infarct size and inflammatory response.

BACKGROUND: Natural IgM antibodies, and anti-phosphorylcholine IgM (anti-PC IgM) in particular, may modulate the pathogenesis of acute myocardial infarction (AMI). OBJECTIVES: An exploratory study was conducted to evaluate the hypothesis that circulating anti-PC IgM and IgM binding to damaged cells increases the infarct size and post-infarct inflammatory response in patients with AMI. MATERIAL AND METHODS: Plasma IgM binding to apoptotic cells (anti-apop IgM) and anti-PC IgM levels were compared in plasma samples from 50 patients with AMI and 46 healthy controls after correction for hemodilution. The cumulative release of cardiac markers LDH (lactate dehydrogenase), CK or CK-MB in human myocardium at 48 hours was used as an indication of infarct size. The circulating levels of mediators such as activated complement, C-reactive protein (CRP), interleukin-6 (IL6), interleukin-8 (IL8) and secretory phospholipase A2 (sPLA2) were used to assess the post-infarct inflammatory response. Patients with low (< median) and high (> median) levels of anti-apop IgM or anti-PC IgM were compared regarding infarct size and post-infarct inflammatory response. An electrocardiographical scoring system (Selvester score) was used to asses myocardial infarct size in patients with a first AMI (n = 24). RESULTS: AMI patients demonstrated lower levels of anti-PC IgM on admission (p < 0.01) and at 48 hours (p < 0.001) when compared to the healthy controls, whereas anti-apop IgM levels were comparable to control levels. In patients with a first infarct, patients with levels of anti-PC IgM above the median demonstrated larger electrocardiographic infarct sizes (p = 0.04) and a more pronounced response of the acute phase protein sPLA2 (p = 0.06), with a similar post-infarct course of LDH, CK and CK-MB. CONCLUSIONS: These findings suggest that anti-PC IgM plasma levels may participatie in amplifying the inflammatory response of the ischemic heart and contribute to infarct size. However, the levels of anti-PC IgM in patients with AMI in this study do not show a significant effect on cardiac markers LDH, CK and CK-MB. Hence, conclusive evidence is not provided by this limited cohort.

Competitive binding of pentraxins and IgM to newly exposed epitopes on late apoptotic cells.

A random distribution of phospholipids among the inner and outer leaflet of the cell membrane occurs during apoptosis and is known as membrane flip-flop. Flip-flopped cells have binding sites for various plasma proteins, such as IgM and the pentraxins C-reactive protein (CRP) and serum amyloid P component (SAP). In this study, we investigated whether pentraxins and IgM antibodies recognize the same binding sites on apoptotic cells, and whether phospholipids constitute these binding sites. Except for SAP which also bound to early apoptotic cells, pentraxins and IgM preferentially bound to late apoptotic cells. Competition experiments with different phosphatemonoesters revealed that CRP and SAP as well as part of the IgM bound to the phospholipids head groups, SAP mainly to phosphorylethanolamine, CRP to phosphorylcholine and phosphorylethanolamine and to a lesser extent to phosphorylserine, and IgM to phosphorylcholine and phosphorylserine. These results were confirmed in experiments in which proteins were adsorbed from plasma with artificial phospholipids particles. IgM and the pentraxins variably competed for the same binding sites on late apoptotic cells, SAP having the highest and CRP the lowest apparent affinity. We conclude that CRP, SAP, and part of the IgM bind to the phospholipid head groups exposed on apoptotic cells. This shared specificity as well as their shared capability to activate complement, suggest that IgM and the pentraxins CRP and SAP exert similar functions in the removal of apoptotic cells.

Levels of natural IgM antibodies against phosphorylcholine in healthy individuals and in patients undergoing isolated limb perfusion.

Natural IgM antibodies against phosphorylcholine (anti-Pc IgM) resemble C-reactive protein (CRP) regarding specificity and have gained increasing attention because of their supposed role in clearance of damaged cells and in cardiovascular disease. In order to quantify these antibodies in human plasma, we have developed an ELISA system, in which p-aminophenylphosphorylcholine (PCH) coupled to human serum albumin (HSA) was coated on microtiters plates. Human plasma or serum samples were incubated in the plates, after which bound anti-Pc IgM was detected with mouse anti-human IgM-HRP. Pre-incubation of plasma with competitors such as phosphorylcholine, phosphorylethanolamine, phosphorylserine or glycine-HSA, confirmed that the ELISA was specific for anti PC IgM. Levels of anti Pc IgM in a cohort of healthy donors differed by more than 100-fold, whereas the fluctuation of anti-Pc IgM levels in individuals over time was small (coefficient of variation between 6% to 25%). Furthermore, there was no correlation between CRP and anti-Pc IgM in this cohort. Levels of anti-Pc IgM in the normal donors correlated significantly with IgM binding to apoptotic cells. To test the hypothesis that anti-Pc IgM can bind to neo-antigens expressed on necrotic or apoptotic cells, anti-Pc IgM was also quantified in patients with tumors undergoing isolated limb perfusion with tumor necrosis factor-alpha (TNF-alpha). Following this procedure, a significant decrease of circulating anti-Pc IgM relative to total IgM was found in all five patients tested. In conclusion, we have developed a specific and reproducible ELISA for anti Pc IgM quantification. Fluctuation of levels of these natural antibodies over time in healthy individuals was limited, although the variation among individuals was large. Significant decreases of levels of anti-Pc IgM were found to occur during tissue damage.

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.

Complement activation by necrotic cells in normal plasma environment compares to that by late apoptotic cells and involves predominantly IgM.

Necrotic cells are generally considered to stimulate inflammation, whereas apoptotic cells should not. However, apoptotic cells have pro-inflammatory properties since they can activate complement. To what extent this activation compares to that by necrotic cells is not known. We compared complement activation by necrotic cells and apoptotic cells in plasma. Jurkat cells were made apoptotic or necrotic by incubation with etoposide or by heat shock, respectively. Cells incubated in recalcified plasma were tested for C3 and C4 fixation and fluid phase generation of complement activation products. Fixation of C3 and C4 to necrotic cells occurred mainly via the classical pathway, independent from the method of necrosis induction and the cell type. Depletion of IgM from plasma almost completely abrogated complement fixation by necrotic cells, which was restored by supplementation with purified IgM. Complement activation by late apoptotic cells was comparable to that by necrotic cells regarding the extent and dependence on IgM. Moreover, incubation of plasma with necrotic or late apoptotic cells led to the generation of comparable amounts of complement activation products. These results indicate that late apoptotic and necrotic cells employ similar complement activation mechanisms in the plasma environment.