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.     

The clearance of apoptotic cells by complement

The complement system is implicated among others in the clearance of apoptotic cells, and deficiency for different components of the complement system is associated with aberrant handling of apoptotic material. As a result of this, it is thought that the complement system is involved in the pathogenesis of autoimmune diseases such as systemic lupus erythematosus (SLE) which affects multiple organs. In this review, the authors highlight the available data on complement and apoptosis, as well as the role of complement in severe autoimmune diseases like SLE.     

Specific binding of L-ficolin and H-ficolin to apoptotic cells leads to complement activation

The serum lectins mannose-binding lectin (MBL), L-ficolin, and H-ficolin are recognition molecules in the lectin complement pathway, which play an important role in innate immunity. To assess involvement of the lectin pathway in the clearance of apoptotic cells, we used flow cytometry to quantify binding of MBL, L-ficolin, and H-ficolin to apoptotic HL60, U937, and Jurkat cells induced by actinomycin D. When apoptotic cells were incubated with normal human serum, MBL and L-ficolin bound to all three cell lines tested; moreover, H-ficolin bound to apoptotic Jurkat cells only. Subsequently, C4 and C3 were deposited on apoptotic cells of all three cell lines. MBL, L-ficolin, and H-ficolin binding to apoptotic cells was confirmed by the use of purified proteins. Purified C4 added to apoptotic cells that had bound pure L-ficolin was deposited on the cell surfaces. In L-ficolin-depleted serum, C3 deposition on HL60 or Jurkat cells decreased to approximately 50% or 70%, respectively, in comparison to the serum before L-ficolin depletion. We conclude that L-ficolin, in addition to MBL, recognizes apoptotic cells and activates complement via the lectin pathway. We also observed in vitro binding of L-ficolin and H-ficolin to cC1q receptor (C1q receptor specific for the collagenous region of C1q)/calreticulin, a candidate receptor for the collagenous region of MBL and C1q. Thus, L-ficolin and H-ficolin as well as MBL participate in the clearance of apoptotic cells through complement activation.     

Anti-CD16 autoantibodies and delayed phagocytosis of apoptotic cells in primary biliary cirrhosis

Primary biliary cirrhosis is characterized by chronic hepatic inflammation and immune mediated apoptosis of bile duct epithelial cells. Delayed macrophage phagocytosis of opsonized apoptotic cells, noted in other autoimmune diseases, may promote inflammation. Recent studies suggest serum anti-CD16 autoantibodies contribute to impaired macrophage phagocytosis by blocking complement receptor 3 (CR3) signaling via CD16. Therefore, serum anti-CD16 levels and the ability of monocyte derived macrophages from individuals with PBC to phagocytosis apoptotic cells were compared to controls. The mean level of anti-CD16 IgM autoantibodies (0.86+/-0.62 v. 0.35+/-0.22, respectively, p=0.031) was increased in PBC compared to control sera, and mean PBC phagocytosis of opsonized apoptotic cells was significantly decreased compared to controls (23.9+/-12.2% v. 43.9+/-14.4%, respectively, p=0.020). However, PBC phagocytosis of opsonized apoptotic cells was not significantly affected by the presence or absence of autologous serum (20.8+/-13.5% v. 23.9+/-12.2%, respectively, p=0.560). PBC phagocytosis of opsonized apoptotic cells inversely correlated with CD16 (and CR3) expression levels on Day 5 after culture in the presence or absence of autologous serum (r=-0.546, p=0.033 and r=-0.519, p=0.042, respectively). Phagocytosis of non-opsonized apoptotic cells did not correlate with CD16 or CR3 expression (p>0.050). In conclusion, PBC macrophage phagocytosis of opsonized apoptotic cells is impaired, irrespective of serum factors and may increase hepatic inflammation.     

Increased sensitivity of early apoptotic cells to complement-mediated lysis

Opsonization of apoptotic cells with complement proteins contributes to their clearance by phagocytes. Little is known about the lytic effects of complement on apoptotic cells. Sensitivity of cells treated with anti-Fas antibody (Jurkat cells), staurosporine or etoposide (Raji cells) to lysis by complement was examined. As shown here, early apoptotic cells are more sensitive to lysis by antibody and complement than control cells. More complement C3 and C9 bound to apoptotic than to control cells, even though antibody binding was similar. Enhanced killing and C3/C9 deposition were blocked by benzyloxy-Val-Ala-Asp-fluoromethylketone, a pan-caspase inhibitor. Complement-mediated lysis of early apoptotic cells was also prevented by inhibitors of caspases 6, 8, 9 or 10. In contrast, caspase inhibitors had no effect on the lysis of non-apoptotic Jurkat and Raji cells. Early apoptotic Jurkat cells were also more sensitive to lysis by the pore formers streptolysin O and melittin. Sensitivity of Jurkat Bcl-2 transfectants to lysis by complement was analyzed. Enhanced Bcl-2 expression was associated with reduced C3 deposition and lower sensitivity to complement-mediated lysis. These results demonstrate that at an early stage in apoptosis, following caspase activation, cells become sensitive to necrotic-type death by complement and other pore formers. Furthermore, they suggest that Bcl-2 is actively protecting Jurkat cells from complement-mediated lysis.     

Endogenous humoral autoreactive immune responses to apoptotic cells: Effects on phagocytic uptake,chemotactic migration and antigenic spread

Enhanced cell death and deficient clearance of cellular debris are thought to contribute to increased self‐antigen exposure in systemic autoimmune disease. To investigate the characteristics of early humoral autoimmune responses, six monoclonal antibodies were generated from two autoimmune prone strains of mice. All antibodies specifically bound the surface of late‐stage apoptotic cells. Similar antibody reactivities were present in the sera of patients with systemic lupus erythematosus. While IgM antibodies significantly reduced the phagocytic uptake of apoptotic thymocytes, IgG antibodies enhanced uptake. Poly‐reactivity was demonstrated in the recognition of ribonucleoproteins and lipids. An antibody reactive towards lysophosphatidylcholine reversed lysophosphatidylcholine‐mediated inhibition of LPS‐induced TNF‐α production and adversely affected the transmigration of phagocytes towards an apoptotic stimulus. In several instances, CDR were characterized by the accumulation of somatic mutations. Anti‐idiotypic antibodies generated upon immunization bound distinct cellular moieties and self‐antigens. Poly‐specific, apoptotic cell‐reactive autoantibodies can therefore directly impact upon the course of disease by influencing phagocytic uptake of apoptotic cells, by inducing a pro‐inflammatory environment through neutralization of bioactive lipids, by blinding phagocytes to the presence of dying cells through the negation of lipidic chemotactic signals, and by mediating diversification of the humoral autoimmune response via the idiotypic network.     

Ficolin-B marks apoptotic and necrotic cells

Ficolins are a group of proteins consisting of a fibrinogen-like and a collagen-like domain. They play a role in innate immunity by activating the complement system via the lectin pathway upon binding to carbohydrate patterns on pathogens. Two types of ficolins have been identified in mice, ficolin A and ficolin B (FcnB). We show in this article that recombinant FcnB binds to late apoptotic cells and to apoptotic bodies as well as to necrotic cells but not to early apoptotic cells. This binding was calcium-dependent and could be competitively inhibited by acetylated BSA, a classical binding substrate of FcnB. In addition, DNA inhibited binding of FcnB to apoptotic and necrotic cells, indicating that DNA exposed by dying cells could also be a ligand for FcnB. Thus, FcnB may play a role in the removal of damaged host cells and maintenance of tissue homeostasis.     

Systemic autoimmune disease induced by dendritic cells that have captured necrotic but not apoptotic cells in susceptible mouse strains

Systemic lupus erythematosus (SLE) is an autoimmune disorder of a largely unknown etiology. Anti-double-stranded (ds) DNA antibodies are a classic hallmark of the disease, although the mechanism underlying their induction remains unclear. We demonstrate here that, in both lupus-prone and normal mouse strains, strong anti-dsDNA antibody responses can be induced by dendritic cells (DC) that have ingested syngeneic necrotic (DC/nec), but not apoptotic (DC/apo), cells. Clinical manifestations of lupus were evident, however, only in susceptible mouse strains, which correlate with the ability of DC/nec to release IFN-gamma and to induce the pathogenic IgG2a anti-dsDNA antibodies. Injection of DC/nec not only accelerated disease progression in the MRL/MpJ-lpr/lpr lupus-prone mice but also induced a lupus-like disease in the MRL/MpJ-+/+ wild-type control strain. Immune complex deposition was readily detectable in the kidneys, and the mice developed proteinuria. Strikingly, female MRL/MpJ-+/+ mice that had received DC/nec, but not DC/apo, developed a 'butterfly' facial lesion resembling a cardinal feature of human SLE. Our study therefore demonstrates that DC/nec inducing a Th1 type of responses, which are otherwise tightly regulated in a normal immune system, may play a pivotal role in SLE pathogenesis.     

Oncostatin M deficiency leads to thymic hypoplasia,accumulation of apoptotic thymocytes and glomerulonephritis

Oncostatin M (OSM) has been implicated in immune regulation, though its precise role remains elusive. Here we show that OSM plays a crucial role in the prevention of autoimmune diseases. OSM‐deficient mice showed normal development of T cells, B cells and DC; however, their thymus showed hypoplasia and altered medullary structure. Autoantibodies against dsDNA accumulated and glomerulonephritis developed in aged OSM‐deficient mice. Apoptotic cells accumulated in the thymus of OSM‐deficient mice, and the administration of dexamethasone in young OSM‐deficient mice resulted in the massive accumulation of apoptotic thymocytes and production of autoantibodies. These results suggest that OSM plays a key role in the prevention of autoimmune disease by regulating the clearance of apoptotic thymocytes.     

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.     

Mannose-binding lectin and innate immunity

Summary:  Innate immunity is the earliest response to invading microbes and acts to contain infection in the first minutes to hours of challenge. Unlike adaptive immunity that relies upon clonal expansion of cells that emerge days after antigenic challenge, the innate immune response is immediate. Soluble mediators, including complement components and the mannose binding lectin (MBL) make an important contribution to innate immune protection and work along with epithelial barriers, cellular defenses such as phagocytosis, and pattern-recognition receptors that trigger pro-inflammatory signaling cascades. These four aspects of the innate immune system act in concert to protect from pathogen invasion. Our work has focused on understanding the protection provided by this complex defense system and, as discussed in this review, the particular contribution of soluble mediators such as MBL and phagocytic cells. Over the past two decades both human epidemiological data and mouse models have indicated that MBL plays a critical role in innate immune protection against a number of pathogens. As demonstrated by our recent in vitro work, we show that MBL and the innate immune signaling triggered by the canonical pattern-recognition receptors (PRRs), the Toll-like receptors (TLRs), are linked by their spatial localization to the phagosome. These observations demonstrated a novel role for MBL as a TLR co-receptor and establishes a new paradigm for the role of opsonins, which we propose to function not only to increase microbial uptake but also to spatially coordinate, amplify, and synchronize innate immune defenses mechanism. In this review we discuss both the attributes of MBL that make it a unique soluble pattern recognition molecule and also highlight its broader role in coordinating innate immune activation.     

Levels of complement in sera from inactive SLE patients, although decreased, do not influence in vitro uptake of apoptotic cells

BACKGROUND: Accumulation of apoptotic cells is considered relevant in the pathogenesis of systemic lupus erythematosus (SLE). Complement factors facilitate the clearance of apoptotic cells and, when decreased, might result in an increased amount of apoptotic cells found in SLE patients. OBJECTIVE: To determine the influence of complement profiles from inactive SLE patients on the in vitro phagocytosis of apoptotic cells. METHODS: Consecutive SLE patients (n=98) with inactive disease (SLEDAI < or =4) and 20 healthy controls (HC) were included. Levels of CH50, C3, C4, C1q, and C1r were measured. Human peripheral blood monocytes were isolated from healthy controls and cultured for 7 days to obtain monocyte-derived macrophages (MDM). Jurkat cells were irradiated with UVB to induce apoptosis. Phagocytosis was tested by incubation of MDM with apoptotic cells in the presence of serum and quantified as phagocytosis index (number of Jurkat cells internalized by 100 macrophages). Serum from 20 patients with CH50<65%, 20 patients with CH50 > or =65%, and 20 HC were used in this assay. RESULTS: All HC and 37% of patients had normal complement levels. CH50 level was decreased in 21% of patients, C3 in 52%, C4 in 29%, C1q in 2% and C1r in 44% of patients. Between patients and HC, differences in level of CH50, C3 and C4 were statistically significant. No difference in phagocytosis index between HC and patients, irrespective of their CH50 level, was detected. No correlation was found between the respective complement levels and phagocytosis index. CONCLUSION: In most SLE patients with inactive disease, levels of one or more complement components are decreased. However, decreased levels of complement do not result in a significantly reduced in vitro uptake of apoptotic Jurkat cells by MDM.     

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.     

A specific intercellular pathway of apoptotic cell death is defective in the mature peripheral T cells of autoimmune lpr and gld mice

Homozygosity for either of the unlinked murine autosomal recessive mutations lpr or gld leads to autoimmunity characterized by peripheral accumulation of CD4^?/CD8^? “double-negative” T cells, autoantibodies and various forms of tissue pathology. Recently, the gene affected by lpr was identified as fas, whose product acts as a trigger for programmed cell death or apoptosis. Data reported here indicate that the Fas receptor and its ligand, the wild-type form of the gld gene product, are essential for antigen-stimulated peripheral T cell apoptosis. Furthermore, the wild-type gld gene product is a non-cell-autonomous protein that is produced by activated T cells. Apoptotic elimination of antigen-receptor-triggered peripheral T cells appears to be abnormal in lpr and gld mice, and this deficiency causes peripheral T cells to accumulate resulting in lymphadenopathy. These findings support the importance of apoptotic regulation of lymphocyte persistence after antigen encounter in vivo.     

Mannose-binding lectin and its genetic variants

Mannose-binding lectin (MBL) is a collagen-like serum protein that mediates activation of the complement system and is of importance for host defence. Common variant alleles situated both in the promoter and structural region of the human MBL gene (MBL2) influence the stability and the serum concentration of the protein. Epidemiological studies have suggested that genetically determined variation in MBL serum concentration influences the susceptibility to and the course of different types of infections, autoimmune, metabolic and cardiovascular diseases, but this is still a subject of debate. The fact that these genetic variations are very frequent indicates a dual role for MBL in host defence. In this survey, we summarize the current molecular understanding of human MBL genetics.     

Mini-review: A pivotal role for innate immunity in the clearance of apoptotic cells

Apoptotic cells can be recognized and taken up by both macrophages and dendritic cells. Phagocytosis of apoptotic cells generally leads to active suppression of cytokine production by professional phagocytes. This is different from the response towards cells that die by necrosis, which induce a pro-inflammatory cytokine profile. Uptake of apoptotic cells involves a large number of receptors and opsonins, which bind to cellular ligands exposed during the various stages of apoptotic cell death. Among the opsonins of apoptotic cells, complement factors, including C1q, and complement-activating members of the pentraxin family play an important role. This is indicated by in vitro phagocytosis studies and supported by the susceptibility to systemic autoimmunity of carriers of genetic deficiencies for early complement proteins. The present review summarizes the role of molecules of innate immunity in the handling of apoptotic cells by macrophages and dendritic cells. It is proposed that C1q and other opsonins prevent autoimmunity and maintain self-tolerance by supporting the efficient clearance of apoptotic material, as well as by actively modulating phagocyte function.     

C5b-9 terminal complement complex assembly on apoptotic cells in human arterial wall with atherosclerosis

Apoptosis plays an important role in atherosclerosis. The factors regulating this process are not well defined. We examined the relation of apoptotic cells with the terminal complement complex C5b-9 in human atherosclerotic lesions. The extent of apoptosis was determined using TdT dUTP nick-end labeling (TUNEL) and immunohistochemistry of apoptosis regulators caspase-3, caspase-9, Bax, and Bcl-2. C5b-9 was localized by immunohistochemistry and immunoelectron microscopy. The apoptotic index was higher in fibrous plaques when compared with intimal fatty streaks and intimal thickenings. Bax expression was present in TUNEL+ apoptotic cells, and Bcl-2 was rarely present in the atherosclerotic wall. Active caspase 9 and caspase 3 deposits were present in the same areas, suggesting an involvement of the mitochondrial pathway. C5b-9 deposits colocalized with TUNEL+ cells, and the percent of double-positive cells was 2% in fatty streaks, 12% in intimal thickenings, and 35% in fibrous plaques. Colocalization of apoptotic cells with C5b-9 was also confirmed by immunoelectron microscopy. In conclusion, some apoptotic cells carry C5b-9 deposits, suggesting that complement might be activated by apoptotic cells and involved in the promotion of apoptosis, contributing to the progression of atherosclerotic lesions.     

早期和晚期凋亡细胞对骨髓源性不成熟DC影响的实验研究

目的 研究早期、晚期凋亡细胞对骨髓源性不成熟DC（imDC）的影响。方法 体外以紫外线照射诱导出早期凋亡细胞；将照射后的细胞在37℃，5％CO2条件下孵育10h，得到晚期凋亡细胞；在-70℃条件下反复冻融得到坏死细胞碎片。提取、纯化并培养骨髓源性imDC；分别以流式细胞仪、ELISA、^3H-TdR掺入的混合淋巴细胞反应等方法分析imDC吞噬早期、晚期凋亡细胞或坏死细胞碎片后在表达共刺激分子、分泌IL-12 p70以及刺激T淋巴细胞增殖等方面的差异。结果 imDC吞噬晚期凋亡或坏死细胞碎片后，明显趋于成熟，表现为MHCⅡ、CD40、CD80、CD86的表达均显著上调，分泌IL-12 p70增强，和T淋巴细胞混合培养后可以充分激活T淋巴细胞。而吞噬早期凋亡细胞后，仍然维持不成熟状态，表现为MHCⅡ、CD40、CD80、CD86的表达均维持于低水平，和培养中的imDC相比差异无统计学意义；分泌IL-12 p70的水平以及刺激T淋巴细胞增殖的能力均显著低于吞噬坏死细胞或晚期凋亡细胞后的DC，和培养中的imDC相比差异无统计学意义。结论 早期凋亡细胞和晚期凋亡细胞的性质完全不同，晚期凋亡细胞可以充分活化抗原提呈细胞，并进一步激活T淋巴细胞，而早期凋亡细胞没有这种活化作用。     

Mannose-binding lectin genotype and invasive pneumococcal infection

Invasive pneumococcal disease is a serious infection that primarily affects young children and elderly or immunocompromised persons, but it also can affect healthy persons. Mannose-binding lectin (MBL) is a mediator of innate host immunity that activates the complement pathway and directly opsonizes pathogens. Variant structural codon and promoter MBL alleles have been associated with susceptibility to infections. Sixty-three Belgian patients with invasive pneumococcal disease and 162 healthy Belgian controls were genotyped for MBL alleles. We found a nonsignificant increased risk between the MBL structural codon variants (52, 54, and 57) and invasive pneumococcal disease. Combining our data with similar data from Kronberg et al. (J Infect Dis 2002;185:1517-20) indicated that MBL structural variants contributed to a small but significant increased risk of invasive pneumococcal disease. On the other hand, the -221 and -550 promoter allele distribution and the prevalence of the combined MBL structural and promoter -221 variant alleles were not significantly different between the patient group and the control group.