Inefficient clearance of dying cells in patients with SLE: anti-dsDNA autoantibodies, MFG-E8, HMGB-1 and other players
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- Kruse, K., Janko, C., Urbonaviciute, V. et al. Apoptosis (2010) 15: 1098. doi:10.1007/s10495-010-0478-8
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Systemic lupus erythematosus (SLE) is a complex disease resulting from inflammatory responses of the immune system against several autoantigens. Inflammation is conditioned by the continuous presence of autoantibodies and leaked autoantigens, e.g. from not properly cleared dying and dead cells. Various soluble molecules and biophysical properties of the surface of apoptotic cells play significant roles in the appropriate recognition and further processing of dying and dead cells. We exemplarily discuss how Milk fat globule epidermal growth factor 8 (MFG-E8), biophysical membrane alterations, High mobility group box 1 (HMGB1), C-reactive protein (CRP), and anti-nuclear autoantibodies may contribute to the etiopathogenesis of the disease. Up to date knowledge about these key elements may provide new insights that lead to the development of new treatment strategies of the disease.
KeywordsClearanceMFG-E8Biophysical featuresPlasma membranePhosphatidylserineHMGB-1CRPANA
Systemic lupus erythematosus (SLE) is a multifactorial genetically predisposed disease resulting from inflammatory responses of the immune system against several autoantigens. Most autoantigens are nuclear antigens often part of subcellular particles or complexes containing protein and nucleic acids. During apoptosis autoantigens like chromatin are modified by endonucleases and proteases and get accessible to the immune system in blebs or at the surfaces of apoptotic cells. The hallmark autoantibodies (AAb) are directed against dsDNA and are detectable in almost all untreated patients. AAb and chromatin fragments form immune complexes which deposit in organs like skin and kidney and cause inflammation and organ damage.
Rapid removal of apoptotic cells is essential to avoid inflammation. In healthy individuals, phagocytosis of apoptotic cells is fast and effective without causing inflammation and immune response. Instead, apoptotic cells have an immunosuppressive effect. However, in part of the SLE patients a clearance deficiency by macrophages of apoptotic cells was to be observed. In addition, lymphocyte apoptosis was reported to be increased in patients with SLE . Consequently, apoptotic cells may undergo secondary necrosis and lose their membrane integrity. Danger signals like HMGB-1, ATP or uric acid and autoantigens leak into the tissue and foster inflammation. The released autoantigens get available for dendritic cells in an inflammatory context. Subsequently, these professional antigen-presenting cells (APC) may challenge the tolerance of T and B lymphocytes.
In healthy individuals there are several mechanisms to remove apoptotic cells. The majority of apoptotic cells are cleared by the recognition of phosphatidylserine (PS) early exposed on their surfaces. PS exposure characterizes apoptotic cells, however, it is not absolutely specific. Other cells, including resting monocytes and activated B lymphocytes transiently expose PS. However, they are not recognized by phagocytes . The reason for this discrepancy may be biomechanical membrane alterations additionally operating on the apoptotic cells enabling co-operative binding of PS ligands . An important adaptor protein involved in PS recognition is MFG-E8, which is necessary for the clearance of apoptotic B cells in the germinal centres .
There are several backup mechanisms to clear late apoptotic cells that have escaped the PS dependent initial clearance processes. Opsonising molecules like the C-reactive protein (CRP) or complement promote phagocytosis by bridging apoptotic cells to receptors on phagocytes [5, 6]. C1q and DNase-1 act together in degrading nuclear material that had been released due to secondary necrosis .
Deficiencies of several complement factors are implicated in the pathogenesis of SLE . In SLE, these serum components are often low or unfunctional, leading to an accumulation of apoptotic debris. In this review we exemplarily discuss how MFG-E8, HMGB-1, CRP and anti-nuclear AAb may contribute to the etiopathogenesis of SLE.
Milk fat globule epidermal growth factor 8 (MFG-E8)
MFG-E8 also called lactadherin in humans was first discovered in mammary glands of lactating mice . There it is specifically expressed by epithelial cells and macrophages associated with milk fat globules. The expression of MFG-E8 during gestation and lactation is regulated stage and tissue specific and is 40 times higher in the mid-lactation phase than before lactation . During the process of weaning it is abundantly present in the mammary glands to increase the phagocytosis of epithelial cells that undergo apoptosis in a high rate .
MFG-E8 gene is located on Chromosome 15 in humans (hMFG-E8) and on Chromosome 7 (mMFG-E8) in mice. In the latter there are two isoforms of the MFG-E8 mRNA, a long and a short form that had been isolated from different tissues . Both mRNA variants result from alternative splicing of a single pre mRNA. To generate the short variant, one exon encoding the proline/threonine rich domain is skipped. The long form is predominantly present in the mammary gland whereas the short form is ubiquitously expressed in many tissues. In contrast to mice only a short form of the MFG-E8 mRNA could be isolated from human tissues. In 2006, Burgess et al.  detected MFG-E8 mRNA in the retina and the retinal pigment epithelium (RPE) of rodents. Therefore this molecule might also play a role in the phagocytosis process of photoreceptor outer segments (POS).
MFG-E8 is expressed in a distinct set of cells
MFG-E8 is a peripheral, secreted glycoprotein and one of the key players mediating phagocytosis. It is secreted by bone marrow-derived immature dendritic cells, generated in the presence of granulocyte–macrophage colony-stimulating factor (GM-CSF), 30 times more than by bone marrow-derived macrophages . Furthermore it is expressed by Langerhans cells in the skin and by follicular dendritic cells in the germinal centre . Peritoneal macrophages express MFG-E8 abundantly after activation. In the absence of MFG-E8 they show a reduced ability to take up apoptotic cells. A molecule that is structurally and functionally homologous to MFG-E8 is DEL-1 (developmental endothelial locus-1) which is also responsible for the phagocytosis of apoptotic cells . Del-1 also binds PS on apoptotic cells and integrins on phagocytes and enhances phagocytic activity. The expression by different macrophage subpopulations of MFG-E8 and Del-1 are mutually exclusive.
Characterization of mice lacking the MFG-E8 gene
MFG-E8−/−. mice were generated by Hanayama et al. by deleting the exons 4 to 6 of the MFG-E8 gene . MFG-E8 is important for the clearance of apoptotic epithelial cells of mammary glands. This mechanism is essential for the involution of mammary glands. MFG-E8−/− mice have small litters of newborn mice. This is due to the impairment of the involution of mammary glands . In the late stage of lactating and weaning epithelial cells of the mammary gland that express MFG-E8 undergo apoptosis. Furthermore, macrophages move into the mammary glands to engulf the apoptotic epithelial cells. The clearance of apoptotic epithelial cells is impaired due to the lack of MFG-E8. The mice suffer from mammary duct ectasia and an impaired redevelopment of mammary glands.
In the spleen and lymph nodes a large number of lymphocytes that form the germinal center undergo apoptosis. In MFG-E8−/− mice the lack of MFG-E8 results in the formation of numerous and enlarged germinal centers. The mice develop splenomegaly and AAb and suffer from an age dependent lupus-like disease. At the age of 40 weeks many MFG-E8−/− mice, especially female mice, produce large amounts of ANA, anti-dsDNA antibodies and suffer from proteinuria. Similar finding can be observed for mice with nonfunctional MFG-E8 proteins. C-domain deletion mutants show a loss of membrane binding activity  and a mutant (D89E) carrying a point mutation in the RGD motive of the EGF domain inhibited the phagocytosis by masking PS on apoptotic cells . This mutant lost integrin recognition and therefore inhibited the clearance by macrophages of apoptotic cells in vitro. Intravenous injection of the mutant D89E into wild type mice resulted in the production of ANA and anti-phospholipid AAb. This effect could be enhanced by co injection of syngeneic apoptotic thymocytes. Hence the inability to recognize and clear apoptotic cells leads to the break of self-tolerance and, consequently, to the development of autoimmune diseases.
Importance of MFG-E8 for the clearance of apoptotic centrocytes
The phagocytosis of dying cells and their removal is one of the most important processes to maintain the function of tissues and to prevent autoimmune diseases like SLE. During infection activated lymphocytes specific for the pathogen migrate from the primary focus into the primary follicle and proliferate strongly. The process of monoclonal expansion of B lymphocytes leads to the formation of the germinal center where somatic hypermutation of the immunoglobulin genes occurs. B cells carrying the mutated B cell receptor are tested for their affinity for the antigen by follicular dendritic cells (FDC) presenting antigen specific immune complexes. During the process of affinity maturation only those B cells get selected that bind the presented antigen with high affinity. This process leads to the apoptosis of thousands of B cells that had lost pathogen specificity. Usually these dying cells get efficiently cleared by tingible body macrophages of the germinal center. In a subgroup of patients with SLE apoptotic cells that are not ingested accumulate in the germinal centres; TUNNEL positive apoptotic remnants were detected outside the macrophages partially decorating the surfaces of FDCs. Apoptotic material concentrated on the surface of FDCs may select B cells specific for nuclear autoantigens that arise by the random process of hypermutation within the germinal center [22, 23]. B cells with high affinity for self-antigens would receive survival signals, differentiate into memory B cells and plasma cells and produce nuclear AAb. In addition they may receive signals to switch the isotype of the antibody.
Healthy individuals show clean germinal centers and the TUNNEL positive apoptotic material is almost exclusively sequestered inside large tingible body macrophages. Therefore, those B cells that have accidentally gained autoreactivity by somatic hypermutations cannot be positively selected and die.
MFG-E8 protein that bridges phagocytes and apoptotic cells was initially thought to be expressed by tingible body macrophages in the germinal centers . In 2008, Kranich et al.  revealed MFG-E8 to be identical to the mouse follicular dendritic cell marker FDC-M1. MFG-E8 rather is secreted by FDCs of the germinal centre licensing tingible body macrophages for the clearance of apoptotic B cells arising within the germinal center. When MFG-E8 is missing, it leads to a defect in the clearance of apoptotic centrocytes and to the accumulation of apoptotic debris in the germinal center.
MFG-E8 in patients with SLE
In humans only a short MFG-E8 variant is expressed (Fig. 1). The human MFG-E8 consists of only one EGF-like domain containing an RGD motive. In addition, the proline/threonine (P/T) rich domain between the EGF-like domain and the two C-like domains is missing . It has been reported that the extra long exon of mMFG-E8 containing the P/T domain offers a higher affinity for PS than the short form.
Human MFG-E8 shares only 65% homology to the factor VIII domain of mMFG-E8 responsible for PS binding. Consequently, hMFG-E8 might have a lower binding affinity for PS than the long variant of mMFG-E8 . Like the murine, the human MFG-E8 binds to PS and to αVβ3 integrin. The human protein shows a bell-shaped dose dependency for the augmentation of the phagocytosis of apoptotic cells with a maximum at 0.8 μg/ml. In the presence of more than 1.6 μg/ml MFG-E8 almost no apoptotic cells were taken up.
Quantifying the levels of MFG-E8 in different cohorts of humans (healthy persons, adult SLE patients and childhood-onset SLE patients) revealed a higher level of MFG-E8 in 17 of 172 adult SLE-patients and in 16 of 72 childhood-onset SLE patients. The range was 3–40 ng/ml in the blood of the positive patients compared to healthy persons never containing more than 3 ng/ml MFG-E8. Medical treatment of those patients harboring high levels of MFG-E8 resulted in a significant lower level in 7/8 patients paralleled by clinical improvement.
A further study on Taiwanese SLE patients (147 patients and 146 controls) investigated the role of MFG-E8 mutants to determine whether genetic variations correlate to genetic predisposition. Single nucleotide polymorphism (SNPs) of the MFG-E8 gene coding region were compared between patients with SLE and healthy controls . Persons carrying the MFG-E8-76Met/Met alleles demonstrated a predisposition to SLE in a recessive mode (odds ratio: 2.1; P = 0.020). In contrast, the allelic variant MFG-E8-76Leu was negatively associated with the disease. The most predisposing genotype was MFG-E8-3Arg/Arg-76Met/Met (OR: 0.29; P = 0.007) when compared to the most protective genotype MFG-E8-3Ser-76Leu. This study implicates that an interference with the MFG-E8-dependent clearance of apoptotic cells comprises a significant though mild risk factor for the development of SLE.
Methods to detect biomechanical cell surface alterations of apoptotic cells
In previous studies the clearance deficiency was solely investigated by analyzing the presence of classical biochemical markers such as the PS exposure on the cell surface of apoptotic cells, e.g. by binding of annexin A5 . The exposure of PS is characteristically elevated on apoptotic cells; however, also viable resting monocytes expose PS constitutively. The major difference between apoptotic and viable PS-exposing cells is that only the former show cooperative binding of the PS ligand annexin A5 and are cleared by macrophages. What might be the signal for macrophages to specifically take up apoptotic cells? During apoptosis focal adhesion and cytoskeletal proteins are cleaved by caspases, resulting in biomechanical weakening of the cytoplasmic membrane . We are currently testing the hypothesis that an increased lateral mobility of PS is the discriminatory feature characterizing apoptotic membranes. This is supported by the findings that Annexin A5 forms a homophilic interaction and crystalline-like structures on apoptotic cells .
Mechanical properties can be measured employing biophysical methods. To detect alterations in the lateral mobility of ligands and receptors, active microrheology methods such as magnetic tweezers can be used . Antibody or ligand-coated superparamagnetic beads get bound to their cognate targets. By applying high forces (up to 30 nN) to these beads, we are able to measure the lateral mobility of their ligands or receptors. In addition, this method enables quantification of cellular stiffness when the beads are bound directly to the actomyosin cytoskeleton using fibronectin or collagen-coated beads . In addition, the usage of beads coated with gangliosid GM1 ligands such as choleratoxin UEB  allows the quantification of the fluidity of lipid micro domains. Indeed, using fibronectin as probe we observed alterations of cell stiffness and fluidity in morphologically apoptotic cells (unpublished).
The mechanical properties of membranes are influenced by the lipid uptake and metabolism. We currently investigate whether biomechanical alterations on the surfaces of apoptotic cells are involved in the impaired clearance of apoptotic cells frequently observed in patients with SLE.
High mobility group box 1 (HMGB1)
HMGB1 is a protein of 215 amino acids highly conserved across mammals . It is a member of the high mobility group (HMG) superfamily of DNA binding, nonhistone architectural chromosomal proteins . HMGB1 together with two additional paralogs HMGB2 and HMGB3, comprises a subfamily of HMGB proteins. HMGB1 contains two basic functional motifs, termed HMG-box domains A and B of approximately 80 amino acid residues each and a long acidic tail containing 30 aspartic or glutamic acid residues, linked to the HMG-boxes by an overall basic region of approximately 20 amino acid residues . In the cell nucleus HMGB1 binds without sequence specificity to the minor groove of DNA and induces bends in the helical structure . This structural change facilitates multiple physical interactions between DNA and various proteins, including steroid hormone receptors [36, 37], p53 , and nuclear factor κB (NF-κB) . HMGB1 interacts with high affinity with nucleosomes and stabilizes their structure. The biological relevance of HMGB1 in vivo was shown in HMGB1-deficient mice. These mice are born with several defects and die soon after birth due to hypoglycemia caused by deficient glucocorticoid receptor function .
Biological effects of HMGB1
Wang et al.  first described extracellularly released HMGB1 as a late-acting mediator of endotoxic shock in mice. It was shown that following stimulation with inflammatory mediators including tumor necrosis factor (TNF) α and Toll-like receptor (TLR) ligands such as lipopolysaccharides, HMGB1 is actively secreted through a non-classical Golgi-independent mechanism by monocytes/macrophages [41–43], dendritic cells (DC) , pituicytes , natural killer  and endothelial cells .
Scaffidi et al.  reported an alternative mechanism for HMGB1 release: HMGB1 normally is loosely bound to chromatin, and if the cells become necrotic, bioactive HMGB1 diffuses into the extracellular space. There HMGB1 triggers inflammation, activates immune responses and promotes tissue repair. HMGB1 is not released during early stages of apoptotic cell death. Hypoacetylation of chromosomal proteins as well as phosphorylation of histone H2B due to apoptotic cell death lead to tight binding of HMGB1 to the chromatin, even during secondary necrosis, preventing its release. Recent findings demonstrated that HMGB1 release may not be an exclusive feature of primary necrosis. At least in certain cells, HMGB1 release occurs also during secondary necrosis , where the HMGB1 is partially released as a stable complex with chromatin .
At least three receptors have been reported to mediate the proinflammatory and immune-activating effects of extracellular HMGB1: (i) the receptor for advanced glycation end products (RAGE), (ii) TLR 2, and (iii) TLR4. Stimulation of these receptors leads to activation of the transcription factor NF-κB, inducing the transcription of multiple proinflammatory genes. Upon (co-)activation with HMGB1, macrophages produce pro-inflammatory cytokines such as TNF-α, IL-1β, IL-6, IL-8, MIP-1α, and MIP-2β. In addition, HMGB1 induces migration and maturation of dendritic cells with expression of HLA-DR, CD83, CD80, and CD86 [50, 51] and regulates T cell activation .
Recent studies highlighted a new aspect of HMGB1 biology: as a cofactor for several TLR ligands to activate TLR-dependent functions. Tian et al. demonstrated that HMGB1 in complex with CpG oligodeoxynucleotides (ODN) binds more efficiently to RAGE than HMGB1 alone. The CpG ODN-mediated TLR 9-dependent cytokine release from B cells and plasmacytoid DC was strongly enhanced by HMGB1 and was dependent on the presence of RAGE and TLR9 . Moreover, Ivanov et al. demonstrated that HMGB1 interacts and pre-associates with TLR9 in the endoplasmic reticulum-Golgi intermediate compartment, and hastens TLR9’s redistribution to early endosomes in response to CpG-ODN . Recently, HMGB proteins were identified to represent universal sentinels for the recognition of virtually all immunogenic nucleic acids, especially of viral and microbial origin. HMGB proteins are critical for triggering innate immune responses by TLR3, TLR7, and TLR9 by their cognate nucleic acids . In addition, HMGB1 binds LPS in a concentration-dependent manner and transfers it to soluble and membrane-bound CD14, which concentrates LPS and increases the sensitivity of LPS signalling via the TLR4-MD2 pathway . These data suggest that HMGB1 in addition to its direct proinflammtory properties augments the response to distinct pathogen associated molecular patterns (PAMPs). Furthermore, the association of HMGB1 with cytokines such as IL-1β markedly enhances its proinflammatory activity .
HMGB1 is subject to multiple posttranslational modifications, including glycosylation, phosphorylation, and acetylation . Recently, we showed that during cell death HMGB1 undergoes reversible oxidative modifications at cysteine residues which may modulate its biological properties, such as its interaction with other proteins. Additionally, oxidation of HMGB1 may lead to generation of neoepitopes prone to promote autoimmune responses to HMGB1 as detected in many autoimmune diseases, but also in healthy controls . Importantly, Kazama et al. demonstrated that HMGB1 that had been oxidized during apoptosis loses its pro-inflammatory properties .
A recent study demonstrated that HMGB1 can also negatively regulate inflammation. HMGB1 interacts with CD24, a glycosylated membrane protein. CD24 in turn, binds siglec 10 in humans and siglec G in mice, which are considered negative regulators of the immune response. Hence, the HMGB1-CD24-Siglec G pathway might protect the host against a lethal response to pathological cell death and discriminate damage- versus pathogen-associated molecular patterns (DAMPs vs. PAMPs). However, the relevance of this finding remains to be further elucidated .
The role of HMGB1 in the pathogenesis of SLE
Because of its immunostimulatory and proinflammatory properties and its abundance in the sera of mice with septic shock , HMGB1 has been proposed to contribute to the pathogenesis of multiple chronic inflammatory and autoimmune diseases. Elevated levels of extracellular HMGB1 have been reported in experimental arthritis models. Similarly, in humans with rheumatoid arthritis (RA) increased concentrations of HMGB1 were detected in the synovial fluid from inflamed joints [61, 62]. Importantly, collagen-induced arthritis in rodents was significantly ameliorated upon systemic application of either an antagonistic A box domain or neutralizing HMGB1-specific antibodies, indicating an important role in the pathogenesis of arthritis . HMGB1 may be also involved in the pathogenesis of lupus. Within the lesional skin of cutaneous lupus, increased amounts of cytoplasmic and extracellular HMGB1 were detected together with high expression of TNFα and IL-1β . A follow up investigation revealed cytoplasmic and extracellular HMGB1 at the peak of clinical activity in experimentally ultraviolet light (UV)-induced lesions of cutaneus lupus .
A serological hallmark of SLE are AAb to double-stranded DNA and nucleosomes. In addition to their high diagnostic specificity, the titres of these AAb correlate with disease activity and may directly contribute to the pathogenesis of lupus nephritis. The antigenic stimulus driving the production of AAb directed to nuclear components such as nucleosomes and dsDNA in SLE is not fully resolved. Naked mammalian DNA as well as purified nucleosomes are weak immunogens. Whereas naked DNA does not bear T cell epitopes which could initiate T-cell help for DNA-recognizing B cells, the immune system should have developed a profound tolerance toward ubiquitous and abundant proteins such as histones.
HMGB1 itself can be a target of an autoimmune response: IgG-AAb to HMGB1 can be traced in patients with SLE and positively correlated with SLE disease activity index [68–70]. Epitope mapping revealed that multiple HMGB1 epitopes were recognized by SLE sera, with the major epitope mapping to box A . Interestingly, we detected antibodies to HMGB1 also in several healthy individuals, displaying no signs of an autoimmune diseases. In healthy individuals the HMGB1 AAb were predominantly of the IgM isotype, and IgG AAb usually displayed a low titre. The presence of anti-HMGB1 IgM in most healthy subjects might be due the sticky nature of HMGB1 or, alternatively to cross-reactivity. The HMGB1-binding IgM may also play a physiological role. It could modulate the proinflammatory activity and the half-live of HMGB1, thereby limiting overwhelming inflammatory responses caused by a massive release of HMGB1.
C-reactive protein (CRP)
CRP is a liver-derived acute phase protein whose serum concentration increases in reaction to stimuli like injury or infection up to 1000 fold [71, 72]. On the transcriptional level CRP synthesis is enhanced by IL-6, IL-1β and TNFα [73, 74]. In contrast, TGF-β has an inhibitory effect . Single nucleotide polymorphisms of the promoter of the CRP gene determine CRP baseline levels .
CRP belongs to the pentraxin family and is composed of five identical non-glycosylated polypeptide subunits non-covalently bound in a cyclic arrangement . Each subunit has a phosphorylcholine binding site located on the same face of the pentamer and binding sites for two calcium ions. The interactions with C1q and FcγR occur on the opposite sites of the pentamer .
For CRP two conformational different forms have been described: the native pentameric form (pCRP) and the denatured monomeric form (mCRP) . The structural modification from pCRP to mCRP occurs experimentally by heat, acid, and urea but also under physiological conditions by binding to plasma membranes [80, 81]. mCRP is located at sites of inflammation and is considered to be the tissue-based form of CRP in contrast to the pentameric serum-based form . pCRP binds to FcγRIIa and to FcγRI and mCRP binds to FcγRIII demonstrating the opsonising capacities of both isoforms [83, 84]. pCRP inhibits various functions of neutrophils like the production of reactive oxygen species, degranulation and chemotaxis to IL-8 . Beside upregulation of complement receptor 3 mCRP activates neutrophils, monocytes and platelets [86, 87].
Binding of CRP to dying cells and CRP-mediated clearance
The plasma membrane of cells undergoes characteristic changes during apoptosis. In viable cells the membrane is asymmetric with PS mainly restricted to the inner leaflet. During apoptosis PS is exposed outside. The freely circulating secretory phospholipase A2 (sPLA2) binds PS of the outer membrane leaflet and converts neighbouring phospholipids into lysophospholipids. Lysophosphatidylcholine enriches on the apoptotic cell surface and represents a target for CRP [88, 89]. Therefore, binding to apoptotic cells of CRP occurs later than binding of Annexin A5. CRP ligands in primary and secondary necrotic cells include a variety of autoantigens like chromatin, histones, small nuclear ribonucleoprotein and nuclear envelope proteins [90–92]. The selective binding of CRP to apoptotic and necrotic cells represents an example of a fine tuning mechanism discriminating between apoptotic and necrotic cells .
Bound CRP is recognized by C1q and amplifies complement activation via the classical pathway. The formation of the membrane attack complex (MAC) is inhibited since CRP recruits the regulatory Factor H. In addition, CRP increases the expression of complement inhibitory proteins like protectin, membrane cofactor protein and decay-accelerating factor . Inactivation of the MAC prevents leakage of autoantigens . CRP promotes opsonisation of apoptotic cells, increases phagocytosis by binding to cellular FcγRs and leads to the secretion by macrophages of the antiinflammatory cytokine TGFß . The fast and efficient uptake of CRP-opsonized apoptotic cells reduces the exposition of potential autoantigens . However, it was reported that CRP does not exert an anti-inflammatory effect if cells have got necrotic .
CRP facilitates the clearance by FcγR mediated uptake in phagocytes of circulating nucleosomes and apoptotic blebs on which nuclear antigens are exposed. CRP also assists in the clearance of nuclear autoantigens by accelerating complement-dependent degradation of chromatin . In this context it is interesting that CRP binds DNase-treated dead cells more effectively than untreated controls . This could also have implications for SLE pathogenesis, where DNase I and complement activity is often low.
Injections of human CRP and expression of CRP as transgen protected mice against experimentally induced lethal apoptosis . CRP was reduced in serum of mice with high rates of apoptotic cells . In CRP transgenic mice in which high rates of splenic and intestinal apoptosis were induced, the amounts of apoptotic cells were significantly decreased compared to wildtype . In addition, it has been shown that CRP inhibits antibody response to phosphocholine-containing epitopes  and the inflammatory potential of LPS in transgenic mice. CRP prevents LPS-induced lethality via induction of the anti-inflammatory cytokine IL-10 [102, 103]. These results suggest CRP being able to reduce autoimmune reactions by masking of autoantigens in the periphery as well as by the induction of anti-inflammatory cytokines .
CRP in SLE
The serum concentration of CRP increases massively during acute inflammation, infection or tissue damage. Despite normally being a sensitive marker of inflammation, CRP serum concentrations often do not accompany acute flares in SLE patients. CRP elevation in reaction to infection is often higher than to disease exacerbations . Metabolism in plasma and whole body turnover rates of CRP showed no evidence for increased clearance or catabolism suggesting the CRP synthesis rate being responsible for low flare-induced CRP activation . A polymorphism at the 3′-untranslated region (UTR) of the CRP-locus results in a disproportionately long 3′-UTR, which has an impact on the stability of the mRNA. The basal CRP expression is lowered and gene carriers are predisposed for SLE . Another possible explanation for the low CRP in SLE may be the formation of complexes with anti-CRP AAb showing an increased clearance from circulation. Posttranslational modifications of the CRP molecule have been discussed to play a role in the clearance of circulating CRP and in the induction of anti-CRP AAb. It has been shown that acute phase CRP from patients suffering from various diseases including SLE differ in their amino acid sequences and carbohydrate contents. Surprisingly, all SLE patients showed the same CRP glycosylation variant . The failure of CRP induction in acute phases might be a key phenomenon in the etiopathogenesis of SLE.
Deficiencies in acute phase pentraxins often predispose humans and mice to lupus . Investigations of lupus-prone NZB/NZW mice have demonstrated the capacity of CRP to ameliorate both clinical and biological disease progression. The onset of SLE and the development of nephritis in NZB/NZW mice were decelerated by CRP injection [110, 111]. In addition, the expression of a human CRP gene in NZB/NZW mice significantly reduced the progression of the disease . These mice have reduced proteinuria and higher life expectancy. Interestingly, CRP did not decrease the anti-DNA AAb titres, but reduced and enhanced the immune complex deposition in the renal cortex and mesangium, respectively. CRP has also been detected in immune complexes isolated from sera of patients with SLE , suggesting that CRP is not able to reduce AAb production but the inflammatory potential of immune complexes .
CRP and anti-CRP autoantibodies in SLE
CRP deposits on nuclei of primary and secondary necrotic cells at sites of inflammation. These necrotic cells are a source of autoantigens triggering inflammation. In this inflammatory context CRP displays a potential autoantigen promoting the development of anti-CRP AAb. In a majority of SLE patients anti-CRP AAb occur [114, 115]. Low levels of CRP (see previous chapter) could also be explained by circulating anti-CRP AAb neutralizing or causing depletion of CRP. However, CRP serum levels did not correlate with anti-CRP AAb [115, 116]. The relevance of the anti-CRP AAb is still under discussion: It has been shown that anti-CRP AAb bind monomeric, tissue-bound mCRP but not free pentameric pCRP . The conversion from pCRP to mCRP increases the C1q binding capacity which might be fostered by autoantibody binding to mCRP . The amount of circulating anti-CRP AAb was reported to correlate with the course of SLE  possibly neutralizing circulating CRP and shifting the CRP-mediated clearance towards inflammation.
During apoptosis many proteins, DNA, chromatin, and RNA are modified by cleavage through apoptosis specific proteases, caspases, and endonucleases [119, 120]. Modified nuclear autoantigens are translocated and clustered in blebs at the surfaces of apoptotic cells [121, 122] allowing the access by the immune system of nuclear autoantigens. Many AAb directed against nuclear autoantigens are produced in SLE and in several other chronic inflammatory autoimmune diseases . Anti-dsDNA is the most prominent AAb and is detected in up to 95% of untreated patients with active disease; their titres are reduced after therapy . Correlation of anti-dsDNA AAb titers with disease activity and the observation of DNA-anti-DNA immune complexes at the sites of tissue damage confer a pathogenic role of anti-dsDNA AAb in SLE .
Quantification in patients with SLE of anti-dsDNA AAb is an important tool in the diagnostic algorithm and in the monitoring of the disease. High titres may predict disease relapses and the efficacy of therapeutics has broadly been assessed for this parameter under various clinical and experimental settings [126, 127]. However, the real significance of high titres of anti-dsDNA AAb should be evaluated considering its biological properties and its ability to cause the pathological changes typically found in patients with SLE.
Classical mechanisms of pathogenicity of anti-dsDNA autoantibodies
Anti-dsDNA AAb from patients with SLE demonstrate high affinity for dsDNA and are usually of IgG isotype. Various human monoclonal AAb of the IgG isotype have extensive somatic mutations, suggestive for an antigen-driven selection in germinal centers [128, 129]. Reversion of the somatic mutations in three of these AAb resulted in a loss of affinity for dsDNA. These particular AAb also recognized autoantigens on the surfaces of apoptotic cells and nucleosomes. The somatic mutations that generate the high affinity dsDNA binding were also required to bind apoptotic cell remnants . Employing the binding site selection method, it has been demonstrated that anti-dsDNA AAb preferentially recognize DNA sequences containing adenosine triplets and long purine stretches typically found in bent-DNA, H-DNA, and triple or quadruple-stranded DNA . These findings suggest that autologous DNA epitopes are responsible for the affinity maturation of IgG AAb.
The presence of anti-dsDNA AAb in damaged kidneys from patients with SLE reported in 1967 substantiated these AAb as major players in the pathogenesis of lupus nephritis [131, 132]. Employing a rat kidney perfusion model it was demonstrated that some murine anti-dsDNA AAb significantly increased the proteinuria . In the severe combined immunodeficient (SCID) mice it was confirmed that the binding of human anti-dsDNA AAb to the kidney causes proteinuria and thus tissue damage, probably through different mechanisms . Mjelle et al. demonstrated that the availability of glomerular chromatin fragments is prerequisite for renal anti-dsDNA AAb binding . Accordingly, the development of lupus nephritis in humans is strongly associated with high titres of anti-dsDNA AAb , which precede the clinical onset of SLE  and of disease flares . Therapeutic interventions targeting B cells with anti-CD20 antibodies and other B cell-specific agents lead in most of cases to a reduction of the levels of pathogenic anti-dsDNA AAb .
The pathogenicity mediated by anti-dsDNA AAb is most likely dependent on the activation of complement by IgG and the recruitment of inflammatory cells via engagement of Fcγ receptors . Antigen affinity and specificity may also be important for the tissue targeting of these AAb. In SLE an impaired uptake by professional phagocytic of apoptotic cells causes an increased release of DNA containing nucleosomes from apoptotic cells . The positively charged histones, which are easily trapped in the glomerular basement membrane, are parts of the nucleosomes. Anti-dsDNA AAb bind, activate complement, recruit inflammatory cells and cause kidney damage . Additionally, the polyreactivity of these AAb may explain the different histological manifestations seen in SLE. For example, cross-reactivity with glomerular α-actinin is strongly associated with development of lupus nephritis .
Mechanism for the pathogenicity of anti-dsDNA autoantibodies
Deficiencies in the complement system, especially of the early components, are considered important for the development of SLE . This has revealed the importance of complement proteins as opsonins of dying and dead cells necessary for their appropriate clearance from tissues . Nevertheless, there is no available data about the clearance status of C1q deficient patients. C1q knockout mice develop glomerulonephritis and show impaired clearance of apoptotic cells . In humans activation of complement by anti-dsDNA-DNA immune complexes causes complement consumption and may, thereby, worsen the clearance status. Murine and human anti-dsDNA AAb have been reported to penetrate living cells in the kidney and they have been related to the pathological changes in the glomerulus. It remains unclear how these AAb are internalized independent of Fcγ receptors and how they translocate to the nucleus [146, 147].
Nucleosomes and secondary necrotic cell-derived material (SNEC) are constantly released into the circulation of patients with SLE [148, 149]; this favours the formation and/or deposition of immune complexes containing nuclei acids, modified proteins, and AAb. Several reports have suggested that SLE AAb promote phagocytosis by macrophages and dendritic cells of apoptotic and secondary necrotic cells. This may shift the immune response towards inflammation and autoimmunity [150–152]. These reports show that AAb are able to opsonize secondary necrotic cells and foster their uptake by professional phagocytes; for some of these experiments the presence of active autologous serum was mandatory.
Considering that anti-dsDNA AAb opsonize SNEC and both are present in the circulation of patients with SLE, we analysed the fate of SNEC in anticoagulated whole blood ex vivo. In the blood of healthy donors, SNEC was enzymatically degraded by the concerted action of C1q and DNase I . It was not phagocytosed by blood-borne phagocytes. In contrast, in blood of SLE patients we observed that anti-dsDNA AAb opsonized SNEC and promoted its uptake by blood-borne phagocytes. Although there are reports that Fcγ receptor ligation on macrophages promote the acquisition of a regulatory phenotype , the autoantibody-dependent phagocytosis of SNEC induced the secretion of high amounts of various inflammatory cytokines like IL-8, IL-1β, TNF-α, IL-18, and IFN-α. We proposed that if dying cells escape regular clearance, anomalous phagocytosis of SNEC in the blood of patients with SLE fuels inflammation and contributes to its chronicity  (Fig. 7).
Once they reach intracellular receptors, nucleic acids are important triggers of cytokine responses. SNEC-immune complexes are shuttled by AAb into the intracellular milieu of innate phagocytic cells triggering inflammatory cytokine responses [149, 154]. The intracellular overload with SNEC of cells that are normally not used to handle this kind of prey represents a novel pathogenic mechanism of anti-dsDNA AAb in SLE. DNase II is an intracellular nuclease present in phagocytic cells necessary to degrade phagocytosed chromatin. In DNase II knock out mice undigested DNA in lysosomes leaks into the cytoplasm and causes the production of lethal amounts of IFN-I . Summarizing, SNEC–IC may be considered binary pyrogens which induce much more inflammation than its single components separately. We propose SNEC–IC being a lupus “pathogen” playing a role in the chronification of inflammation.
The pathological manifestations of SLE are complex and diverse. Research in the last 20 years has brought into light the association among apoptosis, the clearance of cell remnants, and the etiopathogenesis of the disease. In the latter process, various soluble molecules and until now scarcely explored biophysical properties of the surface of apoptotic cells play significant roles in the recognition and further processing of dying and dead cells. MFG-E8 is produced by phagocytic cells to enhance the phagocytosis by bridging phagocytes to apoptotic cells. HMGB1 is released by late apoptotic cells complexed with nucleosomes causing immune activation. CRP is ubiquitous and preferentially opsonizes primary and secondary necrotic cells ameliorating their proinflammatory properties. Finally, AAb arising from a clearance deficiency status perpetuate inflammation precipitating tissue damage. The development of drugs that target one or more of these key elements may provide new treatment strategies that reach beyond the classical amelioration of signs and symptoms of SLE.
This work was supported by the “Deutsche Forschungsgemeinschaft” (HE 4490/3-1 and SFB 643-project B5), by the Training Grant SFB GK 643, by the Interdisciplinary Center for Clinical Research (IZKF) (N2) at the University Hospital of the Friedrich-Alexander University, by an intramural grant (ELAN-fonds M3-09.03.18.1) of the Medical Faculty of the Friedrich-Alexander University and by the K. und R. Wucherpfennigstiftung.