Characterization of murine monoclonal antibodies against the Ro52 autoantigen

Immunization of BALB/c mice with purified recombinant human Ro52 protein resulted in three anti-Ro52 MoAbs termed 2E7, 4C6 and 4F11. All anti-Ro52 MoAbs specifically reacted with recombinant human Ro52 protein, and also with Ro52 protein in total extracts of all human cell lines analysed, including the epithelial cell line HeLa, the B cell line Raji, the bladder carcinoma cell line RT112, and a fibroblast cell line derived from patients with xeroderma pigmentosum. The anti-Ro52 MoAbs were able to immunoprecipitate the recombinant human Ro52 protein expressed in wheat germ extract, but failed to precipitate hY RNAs from cell extracts. The staining pattern of the MoAbs strongly differed between the RT112 cells and the fibroblast cell line. RT112 cells displayed an intense cytoplasmic staining and in addition distinct fine nuclear speckles. In contrast, in the fibroblast cell line no cytoplasmic staining but only staining of distinct nuclear speckles was observed. Using deletion mutants of Ro52 the epitopes recognized by the anti-Ro52 MoAbs 2E7, 4C6 and 4F11 were partially mapped. All three MoAbs appeared to recognize distinct epitopes, that are located in the regions of Ro52 bordered by amino acids 136–164, 208–363 and 136–190, respectively. These MoAbs can be of great use in studying the cellular processes in which the Ro52 protein is involved.     

B cell apotopes of the 60-kDa Ro/SSA and La/SSB autoantigens

Apoptosis has been proposed to influence the initiation and diversification of autoimmunity to the Ro (SSA)/La (SSB) ribonucleoprotein (RNP) particle and serve as a target for autoantibody-mediated tissue injury. We have developed a new approach to B cell epitope mapping which identifies "apotopes," defined as epitopes expressed on the surface of apoptotic cells. Preliminary studies support a role for apotopes as diagnostic markers in systemic lupus erythematosus (SLE) and primary Sj?gren's syndrome. For example, apotopes within the NH(2)-terminal and central regions of La react with the majority of sera from mothers of infants with congenital heart block. Furthermore, a Ro60 apotope is specific for a subset of SLE with isolated anti-Ro60 responses. The mapping of B cell apotopes may prove superior to standard epitope mapping by suggesting novel pathways of autoantibody production and identifying pathogenic species of autoantibodies.     

Detection and occurrence of the 60- and 52-kD Ro (SS-A) antigens and of autoantibodies against these proteins.

The simultaneous detection of anti-La, anti-60-kD Ro and anti-52-kD Ro antibodies by immunoblotting is greatly improved by changing the crosslinking level in the gel to an acrylamide/bisacrylamide ratio of 19:1. Using this method for the analysis of a number of systemic lupus erythematosus (SLE) and Sjögren''s syndrome patient sera it was observed that antibody to the 52-kD Ro protein without anti-60-kD Ro antibody was restricted to Sjögren''s syndrome patients (9/26), whereas antibody to the 60-kD Ro protein without contaminating anti-52-kD Ro antibody was only found in SLE patients (8/38). Moreover, in Sjögren''s syndrome patient sera anti-Ro antibody was found only in combination with anti-La antibody (20/26), whereas in SLE patient sera anti-Ro antibody could be found without detectable anti-La specificity (4/38). Double immunofluorescence microscopy revealed that the 52-kD Ro and the 60-kD Ro proteins co-localize in the cytoplasm as well as in the nucleus, whereas immunoprecipitation of [32P]-labelled HeLa cell extract with monospecific anti-52-kD Ro and anti-60-kD Ro sera showed that both proteins are associated with the Ro RNAs. These data suggest the presence of both the 52-kD and the 60-kD Ro proteins in the same ribonucleoprotein complexes. To study the evolutionary conservation of the 52-kD Ro, the 60-kD Ro and the La proteins, extracts of cell lines derived from various mammalian species were analysed on Western blots using monospecific human antibodies. In contrast to the 60-kD Ro and the La antigens which are well conserved in evolution, the 52-kD Ro antigen could be detected in primate cells only by this immunological approach.     

The Ro 60 kDa autoantigen comes into focus: interpreting epitope mapping experiments on the basis of structure

A conserved RNA-binding protein, the Ro 60 kDa (Ro60) autoantigen, is a major target of autoantibodies in patients suffering from the rheumatic diseases Sjogren's syndrome, systemic lupus erythematosus, subacute cutaneous lupus erythematosus and neonatal lupus erythematosus. In both mice and certain bacteria, Ro60 is important for cell survival following ultraviolet irradiation. Although the function of Ro60 was mysterious for many years, recent experiments have demonstrated that this protein binds misfolded noncoding RNAs in vertebrate cells and likely functions in a pathway by which defective RNAs are recognized and targeted for degradation. Recent structural studies have revealed that Ro60 is shaped like a doughnut with an inner hole. Noncoding RNAs called Y RNAs bind on the outer surface of the ring, while the single-stranded ends of misfolded RNAs likely bind within the hole. Comparison of the Ro60 structure with the results of epitope-mapping studies reveals that many of the currently identified epitopes recognized by patient sera overlap regions of Ro60 that function in RNA binding. Moreover, in some patients with anti-Ro60 antibodies, the initial antigenic epitope corresponds to a loop involved in binding single-stranded RNA in the central cavity.     

Ro ribonucleoproteins contribute to the resistance of Deinococcus radiodurans to ultraviolet irradiation

The genome of the radiation-resistant eubacterium Deinococcus radiodurans contains an ortholog of an RNA-binding protein known as the Ro 60-kD autoantigen. This protein, which was previously identified only in higher eukaryotes, is normally bound to small RNAs known as Y RNAs. We show that the Ro protein ortholog Rsr contributes to the resistance of D. radiodurans to UV irradiation. Rsr binds several small RNAs, encoded upstream of rsr, that accumulate following UV irradiation. One of these RNAs resembles a Y RNA. These results suggest that Ro RNPs could similarly contribute to the recovery of higher cells following UV irradiation.     

An ortholog of the Ro autoantigen functions in 23S rRNA maturation in D. radiodurans

In both animal cells and the eubacterium Deinococcus radiodurans, the Ro autoantigen, a ring-shaped RNA-binding protein, associates with small RNAs called Y RNAs. In vertebrates, Ro also binds the 3' ends of misfolded RNAs and is proposed to function in quality control. However, little is known about the function of Ro and the Y RNAs in vivo. Here, we report that the D. radiodurans ortholog Rsr (Ro sixty related) functions with exoribonucleases in 23S rRNA maturation. During normal growth, 23S rRNA maturation is inefficient, resulting in accumulation of precursors containing 5' and 3' extensions. During growth at elevated temperature, maturation is efficient and requires Rsr and the exoribonucleases RNase PH and RNase II. Consistent with the hypothesis that Y RNAs inhibit Ro activity, maturation is efficient at all temperatures in cells lacking the Y RNA. In the absence of Rsr, 23S rRNA maturation halts at positions of potential secondary structure. As Rsr exhibits genetic and biochemical interactions with the exoribonuclease polynucleotide phosphorylase, Rsr likely functions in an additional process with this nuclease. We propose that Rsr functions as a processivity factor to assist RNA maturation by exoribonucleases. This is the first demonstration of a role for Ro and a Y RNA in vivo.     

Anti-SSA/Ro52 autoantibodies blocking the cardiac 5-HT4 serotoninergic receptor could explain neonatal lupus congenital heart block

The 52-kDa SSA/Ro (Ro52) ribonucleoprotein is an antigenic target strongly associated with the autoimmune response in mothers whose children develop neonatal lupus and congenital heart block. When sera from patients with systemic lupus erythematosus were used as autoimmune controls in an enzyme immunoassay to screen for antibodies against the human serotoninergic 5-HT4-receptor, a high correlation was found between the presence of anti-Ro52 protein antibodies in such sera and antibodies reacting with a synthetic peptide, corresponding to the second extracellular loop of the human 5-HT4 receptor (amino acid residues 165-185). Homology scanning between the 5-HT4 peptide and the sequence of the Ro52 protein indicated two potential common epitopes located between residues 365 and 396 of the Ro52 protein. Cross-reactivity was found between the peptide derived from the 5-HT4 receptor, and a peptide corresponding to residues 365-382 of the Ro52 protein. Autoantibodies, affinity-purified on the 5-HT4 receptor peptide, specifically recognized both the Ro52 protein and the 5-HT4 receptor protein in immunoblots. The affinity-purified antibodies antagonized the serotonin-induced L-type Ca channel activation on human atrial cells. This effect could explain the electrophysiological abnormalities in neonatal lupus.     

The 5S rRNA is associated with Ro60 ribonucleoprotein and is co-precipitated with hYRNAs by anti-Ro antibodies

Ro particles are conserved molecules that contain a YRNA and various Ro proteins, which are recognized by autoimmune sera from patients with lupus erythematosus or Sj?gren's syndrome. The Ro60 ribonucleoprotein (RNP) forms complexes with certain 5S rRNAs, in such a manner that Ro60 could participate in the control of 5S rRNA production. The present studies were carried out to explore the interaction of Ro components, and to address the question whether Ro60 RNP binds simultaneously 5S rRNA and hYRNA. Anti-Ro60 antibodies were used to immunoprecipitate the RNA. Immunoprecipitates were reverse transcribed with specific oligonucleotides and the resulting cDNAs from 5S and hY4 were amplified by PCR. We found that 5S rRNA is complexed with hY4 and hY5 RNAs by means of the Ro60 RNP. Moreover, by in situ hybridization assays we were able to demonstrate that these molecules have a similar nuclear distribution. According to these results, it seems reasonable to assume that the Ro60 protein could be involved in ribosome assembly.     

Interaction of calcium and Ro60: increase of antigenicity

The structural and functional integrity of the cell is largely maintained by protein-protein interactions. Recently, we demonstrated that multiple antigenic peptides (MAPs) constructed from 60 kDa Ro sequence could be used to show intramolecular and intermolecular protein-protein interaction within the 60 kDa Ro ribonucleoprotein particle. We were interested in understanding the mechanism of this binding and hypothesized that this interaction might be mediated through divalent metal ions. The 60 kDa Ro-MAPs failed to interact with purified 60 kDa Ro in the presence of EDTA or EGTA when analyzed by Ouchterlony or surface plasmon resonance (SPR) analysis. When purified 60 kDa Ro was incubated with various metal ions such as Cu2+, Mg2+, Zn2+ and Ca2+, and analyzed by Ouchterlony or SPR for binding to specific 60 kDa Ro-MAPs only Ca2+ ions significantly increased the binding. It was interesting to note that recombinant 60 kDa Ro formed precipitin lines with Ro-MAPs only in the presence of Ca2+ ions. Anti-Ro60 containing SLE sera bound to recombinant Ro60 strongly when incubated in the presence of Ca2+ ions but not in the absence of Ca2+ ions. Using SPR analysis we also found that native Ro60 binds to La only in the presence of Ca2+. These data imply that Ca2+ induces a more native tertiary structure to recombinant 60 kDa Ro and makes it more antigenic. Thus, the observed intramolecular and intermolecular interactions and antigen-antibody interactions could be Ca2+ ion mediated conformational interactions, and we propose that 60 kDa Ro is a calcium binding protein.     

Autoantibody to the leucine zipper region of 52 kDa Ro/SSA binds native 60 kDa Ro/SSA: identification of a tertiary epitope with components from 60 kDa Ro/SSA and 52 kDa Ro/SSA

Anti-Ro (or SSA) is found in the sera of patients with autoimmune rheumatic illnesses. All patients with anti-Ro defined by precipitation bind a 60 000 Da antigen (60 kDa Ro), whereas some patients also bind a 52 000 Da molecule (52 kDa Ro). In general, antibody binding is directed against native 60 kDa Ro and denatured 52 kDa Ro. The mechanism by which anti-52 kDa Ro arises in the setting of anti-60 kDa Ro is unknown. Conflicting data exist as to the existence of a physical interaction between the two proteins in cells and as to cross-reacting antibodies. Antibodies were affinity purified from a peptide within the leucine zipper region of 52 kDa Ro. These purified antibodies binding the 197-207 peptide from 52 kDa Ro (anti-52LZ) bound native 60 kDa Ro as well as denatured 52 kDa Ro. In addition, anti-52LZ also bound up to four regions from the sequence of 60 kDa Ro and a single conformational epitope of 60 kDa Ro. Thus, these primary sites represent components of the tertiary epitope. We hypothesized that if this was the case, these peptides making up a tertiary epitope would show molecular interaction. In fact, peptides from 60 kDa Ro have a molecular interaction with the 52 kDa Ro peptide as well as full-length 52 kDa Ro when assessed by surface plasmon resonance. The leucine-zipper region peptide from 52 kDa Ro bound three of the four peptides from 60 kDa Ro. These data suggest that these two molecular species, 60 and 52 kDa Ro, form a conformational epitope. This relationship may explain why anti-52 kDa Ro is found in association with anti-60 kDa Ro.     

Immunization with peptides from 60 kDa Ro in diverse mouse strains

Antibodies binding the Ro (or SSA) and La (or SBB) proteins are commonly found in a high proportion of sera from patients with systemic lupus erythematosus or Sjögren's syndrome. The mechanism by which these autoantibodies arise is not known. Others and we have shown that immunization of nonautoimmune‐prone mice with short peptides from the Ro ribonucleoprotein particle can induce autoimmunity to 60 kDa Ro and 52 kDa Ro as well as to the 48 kDa La protein after epitope spreading. We have explored the differences in the epitope spreading after 60 kDa Ro peptide immunization in several strains of mice. There is intra‐ and intermolecular diversification of the immune response after immunization of DBA/2J animals with a monomer peptide representing the residues 480–494 of the 60 kDa Ro protein, but this peptide does not induce epitope spreading when used as the immunogen in either C57Bl/6J or PL/J mice. Similar to previously studied BALB/c mice, DBA/2J mice have antibodies binding many epitopes of 60 kDa Ro, and some sera bind 52 kDa Ro as well as La. These mice have antinuclear antibody in their sera. These data demonstrate that Ro peptide immunization results in different outcomes depending upon the strain of mouse used. Furthermore, these data suggest that genetic variation is important with regard to responding towards short peptide immunization by epitope spreading.     

A common autoepitope near the carboxyl terminus of the 60-kD Ro ribonucleoprotein: Sequence similarity with a viral protein

The Ro ribonucleoprotein is composed of hY RNA and a 60.7-kD peptide that is antigenic for autoantibodies produced by many patients with systemic lupus erythematosus or Sjögren's syndrome and mothers of newborns with complete congenital heart block. A major immunoreactive fragment (13 kD) of the 60-kD Ro is bound by 28 of 45 (62%) of the anti-Ro sera tested. Amino acid sequence analysis localizes this fragment to the carboxyl end of the 60-kD Ro peptide. All possible overlapping octapeptides of this 13-kD peptide of 60-kD Ro have been assessed for antigenicity. Sera that bind the 13-kD peptide fragment in immunoblot generally also bind the octapeptides of Ro spanning the sequence AIALREYRKKMDIPA (P<0.01). Inhibition studies with synthetic peptides and purified Ro have established specificity for reference serum antibody binding to an antigenic octapeptide, EYRKKMDI, from this region. The closely related sequence EYRKKLMD is found in the nucleocapsid protein of vesicular stomatitis virus and may portend an immunologic link to this or a related viral antigen. These results also demonstrate that despite fine specificity variation between human sera, there are recurring patterns of anti-Ro binding shared by some patients who have precipitating anti-Ro autoantibodies.     

Post-translational modifications of the major linear epitope 169-190aa of Ro60 kDa autoantigen alter the autoantibody binding

Ro60 kDa is a member of the Ro/LaRNP ribonucleoprotein complex and its major linear B cell epitope, corresponding to the region 169–190aa, has been found to be the initial target of the autoimmune response in patients with systemic lupus erythematosus. This sequence contains one serine and two arginine amino acid residues, which can potentially be modified post‐translationally by phosphorylation or citrullination, respectively. The aim of this study was to develop an immunoassay for anti‐Ro60 kDa epitope antibody detection and to investigate the changes in the antigenicity of the Ro60 kDa epitope when it is post‐translationally modified, by either citrullination or phosphorylation. Peptide analogues corresponding to the unmodified form of the epitope, its phosphorylated form, and a form with both arginine residues citrullinated were synthesized. The peptide coating conditions were investigated and it was found that the use of highly hydrophilic surfaces increase the efficiency of the coating, as well as the sensitivity of the method for anti‐peptide antibody detection. All peptides were tested by the optimized enzyme‐linked immunosorbent assay (ELISA) against 119 sera from patients with primary Sjögren's syndrome, systemic lupus erythematosus and rheumatoid arthritis with anti‐Ro/SSA reactivity, 20 sera from patients with systemic diseases without anti‐Ro/SSA immune reactivity, as well as against 65 sera from normal individuals. A large proportion of the tested sera reacted against all three peptide analogues, although with a preference for the unmodified form of the epitope. In conclusion, post‐translational modifications of the major Ro60 kDa B cell epitope can alter the autoantibody binding.     

Purification of antigenically intact Ro ribonucleoproteins; biochemical and immunological evidence that the 52-kD protein is not a Ro protein.

Anti-Ro sera immunoprecipitate Ro ribonucleoproteins (RNPs) from human cell extracts. Ro RNPs are biochemically heterogeneous particles whose functions are unknown and whose exact composition remains controversial. In addition to 60-kD Ro and to La proteins, a 52-kD polypeptide (p52) has been proposed to be a stable component of the Ro RNPs. To confirm the immunological studies supporting this hypothesis, we have biochemically purified Ro RNPs from HeLa cells using non-denaturing conditions. Ro RNPs segregated into three distinct populations, one of which only contained hY5 RNA (RohY5 RNPs). No p52 co-purified with Ro RNPs. Despite the absence of p52, purified Ro RNPs had biochemical and immunological properties identical to those of unfractionated Ro RNPs. Many anti-Ro sera only recognize p52 in immunoblots, and are said to be monospecific anti-p52. Preincubation with purified RohY5 RNPs (free of p52) of all human anti-Ro (including so-called monospecific anti-p52) sera abolished their capacity to immunoprecipitate Ro RNPs from unfractionated HeLa cell extracts. Conversely, preincubation of anti-Ro sera with purified p52 protein specifically inhibited recognition of p52 in immunoblots, but did not interfere with immunoprecipitation of Ro RNPs. Our data demonstrate that anti-p52 antibodies do not target intact Ro RNPs, nor do they target the native 60-kD Ro protein. Contrary to previous reports, p52 protein is not a stable component of antigenically intact Ro RNPs.     

Protein-protein interactions between native Ro52 and immunoglobulin G heavy chain.

Using a yeast two-hybrid system to search for proteins interacting with Ro52 autoantigen, we identified a novel protein-protein interaction. Two different cDNA clones, which interacted with Ro52 in the yeast two-hybrid system, were identified and isolated from a human B-cell library. Surprisingly, both clones encoded the heavy chain of human IgG1. The expression of both HIS3 and beta-galactosidase reporter genes in yeast suggested that the interaction between Ro52 and IgG occurred in vivo. In vitro studies utilizing recombinant Ro52 and purified immunoglobulins indicated that the interaction was immunoglobulin class and subclass specific. Ro52 interacted with IgG1 and IgG4, but not with IgG2, IgG3, IgA or IgM. Ro52 could also precipitate IgG directly from serum. The identified cDNA clones did not include the variable region of IgG, which suggested a non-classical interaction independent of antibody specificity. We further mapped the domain of Ro52 responsible for this interaction to the C-terminus rfp-like region. In conclusion, our data support an unusual interaction between native Ro52 and IgG. The potential biological significance of this unusual protein-protein interaction is discussed.     

Human Y5 RNA specializes a Ro ribonucleoprotein for 5S ribosomal RNA quality control

Humans express four distinct non-protein-coding Y RNAs (ncRNAs). To investigate Y RNA functional diversification, we exploited an RNA-based affinity purification method to isolate ribonucleoproteins (RNPs) assembled on individual human Y RNAs. Silver staining and mass spectrometry revealed that the Ro and La proteins assemble with all Y RNAs, while additional proteins associate with specific Y RNAs. Unexpectedly, Y5 RNA uniquely copurified ribosomal protein L5 and its binding partner 5S RNA. These findings reveal a contribution of Y5 to 5S surveillance and suggest that interactions between Ro-Y5 and L5-5S RNPs establish 5S RNA as a target of quality control.     

人参皂苷Ro 促进THP-1 细胞向DC 分化的作用

目的:探讨人参皂苷Ro 联合细胞因子(GM-CSF 与IL-4)促进人单核白血病细胞(THP-1)向树突状细胞(DC)分化的作用。方法:筛选细胞因子诱导白血病源DC 敏感细胞株;采用细胞因子联合人参皂苷Ro 诱导筛选的敏感细胞株THP-1 向DC 分化。培养体系中分别加入小(5 μmol/ L)、中(10 μmol/ L)、大(20 μmol/ L)剂量人参皂苷Ro,流式细胞术检测白血病来源DC 表面标志CD1a、MHCⅠ、共刺激分子CD86 表达;逆转录-聚合酶链式反应(RT-PCR)检测白血病来源DCMHCⅠ、CD86 mRNA 转录水平;酶联免疫吸附法(ELISA)检测培养上清TNF-α、IL-6 蛋白水平。结果:THP-1 是细胞因子诱导DC 分化的敏感白血病细胞株,与单纯细胞因子诱导比较,细胞因子联合人参皂苷Ro 作用后,白血病来源DC 表面标志CD1a、MHCⅠ、共刺激分子CD86 比例显著升高(P<0.05),CD1a、CD86 及MHCⅠ转录水平显著升高(P<0.05),培养上清TNF-α、IL-6 蛋白水平显著升高(P<0.05)。结论:人参皂苷Ro 能明显促进细胞因子诱导THP-1 细胞向DC 分化。     

Anti-SSA/Ro antibody determination by enzyme-linked immunosorbent assay as a supplement to standard immunofluorescence in antinuclear antibody screening

The aim of this study was to investigate the frequency and possible clinical relevance of SSA/Ro antibodies, as determined by enzyme-linked immunosorbent assay (ELISA), in patient sera not exhibiting a concomitant positive reaction by the standard immunofluorescence (IF) test using HEP-2 cells as substrate. SSA/Ro reactivity, as shown by ELISA, was found in 285 (7%) of 4025 serum samples consecutively remitted for antinuclear antibody (ANA) screening. Seventy-five of these serum samples (26%), derived from 64 patients, were negative by the IF-ANA screening test. Serum samples from all 64 patients exhibiting SSA/Ro reactivity by ELISA without concomitant positivity by IF-ANA were further investigated by IF using transfected HEP-2 cells hyperexpressing the 60,000 MW SSA/Ro antigen (HEP-2000(R)) and by immunodiffusion (ID) and Western blot. In 55 of these 64 patients, SSA/Ro reactivity could be verified by one or more of the other techniques investigated. Twelve of these patients fulfilled four or more American College of Rheumatology (ACR) criteria for systemic lupus erythematosus (SLE) and another five patients exhibited a histologically confirmed cutaneous lupus erythematosus (LE). In four of the 12 IF-ANA-negative patients with a diagnosis of SLE, the SSA/Ro reactivity was only detectable by ELISA and Western blot. In conclusion, the use of a sensitive ELISA assay could provide a clinically important supplement to the routine ANA screening by IF, which does not detect certain anti-SSA/Ro-containing sera among patients with relevant autoimmune diagnoses. Detection of anti-SSA/Ro antibodies, however, does not alone signify cutaneous LE or SLE but adds weight to these diagnoses that should rely heavily on other clinical information.     

Autoantigen Ro52 directly interacts with human IgG heavy chain in vivo in mammalian cells

Previously, when we used in vivo yeast two-hybrid and in vitro protein-protein interaction analyses, we demonstrated a direct interaction between autoantigen Ro52 and the human IgG heavy chain. This interaction occurred in the absence of antibody-antigen specific interaction. Here, by employing a novel strategy, we further demonstrated that Ro52 co-localized with IgG in transfected mammalian cells. The co-localization was specific to IgG1 but not IgG3. Co-immunoprecipitating IgG with Ro52 from transfected cell lysates suggested that protein complex containing Ro52 and IgG contributed to the in vivo co-localization. In addition, IgG from normal human serum was shown to bind to the surface of apoptotic keratinocytes and the binding could be competitively blocked by 50-fold excesses of IgG1, not IgG3. With a direct binding study, we also demonstrated that IgG1 could bind to the surface of apoptotic cells while IgG3 bound barely. This binding was not competed by Fcgamma fragments indicating a non-Fcgamma receptor mediated interaction. Finally, in a competition analysis the addition of GST-RFP could reduce the IgG binding to the cell surface. Thus, we suggested that the binding of IgG to the apoptotic keratinocytes might be mediated through the interactions with the surface exposed Ro52. The potential role of forming this protein complex on the apoptotic cells will be discussed.