Innate and humoral recognition of the products of cell death: differential antigenicity and immunogenicity in lupus

While apoptotic debris is believed to constitute the original antigenic insult in lupus (which is characterized by a time‐dependent diversification of autoreactivity), whether such debris and autoantibodies specifically recognizing its constituents mediate differential effects on innate and humoral responses in lupus‐prone mice is currently unknown. Apoptotic blebs (as opposed to cellular lysate) enhanced preferentially the maturation of dendritic cells (DCs) from bone marrow precursors drawn from lupus‐prone mice. Murine, somatically mutated, apoptotic cell‐reactive immunoglobulin (Ig)G monoclonal antibodies demonstrated enhanced recognition of DCs and also displayed a prominent lupus strain‐specific bias in mediating DC maturation. Further, immunization of such antibodies specifically in lupus‐prone mice resulted in widespread humoral autoreactivity; hypergammaglobulinaemia (a hallmark of systemic autoimmunity) was observed, accompanied by enhanced antibody titres to cellular moieties. Induced antibodies recognized antigens distinct from those recognized by the antibodies employed for immunization; in particular, nephritis‐associated anti‐double stranded (ds) DNA antibodies and neonatal lupus‐associated anti‐Ro60 antibodies were elicited by a non‐dsDNA, non‐Ro60 reactive antibody, and Sm was a favoured target. Further, only in lupus‐prone mice did such immunization enhance the kinetics of humoral anti‐self responses, resulting in the advanced onset of glomerulosclerosis. These studies reveal that preferential innate and humoral recognition of the products of cell death in a lupus milieu influence the indices associated with autoimmune pathology.     

Phagocytosis of apoptotic cells and immune regulation

The termination of the apoptotic programme occurs in most cases via recognition and clearance by phagocytes, especially the professional phagocytes, such as macrophages and immature dendritic cells. Engulfed cells do not simply disappear from the midst of living tissues. The fine-defined presentation of yielded self-antigens to T cells is a central event in the induction or the maintenance of the peripheral immune tolerance to self. Conversely, abnormality in this pathway may contribute to the pathogenesis of systemic and organ-specific autoimmune diseases. We herein reviewed the relationship between phagocytosis of apoptotic cells and immune regulation, especially the effects of engulfed apoptotic cells on immune tolerance and autoimmune diseases.     

Predominant role of IgM-dependent activation of the classical pathway in the clearance of dying cells by murine bone marrow-derived macrophages in vitro

Soluble molecules including complement components have been shown to facilitate the clearance of dying cells by phagocytes, a process that is important in preventing tissue damage and autoimmunity. However, the extent to which complement is involved in this process and the relative contribution of each of the complement activation pathways is not fully understood. We examined the role of complement in the recognition/uptake of apoptotic thymocytes by murine bone marrow-derived macrophages (BMDM) in vitro using sera from gene-targeted mice. We found this process to be IgM- and complement-dependent, especially when the apoptotic cell-to-BMDM ratio was low, and the level of C3 deposition on apoptotic cells correlated closely with their uptake. The addition of C1q rectified the phagocytic defect seen in the presence of C1q-deficient serum in vitro but had no effect on the phagocytic defect observed with serum deficient in both IgM antibodies and C1q. Similarly, complement activation by IgM antibodies was essential for in vivo C3 deposition on apoptotic cells and their uptake by peritoneal macrophages. Hence, the efficient uptake of dying cells by BMDM requires IgM antibodies and complement.     

Exposure of anionic phospholipids serves as anti-inflammatory and immunosuppressive signal--implications for antiphospholipid syndrome and systemic lupus erythematosus

In contrast to necrotic cells, the clearance of apoptotic ones usually is an anti-inflammatory process which elicits only a marginal immune response. During apoptosis phosphatidylserine (PS) is exposed on the outer leaflet of the cytoplasmic membrane and serves as target for the PS receptor of phagocytes. The latter is responsible for anti-inflammatory signalling and the induction of TGFbeta. We were interested whether the immunogenicity of apoptotic cells can be increased by masking PS. We observed that treatment of xenogeneic apoptotic cells with annexin V (AxV) significantly increased the humoral immune response against surface epitopes of these cells. Furthermore, AxV-coated irradiated tumour cells were able to elicit a long lasting tumour specific cytotoxic T lymphocyte response. AxV efficiently blocked the uptake of irradiated cells by macrophages but not by dendritic cells. Furthermore, AxV skewed the phagocytosis of irradiated cells towards inflammation. Investigation of patients with autoimmune diseases further supported the role of anionic surface phospholipids for anti-inflammatory clearance of apoptotic cells. Impaired clearance and opsonisation with anti-phospholipid-antibodies are discussed to be responsible for the development of systemic lupus erythematosus and anti-phospholipid-syndrome, respectively. Presentation of cryptic epitopes from late apoptotic cells in a proinflammatory context may challenge T cell tolerance. In addition, accumulation of uncleared apoptotic debris in the germinal centres of lymph nodes may result in the survival of autoreactive B cells.     

Experimental Autoimmunity to Rat Male Accessory Glands (MAG): Circulating Antibodies,Immunoglobulins Bound to Target Glands,and Immunoglobulins-Secreting Cells

ABSTRACT: A correlation between spleen B-cell antibody production against MAG antigens and the presence of different antibodies in circulation or antibodies bound to target glands was attempted. The number of 7S and 19S Ig-secreting cells (ISC) found in the spleen and the number of ISC generated after in vitro stimulation of the cells with MAG antigens were evaluated by using the hemolytic plaque assay. Low numbers of 7S and 19S ISC-less than 0.01% of spleen cells—were generated in response to MAG immunization, and no significative increase was observed after in vitro culture of spleen cells with MAG antigens, suggesting that secretory activity of the B-cells can not be improved when liberated from humoral homeostatic mechanisms. The humoral response of MAG-immunized rats, investigated by complement fixation and immunodiffusion assays, has proved negative, and in only two out of 17 rats a weak haemagglutinating activity was observed. Attempts to detect antibodies bound to cellular MAG antigens by immunofluorescence have shown a weak fluorescence in the epithelial cells of the prostate gland in only two rats. In both cases a concomitant tissue damage was observed, but in nine out of 11 cases with histological alterations no fluorescence was observed in the target glands. The medium value of rosette-forming cells (RFC) found in the spleen of MAG-immunized rats did not significantly differ from the value of the HSA-treated control group, although both groups differ in their specific humoral response. Contrary to the depressed humoral response, the MAG-immunized rats showed in all cases a cellular immune mechanism by at least one of the tests in study and in 70.5% of the cases the simultaneous presence of various factors has been demonstrated. In 11 cases the cell-mediated immune response was accompanied by seminal vesicle and/or prostatic lesions with a microscopic aspect close to that of delayed hypersensitivity reactions. These observations suggest that the characteristic lesions of experimental autoimmune vesiculoprostatitis (EAVP) are unlikely to be initiated by antibodies directed against MAG antigens. The mechanism affecting the impaired antibody response is discussed.     

IgG opsonized nuclear remnants from dead cells cause systemic inflammation in SLE

Deficiencies in the recognition and engulfment of apoptotic cells have been reported in patients with systemic lupus erythematosus (SLE). If dying cells are not promptly cleared, they undergo secondary necrosis and release nuclear autoantigens. Secondarily necrotic cell-derived material (SNEC) can be generated in vitro employing various methods. SNEC generated by either methods shows similar DNA content, light scatter characteristics, and binding pattern of dead and dying cell ligands and is readily recognized by autoantibodies (AAb) of many patients with SLE. In whole blood, AAb opsonize SNEC and foster its uptake by blood-borne non-professional phagocytes. We observed a significant secretion of the inflammatory cytokines IL-8 and TNF-α by phagocytes which had engulfed different types of opsonized SNEC. Phagocytosis of SNEC and the subsequent production of inflammatory cytokines were strongly influenced by the presence of DNA in this prey, since DNase I treatment reduced both the uptake of SNEC and the induction of IL-8 and TNF-α production. In conclusion, the proinflammatory phagocytosis by circulating phagocytes of IgG-opsonized cellular remnants fosters systemic inflammation in SLE.     

Enhancement of humoral and cellular immunity with an anti-glucocorticoid-induced tumour necrosis factor receptor monoclonal antibody

Adjuvants, including antibodies to tumour necrosis factor receptor superfamily members, augment immune responses. One member of this family, glucocorticoid‐induced tumour necrosis factor receptor (GITR), is expressed at low levels on naive/resting T cells, B cells and macrophages, but at higher levels on T regulatory cells. The aim of this study was to determine the ability of a rat anti‐mouse GITR monoclonal antibody, 2F8, to stimulate murine humoral and cellular immunity in a prime boost model with particular attention to posology and antigen‐specific effects. 2F8 enhanced the humoral immune response to ovalbumin and haemagglutinin (HA) compared with controls and this enhancement was equal to or greater than that obtained in mice dosed with standard adjuvants. 2F8 F(ab′)₂ fragments were as effective as intact antibody in boosting humoral immunity, indicating that FcR‐mediated cross‐linking of 2F8 is not required for efficacy. Moreover, the enhanced response was durable and antigen specific. Administration of 2F8 shifted the immune response towards a T helper type 1 response with significant enhancement of immunoglobulin G2a‐ and G2b‐specific anti‐HA antibodies, as well as enhanced cellular immunity as measured by ELISPOT. 2F8‐treated mice also generated significantly more neutralizing antibodies to HA than control mice. Our findings show that anti‐GITR is a robust, versatile adjuvant that, unlike commonly used adjuvants that primarily enhance humoral immunity, enhances both humoral and cellular immunity. These results support the continued development of anti‐GITR for such indications as haematological and solid tumours, chronic viral infections, and as a vaccine adjuvant.     

Human IgG isotypes and activating Fcγ receptors in the interaction of Salmonella enterica serovar Typhimurium with phagocytic cells

Several classes and multiple subclasses of immunoglobulins are produced towards protein and polysaccharide antigens in response to Salmonella infection and play a key role in protection against systemic disease. The targeting of Salmonella to Fc receptors (FcR) on phagocytes is a key step in the antibody-mediated antibacterial functions of host cells. We wished to compare the relative efficiency of different human IgG subclasses, which targeted the Salmonella enterica OmpA surface protein in modulating the interaction of bacteria with human phagocytes. To this end, we developed a novel system by tagging OmpA with a foreign CD52 mimotope (TSSPSAD) and opsonizing the bacteria with a panel of humanized CD52 antibodies that share the same antigen-binding V-region, but have constant regions of different subclasses. Our data revealed that opsonization with all the IgG subclasses increases Salmonella uptake by human phagocytes. IgG3 resulted in the highest level of bacterial uptake and the highest average bacterial load per infected cell, which was closely followed by IgG1, then IgG4 and lastly IgG2. Phagocytosis mediated by IgG1, IgG3 and IgG4 had a higher dependency on FcγRI than FcγRIIA, whereas IgG2-mediated phagocytosis required FcγRIIA more than FcγRI. The results show that IgG binding to OmpA increases the uptake of Salmonella by human phagocytic cells and that the efficiency of this process depends both on the subclass of the IgG and the type of FcR that is available for antibody binding.     

Microparticles as antigenic targets of antibodies to DNA and nucleosomes in systemic lupus erythematosus

Systemic lupus erythematosus is a prototypic autoimmune disease characterized by antibodies to DNA and other nuclear molecules. While these antibodies can form immune complexes, the mechanisms generating the bound nuclear antigens are not known. These studies investigated whether microparticles can form complexes with anti-DNA and other anti-nucleosomal antibodies. Microparticles are small membrane-bound vesicles released from dead and dying cells; these particles contain a variety of cellular components, including DNA. To assess antigenicity, microparticles generated in vitro from apoptotic cell lines were tested using murine monoclonal anti-DNA and anti-nucleosomal antibodies as well as plasma from lupus patients. Antibody binding was assessed by flow cytometry. As these studies showed, some but not all of the monoclonal antibodies bound to microparticles prepared from apoptotic HL-60, THP-1 and Jurkat cells. For HL-60 cells, both staurosporine and UV radiation led to the production of antigenically active particles. For the anti-DNA antibody with high particle binding, prior treatment of DNase reduced activity. With plasma from patients with SLE, antibody binding to microparticles was present although a clear relationship with anti-DNA antibody levels was not observed. To determine whether lupus plasma contains immune complexes with particle properties, particle preparations were tested for bound IgG by flow cytometry. These studies indicated that lupus plasma contains particles with IgG binding, with numbers correlated with anti-DNA levels. Together, these findings indicate that microparticles display DNA and nucleosomal molecules in an antigenic form and could represent a source of immune complexes in SLE.     

Soluble CD93 is an apoptotic cell opsonin recognized by αxβ2

Efferocytosis is essential for homeostasis and prevention of the inflammatory and autoimmune diseases resulting from apoptotic cell lysis. CD93 is a transmembrane glycoprotein previously implicated in efferocytosis, with mutations in CD93 predisposing patients to efferocytosis‐associated diseases. CD93 is a cell surface protein, which is proteolytically shed under inflammatory conditions, but it is unknown how CD93 mediates efferocytosis or whether its efferocytic activity is mediated by the soluble or membrane‐bound form. Herein, using cell lines and human monocytes and macrophages, we demonstrate that soluble CD93 (sCD93) potently opsonizes apoptotic cells but not a broad range of microorganisms, whereas membrane‐bound CD93 has no phagocytic, efferocytic, or tethering activity. Using mass spectrometry, we identified α_xβ₂ as the receptor that recognizes sCD93, and via deletion mutagenesis determined that sCD93 binds to apoptotic cells via its C‐type lectin‐like domain and to α_xβ₂ by its EGF‐like repeats. The bridging of apoptotic cells to α_xβ₂ markedly enhanced efferocytosis by macrophages and was abrogated by α_xβ₂ knockdown. Combined, these data elucidate the mechanism by which CD93 regulates efferocytosis and identifies a previously unreported opsonin‐receptor system utilized by phagocytes for the efferocytic clearance of apoptotic cells.     

Autoreactive B‐lymphocytes in SLE and RA patients: Isolation and characterisation using extractable nuclear and citrullinated antigens bound to immunobeads

Systemic lupus erythematosus and rheumatoid arthritis are autoimmune diseases characterised by B‐cell hyperactivation and production of autoantibodies (AutoAbs) against various self‐antigens, including extractable nuclear antigens and citrullinated peptides. Therefore, B lymphocytes and antibody‐secreting cells are considered relevant targets for therapies. However, isolation and characterisation of auto‐reactive specific B lymphocytes are limited, primarily due to technical issues. In this work, we purified extractable nuclear antigen‐specific and citrullinated peptide‐specific auto‐reactive B lymphocytes by magnetic selection with ENA‐ and citrullinated peptide‐bound immunobeads. We obtained blood auto‐reactive B lymphocytes from most patients. Their nature was primarily naïve B cells, some of them in an active status, with low levels of somatic hypermutations in the immunoglobulin heavy‐chain variable regions. Their presence correlated with serum levels of autoAb. Auto‐reactive B lymphocytes were able to differentiate into auto‐reactive antibody‐secreting cells under conditions of stimulation. In addition, based on the presence of circulating auto‐reactive B cells and/or antibody‐secreting cells, four different profiles were described in lupus patients. Thus, tracking auto‐reactive B cells and/or antibody‐secreting cells in patient blood could represent a biomarker for deciding whether to use therapies blocking either B cells, plasma cells or both, as well as a new tool for monitoring minimal residual autoimmune disease in patients.     

Attraction of phagocytes by apoptotic cells is mediated by lysophosphatidylcholine

Apoptotic cells promote the phagocytosis of themselves via the exposure of “eat me” signals. Furthermore, a second, “find me” signal is required to attract phagocytes. In this review the double function of lysophosphatidylcholine for the clearence of apoptotic material as factor leading to attraction of phagocytes as a soluble “find me” signal and opsonisation of apoptotic cells as a membrane bound “eat me” signal is outlined. Lysophosphatidylcholine, exerting this bivalent function, leads to a sufficient clearance of apoptotic cell as a “keep calm” mechanism to prevent activation of the immune system by secondary necrosis.     

Attraction of phagocytes by apoptotic cells is mediated by lysophosphatidylcholine

Apoptotic cells promote the phagocytosis of themselves via the exposure of "eat me" signals. Furthermore, a second, "find me" signal is required to attract phagocytes. In this review the double function of lysophosphatidylcholine for the clearance of apoptotic material as factor leading to attraction of phagocytes as a soluble "find me" signal and opsonisation of apoptotic cells as a membrane bound "eat me" signal is outlined. Lysophosphatidylcholine, exerting this bivalent function, leads to a sufficient clearance of apoptotic cell as a "keep calm" mechanism to prevent activation of the immune system by secondary necrosis.     

Ribosomal versus non-ribosomal cellular antigens: factors determining efficiency of indirect presentation to CD4+ T cells

Proteins released from dying cells can be taken up and presented by antigen‐presenting cells (APC) to T cells. While the presentation of such self antigens may lead to beneficial anti‐tumour responses, in autoimmune disease it leads to pathological immune responses. The sub‐set of self proteins targeted in autoimmune disease is circumscribed, and certain cellular components such as ribonucleoprotein (RNP) complexes are often targeted. Although explanations for this antigen selectivity have been proposed, there has been little direct testing of these hypotheses. We and others previously showed that ribosomal proteins, targeted in autoimmune disease, are also targets of anti‐tumour T‐cell responses. We asked whether particular properties of ribosomal proteins such as incorporation into RNP complexes or sub‐cellular localization enhance ribosomal protein presentation by APC to CD4⁺ T cells. Ribosomal protein antigens within purified intact ribosomes or free of the ribosomes were equally well taken up and presented by APC, demonstrating that inclusion of ribosomal proteins into an RNP complex does not confer an advantage. However, antigens localized to ribosomes within apoptotic cells were less efficiently taken up and presented by APC than the same antigens localized diffusely throughout the cell. This suggests that presentation of ribosomal proteins is somehow down‐regulated, possibly to facilitate presentation of other less‐abundant intracellular proteins. Consequently, the explanation for the frequent targeting of ribosomal proteins by both autoimmune and anti‐tumour T‐cell responses is not at the level of uptake from apoptotic cells and must be sought elsewhere.     

Inflammatory clearance of apoptotic remnants in systemic lupus erythematosus (SLE)

Deficiencies in the recognition and phagocytosis of dead and dying cells have been shown to be one of the main alterations in patients with systemic lupus erythematosus (SLE). Cellular as well as humoral elements play an important role in the clearance of apoptotic and necrotic cells. Non-ingested nuclear material may provide survival signals for autoreactive B-cells and consequently antibodies directed against nuclear structures will be produced. In healthy individuals, nuclear fragments are not phagocytosed in whole blood. Instead, they are mainly degraded by the action of plasma DNases and complement factors. In contrast, the uptake of nuclear fragments by blood-borne phagocytes is increased in most patients with SLE. The phagocytosis of this kind of prey, which might be opsonised by autoantibodies, may contribute to the maintenance of inflammatory responses in SLE.     

Endocytosis by echinoid phagocytes in vitro I. Recognition of foreign matter

Endocytosis, with emphasis on phagocytic recognition was examined in the sea urchin

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. Most cells (about 67%) in the coelomic fluid were phagocytes. There was also a linear uptake of colloidal gold with time in the cultured cells, presumably caused by pinocytosis. No opsonizing activity was found in the cell-free coelomic fluid, and the parallel phagocytic avidity and of different particles also indicates that humoral factors were absent or not essential for recognition of foreign surfaces by phagocytes. Untreated erythrocytes (RBC) were taken up at a low rate, while RBC treated with aldehyde, Fe²⁺, tannin, Con A, or IgM + mouse C5-deficient complement were phagocytosed efficiently. Phagocytosis was reduced when RBC were covered with specific antibodies. Thus, the echinoid phagocyte lacks an Fc receptor, but may have a C3b receptor on its membrane. Other particles with high phagocytic avidities were carbon, Sephadex, latex and . Thus the cell membranes of phylogenetically distant phagocytes may exhibit common structural features responsible for foreign surface recognition.     

Plasma membrane alterations during apoptosis: role in corpse clearance

Apoptosis is a program of cellular self-destruction culminating in the clearance of cell corpses by neighboring macrophages. Studies in recent years have served to characterize a number of structural and molecular plasma membrane alterations that act in concert to mediate efficient engulfment of cell corpses. Hence, "eat me" signals, including the anionic phospholipid phosphatidylserine (PS) and its oxidized counterpart, PS-OX, as well as the PS-binding protein, annexin I, are exposed on the surface of effete cells and function to mediate engulfment by neighboring phagocytic cells. Plasma membrane blebbing (zeiosis), a common feature of the apoptotic program, provides a structural context for the exposition of recognition signals insofar as PS molecules aggregate on the surface of these membrane protrusions. Apoptotic cells also secrete chemotactic factors ("seek me" signals), such as the phospholipid lysophosphatidylcholine, that recruit phagocytes to the site of the apoptotic lesion. Taken together, these events serve to mediate the disposal of effete cells prior to their necrotic disintegration, thus preventing the inflammation and tissue scarring that would otherwise ensue.     

Effects of Actinobacillus pleuropneumoniae hemolysin on porcine neutrophil function.

In an attempt to gain insight into the events that take place during Actinobacillus pleuropneumoniae infection, the present study was designed to ascertain the effects of bacterial toxicity on porcine neutrophil functions and viability. Incubation of phagocytes (2 x 10(6)) with opsonized A. pleuropneumoniae 4074 (2 x 10(7) CFU) resulted in phagocytic uptake of less than or equal to 4%. At the same bacterium-to-phagocyte ratio, levels of lactate dehydrogenase activity of 74 and 81% were detected in the extracellular medium after 1.5 and 3 h of incubation, respectively. Furthermore, the ingested bacteria were not killed by the phagocytes. These effects were ascribed to hemolysin produced by the bacteria, because the presence of hemolysin-neutralizing antibody prevented overt cellular damage, significantly increased phagocytic uptake (P less than 0.001), and resulted in an approximately 10-fold decrease in the number of CFU of the ingested bacteria. Cytolytic doses of isolated hemolysin caused dose-related loss of cell viability, diminished bactericidal activity of toxin-treated phagocytes for Escherichia coli, and decreased the ability of the phagocytes to undergo a respiratory burst upon stimulation with phorbol myristic acetate. In contrast, sublytic doses of the hemolysin activated the phagocytes and caused them to respond to phorbol myristic acetate with increased generation of superoxide anion. Because heated (100 degrees C, 5 min) hemolysin preparations did not produce similar effects, we contend that the observed effects were not due to contaminating endotoxin. The data presented herein indicate that A. pleuropneumoniae hemolysin is a potent antiphagocytic virulence factor by virtue of its leukocidal activity. Sublytic doses of the toxin may have important effects on the oxidative metabolism of phagocytic cells.     

Self‐dsDNA in the pathogenesis of systemic lupus erythematosus

Systemic lupus erythematosus (SLE) is a systemic and poly‐aetiological autoimmune disease characterized by the production of antibodies to autologous double‐stranded DNA (dsDNA) which serve as diagnostic and prognostic markers. The defective clearance of apoptotic material, together with neutrophil extracellular traps (NETs), provides abundant chromatin or self‐dsDNA to trigger the production of anti‐dsDNA antibodies, although the mechanisms remain to be elucidated. In SLE patients, the immune complex (IC) of dsDNA and its autoantibodies trigger the robust type I interferon (IFN‐I) production through intracellular DNA sensors, which drives the adaptive immune system to break down self‐tolerance. In this review, we will discuss the potential resources of self‐dsDNA, the mechanisms of self‐dsDNA‐mediated inflammation through various DNA sensors and its functions in SLE pathogenesis.