Defective Clearance of Self Antigens in SLE

4.1. Evidence from Knockout Mice

Aberrant rates of apoptosis and increased levels of free-circulating chromatin have been reported in human lupus (Amoura et al., 1997), and humans with DNASE1 and C1q gene mutations develop SLE (Kirschfink et al., 1993; Yasutomo et al., 2001), suggesting that efficient removal of chromatin or chromatin-protein complex is crucial to prevent SLE. For example, serum amyloid P component (SAP)-deficient mice develop an SLE-like syndrome (Bickerstaff et al., 1999). Two models are likely to explain the connection between SAP deficiency and SLE. First, SAP participates in dissolution of chromatin, activates complement components, C1-C4, and potentiates hepatic clearance through complement receptors. Thus,

SAP deficiency leads to apoptotic debris accumulation, which activates preexisting autoreactive T and B cells. Second, SAP helps binding of C4b to chromatin and these complexes bind to bone marrow stromal cells by the C4b receptor. In the presence of SAP, immature autoreactive B cells acquire tolerance against chromatin through clonal deletion or anergy or both. In SAP deficiency, chromatin cannot be recruited to the bone marrow, which results in autoreactive B cell escape into the periphery and antigen-driven B cell expansion.

Boes et al. (2000) established mutant mice that cannot secrete IgM but that are able to express surface IgM and IgD, and to secrete other classes of immunoglobulins (Boes et al, 2000). They crossed these mutant mice with lupus-prone lymphoproliferative (lpr) mice and observed elevated levels of IgG autoan-tibodies to double-stranded DNA and histones, and increased deposition of immune complexes in the glomeruli. Furthermore, the absence of secreted IgM also resulted in accelerated development of IgG autoantibodies, even in normal mice. The accelerated autoantibody responses in mice deficient in secreted IgM resemble the effect of complement deficiency on the development of autoimmune disease. In humans and mice, deficiencies in the early components of the complement cascade, including Clq, C2, and C4, are associated with a high incidence of SLE (Kirschfink et al., 1993; Botto et al., 1998). The complement promotes the removal of autoantigens and, hence, could reduce the chance that autoreactive B cells are activated. Intravenous injection of syngeneic apoptotic cells into normal mice induces a rapid response against nuclear antigens (Mevorach et al., 1998b), suggesting that the source of autoantigens for the autoantibody response may be the apoptotic cells (Casciola-Rosen et al., 1994). C1q can bind apoptotic blebs directly, and the activation of complement is required for the clearance of apoptotic cells by macrophages (Mevorach et al., 1998a), suggesting a critical role of complement in the clearance of apoptotic cells.

Mer (a member of the Axl/Mer/Tyro3 receptor tyrosine kinase family)-deficient mice also possess high serum titers of anti-DNA antibodies, although this chapter does not mention whether they develop a pathological autoimmune response (Scott et al., 2001). Since Mer is required for the engulfment and efficient clearance of apoptotic cells, the defective clearance of chromatin in this deficient mouse may induce overt activation of autoreactive lymphocytes.

Hanayama et al. (2004) found that milk fat globule epidermal growth factor 8 (MFG-E8)-deficient mice develop glomerulonephritis as a result of autoantibody production (Hanayama et al., 2004). MFG-E8 binds to apoptotic cells by recognizing phosphatidylserine, and this binding enhances the engulfment of apoptotic cells by macrophages. This finding demonstrates that MFG-E8 has a critical role in removing apoptotic cells and thereby in the accumulation of self-antigens.

Taken together, the defective clearance of nucleosomal antigens can be a causative factor for the generation of anti-dsDNA antibodies, resulting in tissue damage. Since a significant number of autoreactive T cells against self-ligands including nucleosomal antigens are present in the normal body, I would like to postulate that the degree of T cell response against self-ligands is dependent on the duration or amount of TCR signaling as well as on the affinity of interaction between the TCRs and their ligands (Figure 20.1). Normally, because the TCR

Clearance Bacteria Apoptosis Cell

Self-antigens Non self-antigens

Figure 20.1. Accumulation of self-antigens triggers T cell autoreactivity. Mature T cells generally do not respond to self-antigens while proliferating against non-self antigens. However, defective clearance accumulation of self-antigens would allow mature T cells to reach the threshold for activation (black area).

Self-antigens Non self-antigens

Figure 20.1. Accumulation of self-antigens triggers T cell autoreactivity. Mature T cells generally do not respond to self-antigens while proliferating against non-self antigens. However, defective clearance accumulation of self-antigens would allow mature T cells to reach the threshold for activation (black area).

affinity is generally low and self-ligands are rapidly cleared from the body, the duration or amount of TCR signaling does not reach the threshold for activation of sufficient T cells to cause self-damage. However, when accumulation of self-ligands is abnormal, T cells are exposed to them for enough time or in amounts sufficient to induce the differentiation of pathological T cells.

4.2. Mechanisms of Accumulation of Self-Antigens in SLE

The issue of why lymphocytes reactive against nucleosomal antigens generally inside the cells are generated and induce autoimmunity deserves consideration. The role of anti-dsDNA antibodies in the pathophysiology of SLE remains unclear. Over the past decades, the antinuclear specificities of disease-associated autoantibodies have lured investigators to one of the likely sources of autoreactive stimulation in SLE. There are two possible explanations. The first is that cells (either inadvertently damaged [ultraviolet burns] or intentionally targeted [programmed cell death]) die, the condensed chromatin is broken down into nucleo-somes, and the nuclear envelope fragments within vesicles filled with nucleosomal components. This regimented process of apoptosis is often distinguished histologically by pyknotic nuclei and vacuolated cytoplasm. Casciola-Rosen et al. (1994) demonstrated that in the course of death by apoptosis, cells translocate the SLE autoantigens from the nuclear compartment to the cell surface, displaying them to the extracellular environment in membranous blebs, called "apoptotic bodies" (Casciola-Rosen et al., 1994). The physiologic display of nuclear antigens in apoptotic bodies offers a plausible mechanism whereby the autoantigens prominent in SLE are exposed to the immune system, possibly stimulating an autoimmune response.

Another explanation provided by DeGiorgio et al. (2001) is that a subset of anti-DNA antibodies cross-reacts with the N-methyl-D-aspartate (NMDA) subtype of glutamate receptors and induces neuronal cell injury (more generally, that anti-DNA antibody cross-reacts with self-antigens thereby eliciting tissue damage). However, the authors do not directly show that this autoantibody is really responsible for the development of the central nervous damage seen in SLE patients. It would also be very important to identify target antigens expressed in other organs that are recognized by anti-dsDNA antibodies.

A recent study showed that self-IgG2a-reactive B cells are activated by the recognition of immune complexes composed of IgG2a and nucleosomes by cell surface IgM and toll-like receptor (TLR) 9 present on B cells (Leadbetter et al, 2002, 2003). Autoreactive B cells are present in the lymphoid tissues of healthy individuals, but normally remain inactive. Over the decades, it has been demonstrated that to become fully activated, naive B cells must receive signals through Ig receptors and accessory receptors from helper T cells that recognize the same antigen (Fagarasan and Honjo, 2000). However, recent reports have indicated that various constituents of microbes can induce B cells to produce antibody in the absence of such helper T cells (Poeck et al., 2004). For example, lipopolysaccharide (LPS), a major constituent of the cell wall of Gram-negative bacteria, is recognized by a surface receptor present on all B cells, TLR4 (Poltorak et al., 1998). The simultaneous recognition of the LPS-containing bacteria by antigen receptors and TLR4 induces a synergistic signal, and triggers proliferation and production of antibodies by bacteria-specific B cells without T cell help (Poeck et al, 2004). Another toll-like receptor, TLR9, usually serves as a pathogen sensor by detecting unmethylated CpG base pairs that are more common in bacterial than in mammalian DNA, although unmethylated CpG base pairs are found in certain parts of mammalian genes. Leadbetter et al. (2002) have recently reported that immune IgG-nucleosome complexes could activate self-IgG-specific B cells through both the antigen receptor and TLR9, which indicates that TLR9 signaling facilitates the production of rheumatoid factors (Leadbetter et al., 2002). Immune complexes composed of nucleosomes or other self-antigens might have the same effect. Therefore, autoreactive B cells, which recognize nucleosomes directly or indirectly with the help of TLR9 signaling, may not need T cell help to proliferate and produce a variety of antibodies.

Regarding the role of TLR in the induction of autoimmune diseases, Eriksson et al. (2003) reported that dendritic cell activation through both CD40 and TLR4, but not CD40 alone, was needed to induce autoimmune disease in mouse recipients of dendritic cells pulsed with self-antigens. This result indicates that the activation of antigen-presenting cells through TLR plays a central role in the induction of autoimmune diseases. On the basis of these reports, we propose the following model to explain the role of antinucleosome-specific lymphocytes in the induction of SLE. Nucleosomal antigen accumulation due to the defect of DNASE1 increases the formation of immune complexes with methylated DNA and a variety of self-antigens. These immune complexes activate antigen-presenting cells (e.g., dendritic cells and B cells), which, in turn, greatly increase the sensitivity of autoreactive T cells to self-antigens as well as directly stimulate B cell production of autoantibodies.

4.3. Clearance of Self-Antigens as a Therapeutic Strategy

Macanovic et al. (1966) have reported that recombinant DNASE1 reduces the autoimmune response of lupus-prone mice. However, in clinical trials of recombinant DNASE1, the SLE symptoms and laboratory data of patients with SLE did not significantly improve (Davis et al., 1999). According to our studies, DNASE1 mutation could only be detected in 2 out of 100 SLE patients. Thus, it is important to determine whether treatment of recombinant DNASE1 is effective for SLE patients with DNASE1 mutation. Furthermore, in advanced SLE, T and B cells have many nucleosome specificities, whereas, initially, lymphocytes with only a single nucleosomal antigen specificity trigger the disease. Therefore, the treatment using recombinant DNASE1 may have a benefit only at a very early stage of SLE.

Regarding the therapeutic potential of nucleosomal antigens, Lu et al. (1999) reported that CD4+ T cells from lupus patients responded strongly to certain histone peptides (H2B10-33, H416-39, H471-94, H391-105, H2A34-48, and H449-63). These same peptides overlap with major epitopes for helper CD4+ T cells that induce anti-DNA autoantibodies and nephritis in lupus mice. The altered peptide ligands can induce anergy in T cells (Sloan-Lancaster et al., 1993, 1994; Ryan and Evavold, 1998). On the basis of this finding, Kaliyaperumal et al. (1999) tried to inhibit murine lupus using altered histone peptides and succeeded in partially suppressing SLE-like syndrome (Kaliyaperumal et al., 1999). The ability of each peptide associated with human SLE to affect T cell proliferation or cytokine secretion profiles is different, suggesting that each epitope plays a specific role in autoimmune T cell response induction or suppression. Thus, caution should be exercised when applying this technique in the clinic because it would be dangerous to inhibit T cell responses that are inhibiting SLE. Furthermore, because human HLA differs from person to person, it is very difficult to identify the altered ligands in each SLE patient. To overcome this problem, it is essential to establish therapeutic strategies that apply to all SLE patients.

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