Peggy Lymberi

Natural autoantibodies constitute a substantial part of normal circulating immunoglobulins.

According to Burnet’s clonal selection theory,’ in the early 1960s, explanations for tolerance and autoimmunity were attractively simple. Autoreactive lymphocytes were deleted during embryonic life to provide self-tolerance, and autoantibodies, always noxious, were the products of mutant forbidden clones that were able to circumvent this process of immunological homeostasis. However, during the last two decades, several groups succeeded in challenging clonal deletion as a general explanation for tolerance to self’ by: (a) Inducing autoimmune diseases by injecting organ extracts; (b) Demonstrating the presence of numerous autoantibodies in normal serum coming from a normal population;’ (c) Demonstrating the presence of normal autoreactive B cells? (d) Inducing in an animal numerous autoantibodies after challenging with mitogens. The existence of natural antibodies in normal human serum was first reported by Landsteiner in 1900, when he discovered the presence of natural hemagglutinins in serum directed against blood group determinants of the A-B-0 system. Accordingly, Boyden’ defined natural antibodies as a family of molecules present in the body fluids of normal animals that is able to specifically combine with antigens, but not with the immunologically acceptable molecules normally present in the body fluids. Nevertheless, natural antibodies directed against various antigens have been reported in different animal species and are often directed against various autoantigens. Obviously, a clear distinction between autoantibodies and natural antibodies is difficult to establish. During preparation of specific antisera against cytoskeletal proteins, we also detected the presence of natural antitubulin antibodies in the sera of normal humans and several species of normal nonimmunized animals: We subsequently isolated and characterized these antibodies, which were able to interact with autologous tubulin. For several years, we have been working on natural autoantibodies a t the Unit6 d’lmmunocytochimie of Pasteur Institute. The results of these studies are reported in the present work.

Studies on natural antibodiesand autoantibodies

Summary A serum pool from 800 healthy donors and individual samples from three healthy donors were passed through tubulin, actin, thyroglobulin, myoglobin, fetuin, transferrin albumin, cytochrome C and collagen immunoadsorbents. The proteins eluted were mainly composed of the three major Ig classes and were shown to bind specifically to the antigens via the F (ab′)2 fragment. When these isolated antibodies were examined by competitive assay, they fell into three groups. Antibodies in the first group, consisting of anti-tubulin and anti-thyroglobulin antibodies, were inhibited only by their homologous antigens. The second group was comprised of anti-actin, anti-myoglobin and anti-fetuin: these antibodies were inhibited mainly by their homologous antigens, but also to a significant degree by two or three additional antigens; although the reaction between anti-actin antibodies and immobilized actin was inhibited mainly by actin, both tubulin and thyroglobulin inhibited it to a lesser, but still significant extent. The third group consisted of anti-albumin, anti-transferrin, anti-collagen and anti-cytochrome C antibodies, which all bound specically to the antigens but were inhibited only slightly or not at all by their respective antigens. These results prompted us to study 612 monoclonal Ig (MIg) for their antibody activity against the above-mentioned antigens, plus myosin and double-stranded DNA (dsDNA). Of these 612 MIg, 36 (5.7 %) were shown to possess antibody activity. Of these 36, 32 (5.2 % of the total) were directed mainly against actin, but also reacted with tubulin and thyroglobulin, and sometimes with myosin. One MIg reacted mainly with myosin, but also with actin and tubulin, while the 3 others reacted with tubulin, thyroglobulin and dsDNA, respectively. The results produced by the fusion of splenocytes from non-immunized BALB/c mice with a non-secreting myeloma cell line indicated that a significant number of the induced clones produced monoclonal antibodies directed against the above panel of antigens, and that most of the clones obtained bound two or more antigens simultaneously. Three main conclusions emerged: o 1) the above results strongly suggest that natural antibodies againstthe antigens examined are present in normal human serum, and also indicate that natural antibodies directed against a great variety of antigens—often self antigens—might be present in normal serum; 2) at least in some patient sera, MIg seem to share antibody specificities with natural antibodies, and these MIg may consequently correspond to the expansion of a clone producing a natural antibody; 3) results obtained with normal unimmunized mice prove the existence of autoreactive Ig-synthesizing clones which bind to more than two antigens. The above points are discussed in connection with the role of naturalantibodies in terms of self tolerance and recognition, as well as their relationship to induced antibodies.

Sera with high levels of anti-smooth muscle and anti-mitochondrial antibodies frequently bind to cytoskeleton proteins.

Using ELISA methods, 54 sera from chronic active hepatitis (CAH) patients displaying high levels of anti‐smooth muscle antibodies (SMA) and 18 sera from primary biliary cirrhosis (PBC) patients with high levels of anti‐M2 antibodies were examined for the presence of high antibody levels against actin, tubulin, myosin, tropomyosin, troponin. vimenlin and desmin. Our results showed that: (i) in CAH with high SMA activity, increased antibody levels were found in 51‐9% of sera for actin, 31.5% for myosin, 35.2% for tubulin, 34.0% for tropomyosin, 11.3% for troponin. 22.6% for vimentin and 43.4% for desmin, compared with natural antibody levels in 21 normal sera; (ii) Similar high levels of these antibodies were found in the case of PBC; (iii) in most cases, sera simultaneously bound to several antigens of the panel; and (iv) approximately 26% of the CAH sera were found to be negative with the seven antigens examined while 22% were reacted with a cytoskeleton protein (CP) other than actin. These results indicate that current opinion associating SMA with anti‐actin activity in CAH is confirmed for only 50% of cases and that although a good correlation between SMA and anti‐CP antibodies can be obtained, there is still a significant percentage of SMA for which the putative antigen recognized needs to be determined.

Antibodies to acetylcholinesterase cross‐reacting with thyroglobulin in myasthenia gravis and Graves' disease

In the present study we analysed by ELISA the ability of sera from 50 patients with myasthenia gravis (MG), 20 with Hashimotos thyroiditis (HT), 53 with Graves’ disease (GD) and 36 healthy controls (CR) to react with acetylcholinesterase (AChE) from Electrophorus electricus and human thyroglobulin (Tg). Significantly increased anti‐AChE activity was exhibited by a high proportion of MG (IgG 36%) and GD (IgG 21%) sera, while increased anti‐Tg activity was detected in all three patient groups (MG, IgG 26% and IgA 26%; HT, IgG 85% and IgA 40%; and GD, IgG 51%). Interestingly, a significant proportion of MG and GD sera exhibited both IgG anti‐AChE and anti‐Tg activities (MG, 18%; P<0·001; and GD, 15%; P<0·001, versus CR, 0%). This bi‐reactivity was exhibited by anti‐AChE antibodies cross‐reacting with Tg (anti‐AChE/Tg activity); (i) serum anti‐AChE activity was effectively inhibited by soluble Tg, and (ii) affinity‐purified anti‐Tg antibodies cross‐reacted with AChE. Cross‐reactivity seems to be a property of pathological (auto) antibodies; induced (rabbit) antibodies to AChE or Tg were highly mono‐specific. Analysis of clinical data showed that increased IgG anti‐AChE/Tg activity was well associated with: (i) overlapping GD in MG (P<0·02), and (ii) ophthalmopathy in GD (P<0·01). In contrast, no correlation was noted in MG between anti‐AChE activity units and anti‐Tg activity units or acetylcholine receptor antibody titres. The clinical significance of anti‐AChE/Tg antibodies remains to be elucidated.

Increased Natural Autoantibody Activity to Cytoskeleton Proteins in Sera from Patients with Necrobiosis Lipoidica, with or without Insulin-Dependent Diabetes Mellitus

Necrobiosis lipoidica (NL), a skin disease, is associated with insulin-dependent diabetes mellitus (IDDM). Natural autoantibody (NAb) activity in sera from 16 patients suffering from NL, with or without IDDM, was compared to that in sera from 41 patients with IDDM and 43 healthy controls. Isotype-specific enzyme-linked immunosorbent assays (ELISAs) were used to detect NAbs against actin, myosin, keratin, desmin, troponin, tropomyosin, thyroglobulin, insulin, single-stranded DNA and the hapten trinitrophenyl. NAb activity was significantly higher in sera from patients with NL (either with or without IDDM), compared with that detected in sera from patients with IDDM which was similar to that of healthy individuals. High proportion of NL sera exhibited increased IgG anti-tropomyosin (69%), anti-troponin, anti-desmin and anti-keratin (50% each), anti-insulin (44%) and anti-trinitrophenyl (31%) activities, as well as increased IgA and IgM anti-keratin activities (26% and 31%, respectively). The great majority (88%) of positive sera were polyreactive and contained NAbs, polyspecific and monospecific (as demonstrated by immunoadsorption studies), belonging to more than one isotype; there was no predominant serological reactivity pattern. In conclusion, increased NAb activity to cytoskeleton proteins is associated with the dermatological disease NL and not to the overlapping autoimmune disease (IDDM). The origin and significance of these NAbs is discussed.

Natural autoantibodies in nude and normal outbred (Swiss) and inbred (BALBc) mice

Spleen cells from adult unprimed outbred (Swiss) and inbred (BALB/c) mice, either normal (no) or athymic-nude (nu) as well as spleen cells from Swiss nude mice bearing two different human tumors (BUR and PINQ), were fused with the mouse non-secreting myeloma cell line P3X63 Ag8-653. The supernatants of immunoglobulin secreting hybrids, all containing IgM, were screened for antibody activity against macromolecular antigens (autologous: actin, tubulin, myosin, dsDNA) and haptens (TNP, NP, NIP and NBrP). Furthermore, their idiotypic determinants were analyzed using a rabbit anti-idiotype which recognizes a major cross-reactive idiotype (IdD23) of BALB/c natural polyreactive autoantibodies. In all the mice studied, we identified: (1) hybrids reacting strongly with one or more haptens (10.7 to 37.8%) and (2) hybrids secreting natural monoclonal autoantibodies (NMoAb) with broad reactivities (polyreactive and/or oligoreactive) against autoantigens and/or haptens (11.4 to 26.8%). The results indicate that: (1) cells secreting natural autoantibodies with broad reactivities exist in both normal and nude mice, independently of the genetic background (inbred/outbred) of the mouse. However, in nude mice, the natural autoantibodies exhibit a more restricted pattern of reactivity (oligoreactive) compared to those of normal mice, and do not express the common idiotype IdD23 of natural polyreactive autoantibodies. (2) Tumors grafted into nude mice seem to induce the expression of polyreactive autoantibodies bearing the IdD23.

Decreased expression of HLA-DR antigens on monocytes in patients with multiple sclerosis

Immunofluorescence, cell binding assays and enzyme immunoassays were used to investigate the expression of class II major histocompatibility antigens on peripheral blood monocytes in 67 patients with multiple sclerosis. Monocytes from patients with active disease expressed fewer HLA-DR molecules on their surface than normal monocytes; furthermore the percentage of cells which exhibited detectable amounts of surface HLA-DR antigens was decreased in patients with active multiple sclerosis. During the inactive stage of the disease both deficiencies were milder, probably representing secondary pathogenetic phenomena. Quantitation of monocyte surface HLA-DR antigen expression could be valuable in assessing the clinical disease activity. The demonstration of a molecular defect in patients with multiple sclerosis will improve our understanding of the pathogenesis of the disease.

Fine specificity and subclasses of IgG anti-actin autoantibodies differ in health and disease

Current opinions suggest that autoantibodies occurring in autoimmune diseases are generated by B-cells which primarily produce polyspecific natural autoantibodies, through either polyclonal activation or specific antigen selection of these B-cells. In this study, we compared the immunological properties (polyspecificity, fine specificity and IgG subclasses) between natural anti-actin antibodies (N-AAA) and disease-associated AAA (D-AAA). IgG AAA from sera of healthy donors, patients with autoimmune hepatitis type 1 (AIH-1) and patients with primary biliary cirrhosis (PBC) were affinity-purified on actin immunoadsorbent and tested initially for polyspecificity against various cytoskeleton proteins by enzyme-linked immunosorbent assay (ELISA). Fine specificity was studied by Western blotting using proteolytic peptides of actin and by ELISA using synthetic 12 mer peptides, spanning the 221-377 aa sequence of actin. Results showed that both N-AAA and D-AAA are polyspecific. Nevertheless, D-AAA from both diseases showed a specific reactivity pattern as compared to N-AAA, against the 16 kDa C-terminal (229-377 aa) proteolytic peptide of actin and more specifically against the P36 synthetic peptide (351-362 aa). Quantitation of AAA IgG subclasses revealed that IgG1 and IgG3 were specifically increased in D-AAA from AIH-1 and PBC, respectively, as compared to N-AAA. We conclude that D-AAA are differentiated from N-AAA in terms of fine specificity and IgG subclasses, probably through specific antigen selection of B-cells primarily producing N-AAA.

Thyroxine-Binding Antibodies Inhibit T Cell Recognition of a Pathogenic Thyroglobulin Epitope

Thyroid hormone-binding (THB) Abs are frequently detected in autoimmune thyroid disorders but it is unknown whether they can exert immunoregulatory effects. We report that a THB mAb recognizing the 5′ iodine atom of the outer phenolic ring of thyroxine (T4) can block T cell recognition of the pathogenic thyroglobulin (Tg) peptide (2549–2560) that contains T4 at aa position 2553 (T4(2553)). Following peptide binding to the MHC groove, the THB mAb inhibited activation of the Ak-restricted, T4(2553)-specific, mouse T cell hybridoma clone 3.47, which does not recognize other T4-containing epitopes or noniodinated peptide analogues. Addition of the same THB mAb to T4(2553)-pulsed splenocytes largely inhibited specific activation of T4(2553)-primed lymph node cells and significantly reduced their capacity to adoptively transfer thyroiditis to naive CBA/J mice. These data demonstrate that some THB Abs can block recognition of iodine-containing Tg epitopes by autoaggressive T cells and support the view that such Abs may influence the development or maintenance of thyroid disease.

Serological detection of hepatitis B viral infection by a panel of solid-phase enzyme-linked immunosorbent assays (ELISA)

Immunoassays for the detection of hepatitis B virus (HBV) in biological samples were developed. Using recombinant HBV antigens (Ags) and HBV-specific antibodies (Abs), we designed and evaluated a panel of enzyme-linked immunosorbent assays (ELISAs) detecting the main hepatitis B-related viral markers, namely HBV surface Ag (HBsAg), HBV e Ag (HBeAg), Abs to HBsAg (anti-HBs), Abs to HBV core Ag (anti-HBc) and Abs to HBeAg (anti-HBe), in blood serum. The ELISAs were validated using a panel of prescreened, by commercial tests, serum samples. In principle, HBV Ags or anti-HBV monoclonal antibodies (mAbs) were immobilised on microplate wells. Horseradish peroxidase (HRP) or biotin were used to prepare labeled Abs. Specifically for the determination of HBsAg and HBeAg, two-site sandwich immunoenzymometric assays were developed. The useful range was estimated at 20-500 ng/ml and human serum samples assayed were diluted 10- and 4-fold for HBsAg and HBeAg, respectively, with phosphate buffered saline (PBS) containing Tween 20 and gelatin. For the detection of Abs to HBs an indirect ELISA was formulated. Sera were similarly 4-fold diluted in the same buffer. Finally, competitive ELISAs were used for detecting anti-HBc and anti-HBe and sera tested were diluted 20- and 5-fold, respectively. All selected dilutions resulted in the accurate and reliable determination of HBV Ags and anti-HBV Abs. Taken altogether, these ELISAs are highly specific and equally sensitive to the circulating tests. However, their design could be very useful for research and/or preclinical studies of selected HBV-infected individuals.