Brigitte Guilbert

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.

Enzyme-immunoassay for the measurement of antigens using peroxidase conjugates

Summary Human and rat IgG were labelled with peroxidase. Procedures were established using these conjugates and insoluble antibody immunoadsorbents for the quantitation of humoral IgG. The basic principles of these procedures are those already developed for quantitative radio-immunoassay, enzyme activity measurement substituting radioactivity counting. The procedures allowed the determination of 10–200 ngs of antigen. The values obtained in unknown samples using these enzyme-immunoassays were in good agreement with those obtained using the quantitative single radial immunodiffusion technique.

Comparative study of natural autoantibodies in the serum and cerebrospinal fluid of normal individuals and patients with multiple sclerosis and other neurological diseases

Using a panel of antigens (actin, myosin, tubulin, albumin, transferrin, peroxidase, thyroglobulin, DNA, prolactin, TNP and myelin basic protein (MBP], we have tested the antibody activity of serum and cerebrospinal fluid (CSF) from healthy individuals, patients with multiple sclerosis (MS) and individuals with other neurological diseases. No differences in the concentrations and specificities of the serum antibodies were observed among the 3 groups. In contrast, we found that MS patients often had elevated CSF antibody levels against many antigens of the panel. The MS patients with local immunoglobulin production in the central nervous system (CNS) had the highest antibody levels. Restricted antibody activity against a given antigen of the panel was not observed. Compared to the two other groups, the MS group had equivalent titres of anti-MBP antibodies in the CSF. These results suggest that, in MS, a general immune dysregulation exists which leads to a local expansion of B lymphocytes producing autoantibodies with reactivities similar to those of serum natural autoantibodies.

Characteristics of Epstein-Barr virus transformed B cell lines from patients with Alzheimer's disease and age-matched controls

The characteristics of B cell lines isolated from patients with Alzheimers disease (AD) and age-matched controls were investigated after having been transformed by Epstein-Barr virus (EBV). After isolation of mononuclear blood cells and in vivo or in vitro EBV infection, 35 and 21 lymphoblastoid cell lines (LCLs) were generated from 19 patients with AD (mean age 79.4 years) and 21 age-matched controls (mean age 80.0 years), respectively. B lymphocytes from AD patients were immortalised more easily than those from controls; the percentage of in vitro EBV infected LCLs (B95-LCLs) obtained in the AD group was significantly higher (76.2% versus 33.3% in the control group) and the mean time required for establishment was significantly lower (20.2 and 21.9 days versus 26.7 and 60.9 days in the control group). The EBV receptor and surface immunoglobulin (Ig) analyses showed no difference between the two groups. The expression of Epstein-Barr early antigens (EA) and viral capsid antigens (VCAs) revealed a tendency to higher viral replication in LCLs from AD patients; however, VCA expression remained limited to a small number of cells and did not affect overall cell growth. Finally, qualitative and quantitative differences were observed in the pattern of Ig production. Whereas spontaneously established LCLs from AD patients were generally monoclonal (80% of LCLs versus 33% in the control group), B95-LCLs were all polyclonal and secreted more IgM and IgA than those from controls; the mean IgM level was significantly higher in B95-LCLs from the AD group. These results suggest that B cells derived from AD patients seemed to be less differentiated than cells from age-matched controls.

Studies on active immunization with self antigens. I: Production of antibody to unmodified proteins by neonatal immunization

Newborn BALB/c mice were repeatedly injected either with syngeneic (BALB/c) or xenogeneic (bovine) myosin, albumin, or actin in sterile physiological saline. The serum antibody response was evaluated by enzyme immunoassay 1 and 2 months alter birth and after two booster injections. At 1 month, higher antibody titres were found in the sera of mite injected with syngeneic than with xenogeneic antigens. At 2 months and after boosting, anti‐syngeneic actin antibodies were present in equal or higher amounts, anti‐syngeneic albumin antibodies were not detected, and anti‐syngeneic myosin antibodies were considerably decreased. Antibodies produced after booster injections of syngeneic actin were found to be highly specific and to belong mainly to the IgG isotype. These results suggest that newborn mice are better able than adult mice to respond to stimulation with self antigens, and that administration of self proteins during neonatal life may lead to the induction of immunological memory. They also indicate that one of the primary functions of the immune system in newborn mice is the recognition of self antigens.

Anti-tubulin antibodies in rabbits before and after immunization with pig tubulin

Sera from rabbits before and after repeated injections of pig tubulin in complete Freunds adjuvant were examined for antibody activity against pig and rabbit tubulins and against a panel of antigens: actin, myosin, DNA, TNP/BSA. Antibody activity against all the antigens of the panel (PAg) increased moderately after the first but not after subsequent injections. Antibody activity against pig and rabbit tubulins strongly increased after the second immunization when the maximum was reached. Isolation of anti-tubulin antibodies from normal or immune sera on tubulin-immunoadsorbent demonstrated the presence of three different antibody populations: (1) polyspecific IgM reacting with the PAg and the tubulins, present in substantial amounts in normal sera and moderately increased in immune sera; (2) small amounts of polyspecific IgG detected only in immune sera; (3) high amounts of specific IgG reacting with pig and rabbit tubulins, present in immune but not normal sera. Western blot analysis of the specific IgG population showed that it contained antibodies reacting with both native pig and rabbit tubulins, as well as antibodies recognizing only the 30,000 proteolytic fragment of pig, but not that of rabbit tubulin. The results indicate that the immunization of rabbits with heterologous tubulin induced specific IgG anti-tubulin antibodies which recognize the self and non-self antigens differently.

The Epstein-Barr virus-induced production of IgE by human B cells.

B cells, isolated from the blood of healthy individuals and patients allergic to pollen, produced IgE when exposed to the human B-cell polyclonal activator, Epstein-Barr virus (EBV) in vitro and placed in culture. Secreted IgM and IgE were measured using immunoenzymatic assays. No difference was seen between healthy donors and allergic patients in the amount of IgE (or IgM) secreted. Cells were placed in limiting dilution cultures in order to determine the frequency of cells producing IgE or IgM (total and pollen specific) on exposure to EBV. Again, no significant differences in EBV-driven, B-cell precursor frequencies (PF) were seen between normal and allergic individuals. EBV-driven B-cell PF for total IgM and IgE, and pollen-specific IgM and IgE secretion, were 1/450, 1/6500, 1/83,000, and less than 1 per 2,500,000, respectively, for cells from healthy donors, and 1/140, 1/4000, 1/56,000 and less than or equal to 1 per 2,000,000, respectively, for cells from allergic patients. We propose that the increased IgE levels seen in atopic individuals result solely from regulatory defects, rather than an increase in the frequency of B cells committed to the secretion of IgE.

Studies on active immunization with self antigens. II. Production of antibody related to hapten substitution.

BALB/c mice were injected during neonatal life with conjugates in buffered physiological saline, prepared by coupling trinitrophenyl groups (TNP) at various densities to either syngeneic mouse serum albumin (TNP MSA) or xenogeneic bovine serum albumin (TNP USA). Serum samples were obtained on days 30 and 60 after birth, mid on days 75 and 88 after two booster injections, and monoclonal antibodies were prepared from spleens of neonatally treated mice. The antibody titres, isotypes, and specificities were evaluated by enzyme‐immunoassay. It was found that the extent of the anti‐TNP immune response to TNP MSA conjugates depends on the degree of hapten substitution, which is not the case for the anti‐TNP BSA. All the TNP‐MSA conjugates induced mainly IgG and only a few IgM antibodies. These antibodies reacted essentially with the TNP group but seemed to have a higher avidity for the TNP protein conjugate used in their induction. During the course of the immunization, decreasing quantities of TNP‐MSA conjugates were needed to inhibit antibody binding. A large amount of monoclonal anti‐TNP antibodies was found in hybridomas obtained after neonatal treatment either with TNP‐MSA or TNP‐BSA. Therefore, it appears that the anti‐TNP‐immune response obtained after antigenic stimulation with sufficiently substituted TNP‐MSA conjugates possesses all the characteristics of a normally occurring humoral immune response.

Régulation of the humoral immune response by polyspecific natural autoantibodies

Two different BALB/c IgMk polyspecific monoclonal natural autoantibodies E7 and D23 were administered to neonatal BALB/c mice. When adults, these mice were immunized and challenged with calf myosin, BALB/c actin, human transferrin, calf thymus DNA or TNP-coupled bovine serum albumin (TNP/BSA), in complete Freunds adjuvant. The levels of serum antibody were evaluated by enzyme immunoassay. No differences in anti-actin, anti-transferrin and anti-DNA antibody titres were noted between control and antibody-treated mice. However, anti-myosin antibody titres significantly increased in mice treated with either the E7 or D23 antibody, and anti-TNP antibody titres significantly decreased in mice treated with E7 but not with D23. These differences persisted after antigenic challenge and involved only the IgG response of treated mice. These results suggest that polyspecific natural autoantibodies may be involved in the regulation of the humoral immune response.