Autopolyreactivity Confers a Holistic Role in the Immune System.

In this review, we summarize and discuss some key findings from the study of naturally occurring autoantibodies. The B-cell compartment of the immune system appears to recognize almost all endogenous and environmental antigens. This ability is accomplished principally through autopolyreactive humoral and cellular immune receptors. This extended autopolyreactivity (1) along immunoglobulin gene recombination contributes to the immune system's ability to recognize a very large number of self and non-self constituents; and (2) generates a vast immune network that creates communication channels between the organism's interior and exterior. Thus, the immune system continuously evolves depending on the internal and external stimuli it encounters. Furthermore, this far-reaching network's existence implies activities resembling those of classical biological factors or activities that modulate the function of other classical biological factors. A few such antibodies have already been found. Another important concept is that natural autoantibodies are highly dependent on the presence or absence of commensal microbes in the organism. These results are in line with past and recent findings showing the fundamental influence of the microbiota on proper immune system development, and necessitate the existence of a host-microbe homeostasis. This homeostasis requires that the participating humoral and cellular receptors are able to recognize self-antigens and commensal microbes without damaging them. Autopolyreactive immune receptors expressing low affinity for both types of antigens fulfil this role. The immune system appears to play a holistic role similar to that of the nervous system.

Preferential recognition of the phosphorylated major linear B-cell epitope of La/SSB 349-368 aa by anti-La/SSB autoantibodies from patients with systemic autoimmune diseases.

Sera from patients with primary Sjögren Syndrome (pSS) or Systemic Lupus Erythematosus (SLE) often contain autoantibodies directed against La/SSB. The sequence 349-368 aa represents the major B-cell epitope of La/SSB, also it contains, at position 366, a serine amino acid residue which constitutes the main phosphorylation site of the protein. In this study we investigated the differential recognition of the 349-368 aa epitope and its phosphorylated form by antibodies found in sera from patients with systemic autoimmune diseases. Peptides corresponding to the sequence of the unphosphorylated (pep349-368 aa) and the phosphorylated form (pep349-368 aa Ph) of the La/SSB epitope 349-368 aa, as well as to a truncated form spanning the sequence 349-364 aa and lacking the phosphorylation site (pep349-364 aa), were synthesized. Sera from 53 patients with pSS and SLE with anti-La/SSB specificity, 30 patients with pSS and SLE without anti-La/SSB antibodies, 25 patients with rheumatoid arthritis and 32 healthy individuals were investigated by ELISA experiments. Autoantibodies to pep349-368 aa Ph were detected in sera of anti-La/SSB positive patients with a higher prevalence compared to the pep349-368 aa (66%versus 45%). Pep349-368 aa Ph inhibited the antibody binding almost completely (92%), while pep349-368 aa inhibited the binding only partially (45%). Anti-La/SSB antibodies presented a higher relative avidity for the phosphorylated than the unphosphorylated peptide. Immunoadsorbent experiments using the truncated peptide pep349-364 aa indicated that the flow through showed a selective specificity for pep349-368 aa Ph, while the eluted antibodies reacted with both peptide analogues of the La/SSB epitope. These data suggest that sera from pSS and SLE patients with anti-La/SSB reactivity possess autoantibodies that bind more frequently and with a higher avidity to the phosphorylated major B-cell epitope of the molecule.

Changes in the cytokine profile of lupus-prone mice (NZB/NZW)F1 induced by Plasmodium chabaudi and their implications in the reversal of clinical symptoms.

We have previously observed that aged lupus-prone (NZB/NZW)Fl (BWF1) mice when infected with Plasmodium chabaudi show an improvement in their clinical lupus-like symptoms. In order to study the mechanisms involved in the long-lasting protective effect of the P. chabaudi infection in lupus-prone mice we analysed specific aspects of the cellular response, namely the profiles of cytokine mRNA expression and cytokine secretion levels in old BWF1 mice, in comparison with uninfected age-matched BWF1 mice and infected or uninfected BALB/c mice. Two months after infection, cells from BWF1 mice were stimulated with concanavalin A (Con A) and demonstrated a recovery of T cell responsiveness that reached the levels obtained with BALB/c cells. Old BWF1 mice showed high levels of interferon-gamma (IFN-gamma) and IL-5 production and correspondingly low levels of IL-2 and IL-4 secretion before infection with P. chabaudi. Infection did not modify the IFN-gamma levels of BWF1 T cells, whereas it considerably increased the secretion of the Th2-related cytokines IL-4, IL-5 and IL-10. In addition, only BWF1 T cells showed increased mRNA expression of tumour necrosis factor-alpha (TNF-alpha) and transforming growth factor-beta (TGF-beta). This counter-regulatory cytokine network of infected BWF1 mice may be involved in the improvement of their lupus symptoms. The results of our investigations using the complex model of P. chabaudi infection can be extended and, by using more restricted approaches, it may be possible to explain the multiple regulatory defects of lupus-prone mice.

Bulimia nervosa and autoimmunity.

The autoantibodies that react with dopamine and serotonin are of interest in the study of bulimia nervosa. These neurotransmitters play an important role in appetite control, sexual and social behavior, and stress responses, all of which form a part of the clinical picture of bulimia nervosa. Are these autoantibodies involved in the serotoninergic hypofunctioning present in bulimia nervosa? Are they a part of an immunity regulation system essential for the cerebral system's homeostasis? To address these questions, 31 bulimic females (diagnosed according to DSM-III-R criteria) were compared with 10 control subjects (matched to the patients for sex, age, and demographic/psychosocial features). Measurement of the activity of natural autoantibodies reacting with dopamine, dopamine-beta-hydroxylase and serotonin was performed by an enzyme-linked immunosorbent assay (ELISA) for typical immunoglobulins (IgG, IgM, IgA). All of the autoantibodies of the IgG type were lower in the bulimic group than in the control group, a difference that was statistically significant for IgG anti-serotonin and IgG anti-dopamine. There was a trend for the amount of IgM anti-dopamine to be lower in patients than in controls. Dopamine and serotonin are specific components of brain cells. It can therefore be hypothesized that these antigens acting with autoantibodies could be the antigenic cerebral targets reacting with 'anti-brain' antibodies. The study of these specific autoantibodies provides information about the immunological characteristics that may be related to brain disturbances.

Natural antibodies in childhood: development, individual stability, and injury effect indicate a contribution to immune memory.

Natural, often autoreactive antibodies are present in normal sera in large quantity and show alterations in specificity in diverse pathological situations. They have, however, usually not been studied longitudinally. Here we investigated some representative serum reactivities of natural antibodies in 67 normal children and 10 with injury during childhood, followed up for 3 years. Normal children showed an individually characteristic and relatively stable level of most IgM, IgG, and IgA reactivities when measured with ELISA by reference to a standard. Injured children showed some very rapidly enhanced reactivities within 3 days after trauma, which thereafter slowly diminished over years before coming back to a normal level. This period exceeds by far the lifetime of antibodies and plasma cells. We conclude that natural antibodies contribute to the establishment and maintenance of immune memory in a manner that is distinct from classical immune reactions.

A peptide derived from a polyreactive monoclonal anti-DNA natural antibody can modulate lupus development in (NZBxNZW)F1 mice.

In lupus-prone (NZBxNZW)F1 (B/W) mice, elevated levels of polyreactive autoantibodies bearing the D23 idiotype (Id), characteristic of natural antibodies, were detected before and after the appearance of pathological anti-DNA antibodies. While these D23 Id+ antibodies were able to regulate anti-DNA antibodies in the early stage of the disease, we found that during disease evolution they had lost their normal ability to regulate anti-DNA antibodies and furthermore could participate in the lupus-like syndrome. To explore further the role of the D23 Id+ antibodies, we injected young B/W mice with a peptide corresponding to the VH CDR3 region of the D23 monoclonal natural antibody (mNAb). High levels of monospecific antipeptide, as well as polyreactive antibodies, were induced. Among them, the most markedly enhanced antibody population was DNA-reactive immunoglobulin G1 (IgG1). Compared with controls, these immunized mice had a delayed 50% survival rate and proteinuria developed later. Furthermore, IgG1 able to react with IgG2a anti-DNA monoclonal antibodies derived from B/W mice were also produced after peptide immunization. Thus, a peptide corresponding to the CDR3 of the D23 mNAb antibody might play a role in the regulation of murine lupus.

Natural antibody status in patients with Hashimoto's thyroiditis.

Raised levels of circulating natural antibodies (NABS) have been found in association with systemic lupus erythematosus (SLE) and chronic active hepatitis (CAH), indicative of polyclonal B-cell activation associated with these relatively non-organ specific autoimmune diseases. This study examined the natural antibody response in the organ-specific autoimmune disease Hashimoto's thyroiditis. Serum samples obtained from 69 women with newly diagnosed Hashimoto's thyroiditis together with 64 controls were analysed for IgG and IgM NABS directed at DNA, actin, myoglobin, myosin, trinitrophenyl hapten (TNP) and tubulin as the NAB antigen panel using an established ELISA. The same technique was also used to estimate thyroglobulin and thyroid microsomal autoantibody activities. Compared to a reference panel of normal serum samples, 31 of the Hashimoto's samples showed a greater than 2SD elevation of IgG and/or IgM NABS against one or more of the panel antigens together with elevated IgG thyroglobulin and thyroid microsomal antibody levels. The cases positive for one or more of the NAB panel also showed a greater incidence of active Hashimoto's thyroiditis as indicated by the presence of antibodies directed against the thyroid specific antigens. The above findings suggest that raised levels of NABS are also a feature of this organ-specific autoimmune disease. The wide ranging NAB specificities involved are consistent with an underlying or epiphenomenal state of polyclonal B-cell activation.

Immunochemical, structural and translocating properties of anti-DNA antibodies from (NZBxNZW)F1 mice.

Many monoclonal antibodies (mAb) derived from the spleens of (NZBxNZW)F1 mice react strongly with dsDNA and also other self antigens, although more weakly. When added to cell cultures, these polyreactive anti-DNA mAb penetrate into various cell types and accumulate in the nucleus within a few hours. Almost all anti-DNA mAb bind to cell membrane antigens but the extent of their binding does not directly correspond to their penetration capabilities. Sequence analysis of anti-DNA mAb indicated the use of various germ-line VH families. The complementary-determining regions (CDR) 3 differ but they all contain a relatively high number of tyrosines and positively charged amino acids (lysine and arginine). Haptens (biotin, fluor-escein, oligonucleotides) and macromolecules (peroxidase, IgG) covalently coupled to the mAb or their F(ab')2 and Fab fragments were translocated through the cytoplasm and into the cell nucleus. Furthermore, 61% of peripheral blood lymphocytes were labelled when mice were injected with fluorescein-labelled mAb. Peptides corresponding to CDR2, CDR3 and CDR2 linked to CDR3 (CDR2-3) of several penetrating mAb were prepared and their intracellular translocating capacity was assessed. The CDR2-3 peptide, but not the individual peptides, was able to penetrate cells and could be used as a vector to transport macromolecules. Although all CDR2-3 reacted with dsDNA and other self antigens, each one exhibited a distinct polyreactivity profile.

Immunoglobulin double isotype-producing hybridomas isolated from an autoimmune (NZB x NZW)F1 mouse.

In the sera of (NZB x NZW)F1 (B/W) mice that develop a lupus-like syndrome, increased levels of IgG antibodies (Ab) reacting with TNP have been detected before the appearance of IgG anti-DNA Ab and clinical symptoms. A single injection of trinitrophenyl-bovine serum albumin (TNP/BSA) in physiological saline into a young B/W mouse (3 months old), followed by fusion of its splenocytes 3 days later, gave rise to hybridomas simultaneously secreting IgM and IgG Ab with anti-TNP reactivity. Both mu and gamma chains were detected in culture supernatants by ELISA, and double isotype-producing cells were labeled by immunofluorescence. Molecular analysis of two of these double isotype-producing hybridomas showed the presence of mRNA coding for both mu and gamma chains of Ig, and this gamma mRNA could be translated in vitro into a gamma heavy (H) chain. Comparison of the H chain variable-region sequences of IgM and IgG revealed 100% homology between mu and gamma V(H) genes in one clone, while mu and gamma V(H) genes showed only 80% homology in the other clone. Both clones produced a single kappa light (L) chain. These two hybridomas, isolated from a B/W mouse, thus represent two different mechanisms of double isotype expression: the first one corresponds to an IgM to IgG switch, while the second one reflects a lack of allelic exclusion.

Effect of amino acid substitutions in the heavy chain CDR3 of an autoantibody on its reactivity.

In this study, we applied site-directed mutagenesis to the Fab fragment of a mouse IgM (IE12) that was previously shown to inhibit the binding of IgG to autoantigens by interacting with their variable regions. Its native structure was very similar to that of a polyreactive natural IgM (ppc15-30). Indeed, they both use the same light chain and the same VH, D and JH segments. However, the N regions differ and the D is translated in two different reading frames, giving different amino acid compositions of the heavy chain CDR3 (HCDR3). Site-directed mutagenesis modified the HCDR3 of IE12 compared to that of the natural antibody and the resulting effects on its reactivity were analyzed. Because the HCDR3 of IE12 is very rich in aliphatic residues, which are hydrophobic, we replaced them with the more hydrophilic residues of the HCDR3 of the polyreactive IgM. In addition, we evaluated the impact of the proline residues in the HCDR3 of IE12 on its activity, because they are known to restrict backbone flexibility. We found that a more hydrophilic HCDR3 conferred to the IE12 Fab a polyreactive profile. Prolines seem to play an important role in this context, because when they were replaced by glycines, the resulting Fab fragments were highly polyreactive. Our results suggest that, for polyreactivity, hydrophilicity and a certain plasticity of the HCDR3 seem to be necessary. Greater flexibility of the CDR, particularly the HCDR3, might be an important characteristic for polyreactive antibodies.

Polyreactive anti-DNA monoclonal antibodies and a derived peptide as vectors for the intracytoplasmic and intranuclear translocation of macromolecules.

Naturally occurring polyreactive anti-DNA mAbs derived from a nonimmunized (NZB x NZW)F1 mouse with spontaneous lupus erythematosus penetrated and accumulated in the nuclei of a variety of cultured cells. These mAbs and their F(ab')2 and Fab' fragments, covalently coupled to fluorescein, peroxidase, or a 15-mer polynucleotide, also translocated to the cell nuclei. A 30-amino acid peptide corresponding to the combined sequences of the complementary-determining regions 2 and 3 of the heavy chain variable region of one mAb was able to penetrate into the cytoplasm and nucleus of cells of several lines. This peptide recognized DNA and was strongly polyreactive. Streptavidin-peroxidase conjugates complexed with the N-biotinylated peptide were rapidly translocated into cells. Similarly, peroxidase or anti-peroxidase polyclonal antibodies covalently coupled to the N-cysteinylated peptide through an heterobifunctional maleimide cross-linker were also rapidly internalized and frequently accumulated in nuclei. The peptide carrying 19 lysine residues at its N-terminal was highly effective in transfecting 3T3 cells with a plasmid containing the luciferase gene. Thus, penetrating mAbs and derived peptides are versatile vectors for the intracellular delivery of proteins and genes.

[Bulimia and autoimmunity]. / Boulimie et auto-immunité.

In the first part of this study, we investigated the rate of natural autoantibodies, in a sample of 31 female inpatients with bulimia nervosa according to DSM III-R criteria. The control (age and sex matched) group consisted in high school students including 10 females without eating disorders, depressive disorder or immunological disease. We investigated especially natural autoantibodies reacting with compounds of the central nervous system (Dopamine, Dopamine beta Hydroxylase, Serotonin). Our first conclusion is that there is a lower level of these natural auto-antibodies among female patients with bulimia nervosa. In the second part of the study, we have especially investigated the correlation between impulsivity in bulimia nervosa and the rate of natural autoantibodies against serotonin.

Natural polyreactive secretory immunoglobulin A autoantibodies as a possible barrier to infection in humans.

Secretory immunoglobulin A (S-IgA) was investigated in human secretions for the presence of natural antibodies (Abs) acting as the first "immune barrier" to infection before induction or boosting of specific responses. These molecules could be the secretory counterpart of the natural Abs in serum that were previously shown by our laboratory to be polyreactive to autoantigens. Significant levels of S-IgA Abs to human actin, myosin, tubulin, and spectrin were detected in 10 saliva and 8 colostrum samples from normal subjects. Computer-assisted analysis of immunoblots of extracts from human muscles showed these Abs to react with a large number of autoantigens. Their polyreactivity was confirmed by cross-inhibition and by immunoblotting studies of affinity-purified natural Abs, assayed against a large variety of surface or secreted antigens from Streptococcus pyogenes. The thiocyanate elution method showed that functional affinities of some natural Abs can be of the same order of magnitude as those of tetanus vaccine antitoxins. Moreover, nonimmune binding of these natural Abs to the gut protein Fv (Fv-fragment binding protein) can enhance their effector functions. This demonstrates that human secretions contain polyreactive auto-Abs which can also react with pathogens. These secretory Abs of "skeleton key" specificities are possibly produced by a primordial B-1-cell-associated immune system and can be involved in a plurispecific mucosal protection against pathogens, irrespective of the conventional immune response.

Effect of temperature on the reactivities of polyreactive and monospecific monoclonal IgG antibodies.

In this study, the reactivities of two groups of murine monoclonal antibodies (mAbs) of the IgG isotype were compared by ELISA at various temperatures (Ts) (range: 4-56 degrees C). The first group was constituted of 4 polyreactive mAbs that reacted with various antigens (Ags), such as actin, myosin and tubulin. The second contained 3 commercially available monoreactive mAbs specific to actin, myosin or tubulin. The binding of the monospecific mAbs to their Ags was modified only slightly at the other Ts compared with binding at 37 degrees C. In contrast, the activities of the polyreactive IgGs were considerably modified depending upon the T during incubation on Ag. In a second series of experiments, the effects of the T of the washes and conjugate incubation on the mAb/Ag interaction were evaluated. In these experiments, all steps and incubations were carried out at 37 or 4 degrees C. The values were then compared to those obtained when the washes and conjugate incubation were performed at room temperature. These two protocols generated very little difference in terms of monospecific mAbs. For polyreactive IgG, the values were generally lower when the incubations were carried out at 4 degrees C. However, on the whole, the effect of the T of the washes and conjugate incubation was negligible when the mAb/Ag complex had already formed on the polystyrene plate. Furthermore, at 4 degrees C, 2 of the polyreactive mAbs behaved like monospecific antibodies, while the other 2 remained polyspecific. It can be concluded from these experiments that the reactivities of polyreactive mAbs are more T-sensitive than those of monoreactive mAbs. This seems to indicate that they may possess a more plastic structure and thus may more easily undergo deformation when subjected to non-physiological conditions. In addition, the fact that the polyreactive mAbs showed the same variations for the 3 Ag tested suggests that the same paratope could be involved in all reactions.

Isotype and antibody specificity of spontaneously formed immunoglobulins in pig fetuses and germ-free piglets: production by CD5- B cells.

Pig fetuses, colostrum-deprived newborns and germ-free (GF) piglets, animals in which B-cell development is not influenced by maternal regulatory factors, were employed to study the occurrence and specificity of natural antibodies (NAb). Serum immunoglobulins of all isotypes were found in 44-day-old fetuses (the gestation period in pigs lasts 114 days) and their level, with predominating IgM, was increased during fetal ontogeny. In sera of fetuses at the end of embryonic life as well as of newborns and older GF piglets, antibody activity against autoantigens (thyroglobulin, hormones, ssDNA), phylogenetically conserved proteins (myosin), haptens (trinitrophenyl; TNP) and bacterial components (Escherichia coli O86, tetanic anatoxin) was detected by enzyme-linked immunosorbent assay. The antigen-biding activity of IgM NAb increased after isolation of the serum immunoglobulins on a Staphylococcus Protein A (SPA)-Sepharose column. IgM reactivity similar to that detected in serum was found in supernatants from polyclonally stimulated cultures of spleen of 8- and 12-day-old GF piglets. Pig fetal liver IgM+ B cells, which were able to produce IgM after polyclonal stimulation, did not express the CD5 molecule. Our results indicate that pig preimmune repertoire is comparable to that described in humans and mice, although in contrast to these species pig B-1 cells do not express CD5.

Beneficial effect of Salmonella typhimurium infection and of immunoglobulins from S. typhimurium-infected mice on the autoimmune disease of (NZB x NZW) F1 mice.

Various infections can precede or aggravate autoimmune diseases. Yet a beneficial effect of infection has also been described an various mechanisms have been postulated to explain this effect. The aim of this study was to examine the hypothesis that infection can have an immunoregulatory effect on the autoimmune process via the increased production of natural polyreactive antibodies. The effect of Salmonella typhimurium infection on the lupus-like disease of (NZB x NZW)F1 (B/W) mice was therefore studied. The effect of IgM and IgG preparations isolated from the serum of S. typhimurium-infected C57B1/6 and CBA mice on the autoimmune disease of B/W mice was also tested. C57B1/6 and CBA mice were chosen because they are respectively genetically susceptible and resistant to S. typhimurium infection and they differ in their antibody response during the early phase of infection. CBA mice can mount a specific anti-bacterium antibody response, whereas C57B1/6 mice present increased production of polyreactive antibodies. The infection effect was evaluated on several disease parameters, i.e. survival, incidence of high grade proteinuria and serum IgM and IgG antibody activity directed against a panel of autoantigens. Our main findings were: (i) infection of B/W mice with an attenuated strain of S. typhimurium delayed the course of the autoimmune disease when performed before the appearance of autoimmune symptoms; and (ii) IgM and IgG preparations from S. typhimurium-infected C57B1/6 mice had a similar effect whereas the IgM and IgG preparations from infected CBA mice, as well as from normal C57B1/6 and CBA mice, were ineffective. These results suggest that S. typhimurium infection can beneficially influence the development of the autoimmune disease of B/W mice. The immunoregulatory effect of the infection seems to be related at least partially, to the increase of a particular population of antibodies, the polyreactive antibodies.

Differences in the natural autoantibody patterns of patients with schizophrenia and normal individuals.

The expression of IgG and IgM autoantibodies directed against various autoantigens, either part of the central nervous system or not, was investigated in the sera of inpatients with schizophrenia (n = 10). An enzyme immunoassay was used to measure the levels of these autoantibodies in whole sera, IgG-depleted sera, and isolated IgG fractions. IgG and IgM antibodies, reacting with all the antigens tested, were present in the sera of patients with schizophrenia as well as in the sera of normal individuals. Among patients suffering from schizophrenia, IgM natural autoantibody reactivities could be higher (myoglobin, serotonin, tubulin), lower (dopamine), or even identical to those of normal individuals, depending on whether whole or fractionated sera were assayed and on the group of patients with schizophrenia (responders and nonresponders) considered. The isolated IgG fractions of patients suffering from schizophrenia had higher anti-DNA and antiserotonin reactivities than those detected in normal individuals.

Characterization of autoantibody activities in sera anti-DNA antibody and circulating immune complexes from 12 systemic lupus erythematosus patients.

To examine autoantibodies present in patients with active systemic lupus erythematosus (SLE), sera, circulating immune complexes (CIC), and antibodies purified on DNA-immunoadsorbent were tested by enzyme immunoassay. A panel of self-antigens, including DNA, histones (HIS), glomerular basal membrane (GBM), thymus cell extract (TCE), actin (ACT), myosin (MS), and tubulin (TUB), was used to define their specificities. IgM antibodies against all antigens of the panel were detected in sera, CIC, and in antibodies eluted from the DNA-immunoadsorbent and demonstrated a large polyreactivity. IgG antibodies showed restricted activities against DNA, HIS, GBM, and TCE in sera and a large polyreactivity in CIC. Inhibition experiments were performed to assess their mono- or polyreactivities. Among the IgG autoantibody population recognizing DNA, two populations of IgG antibodies were detected in the sera and in the affinity purified anti-DNA: one recognizes DNA, HIS, and GBM, and the other binds to DNA and to cytoskeletal proteins. These autoantibody populations were found in CIC, which also often contained high amounts of IgG antibodies recognizing ACT and MS. A third population of IgG antibody that recognizes only TCE and could not be inhibited by DNA or other antigens was found in serum and CIC. Our data demonstrate the existence of several populations of autoantibody in serum and CIC of SLE patients: (1) IgM polyreactive autoantibodies, (2) IgG polyreactive autoantibodies recognizing DNA and cytoskeletal proteins, (3) IgG specific to DNA, which cross react with HIS and GBM, and (4) IgG specific to TCE antigens.