Blanche Bellon

Mercuric chloride induced autoimmune disease in Brown-Norway rats: sequential search for anti-basement membrane antibodies and circulating immune complexes.

Abstract. Mercuric chloride induces in the Brown‐Norway rat a biphasic autoimmune disease characterized initially by linear IgG deposits along the glomerular basement membrane followed later by granular IgG deposition. In the present study, anti‐glomerular basement membrane antibodies and immune complex‐like material were sequentially assessed in serial serum samples. Both were transiently found at the same period. Glomerular linear IgG deposits were present on day 11 but circulating anti‐glomerular basement membrane antibodies were only found later on day 16. Circulating immune complexes were first detectable on day 8 before the earliest granular IgG deposits were first observed in the spleen vessels on day 16. The disappearance of circulating anti‐glomerular basement membrane antibodies and of circulating immune complexes, although HgCl2 injections were pursued, is in agreement with the self‐limited character of mercuric chloride induced autoimmune disease and suggests the induction of immunosuppressive mechanisms.

Regulatory CD8+ T Cells Control Neonatal Tolerance to a Th2-Mediated Autoimmunity

Exposure of newborn animals to a foreign Ag may result in immunological tolerance to that specific Ag, a phenomenon called neonatal tolerance. We have previously reported that neonatal administration to Brown-Norway rats of mercury, a heavy metal toxicant, induces a dominant tolerance, specific for the chemical otherwise responsible for Th2 cell-mediated autoimmune responses in this susceptible strain of rats. Neonatal exposure to Ags can prime immunity, rather than inactivate or delete responses, and sustain regulatory functions effective against autoreactive T cells. Here, we address whether such a tolerant response is due to the generation of regulatory cells. The results suggest that the CD8+ T cell subset is involved in neonatal tolerance to mercuric salt-induced Th2 autoimmune disease. Thus, we demonstrate that in vivo CD8 depletion breaks tolerance following mercury recall in animals under a neonatal tolerance protocol. Furthermore, adoptive cotransfer of splenocytes from naive and tolerant rats as well as transfer of CD8+ T cells from tolerant animals prevent naive syngeneic rats from developing pathologic Th2 immune responses. These observations indicate that CD8+ T cells are endowed with regulatory functions in neonatal tolerance and mediate active suppression. Moreover, neonatal tolerance induced the expansion of CD8+CD45RChigh T cells and the emergence of a high percentage of IFN-γ-synthesizing CD8+ T cells, which probably reflects the implication of regulatory Tc1 cells. Thus, in vivo induction of neonatal tolerance suppresses Th2 autoimmune responses via generation of a CD8+ cell-mediated regulatory response.

Increased expression of class II major histocompatibility complex molecules on B cells in rats susceptible or resistant to HgCl2-induced autoimmunity.

Administration of HgCl2 to the susceptible Brown‐Norway (BN)rais induces an autoimmune disease characterized by a T‐dependent polyclonal activation of B cells responsible for a dramatic increase in serum IgE concentration. The resistant Lewis (LEW) rats injected with HgCi2. do not exhibit such autoimmune manifestations. We show here that, upon HgCl2 injections, major histocompalibility complex (MHC) class II molecule expression is increased very early in lymph nodes and spleen B cells from both strains. So far. it is the earliest marker (day 3)of the effect of HgCI2on the immune system. In both strains this enhancement is transient, but regulatory mechanisms arc much more efficient in the resistant LEW strain than in the susceptible BN strain, In addition, we observed that MHC class II molecule expression on B cells differs according to the organ and the rat strain tested. All these findings are discussed in an attempt to underline the role of MHC class II molecule expression in the occurrence of mercury‐induced stutoimmunity.

Permeability of the normal rat brain, spinal cord and dorsal root ganglia microcirculations to immunoglobulins G

The distribution of blood-borne immunoglobulins G (IgG) was studied in the cerebral cortex, pineal gland, spinal cord and dorsal root ganglia of normal Lewis rats using the detection of autologous anti-horseradish peroxidase (HRP) antibodies. This detection was performed by means of light and electron microscopy. This study demonstrated that, in the cerebral cortex and the spinal cord microcirculations, endothelial cells are a restrictive barrier against IgG while IgG are able to diffuse into the perivascular parenchyma of the pineal gland and spinal ganglia.

Induction in mucosa of IgG and IgA antibodies against parenterally administered soluble immunogens.

The induction of a mucosal immunity provides an additional principle of vaccination by preventing the entry of pathogens in the body. Albeit the fact that intensive research has been conducted on local vaccines, the major mucosal vaccine commercially available for human use remains the oral polio vaccine. We have previously demonstrated that parenteral vaccination in humans with tetanus toxoid (TT) results in a genital immunoglobulin (Ig)G antibody (Ab) response. Here, we show that injections of TT with no adjuvant induces an anti‐TT response in the mucosal tissues of normal BALB/c mice. The response is multiregional, involves both IgG and IgA isotypes, and is long‐lasting. Similarly, injections of haptens coupled to TT or to other diffusible proteins may induce mucosal Abs. These results led us to immunize normal BALB/c mice with a viral peptide coupled to TT by disulfide bridging. The hapten was a 17 amino acid peptide containing the ELDKWA sequence of human immunodeficiency virus (HIV)‐1 gp41. A significant IgG and IgA Ab response to the immunizing peptide was induced in various mucosal tissues despite the presence of a suboptimal Ab response in the spleen. The results indicate that mucosal immunity to peptides that are candidates for human vaccinations may be achieved by parenteral adjuvant‐free immunization with peptide coupled to TT.

Anti‐Interleukin‐2 Receptor Monoclonal Antibody Therapy Supports a Role for Thl‐Like Cells in HgCl2‐Induced Autoimmunity in Rats

Brown‐Norway (BN) rats injected with HgCl2 develop an autoimmune disease characterized by a T dependent polyclonal B‐cell activation. Increase in major histocompatibility complex class II molecule expression on B cells concomitant with enhancement of serum IgE concentration supports the involvement of the T helper 2 (Th2)‐like subset in the induction of the disease. The mercury disease is autoreguiated and does not develop in Lewis (LEW) rats. Considering the reciprocal regulation, well defined in mice, between the Th1 and Th2 subsets, we addressed the role of the Thl‐like subset in this disease. Brown‐Norway and LEW rats injected with HgCl2 were treated wilh NDS61, a mouse anti‐ral‐IL‐2R MoAb that blocks mainly Th1 cells. Data reported herein show that: (1) HgCl2 treatment does not modify either the percentage of IL‐2R+ cells or IL‐2R expression in both BN and LEW rats; (2) treatment of BN rats with NDS61 MoAb does not modify the induction phase of the mercury disease but delays in parl the regulation phase; (3) such a treatment leads lo some immune abnormalities in LEW rals; (4) HgCl2 markedly potentiates the anti‐mouse Ig antibody response in BN rats which probably limits the effect of this treatment.

IgG Fc membrane receptor on normal human glomerular visceral epithelial cells.

This study demonstrates that human glomerular epithelial cells are able to bind heat aggregated immunoglobulins and antigen-antibody complexes. This has been observed on kidney cryostat sections, on whole glomeruli and on cultured visceral epithelial cells. Binding depends on the presence of the Fc portion of IgG and occurs in the absence of complement, showing that the IgG Fc receptor is different from the C3b receptor. The use of heat aggregated anti-peroxidase IgG and of peroxidase anti-peroxidase complexes allowed us to demonstrate, at the ultrastructural level, that the binding of the reagents at the plasma membrane was followed by their internalization within coated pits of vesicles. These observations strongly suggest that glomerular visceral epithelial cells are capable of receptor mediated endocytosis. The role of this process in glomerular diseases remains to be established.

Increase in polymerized liver tubulin during stimulation of hepatic plasma protein secretion in the rat

Involvement of hepatic microtubules in plasma protein secretion by the liver was investigated by stimulating protein secretion in rat liver and then measuring the different forms of tubulin. Total and free tubulin were estimated in liver supernatants by the [3H] colchicine-binding assay. Polymerized tubulin, assumed to reflect the presence of microtubules, was calculated from the difference between total and free tubulin. To enhance liver plasma protein secretion, an acute inflammatory reaction was induced in one group of rats and a nephrotic syndrome in another. In both cases, total liver tubulin increased significantly compared to normal animals, but free tubulin was unchanged. Accordingly, polymerized tubulin rose by 50% during the inflammatory reaction and by 90% during the nephrotic syndrome. These results support the hypothesis that hepatic microtubules are involved in plasma protein secretion by the liver and also suggest that enhanced secretion requires additional microtubules.

Neonatal tolerance to a Th2-mediated autoimmune disease generates CD8+ Tc1 regulatory cells.

Immunological tolerance can be achieved in animals by exposure of newborn to a foreign antigen. Depending on the dose and timing of the antigenic challenge, tolerance has been reported to result in clonal deletion, anergy or active suppression. In this latter case, regulatory T cells prevent autoimmunity by suppressing the reactivity of pathogenic self-reactive T cells. We have previously reported the generation of a neonatal, mercury-specific, and dominant tolerance to autoimmunity induced by mercury salts in rats. Chronic exposure to mercury salts can lead to SLE-like autoimmune responses, mediated by autoreactive CD4+ Th2 cells, that regulate and are followed by a resistant state mediated by protective CD8+ T cells. The aim of the study was to compare the resistance to the neonatal tolerance to mercury disease, and to further characterize the CD8+ T cells endowed with regulatory capacity in the neonatal tolerance model. We report here that resistance to mercury disease is long lasting and not mercury-specific, suggesting that different CD8+ T cells are involved in resistance and neonatal tolerance, and that regulatory CD8+ Tc1 cells generated in tolerance are required to control the CD8- cell population from developing Th2-mediated autoimmunity. Upon mercury recall, CD8+ CD45RC(high) T cells, that represent the Tc1 subset in the rat, expanded and were polarized towards IFNgamma production. Interestingly, identical results were obtained with the CD8+ CD25+T cell population. Substantial amounts of FasL gene expression were detected in CD8+ T lymphocytes upon recall with the tolerogen. AICD may be one of the regulatory mechanisms used by these regulatory CD8+ Tc1 cells that control neonatal tolerance to a Th2-mediated autoimmune disorder.

Mechanism of allergic cross-reactions. V: High incidence of unanticipated cross-stimulation by natural allergens of rat basophilic leukemia cells sensitized with monoclonal IgE antibodies

The incidence of cross-stimulations by natural allergens was investigated using RBL-2H3 cells sensitized with five different mouse monoclonal anti-DNP IgEs and four mercury-induced rat monoclonal IgEs. Cells sensitized with 3 of the 5 monoclonal anti-DNP IgEs (clones SPE-7, SRT-1, LB4) responded by serotonin release upon stimulation by natural allergens such as Dermatophagoides pteronyssinus, horse dander and mugwort extracts. Serotonin release could be inhibited by monovalent DNP-lysine, indicating the involvement of DNP-binding sites of IgEs. Two of the clones (LO-DNP-30 and LA2) were negative on all tests with allergens. All but one (Hg32) of the mercury-induced rat IgE monoclonal antibodies tested positive with DNP-BSA, and with at least one of the six allergen extracts. IgE clone Hg12 mediated serotonin release with 5 of the 6 allergens tested.