Mechanisms of action of intravenous immunoglobulin in autoimmune and inflammatory diseases.

Intravenous immunoglobulins (IVIg) exert a broad range of immunoregulatory functions that provide a basis for the beneficial effects of IVIg in autoimmune and systemic inflammatory disorders. This review focuses on the effects f IVIg on humoral and cellular immunity that may be of relevance for the treatment of inflammatory neurological diseases.

[Treatment of a septic patient with acquired haemophilia]. / Traitement d'une hémophilie acquise chez un patient septique.

INTRODUCTION: Acquired haemophilia is a rare bleeding diathesis caused by auto-immune depletion of factor VIII. It is characterised by spontaneous haemorrhagic syndrome, which can be fatal sometimes. EXEGESIS: A 71 year-old man presents in a dysimmunitary context (rheumatoid arthritis complicates by an acquired haemophilia) a septicemia with a methicillin resistant staphylococcus aureus. At the time of the hospitalization, the patient is febrile (39 degrees C). The activated partial thromboplastin time is very much increased, the level of factor VIII is lowered by 7% and the title of the inhibitor to factor VIII amounts to 140 Bethesda unities. An haematoma of the right root thigh is also noted. In that case, the concomitant presence of septicemia makes difficult the use of immunosuppressive therapy usually recommended to decrease auto-antibody's level. For the management of the septicemia, an adapted antibiotherapy (vancomycin then teicoplanin) is organized to J1. To control haemorrhagic risk, immunoglobulins are prescribed from d12 to d16, without immediate results. Then prednisone is introduced. We observe a very fast decrease of the anticoagulant circulating title with a neat improvement of the clinical state, allowing so to realize a draining puncture of the psoas. This invasive investigation required the use of prothrombinic complex concentrates ((Feiba) in the dose of 80 UI/kg two to three times a day). Biopsy does not show infection source. CONCLUSION: The infection delayed the prescription of immunosuppressive therapy and the surgery. Use of corticoids, following 5 days of intravenous polyvalent immunoglobulin, was the good choice. After 7 weeks of hospitalization the patient has recovered a normal haemostasis results, and a good general state.

Normal IgG protects against acute graft-versus-host disease by targeting CD4(+)CD134(+) donor alloreactive T cells.

Intravenous immunoglobulin (IVIg) was shown to decrease the severity of acute graft-versus-host disease (aGVHD) in recipients of allogeneic bone marrow transplants. To investigate the mechanisms involved in the protective effect of IVIg, we have used the parent-into-F1 model in which parental lymphocytes are transferred into semi-syngeneic non-irradiated F1 rats. Here we report that IVIg, as well as F(ab')(2) fragments of IVIg, protected (Lewis x Brown-Norway) F1 rats against aGVHD induced by a single injection of Lewis lymphocytes. IVIg was given as five consecutive daily injections, starting on the day preceding that of the transfer of Lewis cells. Protection was associated with a decreased ability of lymphocytes to spontaneously proliferate and to produce NO and IFN-gamma, in the absence of an increased production of IL-10. We further demonstrate that protection was associated with a decrease in CD4(+) T cells bearing the activation marker CD134 in vivo, and with an enhanced apoptosis of activated CD4(+) T cells by IVIg, in vitro. Our observations suggest that the prevention of aGVHD by IVIg in this model is mediated by the induction of apoptosis of activated alloreactive CD4(+)CD134(+) donor T cells. The results further emphasize the role of normal immunoglobulin in modulating alloantigen immune responsiveness.

Immunoglobulin VH-gene usage of autoantibodies in mercuric chloride-induced membranous glomerulopathy in the rat.

Brown-Norway (BN) and Dorus Zadel Black (DZB) rats develop a T-cell-dependent membranous glomerulopathy (MGP) with high proteinuria and antiglomerular basement membrane (GBM) autoreactive antibodies (Abs), upon exposure to mercuric chloride (HgCl2). Laminin is an important autoantigenic target of the anti-GBM Abs, absorbing approximately 30% of the anti-GBM reactivity. Although many anti-GBM Abs have undergone isotype switching, it is currently unclear whether affinity maturation occurs during the HgCl2-induced autoimmune response. To address this question we analysed the rearranged immunoglobulin heavy chain variable-region genes (VHDJH regions) of 15 mAbs that were previously obtained from HgCl2-treated rats. Seven of these mAbs exhibit reactivity towards laminin. Our study showed that the VH-gene usage of antilaminin mAbs is largely restricted to the PC7183 VH-gene family (six out of seven). In addition, we demonstrated that at least three out of six laminin reactive and five out of six non-laminin-binding mAbs are encoded by germline VH genes (a total of eight out of 12 mAbs). Of the eight mAbs that are encoded by germline VH genes, seven are of a non-immunoglobulin M (IgM) isotype, indicating that isotype switching has occurred in these mAbs in the absence of somatic mutations. The mutations observed in the VH genes of the four remaining mAbs do not provide strong evidence for antigenic selection. The data support the notion that B cells in this model of MGP are not subjected to affinity maturation and probably result from polyclonal B-cell activation.

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.

Neonatal induction of tolerance to T(h)2-mediated autoimmunity in rats.

Brown-Norway (BN) rats are highly susceptible to drug-induced immune dysregulations and when injected with mercuric chloride (HgCl(2)) or sodium aurothiopropanolsulfonate (ATPS), they develop a syndrome characterized by a polyclonal B cell activation depending upon CD4(+) T(h)2 cells that recognize self-MHC class II molecules. Since peripheral tolerance of T(h)2 cells might be crucial in the prevention of immunological manifestations such as allergy, establishing conditions for inducing tolerance to HgCl(2)- or ATPS-mediated immune manifestations appeared to be of large interest. We report here that BN rats neonatally injected with HgCl(2): (i) do not develop the mercury disease, (ii) remain resistant to HgCl(2)-induced autoimmunity at 8 weeks of age and later, provided they are regularly exposed to HgCl(2), (iii) are still susceptible to ATPS-induced immune manifestations, and (iv) exhibit spleen cells that adoptively transfer tolerance to HgCl(2)-induced autoimmunity in naive, slightly irradiated, syngeneic recipients. These findings demonstrate that dominant specific tolerance can be neonatally induced using a chemical otherwise responsible for T(h)2-mediated autoimmunity.

Mechanisms of action of intravenous immunoglobulin (IVIG).

Therapeutic preparations of pooled normal polyspecific immunoglobulin G for intravenous use (intravenous immunoglobulin, IVIG) have been shown to be effective in the treatment of a number of autoimmune and systemic inflammatory conditions. IVIG prevents the occurrence of experimental autoimmune encephalomyelitis. Increasing evidence suggests that IVIG is of benefit in patients with relapsing-remitting multiple sclerosis. The present review discusses the immunoregulatory properties and mechanisms of action of IVIG in autoimmune disease.

Grasping at molecular interactions and genetic networks in Drosophila melanogaster using FlyNets, an Internet database.

FlyNets (http://gifts.univ-mrs.fr/FlyNets/FlyNets_home_page.++ +html) is a WWW database describing molecular interactions (protein-DNA, protein-RNA and protein-protein) in the fly Drosophila melanogaster. It is composed of two parts, as follows. (i) FlyNets-base is a specialized database which focuses on molecular interactions involved in Drosophila development. The information content of FlyNets-base is distributed among several specific lines arranged according to a GenBank-like format and grouped into five thematic zones to improve human readability. The FlyNets database achieves a high level of integration with other databases such as FlyBase, EMBL, GenBank and SWISS-PROT through numerous hyperlinks. (ii) FlyNets-list is a very simple and more general databank, the long-term goal of which is to report on any published molecular interaction occuring in the fly, giving direct web access to corresponding s in Medline and in FlyBase. In the context of genome projects, databases describing molecular interactions and genetic networks will provide a link at the functional level between the genome, the proteome and the transcriptome worlds of different organisms. Interaction databases therefore aim at describing the contents, structure, function and behaviour of what we herein define as the interactome world.

Normal immunoglobulin G protects against experimental allergic encephalomyelitis by inducing transferable T cell unresponsiveness to myelin basic protein.

Normal human IgG for intravenous use (IVIg), administered intraperitoneally, protected Lewis rats against experimental allergic encephalomyelitis (EAE) induced by immunization with myelin basic protein (MBP). We demonstrate that protection was associated with an acquired unresponsiveness of lymphocytes to MBP and a decreased ability of the cells to produce IL-2, IFN-gamma and TNF-alpha and, to a lesser degree, IL-4 and IL-10, in the presence of the antigen. Lymph node (LN) cells of protected rats failed to passively transfer EAE to naive syngeneic animals. Our observations indicate that, rather than inducing selective immune deviation, IVIg induces preferential MBP unresponsiveness of Th1 cells. Whereas LN and splenic cells of IVIg-treated rats did not proliferate nor secrete IL-2 in the presence of the antigen, proliferation was restored by adding exogeneous recombinant IL-2. In contrast, LN cells of IVIg-treated rats proliferated normally and produced IL-2 in the presence of concanavalin A, indicating the selectivity for MBP of the anergy induced by IVIg when given at the time of immunization with the antigen. Treatment with IVIg also allowed a resistance to the secondary induction of EAE, indicating that IVIg protects from EAE but does not interfere with the processes that eventually lead to resistance to re-challenge. These data document the immunomodulatory effects of IVIg in T cell-dependent experimental autoimmune disease and further suggest a role for normal Ig in the selection of functional T cell repertoires.

FlyNets and GIF-DB, two internet databases for molecular interactions in Drosophila melanogaster.

GIF-DB and FlyNets are two WWW databases describing molecular (protein-DNA, protein-RNA and protein-protein) interactions occuring in the fly Drosophila melanogaster (http://gifts.univ-mrs.fr/GIFTS_home_page.html ). GIF-DB is a specialised database which focuses on molecular interactions involved in the process of embryonic pattern formation, whereas FlyNets is a new and more general database, the long-term goal of which is to report on any published molecular interaction occuring in the fly. The information content of both databases is distributed in specific lines arranged into an EMBL- (or GenBank-) like format. These databases achieve a high level of integration with other databases such as FlyBase, EMBL, GenBank and SWISS-PROT through numerous hyperlinks. In addition, we also describe SOS-DGDB, a new collection of annotated Drosophila gene sequences, in which binding sites for regulatory proteins are directly visible on the DNA primary sequence and hyperlinked both to GIF-DB and TRANSFAC database entries.

Pooled normal human polyspecific IgM contains neutralizing anti-idiotypes to IgG autoantibodies of autoimmune patients and protects from experimental autoimmune disease.

Normal human serum contains IgM antibodies that regulate the natural autoantibody activity of IgG in autologous serum. In the present study, we show that pooled normal human IgM (IVIgM) purified from plasma of more than 2,500 healthy donors and processed in a similar fashion to that of therapeutic preparations of pooled normal human IgG (IVIg) suppresses activity of IgG autoantibodies purified from the serum of patients with autoimmune diseases in vitro. The inhibitory effect of IVIgM was greater or equivalent to that of IVIg on a molar basis. We show that IVIgM contains anti-idiotypic antibodies directed against idiotypic determinants of autoantibodies, in particular by showing that Sepharose-bound IVIgM selectively retained F(ab')2 fragments of IgG autoantibodies. The infusion of (Lewis x Brown-Norway) F1 rats with IVIgM protected the animals against experimental autoimmune uveitis induced by immunization with the soluble retinal S antigen, as evidenced by clinical scoring and histopathological analysis. The present findings provide a rationale for considering pooled IgM for immunomodulation of autoimmune disease.

A shift in encephalitogenic T cell cytokine pattern is associated with suppression of EAE by intravenous immunoglobulins (IVIg).

Pooled human polyspecific IgG preparations for intravenous use (IVIg) have been used in a number of antibody mediated autoimmune diseases and recently in some T cell mediated disorders including multiple sclerosis, birdshot retinopathy and rheumatoid arthritis. Furthermore, IVIg has been proven beneficial in the corresponding animal models, i.e. experimental autoimmune encephalomyelitis (EAE), experimental autoimmune uveoretinitis and adjuvant arthritis respectively. The exact mechanisms for IVIg action in T cell mediated disorders are still poorly understood. There is evidence that IVIg treatment in vitro and in vivo decreases or changes the kinetics of the secretion by normal PBMC of a number of cytokines and anti-proliferative effect of IVIg on T cells in vitro and in vivo has also been reported. It remains unclear though to what extent the IVIg effects in T cell mediated autoimmunity are related only to non-specific T cell suppression and whether it also reshapes the autoimmune T cell cytokine profile. In this study we demonstrate that IVIg protects against EAE and that this beneficial effect is associated with a decreased proliferation of T cells specific for the immunizing antigen. Moreover, we show that these antigen-specific cells produce low amount of Th1-type cytokines and transfer an attenuated EAE.

Evidence for heterogeneous TCR V beta repertoire expression in mercury-induced immune disorders in rats.

Administration of subtoxic doses of HgCl2 affects differentially the immune system depending on the strain of rats tested. Susceptible Brown-Norway (BN) rats exhibit a CD4+ T cell-dependent polyclonal activation of B cells; in contrast, Lewis (LEW) rats are resistant and develop an immunosuppression mediated by CD8+ T cells recruited by CD4+ T cells. The mechanisms by which mercury induces immune disorders are poorly understood. We were interested in analyzing the diversity and mercury-mediated changes of the TCR Vbeta repertoire in the BN and LEW strains of rats at different times of HgCl2 exposure. Our results obtained after analysis of lymph node T cells by RNase protection assay, flow cytometry or immunoscope assay (i) were not consistent with a superantigen-like stimulus since we observed neither a V beta-selective expansion nor deletion that would have been expected and (ii) showed that in BN rats, as well as in LEW rats, an increase in the number of T cells was associated with the heterogeneous TCR V beta repertoire, thus supporting a polyclonal T cell activation. However, in BN rats the total number of T cells increased very rapidly, whereas in LEW rats only CD8+ T cells accumulated.

GIF-DB, a WWW database on gene interactions involved in Drosophila melanogaster development.

GIF-DB (Gene Interactions in the Fly Database) is a new WWW database (http://www-biol.univ-mrs.fr/ approximately lgpd/GIFTS_home_page. html ) describing gene molecular interactions involved in the process of embryonic pattern formation in the flyDrosophila melanogaster. The detailed information is distributed in specific lines arranged into an EMBL- (or SWISS-PROT-) like format. GIF-DB achieves a high level of integration with other databases such as FlyBase, EMBL and SWISS-PROT through numerous hyperlinks. The original concept of interaction databases examplified by GIF-DB could be extended to other biological subjects and organisms so as to study gene regulatory networks in an evolutionary perspective.

Th1/Th2 cytokine gene expression after mercuric chloride in susceptible and resistant rat strains.

Mercuric chloride (HgCl2) has contrasting effects on different rat strains: susceptible strains, e.g. Brown Norway (BN) develop polyclonal B cell activation, multiple autoantibodies and widespread tissue injury. Lewis (LEW) rats are resistant: no autoimmune response occurs after HgCl2; instead, there is immunosuppression. We have previously shown, by fully quantitative polymerase chain reaction (PCR), up-regulation of interleukin-4 (IL-4) gene expression in HgCl2-treated BN rats, implicating Th2 cells in the autoimmune syndrome. Involvement of the reciprocal Th1 subset, producing interferon-gamma (IFN-gamma), in resistance of LEW rats to HgCl2 has been suggested. We now report extensive analysis of Th1 and Th2 cytokine gene expression in spleen and lymph nodes of susceptible (BN) and resistant (LEW) rats after HgCl2. IL-4 and IFN-gamma were analyzed by quantitative PCR, other cytokines were assessed using semiquantitative PCR: the relative merits of these two techniques are discussed. We show pronounced up-regulation of IL-4 and more modest up-regulation of IFN-gamma in BN rats, but no up-regulation of either in LEW rats. Baseline levels of IFN-gamma were higher in Lew rats. Semiquantitative PCR showed increased expression of IL-2, IL-6 and IL-10 in BN; in LEW rats only IL-10 was increased. There was no marked change in IL-5, IL-13 or transforming growth factor-beta (TGF-beta) in either strain. These data further support the key role of IL-4 in HgCl2-induced autoimmunity, and suggest that failure of up-regulation of IL-4, together with higher baseline IFN-gamma expression, accounts for resistance of LEW rats to HgCl2. However, neither IFN-gamma nor TGF-beta can be implicated in HgCl2-induced immunosuppression in the LEW rat in vivo: our data suggest a role for IL-10 in this phenomenon.

Recombinant IL-4 aggravates experimental autoimmune uveoretinitis in rats.

IL-4 is the critical regulatory cytokine that preferentially promotes a Th2 type of immune response. In certain models of organ-specific autoimmune diseases in which Th1 cells are implicated in the disease process, treatment with IL-4 has been shown to confer protection by deviating the immune response toward a Th2 type. In this study, we addressed the role of IL-4 in experimental autoimmune uveoretinitis, a prototypic Th1-dependent disease induced in susceptible animals following immunization with soluble retinal Ag. Interestingly, treatment of Lewis rats with IL-4 exacerbated experimental autoimmune uveoretinitis, and simultaneous treatment with neutralizing anti-IL-4 Abs attenuated this increase in the severity of the disease. Ex vivo analysis of cytokines produced in response to the immunizing Ag showed an enhancement in the levels of IFN-gamma, TNF-alpha, and nitric oxide following IL-4 treatment. In vitro, IL-4 augmented the production of IFN-gamma by Con A-stimulated splenocytes in a dose-dependent manner. At low concentrations of IL-4, IFN-gamma production was enhanced, while at higher concentrations this production was inhibited. The specificity of the induction of IFN-gamma by IL-4 was confirmed by neutralizing the activity of IL-4 with anti-IL-4. Taken together, the results herein reported demonstrate that IL-4 can induce the production of IFN-gamma and of inflammatory cytokines under certain conditions, and indicate that IL-4 can exert a dose-dependent differential effect on the induction of immune responses and on autoimmunity.

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.

Monoclonal antibodies to human pancreatic procolipase: production and characterization by competitive binding studies.

Hybridomas secreting monoclonal antibodies (MAbs) specific for human pancreatic colipase were established and 11 clones were selected by using a dot immunobinding assay. Characterization of the MAbs was carried out by using direct and competitive epitope mapping methods, including ELISA and inactivation of colipase-dependent pancreatic lipase. Monoclonal antibodies showed four distinct patterns of reactivity. Monoclonal antibody 5.30 (group I) inhibited colipase-dependent lipase activity. The dissociation constant of the inactive antibody-antigen complex was 10(-9) M. Monoclonal antibodies 48.30, 66.24, and 153.23 (group II) had no effect on activity although they bound competitively with MAb 5.30 to antigen as shown by their capacity to displace MAb 5.30 from the antibody-antigen complex and by ELISA additivity test. Dissociation constants calculated from the displacement curves were 0.9 10(-9) M, 0.6 10(-9) M, and 2 10(-9) M, respectively. Noninhibitory MAbs 13.29, 16.25, and 33.30 bound competitively with MAbs of group II but not with MAb 5.30 (group I). Monoclonal antibodies of group IV (MAbs 17.6, 18.1, 37.39, and 169.29) had no effect on activity and did not react with immobilized antigen. None of the MAbs reacted in ELISA with reduced and carboxymethylated human procolipase, indicating that epitopes involved conformationally dependent determinants on protein antigen. Anti-human colipase MAbs showed no cross-reactivity with porcine or equine procolipases. Monoclonal antibodies described here appear to be useful tools for studying surface hydrophobic domain of colipase and/or interaction between colipase and lipase in its active conformation (open lid).