Presence Of Antigen-antibody Complexes Research Articles

Membranous nephropathy: genetics, antigens and antibodies

Membranous nephropathy is a kidney disorder characterized by thickening of the glomerular basement membrane, that causes nephrotic syndrome. It can be caused by various underlying conditions that result in damage to the filtering units of the kidneys, known as nephrons, producing massive proteinuria, hypoalbuminemia, edema and hyperlipidemia. Between 30 to 40% of cases of nephrotic syndrome in adults are due to membranous nephropathy. In recent decades, progress has been made with the discovery of antigens, antibodies and genes involved in the pathophysiology of the disease and a new classification system has been proposed. The presence of antigen-antibody complexes together with genetic factors may influence the susceptibility to such immune dysregulation, and states new information in a what was known between the etiologies of primary and secondary causes. The understanding of the antigens involved in membranous nephropathy is an area of active research, and additional antigens may be identified as our knowledge of the disease continues to evolve. This article summarizes some concepts and recent findings made on this topic.

Dot-immunobinding assay (Dot-Iba).

Dot-immunobinding assay (Dot-Iba) is a simple and highly reproducible immunodiagnostic method. Antibody or antigen is dotted directly onto nitrocellulose membrane (NCM) discs. The diagnostic material to be checked can be incubated on this disc. Presence of antigen-antibody complex in NCM discs can be directly demonstrated with enzyme-conjugated antiglobulins and substrate. Development of a purple-pink colored, insoluble substrate product in the nitrocellulose membrane will be considered a positive result in the assay. This assay allows the processing of multiple specimens at a time and the entire operational procedures required only 4-6 h. Dot-IBA is rapid and the technical steps involved in the assay are much simpler than the other immunoassays such as enzyme-linked immunosorbent assay in detecting circulating antigen and antibody in clinical samples. The Dot-Iba showed an overall sensitivity of 60 % for tuberculous meningitis diagnosis and no false positive results were encountered. Hence this assay is highly specific for the diagnosis of paucibacillary diseases like extrapulmonary tuberculosis. Dot-Iba is best suited to laboratories in developing world where there are constraints in laboratory resources.

Presence and distribution of antigen-antibody complexes in the herniated nucleus pulposus.

Herniated tissue was studied by immunohistochemistry in eight patients with lumbar disc herniation. The results were compared with those of control subjects. To assess the presence and distribution of possible antigen-antibody complexes in herniated disc tissue. It has been suggested that the nucleus pulposus may be recognized as a foreign-body by the immune system and that this will lead to secondary nerve root disturbance. Such immunologic events should be initiated by binding of antibodies to a specific antigen in the disc tissue. However, the presence of antigen-antibody complexes in the herniated disc tissue has not been assessed. Amplification of the peroxidase reaction produced in avidin-biotin-peroxidase complex immunostaining by diaminobenzidine was used to visualize antigen-antibody complexes in the herniated tissue. The authors used herniated tissue from eight patients with lumbar disc herniation and nucleus pulposus from five control subjects with nonlumbar disc herniation. Thin paraffin sections, prefixed in 4% paraformaldehyde, were incubated with anti-human IgG antibody to allow visualization of antigen-antibody complexes in the specimens. A brown deposit, indicating antigen-antibody complexes, could be observed in the pericellular capsule in herniated disc tissue but not in control discs or in the residual discs of the herniation patients. Antigen-antibody complexes seem to be commonly present in herniated disc tissue, but not in healthy discs. However, the pathophysiologic and clinical significance of this observation has to be elucidated further.

Mediators of antigen-induced gastrin release: role of antigen-antibody complexes and the complement system.

That orally administered antigen was shown to induce gastrin release in immunized animals was a new aspect of gastrointestinal physiology. The mediators responsible for this immunological effect are still unclear. In an attempt to discover more about the mechanisms regarding antigen-induced gastrin release, we developed an in vitro system where fragments of rat antral mucosa were challenged. This makes it possible to determine the role of antigen-antibody complexes and the complement system in the mechanism of antigen-induced gastrin release. Wistar rats were immunized in vivo with NIP-OVA and mucosal fragments were challenge, in vitro with NIP-HGG. Gastrin was determined after a preincubation and a challenged incubation period without supernatants. After antigenic challenge, supernatants were used for in vitro challenge in order to rule out the presence of a soluble mediator and activation of complement. In a second group of experiments Wistar rats were used to study in vitro the release of specific antibodies after antigenic challenge. With this experimental design we were able to show increased gastrin secretion after antigenic challenge in vitro in the presence of intact tissue. It is shown that the increased gastrin release is most probably mediated by activation of the complement system in the presence of antigen-antibody complexes. These are built up by specific anti-NIP antibodies and NIP-HGG used for the challenge. The complement system might be the final pathway of the observed increased gastrin release.

A modified Farr technique for the detection of antibody in the presence of antigen-antibody complexes

Antibody bound in an antigen-antibody complex was barely detectable by the standard Farr technique using 10 μg/ml of radiolabeled antigen. When the Farr test was repeated using larger concentrations of antigen the ABC measurements increased several- In separate experiments complexes were dissociated with low pH buffer. The radio-labeled antigen was then added and the mixture returned to neutral pH before the Farr assay was performed. Pretreatment of the complexes with citrate-buffered saline pH 3.1 enhanced antibody measurements 1.5–3.6-fold. Pretreatment with glycine-buffered saline pH 2.2 enhanced complexed antibody measurements 2.1–4.8-fold. The low pH buffers did not affect ABC values on free antibody. Antibody bound in immune complexes can be detected with a modified Farr technique using a high concentration of antigen and/or by pretreatment of the complexes with low pH buffers before the addition of radio-labeled antigen.

Antigen attachment in ELISA

The amount of antigen on the solid phase was studied during various steps of an enzyme-linked immunosorbent assay (ELISA) for chicken anti-bovine serum albumin (BSA) antibodies. Three materials for the solid phase were used: polystyrene, nylon and cyanogen bromide activated paper. There was noticeable leakage of antigen during the assay from both polystyrene (30%) and nylon (60%). This occured during all steps of the assay: during incubation with the sample serum, incubation with the enzyme conjugated antibodies and during the washing procedures. During incubation with the sample serum detachment of antigen was quite rapid and could result in competition between bound and free antigen for antibodies. The presence of antigen-antibody complexes in reaction mixtures could also lead to erroneous results in the antibody assay. With covalent coupling of antigen to solid phase (cyanogen bromide activated paper), batch-to-batch variation in the surface concentration of antigen was strikingly less than in the assay using plastics. Detachment of antigen from activated paper during the assay was less than with polystyrene or nylon. ELISA with activated paper as the solid phase thus seems suitable as a reference method for standardisation of the antigen phase in ELISA systems.

Analysis of cell-mediated hypersensitivity, immune complexes and monocyte-released factor(s) in allergic alveolitis and asymptomatic exposed subjects.

Patients with allergic alveolitis to various antigens, asymptomatic exposed individuals and healthy control subjects were studied. The lymphocyte transformation test and the formation of two lymphokines were shown to be positive in almost all affected subjects and only rarely in asymptomatic exposed individuals. In most symptomatic subjects and only rarely in asymptomatic exposed individuals. In most symptomatic subjects, the presence of antigen-antibody complexes could be demonstrated. However, such complexes were also present in six of the nine asymptomatic exposed individuals having precipitating antibodies. Factor(s) released from sensitized monocytes strongly facilitate the in vitro formation of specific antibodies but are poorly effective when incubated with cells from asymptomatic exposed subjects or normal individuals.

EFFECT OF CYCLOPHOSPHAMIDE AND METHYLPREDNISOLONE ON IN VITRO CELLULAR IMMUNE RESPONSE TO ALLOGENEIC TUMOR CELLS CORRELATION WITH IN VIVO REJECTION

SUMMARY The immunosuppressive effects of methylprednisolone (MP) (5 mg/kg) and cyclophosphamide (CY, 180 and 48 mg/kg) on cellular immune responses to allogeneic tumor cells were evaluated using in vitro tests. The variations in the number of splenocytes and the in vivo response to the injection of allogeneic tumor cells (rejection or facilitation) were recorded. The drugs were administered at different times in relation to immunization. Polyclonal stimulation essentially reflected T cell recovery, whereas monoclonal stimulation was depressed when the drugs were administered at the time of immunization, suggesting a defect at the level of sensitization. MP had little effect on cytotoxicity but was nevertheless associated with allograft facilitation. CY however strongly inhibited the specific activity of cytotoxic lymphocytes when administered at the time of inoculation, but this was associated with normal graft rejection. Reduced numbers of T cells and increased cytotoxicity was recorded when both drugs were administered 2 days prior to immunization. Thus, this study (1) has shown that MP-induced facilitation does not correlate with the levels of in vitro cytotoxicity; (2) has demonstrated a dissociation of the effect of CY on in vitro cytotoxicity and the in vivo response when given at the time of immunization; and (3) confirmed the hypothesis that CY enhances T cell responses when given 2 days before immunization and similar observations being extended to MP treatment. It also suggested that the cytotoxicity observed at that time was responsible for graft rejection. SUMMARY Methylprednisolone (MP) and cyclophosphamide (CY) are widely used as immunosuppressors to induce graft acceptance. Cytotoxic lymphocytes (CTL) are thought to be responsible for graft rejection because they express lytic activity against H-2-incompatible cells (7). However, other components of the immune system play an important role in allograft rejection and facilitation. In particular, B cells and antibodies that may have either cytotoxic or enhancing effects mediated through different mechanisms such as the protection of target cells from cytotoxic effectors (16) or in provoking the formation of antigen-antibody complexes, “defusing” CTL from their targets (24). Suppressor T cells may also participate in graft facilitation and they may be present as such or may be induced by the presence of antigen-antibody complexes (12). However, because of the strong relationship between cytotoxicity and allograft rejection, we wanted to verify whether these immunosuppressive drugs had any effect on the T cell population as evaluated by nonspecific tests (polyclonal stimulation) and specific tests (mixed lymphocyte cultures (MLC) and chromium release test) and to correlate this effect with in vivo graft rejection.

Antigen-reactive cell opsonization: A mechanism of antibody-mediated immune suppression

Antisera against sheep red blood cells (SRBC) specifically suppressed the direct anti-SRBC plaque-forming cell (PFC) response in mice when passively administered with the antigen. The suppressive activity of mouse and rabbit anti-SRBC sera was found to correlate with anti-SRBC opsonic activity but not with hemagglutination or hemolysin titers. Macrophage depletion of mice, using carrageenan treatment, inhibited antibody-mediated immune suppression. When mice immunized with SRBC were given 125I-labeled Udr, radiolabeled spleen lymphocytes were obtained which specifically formed rosettes with SRBC. These radiolabeled antigen-reactive cells (∗ARC) were specifically opsonized in mice treated with antigen-antibody complexes but not in mice treated with antigen or antibody alone. These results suggest that antibody-mediated immune suppression may be due to specific opsonization (and subsequent destruction) of ARC in the presence of antigen-antibody complexes.

Human monocyte spreading in vitro—Inducers and effects on Fc and C3 receptors

Human peripheral blood monocytes undergo cytoplasmic spreading following attachment to a glass surface. The extent of spreading is greater in the presence of antigen-antibody complexes, manganese, subtilisin and dithiothreitol. The human blood monocyte spreads more rapidly than the mouse peritoneal macrophage and is not inhibited by serum. Fc receptor activity is diminished when spreading is induced by antigen-antibody complexes and is not affected by other inducers. The binding of erythrocytes coated with C3 and the ingestion of latex particles are not inhibited during cytoplasmic spreading.

Are the Inflammatory Bowel Diseases Immune Complex Diseases?

Several groups of investigators, using varied tech­ niques, have recentiy demonstrated the existence of circulating antigen-antibody complexes in the inflam­ matory bowel diseases. This phenomenon may be an important clue that can tie together some of the diverse clinical and immunological findings in these conditions. I, 2 As an example, the deposition of these antigen-antibody complexes in other tissues could read­ ily explain some of the extragastrointestinal serum sickness manifestations of the inflammatory bowel diseases (IBD).3 Such localization of inflammatory reac­ tions are common in other diseases in which circulating complexes are found. 4 , 5 The deposition of these com­ plexes in blood vessel walls 3 , 6 may explain the vasculitis that is seen not infrequently in both ulcerative colitis 7 and Crohn's disease. s Even granulomata formation re­ sulting from the deposition of these complexes has been reported. 8 L)- mphocytes obtained from both ulcerative colitis and Crohn's disease patients have been shown to be cytotoxic for colonic cells in tissue culture. 9 - 11 Interest­ ingly, if normal lymphocytes are incubated in the sera obtained from inflammatory bowel disease patients, these lymphocytes will also exhibit this colonic cytotox­ icity; on the other hand, if the cytotoxic lymphocytes obtained from individuals with ulcerative colitis or Crohn's disease are incubated in heterologous IBD sera, these lymphocytes lose their cytotoxicity. 12, 13 Yet, a loss of cytotoxicity does not result if cytotoxic lymphocytes are incubated in autologous sera. 12 , 13 These above results could readily be explained by the presence of antigen-antibody complexes. For instance, Perl mann and Holm have shown that antibody or antibody complexed to target cells can induce lympho­ cyte cytotoxicity to the appropriate target antigen. 14 lED sera, therefore, could induce colon cytotoxicity in normal lymphocytes if those sera contain colon antibody or colon antibodies-antigen complexes. Earlier, it had been demonstrated that IBD sera did contain such an antibody that would react with fetal colon 15 as well as with an antigen which had similar antigenic determi­ nants and is present in many intestinal bacteria. 16. 17 Considerable quantities of this latter, highly immuno­ genic, 18 common heterogenetic antigen must be availa­ ble in the intestinal lumen; furthermore, its absorption into the circulation has been demonstrated in the inflammatory bowel diseases_ 19, 20 The presence, then, of colon antibodies and/or antibodies to the cross-reacting bacterial antigens as well as the bacterial antigen itself

Cell-mediated immunity and immune complexes in thyroid disease

As shown diagrammatically in Figure 12, one might postulate that in vivo several immune mechanisms may play separate or associated roles in the pathogenesis of autoimmune thyroid disease. Although relatively little is known concerning the triggering of an autoimmune response, for the sake of simplicity one might say that the initiating reaction is some sort of breakdown in the control mechanism resulting in the proliferation of autoreactive T and B lymphocytes, i.e., a breakdown in immunological tolerance to self. Specifically sensitised B lymphocytes are involved in the production of antibody. The role of thyroid specific autoantibodies has not been firmly established and much evidence argues against their being primarily responsible for the thyroid lesions with the exception of thyrotoxicosis. Transfusion of large quantities of Hashimoto serum to Rhesus monkeys produces no thyroid alteration (Roitt and Doniach, 1960), and although thyroglobulin antibodies in high titre can cross the placenta, thyroiditis is rarely observed in babies of affected mothers (Parker and Beierwaters, 1961). Complement fixing thyroid microsomal antibody has proved to be cytotoxic to trypsinised thyroid cells in culture suggesting that slight surface injury to these cells may allow access of cytotoxic antibody resulting in cell death (Pulvertaft, Doniach and Roitt, 1961; Irvine, 1962). In vivo cytotoxic antibodies may play a secondary role in the potentiation of the disease following an initial damage to the gland (Roitt, Jones and Doniach, 1962). Morphological and immunological observations suggest that cell-mediated immune mechanisms may be involved in the pathogenesis of autoimmune thyroid disease. In vitro tests for CMI such as the migration inhibition test and lymphocyte transformation test suggest that patients with autoimmune thyroid disease possess circulating lymphocytes capable of recognising and responding to thyroid antigens. In vivo, such sensitised lymphocytes could cause thyroid cell damage either through a direct cytolytic action or via the release of lymphokines. A third mechanism which could be of pathogenic significance in vivo is that involving K cells and antibody. The presence of thyroid specific antigen-antibody complexes on the surface of a thyroid epithelial cell renders it highly susceptible to lysis by K cells. Whether in fact such complexes are freely available on the surface can only be speculative, although there is one report in the literature describing distinct electron-dense deposits in the follicular basement membrane of some follicles in Hashimoto thyroid glands resembling the antigen-antibody deposits of immune complex nephropathies. Similarly, the presence of antigen-antibody complexes in antibody excess in the circulation could result in the recruitment of large numbers of K cells with the specificity to destroy antigen-labelled target cells in the thyroid gland. The extent to which K-cell-mediated cytotoxic mechanisms as opposed to T-cell-mediated phenomena are involved in the production of thyroiditis is again speculative although it is of interest that the level of circulating K cells in Hashimoto patients is elevated, whereas the number of T cells appears to be within the normal range. Studies on the cytotoxic activity of lymphoid cells isolated from an infiltrated Hashimoto gland would be of considerable interest. Recently, Evans and Alexander (1972) have described mechanisms of immunologically specific killing of tumour cells by macrophages. Specific killing of tumour cells can be achieved by (i) macrophages from the peritoneal cavity of tumour-immunised mice, (ii) normal macrophages ‘armed’ in vitro by exposure to spleen cells from tumour-immunised mice and by (iii) normal macrophages ‘armed’ in vitro by exposure to the cell-free supernatant obtained when spleen cells from immunised mice are cultured with specific antigen. The role of macrophages in autoallergic thyroid cell damage has not so far been investigated but studies in this area are well merited. In view of the complexity of the immune system, it would seem unlikely that any one immune mechanism is solely responsible for the production of autoimmune lesions. It is more likely that several mechanisms play associated roles in the pathogenesis, the severity of the disease possibly being dependent on the extent to which one of these mechanisms predominates and this in turn may be related to the underlying triggering reaction. Although it is obviously essential in the first instance to characterise the basic mechanisms involved in the production of autoallergic tissue damage, a clear insight into the pathogenesis of thyroiditis cannot really be achieved until there is a better understanding of the nature of the autoimmune response.