Factors involved in the pathogenesis of neutrophilic vasculitis in MRL/Mp-lpr/lpr mice: a model for human microscopic angiitis.

Anti-neutrophil cytoplasm antibodies (ANCA) directed against myeloperoxidase (MPO) are detected in patients with microscopic angiitis. Human MPO autoantibodies stimulate neutrophil degranulation in vitro and are thought to be pathogenic. We have previously shown that MRL-lpr mice with MPO autoantibodies have a higher incidence of vasculitis than their seronegative littermates. The aim of the present study is to determine the relationship between MPO autoantibodies and microscopic angiitis. The neutrophil binding properties of anti-MPO monoclonal antibodies (mAbs) from MRL-lpr mice were tested using murine heterophils (neutrophils) present in blood and induced peritoneal exudates. MRL anti-MPO mAbs selectively bind activated neutrophils which express MPO in vitro. The pathogenicity of an IgG2b anti-MPO mAb, C6, was investigated in vivo. Anti-MPO mAb, C6 was administered to young MRL mice which had been primed with exogenous TNF alpha to induce neutrophil activation and expression of MPO. Neutrophilic vasculitis similar to microscopic angiitis occurred in 33% of MRL mice which had been treated with anti-MPO mAb. The lesions were mainly restricted to sites of previous endothelial insult which suggests an active role for injured endothelium in this pathology.

Antibody and HIV-1 gp120 recognition of CD4 undermines the concept of mimicry between antibodies and receptors.

It has been proposed that antibodies can mimic the binding of a receptor to its ligand and that anti-idiotype antibodies raised against such antibodies can be used to identify the receptor. A large number of antibodies have been raised against CD4, the receptor on T cells for the envelope glycoprotein gp120 of the human immunodeficiency virus, and the site at which gp120 binds to CD4 has been delineated. It has therefore become possible to contrast the fine specificities of a natural ligand (gp120) and antibodies that interact with the receptor at the same site. Here we report that out of a panel of 225 anti-CD4 antibodies, only one showed fine binding specificity that was broadly like that of gp120, but the evidence was against this being an exact mimic. Thus the data indicate that the production of antibody mimics will occur very rarely or not at all and that the anti-idiotype approach is unlikely to be useful. This contention is supported by a review of the results of attempts to use this approach. Taking strict criteria for success, there is no example for which the anti-idiotype approach has led to the discovery of a previously undescribed receptor or other protein of interest.

A "network antigen" for human CD4. A murine monoclonal anti-idiotype to Leu-3a induces an anti-CD4 response in naive mice.

Previous studies have evaluated anti-CD4 mAb as idiotypic models of the HIV gp120-binding site for CD4. The success of this strategy depends upon the concept of internal image, whereby the binding paratope of the anti-CD4 structurally mimics the equivalent binding surface on HIV gp120. To test this concept of internal image, anti-idiotypic antibodies were raised against the anti-CD4, Leu-3a. If any of these anti-Id detect the paratopic idiotope on the anti-CD4 antibody, their own respective paratopes should structurally model the corresponding binding epitope on CD4 bound by Leu-3a. Consequently, the immunization of naive mice with the selected anti-Id should induce an anti-CD4 response which reflects the binding specificities of Leu-3a. Four anti-Id to Leu-3a were characterized and tested for their ability to induce anti-CD4 responses in naive animals. Although one anti-Id induced an anti-CD4 response in mice, no such response could be detected in other species. Thus the failure to raise anti-Id with internal image characteristics may provide an explanation for the lack of anti-gp120 activity reported in anti-Id antisera raised to multiple anti-CD4 antibodies.

A highly selected panel of anti-CD4 antibodies fails to induce anti-idiotypic antisera mediating human immunodeficiency virus neutralization.

Anti-CD4 antibodies directed to the N terminus of CD4 can inhibit human immunodeficiency virus (HIV) infection. Therefore, it has been proposed that some of these reagents may contain idiotypic determinants which conformationally model the binding site expressed on gp120. In this report, we have selected a panel of anti-CD4 monoclonal antibodies as idiotypic mimics of gp120 by employing cross-blocking techniques, and CD4 epitope mapping using site-directed mutagenesis. These studies suggest that only 4 out of the original panel of 12 would be expected to represent suitable candidates for modelling the gp120 binding site. Nevertheless, anti-idiotypic antisera raised against these antibodies failed to inhibit gp120 binding to CD4. This negative result may reflect the incomplete modelling of the virus binding site by anti-CD4, or the lack of internal image antibody in the anti-idiotypic preparations. Alternatively, the binding site on gp120 may not be accessible to antibody neutralization, excluding the possibility of an idiotypic vaccine to HIV based on anti-CD4 antibody as surrogate antigen.

T cell proliferation induced by monoclonal antibodies to a phosphatidylinositol-linked differentiation antigen of guinea pig lymphocytes.

Monoclonal antibodies (mAb) to differentiation antigens frequently influence the in vitro function of antigen-bearing cells. We characterized a 32-36-kDa membrane protein expressed on guinea pig lymphocytes and Langerhans cells. A series of independently derived mAb to this protein, now called guinea pig T cell activation antigen (gpTAA), induced strong proliferation of T cells in vitro. Cross-linking of the mAb by a secondary antibody (rabbit anti-mouse Ig) and costimulation with phorbol 12-myristate 13-acetate were required for activation. Treatment of the cells with phosphatidylinositol-specific phospholipase C greatly reduced the amount of antigen expressed on the cell surface as measured by flow cytometry analysis. This finding indicates that the antigen is anchored to the cell membrane via phosphatidylinositol linkage as shown similarly for other membrane proteins with T cell activating properties, e.g. Thy-1 and Ly-6. The guinea pig protein differs, however, in its molecular weight and tissue distribution from similar proteins identified in the mouse or in the rat system. Unlike Thy-1, gpTAA is also expressed on B Lymphocytes and Langerhans cells. Considering the previously described involvement in cellular adhesion, and the functional characteristics reported here, gpTAA might represent a new species of differentiation antigen with T cell-activating capacity.

A quantitative immunocytochemical study of the infiltrating lymphocytes in the spinal cord of guinea pigs with chronic relapsing experimental allergic encephalomyelitis.

The production and characterization of an anti-guinea pig B cell monoclonal antibody is described. Immunocytochemical techniques using this antibody and others recognizing a Pan T cell antigen and T cell subsets were employed to study frozen sections of spinal cord from guinea pigs with chronic relapsing experimental allergic encephalomyelitis. T and B cells were found in both perivascular lesions and the central nervous system parenchyma, with the major T cell infiltration occurring by the end of the acute phase of disease. The distribution of T cell subsets suggests a phenotypic selectivity in favour of the transport of CT6 (putative CD8)+ve cells across the blood-brain barrier.

Presentation of myelin basic protein by normal guinea-pig brain endothelial cells and its relevance to experimental allergic encephalomyelitis.

Previous studies have shown that endothelial cells in the central nervous system (CNS) of normal guinea-pigs constitutively express certain MHC class II determinants, whilst the expression of other determinants is apparent during the acute phase of chronic relapsing experimental allergic encephalomyelitis (CREAE). The expression of MHC class II determinants is retained by endothelial cells derived from normal guinea-pig brain tissue and maintained in culture. This present study demonstrates that the MHC class II molecules on these cells can be recognized by allogeneic lymphocytes, resulting in a proliferative response which is enhanced by the addition of exogenous IL-2. The endothelial cells were incapable of presenting either purified protein derivative or ovalbumin, but they could present autologous myelin basic protein (MBP), an encephalitogen implicated in the pathogenesis of EAE. The resulting lymphocyte proliferative response was of the same magnitude as that obtained when a control population of macrophages was used to present MBP. These results, therefore, suggest that cerebrovascular endothelia have the potential to play a role in the pathogenesis of EAE.

The localisation of fibrin, fibronectin and class II major histocompatibility complex antigen in the spinal cord in chronic relapsing experimental allergic encephalomyelitis.

Light and electron immunocytochemistry using antibodies recognising a class II major histocompatibility complex antigen, fibrin, fibronectin, albumin and factor VIII related antigen has been used to stain sections of spinal cord from normal guinea pigs and those with chronic relapsing experimental allergic encephalomyelitis (CREAE). It was found that class II MHC antigens, fibrin and fibronectin were present in normal blood vessels and at high levels in lesions from animals at all stages of the disease. The possible immunological roles of these antigens suggest their participation in the initiation and maintenance of disease state.

An immunoelectron microscopical study of the expression of class II MHC and a T lymphocyte surface marker during chronic relapsing experimental allergic encephalomyelitis.

Immunoelectron microscopy using antibodies recognising Class II MHC antigens and a pan T cell marker was employed to study sections of spinal cord from guinea pigs with chronic relapsing experimental allergic encephalomyelitis (CREAE). It was found that endothelial cells expressed Class II antigens on their luminal surface throughout the course of the disease and that lymphocytes were adherent to these surfaces. In the parenchyma lymphocytes, macrophages and possibly microglia expressed Class II antigens suggesting that they might also be involved in antigen presentation. The different distribution of T cells seen in the individual lesions during the relapse phase may correlate with their respective natural histories.

Behaviour of guinea pig T cells stimulated by antigen, allo-antigen and mitogen.

The expression of major histocompatibility (MHC) antigens on guinea pig T cells was used as a functional marker for lymphocyte activation. Antigen-stimulated lymphocytes were recovered from guinea pigs responding to the contact sensitizer DNFB, and isolated T cells were then phenotyped using a new antiguinea pig monoclonal antibody, MSgp7. The level of expression of MHC class II, as defined by the monclonal antibody, MSgp8, was increased on T cells recovered 4 days after sensitization, as compared with unsensitized controls. The value of this experiment was extended by measuring MHC class II expression on T cells stimulated in vitro by the mitogen concanavalin A, where a clear increase in MSgp8 binding was also observed. Confirmation of the specificity of MSgp8 for guinea pig MHC class II antigens was achieved by studying the inhibitory capacity of this antibody on an MHC class II restricted mixed leucocyte reaction. The combination of antibodies MSgp7 and MSgp8 with flow cytometry could be applied to other guinea pig experimental models to quantitate the expression of MHC class II antigens on T cells to determine their putative value in disease manifestation.

Accessory cell function of dendritic cells from lymph nodes containing Mycobacterium leprae induced granulomas.

Dendritic cells were enriched from guinea pig auricular lymph nodes containing Mycobacterium leprae induced granulomas by immunomagnetic depletion of other cells. These cells were strongly positive for major histocompatibility complex class II antigens and labelled with an antidendritic cell monoclonal antibody, but not with an antimacrophage antibody. Interdigitating dendritic cells were identified in the granulomatous lymph node by staining with the antidendritic cell antibody and by transmission electron microscopy. When cultured in vitro with purified T lymphocytes, these cells acted as accessory cells for both purified protein derivative and concanavalin A induced proliferation. Although previous studies have shown that macrophages from these lymph nodes do not act as accessory cells, the present results indicate that dendritic cells from M. leprae granuloma containing lymph nodes may act as antigen-presenting cells.

Phenotypic analysis of guinea pig Langerhans cells with antibodies directed against leucocyte surface antigens.

The epidermis was stained with a panel of recently produced anti-guinea pig leucocyte antibodies. Guinea pig Langerhans cells were not detectable with antibodies directed against B lymphocytes (MSgp9), T lymphocytes (CT7 and MSgp7), T-helper/inducer (MSgp12) and putative T-suppressor/cytotoxic (CT6 and MSgp6) subsets. Langerhans cell expressed both major histocompatibility complex (MHC) class-I and II antigens and also an epitope (CT4) associated with lymphocyte migration, thus suggesting the migratory potential of this cell. Although the Langerhans cell did not express macrophage specific antigens, MSgp5, which detects lymphoid dendritic cells, was weakly expressed on the Langerhans cell. The Langerhans cell expressed a leucocyte-common antigen detected by H201. Double-labelling studies with anti-MHC class-II antibodies indicated that only 0.4 +/- 0.3% of the pan leucocyte-positive epidermal cells were Ia-negative, indicating that it is unlikely that a guinea pig analogue of the murine Thy-1 + dendritic epidermal cell (Thy-1 + dEC) exists.

Induction of sensitization and tolerance in contact sensitivity with haptenated epidermal cells in the guinea-pig.

Haptenated murine Langerhans' cells (LCs) have been reported to induce contact sensitivity when injected via the subcutaneous, intraperitoneal and, in some instances, the intravenous route. Similar studies were undertaken to elucidate the role of the LC in the induction of contact sensitivity in the guinea-pig. The subcutaneous injection of dinitrophenylated epidermal cells induced hapten-specific contact sensitivity in a dose-dependent fashion. This contrasts with the tolerance that was induced by the intravenous or intraperitoneal injection of similarly haptenated cells. Contact sensitivity by haptenated epidermal cells could be induced in syngeneic and allogeneic recipients and did not require the transfer of viable cells. Using the monoclonal antibody MSgp2, which detects LCs, LC-enriched and LC-depleted populations were prepared by an 'indirect antibody' panning technique. It was found that a haptenated LC-depleted epidermal cell population (0.1% LC) induced the same degree of contact sensitivity or tolerance, depending on the route of immunization, as a haptenated 'freshly isolated' epidermal cell population (1% LC). Whereas, a purified population of haptenated LC (85-90%) induced no significant degree of contact sensitivity or tolerance. These results confirm our previous conclusions based the in vivo depletions of Langerhans' cells, and suggest that the epidermal Langerhans' cell is not essential for the induction of contact sensitivity in the guinea-pig. However, this does not exclude the possibility that the LC is involved in the elicitation of contact sensitivity in a sensitized animal.

Distribution of a guinea pig lymphocyte cell surface antigen of defined function, as detected by the mouse monoclonal antibody MSgp 1.

A mouse anti-guinea pig monoclonal antibody, designated MSgp 1, was derived from a fusion between NS-1 myeloma cells and splenocytes hyperimmunised with guinea pig thymocytes. The MSgp 1 determinant is expressed by a subset of small thymocytes and lymph node T cells which participate in mixed leukocyte reactions. The determinant is modulated by antigen in vivo, and MSgp 1 antibody will prevent MHC class II driven proliferation in vitro. In addition, MSgp 1 reacts with a minor population of lymph node B cells, but not with a chronic B cell leukaemic cell line. Resident peritoneal macrophages express the MSgp 1 determinant, whereas chronic oil-induced peritoneal macrophages do not. The role of MSgp 1 defining guinea pig helper T cells is discussed by comparisons with other documented T helper cell reagents.

An antigenic determinant common to lymphocytes and Langerhans cells of the guinea pig.

A mouse anti-guinea pig monoclonal antibody, designated MSgp 2, was derived by the fusion of NS-1 myeloma cells with mouse splenocytes hyperimmunized with guinea pig B cells. MSgp 2 reacts with an antigen present on the majority of lymphocytes. An unexpected finding was the expression of the MSgp 2 antigen on epidermal Langerhans cells, as defined by cell morphology, expression of MHC class II antigen and presence of Birbeck granules in positive cells. It is suggested that the tissue distribution of MSgp 2 antigen on lymphocytes of the lymph node could indicate a role for this determinant in cell migration.

Characterization of suppressor-inducer cells which control the production of rat erythrocyte-induced anti-erythrocyte autoantibodies.

Mice immunized with rat erythrocytes produce autoantibodies to their own red blood cells, distinct anti-rat agglutinins and autoantigen-specific suppressor cells. Suppressor cells were detected by adoptive transfer of rat erythrocyte-immunized spleen cells to naive recipients. Such recipients failed to make erythrocyte autoantibodies after immunization with rat erythrocytes although their anti-rat erythrocyte response was unimpaired. Depletion and enrichment studies were performed to identify the cell type(s) which transfer suppression. B cell depletion of rat erythrocyte-immunized spleen cells by passage over Ig/anti-Ig-coated bead columns abrogated the transfer of suppression. However, suppression was still transferred after rat erythrocyte immunized spleen cells were passed over beads coated with a complex of 4-azido-2-nitrophenyl (NAP)-mouse IgG-rabbit IgG anti-NAP suggesting that T cells bearing Fc gamma receptors are not responsible for suppression. Positively selected B cells from rat erythrocyte-immunized spleen cells caused some suppression of erythrocyte autoantibodies but only after high numbers of cells were transferred. Neither positively selected Lyt-1+2- nor Lyt-1-2+ T cell subpopulations transferred suppression. By contrast, rat erythrocyte-immunized spleen cells which contained a mixture of B memory and T cells were suppressive and retained their suppressor activity after removal of Lyt-1-2+ but not Lyt-1+2- cells. It is proposed that these Lyt-1+2-T cells belong to a distinct population of suppressor-inducer cells which together with memory B cells stimulate the generation of effector T suppressor cells in naive recipients.