Autoimmune MRL mice express high-affinity IgG2b monoclonal autoantibodies to heparin.

Heparin and heparan sulfate are related glycosaminoglycans which demonstrate high-affinity interactions with a number of proteins, including antithrombin III. The immunogenicity of heparin has been reported previously employing heparin-protein conjugates as immunogens and as antigens in solid-phase assays. Previous studies also demonstrate that anti-heparin antibodies play a role in autoimmune diseases including systemic lupus and anti-phospholipid syndrome and in patients who receive heparin for therapeutic purposes. In the current study, we investigated the expression of monoclonal anti-heparin antibodies in nonimmunized, autoimmune MRL/lpr/lpr++ mice employing a liquid-phase radioimmunoassay. The Kd of monoclonal IgG2b autoantibodies for heparin was approximately 10(-8)M. Anti-heparin antibodies were precipitating, and were not polyreactive. The IgG monoclonal antibodies described in this study represent an immunological instance of a specific, high-affinity heparin-protein interaction.

Cytotoxicity to endothelial cells by sera from aged MRL/lpr/lpr mice is associated with autoimmunity to cell surface heparan sulfate.

Vasculitis is an common clinical feature of systemic lupus erythematosus (SLE) in humans and in animal models of this disease. Humoral autoimmunity against endothelial cells has been previously demonstrated in SLE and other autoimmune disorders, but the precise cell surface antigenic targets involved in the initiation and progression of vascular injury are still essentially unknown. In the current studies, we demonstrate the presence of autoantibodies in the sera of MRL/lpr/lpr mice which bind endothelial cell surface antigens by ELISA and also cause complement-dependent cytotoxicity of these cells. These MRL/lpr/lpr sera induced complement-dependent cleavage and release of 35SO4-labeled material containing primarily cell surface heparan sulfate proteoglycans from these cells, and react with heparin (a glycosaminoglycan related to heparan sulfate) by ELISA and liquid-phase competitive inhibition ELISA. These data indicate that antiendothelial cell autoantibodies present in autoimmune MRL/lpr/lpr mice are directed at least in part against cell surface heparan sulfate proteoglycans. Autoantibodies to cell surface heparan sulfate proteoglycan may play a role in vascular endothelial cell injury in these animals through complement-dependent, autoimmune mechanisms.

Studies of the cellular immune response to heparan sulfate proteoglycan in the tight skin mouse.

As in human scleroderma, tight skin (TSK/+) mice develop cutaneous hyperplasia with over-production of extracellular matrix, including collagen and proteoglycans, associated with autoimmunity to a number of autoantigens. The present study investigated the presence of cellular autoimmunity to basement membrane heparan sulfate proteoglycan (HSPG) in diseased TSK/+mice and their nondiseased littermates, pallid mice (+/pa). Lymphocyte proliferative responses to specific HSPG antigens, including intact HSPG, HSPG protein core (P. Core), and the glycosaminoglycan heparan sulfate (HS) were studied. Splenocytes from young TSK and control pallid mice reacted weakly to intact HSPG and HSPG P. Core antigens and did not respond to HS. However, lymphocytes from old TSK mice were reactive with HSPG and P. Core and demonstrated a de novo response to HS. The proliferative response to intact HSPG and HSPG P. Core in TSK mice was T cell dependent, but the response to HS was T cell independent. The T cell-dependent response was mediated by the CD4-positive subset and required the participation of class II major histocompatibility complex molecules. Cellular autoimmunity to HSPG, a critical cell surface and extracellular matrix component, may play a role in the disease suffered by TSK mice. Further studies are necessary to determine the mechanisms which link autoimmunity to HSPG with the pathology seen in TSK mice, particularly the overproduction of extracellular matrix and fibrosis.

Self-reactive repertoire of tight skin (TSK/+) mouse: immunochemical and molecular characterization of anti-cellular autoantibodies.

The tight skin (TSK/+) mouse has been proposed as an experimental model for progressive systemic sclerosis because of the biochemical alterations in collagen synthesis and pathological similarities to the human disease. Here, we report the analysis of tight skin mice sera for the presence of anti-cytoplasmic and anti-nuclear autoantibodies and determination of the frequency of hybridomas producing anti-cellular autoantibodies. The binding specificity of TSK mAbs to nuclear and cytoplasmic antigens such as keratin, actin, vimentin, and mitochondria was determined. Of 71 monoclonal antibodies that we have studied, only 3 appear to bind to foreign as well as self-antigens, indicating that the majority of these antibodies do not belong to the class of natural autoantibodies. Our results also showed that the frequency of hybridomas producing anti-nuclear and anti-cytoplasmic antibodies was higher in TSK mice than in C57BL/6 pa/pa, the control mouse strain, used in these studies. The results of the analysis of V gene usage showed that the majority of anti-cytoplasmic and anti-nuclear antibodies are encoded by genes from a restricted number of VH and VK genes families. In the sera of TSK mice we have detected the presence autoantibodies specific for cytoplasmic antigens in addition to anti-nuclear and anti-topoisomerase I antibodies which are characteristic of scleroderma. Since the presence of anti-cytoplasmic antibodies has not been described in scleroderma, the significance of their production in tight skin mice is not clear. However, the presence of such autoantibodies in the animal model provides a basis for investigation of this type of antibodies in human disease.

CD5 and immunoglobulin V gene expression in B-cell lymphomas and chronic lymphocytic leukemia.

We studied the expression of CD5 and immunoglobulin variable gene families in a panel of monoclonal Epstein-Barr virus (EBV) transformed lines, chronic lymphocytic leukemias (CLLs) and CD5+ and CD5- B-cell lymphomas. The CD5 gene expression was in all cases identical to that of T-cell malignancies. The utilization of the various VH and VK gene families was roughly proportional to the estimated gene family size in EBV lines obtained from adult healthy subjects. In contrast we found a statistically significant biased usage of VH6 in CLL and VH5 in CD5+ lymphomas as compared with EBV lines, and of VKIII in both CLL and CD5+ lymphomas as compared with EBV lines. Some differences in the variable gene usage were also noted when comparing CD5+ and CD5- lymphomas. These findings are analyzed in the context of possible mechanisms involved in the malignant transformation of CD5+ B cells.

High frequency of autoantibodies bearing cross-reactive idiotopes among hybridomas using VH7183 genes prepared from normal and autoimmune murine strains.

Hybridomas obtained by in vitro stimulation with lipopolysaccharides (LPS) of BALB/c, MRL/lpr, and NZB splenocytes were selected for expression of VH7183 by hybridization using slot blotting. Northern blot analysis showed that the majority of hybrids produce a full length message complementary to the VH7183 probe. The frequency of VH7183 hybridomas was significantly higher in NZB mice as compared with BALB/c mice. Using multiple binding assays, 60% of the total antibodies encoded by VH7183 were specific for self-epitopes. Finally, the vast majority express cross-reactive idiotypes borne by autoantibodies of various specificities.

Malignant fibrous histiocytoma. Expression of monocyte/macrophage differentiation antigens detected with monoclonal antibodies.

Monoclonal antibodies were used in an investigation of the histogenesis of malignant fibrous histiocytoma (MFH), a neoplasm with morphologic features of both fibroblastic and histiocytic differentiation. In 4 cases of MFH studied, the tumor cells were found to react uniformly with antibodies to determinants expressed on monocyte macrophages (T-200, Ia, MoS-1, MoS-39, MoR-17). Both spindle and histiocyte-like tumor cells expressed these markers. In contrast, in 8 non-MFH soft tissue tumors, tumor cell reactivity was not observed. The reactivity of the spindle cells of MFH for determinants of monocyte/macrophages favors their origin from tissue histiocytes (facultative fibroblasts). The results support the view that MFH is a tumor of the mononuclear phagocyte system.

Immunopathology of Hodgkin's disease. Characterization of Reed-Sternberg cells with monoclonal antibodies.

The cellular origin of the Reed-Sternberg cells of Hodgkin's disease is controversial. The authors studied 14 cases of Hodgkin's disease (nodular sclerosis, 9; mixed cellularity, 3; lymphocyte predominant, 2), utilizing a panel of 16 monoclonal antibodies, including 5 new monoclonal antibodies defining differentiation antigens of the monocyte/macrophage system. Reed-Sternberg cells were found to react with antibodies to Ia-like (HLA-DR) determinants (14 of 14 cases), Leu M1, an antigranulocyte antibody (11 of 14 cases), and rarely B-1, an antibody defining an antigen expressed on human B lymphocytes (2 of 14 cases). Reed-Sternberg cells did not react with any of 5 antibodies to differentiation antigens of the monocyte/macrophage system (MoP9, MoS39, MoR17, MoU26, MoU50). In contrast, reactive histiocytes in the Hodgkin's disease infiltrates stained strongly. The findings are evidence against the monocyte-macrophage origin of Reed-Sternberg cells and support the view that the Reed-Sternberg cells of Hodgkin's disease derive from other cell types, such as interdigitating reticulum cells, or as yet uncharacterized cells which do not share antigens of the monocyte/macrophage system.

TdT-positive acute leukemia with monocytoid characteristics: clinical, cytochemical, cytogenetic, and immunologic findings.

Thirteen patients with acute leukemias that were difficult to classify by the use of cytochemical staining and terminal deoxyribonucleotidyl transferase (TdT) activity are reported. The phenotype of the leukemic cells was characterized by the presence of mature or early monocyte lineage antigens and intense Ia antigen expression detected by monoclonal antibodies, terminal deoxytransferase activity, and cytochemical features, including both Sudan black B and periodic acid-Schiff activity. The mean age of this group of patients was 60 years. Five patients had leukemia occurring after chemotherapy or radiotherapy of a prior malignant disease, and two patients had a refractory anemia prior to development of acute leukemia. These patients had a low response rate to chemotherapy. This series of leukemia appears to form a distinct nosologic entity, representing a leukemic transformation among early cells of the monocyte lineage, resulting in a predominant neoplastic cell that is less mature than either the French-American-British M4 acute myelomonocytic leukemia or M5 acute monoblastic leukemia. The presence of terminal deoxytransferase activity was interpreted as indicating the primitive state of the cells in the differentiation sequence, rather than as implying any significance with respect to lineage.

Delineation of four cell types comprising the giant cell tumor of bone. Expression of Ia and monocyte-macrophage lineage antigens.

Giant cell tumors of bone dissociated by collagenase digestion were found to be composed of four different cell types defined by morphology, growth in culture, and pattern of staining with monoclonal antibodies. Giant cells comprised an average of 0.8% of the cells recovered, with the remainder consisting of small stromal cells. Of the giant cells, 20-57% expressed Ia antigens, while all lacked IgG Fc receptors and five differentiation antigens associated with mature members of the monocyte-macrophage lineage (M phi S-1, M phi P-9, M phi P-15, M phi S-39, and 63d3). One antigen, M phi U-50, found on early monocytoid forms was expressed on Ia+ giant cells. 6-36% of the remaining stromal tumor cells formed a second subpopulation that assumed either a rounded or elongated shape in culture. These cells bore Ia antigens, IgG Fc receptors, and five antigens of the monocyte-macrophage lineage usually found on blood monocytes. However, these cells differed from monocytes or macrophages in that the antigen M phi R-17 generally found on tissue macrophages was absent, and the M phi U-50 antigen present on more primitive cells was well expressed. A very limited endocytic capacity was demonstrable. A third population of up to 24% of the tumor cells was defined by the presence of intense staining for Ia antigens but the absence of antigens of mature monocytes. A proportion of these cells expressed M phi U-50 and a minority had IgG Fc receptors. The two Ia(+) populations of stromal cells were not identifiable after 2 wk of culture, nor did tumor cells selected for the presence of Ia antigens proliferate in culture. A fourth population of cells lacked Ia and monocyte lineage antigens, but showed pronounced intracellular staining for acid phosphatase. These cells had a distinctive plump epitheloid to fibroblastoid morphology and were readily established in long-term culture where they gave rise to large multinuclear Ia(-) cells containing acid phosphatase. The possibility is discussed that the cell types of these tumors relate to various stages in the development of osteoclasts from precursors in the mononuclear phagocyte lineage.

The tissue architecture of synovial membranes in inflammatory and non-inflammatory joint diseases. I. The localization of the major synovial cell populations as detected by monoclonal reagents directed towards Ia and monocyte-macrophage antigens.

Utilizing monoclonal reagents directed towards antigens of the monocyte-macrophage lineage and Ia antigens, the tissue architecture of synovial membranes obtained from patients with non-inflammatory joint diseases and patients with rheumatoid arthritis was studied. Emphasis was placed on the localization of the type I, type II and type III synoviocytes that previously had been defined by their cell surface phenotype with regard to the expression of monocyte-macrophage lineage (M theta) and Ia antigens as well as by their phagocytic capacity or the ability to produce glycosaminoglycans. In patients with non-inflammatory joint diseases, cells with the M theta + Ia+ (type I) phenotype constituted the majority of synoviocytes immediately adjacent to the joint cavity; cells with this phenotype were also scattered in the subsynovial tissue and in the perivascular regions. The fibroblastoid type III cells defined by the absence of both M theta and Ia antigens formed the major cell population in the subsynovial tissue in this patient group. In patients with rheumatoid arthritis, the Ia+ M theta + cells were present in a characteristic double configuration forming an intensely positive layer adjacent to the intra-articular space followed by an Ia- M theta - layer that again was succeeded by an intensely Ia+ M theta + layer. Large numbers of synoviocytes bearing M theta + Ia+ antigens were also demonstrated in the diffusely inflamed subsynovial tissue, in the perivascular regions as well as around and within lymphoid infiltrates.(ABSTRACT TRUNCATED AT 250 WORDS)

Identification of three major synovial lining cell populations by monoclonal antibodies directed to Ia antigens and antigens associated with monocytes/macrophages and fibroblasts.

Synovial lining cells obtained from patients with rheumatoid arthritis or non-inflammatory joint diseases were divisible into three populations according to the expression of surface antigens detected by various monoclonal antibodies. A population of cells designated type I was defined by the presence of Ia antigens. Fc receptors, five different monocyte lineage differentiation antigens, and the property of phagocytosis. The greatly increased amounts of both Ia antigens and certain monocyte lineage antigens distinguished these cells from blood monocytes. A second distinctive cell population was non-phagocytic, occasionally binucleate, and had abundant Ia antigens but lacked IgG Fc receptors, monocyte lineage antigens, B or T lymphocyte antigens, and fibroblast-associated antigens detected by reagents raised against synovial cells. This population, designated type II, accounted for approximately one-third of the synovial cells in patients with rheumatoid arthritis but few or no cells in the synovial lining of patients with non-inflammatory diseases. The Ia-positive synovial cells with a dendritic morphology were contained in this population. An additional population, designated type III, contained nearly all of the remaining cells and was defined by the presence of antigens expressed primarily on fibroblasts and by the absence of phagocytosis, demonstrable Ia antigens, and four antigens of the monocyte lineage. This population exhibited proliferative capacity, becoming the predominant cell in long-term cultures. The proportions of each population varied considerably from patient to patient.

Human mononuclear phagocytes differentiation antigens.

Cell surface antigenic analysis using 10 different monoclonal antibodies indicates the following. (a) The membrane antigenic phenotype of various members of the mononuclear phagocyte lineage is characterized by the co-expression of multiple but distinct antigenic determinants. (b) The maturational evolution of the cells is associated with qualitative and/or quantitative modifications of cell surface antigens resulting in the definition of different phenotypic entities. (c) In vitro culture of blood monocytes is accompanied by significant modulation of these antigens. In particular, the expression of MoP-15 appears to be modulated following in vitro activation. (d) The occurrence of these antigens on various normal and leukemic cell populations provides a basis for the establishment of defined patterns of antigenic expression corresponding to successive maturational states of the human mononuclear phagocyte cell lineage. (e) The reactivity of AML cells with various antibodies defines patterns of antigen expression related to the degree of leukemic cell maturity. These patterns change during culture.