A monoclonal antibody (ER-HR3) against murine macrophages. I. Ontogeny,distribution and enzyme histochemical characterization of ER-HR3-positive cells

We describe ER-HR3, a monoclonal antibody directed against bone marrow-derived haemopoietic reticulum cells. ER-HR3-positive cells have the electronmicroscopic and enzyme-histochemical characteristics of macrophages. Additionally, they are able to phagocytoze. The ER-HR3 antigen is expressed by a majority of blood monocytes and is present on a subpopulation of resident macrophages in multiple organs. ER-HR3-positive cells are abundant in the bone marrow, the splenic red pulp, the mesenteric lymphoid paracortex and the interfollicular areas of the Peyer's patch. Few ER-HR3-positive cells have been observed in the thymic cortex and the connective tissues of the gastro-intestinal tract, the dermis and the renal medulla. Moreover, epidermal Langerhans cells express the antigen. No cross-reactivity with other cell types has been found. It is concluded that ER-HR3 has a unique distribution pattern distinct from other macrophage-specific antibodies.     

Role of the L3T4 antigen in T-cell activation. I. Description of a monoclonal IgM antibody to a distinct epitope (L3T4b) of the L3T4 antigen and its effect on interleukin 1-induced thymocyte proliferation

This report describes a new rat monoclonal IgM/k antibody, monoclonal antibody (MAb) 2B6, which reacts with a cell surface antigen present on a subpopulation of both thymocytes (85%) and peripheral T lymphocytes (55-60%). The antigen recognized by MAb 2B6 has multiple properties in common with the L3T4 antigen, as defined by the recently described MAb GK1.5. Thus, MAb 2B6 and MAb GK1.5 give very similar flow cytometry staining patterns on thymocytes, purified spleen T cells and all tested T-cell hybridomas. Depletion of MAb 2B6-positive cells with antibody and complement led to simultaneous depletion of MAb GK1.5-positive cells, and vice versa. Depletion of Lyt 2-positive cells led to enrichment of both MAb 2B6- and MAb GK1.5-positive cells. Both MAb 2B6 and MAb GK1.5 immunoprecipitate the same pattern of cell surface molecules from detergent extracts of radiolabeled thymocytes, the main components being a 55-kDa and a 115-kDa band. We therefore conclude that MAb 2B6 reacts with the L3T4 antigen. Interestingly, MAb 2B6 and MAb GK1.5 do not cross-block and therefore most probably react with distinct epitopes on the L3T4 molecule. The determinant recognized by MAb GK1.5 is called L3T4a. We suggest that the determinant recognized by MAb 2B6 be named L3T4b. As MAb 2B6 was selected for its ability to inhibit the action of interleukin 1 (IL-1) in the thymocyte costimulator assay, it is likely that the L3T4 molecule is functionally involved in the events taking place during IL-1 induction of thymocyte proliferation.     

Detection of phenylalanine hydroxylase protein in human platelets. Immunochemical detection

A protein reactive with anti-phenylalanine hydroxylase monoclonal antibody PH8 has been recovered from human platelet extracts. Two bands corresponding to molecular masses of about 60 kDa and 55 kDa were revealed by immunoblotting after electrophoresis according to Laemmli. Using the same antibody, a single band with a molecular mass of 60 kDa was demonstrated in extracts from human pineal gland; two similar antigens were found in human liver extracts and no antigen was found in adrenal gland extracts. Monoclonal antibodies, PH1 and PH3, did not react with these antigens during immunoblotting. Monoclonal antibodies, PH7 and PH9, reacted with the 55 kDa antigen in platelet extracts. The antigen content in platelet extracts was measured by ELISA with monoclonal antibodies relative to its content in the liver. The antigen content in platelet extracts was about 100 times as low as that in liver extracts and amounted to 100 ng/mg of protein. These findings suggest that the phenylalanine hydroxylase antigen is present in human platelets.     

Immunocytochemical study of lysosomal proteins with a new monoclonal antibody directed against epithelioid macrophages

 A monoclonal antibody, EPI-1, was produced against macrophages in epithelioid granulomas induced in rat foot pads by muramyl dipeptide. This EPI-1 antibody reacted to lysosome-like structures in epithelioid macrophages, peritoneal and pulmonary macrophages, and also in other tissues such as liver, testes, and kidneys. Western blot analysis of epithelioid granulomas, liver, testes, and kidneys revealed the same positive band of 62 kDa. Immunoelectron microscopic study of foot pad granulomas and hepatocytes demonstrated the EPI-1 antigen located in lysosomes and autophagic vesicles, preferentially along their membranes. These findings suggest that the EPI-1 antibody may recognize a novel antigen related to lysosomal membrane proteins in macrophages and other cells, which is useful for identifying lysosomes and their related structures. Accepted: 14 July 1997     

A monoclonal antibody identifies a glycoprotein complex involved in cell-substratum adhesion

The monoclonal antibody CSAT has been reported to perturb the adhesion of chick embryo cells to their substratum (Neff et al. [19]). Evidence is presented here that the antigen recognized by this monoclonal antibody is comprised of three membrane glycoproteins. The antigen is released from cells with non-ionic detergent and purified by monoclonal antibody affinity chromatography. When analysed by SDS-PAGE under non-reducing conditions, the antigen resolves into three components of apparent molecular weights 160 000 (band 1), 135000 (band 2), and 110 000 (band 3). Following reduction of each component, bands 1 and 2 migrate at slightly lower apparent molecular weights, while band 3 migrates at a higher apparent molecular weight, suggesting that band 3 has an internal disulfide bond. All three bands differ from one another as determined by peptide mapping and by immunologic cross-reactivity. It is postulated that the three glycoproteins function as a complex that plays a central role in cell-substratum adhesion.     

A monoclonal antibody to a novel differentiation antigen on human macrophages associated with the down-regulatory phase of the inflammatory process

A monoclonal antibody (RM3/1), raised by immunizing mice with human monocytes, is described which detects a surface antigen on about 20% of freshly isolated peripheral blood monocytes and is increasingly expressed upon cultivation, reaching a maximum between day 2 and 3. By incubation of monocytes with interferon-gamma, 12-O-tetradecanoylphorbol-13-acetate and lipopolysaccharide, antigen expression is decreased but strongly enhanced after incubation with dexamethasone. In cryostat sections of normal tissue, the antibody detects histiocytes in the skin, Kupffer cells in the liver, few alveolar macrophages in the lung, macrophages in the red pulp of the spleen and in the cortex of the thymus, and many macrophages in the placenta. In acute inflammatory tissue, e.g. gingivitis, the antigen is preferentially expressed by macrophages appearing late in the inflammatory process. In chronic inflammation, e.g. BCG granulomas and rheumatoid arthritis, RM3/1-positive macrophages are seen to varying degrees. Double-staining experiments with the antigen 25F9, specific for resting mature macrophages, revealed that RM3/1 and 25F9 are expressed by distinct populations in normal and acute inflammatory tissues. From this it is concluded that the antibody RM3/1 specifically detects a macrophage phenotype which seems to be associated with the healing phase of the inflammatory process.     

A subpopulation of bone marrow cells depleted by a novel antibody, anti-Liv8, is useful for cell therapy to repair damaged liver

We previously reported a new in vivo model named as "GFP/CCl(4) model" for monitoring the transdifferentiation of green fluorescent protein (GFP) positive bone marrow cell (BMC) into albumin-positive hepatocyte under the specific "niche" made by CCl(4) induced persistent liver damage, but the subpopulation which BMCs transdifferentiate into hepatocytes remains unknown. Here we developed a new monoclonal antibody, anti-Liv8, using mouse E 11.5 fetal liver as an antigen. Anti-Liv8 recognized both hematopoietic progenitor cells in fetal liver at E 11.5 and CD45-positive hematopoietic cells in adult bone marrow. We separated Liv8-positive and Liv8-negative cells and then transplanted these cells into a continuous liver damaged model. At 4 weeks after BMC transplantation, more efficient repopulation and transdifferentiation of BMC into hepatocytes were seen with Liv8-negative cells. These findings suggest that the subpopulation of Liv8-negative cells includes useful cells to perform cell therapy on repair damaged liver.     

Marginal zone macrophages in the mouse spleen identified by a monoclonal antibody. Anatomical correlation with a B cell subpopulation

The reactivity of a monoclonal antibody, ER-TR9, that demonstrates heterogeneity among mononuclear phagocytes is described. In the spleen, ER-TR9 exclusively reacts with a population of macrophages located in the marginal zone. ER-TR9 does not react with macrophage antigen 1-positive red pulp macrophages or any other types of splenic stromal cells. ER-TR9+ ve cells localize in anatomical proximity of a subpopulation of B cells, i.e., B cells that are immunoglobulin M positive and weakly positive to negative for immunoglobulin D. The possible significance of this particular interaction between both cell types during the immune response is discussed.     

Preliminary characterization of the urease and a 96 kDa surface-expressed polypeptide of Ureaplasma urealyticum

Analysis by SDS-PAGE of Ureaplasma urealyticum (predominantly serotype 8), propagated in a growth medium containing 10% (v/v) foetal calf serum, revealed a complex series of polypeptides apparently free of medium contaminants. Serological analysis using an immobilized antibody reagent, and immunoblotting using a polyclonal serum, showed the presence of two major and several minor antigens. One major antigen, a putative surface component of apparent molecular mass 96 kDa was shown, with a monoclonal antibody, to be serotype-specific. Growth of the organism was partially suppressed in the presence of the antibody. The second major antigen had an apparent molecular mass of 76 kDa and was presumed to be an internal component since it failed to label with the Bolton and Hunter reagent, in contrast to the 96 kDa antigen. Another monoclonal antibody was characterized which detected the canonical urease enzyme of the organism serotype 8 and of the two other human serotypes tested. Purification of this urease antigen by affinity chromatography and electrophoretic analysis of polypeptides after denaturation revealed a single polypeptide of molecular mass 76 kDa, putatively related to the above major antigen. Enzymic activity could be recovered after purification and demonstrated by in situ techniques only when electrophoretic analysis was done under non-denaturing conditions suggesting that the functional enzyme is a multimeric complex.     

Antigenic relatedness between phospholipases A2 from Naja naja venom and from mammalian cells

Polyclonal antiserum was prepared against phospholipase A2 from Naja naja and used to prepare a purified antibody. It cross-reacted with the antigen, and with intracellular mammalian PLA2. This antibody was immunoreactive and inhibited the PLA2 activity of Naja naja and of guinea pig alveolar macrophages or rat lymphocytes. By immunoblotting, this antiserum revealed one band of PLA2 from Naja naja (14 kDa) and 3 bands for guinea pig alveolar macrophages and rat lymphocytes (30, 45 kDa and a minor band of 14 kDa). These results show an antigenic relatedness between an extracellular PLA2 and membrane-bound PLA2 from two different mammalian species and cell types.     

Immunocytological and biochemical characterization of a new neuronal cell surface component (L1 antigen) which is involved in cell adhesion.

Monoclonal and polyclonal L1 antibodies react by indirect immunofluorescence with the cell surface of cultured tetanus toxin-positive neurons from post-natal cerebella of mice, but not with glial fibrillary acidic protein-positive astrocytes, O4 antigen-positive oligodendrocytes or fibronectin-positive fibroblasts or fibroblast-like cells. During cerebellar development L1 antigen is detectable on tetanus toxin-positive cells as early as embryonic day 13 after 3 days in culture. In sections of the early post-natal cerebellum, L1 antigen is found on pre-migratory neurons in the internal, but not in the external part of the external granular layer. In the adult cerebellum, L1 antigen is predominantly localized in the molecular layer and around Purkinje cells. Fibers in white matter and the granular layer are also L1 antigen-positive. Granule cell bodies and synaptic glomeruli are weakly antigen-positive. Several cell lines derived from neuroblastoma C1300 also express L1 antigen. The antigen is not detectable by enzyme-linked immunosorbent assay in tissue homogenates of liver, kidney, lung, heart, sperm or thymus. With polyclonal L1 antibodies, cross-reactive determinants are found in brains of rat, guinea pig, hamster, chicken, rabbit and man, but not in frog, while monoclonal antibody reacts detectably only with mouse brain. The molecular species recognized by both monoclonal and polyclonal antibodies display two prominent bands by SDS-PAGE under reducing and non-reducing conditions with apparent mol. wts. of 140 and 200 kd. L1 antigen isolated from cultured cerebellar cells consists mainly of a band in the 200-kd range and a faint one at 140 kd. L1 antigen from neuroblastoma N2A shows two bands with slightly higher apparent mol. wts. All molecular forms of L1 antigen can be labeled by [3H]fucose and [3H]glucosamine. Ca2+-independent re-aggregation of cerebellar cells from early post-natal C57BL/6J mice and of the continuous cell line N2A derived from the murine neuroblastoma C1300 is inhibited by Fab fragments of the polyclonal, but not of monoclonal antibody, both of which are known to react with the surface membrane of these cells.     

A novel 57 kDa peroxisomal membrane polypeptide detected by monoclonal antibody (PXM1a/207B)

BALB/c mice were immunized with peroxisomal membranes prepared from rat liver. Spleen cells were fused with myeloma cells (P3/U1) and the hybridomas were selected using peroxisomal membranes. A monoclonal antibody (PXM1a/207B) which recognized peroxisomal membranes was selected. Using the antibody, a novel 57 kDa polypeptide was identified in the peroxisomal membrane fraction. Immunoblot analysis of the subcellular fractions demonstrated that the 57 kDa polypeptide was present exclusively in peroxisomal membranes. The 57 kDa polypeptide was partially digested by trypsin and chymotrypsin under conditions where peroxisomal particles remained intact, indicating that the polypeptide is exposed to the cytosolic face of the peroxisomal membrane. The amount of 57 kDa polypeptide increased in parallel with proliferation of peroxisomes by administration of clofibrate.     

Isolation and characterization of a monoclonal antibody that inhibits HIV-1 infection

To identify a cell surface molecule other than CD4 involved in infection of cultured cells with human immunodeficiency virus type 1 (HIV-1), mice were immunized with the CD4-negative Raji human B-cell line in order to isolate a monoclonal antibody (mAb). We isolated mAb 33A, which inhibited the infection of CD4-positive T cells, B cells, human peripheral blood lymphocytes (PBL), and brain-derived cells with HIV-1. Formation of viral DNA was also blocked when CD4-positive Raji cells were treated with 33A after adsorption of HIV-1, but not before its adsorption. mAb 33A had little effect on syncytium formation induced by cocultivation with HIV-1-producing cells. Flow cytometry revealed that 33A reacted with HTLV-I-positive T-cell lines, Burkitt's lymphoma cell lines, phytohemagglutinin (PHA) -stimulated PBL, brain-derived fibroblast-like cells, and some adherent cell lines, but hardly at all with immature T-cell lines. Immunoblotting experiments showed that 33A recognized an antigen with an apparent molecular mass of 32 kDa, but did not recognize chemokine receptors such as CXCR4, CCR5, or CCR3. The distribution characteristic of the antigen recognized by 33A on various cells and its molecular weight suggest that mAb 33A recognizes a new cellular antigen that is necessary for HIV-1 entry.     

Tyrosine kinases in normal human blood cells. Platelet but not erythrocyte band 3 tyrosine kinase is p60c-src

The major tyrosine kinase from platelets was purified as a 51 kDa active enzyme which was shown to be a degradation product of the protooncogene product p60c-src. Immuno-depletion experiments using a monoclonal antibody recognizing p60c-src failed to remove band 3 phosphorylating activity from red blood cell membranes. The erythrocyte tyrosine kinase was not at all immunoprecipitated by this antibody under conditions where the platelet enzyme was immunoprecipitated.     

Immunoelectron microscopic detection of band 3 protein during erythroid cell differentiation by a monoclonal antibody

We created a monoclonal antibody, designated EB1 (IgM, kappa), that reacts with erythroblasts by fusion of P3-X63-Ag8.653 with splenocytes of rats immunized with erythroblastic islands isolated from mice spleens. Western blotting revealed that EB1 reacted with the band 3 protein of the erythrocytic membrane. It stained erythrocytes and erythroblasts, forming clusters in the bone marrow, splenic red pulp, and fetal liver, but did not stain other tissues in the cryostat sections. The EB1 antigen was detected during dimethyl sulfoxide-induced differentiation of murine erythroleukemia cells. Immunoelectron microscopy revealed that the EB1 antigen was expressed from the basophilic erythroblasts during normal erythroid differentiation. Preferential segregation of the EB1 antigen on the cell membrane of the nucleating erythroblasts was not observed. These results suggest that EB1 is specific for erythrocyte band 3 protein and may be useful for studying erythroid cell differentiation.     

Murine cell surface glycoproteins. Purification of the polymorphic Pgp-1 antigen and analysis of its expression on macrophages and other myeloid cells

We previously reported the initial characterization of a polymorphic major cell surface glycoprotein of about 80,000 daltons from mouse embryo 3T3 cells. This glycoprotein has now been purified 1800-fold to apparent homogeneity by monoclonal antibody affinity chromatography. The purified molecule retained the total antigenic activity of the cell, as determined by antibody binding assays. The quantity of the glycoprotein, 0.06% of the total protein of the crude cell extract, confirmed its presence as a major constituent of the cell plasma membrane. The monoclonal antibody was also used to detect related antigens in cells and tissues of C57BL/6J mice. The antigen was present in high concentration in macrophages and subpopulations of bone marrow and blood polymorphonuclear cells. Much lower concentrations of antigen were detected in spleen cells, thymocytes, and extracts of solid tissues. The apparent Mr of the target antigen of myeloid cells was 92,000. This molecule was a major surface constituent of myeloid cells with 10(6) antibody binding sites per cell containing 10% of total 125I incorporated by the lactoperoxidase procedure. The macrophage glycoprotein labeled on the cell surface with 125I was highly sensitive to trypsin, yielding an antigenically active soluble glycopolypeptide of about 65,000 daltons, that contained all of the incorporated 125I. A similar 65,000-dalton glycopeptide was released from 3T3 cells by trypsin cleavage. These data indicate that a major cell surface constituent of mouse myeloid cells is a 92,000-dalton glycoprotein closely related to the 80,000-dalton glycoprotein of mouse embryo 3T3 cells.     

Identification, immunoaffinity purification and partial characterization of a human decidua-associated protein

A crude extract of pooled early-pregnancy decidual tissue was enriched for soluble decidual proteins by exhaustive affinity absorption with antibodies to human serum proteins immobilized on Eupergit C. The partly purified extract was used to prepare monoclonal antibodies. A monoclonal antibody was obtained recognizing an antigen present in extract of decidual tissue and not in extract of proliferative endometrium. The monoclonal antibody was used for immunoaffinity purification of the decidua-associated protein. By SDS-PAGE analysis, under reducing conditions it yielded 2 bands at apparent molecular weights of 55,000 and 25,000. Under non-reducing conditions a single protein band at apparent molecular weight of 200,000 was observed. The Mr 200,000 protein was named hDP200 and the Mr 55,000 protein was named hDP55. It is suggested that hDP55 is a subunit of the hDP200. The hDP200 did not react with polyclonal antibodies specific for PP12 and PP14. PP14 has been shown to be immunologically indistinguishable from PEP and alpha 2-PEG. Our data therefore suggest that hDP200 is a novel human decidua-associated protein.     

Antigen presentation in the rat. II. An Ia+ radiosensitive T cell can present antigen to primed Ia- T cells

We demonstrated previously the presence of an Ia+ (OX-6+) antigen-presenting cell within the rat T cell fraction that is capable of presenting antigen to antigen-primed OX-6-T cells. This antigen-presenting cell (T-APC) reacted with the monoclonal antibodies W3/25 and W3/13, which is known to react mainly with rat T cells. Further characterization of the T-APC indicated that the cell also reacted with the monoclonal antibody OX-19, which is highly specific for rat T cells. Moreover, the antigen-presenting function of the T-APC was sensitive to treatment with mitomycin C or gamma-irradiation (2000 rad). Under similar conditions, antigen presentation by partially purified dendritic cells or macrophages was totally resistant to these treatments. The antigen-presenting activity of gamma-irradiated T-APC was not reconstituted by the addition of the lymphokines IL 1, IL 2, or Con A supernatants. Although unirradiated T-APC were able to stimulate an MLR response, this function was also sensitive to gamma-irradiation, whereas the MLR-stimulating ability of macrophages and dendritic cells was resistant to gamma-irradiation. These data indicate that Ia+ T cells from the rat are capable of presenting antigen to antigen-primed T lymphocytes and that, in contrast to antigen presentation by macrophages and dendritic cells, the function of T-APC is gamma-radiation sensitive.     

Involvement of Kupffer cells in lipopolysaccharide-induced rapid accumulation of platelets in the liver and the ensuing anaphylaxis-like shock in mice

Intravenous injection of Klebsiella O3 lipopolysaccharide (LPS) into BALB/c mice induces an anaphylaxis-like shock within minutes. Using 5-hydroxytryptamine as a marker for platelets, we previously suggested that a rapid platelet accumulation in the liver and lung precedes the shock, and that a complement-dependent platelet-degradation is involved in the shock. Here, we examined (i) the effect of platelet-depletion (using an anti-platelet monoclonal antibody) on the shock and (ii) the contribution of macrophages to the platelet-accumulation in those organs. LPS-induced platelet-accumulations in the liver and lung were confirmed by immunostaining. In platelet-depleted mice, the shock was largely prevented. The number of F4/80-positive macrophages was much greater in liver than in lung, and the hepatic macrophages were largely lost in mice given clodronate-encapsulated liposomes. In mice treated with such liposomes, both the LPS-induced accumulation of platelets in the liver (but not in the lung) and the shock were largely prevented, and repopulation of hepatic macrophages restored these LPS-induced responses. These results suggest that (i) platelets are indeed involved in the shock, (ii) Kupffer cells mediate the hepatic platelet accumulation, and (iii) preventing this hepatic accumulation can largely prevent rapid shock being induced by LPS (at the dose used here).     

An antigen cross-reactive with gp52 of mammary tumor virus is expressed on a B cell subpopulation of mice

The reactivity of murine lymphoid tissue with biotinylated F(ab')2 fragments of monoclonal antibody VE7 (BIOT VE7), which reacts with gp52 of murine mammary tumor virus (MuMTV), was tested with fluoresceinated avidin in an indirect fluorescent antibody assay on live splenocytes. A small percentage of splenic lymphocytes in C3H/He mice infected exogenously with MuMTV, and in C57BL/6 mice that, like C3H/He, harbor several endogenous MuMTV proviruses in their genomes, were reactive with the monoclonal antibody. The antigen-positive splenocytes were shown to represent a subpopulation of B cells. The possible nature of the B cell-associated antigen recognized by monoclonal antibody VE7 is discussed.