Myeloid-associated antigen 3-alpha-fucosyl-N-acetyllactosamine (FAL): location on various granulocyte membrane glycoproteins and masking upon monocytic differentiation

Seven different granulocyte-reactive murine monoclonal antibodies (mAb) were studied. The antigens recognized by these mAb were immunoprecipitated from lysates of 125I-labeled granulocytes of healthy donors. The isolated antigens were analyzed by electrophoresis on sodium dodecyl sulfate-polyacrylamide gel and autoradiography. All 7 antibodies precipitated the same 6 membrane polypeptides from membrane-iodinated granulocyte lysates: 105 and 150-kDa as most pronounced, together with 260-, 230-, 67- and 52-kDa polypeptides. One of the antibodies studied, B4.3, is directed against 3-alpha-fucosyl-N-acetyllactosamine as shown by absorption with the synthesized carbohydrate molecule. Competition experiments with 125I-labeled B4.3 demonstrated complete inhibition of binding by B4.3 and 3 of the other antibodies (VM D5, UJ308, MI/N1) and partial inhibition by the 3 other antibodies (FMC 10, FMC 12, FMC 13), indicating binding to the same antigenic structure. None of the 7 mAb reacted with monocytes in the immunofluorescence technique, but after neuraminidase treatment of these cells, positive reactions were obtained with all mAb. Immunoprecipitation with lysates of both native and neuraminidase-treated monocytes showed no polypeptide bands. Monocytic differentiation of the cell line HL60 by 12-O-tetradecanoylphorbol-13-acetate (TPA) and of cell line U 937 by dimethylsulfoxide and TPA was accompanied by a decrease in reactivity with these antibodies, which could be recovered by neuraminidase treatment. This indicates that 3-alpha-fucosyl-N-acetyllactosamine is masked for the detection of the antibody upon monocytic differentiation by sialylation.     

Application of a novel immunization protocol to the production of monoclonal antibodies specific for macrophages in human placenta.

A monolayer depletion/adoptive immunization protocol that biased the immune response towards recognition of placental macrophage (pMO) antigens was established. BALB/c spleen cells immune to human pMO were adsorbed onto monolayers of the B-cell line QIMR-WIL. Monolayer-depleted or unfractionated cells were transferred to irradiated recipients, which subsequently were restimulated with pMO then killed for hybridoma production. Screening of hybridomas revealed an increased proportion of pMO-specific hybridomas following transfer and fusion of monolayer-depleted cells. Two monoclonal antibodies (mAb), L9 and L21, which were generated through application of this protocol, are described. L9 recognized an antigen on cells within the villi in sections of term placenta and freshly isolated pMO. With time in culture, expression of this antigen decreased markedly. Macrophages, but no other cell type, in placental cell suspensions expressed this antigen. L9 failed to react with any peripheral blood cells. Immunoprecipitation and SDS-PAGE analyses indicated that two proteins of molecular weight (MW) 40,000 and 43,000 were recognized by L9. Sections of term placenta and freshly isolated pMO failed to react with L21. After 2-3 days in culture, however, most macrophages expressed this antigen. L21 reacted weakly with peripheral monocytes and granulocytes but not other normal peripheral blood cells. Myeloid cell lines reacted strongly with this mAb only after activation with PMA. SDS-PAGE analyses of the L21 immunoprecipitate under non-reducing conditions revealed a single band of 61,000 MW, while two bands of 46,000 and 49,000 MW were detected under reducing conditions. Cellular distribution and molecular weight analyses indicated that the antigens recognized by these two mAb were apparently distinct from previously defined myeloid antigens.     

Monoclonal antibodies to human neutrophil Fc gamma RIII (CD16) identify polypeptide epitopes.

Human neutrophils constitutively express two low-affinity Fc gamma R, Fc gamma RII (CD32) and Fc gamma RIII (CD16). Eleven monoclonal antibodies (mAb) to CD16 were used to identify antigenic differences among Fc gamma RIII-bearing cells, to define functional epitopes of Fc gamma RIII on neutrophils, and to characterize biochemically the epitopes identified by some of these mAb. Flow cytometry demonstrated that 9 of the 11 mAb reacted with neutrophils, 10 of the 11 reacted with natural killer cells, and 9 of 11 reacted with monocytes and monocyte-derived macrophages. These mAb reacted with CD16 positive cells with varying fluorescence intensities. The ability of anti-CD16 mAb to block the binding of 125I-labeled immune complexes to neutrophils was examined. Four monoclonal antibodies strongly inhibited (87-96%) the binding to neutrophils of 125I-labeled immune complexes. Competitive binding assays were performed to determine whether any other anti-CD16 mAb identify the epitope identified by mAb 3G8. Two other mAb, CLBFCGRAN 1 and CLBGRAN 11, blocked binding of 125I-3G8 IgG to neutrophils. Six of the anti-CD16 mAb efficiently immunoprecipitated polypeptides of broad mobility ranging from 45 to 84 kDa from 125I-labeled neutrophils. When Fc gamma RIII, a complex sialoglycoprotein consisting of almost 50% oligosaccharides, was immunoprecipitated from neutrophils with 3G8 Fab Sepharose and subsequently digested with N-glycanase, 5 of the 6 mAb were capable of immunoprecipitating a deglycosylated polypeptide migrating at 29 kDa. These results demonstrate that these 5 mAb identify polypeptide epitopes of Fc gamma RIII, whereas 1 mAb, YFC120.5, may react with a glycosyl moiety or a determinant whose conformation is dependent on the presence of oligosaccharides.     

Monoclonal antibody AMH152 reacts with human monocytes in culture and with inflammatory macrophages.

Monoclonal antibodies (mAb) raised against human peritoneal macrophages were selected for their non-reactivity with freshly sampled blood cells. One of these mAb, AMH152, initially non-reactive, bound to monocytes after 18 h of culture, a property which was not shared by an unrelated antibody of the same isotype (IgG1). The induction of the expression of the antigen detected by AMH152 on monocytes in culture was not influenced by the addition of serum or by the substrate used, plastic that favoured adhesion or teflon bags. Overnight incubation at 4 degrees C in adhesion conditions did not enable antigen expression. A 1-h treatment with phorbol myristate acetate or formyl-methionyl-leucyl-phenylalanine did not increase AMH152 binding. Culturing monocytes with cycloheximide tended to inhibit antigen expression. These observations suggested that antigen expression represents an active phenomenon, requiring protein synthesis. The antigen recognized by mAb AMH152 could be visualized on sections of formalin-fixed and paraffin-embedded tissues. Macrophages of healthy lymphoid organs and tissues that expressed CD68 antigen failed to bind AMH152. In contrast, chronic inflammatory lesions, like those of sarcoidosis, tuberculosis and cat scratch disease, contained epithelioid and multinucleated giant cells that reacted with AMH152. In serous exudates of cancer metastases, 10-40% of macrophages were also stained. The antigenic material was essentially present at the cell periphery. Thus, mAb AMH152 recognized a surface antigen, detectable on paraffin-embedded tissue sections, and which accompanied differentiation of monocytes into inflammatory cells. The expression of this antigen on monocytes in culture suggests that these cells underwent an activation process, even when maintained for some hours in teflon bags and in a serum-free medium.     

Preparation and characterization of murine monoclonal antibodies to swine lymphocyte antigens

A panel of monoclonal antibodies (mAb) was developed by the fusion of Sp2/0 myeloma cells and spleen cells from mice immunized with peripheral blood mononuclear cells (PMNC) or T cells from NIH swine leucocyte antigen (SLA) inbred miniature swine. Twenty stable hybridoma clones were isolated that secreted mAb that reacted with swine PMNC, as determined by an enzyme-linked immunosorbent assay (ELISA). The binding profile to swine PMNC and the ability to fix complement of these mAb were investigated by flow cytometric analyses. The molecular weights of the antigens recognized by six of the mAb were determined by immunoprecipitation of 125I surface-labelled PMNC, followed by SDS-PAGE under reducing conditions. The most interesting mAb, 7-34-1 (IgG2a), precipitated a putative MHC class I molecule composed of a 50,000 MW heavy chain and a 12,000 MW light chain (beta 2m). This is the third SLA class I-reactive monoclonal antibody to be described for swine. Properties of the mAb described in this paper, mAb 7-34-1, are different from the two other SLA class I-specific mAb that have been described elsewhere in the literature (mAb 74-11-10 and mAb PT85). Monoclonal antibody 7-34-1 recognized class I antigens of SLA haplotypes a, c and d in an equivalent manner. This mAb should be especially useful as a general anti-SLA class I reagent for experiments on NIH miniature swine.     

A human renal cancer line as a new antigen source for the detection of antibodies to cytoplasmic and nuclear antigens in sera of patients with Wegener's granulomatosis.

Autoantibodies directed against cytoplasmic antigens of neutrophils (ANCA), especially proteinase 3 (C-ANCA), have proved to be a useful clinical tool to support the diagnosis or to monitor disease activity in Wegener's granulomatosis (WG). Till now, human neutrophil granulocytes have represented the major antigen source used to detect antibodies in WG by the immunofluorescence technique (IFT). We have tested serum samples of 164 patients with different connective tissue diseases (50 suffering from clinically active WG) performing IFT on a human renal cancer line (SK-RC11) and have found antibodies against the nuclear and cytoplasmic antigens in 39 patients. C-ANCA+ sera displayed a characteristic diffuse cytoplasmic staining pattern. Antibody titers measured with human granulocytes were comparable to titers obtained using culture cells. Antibody binding could be inhibited by preabsorption with an extract of human granulocytes or purified proteinase 3. A protein of 29 kDa MW could be isolated by affinity purification using a SK-RC11 extract and a high-titer C-ANCA+ serum and antigenic identity was further confirmed by IFT using a monoclonal antibody to proteinase 3. Treatment of tumor cells with cytokines (interferon, tumor necrosis factor) led to a time dependent translocation of the antigen into the nucleus and back to the cytoplasm. The antigen was also expressed on the surface of live cells colocalized with MHC II. In addition, 21 WG patients had antibodies to cytoplasmic organelles identified by laser scanning microscopy as secretory vesicles of the Golgi complex, and five had antibodies to nuclear antigens. This is, to the best of our knowledge, the first report of proteinase 3 in human non-leukemic cells. Our data demonstrate, that the repertoire of antigens recognized by antibodies in WG sera is not limited to human neutrophils and monocytes and indicates a possible functional role of the antigenic proteins.     

Immunocytochemical characterization of monocyte colonies of human bone marrow: a clue to the origin of Langerhans cells and interdigitating reticulum cells

Human bone marrow was cultured and cells from individual monocyte colonies stained with a variety of polyclonal and monoclonal antibodies. Erythroid colonies and peripheral blood monocytes were used as controls. Both bone marrow cultured and peripheral blood monocytes stained with antibodies to lysozyme, non-specific cross reacting antigen (NCA) and alpha-1-antitrypsin and the cells stained variably for HLA-DR. A small number of cells in each cultured bone marrow colony stained with antibody to human thymic antigen (HTA T6) and most of the cells stained with variable intensity using anti-S-100 protein. No cells reactive with these two antibodies were detected in peripheral blood monocyte preparations and erythroid colony cells were negative for all antigens studied. The presence of cells within individual bone marrow colonies with phenotypic properties of both phagocytic macrophages (lysozyme positive, NCA positive, alpha-1-antitrypsin positive) and Langerhans cells/interdigitating reticulum cells (HTA(T6) positive, S-100 protein positive) suggests that these cells share a common stem cell and are both components of the mononuclear phagocyte system.     

Utility of monoclonal antibodies directed against B and T lymphocytes and monocytes in paraffin-embedded sections

Monoclonal antibodies reactive with B and T lymphocytes, monocytes, and granulocytes were applied to B-5-, Bouin's-, or formalin-fixed paraffin-embedded sections. Most antigens were destroyed or masked by fixation or embedding procedures, or both. However, T200, an antigen present in all lymphoid and hematopoietic cells, and Leu M1, an antigen in granulocytes, were well preserved in formalin-fixed tissues. Leu 1 and BA-1, antigens present on T and B lymphocytes, respectively, were preserved in Bouin's-fixed specimens. With careful selection of fixatives, identification of some T and B lymphocytes and granulocytes by monoclonal antibodies in paraffin-embedded specimens is possible.     

The tissue distribution of the 3 alpha-fucosyl-N-acetyl lactosamine determinant recognized by the CD15 monoclonal antibodies CMRF-7 and 27

Two monoclonal antibodies, CMRF-7 and 27, which react with cells of the granulocytic series, were obtained from hybridomas cloned from separate fusions. Biochemical studies indicate that both antibodies are of the CD15 group and react with the antigenic determinant 3 alpha-fucosyl-N-acetyl lactosamine (hapten X) expressed on some glycolipids and several different granulocyte glycoproteins with a wide range of molecular weights. The antigen was found on some promyelocytes and more differentiated granulocytes, including neutrophils and some eosinophils, but not basophils. Monocytes, lymphocytes, and erythrocytes were negative for CMRF-7 but neuraminidase treatment revealed "cryptic" sites on monocytes and some lymphoid cells. The antibody CMRF-7 reacted with the majority of acute myeloid leukemia blasts in the FAB categories M2-M5 but less frequently with M1 blasts and was positive with only 5/43 acute lymphoid leukemias. Immunoperoxidase staining of other normal human tissues indicates that this determinant is found on a range of epithelial cells in skin, the gastrointestinal tract and the genitourinary system. In addition some parts of the central nervous tissue and some endocrine organs stained with these antibodies.     

Monoclonal antibodies against differentiation antigens on human macrophages

Human macrophages matured in vitro from blood monocytes without additional exogenous activation were used to produce monoclonal antibodies (mAbs). Ten of them were selected for high avidity and the best discrimination between mature macrophages and a panel of other cells including freshly prepared monocytes, B cells, T cells, granulocytes and thrombocytes. Five mAbs (MAX. 1, MAX. 2, MAX. 3, MAX. 11, MAX. 24) were found to detect macrophage differentiation antigens. One mAb (MAX. 26) detects an antigen which is shared by mature macrophages and T cells.     

The monoclonal antibody, UCHL1, recognizes a 180,000 MW component of the human leucocyte-common antigen, CD45.

The leucocyte-common antigen (L-CA or CD45) is a family of high molecular weight glycoproteins, ranging from 180,000 to 220,000 MW that are expressed only on cells of lymphoid and myeloid origin. CD45 monoclonal antibodies (mAbs) recognize epitopes present on all polypeptides of the family, while other mAbs, termed CD45R, recognize determinants found only on the 220,000 MW and 200,000 MW polypeptides. In contrast the mAb UCHL1 recognizes a 180,000 MW antigen. UCHL1-coupled Sepharose beads were used to absorb antigen from lysates of cell lines. CD45 mAbs bound to this immobilized antigen. Antigen immobilized with CD45 mAb-coupled Sepharose beads bound UCHL1. Antigen purified by absorption and elution from the MOLT-4 cell line with CD45 mAb-coupled beads yielded molecules of 180,000 and 190,000 MW. Reprecipitation of the eluted antigen with UCHL1 resulted in a 180,000 MW band only. In a reciprocal experiment, CD45 mAb reprecipitated a 180,000 MW molecule from purified UCHL1 antigen. UCHL1 and the CD45R mAb 2H4 showed a mutually exclusive pattern of reactivity with human T- and B-cell lines, but co-expression of the antigens was seen on two myeloid and one erythroleukaemic cell line. In contrast, epitopes recognized by other putative CD45R mAbs were co-expressed with UCHL1 both on myeloid, erythroid and many T- and B-cell lines. We conclude that UCHL1 recognizes an epitope present only on the 180,000 MW polypeptide of CD45. Expression of this antigen is essentially reciprocal to the epitope detected by the CD45R mAb 2H4.     

Discrimination of Human Macrophages and Dendritic Cells by Means of Monoclonal Antibodies

Two monoclonal antibodies (MoAb), RFD1 and RFD7, have been used to investigate whether human macrophages and dendritic cells represent phenotypically distinct cell types. RFD7 recognizes a 77 kd antigen, and is specific for acid phosphatase positive tissue macrophages, while RFD1 recognizes a unique Class II antigen, which is associated with dendritic cells. With immunohistological/cytological methods it was found that neither of these reagents reacted with granulocytes, monocytes, or lymphocytes, with the exception that a small proportion (less than 20%) of B cells were stained with RFD1. In tissues, RFD7 reacted with mature macrophages only and did not stain Langerhans cells in the skin or the interdigitating (dendritic) cells of the T-cell zones of lymphoid tissue and the thymic medulla. Conversely, RFD1 appeared specific for the interdigitating (dendritic) cells and did not react with macrophage populations identified morphologically, geographically, and histochemically in any tissue studied. When peripheral blood monocytes (RFD1-, RFD7-) were matured in vitro, two distinct populations of RFD1+ RFD7- and RFD7+ RFD1- cells emerged. It is concluded that in normal tissues these two reagents identify phenotypic differences between macrophages and dendritic cells that may have functional significance.     

Mac-1: a macrophage differentiation antigen identified by monoclonal antibody.

We have previously described the derivation of M1/70, a hybrid myeloma line secreting monoclonal rat anti-mouse cell surface antibody (Springer, T., Galfre, G., Secher, D. S. and Milstein, C, Eur. J. Immunol. 1978. 8: 539). We have now investigated the cellular distribution of this antigen using a ¹²⁵I-labeled anti-rat IgG indirect binding assay, the fluorescence-activated cell sorter, autoradiography and precipitation of cell surface molecules. Screening with a tumor cell panel showed strong reactivity with a macrophage-like line but no reactivity with B or T lymphoma lines. In normal tissues, M1/70 antigen was found to be present in small amounts on spleen and exudate granulocytes and a subpopulation of bone marrow cells, in moderate amounts on spleen and blood monocytes and expressed in much larger amounts on spleen histiocytes and peritoneal exudate macrophages. In contrast, M1/70 antigen was found to be absent from erythroid and lymphoid cells. M1/70 antibody precipitated two polypeptides of 190 000 and 105 000 mol. wt. which were present in much greater amounts on peritoneal exudate macrophages than on spleen cells. The expression on phagocytes of two other antigens identified by monoclonal antibodies M1/69 and M1/9.3 was also examined. Monocytes and granulocytes expressed large amounts of M1/69 and low amounts of M1/70 antigen, while in peritoneal exudate macrophages this pattern was dramatically reversed. M1/70 thus defines a differentiation antigen on mononuclear phagocytes and granulocytes, the expression of which is specifically increased during monocyte maturation. This antibody is the first to be described which recognizes a discrete molecule specific to phagocytes.     

A murine cytomegalovirus-neutralizing monoclonal antibody exhibits autoreactivity and induces tissue damage in vivo.

The autoreactivity of murine cytomegalovirus (MCMV)-neutralizing monoclonal antibody (mAb) AC1 was examined in vitro and in vivo. Both mAb AC1 and a human antiserum reactive with U1-small nuclear ribonucleoprotein (U1-snRNP) stained uninfected mouse embryo fibroblasts (MEF) in a speckled nuclear pattern and reacted with 70,000 molecular weight (MW) MEF nuclear antigens by immunoblotting, suggesting that mAb AC1 cross-reacted with the 70,000 MW component of U1-snRNP. However, only mAb AC1 cross-reacted with an additional epithelial cytoplasmic autoantigen present in cultured HEp2 cells. On tissue sections from uninfected mice, mAb AC1 predominantly reacted with a component of central and peripheral nervous systems, although cross-reactivity with the stratum spinosum of the skin and the outer sheath of hair follicles was also observed. Immunoblotting revealed that mAb AC1 reacted with phosphorylated epitopes present on a 98,000 MW MCMV structural protein and the 200,000 MW mouse neurofilament protein (NFP). Treatment of uninfected mice with mAb AC1 resulted in a severe interstitial pneumonia with greatly thickened and congested alveolar septa. Severe oedema of the hypodermis and a mild mesangial proliferative glomerulonephritis were also observed. These results demonstrate that a mAb reacting with a MCMV structural phosphoprotein which can protect mice against the dissemination of MCMV, can also promote the development of autoimmune disease. Therefore, the production of such cross-reactive antibodies may be an important mechanism in the development of autoimmunity following viral infection.     

Subpopulations of guinea-pig T lymphocytes defined by isoforms of the leucocyte common antigen.

This report presents the characterization of three mouse monoclonal antibodies (mAb) reactive with the guinea-pig leucocyte common antigen (LCA); CD45 in the human nomenclature. One, IH-1, reacted with LCA on all leucocytes. The other two were more restricted: IH-2 recognized only the 220,000, 210,000 and 195,000 MW isoforms, and IH-4 the 220,000, 210,000 MW isoforms. Both IH-2 and IH-4 reacted with all B cells and all Kurloff cells [the putative guinea-pig natural killer (NK) cell]. IH-2, but not IH-4, reacted with monocytes and macrophages. Neither reacted with neutrophils. Most thymocytes expressed low levels of the IH-2 and IH-4 epitopes, with those expressing high levels located predominantly within the medulla. Most (90%) CD4+ T cells from newborn guinea-pigs expressed high levels of the IH-2 and IH-4 epitopes; this percentage decreased with age to 70% in 2-year-old animals. We have demonstrated that CD4+ T cells which express low levels of the IH-2 epitope also express low levels of the IH-4 epitope. CD8+ T cells can be divided into two subsets by IH-4 but not IH-2. The reactivities of IH-2 and IH-4 are remarkably similar to those of human anti-CD45RB and anti-CD45RA antibodies respectively. Analogies with man and other species suggest important functional differences for subpopulations of guinea-pig T lymphocytes defined by anti-CD45R antibodies.     

Macrophage heterogeneity in the rat as delineated by two monoclonal antibodies MRC OX-41 and MRC OX-42, the latter recognizing complement receptor type 3.

Two monoclonal antibodies, designated MRC OX-41 and MRC OX-42, have been shown to label subsets of macrophages. Using immunoperoxidase and immunofluorescence analysis, tissue macrophages were shown to be heterogeneous with respect to binding of MRC OX-41 and MRC OX-42 antibodies. Although both antibodies labelled subsets of macrophages, the antibodies also reacted with granulocytes and dendritic cells. The antigens recognized by these antibodies were identified by metabolic and cell surface labelling followed by immunoprecipitation and sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). MRC OX-41 recognized a surface protein of 110,000-120,000 MW, while MRC OX-42 immunoprecipitated three polypeptides with molecular weights of 160,000, 103,000 and 95,000. The Fab fragment of MRC OX-42 antibody inhibited complement-mediated rosette formation between sensitized erythrocytes and rat macrophages and granulocytes. Membrane molecules with similar biochemical and functional properties to MRC OX-42 antigen have been identified in mouse and man as the receptors for iC3b, and it is probable that MRC OX-42 antibody recognizes the rat homologue of the receptors in these other species.     

Phenotypical and biochemical characterization of a variant CD45R expression pattern in human leukocytes.

As a result of a regulatory polymorphism a variant pattern of CD45R expression can be found in 8% of healthy individuals. We have previously reported that all resting T cells of these individuals react with CD45RA monoclonal antibodies (mAb) that are directed to the high-molecular mass isoforms (220 and 205 kDa) of the leukocyte common antigen (CD45). In addition, their T cells remain CD45RA+ after in vitro activation. In this report we studied the regulatory CD45R polymorphism in more detail. Flow cytometry studies revealed that CD45RA antigens are weakly expressed on normal monocytes but are absent from granulocytes. In individuals displaying the variant CD45R pattern both monocytes and granulocytes were CD45RA+. In controls and variant T cells CD45RA mAb precipitated two chains with molecular masses of about 220 and 205 kDa. In activated T cells from controls (CD45RA-) neither p220 nor the p205 chain of CD45RA could be detected. However, activated T cells from variant individuals (CD45RA+) did not express p220 but expressed p205 of CD45RA. These data indicate that there is a failure to down-regulate p205 CD45RA in individuals displaying the variant CD45R expression pattern. Since their T cells lost p220 CD45RA during activation the data also suggest that the two isoforms detected by CD45RA mAb can be regulated independently.     

A novel monoclonal antibody, Mar 1, directed specifically against mononuclear phagocyte system cells in rats.

Three different monoclonal antibodies (mAb), designated Mar 1, Mar 2, and Mar 3, recognizing three distinct novel antigen molecules expressed preferentially in rat macrophages, were produced by the hybridoma technique. Binding of these mAb to isolated cells or fixed cells was detected by radioactive binding assay, immunohistochemical technique and flow cytometry. Mar 1 binds specifically to the cells constituting the mononuclear phagocyte system (MPS), but not to granulocytes nor endocytosis-positive cells from non-lymphoid tissues. Mar 2 and Mar 3 recognize both the former and the latter. The isotypes of Mar 1, Mar 2 and Mar 3 were defined as IgG1, IgG1 and IgG2b, respectively. These mAb were species specific, allo-non-specific and not cytotoxic for rat peritoneal macrophages. Immunoelectron microscopic observation demonstrated that Mar 1-3 antigens are located on both surface membrane and cytoplasmic membrane structures of peritoneal macrophages, particularly on the limiting membrane of phagocytic small vesicles and large phagosomes. Immunoprecipitation experiments demonstrated that the apparent molecular weights (MW) of the reactive antigens of Mar 1, Mar 2 and Mar 3 are 95,000, 100,000 and 55,000 and 27,000, respectively. These findings indicate that all of Mar 1-3 mAb have considerable value in the identification of rat phagocytes and that, of the three kinds of antigens detected with Mar 1-3, Mar 1 antigen is a specific marker for identification of the cells constituting the MPS and may offer the means to assess the functional capability and differentiation process of the macrophage populations.     

Two closely related antigens expressed on granulocytes, macrophages and some reticular elements in rat lymphoid tissues: characterization by monoclonal antibodies

Two distinct novel antigen systems preferentially expressed in rat granulocytes and macrophages were detected using two different monoclonal antibodies (R2-1A6 and R2-2B1). These two antibodies reacted with approximately 50% of rat bone marrow cells, most granulocytes, blood monocytes, alveolar macrophages and peptone-elicited peritoneal macrophages, but not with red blood cells, platelets, thymocytes and T lymphocytes. In addition, R2-2B1 but not R2-1A6 antibody cross-reacted weakly with rat B cells. These two monoclonals also reacted with some reticular elements in rat lymphoid organs including epithelial reticular cells in the thymic medulla and follicular dendritic cells in the lymphoid germinal centre, as well as with the specialized endothelium in the marginal sinuses of the spleen and the post-capillary venules of the lymph node, where lymphocyte recirculation takes place. These antibodies, however, did not label so-called 'dendritic cells' bearing Ia antigens on their cell surfaces, which were found to be located in the thymic medulla, thymus-dependent areas of rat lymphoid tissues and the interstitium of various non-lymphoid organs, suggesting that these dendritic cells, presumably ascribed to those associated with accesory cell function, are separable from the mononuclear phagocyte system in rats by their different reactivities with R2-1A6 and R2-2B1 antibodies.     

Monoclonal antibodies against human granulocytes and myeloid differentiation antigens

Monoclonal antibodies (MCA) were obtained by immunizing BALB/c mice with 99% pure granulocytes from normal donors or with a whole leukocyte suspension obtained from a chronic myelogenous leukemia (CML) patient, and then fusing the mouse spleen cells with a 315–43 myeloma cell clone. Four MCA were selected and studied using ELISA, immunofluorescence, cytotoxicity assays, and FACS analysis. Antibodies 80H.1. 80H.3. and 80H.5 (from normals) and 81H.1 (from CML) detected antigens expressed on neutrophils. Antibodies 80H.1 and 80H.3 (lgG) also reacted with monocytes but not with other blood cell subsets. Antibodies 80H.5 and 81H.1 (lgM) were cytotoxic and reacted strongly with most of the cells of the neutrophil maturation sequence. i.e., myeloblasts, promyelocytes, myelocytes, and mature granulocytes. Antibodies 80H.5 and 81H.1 also inhibited BFU-GM and CFU-E. Antigens recognized by 80H.3. 80H.5, and 81H.1 were expressed both on a proportion of cells from HL.60, KG.1, ML.1, and K562 myeloid cell lines, and on a proportion of blast cells isolated from patients with acute myelogenous leukemia. They were not found on lymphoid cell lines or lymphoid leukemia cells. These MCA recognize either late differentiation antigens expressed on mature neutrophils and monocytes (80H.1 and 80H.3) or early differentiation antigens (80H.5 and 81H.1) specific to the granulocytic lineage. They may be useful for a better definition of those antigens specific to hematopoietic stem cells and their relationship with normal or neoplastic hematopoiesis.