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A cell surface antigen (possibly a receptor) on human myeloid cell lines, that may play a crucial role in the differentiation of promonocytic cells, was detected by a murine monoclonal antibody (MAb 710F). When the myeloid cell lines, HL-60, THP-1 and U937, were cultured with the MAb 710F following pretreatment with phorbol 12-myristate 13-acetate (PMA), they displayed both cellular spreading and a strong adherence to the culture dish substratum. On transforming from their original round shape, the induced cells displayed the well-developed microvilli, spindles, or raffles that are characteristic of macrophages or dendrocytes. Similar morphological changes were not induced by treatment with either PMA or MAb 710F alone. By contrast, the lymphoid cell lines did not respond to these reagents. These results suggested that a signal for cellular adhesion and cytoskeletal reconstitution was triggered by the binding of MAb 710F to an antigen on the PMA-primed cells. Thus, the antigen 710F, which is preferentially expressed on peripheral blood monocytes, may represent a cell surface receptor closely associated with differentiation. The antigen/receptor 710F was shown to be a N-glycosylated protein with Mr. of 35-70k. This MAb may prove useful as a tool for both studies on the differentiation of myeloid lineage cells as well as on monocyte/macrophage adhesion function.

Although the clinical relevance of endothelial cell-monocyte (E-M) antigens has been demonstrated in organ graft transplantation, very limited data exist describing the nature of these antigens. The current study presents biochemical characterization of three different surface antigens of endothelial cells and monocytes that are defined by murine monoclonals produced against gamma-interferon-induced human umbilical vein endothelial cells. The antigens gp150, gp48, and gp24 have molecular weights of 150,000, 48,000, and 24,000, respectively, under reducing conditions. The antibody binding sites of gp150 and gp48 are destroyed by pronase and chymotrypsin, indicating that the molecules are at least partly protein in nature. The inability to label the gp48 molecule with 125I using lactoperoxidase suggests that there is little protein structure exposed to the cell surface or that the molecule lacks sufficient cell surface tyrosine residues to enable detection. Immunoprecipitation of the gp24 molecule under nonreducing conditions shows that a molecule with a higher molecular weight ranging from 40,000-70,000 is detected. Although it is possible that this higher-molecular-weight species is a multimer of the 24,000 Mr species, it is also possible that there is another molecule(s) bound to the 24,000 Mr molecule. All three E-M antigens have some carbohydrate nature as evidenced by lectin-binding studies. The possible relevance of these antigens in the rejection of transplanted organ grafts is discussed.

We have obtained four monoclonal antibodies, IB4, OKM1, OKM9, and OKM10, all directed against the C3bi receptor of human monocytes and macrophages (M phi). Two criteria were used to determine the specificity of these antibodies. First, culture surfaces coated with the antireceptor antibodies caused specific down modulation of C3bi receptor activity on M phi adherent to these substrates. Second, receptor protein purified by using IB4 or OKM1 retained the ability to bind selectively to C3bi-coated erythrocytes. Each of the antibodies recognizes a distinct epitope on the C3bi receptor; they do not compete with one another for binding to monocytes. Further, when immobilized on a solid support, each of the antibodies binds a molecule from M phi lysates that can simultaneously bind one of the other monoclonal anti-C3bi receptor antibodies. OKM10 binds and masks the ligand-binding site of the C3bi receptor, while IB4, OKM1, and OKM9 bind to sites remote from the C3bi binding site. All four antibodies immunoprecipitated polypeptides of Mr 185,000 and 105,000 from 125I-surface-labeled M phi. IB4 also precipitates polypeptides of Mr 185,000, 153,000, and 105,000. We conclude that the C3bi receptor of human M phi is a complex composed of two polypeptides, Mr 185,000 and 105,000. We have identified monoclonal antibodies reacting with four distinct antigenic determinants of this complex. The determinant recognized by antibody OKM10 is at or near the ligand-binding site of the receptor. The determinant recognized by antibody IB4 is shared by at least two other leukocyte surface proteins.

Four monoclonal antibodies, termed AMH-1, AMH-2, AMH-3, and AMH-4, raised against human lung macrophages in bronchoalveolar lavaged fluid, alveolar spaces, and interstitia of lung tissue are described. The antibodies were produced according to hybridoma technique by immunizing mice with bronchoalveolar lavaged cells. All four monoclonal antibodies reacted with macrophages in bronchoalveolar lavaged fluid and alveolar spaces by immunohistochemical staining and flow cytometric analysis, but they gave different reactivity patterns with the monocyte-macrophage lineage. AMH-1 did not react with peripheral blood monocytes, peritoneal macrophages, or pulmonary interstitial macrophages. Although AMH-2 reacted weakly with blood monocytes and with some of the pulmonary interstitial macrophages, it did not react with peritoneal macrophages. AMH-3 did not show reactivities with either blood monocytes or peritoneal macrophages but was positive for most of the pulmonary interstitial macrophages. AMH-4 was reactive with cells from the monocyte-macrophage lineage. There was a correlation between the reactivity patterns of all four antibodies to macrophages in bronchoalveolar lavaged fluid and the patients' smoking habits. Most significantly, epithelioid cells of lung granulomas obtained from patients with sarcoidosis and hypersensitivity pneumonitis were negative for AMH-1 but were strongly stained by AMH-2, AMH-3, and AMH-4. Differences among the four antibodies in their reactivities with macrophages and granulomas in lungs indicate that lung macrophages contain heterogeneous populations which are in various states of differentiation and maturation and that the epithelioid cells and lung macrophages share the same membrane antigens. Therefore, these antibodies would be useful reagents for investigating the subpopulations and functions of macrophages in lungs and for clarifying the pathogenesis of granulomatous lung diseases.

A biotin-avidin horseradish peroxidase system is described for the localisation of membrane-bound monoclonal antibodies by low power transmission electron microscopy. An indirect bridge technique was used, with a biotin-labelled goat anti-mouse immunoglobulin antibody and horseradish peroxidase-conjugated avidin D. The technique provided preparations of high morphological and ultrastructural quality which facilitated accurate cellular identification. Positive immunostaining was specific and precisely defined.

Human blood monocytes (Mo) in medium containing 10% autologous serum were exposed to ethanol (160 mM) at 37 degrees C for 15 min. After being washed, the cells were incubated with murine monoclonal antibodies (MoAb), one binding to the 40 kDa Fc receptor (FcR) (MoAb IV3) and the other to the 72 kDa FcR (MoAb 32). The incubation was performed with and without excess of human or rabbit IgG. The amount of receptor-bound MoAb was evaluated by fluorescein isothiocyanate (FITC)-labelled anti-mouse IgG. Most control Mo bound MoAb IV3 (90 +/- 8% stained cells) while only 52 +/- 2% Mo were positively stained by MoAb 32. The staining increased in the presence of human IgG in the assay mixtures. Pre-incubation with 160 mM ethanol reduced the MoAb IV3 binding (61 +/- 2% stained cells), but had no significant effect on the binding of MoAb 32. Wash-out experiments indicated normalization of receptor function (MoAb IV3 binding) after 4 h. Treatment of medium or serum with ethanol before incubation of the Mo had no effect. It was concluded that a brief exposure of human Mo to ethanol leads to changes in a subpopulation of IgG FcR. Since these changes appeared when MoAb were used against the receptors, they probably represent a decrease in functional receptors and not changes in affinity.

Crude preparations of cytotoxins (CTXs) produced by human peripheral blood mononuclear cells exert a marked cytotoxic effect when applied to cells in the presence of cycloheximide but in its absence can induce resistance to cytotoxicity. To examine the relationship between these cytotoxic and protective activities, we attempted to fully dissociate the CTX from the other proteins secreted by mononuclear cells. Mice injected with preparations of the cytokines secreted by peripheral blood mononuclear cells developed significant titers of serum antibodies to CTX(s). Splenocytes of such immunized mice were fused with NSO myeloma cells; a few among the resulting hybridoma cells secreted CTX-binding antibodies. Immunoadsorbents constructed with a monoclonal antibody produced by one of these hybridomas were used to purify to homogeneity a CTX (Mr approximately 17,500) from crude preparations of cytokines, by a single adsorption and elution cycle. Purified CTX was cytotoxic in the presence of cycloheximide but in its absence induced resistance to cytotoxicity; this resistance was manifested by decreased vulnerability to CTX in a subsequent incubation in the presence of cycloheximide. We conclude that CTX itself can induce certain changes in cells, which are reflected in resistance to its own cytotoxic effect.

A wide variety of monoclonal antibodies (mAb) have now been produced which recognize cell surface antigens on human peripheral blood monocytes. However, few of these mAb demonstrate specificity for monocytes, and fewer still recognize antigens exclusively on monocytes activated by one or more stimuli and/or block specific monocyte functions. The mAb A1-3 binds to lipopolysaccharide (LPS)-stimulated monocytes but not to resting blood monocytes, and inhibits the procoagulant activity of these LPS-activated cells. By using this mAb, we examined the reactivity of monocytes/macrophages (MO) in a broad range of normal and inflammatory tissues by means of a sensitive, four-layer immunoperoxidase technique. Cells of the MO system, in addition to lymphocytes and dendritic cells resident in lymphoid tissues, liver, lung, and other organs, were nonreactive with the A1-3 mAb. In contrast, intense staining of inflammatory MO was found in biopsies from patients with renal allograft rejection, acute glomerulonephritis, or granulomatous diseases. This apparent restriction of A1-3 binding to inflammatory, "activated" MO suggests that A1-3 mAb will be useful for the analysis of MO "activation" in many pathologic processes.

Hamster mAb against activated human monocytes were examined for their reactivities against monocyte activation Ag. One mAb, anti-2.28, stained only monocytes activated with LPS plus IFN-gamma, but not unactivated peripheral blood monocytes, polymorphonuclear leukocytes, lymphocytes, RBC, and platelets. However, it stained peripheral blood T cells activated with PMA plus anti-CD3 and peripheral blood and tonsillar B cells activated with PMA plus anti-mu. Of the 35 cell lines of diverse origin examined for immunofluorescence staining by anti-2.28, only EBV-transformed cell lines showed strong staining by this mAb. One pre-B cell line, Nalm-12, could be induced by PMA to exhibit intermediate staining. Immunoprecipitation studies identified the 2.28 Ag as a 70- to 85-kDa monomer. Immunofluorescence staining, immunoprecipitation, and peptide mapping studies indicated that 2.28 was different from a number of monocyte and lymphocyte surface Ag including Mo3e, B-4 (CD19), B-5, CD39, and the G28-8 Ag Bgp 95. These studies suggest that 2.28 may be a novel hemopoietic non-lineage-specific activation Ag.

Staining of a variety of human tissue sections (lymph node, tonsil, spleen, thymus, kidney, lung, and liver) by the indirect immunoperoxidase method indicated that mAb 44G4, produced against a human pre-B leukemic cell line, was strongly reactive with vascular endothelium. All other cell types observed in these tissues were unreactive. Immunofluorescence staining of endothelial cells isolated from umbilical cord vein and grown in culture confirmed that mAb 44G4 recognized a surface membrane component of vascular endothelium. Granulocytes, monocytes, B and T lymphocytes, and T lymphocytes cultured in the presence of PHA for 72 h did not express the 44G4 Ag. mAb 44G4 reacted weakly with leukemic cells from 28 of 41 patients with non-T cell acute lymphocytic leukemia and 4 of 7 patients with acute myelocytic leukemia, whereas 8 of 10 cases of T cell acute lymphocytic leukemia were negative. Moderate reactivity with leukemic cell lines of pre-B and myelomonocytic origin was also observed. The level of 44G4 Ag on umbilical endothelial cells was three to five times that of leukemic cell lines and 25 times the average levels observed on leukemic cells isolated from patients. Immunoprecipitation of lysates prepared from surface-iodinated endothelial cells and the immunizing pre-B leukemic cell line revealed that the 44G4 Ag from both cell types was composed of two subunits of apparent m.w. 95,000 linked by disulfide bond(s). Comparison of the cellular localization and subunit structure of 44G4 to that of known Ag suggests that it represents a previously undescribed marker of endothelial cells.

Three monoclonal antibodies, P256 , P140, and P112 , react with the 135,000 mol wt IIb component of the glycoprotein IIb/IIa complex. They also react with a 200,000-mol wt protein present at low levels in the complex. Using immunofluorescence techniques, monoclonal antibodies P140 and P112 , but not P256 , can be shown to bind to 80% of human monocytes. However, P256 was able to immunoprecipitate the IIb/IIIa complex from detergent-solubilized monocytes, suggesting that the P256 epitope is less accessible on monocytes than on platelets. The monoclonal antibodies also precipitated molecules of approximately 200,000 mol wt from the monocytes. Two other monoclonal antibodies, J15 (specific for the IIb/IIIa complex) and AN51 (which reacts with the second major platelet glycoprotein complex, I), also react with monocytes. Binding of the monoclonal antibodies to the histiocytic cell line, U937, and promyelocytic cell line, HL-60, reflected the pattern of reaction with monocytes. The presence on monocytes of these glycoproteins, instrumental to the role of platelets in clotting, raises the possibility that monocytes might have similar functions in particular circumstances.

Thirteen monoclonal antibodies (the "VI-series") reactive with B- and T-lymphocytes, monocytes, granulocytes and erythrocytes were tested in sections fixed with formalin, formalin-sublimate or formalin-acetic acid. After fixation and embedding, most of the antigens were not detectable. However, VIE-G 4, a monoclonal antibody selective for glycophorin A, produced a strong reaction with erythroid cells in formalin fixed sections. The binding of the monoclonal antibodies VIM-D 5, VIM-2 and VIM-13 to myeloid, myelomonocytic and monocytic cells became either more intense or was inhibited by preincubation of the sections with pronase, trypsin, papain or neuraminidase. Following enzyme digestion, the monocyte-specific antibody VIM-13 reacted selectively with some of the germinal centre cells.

We report on the characterization of four monoclonal antibodies which were prepared against membrane markers of human myeloid lineage. Fusion, isolation of hybridoma cells and their cloning and testing of the monoclonal antibodies by indirect immunofluorescence and FACS 440 analysis were performed by means of standard procedures. The results indicate that the monoclonal antibodies have a specificity against membrane markers of human myeloid lineage (exactly promyelo-granulocytes). These monoclonal antibodies do not react with human T and B lymphocytes, monocytes, erythrocytes and thrombocytes of peripheral blood. In normal bone marrow reactivity to matured myeloid cells was found to occur in promyelocytes and expressed on all granulocytes. These monoclonal antibodies also react with cells of myeloid cell lines and with other precursor cell lines represented by NALM-1, NALM-16, HEL, K-562, REH no reactivity was detected. The produced antibodies react with some leukaemic cells from patients with more mature myeloid cells (AML with promyelocytes, myelocytes and CML) they do not react with pathological cells from patients with CLL, AML (with myeloblasts), ALL, hairy cell leukaemia, erythroleukaemia and several types lymphoma. All antibodies have a IgM class and express granulocytotoxic and granuloagglutination activity. Using flow cytometry comparative analysis with other monoclonal antibodies was performed to detect the membrane structure (X-haptene) included in standard International classification as CD 15 group.

An IgG2a monoclonal antibody, referred to as 12B1 and raised against the K562 cell line, reacted with adherent monocytes maintained in culture for several days but not with bone marrow or peripheral blood cells including freshly isolated monocytes. Among human leukemic cell lines, 12B1 reacted essentially with the promyelocytic HL60 cell line. 12-O-Tetradecanoylphorbol 13-acetate treatment, but no other differentiation inducer, strongly enhanced its reactivity on K562, HL60 and the histiocytic U937 cell line. Immunoperoxidase staining of sections of normal human tissues showed that 12B1 specifically recognized dendritic reticulum cells in germinal centers of lymph nodes, spleen and tonsils. The 12B1-detected antigen is a highly glycosylated polypeptide of an apparent molecular mass of 93-86 kDa. The 12B1 antigen appears to be a new glycoprotein marker shared by adherent monocytes and dendritic reticulum cells. The association of the 12B1 epitope with cells which present antigen and/or exert accessory function suggests that this molecule could play a role in these activities.

The distributions of lymphocyte subsets and monocytes in the peripheral blood mononuclear leukocytes of 72 normal children from 2 months to 13 5/12 yr were examined using quantitative immunofluorescence analysis with monoclonal antibodies. Distinct decreases with age were found in the total leukocyte counts, the percentages and the absolute numbers of peripheral blood mononuclear leukocytes. The percentages of Leu-2a+ cells, Leu-7+ cells, and Leu-M3+ cells significantly increased with age, whereas the percentages of Leu-3a+ cells, Leu-4+ cells, and 2H7+ cells significantly decreased with age. As a result, ratios of Leu-3a+/Leu-2a+ decreased with age. No prominent differences with age were found in the proportions of Leu-10+ cells and HLA-DR+ cells.

Intraglomerular T cells, monocytes, total leucocytes and other mononuclear subsets were sought in renal biopsies from patients with glomerulonephritis, using monoclonal antibodies and immunoperoxidase techniques. Twenty-four biopsies with no significant glomerular proliferation on optical microscopy, thirty-two with only endocapillary hypercellularity, and twenty-one with extra capillary crescentic glomerular disease were studied. Few intra-glomerular leucocytes were seen in the non-proliferative group. In contrast, when compared with this group, biopsies with glomerular hypercellularity and particularly those with crescents showed increased numbers of intra-glomerular total leucocytes and monocytes/macrophages, as well as an excess of T lymphocytes and T cytotoxic/suppressor cells; T helper/inducer lymphocytes were significantly increased only in the crescentic group. Only small numbers of B lymphocytes and NK cells were found in all groups. The numbers of total glomerular T-cells and monocytes per glomerular cross section were highly correlated in the crescentic group. Only idiopathic IgA nephropathy failed to show a significant increase in the numbers of intra-glomerular leucocytes, in comparison with the non-proliferative group, Henoch-Schönlein purpura biopsies in contrast had an excess of both monocytes and T cell subsets. The finding of T lymphocytes as well as monocytes in glomeruli of proliferative nephritis suggests that cellular immune mechanisms may play a role in their pathogenesis, especially when crescents are present.

OKT3 (10-100 pg/ml) still induces IL-2 responsiveness, while being unable to induce IL-2 production and lymphocyte proliferation, unless exogenous IL-2 is added. This property has been used in order to evaluate and quantify the IL-2 on various cell culture supernatants. Peripheral blood mononuclear cells (MNC) or T cells purified from blood units have been incubated with various concentrations of OKT3 and used as cell target for the quantitation of putative IL-2 containing media. The anti-TAC monoclonal antibody has also been used as internal control in the assay. The procedure suggested seems to be reliable and simpler than the method, currently employed in the IL-2 assay; MNC or T-cells treated with OKT3 proliferate in response to exogenous IL-2 and do not if the anti-TAC monoclonal antibody is previously added. Results are presented and discussed.