Diversity of the human monocyte/macrophage system as detected by monoclonal antibodies

Four monoclonal antibodies against the human monocyte/macrophage system, termed Ki-M1, Ki-M6, Ki-M7, and Ki-M8, are described with regard to their immunohistochemical tissue distribution pattern and their subcellular reactive sites. The differences found applying these analyses are also reflected by the various molecular weights of the recognized antigens. Based on these data it is proposed that the monocyte/macrophage system can be divided into the phagocytosing compartment on one hand and the immune accessory compartment on the other hand; the latter constitutes the interdigitating reticulum cells, the indeterminate dendritic cells, and the Langerhans cells, as well as the follicular dendritic cells (dendritic reticulum cells) as the accessory cells for T- and B-cell immune response, respectively.     

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.     

T-lymphocyte subpopulations identified by monoclonal antibodies after human bone-marrow transplantation

Peripheral T-lymphocyte reconstitution after bone-marrow transplantation, 12 allogeneic and 13 autologous, was studied by indirect immunofluorescence assay using mouse monoclonal antibodies. Abnormal counts were detected in the two major sub-populations of T-cells i.e. helper and T cytotoxic-suppressor lymphocytes, defined by monoclonal antibodies (alpha Leu 3a and B 9.2), and in DR antigen-positive T-cells. The pattern of T-lymphocytes replenishment was identical for both types of transplant, and was not affected by Graft Versus Host disease (GVHD).     

Characterization of human monocyte subpopulations by flow cytometry.

Human monocytes separated from peripheral blood by Ficoll-Hypaque and by adherence to serum-coated dishes show a bimodal volume distribution measured with a fluorescence-activated cell sorter. In the first peak of size distribution histogram of living mononuclear cells, lymphocytes and small monocytes were characterized by latex phagocytosis and non-specific esterase staining, whereas in the second peak the large monocytes dominated. The percentage of esterase stained small monocytes was lower than that of the large ones. Parallel to these data, the rate of the FDA hydrolysis of the small monocytes was lower than that of the large ones. The majority of the large monocytes reacted with sensitized sheep red blood cells (sSRBC) while only the minority of the small monocytes bound sSRBC. Scatchard plots on the binding of fluorescein isothiocyanate (FITC)-labelled human monoclonal IgG1 to the two subpopulations indicated similar association constants. K = 1 . 2 +/- 0 . 3 X 10(5) M-1. The number of Fc receptors was significantly different for the small (3 . 3 +/- 0 . 6 X 10(5)) and the large monocytes (10 +/- 1 X 10(5)).     

Development of lymphocyte subpopulations identified by monoclonal antibodies in human fetuses

We have used a panel of monoclonal antibodies to examine the development of lymphoid and myeloid sub-populations of cells in thymus, bone marrow, and liver of 16 fetuses from 12 to 16 weeks of gestational age. Pre-B and IgM⁺ B cells were present at a ratio of approximately 2:1 in all of the fetal bone marrow and liver samples; cells of both phenotypes were HLA-DR⁺ but did not express the mature B-cell antigen, HB-2. Cells expressing the myelomonocytic antigen, MMA or Leu-M1, were more frequent in bone marrow (40%) than in fetal liver (10%), and cells expressing the HNK-1 or Leu-7 antigen were rare (<1%) in all of the fetal tissues examined. Each of the T-cell antigens, T1, 4, 5, 6, and 8, was expressed by a majority of thymocytes irrespective of the age of the fetal donor. In contrast, cells with the T1, 4, 5, and 8 antigens were not seen in bone marrow and liver before the 13th week of gestation, and T6⁺ cells were never seen in these hemopoietic tissues. These results suggest that fetal liver and bone marrow precursors in humans do not express these T-cell antigens prior to thymic entry and the onset of thymocyte differentiation.     

Monoclonal antibodies detect monocyte/macrophage activation and differentiation antigens and identify functionally distinct subpopulations of human rheumatoid synovial tissue macrophages

Monoclonal antibodies (MAbs) to functionally heterogeneous populations of human rheumatoid arthritis (RA) synovial tissue macrophages and lipopolysaccharide (LPS)-activated U937 cells were generated. These MAbs were used to characterize macrophages in situ in the synovial pannus and to study relative antigen expression on the surface of cells isolated from the synovium and from normal peripheral blood. Monoclonal antibody 3D8, an anti-CD13 MAb, reacts with an antigen expressed on the surface of blood monocytes and is a monocyte activation-related antigen that is upregulated by exposure of monocytes to interferon-gamma (IFN-gamma) and LPS. The expression of the 3D8 antigen increases in parallel with MHC class II antigen expression and also is upregulated in culture as monocytes mature to macrophages. 3D8 antigen is expressed strongly on RA synovial tissue lining cells, which are thought to be composed of macrophages. 8D7 antigen expression, detected by MAb 8D7, increases on blood monocytes on cellular activation with LPS and interferon-gamma, but in contrast to the 3D8 antigen, does not increase with monocyte maturation in vitro. The 8D7 antigen is expressed differentially on density-defined macrophage subpopulations isolated from RA synovial tissue and is expressed more strongly on macrophages that are nonangiogenic than those that are angiogenic.     

Ber-MAC3: new monoclonal antibody that defines human monocyte/macrophage differentiation antigen.

A new monoclonal antibody Ber-MAC3 is reported. It recognises a formol sensitive epitope of a not yet clustered monocyte/macrophage specific 140 kilodalton glycoprotein that is expressed on the cell surface and in the cytoplasm. In 30 cases of acute and chronic leukaemia, Ber-MAC3 staining was restricted to 15 myeloid leukaemias of M4 and M5 types. The tumour cells of two cases of true histiocytic malignancies were Ber-MAC3 positive, whereas those of all 280 malignancies of lymphocytic origin were negative. The latter included 52 cases of Hodgkin's disease and 41 cases of Ki-1 positive anaplastic large cell lymphomas which had previously been classified as true histiocytic lymphomas. Ber-MAC3 therefore seems to be of considerable value for selective identification of monocytes and macrophages at a certain stage of differentiation and seems to be suitable for diagnosing myelomonocytic or monocytic leukaemia and neoplasms of true histiocytic origin.     

Activation of NF-kappa B in human endothelial cells induced by monoclonal and allospecific HLA antibodies

Chronic graft rejection, characterized by a gradual occlusion of grafted vessels, is the most serious complication following heart and kidney transplantation. Although often associated with chronic production of anti-HLA and anti-endothelial antibodies, the precise role of antibodies in chronic rejection remains uncertain. Here we have investigated whether HLA-specific antibodies, either monoclonal or derived from patients, cause endothelial cell activation. Thus we investigated tyrosine phosphorlyation, NF-kappaB activation and cell proliferation in human umbilical vein endothelial cells (HUVEC) or microvascular endothelial cells from adult human heart (CMEC). Ligation of monomorphic determinants of MHC class I molecules (using the mAb W6/32) on the surface of HUVEC caused an increase in tyrosine phosphorylation of proteins of mol. wt approximately 75-80 kDa. Similarly, ligation of monomorphic determinants on both CMEC and HUVEC resulted in increased NF-kappaB binding compared to controls (by 74.4 and 52.5%, P = 0.001) and this was enhanced by addition of secondary antibody. Two HLA-specific mAb resulted in a 277 and 170% increase in NF-kappaB-binding activity compared to controls. Four patient samples containing HLA antibodies were used against HLA-specific HUVEC and four samples were incubated with HUVEC bearing irrelevant antigens. Patient sera alone enhanced NF-kappaB binding by 27-186%, but only when added to HUVEC bearing relevant antigens. W6/32 and allospecific antibodies from patients significantly enhanced HUVEC proliferation, measured by uptake of [(3)H]thymidine. In conclusion, activation of NF-kappaB by human anti-HLA antibodies demonstrates their potential role in pathogenesis of chronic vascular occlusive disease following transplantation.     

Increased amount of serum type IV collagen peptide in human liver fibrosis as determined by enzyme-immunoassay with monoclonal antibodies

Chronic liver injury by various toxic agents causes an increase in collagen biosynthetic activity, resulting in deposition of excessive amounts of collagen and rearrangement of the lobular architecture leading to hepatocellular dysfunction and portal hypertension. The authors have developed sandwich enzyme-immunoassay for human serum type IV collagen peptide using monoclonal antibody as a marker of fibrogenesis and examined the relationship between the amount of this collagen peptide and hepatic disorders including chronic active hepatitis and liver cirrhosis. Sera from patients with chronic active hepatitis or liver cirrhosis were used after confirmation of the histopathological diagnosis. Control sera were obtained from healthy subjects without any serological abnormality in liver function tests. Serum type IV collagen peptide levels were significantly higher in patients with chronic hepatic disorders associated with fibrosis than in healthy subjects. This non-invasive enzyme-immunoassay gave reproducible quantitation, and serum type IV collagen peptide was concluded to be a useful, and reproducible marker for the early detection of fibrogenesis in the liver.     

Reed-Sternberg/lymphocyte rosette: lymphocyte subpopulations as defined by monoclonal antibodies

The Reed-Sternberg cell/lymphocyte rosette characteristic of Hodgkin's disease tissue and cell suspensions was investigated with monoclonal antibodies on fresh viable cell suspensions prepared from nine cases of Hodgkin's disease. The biopsy material comprised six spleens and three lymph nodes. The majority of the rosetting lymphocytes were T cells, primarily of the helper subset. Some of the attached lymphocytes were suppressor T cells. In addition, a few of the rosetting lymphocytes around Reed-Sternberg cells were B cells.     

Murine monoclonal antibodies that identify antigenically distinct subpopulations of human sperm

Murine monoclonal antibodies (MoAbs) were isolated to characterize antigenically distinct subpopulations of human sperm. Spleen cells from Balb/c mice immunized with freshly prepared human sperm, were fused with murine P3-NS1-Ag4-1 myeloma cells by somatic cell hybridization, and supernatants from the IgG-secreting hybridomas were screened by an enzyme immunoassay (EIA) for reactivity against fresh human sperm and a panel of human somatic cells. Two MoAbs, SP1D1 and SP7A7, reacted specifically with human sperm, whereas three others, SP2A9, SP3B3, and SP4F5, cross-reacted with a variety of human somatic cells. The binding of MoAbs were characterized by immunofluorescence, agglutination, and Staphylococcus aureus binding assays. We found that certain MoAbs bound to common antigens of the head and tail, or to tail alone, and had agglutinating activity. However, not all sperm were reactive to antibody, and the binding activity could only be demonstrated in subpopulations of sperm ranging from 5 to 50% of the total number.     

Distribution pattern of lymphocyte subpopulations in human tonsils as analysed by monoclonal antibodies with double immunoenzymatic labeling

Summary The distribution of B- and T-lymphocytes, T-cell subpopulations, including natural killer cells and monocytes/macrophages, was studied in cryostat sections of human tonsils by the avidin-biotin-peroxidase technique using monoclonal antibodies. A double immunostaining procedure was also developed to detect two types of lymphocytes on one single section simultaneously using horseradish peroxidase and alkaline phophatase as labeling enzymes.The primary follicles and the germinal centers of the secondary follicles were mainly found to be positive for B-cells. T-cells were predominantly localized in the follicular caps and in the interfollicular areas. The ratio of helper/inducer cells to suppressor/cytotoxic cells was in favour of helper T-cells. Both subpopulations were also predominant in follicular caps and interfollicular areas.The quantity of natural killer cells was very variable and nearly all were localized exclusively in the germinal centers.Monocytes/macrophages were only seen occasionally in the interfollicular areas. The double-immunoenzymatic labeling was useful for the visualization of combinations of antigens, however, without demonstrating the presence of two different surface antigens on one single cell.     

Functionally different subpopulations of mouse macrophages recognized by monoclonal antibodies

Four rat anti-mouse macrophage monoclonal antibodies are described. Three of them are highly specific for macrophages, and one cross-reacts with granulocytes. All 4 antibodies do not react with membrane antigens shared by all macrophages, but with antigens present only on subpopulations of 20-50% of the cells. All antibodies are directly or indirectly cytotoxic for macrophages. The subpopulations defined by these antibodies can be correlated with certain macrophage functions. Thus, antibody M43 eliminates macrophages that are activated by lymphokine to cytotoxicity. Antibodies M43 and M57 eliminate macrophages that kill antibody-coated tumor targets, and clone 102 (strictly macrophage-specific) eliminates natural killer cells. Only M143, reacting with 10-30% of macrophages, has not yet been correlated with any function. With the use of these antibodies, cells of the macrophage lineage with specific functions can be recognized and eliminated from a given population.     

Immunoregulatory T cell subpopulations in patients with scleroderma using monoclonal antibodies.

Twenty-eight patients with scleroderma were compared with 22 healthy age-matched subjects. Monoclonal antibodies were used to detect the whole T cell population (OKT3), T helper cells (OKT4), and T suppressor/cytotoxic cells (OKT8) by indirect immunofluorescence on isolated peripheral blood mononuclear cells. A subset of scleroderma patients (i.e. 30% or eight of 28 patients) exhibited an elevated ratio of OKT4/OKT8 cells which could be accounted for, mainly by a reduction in OKT8 cells compared with controls. The scleroderma patients with an elevated OKT4/OKT8 ratio tended to be younger, have a shorter disease duration and more extensive skin involvement than patients with a normal OKT4/OKT8 ratio. There was no correlation with the presence of autoantibodies, drug therapy, or HLA-DR type. In order to further determine whether this imbalance in immunoregulatory cell subpopulations was specific for scleroderma, we further studied 16 patients with psoriatic arthritis but without manifest autoimmunity and delineated a similar subset of patients with an elevated OKT4/OKT8 cell ratio (i.e. 38% or six of 16 patients). The results demonstrate similar immunoregulatory T cell imbalances in patients with scleroderma and psoriatic arthritis. These findings suggest that numerical imbalances in lymphocyte subpopulations may not be specific for autoimmune disorders.     

Modulation of expression of mouse macrophage surface antigens by monoclonal antibodies.

Mouse macrophages from peritoneal cavity were exposed to monoclonal antibodies (MAbs) directed against cell surface antigens and the effect on antigen expression was investigated. The two Mabs used, 3A33 and 3A35, were produced by cell fusion between a mouse plasmacytoma and rat lymphocytes immunized against mouse macrophages. The binding of the MAbs to cell surface was measured by immunofluorescence and flow cytometry or by a radioimmunological technique. When injected i.p. the MAbs diminished the expression of the corresponding antigens but did not alter it when added to cultures of adherent macrophages. Antigenic modulation, however, could be produced in vitro either by inhibiting macrophage adherence during incubation with MAbs or by using a second antibody layer. MAb 3A33 (IgG2a) was more effective than 3A35 (IgM) in provoking modulation. The appearance of re-synthesized antigens on cell surface was not affected by macrophage adherence. The modulated antigens were found to internalize into cytoplasmic vacuoles.     

Assay of human IgA subclass antibodies in serum and secretions by means of monoclonal antibodies

Solid-phase radioimmunoassays have been developed for the detection and quantification of human serum and secretory IgA antibodies to a variety of food, bacterial and viral antigens. Monoclonal antibodies specific for IgA1 and IgA2 and capable of binding to serum and secretory IgA were used. The assays were calibrated by reference to standard serum or purified myeloma proteins bound to solid-phase anti-immunoglobulin reagents, and sigmoid calibration curves were constructed by means of computer programs using 4-parameter logistic or weighted logit-log principles. Polymeric and monomeric forms of IgA antibodies were assayed in fractions separated by high performance size exclusion chromatography. These techniques have demonstrated the expected predominance of IgA1 antibodies in serum, and these included polymeric forms. Saliva contained both IgA1 and IgA2 antibodies, and increased proportions of IgA2 antibodies to lipopolysaccharides and lipoteichoic acid were observed.     

The monocyte/macrophage population of the normal human kidney

There is continuing controversy over the role of mononuclear phagocytes in glomerulonephritis. It is, therefore, important to know their distribution in normal subjects. Normal kidneys (34) were assessed using three cytoplasmic markers for macrophages employing a trypsin-immunoperoxidase (PAP) technique with antibodies to alpha-1-antitrypsin, muramidase (lysozyme) and a further oligoclonal antibody, serum 22, developed against highly purified preparations of blood monocytes. Serum 22 detected twice as many monocytes/macrophages as anti-muramidase and four times as many as anti-alpha-1-antitrypsin. Most cells that showed positive staining lay in glomerular and intertubular capillaries and were considered to be blood monocytes. There was wide variation in monocyte numbers throughout different glomeruli and up to 14 monocytes could be present in a glomerulus. Not more than 1 per cent lay within the mesangium. Macrophages were virtually never seen in the interstitium, except in areas of scarring. No macrophages were present in the tubules. Monocytes and macrophages are demonstrated in greater numbers within the kidney by antisera to muramidase than to alpha-1-antitrypsin.     

Identification with monoclonal antibodies of different regions of human plasma fibronectin, including that which interacts with human monocyte fibronectin receptors.

The determinant specificities of five monoclonal anti-fibronectin antibodies, designated BC7, CE9, BD4, AB3 and CPG1, were defined and mapped within intact human plasma fibronectin by immunoblot analyses with defined fragments of fibronectin. The latter were derived by tryptic, chymotryptic or cathepsin D digestion of the intact molecule and fractionated by DE-cellulose chromatography and gelatin and/or heparin affinity chromatography. Monoclonal BC7 recognizes intrachain disulphide-formed determinants within the 27,000 MW N-terminal domain; monoclonal CE9 recognizes determinants within an 18,000 MW fragment immediately adjacent to the carboxyl end of the gelatin-binding domain; monoclonal BD4 recognizes determinants within the cell-adhesive domain and within 150,000 of the N-terminus; monoclonal AB3 recognizes intrachain disulphide-formed determinants within 35,000 of the COOH-terminus of the intact molecule and detectable only on the alpha-chain polypeptide subunit; and monoclonal CPG1 recognizes determinants present on both chains of the intact molecule and immediately adjacent to the interchain disulphide bonds at the COOH-terminus. None of the epitopes recognized by these monoclonal antibodies is present at alternative regions of the intact molecule. Fab fragments of each of these monoclonal antibodies were incubated with gelatin-coated sheep erythrocytes which had been reacted with a fixed amount of intact plasma fibronectin. When these target particles were incubated with monolayers of human monocytes and the resultant rosettes were quantitated, the Fab fragments of BD4 markedly inhibited the proportion of monocytes binding these fibronectin-bearing targets, whereas none of the other Fab fragments had an inhibitory effect. Thus, monocyte fibronectin receptors which mediate adherence of fibronectin bridges to a target via gelatin recognize regions within the cell-adhesive domains of intact fibronectin but not regions at the amino or carboxy terminals.