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The aim of the present investigation was to assay the frequency of primary dyslipoproteinemia in a random sample of one hundred newborns and to describe the minimal methodical requirements for sound diagnosis. After comparison of different methods total lipids were determined by gravimetry, cholesterol and triglycerides by enzymatic methods, nonesterified fatty acids by direct colorimetry; phospholipids were estimated indirectly. All measurements were applied to umbilical cord sera and to lipoprotein fractions separated by selective precipitation. The diagnosis of hyperlipoproteinemia type IV, which is the most frequent one in adults, is highly afflicted with pitfalls in the postnatal period. A primary hyper-alpha-liproteinemia occured in one case and type II-hyperlipoproteinemia in two cases, one of the parents being involved in each case. For mass screening triglycerides should be assayed in serum and cholesterol in precipitated and resolubilized LDL-fraction, for which the minimal requirements are described.

In accordance with the long-range goal to demonstrate the direct derivation of blood monocytes from promyelocytes in human bone marrow, the promyelocytic cell line HL-60 in the present study was differently stimulated with various inducers for the purpose of documenting its capability to evolve into granulocytes or monocytes-macrophages. Each differentiation line was monitored with the use of marker enzymes, antigens, and cell-specific monoclonal antibodies. In addition, studies were done on normal human bone marrow and leukemias. The results provided strong evidence for the close cytogeneic relationship of granulocytes and monocytes and for a common progenitor at the maturation stage of promyelocytes-myelocytes for both cell lineages.

The monocytic lysosomal acid esterase (AcE; EC 3.1.1.6) comprises five isoenzymes, each having specific isoelectric points (pI) as well as antigenicity. In the present study attempts were made to retrace the monocytic origin of human alveolar macrophages (AM) by comparison of their isoenzyme patterns with those of blood monocytes. Resident AM obtained from bronchial lavages lacking any neutrophils and unstimulated monocyte admixture showed in addition to the five monocytic isoenzymes nine additional isoenzyme loci. In vitro stimulation of blood monocytes (BM) using lymphokine-conditioned media led to a gradual transition of the typical monocytic isoenzyme pattern into that of AM. It is concluded that AM originates from blood monocytes by tissue-specific stimulation. This cellular transformation can be modeled in vitro as far as morphology, cytochemistry, and isoenzyme pattern are concerned.

We describe a new monoclonal antibody, termed Ki-M7, which is specific to human myelomonocytic cell lineage and macrophages, as tested by immunohistochemical methods. Ki-M7 recognizes an intracytoplasmic antigen of molecular weight 29,000. Ultrastructurally, the antigen is localized in the lysosome and phagosome compartments and seems to be involved in generation of oxygen radicals during the respiratory burst. Dendritic cells, such as dendritic reticulum cells of lymphoid follicles and interdigitating reticulum cells of lymphoid T-zones, considered as accessory cells of the B- and T-cell immune response, respectively, do not show any reactivity with monoclonal antibody Ki-M7. Ki-M7 seems to be an appropriate reagent to clearly differentiate between the phagocytosing and the immune accessory population of the human monocyte/macrophage system.

A small bowel transplant was performed on a five-year-old boy with a short bowel syndrome. The donor was the child's mother. Despite immunosuppressive therapy, the transplant was acutely rejected and had to be explanted on the twelfth day. Morphologic and immunohistochemical investigations on subsequent biopsies taken from the small bowel transplant were carried out. Besides typical changes in epithelial cells and the presence of T-cell infiltrates and Ig-deposits in vessels, many macrophages were seen. The submucosa in particular was invaded before rejection by numerous macrophages with positive results of antimonocyte/macrophage antibodies Ki-M6 and Ki-M7. The number of the macrophage antibodies Ki-M6 and Ki-M7. The number of the monocyte/macrophage cells and the immunohistochemical characteristics of the same may be important parameters for monitoring small bowel transplantations.

The present paper gives a review of enzyme polymorphism found in individual human blood cells with special reference to human blood monocytes. Evidence is presented indicating that not only different blood cells but also their descendants are characterized by certain constellation of isoenzymes. Considering the typical polymorphism of acid phosphatase (EC 3.1.3.2) and acid esterase (EC 3.1.1.6), we could show that a reliable biochemical marker for blood monocytes can be worked out, which is also detectable in peritoneal and alveolar macrophages as well as epithelioid cells. In different examples we could demonstrate that the isoenzyme patterns of blood monocytes can be modified into that of peritoneal and alveolar macrophages by different stimulation procedures in vitro. Studying the enzyme variants in blood monocytes and different tissue macrophages, additional criteria have been collected, which document the monocytic origin and homogeneity of the human mononuclear-phagocytic system.

The following work was planed to clarify whether enzyme variants were appropriate in disclosing cellular derivation and in indicating defined differentiation stages of stimulated cells. Light and electronmicroscopical studies have revealed a close relationship between monocytes, tissue macrophages, and epithelioid cells. In the present investigation direct evidence was provided for the monocytic origin of epithelioid cells. It could be shown that the monocyte specific isoelectric focusing patterns of acid phosphatase (EC 3.1.3.2) and nonspecific acid esterase (EC 3.1.1.6) were regularly shared by epithelioid cells, obtained from lymph nodes of patients with sarcoidosis. Concerning additional isoenzymes of epithelioid cells in comparison to blood monocytes and peritoneal macrophages, it is concluded that the analysis of enzyme polymorphism might be a reliable tool for tracing cell lineage and for defining functional stages during cell differentiation.

The permanent promyelocytic cell line HL-60 was subjected to stimulation with dimethyl sulfoxide (DMSO) and retinoic acid (RA), as well as 12-O-tetradecanoylphorbol-13-acetate (TPA) and lymphokine conditioned media for the induction of granulocytic or monocytic differentiation, respectively. Cells were investigated cytochemically using alpha-naphthylacetate esterase (acid esterase; AcE), naphthol AS-D chloroacetate esterase, and peroxidase reactions. In addition, the granulocyte or monocyte specific isoenzyme patterns of AcE as an intracytoplasmic property and the immunoreactivity to monoclonal antibodies recognizing granulocytes and monocytes (Ki-M2, Ki-M5) or monocytes alone (Ki-M1) were considered. The results indicated that HL-60 cell line bear the potency to evolve into granulocytes as well as monocytes. Additional studies performed on normal human bone marrow stained for AcE led to the conclusion that the myeloid cell line remains bipolar until the maturation stage of promyelocytes. Myelocytes being AcE positive only in 11.5 +/- 5.0 are heterogeneous and display the first indications of separated monocytic or granulocytic differentiation.

Alveolar macrophages differ from their percursors in blood, monocytes, by expressing strong activity of the tartrate resistant variant of acid phosphatase (TAcP). A study was carried out to analyse the expression of this enzyme cytochemical marker by alveolar macrophages from bronchoalveolar lavage cells from 34 patients with sarcoidosis and 12 control subjects. Alveolar macrophages from control subjects displayed a strong and homogeneous staining pattern and only 0.1% of cells were negative after staining. Macrophages from patients with sarcoidosis showed reduced TAcP activity and up to 7% of the cells were negative. The percentage of TAcP negative macrophages was correlated with the percentage of lymphocytes and with the ratio of CD4 to CD8 lymphocytes among cells recovered by bronchoalveolar lavage. The reduced TAcP activity in alveolar macrophages from patients with sarcoidosis may be due to an increased recruitment of immature precursors from blood.

Cell lines infected by different species of mycoplasma (Mycoplasma orale, Mycoplasma hominis) were decontaminated by co-culture with human blood monocyte (BM)-derived macrophages and pooled human immunoglobulin preparations. Co-cultures with BM-derived macrophages or murine peritoneal macrophages (PM) alone were not successful. The phenotype of infected cell lines did not differ from that of uninfected cell lines as revealed by morphological, enzymecytochemical, and immunocytochemical analysis.

A monoclonal antibody, termed Ki-M6, is described, which shows a restricted reactivity to cells of the monocyte/macrophage system. On light- and electron-microscopic immunoperoxidase staining Ki-M6 recognizes monocytes and the phagocytosing compartment of macrophages residing in different tissue sites; granulocytes and the so-called immune accessories of B- and T-cell immune response as closely monocyte/macrophage related cell populations do not reveal any reactivity. This is shown by comparison with the monoclonal antibodies Ki-M4 and Ki-M1 or OKT6 recognizing immune accessory cells by immunohistochemical methods. Ki-M6 binds to a lysosomal membrane-restricted antigen of 60,000 daltons without influencing significantly lysosome-related functions as far as the chemiluminescence response is concerned.

Although multinucleated giant cells (MGCs) are a known feature of granulomatous reactions, little is known about their destination and function. In this study human blood monocyte (BM)-derived giant cells were generated by lymphokine stimulation in vitro. Their immunophenotype and ultrastructural morphology resembled that of MGCs occurring in vivo. Mitotic activity within MGCs could not be established either in vitro or in vivo. Enzyme equipment of MGCs was elevated in comparison with monocyte-macrophages. In comparison with unfused monocyte-macrophages, MGCs did not reveal a higher level of interleukin-1 production or cytostatic activity. They showed, however, a 20-30-fold increase in the production of oxygen-free radicals in response to zymosan. Transcription of the proto-oncogene c-fms was enhanced in short-term cultivated BM and was rapidly down-regulated in MGCs after fusion had occurred. It is concluded that MGCs represent highly stimulated cells of monocyte-macrophage lineage at a terminal stage of maturation.

The gene product of the c-fms proto-oncogene is a transmembrane protein with tyrosine-kinase activity that is obviously related to the receptor for the colony-stimulating-factor CSF-1. By Northern blot analysis, we investigated the expression of the cellular counterpart of v-fms in purified normal human blood mononuclear cells and different macrophage populations. The proto-oncogene c-fms expression was demonstrable in blood monocytes but not in blood lymphocytes. Short-term cultivated blood monocytes exhibited an increased expression of c-fms in comparison to freshly isolated blood monocytes, possibly due to a temporary down regulation of c-fms during the separation procedure of blood monocytes. A comparably high rate of fms-RNA expression was found in most of the analyzed samples of resident peritoneal macrophages, while resident alveolar macrophages showed a considerably lower level of c-fms expression. In this, alveolar macrophages resembled long-term cultivated adherent blood monocytes, which showed a down regulation of c-fms expression. By correlating these data obtained by Northern blot analysis with phenotypic properties of the analyzed monocyte/macrophage populations, it is concluded that different levels of c-fms expression in monocytes/macrophages correspond to their stage of differentiation and maturity.

Investigating a link between enzyme histochemical and recent immunohistochemical results, the authors studied the activity and the polymorphism of acid esterase (EC 3.1.1.6) in well defined human B-cell lymphomas. TWelve cases of chronic B-lymphocytic leukemia, 18 cases of centroblastic/centrocytic follicular lymphoma, and 17 cases of lymphoplasmacytic/lymphoplasmacytoid lymphoma, as diagnosed according to Kiel classification, were subjected to enzyme assay and isoelectric focusing of acid esterase. Enzyme values revealed no characteristic distribution among the lymphoma entities. The isoenzyme pattern, specific to normal human B-lymphocytes, were regularly detectable in all lymphoma entities. The results document the B-cell origin of the analyzed subsets of B-cell malignancies, although distinctive acid esterase patterns were lacking. The prevalence of three anodal isoenzymes in cases of chronic B-lymphocytic leukemia, hardly detectable in other lymphoma entities, were interpreted as the expression of a clonal proliferation, arrested at a certain differentiation stage of B-cells common for the majority of the tumor cells. Hence, further evidence is provided supporting the view that the studied entities represent B-cell neoplasias at different stages of differentiation expressing a variety of different markers.

Activity and isoelectric focusing (IEF) pattern of lysosomal acid phosphatase (E.C.3.1.3.2.) were investigated in 55 cases of low-grade malignant B-cell lymphoma, classified as chronic B-lymphocytic leukemia (B-CLL), centroblastic/centrocytic follicular lymphoma (CB/CC), lymphoplasmacytic/lymphoplasmacytoid lymphoma (Immunocytoma, IC), and plasmacytoma (PC), applying the criteria of the Kiel classification. The results show (1) that the four lymphoma types present a characteristic range of enzyme activity in an increasing order: B-CLL, BC/CC, IC, and PC. B lymphocytes, germinal center cells, and plasmacytes are the main constituents of these lymphomas. This sequence might reflect one possible mode of B-cell transformation into plasmacytes traversing an amplification stage in germinal centers under normal conditions. (2) All cases showed the basic IEF pattern of normal B lymphocytes with 12 bands localized in three regions between pH 6.1 and 3.9. This finding supports the B-cell origin and the close phenotypical relationship among the investigated lymphomas. (3) The IEF patterns of B-CLL and CB/CC did not differ from that of normal B lymphocytes, whereas two additional isoenzymes were encountered in cases of IC and seven in PC; this suggests that the higher enzyme activity of IC and PC is at least partly due to the appearance of "new" isoenzymes. The results support the validity of the underlying classification and indicate the individually, B-cell origin, and close relationship among the four lymphoma entities investigated.

Using monoclonal antibodies specific for myelomonocytic cells, 40 non-lymphoblastic leukaemias were analysed applying immunostaining to cytospin preparations. Based on the reactivity patterns six groups of acute non-lymphoblastic leukaemias could be determined, mirroring the bimodal differentiation pathway of myelomonocytic cells. Comparative enzyme cytochemical analysis did not render a clear cut correlation and discrimination of the immunocytochemically defined groups. It is concluded that only the application of a broad panel of immunocytochemical and enzyme cytochemical methods allow a sound subdivision and diagnosis of acute non-lymphoblastic leukaemias.

In the present study unstimulated and stimulated human blood monocytes, untreated and phorbol ester treated U-937 cells, as well as human peritoneal and alveolar macrophages were studied with respect to their surface membrane properties. Binding of different lectins and electrophoretic patterns of tritium labeled surface glycoproteins were compared. The analysis of surface glycoproteins could be interpreted as evidence for a common origin of the analysed cell populations. Furthermore, banding patterns of glycoproteins might be useful to define certain activation states within monocyte/macrophage differentiation. In contrast, lectin binding pattern did not clearly discriminate macrophage subpopulations.

Fifteen cases of Hodgkin's disease of nodular and diffuse paragranuloma subtype (nodular and diffuse subtype of lymphocyte predominant type of Hodgkin's disease) were studied by light and electron microscopy, using monoclonal antibodies recognizing T and B-lymphocytes and dendritic reticulum cells. The results were compared with findings on 10 cases of Hodgkin's disease of mixed type with lymphocyte predominance. The present study provides evidence that paragranuloma represents a special variant of Hodgkin's disease different from other subtypes. Paragranuloma is characterized by predominance of B-cells which were demonstrated with a new B cell reagent KiB3 on routinely processed paraffin sections.

The present study was devised to investigate the activity and isoenzymes of lysosomal acid phosphatase in individual normal human blood cells, including the T- and B-population of lymphocytes, with the aim to contribute to the classification of haematopoietic neoplasias on the basis of cell specific isoenzyme patterns. Platelets, erythrocytes, granulocytes, monocytes and T-lymphocytes were isolated from blood by gradient centrifugation or immune adsorption. B-lymphocytes were obtained from human tonsils. After purity control and isolation of lysosomes the concentration of acid phosphatase was assayed using the conventional spectrophotometric method. Isoenzymes were separated by isoelectric focusing on polyacrylamide thin layer slabs. Monocytes revealed the highest activity with 14 mU/10(7) cells, about three times more than granulocytes. T-lymphocytes showed an activity of 2.85 mU/10(7) cells and B-lymphocytes of 1.83 mU/10(7) CELLS. The lowest activity was found in platelets with 0.08 mU/10(7) cells. Granulocytes showed 12 isoenzyme bands, whilst the number for monocyte, B-lymphocytes, T-lymphocytes and platelets were respectively 11, 12, 1 and 4 isoenzyme bands. Thus it became evident that the different blood cell populations can be distinguished on the basis of their acid phosphatase isoenzyme pattern.

The phenotypic heterogeneity of the monocyte/macrophage system is mirrored by the various inflammatory reactions of this cell system. Using monoclonal antibodies functional subpopulations of the monocyte/macrophage system can be observed, though the pathophysiological processes of the various inflammatory reactions still remain mostly unclear. By analysing phenotypic properties and gene expression in cells of the bronchoalveolar lavage it will be shown that the activity and the underlying pathophysiologic mechanisms of reactive processes can be defined.

The cellular homologue of the retroviral oncogene v-fos has been shown to be involved in cell differentiation of hematopoietic cells. By use of the human promyelocyte cell line HL-60, several in vitro differentiation studies suggested a selective activation of c-fos during monocytic differentiation of myeloid precursor cells. In contrast to these observations, we found high levels of c-fos mRNA in purified normal human granulocytes, whereas c-fos was only faintly expressed in blood monocytes. In situ hybridization revealed that the high level of c-fos expression is restricted to neutrophilic granulocytes, whereas c-fos transcription is not detectable in eosinophilic granulocytes. These results indicate that in vitro differentiation systems can be misleading and may not reflect the in vivo situation. The high level of c-fos expression in neutrophilic granulocytes may be caused by superinduction due to the reduced capacity for protein synthesis in these cells.

In this study fresh frozen tissue samples of benign osseous tumors (five non-osteogenic fibromas, one fibrous dysplasia, one chondromyxoidfibroma), tumors of uncertain biological behaviour (eight cases of histiocytosis X, two giant-cell tumors), and of malignant intraosseous tumors (two malignant fibrous histiocytomas, two malignant histiocytosis, four osteosarcomas, one chondrosarcoma and two Ewing sarcomas) were studied with a panel of monoclonal antibodies reactive with monocyte/macrophages and various types of dendritic cells. In addition, tumors were further defined with a broad spectrum of antibodies against filamentous proteins and lymphocyte differentiation antigens. The specimens were stained with a triple-layer immunoalkaline phosphatase protocol. Tumors stained with these antibodies could be roughly divided into two groups. The first group comprised tumors with one predominant cell population reactive with one particular monoclonal antibody. In this group, cases of histiocytosis X were found to be consistently labelled with CD-1 antibodies. The giant-cell tumors showed a very homogeneous staining with certain monocyte/macrophage antibodies (Ki-M8). Nevertheless, even in these tumors, heterogeneity was demonstrated by the occurrence of cells with monocytic differentiation in histiocytosis X and conversely by the occurrence of cells with differentiation antigens of the dendritic cell system in giant-cell tumors. An exception has to be made for the two cases of malignant histiocytosis examined. These tumors were selectively labelled with antibodies against monocyte/macrophages (Ki-M8, IOM-1). The second group comprised tumors showing a high degree of heterogeneity demonstrated by the varying amounts of tumor cells reacting with the applied markers of the monocyte/macrophage and dendritic cell systems. In most cases it was difficult to ascribe labelled cells to the tumor cell population as opposed to an "innocent bystander" inflammatory cell population. This distinction was especially difficult in malignant fibrous histiocytomas underlining the current concept that these tumors are of primitive mesenchymal rather than true histiocytic origin.

Net enzyme activities of normal human blood cells were measured, and isoelectric focusing patterns of acid esterase (AcE) (EC 3.1.1.6) and acid phosphatase (AcP) (EC 3.1.3.2) were compared with corresponding data obtained for two acute T-lymphoblastic leukemia cell lines (JM, Molt-4), one acute B-lymphoblastic leukemia cell line (Ball-1), one acute non-B-non-T-lymphoblastic leukemia cell line (KM 3), and one promyelocytic leukemia cell line (HL-60). The AcE isozymes, found in the individual blood cell types, were regularly expressed by the corresponding cell lines. AcP was regularly expressed by lines HL-60 and KM 3, but lines JM, Molt-4, and Ball-1 showed additional isozymes and/or reduction of the intensity of the typical isozymes found in the presumed normal counterparts. This phenomenon resulted partly in an obscuration of the typical isozyme patterns. Our study documents the applicability of isozyme mapping to the characterization of permanent hematopoietic cell lines. The results suggest that long-term culture conditions can repress phenotypic properties and/or derepress gene activities.

The aim of the study was to establish enzyme-deficient mutants of the human permanent cell line U-937. Following chemical mutagenesis with the use of ethyl methanesulfonate, this cell line was chronically exposed to increasing concentrations of the toxic hypoxanthine analogue 6-thioguanine. Cells surviving hypoxanthine-aminopterin-thymidine selective media were separated by glass adherence with the use of 12-O-tetradecanoyl-phorbol-13-acetate. Three mutant clones were established, which have remained hypoxanthine phosphoribosyltransferase (HPRT) deficient for a period of 7 months, as shown by indirect measurements with the use of autoradiography and scintillation counting of cells exposed to [3H]hypoxanthine. Since the phenotypic properties and growth behavior of U-937 cells have remained unaltered after the induced mutation, a highly restricted chromosomal segment coding for HPRT seems to have been mutated.

Recent studies on the polymorphism of lysosomal hydrolases have shown that all individual blood cell types in the human being possess their own isoenzyme pattern. In the present study acid phosphatase activity of normal human B-lymphocytes and of four different types of low-grade malignant non-Hodgkin's lymphomas according to the Kiel classification was estimated. In addition, the isoenzyme pattern of AcP was investigated by isoelectric focusing. Chronic lymphocytic leukemia of B-cell type (N = 9) and centroblastic centrocytic follicular lymphoma (N = 10) demonstrated significantly lower values than lymphoplasmacytic/lymphoplasmacytoid lymphomas (N = 28) and plasmacytomas (N = 8). The isoenzyme pattern of normal human B-lymphocytes comprised 12 bands between pH 6.3 and 3.85. This basical pattern was shared by all four lymphoma entities. Only lymphoplasmatic lymphoplasmacytoid lymphoma and plasmacytoma revealed additional bands, which probably account for the higher net enzyme activity in these cases.

The aim of the present paper is to detect two different antigens simultaneously in a single slide. In cryostat sections of human tonsils, B-lymphocytes of follicle mantle-bearing surface IgD were immunostained with the alkaline phosphatase method using monoclonal anti IgD. The subsequent staining for T-lymphocyte subsets (T-helper and T-suppressor lymphocytes) was performed again with the alkaline phosphatase method using one of the monoclonal antibodies OKT 4, OKT 8, Leu 3a, Leu 2a. The best results with the alkaline phosphatase method were achieved using naphthol AS phosphate and Fast Blue BB for the revelation of the first antigen and naphthol AS-BI phosphate and diazotized New Fuchsin for the second.

Using morphologic, enzyme-cytochemical, and immunocytochemical methods, the functional diversity of the mononuclear phagocyte system can be studied only to a limited extent. Therefore, enzyme-deficient monocyte/macrophage cell lines have been established as technical prerequisites for the generation of monocyte/macrophage hybrids by applying selective media. After mutation with ethylmethanesulfonate, six clones of U-937 were selected against increasing concentrations of 5'-bromodesoxyuridine; these clones are defective in thymidine kinase (EC 2.7.1.21), as shown by autoradiography and direct measurement of [3H]thymidine uptake. A broad marker panel indicates that the clones could be appropriate for the establishment of human monocyte/macrophage hybrids.

A monoclonal antibody, Ki-M1, was produced, and its immunoreactivity was tested by light and electron-microscopic immunohistochemistry. Ki-M1 was found to react with monocytes, cells of the mononuclear phagocyte system (MPS), interdigitating reticulum cells (IDC), and the so-called indeterminate dendritic cells of lymphoid tissue. No reactivity was seen in other human tissues or other hematopoietic cells, including granulocytes and cells of the unstimulated promyelocyte cell line HL-60. Thus, Ki-M1 is the first of the monoclonal antibodies to MPS cells to react with both human IDC and MPS cells. This suggests that IDC and MPS cells may have a common cytogenesis.

Lysosomal acid phosphatase and acid esterase from highly purified, viable human thymocytes, T lymphocytes, B lymphocytes, granulocytes, monocytes, platelets, and erythrocytes were subjected to direct measurement of activity and to isoelectric focusing in polyacrylamide thin-layer slabs. The typical patterns for each cell line were found to be distinctly different from the results recently presented in this journal. The study described here opens a new possibility for the biochemical identification of cells and their functional derivatives and neoplastic variants.

The aim of this study was to test the hypothesis of the homogeneity and monocytic origin of the normal unstimulated (resident) peritoneal macrophages in man. The isoelectric focusing pattern of one of the lysosomal marker enzymes, acid esterase (EC 3.1.1.6), was studied in highly purified samples of normal human blood cell fractions. All types of blood cells showed a cell-specific constellation of their enzyme variants as far as blood cells were concerned. Comparisons were made with resident peritoneal macrophages of healthy subjects. Only blood monocytes shared all their isoenzymes with peritoneal macrophages. Two additional bands, not seen in monocytes, occurred, however, in peritoneal macrophages. During exposure of blood monocytes to prolonged glass adherence there was a gradual transition of the monocytic pattern into that of peritoneal macrophages. The results document the homogeneity and monocytic origin of resident peritoneal macrophages in man.

Human blood monocytes (BM) were stimulated with various immune modulators in short-term cultures. Tartrate-resistant acid phosphatase (TAcP) activity was demonstrated with an enzyme cytochemical method. Other members of the mononuclear phagocyte system (MPS), such as peritoneal (PM) and alveolar macrophages (AM), were also tested. Unstimulated BM and physiologic functional forms of macrophages, with the exception of AM, were invariably TAcP negative. On appropriate stimulation, particularly with media containing lymphokines, cultured BM became TAcP positive. The results suggest that TAcP is an inducible differentiation marker that indicates transformation of monocytes into cells belonging to a distinct subset of the MPS.

Dipeptidylpeptidase IV (DPP IV) occurs among others in exocrine epithelia, hepatocytes, renal tubuli, endothelia, and myofibroblasts of man and laboratory animals. Also T mu lymphocytes and their varying differentiated neoplastic counterparts reveal this enzyme activity. The present paper describes a new monoclonal antibody recognizing DPP IV. Additional efforts have been taken to detect the subcellular localization of DPP IV and its isoelectric focusing pattern in different tissue types. The monoclonal antibody anti-DPP IV (clone II-19) shows a reaction pattern indistinguishable from the corresponding enzymehistochemical reaction. These findings were further substantiated by immunoblotting analysis. In line with the results of direct enzyme measurements in different subcellular fractions a considerable portion of the enzyme is localized in the membrane fraction.

U-937 represents a well established permanent human hematopoietic cell line. Electron microscopical and enzyme cytochemical studies as well as the analysis of surface glycoproteins have provided ample evidence for the monocytic origin of U-937. Upon stimulation with the tumour promotor 12-O-tetradecanoylphorbol-13-acetate (TPA), U-937 cells evolve into macrophage-like cells with phagocytic capacities. Since human blood monocytes (BM) are characterized by five acid esterase (AcE; EC 3.1.1.6) isoenzymes which are cell-specific in terms of isoelectric points (pI) and antigenicity, attempts were made in the present study to identify identical isoenzyme patterns in non- and TPA-stimulated U-937 cells. BM, cultured BM, non- and TPA-stimulated U-937 cells, as well as samples of resident human peritoneal macrophages (PM) as clearly-defined functional derivatives of BM, were subjected to isoenzyme analysis using isoelectric focusing (IEF). The five monocyte specific isoenzymes of AcE were identified in both populations of U-937. TPA-stimulated samples showed two additional bands, identical to those appearing in cultured BM after 4 days of glass-adherence and characteristic of resident human PM. Antisera raised against AcE of BM immunoprecipitated the two additional isoenzymes of TPA-stimulated U-937. It is concluded (1) the isoenzyme mapping of AcE documents the monocytic origin of U-937. (2) TPA-stimulation caricatures transformation of BM into resident tissue macrophages as far as pure morphology and AcE isoenzyme patterns are concerned. Thus, AcE isoenzyme mapping is apt for establishing reproducible and standardized criteria of different activation/differentiation states within the monocyte-macrophage lineage.

In this report, we provide evidence that suggests the dendritic reticulum cells (DRC) occurring in germinal centers of lymphatic follicles originate from a distinct endogenous peroxidase-positive mononuclear blood cell subset. The new monoclonal antibody Ki-M4 showed a highly restricted reactivity, tested by immune histochemistry, being confined to DRC, lining cells of lymph node sinuses, and to 0.001% of nonadherent mononuclear blood cells separated at a density of 1.077 g/ml; the latter exhibited endogenous peroxidase activity in cytoplasmic granules. Such granules being specific for myelomonocytic cells implies that DRC may derive from a committed nonadherent subpopulation of this cell line, which in turn originates from bone marrow. The Ki-M4-reactive antigen was found to be destroyed by the majority of fixatives and to resist 4% paraformaldehyde for 10 min and cold acetone for 30 sec. In cell suspensions from human tonsils, Ki-M4 showed strong reactivity with the outer surface of DRC plasma membrane. This observation demonstrates the possibility of using Ki-M4 to separate DRC from cell suspensions in functional tests.

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.

Experiments were devised to clarify the cellular origin of human peritoneal macrophages. As a cell marker, the specific isoenzyme pattern of acid esterase was studied in separated normal blood cells and resident (sessile) peritoneal macrophages using isoelectric focusing. The enzyme pattern of peritoneal macrophages differed from that of all blood cells with the exception of blood monocytes. They showed all five specific monocytic isoenzymes in addition to two further bands. The results show that resident peritoneal macrophages, like exudate peritoneal macrophages, are derived from blood monocytes.

Alveolar macrophages from 28 patients with pulmonary sarcoidosis, from 6 patients with idiopathic pulmonary fibrosis and from 8 control subjects were tested for their spontaneous interleukin-1 release. Whereas alveolar macrophages from the controls and from the patients with idiopathic pulmonary fibrosis did not show a significant release of interleukin 1, 14 of the sarcoidosis patients displayed a considerable spontaneous interleukin-1 secretion. Besides radiographic signs of an increased disease activity these patients revealed a significantly elevated number of lymphocytes and a significantly increased T4/T8-ratio in the bronchoalveolar lavage indicating an enhanced inflammatory activity of sarcoidosis. The spontaneous interleukin-1 release is possibly due to a raised number of immature alveolar macrophages in the lung of patients with sarcoidosis.

8 cases of Hodgkin's disease (HD nodular sclerosis (n = 3), HD mixed type (n = 2), and nodular paragranuloma (n = 3] were investigated with monoclonal antibodies against histiocytes (Ki-M1, Ki-M3, Ki-M4, Ki-M6, and Ki-M7). Reactivity of H- and SR-cells with the monoclonal antibodies Ki-M3 and Ki-M7 could be shown at the ultrastructural level in cases of HD of nodular sclerosis and mixed type. In nodular paragranuloma no positive H-, SR- nor L & H-cells could be demonstrated. The reaction site of the monoclonal antibodies in H- and SR-cells was comparable to those in histiocytes.

In the present study phenotypic properties of non-stimulated and stimulated blood monocytes and of their normal macrophage derivatives were studied applying enzyme cytochemistry, isoenzyme analysis of acid esterase (EC 3.1.1.6), and immunohistochemical staining using a panel of newly established monoclonal antibodies specific for the monocyte/macrophage lineage. Certain marker profiles could be established for the various normal subpopulations within the monocyte/macrophage system, which were also observable in epithelioid cells and U-937 cell line considered as reactive and neoplastic differentiation variants of monocytes, respectively. Alveolar macrophages, in contrast to the other analysed monocyte/macrophage populations, showed a highly activated phenotype comparable to lymphokine stimulated blood monocytes and epithelioid cells. The results underline the concept that the adaptation of monocytes/macrophages to their particular microenvironment is of decisive importance for their definitive differentiation.

Utilizing rat peritoneal macrophages as the immunogen, a new monoclonal antibody enabling differential monitoring of the mononuclear phagocyte system (MPS) by immunohistochemistry has been raised. Designated Ki-M2R, this antigen could be detected with the immune alkaline phosphatase reaction on all macrophages including those of bone marrow, lymphatic sinuses, lymphoid follicles, splenic red pulp, and von Kupffer cells of the liver, as well as on macrophages of connective tissue, renal interstitial tissue, serous cavities, and gastrointestinal tract. Langerhans cells--the MPS-derived reticulum cells of the epidermis--interdigitating reticulum cells, and dendritic reticulum cells of lymphoid follicles were invariably negative. Blood monocytes were rendered positive only after evolving into macrophages upon appropriate stimulation. Thus, Ki-M2R selectively labels monocytes after transformation into macrophages.

Monoclonal antibodies (MAbs) Ki-M3 and Ki-M7, which detect cells of the human monocyte/macrophage lineages, were tested using immunohistochemistry on a series of benign and malignant human tumors (n = 43). Positive staining was detected in all renal-cell carcinomas (n = 19 positive for both Ki-M3 and Ki-M7), a few cases of breast carcinoma (n = 6 for Ki-M3; n = 2 for Ki-M7), in 1 case each of prostatic carcinoma (Ki-M3) and prostatic adenoma (Ki-M3), and in 1 case of endometriosis (Ki-M3). In normal human kidney Ki-M3 and Ki-M7 reacted with proximal tubules and parts of the distal tubules and with the loop of Henle. The Ki-M3 and Ki-M7 MAbs proved useful in detecting renal-cell carcinomas and in distinguishing these tumors and their metastases from morphologically similar neoplasms: in addition, our study further hints that renal cell carcinomas are derived from tubular epithelial cells.