H.J. Radzun

Combined immunohistochemical staining for surface IgD and T-lymphocyte subsets with monoclonal antibodies in human tonsils

SummaryThe 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.

A monoclonal antibody detecting dipeptidylpeptidase IV in human tissue

Dipeptidylpeptidase IV (DPP IV) occurs among others in exocrine epithelia, hepatocytes, renal tubuli, endothelia, and myofibroblasts of man and laboratory animals. Also Tµ 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.

Lysosomal acid phosphatase: Activity and isoenzymes in separated normal human blood cells

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.

Alternative myelomonocytic differentiation of HL-60 reflects dual prospective potency of promyelocytes in human

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.

Selective Recognition of Rat Follicular Dendritic Cells (Dendritic Reticulum Cells) by a New Monoclonal Antibody Ki-M4R In Vitro and In Vivo

Using unstimulated rat peritoneal cells as immunogen a new monoclonal antibody KiM4R was produced. Ki‐M4R recognizes follicular dendritic cells (dendritic reticulum cells) in germinal centers of lymphoid follicles in lymphatic tissue. In addition, sinus lining cells, endothelia of postcapillary venules, as well as mesangial cells of the renal glomerula immunoreact with Ki‐M4R in vitro as well as in vivo. This antibody might be useful for studying the interaction of follicular dendritic cells and B‐cell immune response.

Lectin binding and surface glycoprotein pattern of human macrophage populations

SummaryIn 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.

Heterogenous expression and putative structure of human monocyte/macrophage serine esterase l

Human monocyte serine esterase 1 (HMSE1) was purified from U937 cell extract. Since the N terminus of the enzyme was blocked, cleavage with trypsin was used to obtain several peptides accessible to amino acid sequencing. Based on partial amino acid sequence information, an oligonucleotide probe was synthesized and used to screen a U937 cDNA library. One clone was isolated and sequenced by us which contains an open reading frame of 503 amino acids that lacks about 50 amino acids at the N terminus relative to the protein. Computer analysis revealed an active site characteristic of known carboxylesterases with a catalytic active serine. Northern blot hybridization analysis revealed that the expression of HMSE1 is restricted to cells of the monocyte/macrophage system. In contrast to the moderate expression of HMSE1 in monocytes, alveolar macrophages showed very high amounts of the transcript. With the sequence features detected by computer analysis a structure model of HMSE1 as a dimeric, membrane-bound ectoenzyme was developed.

Cell-specific polymorphism of acid phosphatase in human blood cells: their functional and leukemic variants.

Abstract The observation that the cytochemical patterns of certain enzymes represent a stable marker for the identification of blood cells and resist functional stimuli as well as malignant transformation prompted the study of cell-specific polymorphism of acid phosphatase (EC 3.1.3.2) in human blood cells. Normal and leukemic human T and B lymphocytes as well as normal and leukemic blood monocytes and human alveolar macrophages as a functional derivative of blood monocytes were separated and subjected to purity control. Lysosomes. collected after cell cavitation, were solubilized and the particle-free supernatant was subjected to a direct assay of acid phosphatase using p-nitrophenylphosphate. Aliquot samples were analyzed by thin-layer isoelectric focusing and the banding of the acid phosphatase was recorded on gels using naphthol AS-BI phosphate after having demonstrated that on a quantitative term a positive correlation exists between the results with both substrates. The banding patterns proved to be highly cell specific as far as blood cells were concerned. The cell specificity of the acid phosphatase polymorphism defied functional as well as neoplastic transformation allowing for a clear recognition and classification of the individual cell lines. The cell-specific polymorphism of lysosomal enzymes seems to bear the potentiality of a further biochemical cell marker as exemplified by the results obtained with acid phosphatase.

Isolation of monocytic serine esterase and evaluation of its proteolytic activity.

Monocytes are characterized by high activity of α‐naphthyl acetate esterase (ANAE), distinguishing them from all other blood cells. The physiological function of this monocyte marker enzyme has not yet been elucidated. In this study ANAEs potential proteolytic activity was anlayzed because serine esterases/proteases can function as effector molecules in cell‐mediated cytotoxicity and because monocytes‐macrophages are known to exert cytotoxic effects on tumor cells. This enzyme was purified from the monocytic cell line U‐937 by preparative isoelectric focusing and a three‐step high‐performance liquid chromatography that conserved its catalytic activity. It has a molecular mass of 60 kd, and partial amino acid sequence revealed that the enzyme is not identical to known serine esterases/proteases. The purified enzyme failed to digest a couple of peptides, indicating lack of protease activity. In addition, the esterolytic activity of ANAE was not inhibited by protease inhibitors. The isolation and purification of ANAE enable further studies concerning its function in monocytes‐macrophages and its relation to monocytic cytotoxicity.

An improved method for elimination of mycoplasmas from cell cultures

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.