Increased expression of highly branched N-linked oligosaccharides terminating in N-acetylglucosamine residues in neoplastic and sclerodermal chicken fibroblasts.

Although neoplastic cells often show a shift towards the expression of larger N-linked oligosaccharides compared to their normal counterparts, little consideration has been given to the possibility that these changes might be a more general phenomenon characteristic of certain neoplastic and non-neoplastic proliferative disorders. Terminal N-acetylglucosamine (GlcNAc) cluster antigen (TGCA) is an immunoreactive epitope(s) of highly branched N-linked oligosaccharides terminating in GlcNAc residues. Here we have compared the expression of this antigen in normal, neoplastic and sclerodermal chicken fibroblasts by immunomorphological methods. TGCA was detectable in only a few, if any, fibroblasts of normal chicken skin or those cultured from chicken embryos. In contrast, the antigen appeared in 15 to 30% of chicken embryo fibroblasts transformed with avian sarcoma viruses and about 50% of neoplastic fibroblasts of both Rous sarcoma virus-induced fibrosarcomas and carcinogen-induced transplantable fibrosarcomas. Significantly, TGCA was also found in most activated fibroblasts in the skin of chickens with hereditary scleroderma. These results indicate that increased expression of highly branched N-linked oligosaccharides terminating in GlcNAc residues is characteristic of both neoplastic and sclerodermal chicken fibroblasts. Investigation of this phenomenon may thus provide insight into biochemical pathways involved in neoplastic transformation and pathogenesis of a number of non-neoplastic proliferative connective tissue disorders such as scleroderma. Moreover, changes in the expression of TGCA-positive oligosaccharides (or their modified biochemical counterparts in mammalian species) may have considerable value for diagnosis of several connective tissue diseases.

Immunohistochemical study of ontogeny and phylogeny of a terminal N-acetylglucosamine cluster antigen.

In this work an immunohistochemical method was used to study the ontogeny and phylogeny of a terminal N-acetylglucosamine (GlcNAc) cluster antigen which is an epitope(s) of highly branched N-linked oligosaccharides terminating in GlcNAc residues. The ontogenic studies demonstrated that expression of the antigen is developmentally regulated in lymphocytes, epithelial cells of endodermal origin and kidney mesangial cells of the chicken. The antigen was found in several other avian species studied, namely, the Japanese quail, duck, goose and turkey. Furthermore, the distribution of the antigen in all these species was similar. In adult animals, it was found in bursal and thymic lymphocytes, macrophages, spleen reticulum cells, epithelial cells of the intestine and bronchioles and capillary endothelial cells. The antigen was also detected in epithelial cells of the gastrointestinal tract of several lower vertebrates studied: the amphibian (frog), reptile (chameleon) and fish (rainbow trout). It was undetectable in various organs of the human, African green monkey, calf, pig, rat and guinea-pig, but was found in the intestinal epithelial cells of ten mouse strains. It is likely that biosynthetic processing leading to the formation of highly branched N-linked glycans terminating in GlcNAc residues is conserved during evolution in birds and other lower vertebrates.

Immunochemistry of highly branched N-glycans. Terminal N-acetyl-D-glucosamine (GlcNAc) cluster antigens contain epitopes composed of terminal GlcNAc residues linked to mannose.

We have previously demonstrated by the immunoperoxidase method the presence of a chicken heterophile antigenic determinant (CHAD-1) in medullary lymphocytes of the bursa of Fabricius and thymus as well as in some nonlymphoid cells. It has been found that the anti-CHAD-1 antibody could be neutralized by absorption with several glycoproteins or glycopeptides containing highly branched, asparagine-linked oligosaccharides terminating in N-acetylglucosamine residues. In the present study, fetuin, desialo-fetuin, and a series of 27 highly purified oligosaccharides with well-defined structures were used to investigate the chemical composition and fine structure of the CHAD-1 epitope. It was shown that anti-CHAD-1 antibody binds to oligosaccharides with at least three terminal N-acetyl glucosamine residues at the nonreducing end. These residues may be linked beta 1-2, beta 1-4, or beta 1-6 to one, two, or three different mannose residues. The antibody combining site accommodates at least four carbohydrate residues. Oligosaccharides containing five or six terminal N-acetylglucosamine residues at the nonreducing end demonstrated the highest immunoreactivity with the anti-CHAD-1 antibody. Substitution of terminal N-acetylglucosamine residues with galactose, or with galactose and sialic acid, masks CHAD-1. On the basis of this work, epitopes that react with the anti-CHAD-1 antibody will be renamed terminal N-acetylglucosamine cluster antigens (TGCA). Anti-TGCA antibody has potential use in the monitoring of biosynthetic processing of asparagine-linked oligosaccharides and in studies of their cellular distribution and functions.

Chicken mucin-cross-reactive antigen.

We have previously described a chicken heterophile antigenic determinant (CHAD-1) shared by Mycobacterium smegmatis and chicken tissues. We then demonstrated that CHAD-1 is present on several chicken glycoproteins and that its immunoreactive domains are highly branched asparagine-linked oligosaccharides terminating in N-acetylglucosamine residues. In the present study, we have shown that CHAD-1 is also expressed by mucin purified to homogeneity from a soluble mucus of chicken intestine. Another antigen found on chicken mucin is a chicken mucin-cross-reactive antigen (CMCRA). Antisera to this antigen were produced by immunization of rabbits with an enriched preparation of CHAD-1 isolated from the bursa of Fabricius. These antisera were absorbed with Mycobacterium smegmatis (to block the anti-CHAD-1 antibody) and with chicken serum, and then used for immunoperoxidase staining of chicken tissue sections for CMCRA. The latter antigen was detected in most medullary cells of the bursa, in epithelial cells and Hassal's corpuscles of the thymus, and in mucus-producing cells of the intestine, esophagus, trachea, and bronchi. Using Western immunoblot analysis, we demonstrated that CMCRA is expressed by a number of polypeptides extracted from bursal lymphoid cells. These polypeptides could not be detected in extracts of thymus, spleen, peripheral blood or bone marrow mononuclear cells.

Identification of terminal N-acetylglucosamine residues of highly branched asparagine-linked oligosaccharides as immunoreactive domains of a chicken heterophile antigenic determinant.

Chicken heterophile antigenic determinant (CHAD-1) has been previously found in medullary lymphocytes of the bursa and thymus as well as in some non-lymphoid cells by the immunoperoxidase method, using rabbit antiserum to a complete Freund's adjuvant (CFA) as the first antibody. In this work we demonstrated that absorption of anti-CFA serum with highly purified preparations of hen egg white glycoproteins (ovomucoid, ovoinhibitor, ovalbumin) or chicken orosomucoid completely blocked immunoperoxidase staining for CHAD-1. Treatment of these glycoproteins with beta-N-acetylglucosaminidase suppressed their capacity to inhibit this staining. Absorption of anti-CFA serum with asparagine-linked glycopeptides which have the mannose alpha 1,3 arm disubstituted by GlcNAc residues and which have another GlcNAc residue linked beta 1,4 to the beta-linked mannose of the core also inhibited staining for CHAD-1. These data indicated that highly branched asparagine-linked oligosaccharides with terminal GlcNAc residues beta-linked to mannose represent immunoreactive domains of CHAD-1.

Novel heterophile chicken antigen: immunohistochemical localization using antisera to Mycobacterium smegmatis and possible association with lymphocyte maturation.

A novel heterophile antigen shared by Mycobacterium smegmatis and chicken tissues was demonstrated by the indirect immunoperoxidase method using antisera raised in rabbits immunized with a complete Freund's adjuvant containing killed Mycobacterium smegmatis as an immunostimulating component. This antigen was strongly expressed in medullary lymphocytes of the thymus and bursa of Fabricius, but was undetectable in lymphoid cells of the cortical regions of these organs. Only a few lymphocytes stained positively for the antigen in T- and B-cell areas of the spleen. These data suggest that the heterophile antigen is associated with the intrathymic and intrabursal maturation of chicken lymphocytes. The antigen was also detected in some nonlymphoid cells. It was not found in sheep erythrocytes, human and rat tissues or in killed bacillus Calmette--Guerin.

A flow cytometric method for the detection of adenosine deaminase in mononuclear cells.

The purpose of this study was to develop a flow cytometric method for the detection of adenosine deaminase (ADA) in a single cell suspension of mononuclear cells. Anti-human ADA antibody was purified by affinity chromatography on a column of Sepharose 4B to which calf ADA was covalently linked. This antibody was used for indirect immunofluorescent staining of cells fixed in 4% paraformaldehyde. The specificity of staining was proved by substitution of anti-human ADA with normal rabbit IgG and by absorption experiments. The fluorescence profile of the cells was then analyzed by flow cytometry. Two groups of cells were studied: (a) thymocytes, tonsil cells and peripheral blood mononuclear cells (PBMC), (b) ADA-positive and ADA-deficient cell lines. In each of these populations of cells a peak of specific immunofluorescence staining for the enzyme could be easily distinguished from weak background staining of control preparations. Within each group, the cell population with higher ADA activity also displayed a greater intensity of cell fluorescence. Flow cytometry provides a means for quantitation of ADA in individual mononuclear cells.

Immunomorphological localization of adenosine deaminase in rat tissues during ontogeny.

Immunomorphological methods were used to localize adenosine deaminase in tissues of the rat at different stages of ontogeny. In the thymus, lymphocytes began to express significant amounts of the enzyme with the appearance of demarcation between the cortex and medulla at 17 days of gestation. At any stage of ontogeny studied, strong adenosine deaminase staining was seen predominantly in cortical thymocytes. In the spleen and lymph node, the enzyme was initially detected in T cell areas, whereas primary follicles did not show positive adenosine deaminase staining. During further development, the enzyme was demonstrated in some lymphocytes of germinal centres and plasma cells. In the duodenum, epithelial cells of villi and the neck of crypts showed positive adenosine deaminase staining whereas no staining for the enzyme was observed in the epithelial cells of the base of crypts. Strongly positive staining for adenosine deaminase appeared in plasma cells of the lamina propria by four weeks after birth. The transient positive reaction for the deaminase could be recognized in epithelial cells of tubules of the kidney during late foetal and early postnatal development. The tubules of adult rats did not stain for the enzyme. In the cartilage of 15-day foetuses, positive adenosine deaminase staining was seen only in perichondrial cells and hypertrophic cells. Kupffer cells in the liver and endothelial cells of blood vessels stained positively for the enzyme at every stage of ontogeny studied.

Ecto-5'-nucleotidase. I. Expression in human normal and neoplastic lymphoid cell lines representing sequential stages of B-cell differentiation.

The possible association of ecto-5'-nucleotidase (5'-NT) with differentiation of B-cells was explored with the use of normal and neoplastic lymphoblastoid cell lines representing sequential stages of B-cell maturation. There was no relationship between patterns of enzyme expression in the cell lines and immunoglobulin (Ig) secretion, chromosome constitution, proliferative rate, cell volume, or the presence of B1 and B2 antigens. Pre-B-cell lines, which were negative for surface Ig or Ig secretion but positive for cytoplasmic mu-chains, showed the presence of 5'-NT, whereas 9 of 11 lymphoma cell lines, Burkitt's or non-Burkitt's type, both secreting and nonsecreting, did not exhibit enzyme activity. Four myeloma cell lines and 13 of 15 normal B-cell lines were positive for 5'-NT. These results suggested that 5'-NT was present in pre-B-cells and in some very early B-cells. 5'-NT usually disappeared from early and some intermediate B-cells and reappeared in mature B-cells and plasmacytoid cells.

Ecto-5'-nucleotidase. II. Effect of 12-O-tetradecanoylphorbol 13-acetate on the expression of enzyme in normal and neoplastic B-cell lines.

Immature B-cells, including B-cell lymphoma lines, are often deficient in ecto-5'-nucleotidase (5'-NT) activity. 12-O-Tetradecanoylphorbol 13-acetate (TPA) was shown to be capable of inducing maturation toward plasmacytoid-like cells in immunoglobulin (Ig)-secreting B-cell lines. An attempt was made to induce the enzyme in 5'-NT-negative B-cell lymphoma lines with TPA to clarify the relationship between 5'-NT and B-cell differentiation. After 3 days in the presence or absence of TPA, these cell lines were examined morphologically, and their 5'-NT activity, Ig secretion, surface Ig, and Ia, B1, and B2 antigens were estimated. Neither Ig secretion nor 5'-NT activity was induced by TPA in any of 4 nonsecreting cell lines studied. Ig secretion was significantly increased in 4 of 5 lg-secreting cell lines. Two of these inducible cell lines, JD 38 and ST 486, became positive for 5'-NT activity and acquired morphologic characteristics of plasma cells after culture with TPA. The lymphoma cell line JD 38 was transplanted into nude mice and gave rise to a solid tumor. Although the tumor cells remained negative for 5'-NT, they could be induced by TPA to express both the enzyme activity and plasmacytoid-like appearance. These data suggested that in the Ig-secreting B-cell lymphoma lines, there was an association between the inducibility of 5'-NT and the capacity of these cell lines to undergo plasma-cytoid-like transformation in response to TPA.

Immunohistochemical localization of adenosine deaminase in human benign extrathymic lymphoid tissues and B-cell lymphomas.

Immunomorphologic methods were utilized to localize adenosine deaminase (ADA) in extrathymic benign lymphoid tissues and B-cell lymphomas. In reactive lymph nodes, tonsils and appendix, germinal centers displayed strong ADA-positive nuclear staining in small cleaved lymphocytes and weak nuclear and/or cytoplasmic staining in large lymphoid cells. A significant proportion of ADA-positive lymphocytes in the germinal centers were B-cells. The mantle zone of secondary follicles did not stain for ADA. The plasma cells in the medullary cords demonstrated mainly cytoplasmic staining. In the spleen, ADA-positive lymphocytes were located in the periarteriolar sheath and paratrabecular white pulp. In lymphoma B-cells, patterns of ADA staining were similar to those observed in normal B-lymphocytes of similar morphology. This study demonstrated that human normal and lymphoma B-lymphoid cells are heterogeneous with respect to ADA expression. This heterogeneity appears to be associated with differentiation and/or proliferation of B-lymphocytes.

Localization and identity of adenosine deaminase-positive cells in tissues of the young rat and calf.

Rabbit antibody to calf adenosine deaminase (ADA) was used to localize this enzyme in tissues of the young rat and calf by the immunoperoxidase method. The distribution patterns of ADA in most tissues were similar for both species. Within the thymus gland, the enzyme was strongly expressed predominantly in cortical lymphocytes. In the spleen and lymph nodes, most lymphocytes of T-cell areas stained weakly for ADA, whereas only a small number of ADA-positive cells were found in B-cell areas. Clumps of strongly ADA-positive mononuclear blastoid and plasma cells were observed in the medullary regions of lymph nodes, around peri-arteriolar lymphocyte sheaths and in the red pulp of the spleen, and in the lamina propria of the intestine. Double immunofluorescence staining studies in the rat showed that some of these blastoid cells contained both ADA and immunoglobulins and appeared to be plasmablasts. Strong staining for ADA was also found, in both the rat and calf, in as yet unidentified mononuclear blastoid cells in the interstitium of non-lymphoid organs (kidney, heart, lung), in endothelial cells of some arterioles and capillaries, and in Kupffer cells of the liver. In addition, ADA was strongly expressed in calf bile canaliculi. These studies define areas in rat and calf tissues which contain ADA-positive cells and provide a model system for investigations of the relationship between ADA and the function and development of these cells.

Purification and some properties of adenosine deaminase from human thymus.

Two fractions of adenosine deaminase (ADA) were separated by ion-exchange chromatography and purified to homogeneity from human thymus tissue by a combination of conventional biochemical methods and affinity chromatography. Some of the physical, chemical and serological properties of the two fractions were compared to those of erythrocyte ADA. All three proteins had apparent molecular weights of about 45,000. They exhibited similar amino acid composition, specific enzymatic activities, Km values for adenosine and antigenic activities as determined by radioimmunoassay. A small portion of ADA isolated from thymus did not bind to complexing protein whereas all of the erythrocyte ADA was bound by this protein. So far, this has been the only difference found between thymic and erythrocyte ADA.

Modulation of human T-cell differentiation markers by 12-O-tetradecanoylphorbol-13-acetate.

The analysis of seven differentiation markers following incubation with the tumor promotor 12-O-tetradecanoylphorbol-13-acetate (TPA) was examined in the human leukemic T-cell line MOLT-3. Significant changes were observed in the activity of the markers terminal deoxynucleotidyl transferase (TdT). spontaneous proliferation and the ability of these cells to bind sheep erythrocytes. Levels of human thymus-leukemia-associated antigen (HThy-L) recently identified as a low molecular weight form of adenosine deaminase (ADA), were reduced by about 50%. No significant changes were observed in ecto-5'-nucleotidase [5'-NT) activities, in the proliferative response to PHA, or in the expression of IA-like antigens. These data and the time kinetics of the changes suggest that following incubation of these T-lymphoblasts with TPA there is a sequential loss of TdT, loss of the capacity for spontaneous proliferation, and the appearance of receptors for sheep erythrocytes. Subsequently there is a decrease in the level of HThy-L/ADA. This sequence appears to follow that proposed for prethymic precursor T-cell differentiation following activation with thymic epithelium.

Heterogeneity of human thymocytes and a malignant T-lymphoblast cell line, MOLT-3.

The purpose of this paper was to study the heterogeneity of human thymocytes and leukemic cells of the T-cell line MOLT-3 by velocity sedimentation. Analysis of the subpopulations of thymocytes demonstrated that they represent a heterogeneous population of cells with respect to their size, proliferative activity, and presence and quantities of terminal deoxynucleotidyl transferase and human thymus leukemia-associated antigen, a thymic isozyme of adenosine deaminase (HThy-L/ADA). Only a minor subpopulation of thymocytes (large cells) was in active cycle. The highest level of HThy-L/ADA was associated with the main subpopulation of thymocytes sedimenting at 3 to 4 mm/hr while low amounts of the HThy-L/ADA antigen (enzyme) were found in the minor fractions of the small and large cells. The distribution of terminal deoxynucleotidyl transferase-positive cells indicated that most, but not all, thymocytes contain the enzyme. Analysis of the T-cell line MOLT-3 showed that these cells could be separated into subpopulations with different biochemical and biological properties. More than one subpopulation of cells was capable of DNA synthesis. In contrast to the thymocytes, all fractions of MOLT-3 cells contained high amounts of HThy-L/ADA. The proportion of terminal deoxynucleotidyl transferase-positive cells as a function of sedimentation velocity was also quite constant although there was a slight but reproducible drop in the percentage of these cells in the slowly sedimenting fractions. The percentage of cells with receptors for sheep erythrocytes also remained high in fractions separated on the basis of size, although a consistently higher percentage was found in smaller cells. These studies indicated that thymus cells as well as the malignant T-cell line MOLT-3 can be separated on the basis of sedimentation velocity into subpopulations with different biological and biochemical properties. The data also indicated that the heterogeneity of MOLT-3 line cannot be explained solely on the basis of volume changes due to cell cycle, suggesting that they may represent heterogeneous populations of cells.

An immunomorphologic study of adenosine deaminase distribution in human thymus tissue, normal lymphocytes, and hematopoietic cell lines.

Adenosine deaminase (ADA) has been detected immunohistochemically in human thymus. The enzyme was localized predominantly in cortical thymocytes. Occasional lymphocytes in the medulla were also positive for ADA. Blood vessels, connective tissue, and Hassall's corpuscles were not stained for the enzyme. Using single-cell immunofluorescence and immunoperoxidase assays, we found that thymocytes and lymphoid cells of peripheral blood (PBL) and tonsils were heterogeneous with respect to ADA expression. About 70% of thymocytes were strongly stained for the enzyme whereas weak staining was seen in 20% of cells. About 10% of thymocytes were ADA negative. Twenty percent of PBL and tonsil cells were strongly positive for ADA, 10% of cells were negative for the enzyme, and weak staining was seen in the remainder. Bone marrow mononuclear cells were not stained for ADA. One hundred percent of lymphoblasts of 3 T cells leukemia lines were strongly stained for the enzyme whereas weak staining was seen in a pre-B cell leukemia line, 4 B cell lymphoma/normal lines, 2 non-T, non-B cell leukemia lines and 2 myeloid cell leukemia lines. There was a good correlation between intensity of cellular staining and quantity and activity of ADA detected in cell extracts by radioimmunoassay and enzymatically. The development of immunomorphologic methods for the detection of ADA provides a tool to study the role of the enzyme in function(s) and differentiation of normal and leukemic cells.

Detection of human, rat and mouse adenosine deaminase by immunochemical and immunomorphologic methods using antiserum to calf enzyme.

A commercial preparation of calf adenosine deaminase (calf ADA) was further purified by affinity chromatography and used for immunization of rabbits. The resulting anti-calf-ADA sera reacted by immunodiffusion with both calf and human ADA, and precipitated about 90% of radiolabeled enzyme isolated from human thymus tissue. Moreover, ADA activity was detected in the pellets formed by immunoprecipitation of unlabeled human enzyme by anti-calf-ADA sera. These antisera were successfully used for the immunomorphologic localization of ADA in human thymus tissue and in lymphoid cell preparations. The anti-calf ADA sera could also be used for the immunofluorescent detection of enzyme in rat and mouse thymocytes. The utilization of anti-calf-ADA serum for immunochemical and immunomorphologic detection of enzyme provides a valuable and sensitive reagent for the identification of ADA-positive cells in humans and several other species.

Identification of human thymus-leukemia-associated antigen as a low-molecular-weight form of adenosine deaminase.

In the determination of whether human thymus-leukemia-associated antigen (HThy-L) is a low-molecular-weight form of adenosine deaminase (ADA), both HThy-L and ADA were found to have the same molecular weight of 45,000 as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The antigen and enzyme displayed a phenomenon of complete identity in immunodiffusion and a high degree of cross-reaction in a competitive radioimmunoassay for HThy-L or ADA. When tested for adenosine-deaminating activity, HThy-L was nearly as active as purified low-molecular-weight ADA from erythrocytes. However, HThy-L and ADA differed in their capacity to combine with the complexing protein isolated from human kidney. Apparently, HThy-L represents a thymic isoenzyme of ADA and is the first antigen to be associated with differentiation of hematopoietic cells for which a functional activity is established.