Lymphoid Cell Membranes Research Articles

Electron spin resonance spectroscopy in the study of lymphoid cell receptors

ESR spectroscopy is a sensitive method which can be used to monitor a wide range of changes in and near the plasma membranes of lymphoid cells during such events as ligand-induced receptor patching and capping, activation and endocytosis and phagocytosis. The advantages of the technique are rapidity, sensitivity, small sample size (10(6)-10(7) cells), and nondestructive nature. Spin labels that are attached to a range of intrinsic and extrinsic molecules give information about the fluidity and polarity of the environment in which they are located. Because of the occurrence of interaction when probes are sequestered in restricted regions of the membrane, ESR spectroscopy is also a valuable technique for measuring the formation of domains in the cell membrane.

Effects in vitro of cadmium ions on some membrane and nuclear parameters of normal and irradiated thymic lymphoid cells.

Effects of cadmium chloride upon 3H-Con A binding, number of autologous rosette-forming cells (ARFC), cell viability and the degree of DNA supercoiling were studied in normal and irradiated thymic lymphoid cells, isolated from rats and incubated up to 6 h in vitro. Cd (10-100 microM) did not significantly alter the patterns of surface markers and viability of normal thymocytes, as measured by supravital staining or nuclear pyknotic criteria. The following effects of Cd were noted for irradiated thymic cells: 1) Cd ions (25 microM) caused elimination of radiation-induced increase of Con A binding; 2) the characteristic loss of ARFC receptors, like development of nuclear pyknosis, was prevented in the presence of CdCl2 (10-100 microM); 3) the postradiation relaxation of nuclear supercoiled DNA was distinctly less pronounced with Cd. Possible reasons for these effects of Cd are discussed. Irradiated lymphoid cells are proposed as a suitable experimental model for the studies of different toxic actions of Cd and other heavy metals.

Inhibition of lymphoid cell growth by a lipid-like component of macrophage hybridoma cells.

Previous studies have shown that plasma membranes of murine lymphocytes and lymphoid tumor cells can reversibly inhibit the growth of both normal and transformed lymphocytes. The inhibitor can be extracted with organic solvents and has properties consistent with it being a lipid or lipid-like component of the membrane. This report identifies a series of cloned macrophage hybridoma cell lines, obtained by fusion of splenic adherent cells and the P388D1 line, which have very high levels of lipid-like growth-inhibitory molecules. Furthermore, a survey of seven cloned lines indicated that the macrophages fell into two distinct groups with regard to their level of growth-inhibitory activity. Group 1 lines had little or no inhibitory activity when cells were examined for their effect on a B lymphocyte proliferative response. Organic extracts from these macrophages had inhibitory activity (on a per cell basis) comparable to that seen with extracts of the P388D1 parental cell line and lymphoid tumor cells. In contrast, relatively low numbers of Group 2 macrophages could profoundly inhibit B macrophage proliferation. The growth-inhibitory activity was quantitatively recovered in organic extracts of the macrophages. Although the precise nature of the lipid moiety remains undefined, the data argue against the involvement of oxidized cholesterol. These findings indicate that lipid-like inhibitors of cell growth are present and functional in these macrophage cell lines. In addition, the results demonstrate that the inhibitory activity found in plasma membranes and liposomes is present and active in the membranes of intact cells, which is in contrast to the possibility that the inhibitor is an artifact generated during subcellular fractionation. Thus, the inhibitor is likely to have a physiologic role in growth control and in macrophage-mediated immunoregulation, probably acting via a mechanism involving cell-cell contact.

Triton X-100 extraction of P815 tumor cells: evidence for a plasma membrane skeleton structure.

It has been shown that a Triton X-100-insoluble protein matrix can be isolated from the plasma membranes of P815 tumor cells and murine lymphoid cells (Mescher, M. F., M. J. L. Jose and S. P. Balk, 1981, Nature (Lond.), 289:139-144). The properties of the matrix suggested that this set of proteins might form a membrane skeletal structure, stable in the absence of the lipid bilayer. Since purification of plasma membrane results in yields of only 20 to 40%, it was not clear whether the matrix was associated with the entire plasma membrane. To determine if a detergent-insoluble structure was present over the entire cell periphery and stable in the absence of the membrane bilayer or cytoskeletal components, we have examined extraction of whole cells with Triton X-100. Using the same conditions as those used for isolation of the matrix from membranes, we found that extraction of intact cells resulted in structures consisting of a continuous layer of protein at the periphery, a largely empty cytoplasmic space, and a nuclear remnant. Little or no lipid bilayer structure was evident in association with the peripheral layer, and no filamentous cytoskeletal structures could be seen in the cytoplasmic space by thin-section electron microscopy. Analysis of these Triton shells showed them to retain approximately 15% of the total cell protein, most of which was accounted for by low molecular weight nuclear proteins. 5'-Nucleotidase, a cell surface enzyme that remains associated with the plasma membrane matrix, was quantitatively recovered with the shells. Included among the polypeptides present in the shells was a set with mobilities identical to those of the set that makes up the plasma membrane matrix. The polypeptide composition of the shells further confirmed that cytoskeletal proteins were present to a very low extent, if at all, after the extraction. The results demonstrate that a detergent-insoluble protein matrix associated with the periphery of these cells forms a continuous, intact macrostructure whose stability is independent of the membrane bilayer or filamentous cytoskeletal elements, and thus has the properties of a membrane skeletal structure. Although not yet directly demonstrated, the results also strongly suggest that this peripheral layer is composed of the previously described set of plasma membrane matrix proteins. This article discusses possible roles for this proposed membrane skeletal structure in stabilizing the membrane bilayer and affecting the dynamics of other membrane proteins.

Growth-inhibitory activity of lymphoid cell plasma membranes. I. Inhibition of lymphocyte and lymphoid tumor cell growth.

Membranes isolated from normal spleen cells or lymphoid tumor cells were found to inhibit in vitro growth of several murine tumor cell lines including a B cell hybridoma, a thymoma, and a mastocytoma. 50% inhibition occurred at membrane protein concentrations of 60-100 micrograms/ml. A similar concentration dependence was found for inhibition of [3H]-thymidine incorporation by tumor cells and for the lipopolysaccharide-induced mitogenic response of normal spleen cells. The inhibitory activity co-purified with the plasma membrane upon fractionation of crude membranes. Membrane solubilization with deoxycholate followed by dialysis to remove the detergent gave good recovery of inhibitory activity in the resulting reconstituted membranes. Membrane-mediated growth inhibition resulted from a decreased rate of proliferation and not from increased cell death. A toxic effect of the membranes was further ruled out by the finding that increasing the fetal calf serum content of the medium could substantially reverse the growth inhibition. Thus, the plasma membrane of lymphoid cells contains a component that can slow or stop the growth of cells in culture. This membrane component may have a role in cell contact-mediated regulation of growth.

Distribution of Leu 7 (HNK-1) antigen in human digestive organs: an immunohistochemical study with monoclonal antibody.

Leu 7 immunoreactivity was demonstrated with the indirect peroxidase-labelled antibody method on frozen and paraffin-embedded tissue sections of human digestive organs. Anti-Leu 7 monoclonal antibody, which allegedly detects mononuclear cells with natural killer or killer activity, recognized lymphoid cells among intestinal epithelial cells and in the germinal centres of solitary lymphoid follicles of small and large intestine, and a few in gallbladder, liver and the lamina propria of the intestine. In addition, peripheral nerve fibres, endocrine cells in the gut and pancreas and carcinoid and islet cell tumours were also positively stained. At the ultrastructural level, Leu 7 antigen was localized on the plasma membrane of granulated lymphoid cells in the gut mucosa and on the secretory granules of intestinal endocrine cells. In normal pancreas, Leu 7 immunoreactivity was demonstrated in most cells containing pancreatic polypeptide and in many cells containing somatostatin or glicentin. Insulin-containing cells, however, lacked Leu 7 immunoreactant. These findings were obtained in both frozen sections and paraffin-embedded sections. The possible cross-reactivities of monoclonal antibodies are discussed as they raise an important caveat in immunohistochemical studies using these antibodies.

Introduction of HLA-A/B antigens into lymphoid cell membranes by cell-liposome fusion.

Conditions were optimized for surface binding by mouse lymphblastoid cell lines of liposomes containing purified human histocompatibility antigens. Antigens were reconstituted into lipid vesicles by detergent dialysis, and were incubated with the acceptor cell lines. After washing, the amount of HLA antigen on the cell surface was quantitated by double antibody radioimmunoassay. Uptake and surface expression was shown to be dependent upon vesicle phospholipid composition, vesicle:cell ratio, acceptor cell line, and stage of cell growth. These studies indicate that by using vesicles composed of 50% phosphatidylethanolamine/50% phosphatidylserine it is possible to introduce into the mouse EL4 cell surface about 70% of the amount of HLA expressed normally on the human lymphoblastoid line JY. The antigen is stably expressed on the surface for several hours and appears to be integrated into the cell membrane, as assessed by both fluorescence microscopy and susceptibility to lysis by using anti-HLA antibody and complement. The method used has the advantage over previously described procedures of not requiring the use of fusogenic proteins or agents such as polyethylene glycol or lysophospholipids, which might perturb overall membrane structure or properties.

Technical considerations in immunohistology of lymphoid cell membrane antigens.

Technical considerations in immunohistology of lymphoid cell membrane antigens.

42] Use of fluorescent antibodies in the study of lymphoid cell membrane molecules

This chapter explores that visualization of antigen-antibody reactions at the level of light microscopy is made possible by covalent coupling of fluorescent substances to antibody molecules. It discusses the large variety of reagents, which are built in the antibody system and have been recently made practical by the hybridoma technique, the specificity of the reactions, and the simplicity of the technique, making immunofluorescence the method of choice for the localization of macromolecules in cells and tissues. The chapter reviews that application to the study of membrane-bound components of living cells provides information on basic processes of cell physiology and on the organization of biological membranes, together with the possibility of detecting two antigens at the time by using antibodies of different specificity coupled to different fluorochromes, makes it possible to study association, independence, and interactions of various membrane structures at the single cell level.

Transplantation in miniature swine. XII. N-terminal sequences of class I histocompatibility antigens (SLA) and beta 2-microglobulin.

Purified class I histocompatibility antigens (SLA) from three haplotypes were prepared by papain treatment of lymphoid cell membranes obtained from spleens and lymph nodes of miniature swine homozygous at their major histocompatibility complex. Antigens were purified by ion-exchange chromatography followed by gel filtration. Purity was analyzed by SDS-PAGE, and antigenic specificity by inhibition of complement-dependent, alloantiserum-mediated cytotoxicity. The SLA antigens were reduced and alkylated, and the component heavy and light chains were isolated by gel filtration under dissociating conditions. N-terminal amino acid sequences were obtained for SLAaa, SLAcc, and SLAdd heavy chains, as well as for the light chain, beta 2-microglobulin. The swine antigens showed high levels of homology with class I antigens from other animal species. Heterogeneity was observed among the swine haplotypes, and several of the positions at which substitutions were found are apparently invariant in other animal species. In contrast, only minimal sequence heterogeneity was detected within haplotypes, the basis of which may be of relevance to understanding the evolutionary development of these molecules.

Genetic control of contract hypersensitivity. II. Suppressor cells induced by intravenous injection of DNP-coupled syngeneic spleen cells possessing thy-1 antigen, I-J determinant, Lyt-2+, 3+ antigen, FcR- and DNP-membrane binding receptor on their surfaces.

AbstractThe characteristics of suppressor cells induced by 2,4‐dinitrophenyl (DNP)‐coupled syngeneic lymphocytes (syninduced suppressor cells) were studied. 2,4‐dinitro‐1‐fluorobenzene (DNFB) contact hypersensitivity was completely suppressed when the syninduced suppressor cells were transferred intravenously. These syninduced suppressor cells had surface markers of Thy‐1, FcR− and Lyt‐2+, 3+ antigens, as well as I‐J gene products on their cell surfaces. The suppression of DNFB contact hypersensitivity was abrogated when these suppressor T cells were incubated in Petri dishes coated with the DNP‐syngeneic lymphoid cell membrane, which suggests that these suppressor T cells had the specific antigen‐binding receptors on their cell surfaces.

Actin-containing matrix associated with the plasma membrane of murine tumour and lymphoid cells.

A detergent-insoluble matrix has been isolated from murine tumour cell and lymphocyte plasma membranes. The major components of this matrix include actin and four additional proteins not previously identified as cytoskeletal proteins. Labelling studies indicate that the matrix is located on the inner face of the plasma membrane. A cell-surface glycoprotein, 5'-nucleotidase, remains associated with the isolated matrix.

Lipid analysis of isolated plasma membranes of lymphoid cells in benign thymomas of Buffalo/Mna rats.

Plasma membranes were isolated from lymphoid cells of benign thymomas obtained from inbred BUF/Mna rats (21 mo old) and from normal thymocytes obtained from young rats (7 wk old) of the same strain. The isolated plasma membranes were electron microscopically pure, and the specific activities of Na+, K+-ATPase, and 5'-nucleotidase were enhanced. The lipid compositions of the plasma membranes from these two sources were analyzed and compared. The cholesterol and plasmalogen contents of membranes from both sources were similar, but the phospholipid content of the benign thymoma lymphoid cell membranes was slightly lower than that of the normal thymocytes, resulting in a somewhat higher molar ratio of cholesterol to phospholipid. The plasma membranes of the thymoma lymphoid cells also exhibited a slightly higher microviscosity as measured with fluorescence polarization. No significant differences were observed in the phospholipid compositions of the two membrane preparations.

Suppression of the antibody response to type III pneumococcal polysaccharide with antigen coupled to syngeneic lymphoid cells

Numerous models of immunologic nonresponsiveness or tolerance have been developed in order to investigate the mechanisms involved in the regulation of selftolerance and the immune response to foreign antigens. One very efficient means of inducing tolerance with haptens involves coupling of the hapten to lymphoid cell membranes (l-6). Such hapten-modified lymphocyte membranes induce a potent and long-lasting state of nonresponsiveness in T cells which mediate delayed hypersensitivity (l-3, 6) and also in hapten-specific B cells (4, 5). Mice injected with hapten-modified lymphocytes may appear tolerant by either of two mechanisms, namely clonal inhibition and the induction of specific suppressor T cells (Ts)~ (3,5,6). Both of these mechanisms may be operative in a tolerant animal although detectable Ts activity apparently appears later and is of shorter duration than clone inhibition (3). Studies of the mechanisms by which tolerance is induced by antigen-modified lymphocytes may have considerable significance for understanding the mechanisms of induction of tolerance to self and for understanding the role of cell surfaceassociated antigens in regulation of the immune response. In this regard it would be of interest to know whether other more complex types of antigens might similarly induce tolerance if they are coupled to lymphocytes. That the induction of tolerance with antigen-coupled lymphocytes may be a general phenomenon is suggested by very recent reports which showed that an amino acid terpolymer GLO (7) as well as several protein antigens (8,9) could induce tolerance and activate Ts in mice. In this paper we report that this concept may be further generalized to include a thymusindependent (TI) polysaccharide antigen, Type III pneumococcal polysaccharide (S3). The parameters of tolerance induction by S3-coupled spleen cells (S3-SC) and its dependence on T cells will be described.

Cotranslational cleavage and glycosylation of the mineral oil-induced plasmacytoma-46B κ chain precursor by plasmacytoma microsomes

The mineral oil-induced plasmacytoma-46B light chain precursor, pL M46B, is a cotranslational acceptor for the transfer of the endogenous lipid-linked core oligosaccharides present in microsomal membrane preparations from plasmacytoma tissue as well as those from canine pancreas and Krebs II ascites tumor cells. The membranes tested appear to first cleave then glycosylate pL M46B. All the membrane preparations tested were capable of cleaving and glycosylating only a fraction of the pL M46B synthesized. The predominant new form observed in the presence of ascites membranes was the cleaved, glycosylated species, L Gly M46B, while the plasmacytoma and pancreas membranes produced both L Gly M46B and the cleaved species L M46B. These studies describe a system suitable for the study of the core glycosylation activity of lymphoid cell membranes.

Membrane Receptors and Their Redistribution in Lymphoproliferative Disorders

AbstractLymphoid cells from 20 patients with lymphoproliferative disorders, including chronic lymphocytic leukemia, hairy cell leukemia, Sezary syndrome, lymphoma, and lymphadenitis, were studied for redistribution of surface membrane immunoglobulins (SmIg) and concanavalin A (Con-A) receptors. Fluorescein-labeled polyvalent goat anti-human immunoglobulin and fluoresceinated concanavalin A were used as ligands. Results were similar with both ligands. The highest percentage of capping of ligand-membrane receptors was noted in mononuclear cells from patients with “hairy” cell leukemia: from 24% to 90%. These cells showed moderate to marked fluorescein activity and were able to cap within 15 min at 4°C. Chronic lymphocytic leukemia cells showed a weak fluorescein stain with a very low percentage of cells (0%–16%) capping. Lymph node cells from patients with lymphoma demonstrated moderate to strong fluorescein activity with only an average of 3% of the cells capping; while lymphoid cells from patients with lymphadenitis showed an average of 27.5% capping and moderate fluorescein activity. Capping of Con-A receptors in mononuclear cells from patients with Sezary syndrome was poor (0%–14%) with moderate fluorescein intensity. This report demonstrates difference in density and mobility of binding sites for SmIg and Con-A on the surface membrane of lymphoid cells from various subclasses of lymphoproliferative disorders. These differences may assist in the differential diagnosis and classification of these conditions.

Membrane receptors and their redistribution in lymphoproliferative disorders

Lymphoid cells from 20 patients with lymphoproliferative disorders, including chronic lymphocytic leukemia, hairy cell leukemia, Sezary syndrome, lymphoma, and lymphadenitis, were studied for redistribution of surface membrane immunoglobulins (SmIg) and concanavalin A (Con-A) receptors. Fluorescein-labeled polyvalent goat anti-human immunoglobulin and fluoresceinated concanavalin A were used as ligands. Results were similar with both ligands. The highest percentage of capping of ligand-membrane receptors was noted in mononuclear cells from patients with “hairy” cell leukemia: from 24% to 90%. These cells showed moderate to marked fluorescein activity and were able to cap within 15 min at 4 degrees C. Chronic lymphocytic leukemia cells showed a weak fluorescein stain with a very low percentage of cells (0%--16%) capping. Lymph node cells from patients with lymphoma demonstrated moderate to strong fluorescein activity with only an average of 3% of the cells capping; while lymphoid cells from patients with lymphaedenitis showed an average of 27.5% capping and moderate fluorescein activity. Capping of Con-A receptors in mononuclear cells from patients with Sezary syndrome was poor (0%--14%) with moderate fluorescein intensity. This report demonstrates difference in density and mobility of binding sites for SmIg and Con-A on the surface membrane of lymphoid cells from various subclasses of lymphoproliferative disorders. These differences may assist in the differential diagnosis and classification of these conditions.

Distribution of actin and myosin in muscle and non-muscle cells

Specific anti-actin and anti-myosin antibodies were shown to react in single and double immunofluorescence sandwich tests with identical sites in non-muscle cells in frozen sections of tissues and in cultured cells. In tissues, both antibodies reacted with liver cell membranes, parts of renal glomeruli, brush borders and peritubular fibrils of renal tubules, brain synaptic junctions, and membranes of lymphoid cells in thymic medulla, lymph nodes and spleen. Both antibodies reacted strongly with long parallel cytoplasmic fibrils in cultured fibroblasts, and with disrupted fibrils in cytochalasin-B treated cells. In neuroblastoma cells both antibodies gave prominent staining of growth cones and microspikes. The observation that the distribution of myosin parallels that of actin in non-muscle cells argues strongly in favour of a functional interaction between the two molecules in the generation of contractile activity in non-muscle cells.

The distribution of HLA on human lymphoid, bone marrow and peripheral blood cells.

The cellular distribution and the differential expression of HLA on cell suspensions and tissue sections has been investigated using the monoclonal antibody W6-32, which reacts with the high molecular weight chain of the major histocompatibility antigen. Lymphocytes and platelets, as assessed by autoradiographic and immunoperoxidase labeling, were the most densely labeled cells. Myeloid precursors showed more labeling than mature neutrophils. Electron microscopic immunoperoxidase labeling showed a continuous distribution of HLA antigen on lymphoid and myeloid cell membranes. Erythroid precursors (including reticulocytes), although very weakly labeled, were clearly positive, in comparison with mature erythrocytes. In the thymus, HLA-negative, thymocyte antigen-positive cells (85%) can be distinguished from HLA-positive, thymocyte antigen-negative cells (15%). By using immunofluorescence techniques on tissue sections, the former cells were shown to be cortical thymocytes and the latter medullary cells.

Genetic basis of gluten-sensitive enteropathy

Families of patients with gluten-sensitive enteropathy (GSE) were typed for HLA-A and -B antigens as well as for HLA-DW3 antigen(s) using appropriate typing alloantisera. In addition, patients and families were typed for GSE-associated B cell antigens using alloantisera obtained from mothers and wives of patients. The data obtained suggest that disease occurs within families when two conditions were fulfilled: (1) the family member is homozygous for the GSE-associated B cell antigens and (2) the family member also bears the HLA-DW3 antigen(s) or an antigen usually associated with DW3. In addition, with the family studies it was possible to show that the gene(s) controlling the DW3 antigen(s) and those controlling the GSE-associated B cell antigens are separate nonlinked genetic loci, a fact which leads to the conclusion that GSE has a genetic basis in at least two genes. It is speculated that the genes responsible for GSE code for surface proteins which are physically associated on lymphoid cell membranes and which form receptors important to the initiation of disease.