Changes In Surface Markers Research Articles

Decreased oxidative burst activity of monocytes from asymptomatic HIV-infected individuals

Although changes in function of monocytes from AIDS patients have previously been reported, the functional deficits that could occur in monocytes from asymptomatic HIV-seropositive individuals has not been extensively investigated. The goal of this study was to evaluate the oxidative burst response and cell surface marker expression of monocytes from this group. Both the oxidative burst, as measured by 2′, 7′-dichlorofluorescin diacetate oxidation, and cell surface markers were evaluated on CD14 + (Leu-M3) monocytes by two-color flow cytometry. A significantly lower oxidative burst capacity was observed in asymptomatic, seropositives after phorbol myristate acetate and calcium ionophore stimulation when compared to seronegative controls. However, differences in oxidative burst between the two groups were not observed after stimulation with heat-aggregated IgG. The oxidative burst of monocytes from seropositive HIV antigen + or antigen − subjects was not significantly different. The most significant difference in monocyte surface markers between seropositive and seronegative controls was a decrease in the proportion of complement receptor 3 + (CD11b +) cells while slightly decreased numbers of CD13 + and CD33 + monocytes were observed. The decreased expression of CD11b + cells in seropositives was found entirely within the seropositive, HIV antigen + group. Similarly, when monocytes from seropositive HIV antigen + and antigen − were compared, significantly lower numbers of CD4 + and HLA-DR + cells were noted. These results indicate that significant changes in monocyte function and surface markers occure early during the course of HIV infection and are associated with the expression of serum HIV antigen.

Analysis of the topological changes induced on cells exposed to adhesive or mechanical stimuli

Fluorescent probes are widely used to study cell structure and function. However, few reports were devoted to a quantitative analysis of the intracellular distribution of fluorescent markers. In the present work, we describe the topographical changes of surface and cytoskeletal markers on individual cells subjected to adhesive or mechanical interaction. Conjugates were prepared with a cytotoxic T-lymphocyte clone and target cells. Specific antigens, membrane phospholipids, surface glycoconjugates, and polymerized actin were labeled with fluorescent antibodies or biochemical probes. The analysis of fluorescence distributions in conjugates demonstrated a selective reorganization of the plasma membrane with a gathering of some molecular species in the intercellular adhesion area. Furthermore, individual phagocytic cells were sucked into glass micropipets, then stained with fluorescent phallacidin to analyze the effect of mechanical efforts on the cytoskeleton organization. The concentration of polymerized actin was found to be similar in mechanically-induced protrusions and whole cells. It is concluded that adhesive interactions may result in marked cell polarization and formation of membrane zones with a particular biochemical composition. The submembranar cytoskeleton might play a role in this process.

Modulation of interferon-γ-induced major histocompatibility (MHC) and CD14 antigen changes by lipophilic muramyltripeptide MTP-PE in human monocytes

The lipophilic muramylpeptide derivative muramyltripeptide-phosphatidylethanolamine (MTP-PE, 0.05 to 5 μg/ml) and human recombinant interferon-γ (rIFN-γ, 1 to 100 U/ml) were applied singly or in combination to fresh human mononuclear blood leucocytes in vitro. After 15 to 72 hr incubation, culture- and drug-induced changes in β2-microglobulin (MHC class I associated), HLA-DR (MHC class II), and Leu-M3 (CD14) antigen expression were investigated by flow cytometry; changes in monocyte morphology (forward light scatter and side scatter) were assessed by scatter analysis. It was found that (1) rIFN-γ caused a simultaneous down-regulation of the CD14 antigen and an up-regulation of MHC class I and class II molecules on the surface of cultured monocytes; (2) MTP-PE, which by itself failed to influence the expression of these antigens, synergized with rIFN-γ in increasing MHC antigens and reducing CD14; (3) at high concentrations rIFN-γ reduced monocyte viability to a small but significant extent and this effect was further potentiated by MTP-PE; and (4) untreated monocytes in culture showed an apparently MTP-PE-insensitive increase in size, density, and β2-microglobulin, HLA-DR, and CD14 antigen expression. The influence of MTP-PE on rIFN-γ-induced surface marker changes may contribute to its immunoadjuvant activity in vivo.

Analysis of heavy metal immunotoxicity by multiparameter flow cytometry: Correlation of flow cytometry and immune function data in B6CF1 mice

Bone marrow and spleen cells obtained from female B6C3F1 mice given a single i.p. exposure to cadmium acetate (0.9 mg/kg), lead acetate (12 mg/kg), or sodium acetate (12 mg/kg), were studied using flow cytometry, immunologic, and hematologic assays. Significant changes were detected in subpopulations of bone marrow cells using multiparameter flow cytometry within 1 day following treatment with cadmium or lead. Bone marrow cells obtained from B6C3F1 mice 5 days after treatment with cadmium or lead were found to have a decreased number of cells expressing Mac-1, 55-7.2, 14.8, and Lyt-1 antigens, suggesting a shift to immature cell types. An increase in the number of progenitor cells (CFU-C) obtained from the bone marrow of mice treated with heavy metals was also noted 5 days after exposure to cadmium or lead. A time-dependent suppression of the in vitro primary humoral immune response of spleen cells to SRBCs, TNP-Ficoll and TNP-LPS was produced by cadmium or lead treatment. Suppression of the mitogenic response of spleen cells to Con A, PHA, and LPS was also found to be time-dependent. Spleen cell surface marker expression (Mac-1, Lyt-1, Lyt-2 and 14.8) was altered in response to cadmium or lead treatments, but these changes did not appear to correlate with the humoral immunity or mitogen-induced proliferation data. These studies demonstrate that changes in cell surface markers on discrete subpopulations of lymphoid cells present in the spleens of heavy metal exposed mice may not correlate with alterations in the functional activity of these cells. However, changes in murine bone marrow surface markers in response to cadmium or lead treatment predicts a shift to immature cell types, which appeared to correlate with the increase in CFU-C activity.

Phenotypic and karyotypic properties of hyperdiploid acute lymphoblastic leukaemia of childhood.

The DNA/cell content was measured by flow cytometry in samples obtained from 98 unselected children with acute lymphocytic leukaemia (ALL) at diagnosis. The frequency of anomalies in modal DNA content was compared to that encountered in acute childhood non-lymphocytic leukaemia (ANLL) and disseminated non-Hodgkin's lymphoma (NHL). In ALL the most frequent (35%) aberration in DNA content was an increase by 20% relative to the modal value of normal white blood cells. This subcategory, referred to as hyperdiploid ALL (HD-ALL), was characterized by a close association with the expression of the c-ALL surface marker (20/20 patients) and characteristic numerical chromosome changes, including tri- or tetrasomy of chromosome 21. Moreover, patients with hyperdiploid ALL had a much lower peripheral leucocyte count (P = 0.001) than those with diploid disease and a varying proportion of their leukaemic cells existed in the peripheral blood as morphologically normal lymphocytes expressing the c-ALL antigen. Within the standard risk category, patients with HD-ALL had a longer disease-free survival than those with diploid disease (P = 0.058). It is concluded that routine analysis by flow cytometry can conveniently and consistently detect ALL patients with hyperdiploid chromosome numbers. Hyperdiploid ALL constitutes a fairly large subtype of childhood ALL with specific biological and karyotypic properties, possibly associated with favourable prognosis.

Cellular basis for neonatally induced T-suppressor activity. Primary B cell maturation is blocked by suppressor-helper interactions restricted by loci on chromosome 12.

The cellular mechanism and genetic restriction of neonatally induced HA-specific suppressor T (Ts) cells have been examined. The in vivo effect of these Ts cells on antibody production, primary B cell proliferation, B cell surface marker changes, and helper T (Th) cell priming during primary responses to HA have been determined. The results indicate that, although antigen-induced B cell proliferative responses and surface marker changes occur in the presence of Ts cells, differentiation to Ig secretion, and long-lived memory B cell production are prevented. Further, antigen-specific Th cell priming is completely ablated by Ts cells, suggesting that Ts act by preventing the delivery of Th signals required for both the later stages of primary B cell maturation, and the formation of memory B cell populations. Finally, in vivo cell mixing experiments using congenic mice indicate that this Ts-Th interaction is restricted by loci on mouse chromosome 12.

Mode of cell surface marker changes during retinoic acid-induced differentiation in pluripotent embryonal carcinoma cells

Pluripotent teratocarcinoma cell line, 311, was cultured in the presence of retinoic acid (RA) and studied for the processes of early marker changes associated with cell differentiation. The cell populations that have lost peanut agglutinin (PNA), Lotus tetragonolobus agglutinin (LTA) or wheat germ agglutinin (WGA) receptor increased in proportion to the period since the start of RA treatment. The kinetics of the appearance of these receptor-negative cell populations suggests that the differentiating cells lose lectin receptors in the order of PNA, LTA and WGA. However, the changes in F9 antigen(s) and LTA receptor occurred at an equal frequency in PNA + and PNA − cells, indicating that, although the loss of lectin receptors takes place in a distinct order, the change in each receptor itself proceeds independently of the state of other lectin receptors.

Effect of thymosin and phorbol ester on purine metabolic enzymes and cell surface phenotype in a malignant T-cell line (MOLT-3)

Previous studies have shown that thymosin (fraction 5) is able to induce surface differentiation markers on normal murine bone marrow T-cell precursors. Phorbol ester (TPA) promotes differentiation of human leukaemic lymphoblasts as assessed by changes in phenotypic surface markers and terminal deoxynucleotidyl transferase (TdT) activity. Changes in levels of purine degradative enzymes occur during T-cell maturation with a fall in adenosine deaminase (ADA) and a rise in purine nucleoside phosphorylase (PNP) and 5′ nucleotidase (5 'NT) activities. We have now investigated the effect of both thymosin (fraction 5) and TPA on a human leukaemic T-cell line (MOLT-3) in expression of the surface antigenic markers NAI/34 (a marker of immature thymocytes) and OKT11 (which corresponds to the sheep erythrocyte receptor) and of TdT, ADA, PNP and 5 'NT. Thymosin (after 96 h incubation) significantly reduced the number of cells positive for NAI/34 from 76.0 ± 5.0% (mean ± S.D.) to 51.3 ± 9.3% ( p < 0.01) but caused no significant change in the percentage of TdT or OKT11 positive cells. ADA levels were significantly reduced ( p < 0.02) and 5 'NT levels were significantly elevated (mean increase 303 ± 142% of control, ( p < 0.001) but the increase in PNP level (108 ± 16.8% of control) was not significant ( p > 0.05). On the other hand, TPA (after 96 h incubation) significantly reduced cells positive for NAI/34 from 76.0 ± 5.0 to 30.0 ± 9.8% and for TdT from 81.7 ± 6.5 to 17.3 ± 4.4% and increased OKT11 positive cells from 67.3 ± 5.5 to 89.0 ± 2.8% ( p < 0.001). TPA caused no significant change in 5 'NT or ADA levels ( p > 0.05), but an increase in PNP level to 158 ± 12.9% of control ( p < 0.02). The present study demonstrates for the first time that the normal thymic hormone, thymosin is capable of inducing differentiation changes in thymic derived human leukaemic cells. In addition, it shows that different inducing agents may cause different patterns of differentiation changes in leukaemic cells.

Changes in surface markers of human monocytes following their in vitro maturation to macrophages.

Peripheral blood monocytes are a transitory population between bone marrow promonocytes and tissue macrophages (1,2). Blood monocytes have several characteristics which assign them to the macrophage lineage. However, each of the macrophage markers is not always expressed on the entire monocyte population (3,4) and some markers and functions are altered during monocyte maturation or activation (5,6,7). In vitro cultivation of human monocytes is accompanied by a series of changes which results in their transition to macrophages (8). In the present study, we analyzed membrane markers on freshly obtained monocytes and on mature macrophages. These were the receptor for peanut agglutinin (PNA), which is associated with cell maturation in T lymphocytes (9) and the HLA-DR which is associated with some immune functions (10–12).

Induction of cytotoxic activity in human lymphocytes against autologous and allogeneic melanoma cells in vitro by culture with interleukin 2.

The influence of interleukin 2(IL2) on the cytotoxic activity of lymphocytes from patients with melanoma against autologous and a variety of allogeneic melanoma cells was studied. IL2 was produced from blood lymphocytes cultured for 24 h with phytohaemagglutinin (PHA) and purified by membrane chromatography to exclude PHA. Lymphocytes from 13 patients with melanoma at various clinical stages were cultured fro 6 days with IL2 (2 U/ml) and then tested for cytotoxic activity against autologous melanoma cells, three allogeneic melanoma and three non-melanoma cells. Autologous cytotoxicity was generated by culture with IL2 alone and was not increased by culture with both IL2 and autologous tumour cells. Marked increases in cytotoxic activity were also generated against the allogeneic target cells and were maximal against the NK-insensitive Chang target cells. Similar degrees of cytotoxicity were induced by IL2 stimulation of lymphocytes from melanoma patients, patients with nonmelanoma carcinoma and normal subjects against the allogeneic target cells. Cold target inhibition studies were carried out against IL2 induced autologous cytotoxicity in five patients. In four of five studies the autologous target cells inhibited more than the allogeneic target cells. There was no significant difference between the inhibition produced by allogeneic melanoma cells and that produced by non-melanoma cells. Similarly, in studies against allogeneic target cells, there was no significant difference in the inhibition produced by allogeneic melanoma compared to non-melanoma target cells. This applied irrespective of whether effector cells were from melanoma or non-melanoma subjects. These results suggest that lymphocytes from patients with melanoma are primed against autologous antigens in vivo and that provision of a second signal, IL2, in vitro can induce cytotoxicity against the autologous tumour. The cytotoxicity generated against the allogeneic target cells did not appear to have specificity to melanoma. Several results, such as the pattern of cytotoxicity against the target cells and change in cell surface markers on the lymphocytes during culture, suggested that cytotoxicity was mediated by activated T cells rather than by nature killer cells. These findings appear to have important implications both in the understanding of tumor host relationships and for the use of IL2 in therapy.

Immunological studies in chronic lymphocytic leukemia—I. Elucidation of subset heterogeneity

The interrelations between subsets of malignant as well as non-malignant B- CLL lymphocytes were investigated by surface marker assays performed on (1) unfractionated mononuclear cells, (2) rosette sedimentation purified lymphocytes, and (3) lymphocytes obtained from Percoll density gradients. The most stable marker of the malignant B-cells was their ability to form rosettes with mouse RBC, while surface membrane immunoglobulins, Fc IgG- and complement receptors were present only on a minor proportion of the CLL cells. By density gradient experiments it could be shown that large CLL cells, while having lost all surface markers, had retained their receptor for mouse erythrocytes. By analysis of purified T-cell fractions increased amounts of high-avidity (“active”) sheep rosette forming cells and of Fc IgG-receptor carrying T G cells could be demonstrated, while the amounts of Fc IgM-receptor carrying T M cells were normal. Based on these experiments in CLL patients we suggest a sequence of surface marker changes taking place during the development of the disease with a blastoid mouse rosette forming cell, devoid of other markers, as the end product.

Human and murine tumours: changes in cell surface structures coded by the major histocompatibility complex region.

Serological and structural changes of surface markers involved in immune reactions may occur in human and murine tumour systems. Thus nine out of twenty-one human tumour cell lines and SV40-transformed fibroblasts differed from autologous lymphoblastoid cells or fibroblasts in their reactivity with HLA alloantisera. H-2 antigens isolated from the murine tumour cells 6C3HED and TP1422, undergo structural changes. An alien HLA-B7 was detected in sera from two melanoma patients. The serologic activity of H-2 antigens was significantly increased in the serum and ascites fluid of tumour bearing mice. Additionally, human SV40-transformed fibroblasts acquire receptors for monkey red blood cells and the murine lymphosarcoma cells 6C3HED express receptors for sheep red blood cells.

Simultaneous Presence of Receptors for Complement and Sheep Red Blood Cells on Human Fetal Thymocytes

The cells of four thymus glands, two at 12 weeks, one at 16 weeks and one at 22 weeks gestational age, were examined cytochemically for enzymic activity and for surface markers. The reactions for chloroacetate esterase, peroxidase, and alkaline phosphatase were negative and the reaction for nonspecific esterase was only weakly positive in some of the cells. In contrast, nearly all thymocytes at the 12th gestational week showed strong focal acid phosphatase (acP) activity. The number of acP-stainable cells and the staining intensity declined progressively. A high percentage of thymocytes in early fetal life express a complement receptor (CR); the proportion of CR-bearing cells decreased while cells forming rosettes with sheep erythrocytes (possessing erythrocyte receptors: ER) correspondingly increased during fetal life. Using a mixed rosette assay, up to 30% of the thymocytes at 12 weeks gestation were found to bear CR and ER simultaneously. The number of CR- and ER-positive cells also declined progressively with fetal age. These findings show that CR-positive, ER-negative thymus cells mature into CR-negative, ER-positive thymocytes via a CR-positive and ER-positive intermediate cell, indicating that mature ER-positive thymocytes do not originate from another cell line lacking CR. The changes in surface markers during early T cell maturation is relevant to certain lymphomas whose cells also show strong focal acP activity and also express both CR and ER. These lymphoma cells may correspond to fetal thymocytes or T precursor cells present in small numbers after birth.

Mitogen Activation of Human Chronic Lymphatic Leukemia Cells

Abstract The response of CLL (chronic lymphatic leukemia) lymphocytes to PHA, PWM, and Con A with respect to changes in surface markers and synthesis and secretion of immunoglobulin were examined. After PHA stimulation the percentage of cells bearing readily detectable surface immunoglobulin (SmIg) diminished rapidly whereas cells forming rosettes with sheep erythrocytes (E-rosettes) increased from &amp;lt;1% to 30 to 50%. The great majority of blast-transformed cells were E-rosette-positive cells with a small population of SmIg-positive blast cells also observed. Ig production in four of seven CLL lymphocyte populations was increased 2.5 to greater than 40-fold after 4 to 6 days of culture in the presence of PHA. In contrast, pokeweed mitogen did not affect Ig synthesis. Furthermore, the Ig secreted into the culture supernatant fluids from seven of eight CLL cases examined consisted almost entirely of free light chain molecules. In contrast, the cell lysates contained a significant proportion of intact Ig molecules. These results indicate that CLL cells can, under certain circumstances, be stimulated to synthesize and secrete increased amounts of Ig, but that there is a basic defect in the biosynthetic mechanism of these cells which result in the secretion of free light chains rather than intact immunoglobulin molecules.

The effect of uremia and transplantation on lymphocyte subpopulations

The in vitro immune response of uremic and transplanted patients was studied by determining lymphocyte surface and functional markers. Because of lymphopenia, the absolute number of T and B cells are diminished in uremia and transplantation. The uremic state had a profound effect of T cell mitogenic response, while the relative number (%) of T cells was not reduced. B cell responses and relative numbers were significantly diminished. The early posttransplant period was associated with a significant reduction in both T and B cell response and relative numbers. All indexes returned toward normal late in the posttransplant course with reduction of immunosuppression and cessation of the uremic state. Horse anti-human lymphocyte globulin treatment caused a significant reduction in T cells and an increase in B cells. Prednisone therapy did not appear to influence the relative number of the lymphocyte subpopulation but did reduce the mitogenic responses. Changes in surface or functional markers did not prove useful in predicting rejection episodes in transplanted patients.