Ox Erythrocytes Research Articles

Fc gamma receptors on lymphocytes from normal donors and patients with lymphoproliferative diseases. Influence of incubation conditions.

The antibody-coated human erythrocyte and the antibody-coated ox erythrocyte rosette assays (EAHu and EAOx) were compared to detect Fc gamma receptors on human peripheral blood lymphocytes. Two incubation conditions were examined: 1 h at room temperature and overnight at 4 degrees C. In healthy persons, in patients with Hodgkin's disease and in patients with non-Hodgkin lymphoma (NHL) the mean percentage of EAHu-rosette-forming cells (EAHu-RFC) increased significantly when the incubation was carried out overnight at 4 degrees C instead of 1 h at room temperature. This increase was caused by Fc gamma receptor-bearing T cells. In the case of EAOx-RFC only a slight increase was found. The percentage of EAHu-RFC and EAOx-RFC differed significantly in the healthy group after the overnight incubation and in the NHL group after the 1-hour incubation. When comparing the mean percentage of EA-RFC in the patient groups with that of the healthy persons significant increases were observed: EAHu-RFC in patients with Hodgkin's disease in the overnight incubation and EAOx-RFC in patients with Hodgkin's disease and NHL in both incubation conditions. In patients with chronic lymphocytic leukemia (CLL, B-cell type) the mean percentage of EAHu-RFC was very low, however that of EAOx-RFC was moderate to high. It is concluded that in the two rosette assays the antigen-antibody complexes may have different avidities to different lymphocyte subpopulations, and that incubation conditions may have an influence on this avidity.

Inhibition of T gamma rosette formation by the sera from patients with systemic lupus erythematosus. Relation to T gamma-specific antilymphocyte antibody.

The cytotoxic activities of lupus sera were measured against IgG Fc-receptor-bearing T lymphocytes (T gamma cells) and T lymphocytes lacking this receptor [T gamma (-) cells], and the activities were compared with the inhibitory activities of the sera for the formation of rosettes (T gamma rosettes) between T lymphocytes and ox erythrocytes sensitized with IgG antibody. The cytotoxic activities of the sera against both T gamma and T gamma (-) cells well correlated with their T gamma rosette inhibitory rates. Also, the cytotoxic activities after the removal of IgM antibodies strongly correlated with the inhibitory rates. Among them, the highest correlation was observed between IgG T gamma-specific cytotoxic antibodies and T gamma rosette inhibitory rates of the sera. Gel filtration, ultracentrifugation, pepsin digestion, and reduction and alkylation of the sera revealed that main inhibitory activities were contained in IgG fractions. These results suggested that IgG T gamma-specific antibody suppressed T gamma rosette formation and might contribute to the reduction of T gamma cell number in patients with systemic lupus erythematosus.

An Epstein-Barr virus-negative B lymphoma cell line (THP-2) from a Burkitt's lymphoma of a Japanese patient.

An Epstein-Barr virus-negative lymphoma cell line, THP-2, was established from a Burkitt's lymphoma taken from a Japanese patient. THP-2 cells grew in single cell suspension with a doubling time of 24 hr; the cells carried surface-bound mu-lambda immunoglobulins and formed rosettes with IgG antibody-coated ox erythrocytes. THP-2 cells expressed B1, common acute lymphoblastic leukemia (ALL) (J5) and Ia-like antigens of their surface as defined by monoclonal antibodies. Epstein-Barr virus-associated nuclear antigen (EBNA) was not detected. The cell line THP-2 has been maintained for over 5 years. Among the markers examined, only common ALL antigen has been present on the cell surface of THP-2 for these 5 years.

Isolation of Ia positive human leukocytes by a direct rosette assay

A direct rosette assay (DRA) using ox erythrocytes coated with monoclonal antibody (AF-10) to human Ia-like antigen (OE-anti-Ia) is a rapid and convenient method for detection and isolation of Ia-like antigen-bearing cells. Various cell lines and peripheral blood lymphocytes were tested by DRA. Cell lines expressing Ia-like antigen formed rosettes, while cell lines lacking Ia-like antigen did not. In a highly purified T cell fraction, a small number of T cells formed rosettes; but in a Con A-stimulated T cell fraction, about 40% of the cells formed rosettes. The majority of purified B cells and monocytes formed rosettes. Rosette-forming and non-rosette-forming mononuclear cells could be separated by centrifugation on Ficoll-Urografin. Rosette-forming cells reacted positively by indirect immunofluorescence (IIF), while non-rosette-forming cells reacted negatively. The results obtained by DRA were consistent with those obtained by IIF with OKIa ∗1 and AF-10. DRA is thus suitable not only for detection of Ia-like antigen-bearing cells, but also for separation of Ia-like antigen-positive (Ia +) and -negative (Ia −) cells.

A rapid rosette technique for quantitation and separation of mononuclear cell subsets using monoclonal antibodies

We present a rapid and simple method for simultaneous quantitation and separation of mononuclear cell (MNC) subsets. When lymphoid cells are sensitized with monoclonal antibodies of the OK and Leu series, they rapidly form rosettes with ox erythrocytes (ORBC) coated with affinity-purified rabbit IgG against mouse IgG. Rosette-forming cells (RFC) may then be counted and separated from non-rosetting MNC by Isopaque-Ficoll gradient centrifugation. The yield and viability are close to 100% after ORBC lysis. Adherent cells do not interfere. Isolated T8 + and Leu3a + cells were further tested: the purity was 97–99%, and the cells were functionally intact with respect to their modulating activity on the generation of immunoglobulin-secreting cells by MNC after stimulation with pokeweed mitogen.

THE ONTOGENY OF IMMUNOLOGICAL RECEPTORS IN THE THYMUS

Receptors for sheep erythrocytes, for the Fc part of IgG (Fc gamma R) and IgM (Fc mu R), and for components of activated human complement C3 (C3bR and C3dR) in thymus tissue from fetuses, infants and children were studied using haemadsorption to cryostat sections in a closed chamber. Sheep erythrocytes (SE) did not adhere to sections of thymus from fetuses at 10-11 weeks of gestation, adhered weakly between 14 and 20 weeks, while they adhered in a dense monolayer in older fetuses, in infants and in children, denser in the cortex than in the medulla. Ox erythrocytes (OE), sensitized with IgG to demonstrate Fc gamma R, adhered focally to both cortical and medullary areas. The adherence was more pronounced to the sections of fetal thymus than to the sections from infants and children. OE, sensitized with IgM to demonstrate Fc mu R, adhered focally to all thymus tissue sections, preferentially to the cortex. The adherence was most pronounced in fetal thymus. SE, sensitized with IgM and coated with complement, adhered to sections of fetal thymus, but the density decreased markedly during fetal life. Indicator cells demonstrating C3bR adhered focally also to sections from infants and children, while indicator cells demonstrating C3dR did not adhere to sections from individuals older than 38 weeks of gestation. Indicator cells demonstrating C3bR and C3dR adhered both to cortex and medulla.

Asynchronous expression of granulocyte membrane receptors in megaloblastic anaemia.

The expression of Fc(IgG) and C3b membrane receptors by granulocytes and their precursors was examined in 23 cases of megaloblastic anaemia which were graded I-III according to morphological severity. Fractionated bone marrow and peripheral blood granulocyte receptors were assessed by rosette formation with optimally sensitized ox erythrocytes and the results compared with those found in 14 normal marrows. Promyelocyte Fc and C3b receptor activities in megaloblastic anaemia did not differ from normal whilst the number of Fc receptor positive myelocytes and later cells showed a significant increase (P less than 0.05 in Grade I, P less than 0.01 in Grades II and III) proportional to the severity of megaloblastosis. An increase in the number of C3b receptor positive granulocytes was seen in early megaloblastic anaemias and, in contrast to Fc receptor expression, showed the highest receptor activities in the Grade II cases. The most significant changes in receptor expression were seen at the metamyelocyte stage and appear to be related to the numbers of these cells found in the megaloblastic marrows. It is suggested that these alterations are primarily related to asynchronous granulocyte maturation and the application of these findings to the study of granulocytic disorders is discussed.

Small-scale isolation and characterisation of human peripheral blood monocytes.

A technique is described for the isolation and characterisation of monocytes from 15 ml of human peripheral blood. After density gradient centrifugation over Ficoll-Hypaque, monocytes are purified further by substrate adherence under carefully defined conditions. The use of a simple microwell slide system permits the production of multiple small-scale monolayer populations which can be characterised further in terms of their histochemical reactivity (combined stain for chloroacetate and non-specific esterases); endocytic capacity (latex particles); Fc (sensitised ox erythrocytes) and C3 (serum-coated yeast) receptor expression; and precursor maturation potential (7-day cultures). Evidence of considerable cellular heterogeneity is presented.

Erythroid Fc-IgG and Complement Receptor Expression: A Study of Normoblastic and Megaloblastic Human Bone Marrows

Using a standardized rosetting technique with IgG-coated ox erythrocytes, avid IgG (Fc) receptors were demonstrated on red cell precursors. The proportion of receptor-positive cells in normal marrows was highest in early precursors and appeared to be lost with maturation. In megaloblastosis, the absolute percentage of early precursors increase, but there is an even greater increase in the proportion of receptor-positive cells. It is proposed that this reflects the degree of maturation arrest. The specificity of the receptor was confirmed by inhibition studies with aggregated human IgG. In contrast, the expression of the C3b "immune adherence" receptor, assessed by IgM-C3b-coated ox erythrocytes, was seen to increase with erythroid maturation. Early megaloblasts, especially in severe megaloblastosis, showed a marked decrease in C3b receptor activity, again in proportion to the level of maturation arrest. The significance and possible function of these receptors is discussed.

Erythroid Fc-IgG and complement receptor expression: a study of normoblastic and megaloblastic human bone marrows

Using a standardized rosetting technique with IgG-coated ox erythrocytes, avid IgG (Fc) receptors were demonstrated on red cell precursors. The proportion of receptor-positive cells in normal marrows was highest in early precursors and appeared to be lost with maturation. In megaloblastosis, the absolute percentage of early precursors increase, but there is an even greater increase in the proportion of receptor-positive cells. It is proposed that this reflects the degree of maturation arrest. The specificity of the receptor was confirmed by inhibition studies with aggregated human IgG. In contrast, the expression of the C3b “immune adherence” receptor, assessed by IgM-C3b-coated ox erythrocytes, was seen to increase with erythroid maturation. Early megaloblasts, especially in severe megaloblastosis, showed a marked decrease in C3b receptor activity, again in proportion to the level of maturation arrest. The significance and possible function of these receptors is discussed.

Two distinct Fc receptors for IgG on human peripheral T lymphocytes.

The proportion of human peripheral T lymphocytes forming rosettes with IgG-coated ox erythrocytes (ORBC) is increased after controlled hypotonic treatment. This increment may be as high as 40% of total T cells, depending on the lymphocyte donor. Such treatment is shown not to result in selective cell loss. Induced rosetting is mediated by a receptor specific for the Fc portion of human IgG (Fc gamma R). Inhibition of induced Fc gamma R activity is equally well accomplished by monomeric and by aggregated IgG of defined size. This is in contrast to the Fc gamma R detected before hypotonic treatment, which is not significantly inhibited by monomeric IgG. Capping studies established the structural independence of these two types of Fc gamma R in the lymphocyte membrane by virtue of selective cross-linking of either receptor while leaving the respective counterpart unaffected. The biochemical basis of the hypotonic effect is not yet resolved. However, the data presented suggest that hypotonicity results in removal of Fc gamma R-bound cytophilic IgG. Operationally, we propose the term induced Fc gamma R (Fc gamma R-I) for the here-described new type of receptor with high affinity for monomeric IgG.Fc gamma R that are directly assayable without hypotonic induction and not inhibited by monomeric IgG are termed free Fc gamma R (Fc gamma R-F).

Rapid replicate reverse haemolytic plaque assay directly in microwells used for tissue culture

The reverse haemolytic plaque assay was adapted as a micromethod using microwells of flat-bottomed microtitre trays. To microwells containing the lymphocytes under test were added protein A-coupled ox erythrocytes, developing antisera directed against any class of immunoglobulin, and guinea pig complement absorbed with protein A-coupled erythrocytes. Plaques were scored by counting with a stereoscan microscope. The method is applicable both to spontaneous and mitogen-induced plaque formation and, as a further development of the technique to differentiated B cells in pokeweed mitogen-driven cultures, plaques were enumerated directly in the same wells of the microtitre tray as were employed for culture of the B cells. The method allows for several hundred plaque assays at one sitting.

The role of autologous helper and suppressor T cells in the regulation of human granulopoiesis.

Helper and suppressor T cells play a physiological role in immunoregulation and possibly in other biological systems. Previous studies have suggested that unfractionated T cells influence human blood cell formation in vitro. Subpopulations of such cells (TM and TG cells) were prepared by immune ox erythrocyte rosetting techniques. Co-cultures were established in semi-solid agar with autologous null lymphocytes as a source of granulocyte-macrophage progenitor cells (CFU-GM). TM(helper) cells produced an increase and TG (suppressor) cells a decrease in the expression of CFU-GM as reflected in the number of GM colonies generated by comparison with cultures of null lymphocytes alone. These data point to a contribution from T lymphocyte subpopulations to the physiological regulation of human granulopoiesis.

Effects of Staphylococcus aureus Cowan I bacteria on the immunoglobulin production from human B-cell subsets

Staphylococcus aureus Cowan I bacteria (SpA CoI) is known to be a polyclonal B-cell activator of human lymphocytes. In this study, we investigated which of the B-cell subsets SpA CoI could stimulate and induce immunoglobulin (Ig) production. B-Cell subsets were separated from peripheral blood and tonsil lymphocytes by rosette formation with E, EA IgG, EAC, anti-Ig-conjugated ox erythrocytes (OE-anti-Ig), and protein A-conjugated OE (OE-Pro A), or on a bovine serum albumin (BSA) discontinuous density gradient. The cells responding to SpA CoI included E −, C3 receptor-positive (C3R +), Fc receptor-negative (FcR −), and surface Ig-positive (SIg +) B-cell subsets. These B-cell populations responded well to SpA CoI and produced significant amounts of IgG, IgM, and a lesser amount of IgA. Among SIg + B cells, IgG, IgA, and IgM + B-cell subsets responded to SpA CoI and produced large amounts of Ig belonging to each corresponding Ig class. IgD + B cells failed to produce Ig of any class, except for minimal amounts of IgG and IgM. While both the protein A receptor-positive (Pro A · R +) and negative (Pro A · R −) cells responded well to SpA CoI, Pro A · R + B cells produced IgG mainly and Pro A · R − B cells produced IgM. Fractionation of B cells on a BSA gradient revealed that comparatively small-sized and denser B-cell subsets responded well to SpA CoI and produced every class of Ig.

Regulation of Human Blood Erythroid Burst-Forming Unit (BFU-E) Proliferation by T-Lymphocyte Subpopulations Defined by Fc Receptors and Monoclonal Antibodies

To determine whether normal T-cell subpopulations influence human blood BFU-E proliferation variably, sheep erythrocyte rosettable cells (unseparated T cells) were fractionated into subpopulations based on their differential binding to IgM (Tμ cells) or IgG-coated ox erythrocytes (Tγ cells). For comparative purposes, T cells, Tγ cells, and T cells depleted of Tγ cells (T-non-γ cells) were further characterized by their reaction with the OK panel of monoclonal antibodies (OKT3, OKT4, OKT8, OKM1). Quantities of 2.0 × 105 Tμ, Tγ, or T-non-γ cells were mixed with 2.0 × 106 autologous BFU-E-enriched null cells in a methyl-cellulose culture system with 2.0 IU erythropoietin. Tμ cells or T-non-γ cells consistently increased BFU-E numbers above that observed with either unseparated T cells or Tγ cells (p

Modulation of human B-lymphocyte receptors for IgG does not affect HLA-DR antigens.

Receptors for the Fc portion of immunoglobulin G (Fc gamma R) on the surface of human peripheral T cells can be modulated on contact with antigen-antibody immune complexes (IgG-IC). Our results demonstrate that the Fc gamma R present on the surface of human B cells are also modulated on exposure to IgG-IC. Furthermore, the expression of HLA-DR molecules is not affected by the IgG-IC induced disappearance of Fc gamma R. In fact, the percentage of HLA-DR-positive cells and the relative amount of HLA-DR molecules on individual B cells analysed by flow microfluorometry was shown not to be influenced by Fc gamma R modulation. Moreover, pretreatment of cells with monoclonal antibodies directed against non-polymorphic determinants of HLA-DR antigens did not prevent the binding of IgG-coated ox erythrocytes. These results argue against a structural relationship between Fc gamma R and HLA-DR molecules on the surface of the B-cell populations investigated here.

A general method for the cytochemical and ultrastructural studies of human lymphocyte subsets defined by monoclonal antibodies

After incubation with monoclonal mouse antisera raised against membrane differentiation antigens, subsets of human lymphocytes can be detected by means of latex microspheres or ox erythrocytes coated with anti-mouse Ig antibodies. Optimal conditions are described using a panel of monoclonal antisera (Ortho Pharmaceuticals and New England Nuclear). The method is as sensitive as the complement-mediated microcytotoxicity assay, is rapid, easy to perform and permits exhaustive light and electron microscopic studies of lymphocytes bound to the carrier particles. The advantages of the simultaneous analysis of cytological features and monoclonal antibody-defined surface markers in normal and pathological individual lymphocytes are discussed with regard to lymphoid cell maturation and neoplastic lymphocyte classification.

Parallel tubular structures in T,B and null lymphocyte subpopulations.

T, B and Null lymphocyte-enriched subpopulations were isolated by means of sheep red blood cell (E SRBC) sedimentation and nylon wool adherence. In these subsets the proportion of Fc receptor-bearing cells was determined by the antibody-coated human and ox erythrocyte rosette assays (EA Hu and Ox). In the T-cell fraction the proportion of Fc-bearing lymphocytes was dependent on the procedure by which the sheep erythrocytes were removed from the E SRBC rosette-forming cells. Mechanical vibration resulted in considerably higher percentages of EA rosette-forming cells (EA-RFC) than osmotic shock or lysis with ammonium chloride. In all three procedures the number of EA Hu-RFC was slightly higher than the number of EA Ox-RFC. In the B-cell fraction the proportion of EA Ox-RFC was higher than that of EA Hu-RFC, mean values 43 and 33%, respectively. In the Null cell fraction the reversed was seen: mean values 54% EA Hu-RFC and 45% EA Ox-RFC. In all three lymphocyte fractions the majority of the EA-RFC contained parallel tubular structures and/or associated amorphous granules. Non-Fc receptor-bearing lymphocytes only occasionally showed parallel tubular structures or the amorphous granules.

Complement components on human lymphocytes

Lymphocytes isolated from normal human EDTA-blood were tested for the presence of cell membrane-bound complement components by reaction with specific antibodies coupled to trypsin-treated ox erythrocytes. The highest proportions of positively reacting cells were found to have C4 and β1H-globulin, which were present on 11 and 9% (mean values) of lymphocytes, respectively. Other complement components, C1q, C3, and C6, were detected on approximately 3% lymphocytes. C4 was also demonstrated on tonsil and lymph node lymphocytes. All of the cells carrying complement were C3-receptor-bearing, Ig-positive, B lymphocytes, a proportion of which also had receptors for IgG-Fc.

Anti-Ia reactivity in sera from patients with systemic lupus erythematosus.

Antileukocyte antibodies in sera from patients with systemic lupus erythematosus (SLE) were characterized by determining cross-reacting specificies with the antigens defined by OKT3, OKT4, OKT8, OKM1 and anti-Ia hybridoma antibodies (Abs). T cells were prepared by sheep erythrocyte (E) rosetting after removal of adherent cells. T cells, or non-T cells, were preincubated with SLE sera at 4 degrees C and then with monoclonal Abs. Binding by specific monoclonal Abs was assessed by two methods: rosetting with ox erythrocytes conjugated with goat anti-mouse IgG and also in the fluorescence-activated cell sorter using fluorescein isothiocyanate-conjugated goat anti-mouse IgG. Using the rosetting method, we found that sera from SLE can block the binding of monoclonal mouse hybridoma anti-Ia Abs to T cells; the blocking of other monoclonal Abs was not consistent. Using fluorescence-activated cell sorter analysis, preincubation with SLE sera lowered the intensity of staining and total percentage of either T or non-T cells stained by monoclonal anti-Ia Abs. Blocking of anti-Ia Abs binding by SLE sera was not histocompatibility leukocyte antigen (HLA)-DR restricted and was not due to Fc receptor binding. These results indicated that antibodies in SLE sera react with structures contiguous to or identical with Ia determinants. Anti-Ia activities in SLE sera correlate with SLE disease activity. In addition, there was a significant negative correlation between anti-Ia blocking activity in the sera and the percentage of Ia-positive T cells in the blood of SLE patients. Antibodies in SLE sera with anti-Ia blocking activity may play an important role in immune dysregulation in SLE patients.