EA Rosette Formation Research Articles

Study on EA-agglutinating factor released by cells with Fc receptors

The supernatant fluids from activated lymphocytes or from herpes simplex virus-infected cells agglutinated EA but not E. The effect of preincubation of various “Fc receptornegative” cells with these supernatant fluids on the formation of EA rosettes was investigated. Following such preincubation lymphoblasts, brain cells, and cells from methyl-cholanthrene-induced murine sarcoma formed EA rosettes.

Interaction of anti-staphylococcal protein A antisera with Fc receptor-bearing human normal lymphocytes.

Staphylococcal protein A (SpA) is known to bind the Fc region of IgG of most mammalians and to possess biologic activity both in vivo and in vitro, where it acts as a lymphocyte polyclonal mitogen. Its binding to the Fc gamma portion bears many features of the antibody-antigen interaction, such as the dissociation constant, lattice formation, and complement activation. Moreover, SpA seems to compete with membrane Fc receptors for IgG so that the possibility of an interaction with the same CH domain(s) of IgG can be considered. In the present study, evidence is given that anti-SpA antisera obtained from chickens and rabbits are able to inhibit EA rosette formation by normal human lymphocytes and that they are able to recognize, with immunofluorescent staining, a subpopulation of normal human peripheral blood lymphocytes (PBL) that closely resembles that of EA rosette-forming cells (RFC). Moreover, the depletion of EA RFC by means of a single gradient centrifugation is accomplished by the parallel depletion of PBL stainable by anti-SpA antisera. The relevance of these results in the hypothesis of a similarity between the combining sites of SpA and membrane Fc receptor(s) for IgG is discussed.

Differential effects of propranolol on lymphocyte rosette formation and response to plant mitogens.

The effects of propranolol on various lymphocyte functions were studied to gain a better understanding of the recently demonstrated suppressive effect of propranolol on rheumatoid factor production. D- and L-propranolol at a concentration of 1 X 10(-4)M inhibited the formation of human EA rosettes. The inhibition occurred within one minute of adding the compounds, was reversible, and did not affect cell viability. Addtion of propranolol to preformed EA rosettes failed to disaggregate them. Patching and capping of SIg by an Fab'2 anti-IgG were inhibited at 2.5 X 10(-5)M and above. Propranolol at 2.5 X 10(-5)M also inhibited lymphocyte response to phytohemagglutinin and pokeweed mitogen without evidence of cell toxicity by trypan blue staining or absolute numbers of surviving cells. Congeners of propranolol with mainly beta adrenergic blocking properties did not show inhibitory effects. The inhibitory activities of propranolol are interpreted in terms of propranolol's membrane stablizing effects and ability to interfere with membrane receptor movement.

Binding of circulating immune complexes to human peripheral blood lymphocytes: Effect of complement

The mode of binding of immune complexes, with and without bound complement components, to normal human peripheral blood lymphocytes was examined. Heat-aggregated human IgG (agg IgG) was employed as an in vitro model of immune complexes. Immune complexes without bound complement or with bound C1, C4, or C2 inhibited antibody-dependent cellular cytotoxicity (ADCC) and EA rosette formation but not rosette formation with EAC. In contrast, immune complexes with bound C3b blocked EAC rosette formation but inhibited ADCC and EA rosette formation little if at all. These findings demonstrate that agg IgG, and thus immune complexes, without fixed complement or those with bound C1, C4, or C2, preferentially interact with the lymphocyte Fc receptor and that C3b-bound immune complexes bind to the lymphocyte surface C3b receptor.

Immunobiology of the human placenta: I. IgGFc receptors in trophoblastic villi

Qualitative and quantitative studies were performed on IgGFc receptors in the trophoblastic villi of human placentae ranging in gestational age from less than 4 weeks to full term. IgGFc receptors were detected on cells of the syncytiotrophoblast (ST) using two different assay systems; EA rosette formation and direct immunofluorescence with deaggregated human IgG. The ST IgGFc receptors had high affinity for native IgG molecules (deaggregated IgG) as well as affinity for antigen-antibody complexes (EA). The receptors for deaggregated IgG were present on a majority of ST cells in first and second trimester trophoblast, but were significantly less frequent on ST cells in older placentae. Similar receptors also were detected on cells lining some fetal vessels in the trophoblastic villi. Not only were the IgGFc receptors expressed on ST cells, but in vivo bound IgG was detected in association with the ST and the two patterns of IgG binding were essentially identical. In contrast to the qualitative nature of the receptors on ST cells, cells in the stromal (central) region of the trophoblastic villi expressed IgGFc receptors that had high affinity for EA but failed to bind the deaggregated IgG except at a high concentration. The results are discussed with respect to the possible role of the IgGFc receptors in the specific transfer of IgG from maternal to fetal circulations.

Presence of “la‐like” antigens on human T lymphocytes bearing receptors for IgG

Human peripheral blood T cells bearing receptors for the Fc portion of immunoglobulins were characterized by the formation of double rosettes with sheep red blood cells and IgG-coated chicken erythrocytes (EA). Treatment of cells by anti-"Ia-like" antisera inhibits the binding of EA. Inihbition appeared to be specific since anti-HLA-A and HLA-B antibodies did not inhibit the formation of EA rosettes, whereas F (ab')2 fragments of anti-Ia-like IgG were found to be as potent inhibitors as the whole IgG. This result shows that in human and in mice, Ia-like determinants arepresent, close to Fc gamma receptors, on the surface of the T lymphocytes bearing such receptors.

Association between HLA determinants and complement receptors on human lymphocytes.

The relationship between the major histocompatibility complex (MHC) and functional receptors on the surface of human lymphocytes was studied. HLA antisera were tested for their effect on the formation of E-, EA-, and EAC'-rosettes by human peripheral blood lymphocytes (PBL). Antisera to various HLA specificities inhibited the formation of EAC'-rosettes, but had no effect on the formation of E-rosettes. The formation of EA-rosettes was inhibited by HLA antisera only in part among the individuals tested. Anti beta2-microglobulin serum resembled HLA antisera in its effect on the formation of the various rosettes. HLA determinants and complement receptors are different entities on the cell surface since elimination of complement receptors by trypsin treatment does not seem to affect the expression of HLA antigens on the cell surface. It is suggested that EAC' receptors are located close to HLA determinants.

Fc Receptors on Mouse Effector Cells Mediating Natural Cytotoxicity Against Tumor Cells

Mouse effector cells mediating natural cytotoxicity against tumor cells have been previously thought to be lymphocytes that lack any detectable cell surface markers. The present study presents evidence for receptors for the Fc portion of IgG on these cells. By adsorption of cytotoxic spleen cells on monolayers of sheep erythrocytes (E) plus IgG antibodies to sheep erythrocytes (EA), 50 to 96% of the total cytotoxic reactivity could be removed. Parallel adsorption of cells on E monolayers or on EA monolayers coated with protein A, to block the Fc portion of IgG, resulted in little or no depletion of cytotoxic activity. The presence of Fc receptors on the NK cells was confirmed by combining EA rosette formation with velocity sedimentation at unit gravity. Peak cytotoxicity occurred at the same sedimentation velocity as the peak of Fc-positive cells. After EA rosette formation, there was a shift to a higher sedimentation velocity in the Fc-positive cells and in the natural cytotoxic activity. The increase in sedimentation velocity of NK activity that was observed in these experiments indicated that most of the cells had only bound a small number (three or four) of antibody-coated erythrocytes. Together, these data indicate that cells with Fc receptors account for most of the total lytic activity of normal mouse spleen cells.

Control of normal differentiation of myeloid leukemic cells. XII. Isolation of normal myeloid colony‐forming cells from bone marrow and the sequence of differentiation to mature granulocytes in normal and D+ myeloid leukemic cells

AbstractAn enriched population of early myeloid cells has been obtained from normal mouse bone marrow by injection of mice with sodium caseinate and the removal of cells with C3 (EAC) rosettes by Ficoll‐Hypaque density centrifugation. This enriched population had no EAC or Fc (EA) rosettes and contained 87% early myeloid cells stained for myeloperoxidase and/or AS‐D‐chloroacetate esterase, 7% cells in later stages (ring forms) of myeloid differentiation and 6% unstained cells, 2% of which were small lymphocytes. After seeding in agar with the macrophage and granulocyte inducer MGI, the enriched population showed a cloning efficiency of 14% when removed from the animal and of 24% after one day in mass culture. Both the enriched and the unfractionated bone marrow cells gave the same proportion of macrophage and granulocyte colonies.The normal early myeloid cells were induced to differentiate by MGI in mass culture in liquid medium to mature granulocytes and macrophages. The sequence of granulocyte differentiation was the formation of EA and EAC rosettes followed by the synthesis and secretion of lysozyme and morphological differentiation to mature cells. D+ myeloid leukemic cells with no EA or EAC rosettes had a similar morphology to normal early myeloid cells and showed the same sequence of differentiation. The induction of EA and EAC rosettes occurred at the same time in both the normal and D+ leukemic cells, but lysozyme synthesis and the formation of mature granulocytes was induced later in the leukemic than in the normal cells. The results indicate that selection for non‐rosette‐forming normal early myeloid cells also selected for myeloid colony forming cells, that these normal early myeloid cells can form colonies with differentiation to macrophages and granulocytes, that normal and D+ myeloid leukemic cells have a similar sequence of differentiation and that the normal cells had a greater sensitivity for the formation of mature cells by MGI.

Inhibition of antibody-dependent cellular cytotoxicity and immunoglobulin synthesis by an antiserum prepared against a human B-cell Ia-like molecule.

Rabbit antisera to the human B-cell-specific antigen complex, p23,30, was used to define further the functional heterogeneity of isolated human lymphocyte subpopulations. Specific depletion of p23,30-bearing cells from Ig-negative cell populations and Ig-negative, E rosette-negative (Null) populations by either complement-mediated lysis or by physical separation on goat antirabbit Fab immunoabsorbent columns, eliminates the antibody-dependent cellular cytotoxic (ADCC) function. Furthermore, binding of anti-p23,30 serum to the effector cell surface inhibits ADCC but does not interfere with EA rosette formation. Apparently p23,30 represents a cell surface site which is distinct from the Fc receptor but which is important in the triggering of ADCC. In addition, depletion of p23,30-bearing cells from unfractionated cell populations, Ig-positive B-cell populations and Ig-negative, E rosette-negative (Null) populations eliminates the capacity of these populations to secrete immunoglobulin during subsequent culturing. Thus both the Ig-secreting cells and the ADCC effector cells within the Ig-negative, E rosette-negative (Null) population reside in the same population of cells which bears the p23,30 antigen.

Evidence for clustering of H-2K, H-2D, and the Fc receptor on the membranes of B cells.

Alloantisera to H-2K, H-2D, and Ia antigens markedly inhibited the binding of EA but not FITC-IgG by the B cell Fc receptor. EA rosette formation approached normal levels when masked H-2 but not Ia specificities were allowed to cap on the membranes of B cells. beta2-mu coated SRBC were bound by the Fc receptor, and high concentrations of soluble beta2-mu were found to moderately inhibit EA rosette formation while lower concentrations enhanced binding. The data support the concept of Fc/Ia identity, and they suggest that H-2K, H-2D, and the Fc receptor may be closely grouped on the membranes of B cells. Further, these observations suggest that the beta2-microglobulin associated with H-2 could serve to link T cells with the Fc receptor of B cells during the inductive phase of antibody synthesis.

Resistance of activated macrophages to H-2 antibody-mediated cytotoxicity and Fc rosette inhibition.

MACROPHAGES can have critical roles in repair, inflammation and a large variety of immunological phenomena1,2. The biological roles of these cells often occur subsequent to their ‘activation’, a subjective term which refers to a greatly heightened state of reactivity1–3. Activation can be induced by a variety of stimuli including ingestion of certain macro-organisms, contact with immune complexes, bacterial lipopolysaccharides and lymphokines3. Activated macrophages usually show an increase in cell size, increased adherence properties and cellular processes, lysosomal enzyme activity, and phagocytic ability1,2. In addition, alteration in characteristics and functions of various macrophage receptors can apparently take place as a consequence of activation4,5. Such a change is described in this paper: it involves Fc receptors as detected by Fc or EA rosette formation (see Table 1), and their relationship to surface mouse major histocompatibility (H–2) antigens as detected by anti-H–2 alloantisera. Aside from the general relevance this change has with regard to membrane alterations which occur in activated lymphoreticular cells, it can also be exploited as a marker for activated macrophages.

The specificity and significance of the inhibition of Fc receptor binding by anti H-2 sera

s. The possibility that Ia antigens are unique among H-2 antigens in their relationship to the Fc receptor was investigated in an EA rosette assay. Antibody specific for antigens in various regions of the H-2 complex was incubated with mouse cells, and the ability of the cells to form rosettes with antibody-coated chicken erythrocytes was tested. Antibody raised against the H-2 antigens of Ia-negative tumor cells was highly effective in inhibiting rosette formation. A variety of antisera against K-, 1-, and D-region antigens tested in recombinant mice inhibited EA rosette formation, suggesting that antigens in each of these regions could be detected in rosette inhibition. The F(ab')2 fragments of all antisera tested also produced specific EA rosette inhibition. Finally, antibody against la antigens failed to inhibit bone marrow RFCs, although antibody against H-2 K and H-2D antigens did inhibit. Although H-2 serology is in a state of rapid change at present, it must be concluded that in this assay, antibody against antigens in the K and D regions as well as the 1 region can inhibit EA rosette formation. Inhibition of these rosettes by anti H-2 sera is therefore not due to a special association of Ia antigens with Fc receptors.

THE EXPRESSION OF H‐2K, H‐2D AND Ia ANTIGENS IN VARIOUS TISSUES AS ASSESSED IN Fc RECEPTOR INHIBITION SYSTEMS

SUMMARYThe ability of mouse alloantibody to inhibit EA rosette formation and antibody‐dependent cell‐mediated cytotoxicity (ADCC) was used to study the expression of H‐2K, Ia and H‐2D antigens in various tissues. As previously reported antisera against each of these groups of antigens inhibited B lymphocyte EA rosette formation. Continuing studies confirmed these observations but established that quantitative differences may exist in the ease with which antibody against antigens in each region can inhibit EA rosettes: anti H‐2D and anti‐Ia seemed stronger relative to their cytotoxic titres than anti H‐2K. Possible reasons for this are discussed. When rosette forming cells from other tissues were studied, (bone marrow cells, peritoneal macrophages and tumour cells), they were inhibited by anti H‐2K and anti H‐2D sera but not by anti Ia sera, presumably reflecting the restricted distribution of Ia antigens in those tissues.Inhibition of ADCC by various antisera reflected qualitatively and quantitatively the expression of H‐2 antigens in various tissues: whereas effector cell activity in spleen, bone marrow, or peritoneal cell populations was inhibited by anti H‐2 or anti‐Ia sera, the amount of inhibition observed with anti‐Ia was much less when the tissue expressed little Ia antigen (bone marrow) than when it expressed abundant Ia antigen (spleen). The ability of cytotoxicity inhibition to detect antibody coated cells was used to assess the relative amount of Ia antigen on thymus and on lymph node cells, showing significant amounts of Ia antigen on thymus cells. Fc receptor inhibition studies may thus be useful as new approaches to the study of the expression of the antigens of the major histocompatibility complex.

Expression of a receptor for IgM by human T cells in vitro.

The capacity of human peripheral blood lymphocytes (PBL) to bind antigen-IgG antibody (EA(IgG)) and antigen-IgM antibody (EA(IgM)) complexes was investigated using a rosette technique with ox erythrocytes coated with rabbit IgG or IgM antibody. It was found that while EA(IgG)-rosette-forming cells (RFC) were detected on PBL freshly drawn from normal individuals, EA(IgM)-RFC were present only in suspensions kept in culture for 24 h in mediá supplemented with sera containing very low or no amounts of IgM. Experiments of simultaneous detection of EA(IgG)-RFC or EA(IgM)-RFC and other membrane markers for human T or B cells together with experiments on purified T or B cell populations indicated that EA(IgG)-RFC were formed by both T and B cells, while T cells only were capable of EA(IgM) rosette formation. The specificity of the receptors for IgG and IgM was determined by studying the inhibitory capacity of purified human IgM and IgG in the rosette assay. The receptor for IgG was inhibited by IgG and not by IgM, while the reverse was true for the receptor for IgM.

Effects of Anti-β2 microglobulin antibodies on human lymphocytes

Anti-β2 microglobulin antisera prepared in rabbits immunized with β2m purified from the urine of a single patient were cytotoxic for human T and B lymphocytes of all donors tested; lymphocytotoxicity could be fully inhibited by all human sera tested, not by serum from other animal species. Anti-β2 microglobulin antibodies and their F(ab′)2 fragments had little effect on E and EAC rosette formation, suggesting that β2m is not closely associated with receptors for sheep erythrocytes on T lymphocytes or receptors for C3 on B cells. Anti-β2m IgG and F(ab′)2 fragments inhibited EA rosette formation though the latter did not impair lysis of antibody-coated xenogeneic erythrocytes by lymphocytes bearing receptors for the Fc portion of IgG. Some of the antisera had a mild mitogenic effect, all of them inhibited mitogen and antigen-induced lymphocyte proliferation at high concentrations whereas they potentiated these responses at low concentrations. In mixed lymphocyte cultures pretreatment of responding cells markedly depressed the response whereas coating of stimulating cells with β2m antibodies had little or no effect.