EA Rosette Formation Research Articles

Immunomodulating Effects of Various Phenothiazines and Related Tricyclic Compounds in Vitro and in Vivo

A biotechnological approach to the immunomodulating ability of various phenothiazines and related tricyclic compounds in vitro and in vivo was made. The effect of these compounds was studied on various mononuclear leukocyte functions such as antibody-dependent cell-mediated cytotoxicity, blast transformation of lymphocytes by three mitogens, phytohemagglutinin, concanavalin A and pokeweed mitogen (PWM), rosette formation with erythrocytes and with antibody coated erythrocytes (EA), as well as on polymorphonuclear leukocyte functions including phagocytosis and chemiluminescence emission. Moreover, the ability of these compounds to modulate the bioluminescence emission capacity of Escherichia coli cloned with lux genes was measured. Also in vivo studies were made by measuring the number of haemolytic plaque forming cells in the spleen of mice after drug administration. The electrochemical structure of these tricyclic compounds was shown to be in good correlation with their immunomodulating activity on all the functions except EA rosette formation and on blast transformation by PWM showing the role of charge transfer complex formation in the drug-target cell interaction. These results suggest that the biological effectiveness of phenothiazines and related tricyclic compounds can be presumed from their electrochemical properties and a tricyclic compound with a given biological effect can be theoretically designed.

Fine specificity of a rabbit antibody interacting with human IgG Fc receptor-like molecules

A polyclonal rabbit antibody raised against an Fc receptor (FcR)-like membrane glycoprotein fraction of chronic leukaemic lymphocytes has previously been prepared and partially characterized. This antibody, called AbA, was found to precipitate a 70-kDa and a 45-kDa fraction of the detergent lysate of U937 cells and to inhibit ligand binding to Fc gamma R on the P388D1 murine macrophage cell line. In the present work we have characterised this antibody further. All Fc gamma RII-positive B lymphoblastoid cell lines, as well as resting human B lymphocytes, were positively stained with the AbA antibody. U937 cells were found to be negative, but after stimulation with phorbol ester (PMA), 50% of the cells became positive. AbA antibody did not react with human T cell lines or with the T + 0 cell subset of peripheral blood. Monocytes were also negative. On the other hand, AbA antibody exhibited a dose-dependent inhibition of antibody-mediated cytotoxic reaction (ADCC) of monocytes, while not affecting K cell-mediated ADCC. It had an inhibitory effect of EA rosette formation of B cells and stimulated U937 cells. Furthermore, it interacted with the soluble form of Fc gamma RII released by activated B lymphocytes, and--similarly to IgG--precipitated a 33 kDa fraction from the supernatant of B cells.

Comparative study of alloimmune reactions induced by leukocyte and platelet transfusions in humans: characteristic changes of activation markers, gamma interferon, and FcR blocking antibody production

This study was undertaken to define immune responses induced by leukocyte and platelet transfusions. We offer evidence that activation by alloimmunization with intravenously administered “buffy coat” cells results in immune alterations similar to the early rejection episodes following kidney transplantation. Thus, IL-2 and HLA-DR expression increased on PBL 2 and 3 days after antigen exposure and paralleled elevations of IFN-γ, neopterin, and beta 2 microglobulin levels. No significant changes were detected in the number of T-cell subpopulations. Alloimmunization by purified platelets alone that express only class I MHC antigens failed to induce the above alteration, even after repeated exposure. Repeated alloimmunization with “buffy coat” cells or isolated platelets stimulated a progressive increase up to 100% blocking activity on EA rosette formation. We conclude that the expression of activation markers on PBL and the production of IFN gamma and neopterin reflect the early phase of alloimmune response induced by leukocytes. This type of alloactivation is not a prerequisite for the development of FcR-blocking antibody, which is produced after pure platelet transfusion as well.

MHC antigen expression on bulk isolated macrophage-microglia from newborn mouse brain: induction of Ia antigen expression by γ-interferon

Macrophage-microglia were isolated from primary mixed brain cell cultures of normal newborn mice. They were successfully maintained in vitro for at least 8 weeks. Purity of the cultures was 97–100%, as determined by endocytosis of latex beads, non-specific staining through Fc receptors, EA and EAC rosette formation. These cells were non-specific esterase-positive, but peroxidase-negative. Electron-microscope observations revealed morphological similarities to mature macrophages. Isolated macrophage-microglia seldom incorporated [ 3H]thymidine in vitro. By means of 51Cr release assay, using monoclonal antibodies against mouse major histocompatibility complex (MHC) antigens and complement, we detected class I MHC (H-2) antigen on unstimulated macrophage-microglia, and both class I and class II (Ia) antigens on γ-interferon-treated cells. These observations suggest possible immunoregulatory functions of macrophage-microglia in the central nervous system, as is characteristics of other cells of monocyte lineage.

Further investigation of nonspecific biological substance in anti-Rh(D) preparations.

Fc receptor-blocking activity, based on the EA rosetting inhibition of anti-Rh(D) gamma-globulin preparations, was determined during biological analyses. After sample purification by means of precipitation with a low concentration of ammonium sulphate, fractions were obtained containing anti-D activity and Fc receptor-blocking activity separately. Fc receptor-blocking antibodies inhibited both the formation of EA rosettes and the antibody-dependent cellular cytotoxicity activity. It is suggested that Fc receptor-blocking antibodies obtained during hyperimmunization probably result in the immunosuppressive effect of Rh immune globulin (via the blocking of Fc receptors of activated lymphocytes) that decreases in turn the progress of Rh immunization.

Modulation of IgG effector functions by a monovalent fragment of staphylococcal protein A

The monovalent V-1 fragment of protein A (fSpA) with a mol. wt of 13,000 obtained from an u.v. mutant of Staphylococcus aureus Cowan I strain was proved to be able to modulate significantly some of the effector functions of IgG, such as complement fixation, catabolism, attachment to Fc receptors and antibody-dependent cell-mediated cytotoxicity. Moreover fSpA-like protein A obtained from the A676 strain is mitogenic and enhances NK activity of human peripheral lymphocytes. The efficiency of fSpA was found to be lower than that of protein A with regard to its ability to inhibit complement fixation, EA rosette formation and antibody-dependent cell-mediated cytotoxicity. Both protein A and fSpA had the same efficiency in activation of the complement system after reaction with human or guinea pig IgG, and in increasing the IgG catabolism. Unlike fSpA the monovalent B fragment of protein A (with mol. wt of 7000) was not able to inhibit EA rosette formation and antibody-dependent cell-mediated cytotoxicity. The results recommend fSpA, substituting for protein A, as a molecular probe for the investigation of IgG antibody and lymphocyte effector functions.

Further Investigation of Nonspecific Biological Substance in Anti-Rh(D) Preparations

Fc receptor-blocking activity, based on the EA resetting inhibition of anti-Rh(D) gamma-globulin preparations, was determined during biological analyses. After sample purification by means of precipitation with a low concentration of ammonium sulphate, fractions were obtained containing anti-D activity and Fc receptorblocking activity separately. Fc receptor-blocking antibodies inhibited both the formation of EA rosettes and the antibody-dependent cellular cytotoxicity activity. It is suggested that Fc receptor-blocking antibodies obtained during hyperimmunization probably result in the immunosuppressive effect of Rh immune globulin (via the blocking of Fc receptors of activated lymphocytes) that decreases in turn the progress of Rh immunization.

The mouse Ly-17 locus identifies a polymorphism of the Fc receptor

The mouse Ly-17.2 alloantigen has recently been defined with both conventional and monoclonal antibodies; it identifies a locus, sited on chromosome 1, the products of which were considered to be specific for B cells. Using another Ly-17.2-specific monoclonal antibody (described herein), the tissue distribution of the Ly-17.2 antigen was shown to extend to a subpopulation of T lymphocytes and to neutrophils. This distribution is remarkably similar to that of the Fc receptor for immunoglobulin. Indeed, we now demonstrate that the Ly-17 locus codes for a polymorphism of the Fc receptor, a conclusion based upon (a) an identical tissue distribution of Ly-17.2 and FcR on both normal and tumor tissue; (b) specific inhibition of EA rosette formation by F(ab')2 fragments of anti-Ly-17.2; (c) inhibition of the binding of the 2.4G2 monoclonal rat antimouse Fc receptor antibody by Ly-17.2 antibody; (d) precipitation of an identical series of molecules by our Ly-17.2-specific antibody and by the recognized Fc receptor-specific antibody (2.4G2); and (e) the demonstration by coprecipitation that the Ly-17.2 specificity is present on Fc receptor molecules. The studies suggest that the xenogeneic monoclonal antibody (2.4G2) which recognizes an invariant site on the FcR molecule and the polymorphic site are closely associated. In addition, the studies firmly map a gene coding for or regulating the expression of the FcR to chromosome 1.

Structural requirements of immunoglobulin G for binding to the Fc gamma receptors of the human tumor cell lines U937, HL-60, ML-1, and K562.

Fc receptors (FcR) on U937, HL-60, ML-1, and K562 cells were characterized by using competitive binding assays and EA rosette inhibition studies. Like human monocytes, U937, HL-60, and ML-1 cells bound monomeric human IgG Fc with high affinity and mildly reduced and alkylated human IgG and Fc with a somewhat diminished affinity. In contrast, K562 cells had a much lower affinity for monomeric human IgG or Fc. Concentrations of these proteins as high as 10(-5) M were needed to completely block EA rosette formation. There was no binding of reduced and alkylated IgG and Fc. We assessed the influence of various segments of IgG on FcR interactions by using human pFc', rabbit Facb, mouse IgG2b-IgG2a hybrid Ig, and also studied the effect of reduction of the interchain disulfide bonds. The FcR on all four cell types bound rabbit IgG but not rabbit Facb or human pFc', suggesting that rabbit C gamma 3 domains are required for FcR interaction and that isolated human C gamma 3 domains do not have a human FcR binding site. Murine IgG2a, but not IgG2b, was cytophilic for U937, HL-60, and ML-1 cells. Binding studies with the use of several mouse myeloma variant Ig molecules having hybrid gamma 2b-gamma 2a heavy chains showed that variants with a complete gamma 2a Fc region bound to these FcR-like IgG2a, whereas those having gamma 2a sequences only in the C gamma 3 region and in a short adjacent segment of the C gamma 2 region behaved like IgG2b and did not bind. These results suggested that additional murine gamma 2a sequences are required for FcR binding. Interestingly, the Fc fragments from murine proteins with a complete gamma 2a Fc region bound only to a limited extent. These fragments are shorter than the human IgG1 Fc fragments, and they lack that segment of the hinge region that includes the interchain disulfide bonds. Cleavage of the interchain disulfide bonds of murine Ig having a complete gamma 2a Fc region diminished binding to a similar extent as that observed with human IgG. Together, these findings provide additional insight into the roles of the hinge, C gamma 2, and C gamma 3 regions of human, rabbit, and mouse IgG in their interaction with the FcR of human tumor cells.

Acute undifferentiated leukemia: Induction of partial differentiation by phorbol ester

Expression of lineage-associated surface antigens, was studied in 7 patients with acute undifferentiated leukemia (AUL), 3 patients with acute myeloid leukemia (AML), 4 patients with acute lymphoid leukemia (ALL) and bone marrow from 2 healthy donors, before and after exposure to the differentiating agent 12- O-tetradecanoylphorbol-13-acetate (TPA). The surface antigens were identified by monoclonal antibodies (My4, My8, My9, MO1, B 1, CALLA, T 11) and formation of EA and EAC rosettes. Adherence to plastic was also assessed. Cells from the AML patients responded to TPA with an increase in myeloid antigen positive cells and other markers of differentiation. Four of the AUL patients showed, also, a large increase in the fraction of cells expressing one or more myeloid markers, in correlation with formation of EAC rosettes. In contrast, the percentage of cells expressing myeloid antigens, did not increase in the 4 ALL patients, or in the normal donors. These findings confirm the heterogeneity of undifferentiated leukemias, and suggest the hypothesis that some AUL's can be induced to express markers of early myeloid cells.

ABNORMAL PMN FUNCTIONS ASSOCIATED WITH A HERITABLE DEFICIENCY OF A HIGH M.W. GLYCOPROTEIN (GP138)

A 4 y/o female with delayed umbilical cord severance, granulocytosis, periodontitis & recurrent soft tissue infections demonstrated severely diminished leukocyte mobilization (Rebuck window). SDS-PAGE of patient PMN homogenates revealed deficient (<5% normal) GP138, a major surface glycoprotein complex of normal PMNs. As shown with a NaB3H4 labelling technique, GP138 was undetectable on the surface of patient PMNs & was diminished (20% normal) on maternal PMNs. Adhesion-dependent functions of patient PMNs including directed migration, attachment & spreading (plastic or glass), aggregation (C5a, fMLP or PMA), orientation in chemotactic gradients, antibody-dependent cellular cytotoxicity (due to diminished target cell binding), & phagocytic ingestion of particles selectively opsonized by C3b (Oil-Red-O, 14C Staph. & zymosan) were profoundly diminished (p <.001 in each assay). Fc receptor "facilitated" phagocytosis & spreading & EA rosette formation by patient PMNs were normal. Adhesion independent patient PMN functions including specific binding of f-Met-Leu-3H-Phe & fMLP, C5a &/or PMA mediated shape change, O2- generation, degranulation (MPO & lactoferrin), microtubule assembly (tubulin IF) & alterations of membrane fluidity (ESR) or surface charge (cell electrophoresis) were normal. Thus, GP138 represents a critical surface glycoprotein(s) which is functionally related to the C3b receptor & is required for adhesion-dependent functions facilitating the inflammatory response in host defense.

Ligand inhibition studies on the role of Fc receptors in antibody-dependent cell-mediated cytotoxicity

Subjection of human peripheral blood lymphocytes to a temp shift from 4 to 37°C resulted in a shedding of Fc receptors (termed FcRI) from 40–50% of FcR-positive cells followed by their re-expression within 4hr; a phenomenon which had no effect on the cells' antibody-dependent killing capacity. Removal of lymphocytes having an immobile form of the Fc receptor resistant to the effects of the temp shift (termed FcRII), or removal of lymphocytes bearing both FcRI and FcRII, resulted in a similar amount of reduction in ADCC activity. This was attributed, therefore, to the loss of FcRII-positive cells. The influence of isolated (shedded) FcRI and C1q on ADCC activity was investigated. Soluble FcRI was shown to inhibit ADCC mediated through the immobile Fc receptors (FcRII), despite its lack of an ability to block EA rosette formation through these receptors. C1q also had a dose-dependent inhibitory effect on ADCC. These observations are consistent with earlier findings that FcRII possesses two active binding sites; and suggest that a prerequisite for killing in ADCC is the interaction of these with the Cγ2 and Cγ3 domains. The ability of synthetic peptides representative of human γ 1-chain sequences to inhibit ADCC was determined, in an attempt to locate those sites within the IgG antibody Fc region involved in interaction with two FcR binding sites. Preliminary evidence was obtained to suggest that one of these is situated within the Cγ2 domain, in the region of residues 274 (Lys)-294 (Glu).

Feedback suppression of the immune response in vivo: III. Lyt-1 + B cells are suppressor-inducer cells

We previously demonstrated that injection of a high dose (4 × 10 9) of sheep erythrocytes (SRBC) into C57L/6 mice results in the generation of splenic B cells (plastic nonadherent, Thy-1 − and Ig +) which, when transferred to normal syngeneic recipients, subsequently induce antigenspecific suppressor T cells to suppress the recipient's plaque-forming cell (PFC) responses to SRBC. In the present study we characterized the suppressor-inducer B cells phenotypically. Cytotoxic treatment of the donor's immune spleen cells with anti-Lyt-1 antibody plus complement (C′) but not with anti-Lyt-2 antibody plus C′, relieved the suppression of PFC responses in recipients. The FcR r + population separated by EA-rosette formation showed enriched suppressorinducing activity, whereas the FcR r − population showed no activity. Our findings, taken together with the previous ones, suggest that suppressor-inducer cells are Thy-1 −, Lyt-1 +, Lyt-2 −, FcR r +, and Ig +.

Decrease of E and EA but Not EAC Rosette Formation after Incubation of Blood Lymphocytes in Cerebrospinal Fluid

Peripheral blood lymphocytes (PBL) were incubated in autologous cerebrospinal fluid (CSF) or Hanks' balanced salt solution (HBSS) for between 5 and 60 min at 37 degrees C. Incubation in CSF caused an increase in cell death as measured by trypan blue inclusion. Incubation in CSF also resulted in a decreased percentage of E and EA rosette-forming cells (RFC)--but not of EAC-RFC--as compared with preincubation levels. Incubation in HBSS did not cause significant changes in rosette formation. Although incubation in CSF resulted in decreased E and EA rosette formation by PBL, lymphocytes isolated from CSF showed increased E and EAC rosette formation as compared with PBL.

Effects of urethan on lymphokine-producing activity of lymphocytes and on some functions of peritoneal macrophages in rats.

Effects of urethan on some functions of blood lymphocytes and peritoneal macrophages (PMs) of rats were studied in in vivo and in vitro experiments. The in vitro lymphokine (LK) producing activity of lymphocytes in the presence of specific antigen was depressed by urethan administered 1-5 days before the BCG sensitization. However, the drug injected after the BCG sensitization was not effective on the LK production. Urethan added to the cultures of previously BCG-primed lymphocytes did not influence the LK production. The sensitivity of glycogen-provoked PMs (pPM) to LK-induced activation and, at the same time, the 125I-IgG2a binding capacity as well as the EA rosette formation of the provoked PMs were depressed by urethan administered 1-5 days before the lavage of PMs. These functions of resident PMs (rPM) were not altered by the drug treatment. Urethan added to the cultures of resident or provoked PMs proved to be ineffective. These results led to the conclusions that urethan, after its in vivo metabolic conversion, causes an impairment of the macrophage functions in the inductive phase of the immune response and this event may be the crucial point in the immunosuppressive effect of urethan.

IgG Fc Receptor-bearing cells during early lymphoid cell development in the chicken

Cells from intraembryonic mesenchyme, yolk sac, bursa of Fabricius, and thymus from chicken embryos at different stages of development were studied for the presence of IgG Fc receptors by EA-rosette formation and binding of heat-aggregated chicken IgG (agg IgG). Cells with Fc receptors were found in high frequency in the intraembryonic mesenchyme as early as on the third day of incubation, in the yolk sac on the 7th day, in the bursa on the 10th day, and in the thymus on the 16th day of embryonic development. In the bursa the number of agg IgG binding cells increased with the age of the embryo and remained high after hatching, whereas in the thymus the peak value (76%) was observed on the 16th embryonic day, and after hatching only about 10% of the cells expressed the agg IgG receptors. The results also suggest that the appearance of IgG Fc receptors precedes the expression of B-L (Ia-like) antigens and of cytoplasmic and surface immunoglobulins on early lymphoid cells of the chicken embryo.

The effect of the chemical modification in human Fc γ fragment on protein A and Fc receptor binding

The role of acidic side-chains on Fc γ fragment in granulocyte receptor binding and in S. aureus protein A binding has been investigated by means of chemical modification. Alteration of a restricted number of carboxyl groups after 5 min of reaction is sufficient to abrogate the capacity of Fc to inhibit EA rosette formation by human neutrophils. More limited modification, which affects mainly the most exposed acidic chains, does not change receptor binding activity. In contrast, the interaction with protein A is largely unaffected, even under reaction conditions which are able to induce significant changes in the circular dichroism spectrum of Fc fragment. The results suggest that some acidic groups on Fc may be involved in the interaction with neutrophil receptor and that the binding to protein A and Fc receptor involves different sites.

A reevaluation of the putative association between the Fc gamma receptor and histocompatibility antigens on human mononuclear leukocytes.

The controversial question of an association between human Ia antigens and the FcR for immunoglobulin G has been examined. The experimental basis upon which such a relationship has been predicted was reevaluated. Human allosera with specificity for classical transplantation antigens, HLA-DR, and the related supertypic specificities MB and MT have been tested for their capacities to inhibit homologous EA rosette formation by PBM and B-CLL cells. Both immune and "nonimmune" sera inhibited rosette formation. Sera were normalized with respect to IgG concentration, and no significant quantitative differences in the observed inhibition were seen. Positive reactivity of the antisera with Ia, HLA-A, or HLA-B antigens did not influence EA binding to FcR. Furthermore, F(ab')2 of IgG from reactive sera did not inhibit rosette formation. These observations have been extended using murine reagents. A.TH anti-A.TL serum, which reacts with human Ia in binding and immunoprecipitation studies, and five monoclonal antibodies, which have been raised against human Ia and bind to monomorphic determinants, were tested for an effect on EA rosette formation. When these reagents were tested at concentrations at which they could be shown to bind to antigens on the target cell population, no inhibition of EA rosette formation by human PBM, MON or B-CLL cells could be demonstrated. The studies provide no evidence to suggest the existence of an association between Ia and the human FcR.

Proteinase-induced modulation of the activity of rat macrophages to bind rabbit-IgG-sensitized sheep erythrocytes

Rat alveolar and peritoneal macrophages were tested in a rosetting assay for their capacity to bind rabbit IgG antibody sensitized sheep erythrocytes (EA) before and after various times of incubation with proteolytic enzymes. With most enzymes, a biphasic effect on EA rosette formation was observed: an initial enhancement was followed by a reduction of the number of rosette-forming cells (RFC). The extent and rate of these changes depended on the type and concentration of the enzyme and on the amount of IgG antibodies used to sensitize the sheep erythrocytes. At low enzyme concentrations and with lowly sensitized EA, the phase of RFC enhancement was more prominent and prolonged. With an increase of enzyme concentration, the rate of reduction of RFC was increased. With regard to different enzymes, the highest rate of receptor degradation was found with pronase, followed by trypsin, whereas α-chymotrypsin had little receptor-degrading activity. A slight enhancement not followed by a loss of EA-rosetting activity was induced by granulocyte elastase at the concentrations studied. The results may give an explanation to contradicting reports in the literature, in which a single dose or just a few doses of proteolytic enzymes were employed and no kinetic studies were performed. In addition, EA-rosette-inhibiting material was found in supernatants of macrophage cultures, suggesting that receptor-like material was shed during incubation in serum-free medium. This material lost its EA-rosette-inhibiting capacity after proteinase treatment.

Activation of human blood lymphocyte subsets for cytotoxic potential

Blood lymphocytes of individuals differ in the spontaneous cytotoxic potential exerted in vitro against certain cell lines (natural killing, NK). In the low NK donors, the activity can be enhanced by short-term IFN pretreatment of the effectors (interferon activated killing, IAK) and by addition of PHA to the short-term assay (lectin-dependent cellular cytotoxicity, LDCC). Lymphocyte subpopulations fractionated on the basis of nylon adherence, SRBC, and EA rosette formation differ in their response to these measures. The results obtained with IFN-treated lymphocytes of low NK donors were similar in strength to the spontaneous activity of the high NK donors. Therefore, the distinction between NK and IAK is only operational. The nylon passed E receptor-negative and low-avidity E receptor-positive cells had the strongest NK activity. These subsets can be triggered for enhanced activity by IFN. In the majority of the cases the high-activity E-receptor-positive subset which did not sediment with EA indicators had low NK effect and was not triggered by IFN. Addition of PHA to the lytic assay, however, induced activity in the subset. Realization of DNA synthesis was not necessary for the lytic performance. The PHA-imposed triggering event was not dependent on IFN production nor on induction of the competence for IFN response. The results showed that all non-B lymphocyte subsets separated on the basis of nylon wool adherence, SRBC, and EA rosetting contain cells with lytic potential if the appropriate stimulus is used. The relative activities of the subsets against K562 and Daudi differed. Cells which rosetted readily with EA indicators had weak effect against Daudi.