Formation of Paul-Bunnell antibodies by cultures of lymphocytes from infectious mononucleosis.

Short-term cultures of peripheral blood lymphocytes obtained from 20 infectious mononucleosis patients 2–4 weeks after the onset of the disease were studied for formation of heterophile antibodies. In studying pooled supernatant fluids of lymphocytes from three patients cultured for 3–20 days, lytic antibodies for red blood cells of bovine (BRBC) and sheep (SRBC) origin were demonstrated. These hemolysins were shown to be of IgM nature and Paul-Bunnell specificity. Subsequently, plaque-forming cell (PFC) assays were performed with lymphocyte cultures of 15 patients. Significant numbers (60–750/2 × 10⁷ cells) of PFC secreting antibodies against BRBC were demonstrated in lymphocyte cultures of 12 patients. The number of PFC apparently reached its peak after 5 to 10 days of culturing. No or a very few PFC were observed in the lymphocytes that were not cultured or in lymphocytes cultured for 3 weeks or longer. Lymphocyte cultures prepared in a similar fashion from normal individuals or patients suffering from sore throat and submandibular lymphadenopathy of other than infectious mononucleosis origin did not produce PFC. Production of lytic zones by antibodies to BRBC secreted by PFC was inhibited by preincubation of lymphocytes of infectious mononucleosis patients with solubilized Paul-Bunnell antigen but not with other heterophile antigens, indicating that antibodies involved in the PFC formation are of Paul-Bunnell specificity. An increased number of PFC against BRBC were obtained in two of three lymphocyte cultures after cultivation with BRBC or solubilized Paul-Bunnell antigen.     

Morphological differences in the interactions between human mononuclear cells and coated or uncoated sheep red blood cells

Summary Enriched preparations of E, EA and EAC rosettes formed by human peripheral blood mononuclear cells were freeze-etched and examined electron-microscopically. In E rosettes only lymphocytes were involved, whereas in EA and EAC rosettes lymphocytes and mononuclear phagocytes participated as rosette-forming cells. In EA and EAC rosettes, cytoplasmic extensions of the rosette forming cell were seen to penetrate the sheep red blood cell, whereas E rosettes showed a broad zone of adherence without penetration. None of the three types of rosettes showed an interspace between the membranes. Unlike E rosettes, EA and EAC rosettes showed polarity in the adherence of sheep red blood cells. These observations made by freeze-etch electron microscopy indicate distinct morphological differences between rosettes formed with coated or uncoated erythrocytes.The authors wish to thank Prof. Dr. A. Cats, Dr. P.C.J. van Breda Vriesman and Dr. J.C.H. de Man for helpful discussion and criticism; the assistance of Miss R. Kleinjan and Mrs. A.C. Scheurkogel-van Efferen is gratefully acknowledged. This work was supported in part by a grant of the Praeventiefonds     

A human lymphoid cell line with receptors for both sheep red blood cells and complement

The human lymphoid cell line MOLT 4, from a patient with acute lymphocytic leukemia, was initially considered to be derived from T lymphocytes, on the basis of rosette formation with sheep erythrocytes (E). This cell line has now also been found to form rosettes with sheep erythrocytes sensitized with rabbit antibody and mouse complement (EAC). Evidence is presented that the formation of both E and EAC rosettes is due to two separate receptors on the MOLT cells: (a) EAC rosettes were formed more rapidly and were more stable than E rosettes; (b) preincubation of MOLT with an EAC membrane preparation inhibited resetting with EAC and not with E; (c) MOLT formed rosettes with EAC prepared from trypsinized E, but did not bind to trypsin-treated E alone. The implications of this finding, in regard to the derivation of this cell line, are discussed.     

Redistribution of the Fc receptor on human blood monocytes and peritoneal macrophages induced by immunoglobulin G-sensitized erythrocytes.

The Fc receptor is a plasma membrane component exhibiting an affinity for the C gamma 3 domain of certain subclasses of immunoglobulin G. Using anti-Rho (D)-sensitized red cells (EA) in a slide rosette technique, we have demonstrated a translational mobility and polar redistribution of this receptor on the human blood monocyte and peritoneal macrophage. These cells, isolated from venous blood and malignant ascites and identified histochemically, showed a time- and temperature-dependent receptor capping defined by binding six or more EA confined to the cell half-perimeter. Caps formed in serum were mainly extreme caps in which bound EA appeared as a morula contiguous with the adherent cell. At 21 degrees C and 37 degrees C in serum 80% live rosettes formed caps; virtually none formed at 4 degrees C and about 25% were seen in PBS at 21 degrees C. Similarly, 10% extreme caps in PBS and 60 and 70% in serum were seen at room temperature and 37 degrees C, respectively, suggesting a serum factor(s) was important in promoting live rosette capping. Capping was reversibly inhibited by sodium azide although inhibition was incomplete even at 0.1 M, a concentration 10-fold higher than that giving complete inhibition of lymphocyte antigen-receptor capping. Azide also induced reversion of capped rosettes to diffuse, noncapped rosettes, and to levels comparable to those seen in inhibition studies. Re-exposure to EA after rosette capping failed to increase either the proportion of cells forming rosettes or the fullness of such rosettes indicating a critical number of receptors had been capped. Live rosettes induced a spherocytosis in bound EA irrespective of capping status; such changes appeared early in PBS where capping was minimal. Dead cells bore EA as normal biconcave discs. Some rosetting EA were ultimately hemolyzed upon prolonged incubation, and erythrophagocytosis was seen in about 15% of capped rosettes at 37 degrees C.     

Proportion of lymphocytes forming E, EA, and EAC rosettes following treatment with human interferon preparations in vitro

The proportion of lymphocytes forming E, EA, and EAC rosettes after treatment with human interferon preparations in vitro was measured. While interferon increased the percentage of lymphocytes forming E rosettes, the percentage of cells forming EA rosettes was diminished. The proportion of lymphocytes forming EAC rosettes was not altered to any major extent by interferon treatment. The same effects were observed when fibroblast interferon, purified to homogeneity with regard to molecular weight, was used.     

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

An 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.     

Membrane markers of the lymphocytes of human peripheral blood: techniques of study and normal values in adults]

Peripheral blood lymphocytes from normal subjects were evaluated for their cell surface markers. Normal values were determined in females and males for T-cells (E rosettes and active E rosettes) and for B cells (surface immunoglobulins, EA rosettes and EAC rosettes). Means expressed in percentage and in absolute number per cubic millimeter +/- standard error in normal subjects males and females.     

Release of E-rosette augmenting factor (E-RAF) after stimulation of human leukocytes with mitogens or antigens.

Stimulation of human peripheral blood leukocytes (HPBL) with mitogens such as phytohemagglutinin or concanavalin A increases the total number of lymphocytes that form rosettes with sheep erythrocytes. A similar effect is seen when HPBL from skin test-positive, but not skin test-negative, donors are stimulated by a specific antigen. It was also found that the culture fluids from mitogen-stimulated lymphocytes contained a substance that significantly increased the percentage of E-rosette forming cells (85 to 95%) over that of control culture fluids (40%). A similar phenomenon was also observed with supernatant fluids derived from antigenic stimulation of cells from skin test-positive donors, but not from skin test-negative subjects. The factor that produces this effect has been designated E-rosette augmenting factor (E-RAF). It is nondialyzable; it appears in the supernatants of antigen or mitogen-stimulated cells within 12 hr; and its production is not blocked by mitomycin C. It is produced by cells that do not adhere to glass wool columns and by mitogen-stimulated thymocytes. Sephadex G-100 chromatography showed that mitogen-induced E-RAF eluted from the column in advance of antigen-induced E-RAF. E-RAF has many properties that are characteristic of lymphokines.     

Activated T lymphocytes within human solid tumors

Summary The majority of lymphocytes separated from tumor cell suspensions were T cells. Conjugates of T lymphocytes and tumor cells were often seen. Variable numbers of T cells exhibited signs of activation such as the ability to form stable E rosettes and attachment to normal and malignant cells (a phenomenon designated natural attachment: NA). A proportion of T cells activated in vitro by allogeneic stimulation regularly exhibit these properties. The T cell-tumor conjugates in the suspensions may represent the NA phenomenon, but they could also be the product of T cells that adhere on the basis of specific recognition of cell surface antigens.Abbreviations BBS balanced salt solution - E rosettes rosettes formed with sheep erythrocytes - EA rosettes rosettes formed with ox erythrocytes coated with anti-ox IgG - FCS fetal calf serum - MLC mixed lymphocyte cultures - NA natural attachment - PBL peripheral blood lymphocytes - SRBC sheep erythrocytes - T lymphocytes thymusderived lymphocytes     

Differential ability of mitogen-stimulated human leukocyte-conditioned media to induce Fc receptors in human leukemia cells

The ability of mitogen-stimulated human leukocyte-conditioned media (M-CM) to induce the in vitro differentiation of various human leukemic cell lines was evaluated by measuring the appearance of Fc receptors (FcR) through their ability to form EA rosettes. Only cells of myeloid lineage were induced by M-CM to express FcR; T-, and B-, and non-T/non-B cells failed to respond. As determined with ML-1, a line of human myeloblastic leukemia cells, pokeweed mitogen-conditioned medium, at concentrations of 1-10%, stimulated the expression of FcR in 70-98% of the cells within 1 day after treatment. Phytohemagglutinin-, concanavalin A-, and lipopolysaccharide-conditioned media were less active. The FcR-inducing activity was partially separated from M-CM by chromatography on Sephadex G-75. It was stable between pH 4 and 10, and lost activity at temperatures above 40 degrees C.     

Cannabinols and the rosette forming properties of lymphocytes in vitro.

Preincubation of lymphocytes, for 60 minutes at 37°C with tetrahydrocannabinol (THC), obtained from controls produced reduction in early but not total T cell rosettes compared to aliquots treated with vehicle alone. Similar reductions in early rosettes were seen after preincubation with cannabinol, cannabidiol, and olivetol. The marijuana smokers' lymphocytes were less affected by preincubation with THC than those from controls. Presumably their lymphocytes may already have been affected by prior exposure to cannabinols. These data show that THC and other cannabinols can affect the rosetting properties of T cells $\text{in vitro}$ and may provide a model for the study of THC effects on these immunocompetent cells.     

Eosinophils and immune mechanisms: V. Demonstration of mouse spleen cell-derived chemotactic activities for eosinophils and mononuclear cells and comparisons with eosinophil stimulation promoter

The chemotactic response of mouse eosinophil-rich peritoneal exudative cells to the lymphokine eosinophil stimulation promoter (ESP) was examined. Both eosinophils and monocytes are chemotactically attracted across a 3-μm-pore size polycarbonate filter toward a concentration gradient of ESP-containing culture supernatant fluids. Deactivation of both cell types occurs following preincubation of the responding cells in culture supernates containing ESP activity. The chemotactic activity for both eosinophils and mononuclear cells is stable when incubated at 60 °C for 30 min but is labile at 80 °C, is nondialyzable, and at peak activity exhibits an apparent molecular weight of approximately 25,700 daltons, based on Sephadex G-100 gel chromatography. Production conditions required for the generation of chemotactic and ESP activities are identical, and fractions of culture supernatant fluids possessing one activity are also positive for the other. Preliminary results therefore indicate that the lymphokine ESP attracts both eosinophils and monocytes in a gradient-induced chemotaxis assay.     

Rabbit T cells with and without Fc receptors

Earlier, indirect evidence for rabbit subpopulations differing in Fc receptors and in response to mitogen has been directly tested. T cells were purified from spleen suspension by removal of adherent cells, followed by removal of Ig-bearing cells on petri dishes coated with antibody, directed against the light chain allotype of Ig receptors. The purified cells were further fractionated by formation of EA rosettes and separation on Ficoll-Hypaque. T cells which lacked Fc receptors had a larger response when stimulated with Con A or PHA than did T cells which possessed Fc receptors. Both subpopulations responded more when irradiated nonadherent B cells were added to the mixture, but the extent of help was the same for both cell populations. T cells which contained both Fc receptor-bearing cells and cells which lacked the receptor had a response which was intermediate between that of the two separated subpopulations.     

Isotype Specificity and Heterogeneity of Fcγ-Receptors on Guinea Pig Splenic B and T Lymphocytes

The isotype specificity of Fc-receptors for IgG (Fcγ-Rs) on normal guinea pig splenic B and T cells was determined by a rosette assay using sheep erythrocytes sensitized with either homologous IgG1 or IgG2 anti-sheep erythrocyte antibody [EA(IgG1) or EA(IgG2)]. Approximately 70% of the lymphocytes in the highly purified B-cell fraction could form rosettes with EA(IgG2), and 55% of the cells with EA(IgG1). Inhibition experiments with soluble complexes of IgG1 or IgG2 antibody with ovalbumin demonstrated that approximately 20% of the EA(IgG2) rosette-forming B cells bore the Fcγ-R monospecific for IgG2, whereas 80% of the cells had two distinct Fcγ-Rs simultaneously; one monospecific for IgG2 and the other bispecific for IgG1 and IgG2. The existence of a B cell bearing the Fcγ-R monospecific for IgG1 was not definitively demonstrated in the B-cell fraction. In the T cell-enriched fraction, approximately 40% of the cells could form rosettes with EA(IgG2). The EA(IgG1)rosette-forming cells, however, comprised only 6% of the total cells, indicating that most of the EA(IgG2) rosette-forming T cells bear essentially the Fcγ-R monspecific for IgG2 alone. The results obtained revealed that guinea pig splenic lymphocytes bear two distinct Fcγ-Rs, which are not equally distributed on the B- and T-cell populations and also on their respective subsets.     

Tissue hemolysins as lysin-inhibitor complexes

1. Lytic substances are enzymatically produced at 37°C. from tissue slices or homogenates (mouse liver, kidney, etc.) and appear in the medium in which the tissue fragments are suspended. Their concentration increases with the time during which the tissue is kept at 37°C. (preincubation), and is accompanied by pH changes, so that the lytic activity as finally measured is a function of both the time of preincubation and of the pH. The optimum pH for lysin production is above 7.0, but the lysins, once produced, hemolyze red cells more rapidly at low pH's than at high ones. The enzyme system which produces the lysins is inactivated by heating to 100°C. for 5 minutes. Sodium iodoacetate and fluoride interfere with lysin production principally by reducing the concomitant pH shift; KCN accelerates the production of lytic material in mouse liver homogenates. 2. Comparison of the lytic activity of the supernatant fluid of a preincubated homogenate with the much greater lytic activity of the substances which can be extracted from the same supernatant fluid by alcohol and ether points to these extractable substances existing in the supernatant fluid as lysin-inhibitor complexes of relatively low lytic activity. These complexes are formed enzymatically during preincubation from non-lytic complexes in the tissue. The latter may be lipoproteins, and the highly lytic ether-extractable substances may be fatty acids or their soaps. 3. The diffusibility of the lysin-inhibitor complexes is small. 4. Lytic substances which are ether-insoluble can be extracted with alcohol from tissues as well as from serum. These "lysolecithin-like" substances exist in the supernatant fluids of homogenates as lysin-inhibitor complexes. 5. Lysis of mouse red cells by substances contained in mouse tissue (liver and kidney) is often accompanied by the formation of methemoglobin and choleglobin. Mouse red cells containing choleglobin are abnormally fragile both osmotically and mechanically, and it is possible that a process involving the production of choleglobin, accompanied or followed by globin denaturation, is one which contributes towards the hemolysis which occurs in systems containing tissue slices or homogenates.     

Studies on mouse autoreactive cells: I. Role of H-2 antigens in mouse autologous rosette formation

A minority (1–2%) of normal mouse lymphoid cells bind autologous erythrocytes and form rosettes. In this study we examined the antigenic specificity involved in the formation of such rosettes. A significant difference in the incidence of rosettes formed, respectively, with autologous and allogeneic mouse erythrocytes is found. Moreover, preincubation of lymphoid cells with low concentrations of syngeneic erythrocytic ghosts causes significant competitive inhibition of subsequent rosette formation. Allogeneic ghosts obtained from nonrelated or from congenic resistant strains of mice do not display this inhibitory effect under the same conditions. It is thus suggested that mouse autologous rosette-forming cells bear receptors for syngeneic H-2 antigens that are involved in the binding of autologous erythrocytes. More precisely, compatibility between lymphocyte and erythrocyte restricted to K or D only is sufficient to ensure a level of rosettes similar to that obtained when complete identity occurs for K, I, and D regions.     

Involvement of human membrane-associated complement components in the rosette formation between marmoset red blood cells and human leukocytes

The majority of human monocytes, a subpopulation of B lymphocytes, and all B lymphoblastoid cell lines (B-LCL) tested but one form rosettes with Marmoset red blood cells (MaRBC). None of the human peripheral T cells, T-LCL, and B chronic lymphoid leukemia cells (B-CLL) used bind to MaRBC. The binding could not be correlated with any membrane markers or antigens present on cultured cells or peripheral blood leukocytes (PBL). Blocking of the rosette formation by preincubation of MaRBC with purified human complement (C) components and cobra venom or by pretreatment of leukocytes and cultured cells with antisera to human C components suggested that membrane-associated C components present on PBL or B-LCL are involved in the binding to MaRBC.     

Platelet release products modulate some aspects of polymorphonuclear leukocyte activation.

The aim of this research was to evaluate in vitro interactions between platelets and polymorphonuclear leukocytes. The effects of supernatant from thrombin-activated platelets and two platelet release products (adenosine triphosphate and beta-thromboglobulin) were tested on the following features of polymorphonuclear leukocytes activation: opsonized zymosan and phorbol myristate acetate stimulated chemiluminescence, release of membrane bound calcium, NADPH-oxidase activity, and membrane fluidity (fluorescent polarization). The results showed that the addition of platelet supernatant to polymorphonuclear leukocytes induces a significant activation of cells. On the other hand, after three hours of preincubation of polymorphonuclear leukocytes with platelet supernatant, a decreased response of polymorphonuclear leukocytes to stimulation with phorbol myristate acetate, a significant decrease in NADPH-oxidase activity, and a lowered membrane fluidity were observed. Adenosine triphosphate modulated only opsonized zymosan stimulated chemiluminescence, with and without preincubation with polymorphonuclear leukocytes. Beta-thromboglobulin caused a decrease of the chemiluminescent response of polymorphonuclear leukocytes, using both agonists, with and without preincubation with polymorphonuclear leukocytes. Moreover beta-thromboglobulin only caused a decrease of the polymorphonuclear leukocytes membrane fluidity without preincubation with the cells. These results support the thesis that platelets have a "time-related" modulating activity on polymorphonuclear leukocytes.     

Interaction of antigen-specific T cell factors with unique "receptors" on the surface of mast cells: demonstration in vitro by an indirect rosetting technique

Picryl (trinitrophenyl) chloride (PCL) contact sensitization of mice induces T cells that release an antigen-binding T cell factor (PCLF) that plays an important role in the initiation of contact sensitivity responses, in part via activation of mast cells. The current study employs an in vitro indirect rosette assay to demonstrate that PCLF can interact with the mast cell surface. Sheep red blood cells (SRBC) were hapten conjugated with trinitrophenyl (TNP), dinitrophenyl (DNP), or oxazolone (OX). When TNP-conjugated SRBC were coated with PCLF, monoclonal anti-DNP IgE, or anti-DNP IgG1, they produced 40 to 50% rosettes with purified normal mouse peritoneal mast cells. Analogous antigen-binding factors, from lymphoid cells of OX and dinitrofluorobenzene contact-sensitized mice, gave similar mast cell rosetting levels with OX-SRBC and DNP-SRBC, respectively. PCLF demonstrated a high degree of hapten specificity in that it formed rosettes with TNP-SRBC but not with DNP-SRBC, unlike IgE and IgG1, or DNPF, which formed rosettes with either SRBC type. Similarly, soluble TNP-BSA could inhibit PCLF rosette-forming capacity, but soluble DNP-BSA could not. In addition to mouse mast cells, PCLF formed rosettes with rat basophil leukemia cells, mouse peritoneal exudate macrophages, mouse alveolar macrophages, and J 774 cultured mouse macrophages; it did not form rosettes with rat mast cells, rat alveolar macrophages, or mouse spleen cells. Thus, PCLF-formed rosettes were antigen specific, relatively species specific, and mast cell/macrophage specific. PCLF-mediated rosette-forming activity could be detected in the presence of nanogram quantities of PCLF. More than 10 times greater IgE was needed to produce IgE-mediated rosettes. Reduction and alkylation eliminated the rosetting activity of IgE, but the rosetting activity of PCLF was not affected. PCLF, but not IgE rosette-forming activity, could be removed by and eluted from affinity columns linked with a monoclonal antibody specific for T cell-derived antigen-binding factors, whereas PCLF rosetting activity was not retained by an anti-immunoglobulin affinity column. Preincubation of mast cells with rat myeloma IgE or mouse monoclonal IgE of various specificities blocked IgE rosettes but not PCLF-induced rosettes. Other immunoglobulin isotypes likewise did not block PCLF rosettes. However, PCLF rosettes could be blocked by preincubation of mast cells with OX factor (OXF),and OXF-mediated rosettes could be blocked similarly by PCLF. These results suggest that the antigen-binding T cell factor PCLF interacts with a unique receptor on the surface of mouse mast cells.     

Mitogen activation of human chronic lymphatic leukemia cells. I. Synthesis and secretion of immunoglobulin.

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 less than 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.