Spontaneous rosette formation of rat thymus cells with guinea pig erythrocytes.

C57 black mice were immunised intraperitoneally with a DBA2 lymphoma (SL2). Fourteen days later spleen cells were prepared. These cells lysed the specific target (SL2) in vitro. Spleen cells were cultured for 24 hr at 37 ° C. Cell-free culture supernatants contained IgG and lysed SL2 cells either in the presence of a source of complement or in the presence of a monolayer of macrophages (a good source of antibody-dependent effectors). The cells producing cell-dependent antibody adhered to nylon wool and were unaffected by anti-theta serum. It was found that the production of antibody in vitro did not make a significant contribution to the observed cytolysis of SL2 by sensitised spleen cells. This effect was mediated by thymus-derived lymphocytes.     

Detection of human B cells and chronic myelocytic leukemic cells by rosette formation with sheep erythrocytes and fresh human sera.

A new method of detecting the C3b receptor is reported. A particular merit of this method is that anti-RBC rabbit antiserum is not required. Rosettes were formed with human B lymphocytes, B lymphoblasts and granulocytes, using sheep erythrocytes (SRBC) sensitized with fresh human serum (FHS). T lymphocytes and T lymphoblasts did not form rosettes. The percentage of cells forming rosettes with this method approximated the percentage of rosettes formed with EACm. However, FHS coated SRBC did not react with most cells of B cell type chronic lymphocytic leukemia (CLL), whereas EACm rosette formations showed a definite reaction. On the other hand, 34--58% of cells of chronic myelocytic leukemia (CML) bound with the indicator red cells. SRBC sensitized with fresh rabbit or guinea pig serum formed rosettes with PBL, tonsil cells, B lymphoblasts and granulocytes. Complement and IgM antibody were required for this reaction, as in EAC rosette formation.     

Characteristics of spontaneous rosette formation by mouse lymphocytes with autologous erythrocytes

A substantial proportion of mouse spleen and thymus cells form spontaneous rosettes with syngeneic and allogeneic erythrocytes. Rosette formation is independent of the metabolic state of the cell: Inhibitors of ATP formation (NaCN, NaN₃) and of cell motility (cytochalasin B, colchicine, vinblastine) have little or no effect. Erythrocytes can be bound by living or dead lymphocytes. The stability of rosettes against high temperature and shearing forces is weaker than that of immune rosettes. The increase or decrease of electrostatic repulsion between cells will enhance or reduce rosette formation. Membrane proteins are involved in cell-to-cell contacts, since several proteases destroy relevant structures on the cell surfaces of lymphocytes, erythrocytes, or both.     

Inhibition of rosette formation of IgE-bearing rat mast cells by disodium chromoglycate.

The interaction of reaginic antibody with specific antigen was studied by the rosette formation of peritoneal rat mast cells. The mast cells were obtained from actively sensitized rats or were passively sensitized in vitro. Rosette formation was of a higher degree with mast cells of actively sensitized rats; in this case 58% of the cells showed a strong rosette-forming effect (blinding more than 5 SRBC). No rosette formation was detected in 18% of the cells. With passively sensitized rat mast cells, rosette formation was 45% and 22%, respectively. Rosette formation of both actively and passively sensitized mast cells could be inhibited by disodium chromoglycate (DSCG); the inhibitory effect of 20 micrograms and 200 micrograms of the drug was the same, and neither dose caused a full inhibition. It is suggested that the linkage of specific antigen to the surface of sensitized mast cells can be inhibited by DSCG in vivo.     

Distinction between malignant L cells and normal mouse fibroblasts by rosette formation with sheep red blood cells.

Murine L cell fibroblasts, and derivatives were found to rosette with sheep red blood cells (SRBC). Primary fibroblast explants from the parent murine strain, C3H, did not possess this potential. No rosettes were observed with primary fibroblast explants from C57BL and B10Br mice, with a human fetal lung fibroblast, with baby hamster fibroblasts or their polyoma transormed derivative, or with a cell line, 1T-22, derived from BALB/c mice. Hybridization of 1T-22 and L cells, by Sendai virus-mediated cell fusion, suppressed the rosette potential of the L cell parent. The receptor for SRBC on L cells appears to result from the expression of a recessive characteristic.     

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.     

Types of cells involved in antigen-stimulated and spontaneous rosette formation with Toxoplasma gondii.

Antigen-binding cells were identified by using rosette formation of Toxoplasma gondii and defined lymphoid populations under different experimental conditions. Treatment of immunized spleen cell suspensions with anti-Thy 1 serum plus complement inhibited 5 to 29% of the rosette-forming cells (RFC). Higher numbers of thymus-derived lymphocyte-RFC were obtained after incubation at 4 degrees C and by the centrifugation method than by simple incubation at 20 degrees C. RFC were also observed with thymocytes. Combined treatment with anti-Thy 1 serum plus complement and depletion of adherent cells indicated that the major proportion, 46 to 70%, of RFC were B cells. Spleenocytes of nu/nu mice formed similarly high numbers of rosettes. Spontaneous RFC were observed in nonimmunized mice with both spleen and thymus populations. Numbers of rosettes varied considerably depending on the method and the source of cell population used. Removal of adherent cells from spleen suspensions resulted in RFC reduction of 14 to 25% in immunized and 14 to 33% in nonimmunized animals. Pretreatment with anti-mouse immunoglobulin inhibited completely the spleen and spontaneous thymus RFC and partially the thymus RFC in immunized animals.     

Phenotypic markers for the feline monocyte: rosette formation with human erythrocytes and a monoclonal antibody which binds myeloid cells

A small population of cells with the ability to form rosettes with human erythrocytes was found in feline peripheral blood leukocytes (PBL) (10%) and bone marrow (9%), but not in purified granulocyte preparations, thymus, and lymph node tissues. The morphologic appearance and ability to phagocytize latex beads indicated these cells were monocytes. A monoclonal antibody, CM277, with a binding specificity for feline peripheral blood phagocytes was also characterized. Immunofluorescent microscopy revealed CM277 to bind specifically to monocytes and polymorphonuclear neutrophils. The binding of CM277 to monocytes was also shown by human erythrocyte-rosette formation wherein there was a high degree of correlation between these two phenotypic markers for cells ingesting latex beads. Monocytes, polymorphonuclear neutrophils, and T lymphocytes of the cat rosette with guinea pig erythrocytes (GPE) and using CM277 we were able to determine the contribution of the former two cell types to the GPE-rosetting population. Monocytes and polymorphonuclear neutrophils comprised the majority of the GPE-rosetting cells in fresh PBL (greater than 60%), but after culturing overnight, there was a substantial decrease in these cells (less than 35%). In contrast, GPE-rosetting T lymphocytes comprised approximately 10% of the cells in fresh PBL, and after in vitro culture for 1 day they constituted 35-45% of all cells. The removal of monocytes by human erythrocyte-rosetting did not affect the pokeweed mitogen-induced synthesis of Ig, but did lead to an increased production of interleukin 2. Removal of the GPE-rosetting population from PBL resulted in a marked decrease in interleukin 2 production, pointing to a positive contribution of GPE-rosetting T lymphocytes to the synthesis of this lymphokine.     

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.     

Complement inhibitor(s) released by leukocytes. I. Pretreatment of sheep erythrocytes with supernatants of mouse spleen and thymus cells inhibit whole complement activity and C2 utilization.

Sheep erythrocytes pretreated with supernatants of mouse spleen or thymus cells become resistant to lysis by guinea pig complement. The inhibitory activity (IA) reduces the utilization of C2 by EAC14. Because IA binds to the surface of sheep erythrocytes and does not inhibit C1 irreversibly, it is probably a hitherto undescribed inhibitor of complement.     

Modulation of cellular immune function in vitro by histamine receptor-bearing lymphocytes: mechanism of action.

When soluble histamine is added to guinea pig lymphocytes in vitro, antigen-induced cellular proliferation and the production of migration inhibitory factor is suppressed. The inhibitory effects that are produced by histamine have been shown to be mediated by the histamine-type 2 receptors of the involved cells, but the exact nature of this suppression has not been fully explored. The present studies have evaluated, following immunization, the effect of histamine on macrophage function in vitro, and affinity chromatography to delete a subpopulation of cells bearing histamine receptors. When we treated monolayers of peritoneal exudate cells with histamine (up to 10^?3M) we found that histamine did not interfere with antigen binding by macrophages, macro phage presentation of antigen to lymphocytes, nor the antigen-independent or antigen-dependent lymphocyte-macrophage rosetting. Columns containing insolubilized conjugates of histamine and rabbit serum albumin depleted a subpopulation of cells responsive to histamine i.e., the non-adherent cells made migration inhibitory factor and proliferated in the presence of histamine. The latter finding suggested that the retained cells might have suppressor function and if so, might mediate their effect through the release of a soluble factor. Preliminary data obtained in these studies supports this hypothesis. We conclude that cells bearing histamine receptors may serve a regulatory role in cellular immunity after their activation by histamine by producing a non-dialyzable factor with immunosuppressive properties.     

Enhancement of early human E rosette formation by cholinergic stimuli.

The effects of the cholinergic stimuli carbamylcholine (carbachol) and dibutyrl cyclic guanosine monophosphate (DBCGMP) were determined on both 'early' and 'total' E rosette formation. Ficoll-Hypaque-separated lymphocytes were preincubated with either carbachol or DBCGMP over a 10(-3) M to 10(-13) M dose range. Both agents significantly enhanced 'early', but not 'total' E rosette formation. Peak enhancement above control values occurred at 10(-7) M (72%) and 10(-9) M (69%) for carbachol and 10(-5) M (70%) and 10(-7) M (70%) for DBCGMP. Kinetic studies showed a rapid onset of enhancement (2.5 min) for carbachol, whereas DBCGMP required 15 min for significant enhancement to occur. The muscurinic nature of carbachol enhancement of E rosettes was demonstrated. Atropine at 10(-7) M completely abolished the carbachol effect while showing little inhibition of the DBCGMP effect on rosette formation. These studies indicate that the cholinergic stimuli carbachl and DBCGMP significantly enhance the 'early' E rosette former in man. Human T lymphocytes appear to have functional cholinergic receptors that can be blocked by the muscurinic antagonist atropine. The role of the cyclic nucleotides and their stimulants on the immune system is incompletely understood, but it would appear that they are extremely important in the differentiation and function of the T lymphocyte. E rosette formation may be a useful model in man for studying the effects of the cyclic nucleotides on the human T lymphocyte.     

Functional subsets of human T cells defined by "active" rosette formation

Experiments were conducted defining the possible basis for increased susceptibility of alloxan-treated and genetically diabetic C57Bl/KsJ mice to infections with Candida albicans. Alloxan monohydrate (175 mg/kg) produced a prolonged state of hyperglycemia, which persisted through 31 days. Parameters of immune responses varied depending upon the interval between alloxan administration and testing. In the period immediately following alloxan treatment (1–14 days), the numbers of lymphocytes in the thymus and spleen were reduced, the numbers of recoverable peritoneal macrophages were decreased, and the mice showed an increased susceptibility to intravenous infection with C. albicans. In contrast, splenic lymphocytes responded normally to stimulation with Con A, and in vitro phagocytosis of yeast cells by peritoneal macrophages was normal. Also, in vivo production of such lymphokines as migration inhibitory factor (MIF) and macrophage activating factor (MAF), as well as delayed hypersensitivity footpad responses, was generally within the normal range. In the later phase of alloxan diabetes (21–28 days) after administration of alloxan, lymphoid cellularity recovered progressively and the numbers of recoverable peritoneal macrophages were normal. However, these mice still showed an increased susceptibility to C. albicans infection. Genetically diabetic mice (C57Bl/KsJ, db/db) were abnormal in virtually all the assays involving cell-mediated immunity. The numbers of lymphocytes and peritoneal macrophages were markedly decreased, lymphoid cells responded poorly to Con A, and the phagocytosis of yeast cells by macrophages was depressed. The in vivo production of lymphokines and footpad responses of the delayed-type hypersensitivity were depressed. In addition, these mice were highly susceptible to intravenous infection with C. albicans.     

Inhibition effect of rosette formation between human lymphocytes and sheep erythrocytes by specific heptapeptide isolated from uremic fluid and its analogs.

One of middle molecular substances (H-His-Pro-Ala-Glu-Asn-Gly-Lys-OH) and its two analogs, in which the proline residue in position 2 was replaced by glycine and the alanine residue in position 3 was replaced by valine exert a inhibition effect on $\text{in vitro}$ E-rosette formation. Its synthetic two analogs showed diminished biological activity compared to native heptapeptide.     

Histamine-releasing activity (HRA). I. Production by mitogen- or antigen-stimulated human mononuclear cells.

Supernatants from 1- to 2-day cultures of human mononuclear cells induced the release of histamine from basophils. Generation of this histamine-releasing activity (HRA) was stimulated by addition of concanavalin A to the cell cultures. Mononuclear cells were also cultured with SKSD and Candida albicans antigens. Stimulation of HRA production by these antigens was correlated with positive delayed skin reactions. Serial dilutions of supernatants assayed for HRA provided a semiquantitative determination of the level of HRA in mitogen- or antigen-stimulated samples. Antigen increased HRA production when added during the first or second day of culture. Generation of HRA probably requires active protein synthesis, since puromycin was inhibitory, and since preformed HRA could not be recovered from lysed cells. HRA was detected in supernatants after 4 hr, and the effects of antigen stimulation were apparent after 8 hr of culture. Replacement of supernatants with fresh culture medium allowed continued synthesis of substantial quantities of HRA during the second day of culture. A linear correlation was observed between the amount of HRA produced and the mononuclear cell concentration. Our findings provide evidence for the interaction of lymphocytes and basophils via a soluble mediator.     

Rosette formation between rat thymocytes and guinea pig erythrocytes requires "active" fetal calf serum. II. Characterization of the receptor-bearing thymocytes.

High proportions of thymocytes from many rat strains participate in rosette formation (RF) with guinea pig erythrocytes only in the presence of non-heated fetal calf serum. Adult Lewis rat spleens, lymph nodes, peripheral blood and bone marrow contain very few cells capable of participating in RF. Adult levels of cells participating in RF are present in Lewis rat thymus from the perinatal period onward. Essentially, no cells participating in RF are found in the spleens or bone marrow of Lewis rats during the perinatal period. Thymocytes participating in RF are sensitive to high doses of cortisone and low doses of irradiation in vivo. After sublethal irradiation, thymuses are rapidly repopulated by cells participating in RF. After lethal irradiation followed by bone marrow transplantation, thymuses are repopulated by donor cells that can participate in RF. Of interest, a moderate proportion of thymocytes from many mouse strains also can participate in RF. This subpopulation of murine thymocytes are highly cortisone sensitive.     

Human autologous rosettes. I. Mechanism of binding of autologous erythrocytes by T cells.

A population of human peripheral blood lymphocytes is able to bind autologous erythrocytes in the presence of autologous serum. Erythrocyte binding is found to be more efficient at 4 °C after preincubation of lymphocytes in autologous serum for 30 min followed by overnight incubation. The overall cellular concentration and erythrocyte/lymphocyte ratio are also crucial in determining the weak erythrocyte binding to autologous lymphocytes. Membrane proteins are involved since the binding structures are sensitive to protease treatment. The 26% RFC obtained with an optimized assay are related to the T-cell lineage.     

Functional characteristics of histamine receptor-bearing mononuclear cells. II. Identification and characterization of two histamine-induced human lymphokines that inhibit lymphocyte migration

Although functional histamine receptors have generally been restricted to those human T lymphocytes expressing suppressor cell functions, more recent evidence suggests that histamine receptor-bearing human T lymphocytes are functionally heterogeneous and capable of other immunomodulatory activities. Lymphocyte chemoattractant factor (LCF) is a cationic sialoprotein with an apparent m.w. of 56,000, whose production is limited to histamine-type 2 receptor-bearing human T cells. LCF is selectively chemokinetic for T lymphocytes, and presumably contributes to the recruitment of unsensitized effector lymphocytes at inflammatory sites. In addition to LCF, Sephadex G-100 gel filtration of histamine-induced lymphocyte supernatants revealed two regions of migration inhibitory activity for human blood T and rat splenic lymphocytes. These regions corresponded to m.w. of 70,000 to 80,000 (LyMIF75K) and 30,000 to 40,000 (LyMIF35K). LyMIF75K had a single pI of 7.5 to 8.0, and its biologic activity was sensitive to trypsin but not to neuraminidase or heat (56 degrees C). LyMIF35K had a single pI of 8.5 to 8.8, and its biologic activity was sensitive to neuraminidase and heat but not to trypsin. These LyMIFs therefore appeared to be distinct from one another and physicochemically different from other migration inhibitory lymphokines. All three lymphokine activities appeared within 4 hr of incubation. The minimum concentration of histamine required to stimulate production of the LyMIF was 10(-6) M. Lymphocytes that did not adhere to a histamine affinity matrix were unable to produce either LyMIF upon subsequent stimulation with histamine or concanavalin A (Con A). Lymphocytes incubated with histamine and diphenhydramine produced LCF but neither LyMIF, whereas cells incubated with histamine in the presence of cimetidine produced both LyMIF but not LCF. These data suggest that a subset of lymphocytes defined by the presence of histamine-type 1 receptors are capable of producing two distinct species of lymphocyte migration inhibitory activity. These cells may contribute to the immobilization of effector T lymphocytes chemokinetically attracted to certain inflammatory sites.