An appraisal of Fc receptors on human peripheral blood B and L lymphocytes.

Human circulating lymphocytes with easily detectable surface immunoglobulin have been divided into two populations, B cells and L cells. This second population lacks membrane-incorporated Ig, but has a receptor for membrane-labile cytophilic IgG. In this study purified B and L lymphocytes were examined for Fc receptors that bind aggregated IgG and IgG complexed to erythrocytes. Purified lymphocyte populations were prepared by nylon columns and by negative selection with rosetting techniques. L lymphocytes bound aggregated guinea pig and human IgG, and formed rosettes with human erythrocytes sensitized with Ripley IgG (EA). Treatment of L lymphocytes with trypsin had no effect on the receptors for IgG. B lymphocytes did not bind EA and attachment of aggregated IgG was variable; up to one-third of these cells fixed aggregated human IgG to the cell membrane. Trypsin treatment abolished binding of Agg-IgG to B cells in sharp contrast to its effect on L cells. Furthermore, double-label immunofluorescence studies showed that cells with both membrane-incorporated Ig and receptors for aggregated guinea pig IgG were rare. These studies indicate that human peripheral blood B lymphocytes lack a high affinity, trypsin-resistant Fc receptor that is present on L lymphocytes.     

Protein tyrosine phosphorylation is induced in murine B lymphocytes in response to stimulation with anti-immunoglobulin.

Activation of both T and B lymphocytes through their membrane receptors for antigen is known to induce breakdown of inositol phospholipids. In addition, T cell activation by antigen is accompanied by increased protein tyrosine phosphorylation of components of the T cell antigen receptor. We now provide evidence that B cell activation through membrane immunoglobulin is also coupled to stimulation of protein tyrosine kinase activity. One potential candidate for a B lymphocyte protein tyrosine kinase is an 80 kd molecule that is itself phosphorylated at tyrosine residues in response to stimulation with anti-immunoglobulin antibodies.     

Proliferation of human malignant lymphocytes induced by anti-IgM independent of B cell growth factor

Human malignant B lymphocytes were identified that proliferate in response to small doses of anti-immunoglobulin. Proliferation was induced by monoclonal mouse anti-HIgM, polyclonal goat anti-HIgM, and F(ab')2 fragments thereof, in vitro, and was not accompanied by immunoglobulin secretion. Proliferation was found to be unaffected by T cell depletion and was not enhanced by supplementation with B cell growth factor. Culture fluids from unstimulated malignant lymphocytes as well as from malignant lymphocytes stimulated with anti-HIgM contained no measurable B cell growth factor activity. Thus, proliferation of these malignant lymphocytes was not dependent on the presence of T lymphocytes and was independent of the presence of B cell growth factor. These results imply that B cell stimulatory factors may not be required for proliferation of all human B lymphocytes. Moreover, these results imply that treatment with anti-immunoglobulin reagents may be inappropriate for some B lymphocyte malignancies.     

Anti-immunoglobulin in combination with cytochalasin stimulates proliferation of murine B lymphocytes

The ability of cytochalasin to influence the stimulation of murine B lymphocytes through surface immunoglobulin was assessed during short term cultures. Modest doses of anti-immunoglobulin alone did not stimulate proliferation of mouse spleen cells at 2 days. Cytochalasin B alone also had no effect. However, anti-immunoglobulin in combination with cytochalasin B stimulated substantial proliferation as judged by [3H]thymidine incorporation. Cytochalasins A, E, and D, and dihydrocytochalasin B were all effective in promoting B cell proliferation. Spleen cells from xid-defective (CBA/N X DBA/2)F1 male mice failed to proliferate in response to anti-immunoglobulin plus cytochalasin, suggesting that this treatment affects the same subset of B cells as anti-immunoglobulin plus B cell growth factor. Moreover, proliferation that was stimulated by anti-immunoglobulin plus cytochalasin B was not affected by T cell depletion. Cytochalasin may circumvent the need for, or replace, a second signal for proliferation.     

Induction of high affinity IL 2 receptors on B cells responding to anti-Ig and T cell-derived helper factors

We have reported that IL 2 is one of the essential helper factors in culture supernatants from concanavalin A-activated spleen cells or T cell hybridomas that support proliferation and immunoglobulin secretion in B cell cultures responding to anti-immunoglobulin. Here we show that cells in such cultures consume IL 2 and bear high affinity IL 2 receptors detected by binding of purified, radiolabeled IL 2. Induction of high affinity IL 2 receptors depends on addition of both anti-immunoglobulin and helper factors, and does not occur in cultures given only anti-Ig, only helper factors, concanavalin A plus helper factors, or LPS. The majority of IL 2 receptors are on cells that also bear endogenous membrane immunoglobulin, because they are found in the membrane immunoglobulin-positive fraction when cultured cells are separated by fluorescence sorting after overnight culture in the absence of anti-immunoglobulin to allow reexpression of membrane immunoglobulin.     

Cell membrane IgD: demonstration of IgD on human lymphocytes by enzyme-catalyzed iodination and comparison with cell surface Ig of mouse, guinea pig, and rabbit.

A substantial fraction of human cord blood and peripheral blood lymphocytes have recently been shown to bear IgD. Although IgD has not been identified in mice, it has been suggested that it is also a major surface immunoglobulin of murine lymphocytes. Thus, lactoperoxidase-catalyzed iodination of surface immunoglobulin of mouse spleen and lymph node cells reveals the existence of an IgH chain differing from mu, gamma, and alpha-chain both antigenically and by mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This new H chain class has been previously proposed to be the mouse homologue of delta-chain. In this paper, we analyzed human, mouse, guinea pig, and rabbit lymphoid cell membrane Ig by lactoperoxidase-catalyzed iodination, extraction with non-ionic detergent precipitation with a variety of specific anti-Ig sera, and electrophoresis of dissolved reduced precipitates on sodium dodecyl sulfate-polyacrylamide gels. Our studies confirm the previous reports of a new mouse cell membrane H chain with a mobility more rapid than that of mu-chain. However, we fail to detect a molecule with this electrophoretic mobility on the surface of guinea pig or rabbit lymph node and spleen cells. Moreover, neither anti-kappa nor anti-delta antibody precipitates a molecule with an H chain of this mobility from labeled extracts of human cord blood or peripheral blood lymphocytes. Cell surface delta was identified on both human cord blood and peripheral blood lymphocytes, but it proved to have mobility similar to human and mouse mu-chain. This result indicates either that mouse delta-chain has an electrophoretic mobility on sodium dodecyl sulfate-polyacrylamide gels which differs appreciably from that of human membrane delta-chain or that the newly described mouse H chain is not the homologue of human delta-chain.     

Anti-immunoglobulin-induced proliferation of B cells : Parallelism in the inhibition by chloroquine, monensin and immunoglobulin

Proliferation of rabbit lymphocytes was induced with goat anti-rabbit immunoglobulin. Chloroquine and monensin, known to inhibit internalization-related events, yielded inhibition of proliferation that paralleled the inhibition by a specific competitive ligand, rabbit immunoglobulin (IgG), whereas inhibition by puromycin did not. Moreover, virtually all of the cells that can be activated in freshly isolated populations adhered to anti-immunoglobulin-coated Petri plates, whereas all of the activatable population was recovered in the non-adherent fraction after a brief incubation of the cells with anti-immunoglobulin to induce internalization of surface membrane immunoglobulin. Using immunofluorescence it was further observed that monensin and Chloroquine inhibit the reappearance of surface immunoglobulins on the cell surface to some extent subsequent to their removal induced by anti-immunoglobulin.     

Cyto-immunological studies of guinea pig sperm antigens

Summary Antigenic localization in guinea pig epididymal sperm and testicular imprints as well as in viable, motile guinea pig epididymal sperm was studied by means of fluorescent labelled antibody techniques. Globulins from rabbits and chickens immunized with guinea pig epididymal sperm were used in the direct procedure while sera from sheep and fowl injected with rabbit globulins were used in the indirect procedure. The main findings were: 1) spermatozoa from the distal portion of the epididymis displayed brilliant fluorescent acrosomes and less intensely stained midpieces and principal pieces when treated as dried smears in both the direct and indirect methods; 2) testicular spermatozoa were similarly stained but whereas in epididymal spermatozoa the whole acrosome stained intensely, the testicular spermatozoal acrosome displayed intense fluorescence of the inner acrosome; 3) protoplasmic droplets fluoresced strongly; 4) cross-reactivity was observed between human and guinea pig sperm but not between rat and guinea pig sperm, indicating an antigenic relationship between human and guinea pig but not between guinea pig and rat; 5) treatment of viable, motile guinea pig spermatozoa with fluorescent globulins resulted in agglutination and immobilization as well as formation of antigen-antibody aggregates adherent to the cell membrane of the head, midpiece and principal piece; the formation of such fluorescent aggregates in the medium surrounding the treated motile sperm was indicative of leaching of antigenic material from the sperm cells.This investigation was supported by funds from United States Public Health Service grant HE-05798-03, The Ford Foundation and National Science Foundation.     

Synthesis of immunoglobulin by rabbit lymphocytes in vitro in response to anti-immunoglobulin antiserum

Stimulation of synthesis of immunoglobulin (Ig) in vitro by Con A and anti-Ig in cultures of rabbit lymphoid cells has been analyzed qualitatively using an assay that measures the incorporation of [³H]leucine into newly synthesized proteins, followed by the specific absorption of tritiated immunoglobulin by staphylococcal protein A. Whereas Con A stimulates Ig production by spleen cells only if T lymphocytes are present, anti-immunoglobulin serum enhances Ig synthesis in the absence of T lymphocytes. In contrast, neither Con A nor anti-immunoglobulin serum stimulates peripheral blood lymphocytes to produce enhanced levels of Ig. It is concluded that both Con A and anti-immunoglobulin serum do not activate resting B cells but drive differentiation of B cells which are already synthesizing Ig. Anti-Ig acts directly whereas stimulation of B-cell Ig synthesis by Con A occurs indirectly through stimulation of T cells.     

The role of immunoglobulin receptors on lymphocytes in production of MIF in guinea pigs

The effect of anti-guinea pig IgG sera and anti-rabbit light kappa chain serum on the capacity of sensitized lymphocytes of guinea pigs to production of migration inhibitor factor (MIF) was investigated. The lymph node cells, thymocytes and circulating lymphocytes taken from dinitrophenyl- (DNP) sensitized guinea pigs were preincubated with antisera against gamma₁ + gamma₂ globulins, gamma₁ globulins, gamma₂ globulin, light kappa chains or normal rabbit serum as control and stimulated with antigen in vitro to production of MIF. The inhibitory effect of lymphocyte culture supernates on the migration of guinea pig normal macrophages was determined by capillary tube test. It was found that all the anti-immunoglobulin sera used suppressed, in varied degree, the release of MIF by sensitized lymphocytes. It is suggested that the suppressive influence of anti-IgG sera reflects their blocking effect on surface receptors specific for antigen.     

T-cell-activating monoclonal antibodies, reacting with both leukocytes and erythrocytes, recognize the guinea pig Thy-1 differentiation antigen: characterization and cloning of guinea pig CD90

A glycophosphatidylinositol (GPI)-linked differentiation antigen expressed on guinea pig T and B lymphocytes was identified by several monoclonal antibodies; it has been shown previously that this membrane protein induced strong polyclonal T cell proliferation upon antibody binding and costimulation by PMA. Purification by immunoadsorption and microsequencing revealed that this T-cell-activating protein is the homologue of Thy-1 or CD90. In contrast to the Thy-1 antigen of most other species, guinea pig Thy-1 has a much higher molecular weight, which is due to a more extensive N-linked glycosylation, bringing the molecular weight of the total antigen up to 36 kDa. Molecular cloning of guinea pig Thy-1 indicated that the deduced molecular weight of the protein backbone is 12,777 after removal of an N-terminal 19-amino-acid leader peptide and cleavage of the 31 amino acids for GPI anchoring the C-terminal end. Sequence comparison showed that guinea pig Thy-1 has an 82% homology to human and a 72% homology to mouse Thy-1 on the amino acid level. Immunohistological staining of cryostat sections revealed intensive staining with the monoclonal antibody H154 on fibroblasts, fibrocytes, Kupffer cells, alveolar macrophages, and mesangial cells. As observed in the human, mouse, and rat, Thy-1 is abundant in the guinea pig brain. Unlike Thy-1 expression in other species, guinea pig Thy-1 is strongly expressed on most resting, nonactivated B cells and, to a lesser extent, on erythrocytes. While treatment of erythrocytes and lymphocytes with GPI-specific phospholipase C largely decreased reactivity with mAb H154, T cells retained the proliferative response to antibody and phorbol esters.     

Partial purification, physicochemical characterization and biological function of a rosette-decreasing factor (RDF) extracted from guinea pig thymus.

A factor that decreases rosette formation between guinea pig T-cells and rabbit red blood cells (RRBC) was extracted from the thymus of the guinea pig. The active factor could be extracted from the spleen as well as the thymus, but not from the liver or kidney. The active factor of the thymic extract was found in the precipitates produced by 80% saturated ammonium sulfate and it was separated from the water-soluble fraction of the precipitates. The molecular weight of the partially purified substance was estimated to range between 10,000 and 30,000 by filtration through a diaflow membrane. From the studies on physicochemical characterization, it might be a heat-resistant basic peptide probably bound to a ribonucleotide moiety. This factor reduced rosette formation between RRBC and guinea pig T-cells, but did not reduce erythrocyte-antibody-complement rosette formation. This factor also inhibited mitogen (concanavalin A, phytohemagglutinin-P)- induced DNA synthesis of guinea pig lymphocytes and antigen-induced DNA synthesis of sensitized guinea pig lymphocytes.     

Heterogeneity in cellular antigen retention structures

The mechanism of presentation of foreign antigens to helper T lymphocytes and the nature of the structures involved in this process are not totally understood. It is well documented that this event is carried out by antigen-presenting cells (APC) (e.g., macrophages, dendritic cells, and B lymphocytes) that internalize the antigen, process it, reexpress it on their membrane surface, and present it to the T cell in the context of major histocompatibility complex class II (Ia) molecules. Recent evidence supports the hypothesis that peptide antigens associate directly with Ia molecules on the APC surface membrane. However, the characteristics of other APC membrane structures potentially involved in antigen presentation are not entirely clear. Previous studies in our laboratories identified a guinea pig macrophage membrane-bound, non-Ia-containing antigenic complex (peak A) formed upon incubation of APC with the octapeptide antigen angiotensin (AII). This complex was capable of stimulating AII-immune guinea pig T cells and thus appeared to contain the immunologically relevant form of the antigen. For this reason it was important to establish whether such complex formation with peptides occurs with other cell types and with other peptide antigens. In the present study we found that other types of cells are also capable of forming such a membrane complex with antigen (peak A) and that this event is not unique to AII. Two other peptides, alpha-melanocyte-stimulating hormone and human fibrinopeptide B, both of which are antigenic in mice, were found to form peak A with a number of murine cell lines. As in our earlier studies with guinea pig macrophages, there was no evidence from these experiments for a role for major histocompatibility complex Ia antigens in the peptide binding observed. Differences in both the amount of peak A formation and the pattern of peptide antigen degradation were found from cell line to cell line for a given peptide, and from peptide to peptide for a given cell line, suggesting cellular heterogeneity in peptide processing and retention. In addition, cross-inhibition studies indicated that there was peptide specificity in the formation of peak A perhaps suggestive of molecular heterogeneity in the structure of peak A. These results indicate that there may be several types of cell surface molecules that specifically bind and retain peptide antigens.(ABSTRACT TRUNCATED AT 400 WORDS)     

Macrophage-lymphocyte interaction: antigen-independent binding of guinea pig lymph node lymphocytes by macrophages.

The antigen independent binding of guinea pig lymph node lymphocytes by glass-adherent macrophages was investigated. Binding was found to be mediated by a trypsin digestible, divalent cation-dependent, temperature-sensitive macrophage receptor mechanism that was not competitively inhibited by excess immunoglobulin. Data are presented to indicate that in the absence of antigen, macrophages were capable of binding both thymus-derived and bone marrow-derived lymphocytes without apparent selectivity, and further, that the binding of neither cell was mediated by surface membrane-associated immunoglobulin.     

The role of lymphoid cells in antibody-induced suppression of the fourth component of guinea pig complement

Many laboratories have demonstrated that immunoglobulin production by B cells is controlled by networks of interacting lymphocytes and their products. Our laboratory has demonstrated that complement components produced by macrophages are also regulated by networks of interacting cells and humoral factors. Treatment of mice in vivo or guinea pig cells in vitro with anticomponent antibody specifically inhibits synthesis and secretion of the component by macrophages. We have further characterized the cellular basis for in vitro suppression of the fourth component of guinea pig complement. C4 suppression has been accomplished with dispersed spleen cells as well as intact splenic fragments. This facilitated examination of the cells responsible for long-term C4 suppression. The data suggested that C4 suppression required either cell contact or sufficient concentrations of soluble factors. Long-term suppression of C4 depends upon a lymphoid cell contained in the spleen and in lymph nodes but absent or in insufficient concentration in the peritoneum. The lymphocyte that actively maintains suppression was negative for the guinea pig T-cell marker detected by the monoclonal antibody mc8BE6. Therefore, the critical cell is either another T-cell subset or non-T lymphocyte. These data demonstrate that a network of interacting cells analogous to that proposed to regulate antibody synthesis is also involved in regulating some nonlymphoid cell products.     

Studies on the effect of soluble lymphocyte products (lymphokines) on macrophage physiology. I. Early changes in enzyme activity and permeability.

Various cytochemical techniques have been used to quantitate the rapid effect of a partially purified, soluble product from lymphocytes (lymphokine) on normal guinea pig macrophages in vitro. Early changes in the utilisation of hydrogen liberated from the hexose monophosphate shunt and on cellular permeability were observed. The ability of the lymphokine to alter hydrogen utilisation was also seen in experiments on cryostat sections of guinea pig liver, suggesting that the cytochemical effects were not predetermined by changes at the membrane level. It is suggested that lymphokine-induced changes within the cell may reduce some biosynthetic activity affecting the cell membrane and this may in part reflect the decreased migrating ability of the cells. Increases in NADPH oxidation after lymphokine contact are discussed in relation to the bactericidal capacity of the cells.     

Dynorphins Modulate DNA Synthesis in Fetal Brain Cell Aggregates

Abstract: Previously, opioid peptide analogues, β-endorphin, and synthetic opiates were found to inhibit DNA synthesis in 7-day fetal rat brain cell aggregates via κ-and μ-opioid receptors. Here dynorphins and other endogenous opioid peptides were investigated for their effect on DNA synthesis in rat and guinea pig brain cell aggregates. At 1 µ M , all dynorphins tested and β-endorphin inhibited [³H]thymidine incorporation into DNA by 20–38% in 7-day rat brain cell aggregates. The putative ε-antagonist β-endorphin (1–27) did not prevent the effect of β-endorphin, suggesting that the ε-receptor is not involved in opioid inhibition of DNA synthesis. The κ-selective antagonist norbinaltorphimine blocked dynorphin A or B inhibition of DNA synthesis, implicating a κ-opioid receptor. In dose-dependency studies, dynorphin B was three orders of magnitude more potent than dynorphin A in the attenuation of thymidine incorporation, indicative of the mediation of its action by a discrete κ-receptor subtype. The IC₅₀ value of 0.1 n M estimated for dynorphin B is in the physiological range for dynorphins in developing brain. In guinea pig brain cell aggregates, the κ-receptor agonists U50488, U69593, and dynorphin B reduced thymidine incorporation by 40%. When 21-day aggregates were treated with dynorphins, a 33–86% enhancement of thymidine incorporation was observed. Because both 7- and 21-day aggregates correspond to stages in development when glial cell proliferation is prevalent and glia preferentially express κ-receptors in rat brain, these findings support the hypothesis that dynorphins modulate glial DNA synthesis during brain ontogeny.     

B lymphocytes in the guinea pig thymus are specifically localized in the central area.

Since the central area is an integral part of the guinea pig thymus, the cells in this area were compared with those in the thymic cortex and medulla in cryostat-sections by using methods for demonstration of E-, EA-, EAC-adherence and surface membrane immunoglobulins. In the extra cortical central area (ECCA) 15 to 25% of the lymphocytes showed EAC-adherence and 5 to 10% appeared to bear surface membrane immunoglobulins (SIg). In the lymph sinuses up to 70% of the lymphocytes were EAC- and SIg-positive. A small amount of EAC-adhering cells was present in the medulla of the central area. Cortical lymphocytes were EAC- and SIg-negative. From these results we conclude that in the guinea pig thymus B lymphocytes are specifically localized in the central area.     

Pronase treatment of lymphocytes reduces the time required for onset of S phase in response to anti-immunoglobulin

Murine B lymphocytes in the presence of antibody specific for surface membrane immunoglobulin begin to synthesize DNA at about the 36th hr of culture, although the onset of synthesis in response to other B cell-reactive mitogens occurs at approximately 18 hr. In contrast, the onset of DNA synthesis by Pronase-treated cells in response to anti-immunoglobulin required only 18 hr. This earlier onset of S phase was not observed when Pronase treatment was performed in the presence of ovalbumin or the protease inhibitors phenylmethylsulfonyl fluoride or aprotinin. It is unlikely that simple carryover of Pronase from the treatment procedure to the cell culture process was involved, because Pronase treatment for 1 hr at 3 degrees C rather than at 37 degrees C did not result in early onset of DNA synthesis. Cells treated with Pronase and then with mitomycin C to irreversibly inhibit their capacity to synthesize DNA were incapable of inducing early onset of S phase on co-culture with untreated cells, suggesting that Pronase may act directly on B cells rather than indirectly via other cells in the splenocyte population.     

Antigen recognition: the specificity of an isolated T lymphocyte population.

Guinea pig lymph node lymphocytes were separated into T and B cell fractions on immunoabsorbent columns. Separated cells were functionally distinct: T cells proliferated in response to ConA, PHA, soluble and alloantigen, whereas anti-Ig reagents only stimulated B cells. The in vitro proliferative response of guinea pig lymph node T lymphocytes was then shown to be highly discriminating when elicited by a series of structurally similar synthetic DNP-oligolysine antigens. Proliferation was always most extensive in response to the homologous, immunizing antigen, and less intense to cross-reacting DNP-oligolysines. Specificity of proliferation was maintained in the absence of both B lymphocytes and antibody secreting cells, suggesting that T cell recognition is not "acquired" from B cells or secreted antibody, but is a property inherent to the T cell.