Emil R. Unanue

The Regulatory Role of Macrophages in Antigenic Stimulation Part Two: Symbiotic Relationship between Lymphocytes and Macrophages

Publisher Summary Changes in antigen molecules that resulted in enhanced or decreased uptake by the phagocytes resulted in higher or lower immune responses, respectively. After the development of methodologies for obtaining live exudate cells rich in phagocytes and for pulsing these with antigen, the immune response to macrophage-associated antigens was possible to assay, using combinations of in vivo and in vitro methods. The presentation of antigen bound to live macrophages to the lymphocytes was a highly efficient mode of generating an immune response. In the marine system, the requirement for phagocytes or other acc|essory cells was such that as little as 1% contamination with phagocytes still enabled a T-cell proliferative response to develop. Thus, depletion procedures had to be extremely efficient. Macrophages are definitely involved in some response to many of the conventional polyclonal stimuli. Involvement of the macrophage may be by way of secreted active molecule. Fc fragments of Ig act as a polyclonal stimulant only after a processing of the fragment by the macrophages. The developments in macrophage biology over the past few years were not predicted till 1972.

The Regulatory Role of Macrophages in Antigenic Stimulation

Publisher Summary This chapter focuses on the role of the macrophage in removing antigen from extracellular fluids, degrading the larger part of this antigen while presenting a small part of it in persisting immunogenic form. This handling of antigen is done without contributing to the specificity of the immune response, which is determined by the antigenreactive T and B lymphocytes. During the process of uptake of antigen, macrophages appear to retain a few molecules of antigen, undegraded or with few chemical changes. Macrophage-associated antigen becomes an effective immunogenic stimulus mainly in conditions that require two types of lymphocytes to meet with antigen molecules. These lymphocytes are specifically antigen-committed and few in number. The positive immunogenic role of macrophages is related to their capacity to (1) remove extracellular antigen, which might be capable of interacting with and eliminating isolated T or B lymphocytes and (2) retain antigen in lymphoid tissues and promote its necessary meeting with both T and B cells.

Membrane and Cytoplasmic Changes in B Lymphocytes Induced by Ligand-Surface Immunoglobulin Interaction

Publisher Summary This chapter discusses membrane and cytoplasmic changes in B lymphocytes induced by ligand–surface immunoglobulin (Ig) interaction. Surface Ig is the receptor for antigen molecules on B lymphocytes. Surface Ig has been identified on the B-cell surface by using immunocytochemistry or direct biochemical analysis. The number of Ig molecules presumably serving as antigen receptors on the B cell surface is on the average of 105 molecules per B cell. The amount of surface-bound Ig is large comparing to the number of hormone receptors needed to trigger certain cellular responses. One could speculate that many of the surface Ig–antigen interactions result in nonfunctional events; therefore, many receptors are needed. The B cell requires such a high number of receptors to effectively bind antigen molecules of large size and variable epitope densities or a number of successive hits are needed in these cells to reach an effective threshold of stimulation. The distribution of surface Ig and other surface macromolecules has been studied at the ultrastructural level by using various methods. One method was the ultrastructural analysis of regular thin section of cells exposed to antibodies conjugated to a large visible molecule.

The Regulation of Lymphocyte Functions by the Macrophage

Macrophages may exert a regulatory influence at various stages in the life of the lymphocyte - they may influence the non-antigen-driven differentiation of lymphocytes - as exemplified by the effects on thymic differentiation; they may establish the mode and form of antigen to be presented or recognized by the lymphocyte; may regulate the lymphocytes antigen-driven functions. Each of these critical regulatory steps needs explaining in molecular terms and integrated and placed in the context of the other regulatory functions of lymphocytes. The control of secretion of MP is an eloquent example of the molecular complexities and the intricate congrol mechanisms - internal and external - operating at each step of each regulatory process. A final comment concern the question of macrophage heterogeneity. Is the same cell performing all the functions of degradation, presentation, and secretion - or cytotoxicity? Or do we have subpopulation, each with a different role? This issue is not settled. The unitarians argue that the phagocytes pass through different stages of differentiation and that each function may become more or less prominent at each stage. Certainly, the manner in which each macrophage function is assayed can condition the outcome: for antigen presentation, one adds 1 % of macrophages to cultures of spleen cells; for cytotoxic assays, the figure is 50 to 100 macrophages per tumor cell! It is our feeling that until such time as membrane molecules are identified and used as probes for differentiation or for identification of subsets we will not resolve this issue. Along these lines, macrophages have been found to have Ia antigens (Hämmerling et al. 1975, Schwartz et al. 1976) and can be divided into two sets on the basis of the presence or absence of Ia. Dorf and I have found - by cytotoxicity - that only about 35 to 50% of peritoneal macrophages bear Ia molecules (Dorf & Uanue 1977). Exceptionally, some exudates will bear up to 75% positive cells. Neither Ia-positive nor Ia-negative macrophages change significantly after prolonged periods of culture. Whether these results indicate two defined subsets of macrophages is now being investigated.

The binding of clathrin triskelions to membranes from coated vesicles

We have developed a quantitative method to study the interaction of radiolabeled clathrin triskelions with membranes from brain coated vesicles. Clathrin triskellions do not bind to coated vesicles unless these are stripped of their clathrin coat. The binding of triskelions is of high affinity (KD congruent to 2 x 10(-9) M), is saturable and depends on vesicle concentration. Triskelions bind to protein-sensitive structures of the stripped vesicles. Polypeptides of about 110,000 daltons appear to be involved in the binding structure. The association of triskelions to stripped vesicles was confirmed by electron microscopy.

Spontaneous T-cell lymphokine production and enhanced macrophage la expression and tumoricidal acitivity in MRL-Ipr mice

Abstract Three macrophage functions were studied in MRL-Ipr mice with autoimmune lymphoproliferative disease: surface expression of I-region-associated (Ia) antigens, tumor cytotoxicity, and interleukin-1 (IL-1) production. MRL-Ipr mice had a significantly increased representation of Ia-positive macrophages in the peritoneal cavity, compared to all normal strains of mice. In order to study the basis of this increase, thymocytes or splenocytes from MRL-Ipr mice were transplanted intraperitoneally into normal mice. Three days later the recipient mice had peritoneal exudates rich in Ia-positive macrophages. The cells which induced this response were T cells which elaborated a lymphokine responsible for the recruitment of Ia-positive macrophages. In previous studies from our laboratory using normal mice, lymphokine was secreted only following the interaction of immune T cells with antigen. The resident macrophages of MRL-Ipr mice were activated and killed tumor cells if triggered by an interaction with bacterial products, even without the addition of lymphokines. Secretion of IL-1 was normal. Our results indicate that the diseased MRL-1pr mice are characterized by (i) activated T cells that spontaneously secrete macrophage stimulatory molecules; and (ii) activated macrophages that show both an increased expression of Ia and lymphokine-independent triggering of tumoricidal activity.

B lymphocyte biology studied with anti-Ig antibodies.

One of the main areas of research in our laboratory is the study of B cell activation by ligands. We have focused our work on the function of membrane immunoglobulin (Ig) because, as the antigen-binding receptor, it is the initial molecule involved in modulating antigen-specific proliferation and differentiation. In view of the paucity of specific antigen-binding cells, even after enrichment procedures, most of our experiments on B cell activation have used anti-Ig antibodies as a polyclonal ligand specific for membrane Ig. When a ligand has bound a receptor protein, this event is transmitted to the cell interior, where cytoplasmic structures initiate the biochemical pathways underlying the cell response. A receptor protein can trigger cytoplasmic processes in a limited number of ways: it can produce a structural membrane change that alters ion conductance, resulting in the modification of cell physiology through ion-dependent processes; or it can activate local enzymes that alter the properties of key cytoplasmic proteins either directly, through covalent modification (such as phosphorylation or methylation), or indirectly, through the generation of biologically active messenger products (such as cyclic nucleotides or prostaglandins). Depending upon a number of conditions, the leceptor-ligand interactions can modify the physiological activity of the cell, as well as its growth and differentiation.

Accessory cell requirement in the proliferative response of T lymphocytes to hemocyanin

Abstract T cells were obtained from mice immunized to keyhole limpet hemocyanin (KLH) and tested in culture for their proliferation to KLH or to concanavalin A. The T-cell proliferation to KLH depended on the presence of Ia-bearing macrophages. This was shown only after extensive manipulations of T cells depleting accessory cells by adherence to dishes and passage through nylon-wool columns. The macrophage dependency required homology with macrophages from strains of mice sharing the left side of H-2. The macrophage dependency was not bypassed by conditioned media from macrophages. In contrast, the proliferative response to concanavalin A, which was also macrophage dependent, showed no H-2 restriction and could be replaced by a macrophage-conditioned medium. Nonadherent spleen cells, extensively devoid of macrophages, could substitute for macrophages but at a hundredfold excess. Cells isolated in the fluorescence-activated cell sorter with anti-Ig reagents also showed an antigen-presenting function. We raise the question whether our results imply an antigen-presenting function of B cells.

The disruption of immunoglobulin caps by local anesthetics

Abstract Surface complexes of Ig-anti-Ig (Ig, immunoglobulin) readily redistribute into caps. Local anesthetics (lidocaine and chlorpromazine) disrupt the caps, the complexes disseminating over the entire surface. Removal of the anesthetic then allows the complexes to reform into caps. The effects of the anesthetics on cap disruption were counteracted by an increase in extracellular Ca2+ and were not energy dependent. The inhibition of cap formation by anesthetic is also counteracted by increased extracellular Ca2+.

Redistribution and Fate of Ig Complexes on Surface of B Lymphocytes: Functional Implications and Mechanisms

This is a brief review of our recent work dealing with immunoglobulin (Ig) receptors on B lymphocytes. It emphasizes our research on the topography of surface Ig molecules and on their redistribution and fate after combination with a suitable Iigand; and discusses the possible relationship of these events with the stimulation of the lymphocyte. As a result of studies involving Ig and other surface molecules, a picture starts to develop concerning the distribution and fate of different immune complexes on eell surfaces. These results will obviously be of importance when studying complexes involving receptor Ig and various forms of antigen.