Immunologic specificity of antigen-binding T cell-derived factors that transfer mast cell-dependent, immediate hypersensitivity-like reactions.
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Biomedical subjects
W Ptak
Publications and source records attributed to W Ptak.
Alleviation of IgE-mediated immune reactions in hypoinsulinaemic and hyperglycaemic mice.
Alloxan diabetic mice do not easily develop anaphylactic shock, and formation of IgE antibodies to sensitizing antigen almost ceases in these animals. Also, mice sensitized as normoglycaemic and then made diabetic are to a great degree protected from anaphylaxis, although they form minute but measureable amounts of IgE antibodies. Immune cells transferred into diabetic mice lose their ability to form IgE, while cells from immune diabetic mice, which in the hypoinsulinaemic milieu of donors do not synthesize appreciable amounts of IgE, regain this ability upon transfer into normal recipients. We conclude that the IgE response is very insulin-dependent and that insulin deficiency affects IgE-forming cells directly or indirectly via its influence on T helper cells. In the current study we also considered whether hyperglycaemia may influence the effector stage of anaphylactic reactions. To test this, massive doses of glucose were given to normoglycaemic mice, which increased their blood glucose level to that seen in diabetic mice and prevented local anaphylactic reactions when these mice were injected with monoclonal IgE antibody and challenged locally with antigen.
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Defective elicitation of delayed-type hypersensitivity in W/Wv and SI/SId mast cell-deficient mice.
Previous studies have indicated that cutaneous mast cells are involved in the elicitation of delayed-type hypersensitivity (DTH) in mice. Mast cells are thought to be required in DTH to release serotonin to open gaps between endothelial cells, allowing entrance of effector cells into the tissue. Two different strains of mice with independent genetic defects that lead to a substantial mast cell deficiency (W/Wv and SI/SId), and their normal littermate +/+ controls, were studied for their ability to express DTH. Both strains were shown to be deficient in serotonin-containing mast cells at skin sites of preferential elicitation of DTH in normal mice, such as the ear or footpad. Defective DTH was found in both mast cell-deficient strains by using two different systems: 1) sheep erythrocyte-induced footpad DTH, and 2) picryl chloride-induced contact sensitivity ear swelling responses. Adoptive transfer experiments demonstrated that abnormal DTH in mast cell-deficient mice was due to a defect in the elicitation of DTH, rather than a defect in the induction of effector T cells. In these experiments, the ability to elicit DTH could be transferred to normal +/+ mice with sensitized cells from mast cell-deficient mice, but sensitized cells from +/+ mice could not transfer DTH responsiveness to mast cell-deficient mice. In addition, no defects in numbers of epidermal Langerhans cells or in antigen-presenting cell function were found in W/Wv or SI/SId mice. We therefore concluded that abnormal elicitation of DTH in W/Wv and SI/SId mice was probably due to their mast cell deficiency. The inability of mast cell-deficient mice to express DTH was overcome when sensitized T cells and specific antigen were placed in the extravascular tissues by local passive transfer. These results suggest that mast cell release of vasoactive mediators, such as serotonin, is required in DTH to allow effector T cells to leave the intravascular space, enter the tissues, and become activated by antigen to release chemoattractant lymphokines that recruit a nonspecific infiltrate of inflammatory cells.
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Purification and characterization of TNP-specific immunoregulatory molecules produced by T cells sensitized by picrylchloride (PC1F).
T cell-derived TNP-specific factors associated with immunoregulatory activity were obtained by culture of T cells obtained from mice sensitized by skin-painting with picrylchloride. Culture medium was absorbed to TNP-Sepharose and TNP binding proteins were prepared by elution with TNP. The hapten affinity-purified proteins were characterized by size and charge and were found to be acidic 70,000 m.w. polypeptides that occur as monomers or oligomers. Oligomeric proteins interact with factors produced by mice injected with trinitrobenzenesulfonic acid to form factors that suppress specifically the ability of TNP-sensitized T cells to transfer contact sensitivity to TNP. Monomeric (no more than 70,000 m.w.) molecules do not form suppressor factors but can transfer contact sensitivity to TNP. Moreover, reduction and alkylation of oligomeric molecules inactivates their suppressor activity but causes them to be able to transfer contact sensitivity. The results suggest that T cell-derived antigen-specific molecules may have different effector functions dependent on their oligomeric state.
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Specialized antigen-presenting cells. Splenic dendritic cells and peritoneal-exudate cells induced by mycobacteria activate effector T cells that are resistant to suppression.
We have tested the ability of several types of trinitrophenyl (TNP)-labeled Ia+ cells to induce contact hypersensitivity (CS) after intravenous injection. Most labeled cell types (spleen cells, splenic macrophages, various types of peritoneal-exudate cells) not only fail to induce CS after this type of inoculation but, rather, activate T suppressor cells leading to specific immunological tolerance. Occasionally, some of these immunizing cells managed to bypass the T suppressor system and induced CS. In those cases the response was short-lived and could be blocked by concomitant injection of trinitrobenzelsulphonic acid (TNBS), a potent inducer of T suppressor cells. In sharp contrast to these results, TNP-labeled splenic dendritic cells and TNP-labeled peritoneal-exudate cells induced by complete Freund's adjuvant had the following distinctive features: (a) They were always able to sensitize when injected intravenously, and the degree of sensitization they produced was roughly equivalent to that achieved by cutaneous application of picryl chloride, the chemically reactive form of TNP. (b) The response they elicited was long lived (i.e., lasted for greater than 3 wk). (c) Their sensitizing capacity could not be blocked by the concomitant injection of TNBS. (d) They elicited a response that could be adoptively transferred to untreated, normal recipients. These results indicate that the type of cell that first presents antigen to the immune system plays an important, even essential, role in determining the strength and duration of the subsequent immune response. In particular, the results suggest that some special antigen-presenting cells can induce a response that is relatively resistant to host suppressor mechanisms. Evidence that they do so by activating contrasuppressor cells is discussed.
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Transfer of an antigen-specific immediate hypersensitivity-like reaction with an antigen-binding factor produced by T cells.
The fact that T cell-dependent activation of mast cells occurs in delayed-type hypersensitivity led us to investigate whether a T cell product could mimic some of the functions of IgE. We report that 24- or 48-hr cultures of T cells from mice immunized optimally for delayed-type hypersensitivity resulted in release of an antigen-binding factor that transferred the ability to elicit an antigen-specific immediate hypersensitivity-like skin reaction in normal recipients. The responsible factor was concentrated and purified by affinity chromatography on antigen columns and was distinguished from immunoglobulin by several criteria: (i) it was released by purified T cells (anti-immunoglobulin plate depletion of B cells); (ii) it expressed no known antigenic markers of immunoglobulins (enzyme-linked immunosorbant direct binding assay); (iii) it had a molecular weight of 70,000 or less (sucrose gradient ultracentrifugation); and (iv) it had serological markers associated with antigen-specific T cell factors from other experimental systems. We suggest that, at sites of delayed-type hypersensitivity, antigen-reactive T cells may release antigen-specific factors that lead to mast cell activation and release of vasoactive amines, which is required for elicitation for these responses.
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Interactions between molecules (subfactors) released by different T cell sets that yield a complete factor with biological (suppressive) activity.
T cells that have been immunized to express optimal levels of contact hypersensitivity upon adoptive transfer to normal animals can be inhibited from doing so by incubating them with an antigen-specific T suppressor factor. This factor is composed of at least two subunits which come from cells expressing different Ly phenotypes; an antigen-specific antigen-binding "subfactor" is made by an Ly-1 cell and a non-antigen-binding one is made by an Ly-2 cell. Neither of these cells nor their products express detectable amounts of major histocompatibility gene products. The mode of immunization plays an important role in determining which of these subfactors will be produced. Painting the skin with a reactive hapten immunizes Ly-1 cells that secrete antigen-binding material, whereas intravenous injection of trinitrobenzenesulfonic acid activates Ly-2 cells to produce a second subunit that does not see antigen. There is reason to believe that the molecule that does not bind to antigen does have some antigen specificity. An analysis of the data at hand suggests that the antigen specificity stems from an interaction of the two subunits described with yet another subunit and that biological activity is dependent upon three macromolecules. Thus, the complex level of cellular interactions that regulate immunity may also be reflected in a similar type of complexity in the interaction between their biologically active cell-free products.
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Immunological agnosis: a state that derives from T suppressor cell inhibition of antigen-presenting cells.
The biologically active mediators of antigen-specific T suppressor cells can combine with antigen on cells that are specialized to present antigen (APC) and render these APC incapable of presenting not only the specific antigen that the product of the T suppressor cell sees but also any other antigen in or on the APC. Thus, antigen-bearing suppressed APC fail to activate either the helper or suppressor system involved in the regulation of contact sensitivity responses. These results demonstrate that APC are targets of T suppressor cells. They also imply that a metabolic event is required for functional antigen presentation and that T suppressor cells can block that metabolic pathway.
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Immunoregulatory circuits which modulate responsiveness to suppressor cell signals: contrasuppressor cells can convert an in vivo tolerogenic signal into an immunogenic one.
The intravenous injection of 2,4,6-trinitrophenyl (TNP)-labeled peritoneal exudate cells (TNP-PEC) into CBA mice fails to produce a state of hypersensitivity; rather, it renders recipient mice incapable of mounting a contact hypersensitivity response when they are subsequently immunized with a reactive form of the specific hapten. However, if precultured neonatal spleen cells are injected along with the cells that induce tolerance (TNP-PEC), not only is the development of tolerance inhibited but sensitization to TNP develops. The neonatal spleen cell responsible for turning the tolerogenic signal into an immunogenic one is I-J+ and adheres to the Vicia villosa lectin. Thus, it expresses markers that distinguish contrasuppressor effector cells from helper cells (D. R. Green et al., Eur. J. Immunol. 1981. 11:973), indicating that activated contrasuppressor cells can act as potent, helpful regulatory cells in vivo.
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Ease of induction of cells producing TNP-specific suppressor factor in hypoinsulinemic mice.
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Characterization of T-cell surface proteins bound by heterologous antisera to antigen-specific T-cell products.
Heterologous antisera specific for murine T-cell antigen-recognition molecules were prepared by immunization of rabbits with dinitrophenyl-specific murine T-cell suppressor factors that had been purified by hapten-affinity chromatography. The antisera (i) bind to antigen-specific T-cell products that differ in their antigen-recognizing specificity; (ii) absorb the specific suppressor activity in preparations containing suppressor factors; (iii) stain all Lyt2+ T cells brightly in indirect immunofluorescence examination, stain some Lyt1+ cells (with low intensity), and do not stain B cells; (iv) precipitate cell membrane proteins from T cells that bear striking structural resemblance to the antigen-specific molecules used for immunization. These results suggest that, like B cells, there is a commonality between antigen-specific effector molecules released by T cells and their membrane-associated receptors.
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Isolation and partial characterization of an antigen-specific T-cell factor associated with the suppression of delayed type hypersensitivity.
Antigen-specific factors associated with immunosuppressive activity, released by cultured T cells from mice tolerant to the haptens trinitrophenyl, dinitrophenyl and oxazolone, were purified by hapten affinity chromatography. Their binding specificity for antigens paralleled their immunoregulatory activity. Like some immunoglobulin molecules, these factors had blocked NH2 termini and could be bound to Fc-like receptors on macrophages. However, neither immunoglobulin constant region determinants (isotypes) nor antigens encoded by the major histocompatibility complex were detected on the suppressive factors. The purified factors occurred as 68,000-dalton proteins and non-covalently linked dimers. No associated immunoglobulin light chain molecules were detected. The factors showed a marked propensity toward degradation with major breakdown products of 45,000-50,000 and 25,000-30,000 daltons. These results suggest that these molecules are the T-cell products analogous to B-cell immunoglobulin (equivalent to heavy chains) and that they may be the antigen-specific components which act in conjunction with major histocompatibility-controlled gene products to perform antigen-specific suppression.
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Inhibition of contact sensitivity by macrophages.
Lymphoid cells of mice injected with picrylsulphonic acid and then painted with picryl chloride produce a specific T suppressor factor (TSF) in vitro. This factor arms peritoneal exudate cells, which then produce a nonspecific factor which inhibits the transfer of contact sensitivity by immune cells incubated in it. An adherent, theta-negative cell, which is presumably a macrophage, is responsible. This justifies the use of the term macrophage suppressor factor. As a separate phenomenon, passive transfer cells lose their activity when incubated on high density monolayers of normal peritoneal exudate cells. However, this is not associated with the production of a supernatant factor. The inhibition of transfer when immune cells are incubated with specific TSF is unaffected by nylon wool filtration (which removes macrophages). This suggests that TSF is able to depress the passive transfer of contact sensitivity by a macrophage-independent process.
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Macrophage function in alloxan-diabetic mice: expression and activity of Fc receptors.
The expression of Fc receptors (FcR) on macrophage surfaces is dependent on the in vitro insulin level. Macrophages (Mø) of alloxan-diabetic animals have more FcR and phagocytose heavily opsonized sheep erythrocytes (SRBC) better, than normal Mø. The reverse is, however, true when suboptimally opsonized SRBC are used. No differences were found between normal and diabetic macrophages in the rate of catabolism of engulfed antigen. We regard it likely that the generation and/or transmission of Fc-dependent signal from the cell surfaces may be impaired in hypoinsulinaemic environment.
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Role of antigen-presenting cells in the development and persistence of contact hypersensitivity.
Three outcomes pertinent to contact sensitivity (CS) follow immunization with various forms of trinitrophenylated (TNP) substrates: (a) specific immunological unresponsiveness for CS is induced when immunization favors activation of splenic suppressor cells. This state is achieved by intravenous injection of trinitrophenyl-conjugated to various types of cells, such as peritoneal exudate cells (PEC). (b) A short-lived or evanescent form of CS is induced when immunization reduces activation of the suppressor circuit. This can be achieved by subcutaneous immunization with trinitrophenyl conjugated to syngeneic PEC, by pretreatment with cyclophosphamide to diminish suppression before intravenous immunization, or by altering the mode of antigen presentation by using TNP-substrate that has undergone phagocytosis. (c) A long-lived form of CS is induced when trinitrophenyl is presented to the immune system on skin cells either by contact skin painting with reactive trinitrophenyl, or by subcutaneous, or even intravenous injection of trinitrophenyl-conjugated epidermal cells. In fact, trinitrophenyl-conjugated epidermal cells induced CS even when the suppressor circuit was activated by intravenous coadministration of TNP-PEC. This implies that antigen presentation on epidermal cells induces sensitized cells that are relatively resistant to suppression. The cell type(s) in the skin that are primarily responsible for this potent form of antigen presentation are most likely Langerhans cells, because they can be concentrated by virtue of their Fc receptors and they are Ia positive. Thus, both the anatomical site where antigen is first encountered by the immune apparatus, as well as the nature of the cells which present the antigen, determine whether a CS response will ensue, as well as whether it will be evanescent or long-lasting.
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Development of specific suppressor cells in hypoinsulinaemic mice.
Mice and rats injected with alloxan or streptozotocin develop permanent diabetes, characterised by deficient insulin production. It has been demonstrated that hypoinsulinaemia in mice leads to significant loss of lymphatic tissue, and these diabetic animals cannot develop contact sensitivity or efficient graft rejection. Administration of insulin partially restored these responses and also caused an increase in the weight of the thymus and spleen. Similar suppression of T cell-dependent phenomena has been observed in surgically pancreatectomised rats. Lymphocytes of these hypoinsulinaemic animals show significantly decreased in vitro responses to plant lectins and generate only low levels of cytotoxic effector cells. We previously showed that cells of normoglycaemic oxazolone-sensitised mice cannot transfer significant contact sensitivity reactions into diabetic recipients indicating that the milieu of hyperglycaemic insulin deficient animals cannot support all the activity of immune T cells. By mixing immunised T cells from control and diabetic mice and transferring the mixtures into normal recipients we now show that the non-supportive millieu in diabetic animals may be due to active suppression rather than to athrepsis.
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Induction of suppressor cells and cells producing antigen-specific suppressor factors by haptens bound to self carriers.
Injection of TNP-, DNP- or oxazolone-substituted syngeneic cells into mice causes the development of hapten-specific T suppressor cells which prevent the animals from being activity sensitized with homologous hapten. These cells injected together with immunized cells abrogate the latter's ability to transfer passively the contact sensitivity (CS) reaction into normal recipients. T lymphocytes from animals made unresponsive and sensitized with homologous hapten synthesize in vitro antigen-specific suppressor factors (SF) which when incubated with immune lymphocytes prevent them transferring adoptively the CS reaction. The type of cell used to induce suppression or production of suppressor factor (haptenated erythrocytes, thymocytes or macrophages) is not critical suggesting that a hapten-substituted common membrane structure is recognized as a tolerogen. The present work demonstrates that while the specific unresponsiveness induced by cell-bound hapten in vivo is long lasting, cells from tolerized animals are able to suppress the immunized cells in passive transfer or produce in vitro antigen-specific suppressor factors only when tested several days after tolerization.
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Cell interactions that regulate the T-cell dose-response profile to concanavalin A.
We have tested the ability of structures on macrophage (M phi) membranes (M phi-MEM) and on several other types of cells with Fc receptors to affect the DNA synthetic response of concanavalin-A-stimulated T cells. Of the cells tested, only M phi-MEM have the capacity to relieve the suppression produced by supra-optimal doses of the mitogen. The M phi-MEM do not increase Con A responses by altering the stimulatory capacity of the Con A. These observations, analysed together with previous results, which have indicated that live intact M phi are required for the transfer of information between lymphocyte sets and that M phi-MEM preparations can act as competitive antagonists for this function, are interpreted as follows: some supraoptimal doses of Con A activate suppressor cells, which are responsible for limiting the DNA synthetic response to the mitogen; the M phi-MEM abrogate this suppression by absorbing the signal that activates the suppressor cell. Kinetic studies suggest that the M phi-MEM do not affect the activity of already activated suppressor cells. We also found that Con A usually activates two separately responding T-cell populations with different sensitivities to dose and to time of contact with mitogen and that the suppression of both populations can be relieved by M phi-MEM. These results support the notion that the overall immunological circuit is composed of multiple independently regulated mini-circuits, with M phi acting as transmission posts for intra- and perhaps also inter-circuit communication.
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