Activation in vivo of a major antisuppressor T-cell pathway immediately after immunization. III. T-cell requirements for its induction

Immunogenic stimuli rapidly induce a potent mediator with antisuppressor activity which represents complexes of Ig and antigen. The formation of the complexes depends on the interaction of two T cells both of which bear the Ly1 phenotype. The two T cells can be separated on the basis of their sensitivity to antilymphocytic serum and dependency on the presence of thymus. T cells bearing I region coded determinants are essential for the formation of the mediator.     

The role of macrophages in the generation of T helper cells. V. Evidence for differential activation of short-lived T1 and long-lived T2 lymphocytes by the macrophage factors GRF and NMF.

The generation of T helper cells in vitro requires macrophages or macrophage-derived factors such as genetically related macrophage factor (GRF) or nonspecific macrophage factor (NMF). However, there is a basic difference of T helper cell induction when using particulate antigens. The present study demonstrates that this difference is based on the activation of two different T cell subsets. GRF activates short-lived 'T1' cells which amplify the induction of T2 cells, which are the helper cell precursors. Thus, the genetic restriction of T helper cell induction seen with soluble antigen or GRF lies on the level of macrophage or GRF interaction with T1 cells. NMF (or macrophages) and particulate antigens directly activate the helper cell precursor (T2) indicating no requirement for T1-T2 cooperation. The direct activation of the helper cell precursor with particulate antigens does not require histocompatible macrophages or NMF from histocompatible macrophages. The present results may explain some of the discrepancies reported in the literature concerning the genetic requirements and specificity of T cell activation.     

Activation in vivo of a major antisuppressor T-cell pathway immediately after immunization. II. T-cell requirements for its expression

Immunogens activate in vivo within 3-6 hr after injection a new and hitherto unrecognized T-cell pathway which interferes with T-cell suppression, therefore called antisuppression. An important soluble mediator with antisuppressor activity is detected in the serum of immunized animals within 3-6 hr. The mediator represents a unique form of complexes of Ig and antigen. The antisuppressor function of the complexes does not represent a direct "neutralizing" effect of the complexes on the effector T suppressor cells. The antisuppressor complexes activate an Ly2+ T cell which, with the interaction of an Ly123+ T cell, blocks completely T-suppressor-cell function. The biological significance of the T antisuppressor pathway is discussed.     

Induction in vivo of enhancing factors for antibody production: II. Ig-Antigen complexes formed within 6 hours after immunization markedly enhance antibody formation

Serum collected 6 hr after injection of SRBC contains helper factor(s) which enhances antibody formation. The factor which markedly enhances the 7s response is an Ig-Ag complex. The complexes contain Ia antigens as shown by the ability of proper immunoadsorbents to remove both the antibody-enhancing activity for 7s, as well as the “cytophilic Ig” which is taken up by T cells. The enhancement of 19s is due either to a different factor which contains neither Ig nor antigen, or to the same Ig-Ag complexes which act at lower concentrations. Both 19s and 7s enhancing activities show no H₂ restriction in the presence of T cells. However no antibody enhancement was obtained when allogeneic 6HS was injected in thymectomized, lethally irradiated, and bone-marrow-reconstituted Balb/c mice, which suggests that the presence of T cells overcomes the H₂ barrier which exists when the complexes act directly on B cells. Since the complexes are formed through the mediation of a T-cell factor they may be considered a T-cell replacing factor and are predominantly concerned with the regulation of 7s response.     

Antigen handling by guinea pig macrophages: further evidence for the sequestration of antigen relevant for activation of primed T lymphocytes.

Guinea pig macrophages can take up sufficient 2,4 dinitrophenyl guinea pig albumin during a brief in vitro exposure at 37 degrees C to trigger proliferation and lymphokine production with primed T lymphocytes on subsequent co-culture. Treatment of such antigen-bearing macrophages with trypsin, a procedure which removes surface antigen, does not alter the ability of such macrophage to initiate the release of migration inhibition factor from sensitized T lymphocytes. In addition, formation of antigen-specific rosettes between primed T cells and antigen-bearing macrophages is not blocked by high concentrations of antibody directed against the antigen mediating this interaction. Similarly, primed T lymphocyte DNA synthesis induced by antigen-bearing macrophages is not inhibited by specific antibody to that antigen. These data support the conclusion that the fraction of macrophage-associated antigen which is relevant to T lymphocyte activation does not reside on the macrophage surface but rather remains in a restricted compartment from which it is accessible to the T cell but unavailable to either blockade by specific antibody or removal by proteolytic enzymes.     

Mechanism and specificity of macrophage-mediated cytotoxity.

The cytotoxic effect of macrophages derived from alloimmunized mice (immune macrophages) was found to be immunologically specific. The immune macrophages killed only target macrophages carrying the alloantigens used for immunization in mixed macrophage cultures (MMC) under optimal conditions of contact between effector and target cells. T-sensitized lymphocytes, but not B cells, were capable of arming nonimmune macrophages and conferring upon them cytotoxic activity; the arming factor, which seemed to be a T mediator or T-cell receptor (membrane component) was removable by trypsin. Frequent rinsing or addition of hydrocortisone significantly decreased the cytotoxicity of the MMC. Pretreatment of peritoneal cells with anti-θ antisera and complement markedly decreased immune macrophage cytotoxic activity. It is suggested that the presence of a very small number of T-sensitized lymphocytes is required for strong cytotoxic activity to be manifested by the macrophages.     

Macrophage-T cell interactions. IV. A new receptor on T cells: Ia antigens as receptors for Ig-antigen complexes which appear early after immunization.

The Fc-receptor-positive T cells have been shown to bear a new receptor which is involved in the uptake of cytophilic complexes of Ig and antigen which are detected in the serum of mice within 6 hr following immunization. It was shown that the Fc receptor on T cells is not necessary for the uptake of these complexes. This new receptor is labile during culture of the T cells in vitro and disappears together with the Fc receptors. The new receptor is taken up by the T cells from a macrophage supernate. The evidence presented suggests that the receptor for these complexes bears determinants coded by the I region of the major histocompatibility complex but is distinct from conventional Fc receptor in that it does not bind to Fc fragments of Ig.     

Human lung macrophage-derived histamine-releasing activity is due to IgE-dependent factors

Human lung macrophages obtained from surgical specimens spontaneously secreted a factor(s) (which we term macrophage factor) during 24-hr culture that induced calcium-dependent histamine release from human basophils and lung mast cells. Macrophage factor induced noncytotoxic histamine release from purified (85%) basophils. The kinetics of release were relatively slow and similar to that of anti-IgE. We performed a series of experiments to test the IgE dependence of macrophage factor-induced release. Preincubation of basophils with anti-IgE in calcium-free medium resulted in complete desensitization to macrophage factor-induced histamine release (i.e., when calcium and macrophage factor were added to the basophils, no histamine release occurred), and preincubation with macrophage factor in calcium-free medium resulted in partial desensitization to anti-IgE-induced histamine release. Pretreatment of basophils with pH 3.9 lactic acid buffer, which dissociates basophil IgE from its receptors, markedly reduced the capacity of basophils to release histamine in response to macrophage factor. Basophils that were incubated with IgE myeloma (but not with IgG) after lactic acid treatment partially or completely regained their capacity to release histamine in response to macrophage factor. Fluid-phase IgE myeloma (15 micrograms/ml) (but not IgG) inhibited basophil histamine release induced by two macrophage-derived supernatants, whereas IgE myeloma (200 micrograms/ml) did not inhibit release due to other supernatants. IgE-affinity columns removed the histamine-releasing activity of five macrophage-derived supernatants, and IgG-affinity columns had similar effects. However, neither affinity column removed the histamine-releasing activity of three other macrophage-derived supernatants. On Sephadex G-75 chromatography, nearly all of the histamine-releasing activity migrated as single peak with an apparent m.w. of 18,000. These results suggest that, although macrophage factor are heterogeneous, they are related, as they are a IgE-dependent factors that induce histamine release by interacting with cell surface IgE. These macrophage factors may be responsible for stimulation of basophil/mast cell mediator release in chronic allergic reactions.     

Macrophage migration stimulation factor, migration inhibition factor and the role of suppressor cells in their production.

Guinea pig lymph node lymphocytes and human peripheral blood lymphocytes when stimulated by specific antigen or mitogen will release factors that affect in vitro macrophage migration. Migration inhibition factor production appears to be under the control of suppressor cells which are T lymphocytes. When suppressor cells are generated by stimulation with Con A for 4 days, migration stimulation factor (M.St.F.) activity is found. In other situations where M.St.F. is found this is thought to be due to increased suppressor cell activity. For example, young adults produce this lymphokine when stimulated with Con A, whereas aged individuals produce MIF. Concanavalin A appears to be the mitogen of choice for M.St.F. production, and phytohemagglutinin for MIF production. The release of this putative factor M.St.F. from suppressor T cells helps to explain some of the difficulties that have existed in studies of macrophage migration inhibition.     

Adherent cell function in murine T lymphocyte antigen recognition. II. Definition of genetically restricted and nonrestricted macrophage functions in T cell proliferation.

The mechanisms by which adherent cells, presumably of mononuclear phagocytic lineage, influence in vitro antigen-specific activation of murine T lymphocytes was examined. Two distinct functions for macrophages could be discerned. One macrophage function is dependent on a soluble factor produced by cultured adherent cells and is most easily studied with complex multideterminant antigens. This factor is neither antigen-specific nor MHC-restricted in its action in that PEC, regardless of haplotype, produce factor in the absence of antigen. A second function, antigen-specific T cell activation, is seen when antigens of more restricted heterogeneity are used, such as those under the control of Ir genes. This latter activity demands identity or partial identity between the antigen-presenting cell and the primed T cell, thus suggesting an additional specific, genetically restricted function for macrophages in in vitro antigen recognition. Whether these adherent cell functions are mediated by all or distinct subsets of cells was not established.     

Leukocyte adherence inhibition response to murine sarcoma virus-induced tumors. II. Requirement for T-cell subpopulations and macrophages

Murine sarcoma virus (MSV)-immune T cells from C57BL/6 mice respond to intact RBL-5 tumor cells with the production of leukocyte adherence inhibition factor (LAIF), which mediates an adherence inhibition response of macrophages. LAIF is elaborated by isolated Lyt-2+ cells incubated with RBL-5 cells, whereas Lyt-1+ cells elaborate a substance that enhances macrophage adherence. Spleen macrophages or peritoneal exudate macrophages from MSV-immune mice when present at concentrations of 0.1% changed the response of Lyt-1+ cells from the formation of an adherence enhancing factor to the formation of an adherence inhibiting factor. Migration inhibition factor (MIF) was formed by Lyt-1+ cells, but not by Lyt-2+ cells under identical culture conditions. Addition of either spleen macrophages from mice with progressively growing tumors or tumor-infiltrating macrophages suppressed LAIF formation by both Lyt-1+ and Lyt-2+ cells. Tumor-infiltrating macrophages elicited an adherence enhancing factor from Lyt-2+ cells when present at high concentrations. The results suggest that the extent of macrophage adherence in vitro is the outcome of an interaction of macrophages with mediators that have opposing effects.     

Stimulation of macrophage H2O2 release by resident thymocytes: effect of a soluble factor distinct from interferon-gamma

Activated T cells are known to stimulate macrophage oxidative metabolism and antimicrobial activity through release of interferon-gamma (IFN-gamma). In contrast, the role of nonactivated T cells in regulating macrophage effector functions is less well defined. We have previously reported that a low molecular weight soluble factor derived from resident (nonactivated) thymocytes enhances macrophage receptor-mediated phagocytosis. In the present study, we examined the capacity of resident murine thymocytes to stimulate the respiratory burst and microbicidal activity of peritoneal macrophages. Macrophages cultured for 1-2 days with cell-free thymocyte supernatant (TS) released two to three times more H2O2 in response to PMA or opsonized zymosan than did control macrophages. The H2O2-stimulating factor in TS was distinguished from IFN-gamma by its heat stability (100 degrees C, 20 min), approximate MW of 2400 Da (gel filtration high-pressure liquid chromatography), and absence of interferon activity in both antiviral and enzyme-linked immunosorbent assays. TS-treated macrophages, however, did not exhibit a greater capacity to kill or inhibit the intracellular growth of Toxoplasma gondii, indicating that the thymocyte factor did not fully activate macrophage microbicidal mechanisms. These data suggest that thymocytes can increase the respiratory burst capacity of macrophages in the absence of antigen-specific immune responses.     

Transfer of agammaglobulinemia in the chicken. I. Generation of suppressor activity by injection of bursa cells

Spleen cells from adult agammaglobulinemic (bursectomized) chickens taken 1 to 3 weeks after an injection of histocompatible bursa cells can inhibit the adoptive antibody response to B. abortus of normal spleen or bursa cells in irradiated recipients. Spleen cells from Aγ chickens not injected with bursa cells generally do not. Moreover, bursectomized chickens which have been reconstituted with spleen cells within the first week after hatching do not respond with suppressor cell formation upon bursa cell injection. This apparent “autoimmunization” with bursa cells induces suppressor T cells which are only minimally sensitive to treatment with mitomycin C or to 5000 R γ irradiation. The suppressor activity is neither induced nor potentiated by concanavalin A in vivo. It is much stronger in spleen than in thymus cells and appears to be macrophage independent and to require intact cells. The cell component which stimulates the suppressor activity is more pronounced on bursa than on spleen cells, and is at most present to a very limited extent on bone marrow, thymus, or peritoneal exudate cells. It is better represented in comparable cell numbers of Day 17 than of Day 14 embryonic bursa. The inducing cell component is present in the membrane fraction of disrupted bursa cells. Immunization with bursa cells from B locus histoincompatible chickens leads to suppressor activity against histocompatible bursa cells. Although the removal of Ig-bearing cells from bursa greatly diminishes its immunizing capacity, injection of serum IgM and IgG does not induce suppressor cells. It is suggested that tolerance to a B-cell antigen is lacking in adult Aγ chickens, resulting in an autoimmune response upon exposure to B cells. The B-cell antigen may be a cell surface-specific form of Ig, a complex of Ig and a membrane component, or a differentiation antigen which appears simultaneously with Ig during ontogeny.     

T cell induction of membrane IL 1 on macrophages

We have studied the role of T cells in the induction of a membrane-associated form of interleukin 1 (mIL 1) in murine macrophages. T helper cell clones and a T cell hybridoma induced macrophages to express mIL 1 after an antigen-specific, Ia-restricted interaction. Induction of mIL 1 was proportional to antigen concentration and was increased in the early course of the response in macrophages pretreated in culture with interferon-gamma. mIL 1 activity was detectable 4 hr after interaction with T cells. mIL 1 induction was inhibited by antibodies to either class II molecules or the T cell receptor. Two pathways of T cell-mediated mIL 1 induction could be defined. In the first, T cells, whose protein synthesizing capacity was completely eliminated by pretreatment with the irreversible protein synthesis inhibitor emetine, induced levels of mIL 1 expression indistinguishable from controls. In the second, T cells stimulated by paraformaldehyde-fixed macrophages in the presence of concanavalin A or antigen secreted a soluble factor that induced macrophage mIL 1 expression. Thus, it appears that T cells may induce macrophages to express mIL 1 both by direct cell-cell contact mediated through binding of T cell receptor to the Ia/antigen complex, and through the release of a lymphokine after activation. This lymphokine does not appear to be IL 2, IFN-gamma, BSF-1, or CSF-1.     

B cell stimulatory factor-1 (interleukin 4) activates macrophages for increased tumoricidal activity and expression of Ia antigens

Macrophages are activated by lymphokines (LK) to kill tumor cell and microbial targets. Interferon-gamma (IFN) is the major LK activity in conventional, antigen or mitogen-stimulated spleen cell culture fluids for induction of these macrophage effector functions. In view of the recent demonstration that murine macrophage-like cell lines have receptors for B cell stimulatory factor-1/interleukin 4 (BSF-1), a possible role for BSF-1 in regulation of macrophage function was considered. In this communication, thioglycollate-elicited murine peritoneal macrophages were shown to express about 2300 high affinity (Ka approximately 2 X 10(10) M-1) BSF-1 receptors/cell. Peritoneal macrophages treated with purified, T cell-derived BSF-1 developed potent tumoricidal activity against fibrosarcoma target cells. The concentration of BSF-1 that induced 50% of maximal tumor cytotoxicity was 38 +/- 4 U/ml for seven experiments; similar dose-responses were observed with recombinant BSF-1. That BSF-1 dose-responses for induction of macrophage-mediated tumor cytotoxicity were not affected by 5 micrograms/ml polymyxin B suggested that contaminant endotoxins played little or no role in cytotoxic activity. BSF-1 alone (less than or equal to 500 U/ml) was not directly toxic to tumor cells or macrophages. Macrophage tumoricidal activity induced by BSF-1 but not by IFN was inhibited greater than or equal to 90% with monoclonal anti-BSF-1 antibody. BSF-1 induced Ia antigen expression on peritoneal macrophages and increased (twofold to threefold) FcR(II)-dependent binding of murine IgG immune complexes to bone marrow-derived macrophages (greater than 98% macrophages). Based on these findings, it was concluded that BSF-1 is a potent macrophage activation factor.     

Generation of T helper cells in vitro. IV. F1 T helper cells primed with antigen-pulsed parental macrophages are genetically restricted in their antigen-specific helper activity.

A sequential culture technique for the in vitro induction and subsequent assay of T helper cells is employed to examine the histocompatibility requirements for antigen recognition by murine T helper cells. F1 T cells are primed in vitro with antigen-pulsed parental strain macrophages and tested for antigen-specific helper activity in cultures containing anti-Thy 1.2 serum and C treated spleen cells from hapten-primed parental or F1 mice. A semiallogenieic system is used and appropriate controls are included to avoid possible complicating effects of allogeneic interactions. The results indicate that F1 T helper cells preferentially stimulate carrier-specific anti-hapten plaque-forming cell responses in spleen cells which are H-2 identical with the macrophage used initially to prime the T cells. Parental spleen cell cultures do not respond to F1 T helper cells which were primed with the other parental strain macrophage. Supplementing this culture with macrophages which are histocompatible with those used to prime the F1T cells is sufficient to restore T helper cell activity. Thus, the genetic restriction described here is between the primed T cell and the macrophage used to elicit secondary responses and not between the T cell and B cell. The results in this semiallogeneic system, however, do not rule out the possibility of additional allogeneic genetic restrictions in the subsequent interaction of T cells with B cells.     

Macrophages and T cells control distinct phases of B cell differentiation in the humoral immune response in vitro

The differentiation of B cells in the in vitro PFC-response to red blood cell antigen proceeds through 2 phases. Antigen-reactive B cells acquire the ability to interact with helper T cells in the first phase. This phase is controlled by macrophages through a mediator that they release (Interleukin 1 ([Il-1]). B cells convert into antibody-secreting cells (PFC) in the second phase, which is controlled by helper T cells or by a mediator that they release (T cell-replacing factors [TRF]). This is demonstrated in experiments in which Il-1 increases the number of B cells capable of responding to T cell help. The majority of antigen-reactive B cells reaches that state of differentiation within 40 hr of incubation with Il-1. After this time, the response of B cells depends no longer on the presence of Il-1 but on the presence of T cells or TRF. The presented data suggest that antigen-primed helper T cells (but not unprimed T cells) induce the release of Il-1 by macrophages, thereby also influencing the early phase of B cell differentiation.     

Macrophage requirements of CR- and CR+ B lymphocytes for antibody production in vitro.

A Sephadex G-10 column coated with antigen-antibody complexes and complement retains complement receptor-bearing (CR+) mouse spleen cells. The effluent is rich in thymus-derived cells (T cells), and contains bone marrow-derived cells (B cells) which carry surface immunoglobulin (Ig), Ir-associated antigen (Ia), and Fc receptors, but no complement receptors (CR-). Although both unfractionated and CR- B cell populations are capable of producing antibody to red cell antigens, they differ in their requirements for the initiation of the response. Unfractionated B cells cooperate with primed as well as unprimed helper T cells; macrophages are required for this cooperation but can be replaced by 2-mercaptoethanol. CR- B cells cooperate with primed but not with unprimed T cells provided macrophages are added to cultures. After addition of culture supernatant from BCG-activated macrophages CR- B cells cooperate with both unprimed and primed T helper cells.     

IgE immune complexes induce immediate and prolonged release of leukotriene C4 (LTC4) from rat alveolar macrophages

Alveolar macrophages obtained by lung lavage from rats were incubated with monoclonal mouse anti-DNP IgE and specific antigen (DNP-HSA) and were found to release a slow reacting substance (SRS), which was characterized by high performance liquid chromatography as leukotriene C4 (LTC)4. Alveolar macrophages incubated with 1 microM A23187 (calcium ionophore) released similar amounts of SRS (6.0 +/- 2.2 and 5.7 +/- 3.7 X 10(-10) mol of LTC4 per 5 X 10(6) alveolar macrophages, respectively). The optimal conditions and mechanism of LTC release by IgE and antigen were examined. LTC4 release was maximal when freshly retrieved alveolar macrophages were incubated for 20 min with 10 micrograms/ml IgE and then for 20 min with 100 ng/ml antigen or for 20 min with IgE and antigen that had been preincubated together for 30 min at room temperature. In addition, LTC4 release was maximal when cells were challenged with IgE and antigen in a protein-free balanced salt solution and when the cells were tumbled to prevent adherence. Dose response experiments revealed that macrophages released LTC4 when stimulated with as little as 10 ng IgE and 100 ng DNP-HSA. Alveolar macrophages did not release LTC when challenged with IgE or DNP-HSA alone. Activation of LTC4 release by IgE and antigen was rapid in onset (2.5 to 5 min), and washing to remove fluid phase IgE and antigen revealed that once activated, alveolar macrophages were capable of prolonged and continuous release of LTC4. Peritoneal lavage cells stimulated with IgE and antigen did not release SRS but could release SRS when incubated with A23187 (5.7 +/- 1.3 X 10(-10) mol LTC4/5 X 10(6) macrophages). A large variability existed between individual rats in the ability of their alveolar macrophages to be activated by IgE and antigen to release LTC4. DNP-HSA labeled with 125I was used to show formation of immune complexes of IgE and antigen when IgE and antigen were incubated together before macrophage challenge. IgE immune complexes containing as little as 2 ng of antigen elicited the release of LTC4 from alveolar macrophages. These data indicate that rat alveolar macrophages release primarily LTC4 when challenged with IgE immune complexes, and that the alveolar macrophage may differ in this respect from peritoneal macrophages that do not release detectable quantities of LTC4 when challenged under identical conditions.     

Radiation leukemia virus-transformed immunocompetent T cells. II. Antigen-induced macrophage migration inhibition factor and leukocyte migration inhibition factor production

OVA-specific T cells were immortalized by infection with radiation leukemia virus (RadLV). Some clones derived from such population were shown to exhibit helper activity. We then tested clones without such function and found among them some that secreted macrophage migration inhibition factor (MIF) and leukocyte migration inhibition factor (LIF) upon exposure to the antigen in vitro. The lymphokine-producing clones, which were Thy-1+, Ly-1+ and Ly-2-, did not secrete MIF and LIF constitutively. Like other antigen-specific T cells, the immortalized clones could not be stimulated by free soluble antigen but required macrophages for presentation and for triggering the lymphokine production. The antigen-activated clones exclusively produced MIF and LIF, but not interleukin 2 or colony-stimulating factor. They neither provided helper activity nor induced delayed-type hypersensitivity. The data suggest that the T-cell clones carry the antigen receptors and that their antigen-inducible biological function is restricted to the migration inhibitory factor production.