Functional activation of immune lymphocytes by antigenic stimulation in cell-mediated immunity. IV. Role of macrophage and its soluble factor in antigen-induced MIF production of immune T lymphocytes.

Histocompatibility-linked restriction of macrophage-T lymphocyte interaction in antigen-induced MIF production by sensitized lymphocytes was examined, by using combinations of inbred strain 2, strain 13, and JY-1 guinea pigs. The effective interaction of the antigen-bearing macrophages with the immune T lymphocytes was observed when the donor of the antigen-bearing macrophages and that of the immune lymphocytes shared Ia antigens of the major histocompatibility complex. Identities of B antigens and S antigens were not important for this cooperation. It was further demonstrated that the previously reported soluble factor derived from LPS-stimulated peritoneal adherent cells (macrophages) could help antigenic activation of the immune lymphocytes across the strain barrier provided a small number of macrophages (0.01%) from syngeneic strain were present. These results show that the presence of macrophages is absolutely required to present antigen to immune T lymphocytes in a genetically restricted manner and the soluble factor from macrophages appears to give a nonspecific effect on the lymphocyte activation in addition to or in collaboration with antigenic stimulation.     

Macrophages in resistance to rickettsial infection: susceptibility to lethal effects of Rickettsia akari infection in mouse strains with defective macrophage function

We have confirmed the requirement of macrophages in the antigen-induced T-lymphocyte proliferative response and in the generation of migration inhibition factor (MIF) by immune lymphocytes. Extending these observations, we have found that autologous and non-syngeneic, oil-induced peritoneal exudate macrophages were equally effective in restoring the proliferative response and MIF production by column-purified lymph node T cells. MIF activity was optimally restored when T cells were reconstituted with 1 to 40% exudate-derived macrophages whereas 10 to 30% macrophages were needed to optimally restore the T-cell proliferative response. Normal resident macrophages from the peritoneal cavity were also capable of restoring T-cell reactivity as were normal or BCG-activated pulmonary alveolar macrophages. It was also found that the addition of as few as 1.0% glycogen-elicited peritoneal exudate cells restored the production of MIF by T cells. Quantitative considerations demonstrated that the responsible cells in these preparations were polymorphonuclear cells rather than macrophages. In contrast, neither MIF production nor the proliferative response by T cells were restored by the addition of red blood cells. In these studies we were able to demonstrate that freeze-thawed macrophages could restore antigen-induced MIF production, but not antigen-induced cellular proliferation. The ability of freeze-thawed macrophages to stimulate T cells to produce MIF was apparently associated with the macrophage membranes and not with a soluble factor in the macrophage extracts. These results demonstrate that multiple sources of phagocytic cells may interact cooperatively with lymphocytes in reactions of cell-mediated immunity. Further, at least in the case of MIF production, this interaction involves a membrane-bound determinant that is effective even in the absence of viable macrophages.     

Macrophage-lymphocyte clusters in the immune response to soluble protein antigen in vitro. IV. Synergy of lymphocytes in the formation of clusters.

T-lymphocyte-enriched lymph node lymphocytes from guinea pigs immunized with Mycobacterium tuberculosis produce clusters with macrophages when cultivated on monolayers of syngeneic purified protein derivative of tuberculin (PPD)-pulsed peritoneal macrophages. The clusters consist of a macrophage with a central lymphocyte attached to it, and several peripheral lymphocytes attached to the central one. By mechanical manipulation immune lymphocytes incubated on monolayers of PPD-pulsed macrophages were separated into those which adhered firmly to the macrophages after 4 hr of culture and those which did not adhere. While neither of the two populations was able to produce significant numbers of clusters alone, they did so in combination. The number of macrophage-lymphocyte clusters which are produced in a culture depends not only on the absolute number of immune lymphocytes in the culture, but also on the concentration of lymphocytes per area of the macrophage monolayer, with high concentrations resulting in high numbers of clusters. Autoradiographic studies showed that the DNA-synthesizing lymphocytes physically associated with macrophages were located mainly inside the clusters in cultures with high concentrations of lymphocytes but mainly outside the clusters in cultures with low concentrations of lymphocytes.     

Studies on the mechanism of suppression of delayed hypersensitivity by the antischistosomal compund niridazole.

Niridazole given in a single oral dose of 100 mg/kg to guinea pigs sensitized to ortho-chlorobenzoyl chloride-bovine gamma-globulin (OCB-BGG) regularly abolished delayed cutaneous reactivity. Little effect was observed, however, when cells from these animals were tested in vitro with either direct or indirect assays for migration inhibitory factor (MIF). On the other hand, sera taken from nonsensitized guinea pigs after they had received 100 mg/kg of niridazole markedly diminished antigen-induced inhibition of migration of sensitized peritoneal exudate cells in vitro. The immunosuppressive effects of such sera could not be produced by niridazole itself, thereby suggesting an effect of niridazole metabolites. This suppressive activity was readily removed from the serum by dialysis. The active serum blocked the production of MIF by sensitized lymph node cells but did not affect the action of preformed MIF on macrophages. The effect of this serum was reversible; lymph node cells incubated for 24 hr with active serum, then washed and reincubated with antigen in normal serum, produced normal amounts of MIF. These studies suggest that metabolites of niridazole, but not the parent compound itslef, suppress delayed hypersensitivity in guinea pigs and prevent MIF production by lymphocytes without affecting the macrophage response to MIF.     

Functional activation of immune lymphocytes by antigenic stimulation in cell-mediated immunity II. Analysis of the macrophage-replacing activity of the LPS-stimulated macrophage culture supernatant in the antigenic activation of purified immune lymphocytes

A requirement for the cooperation of macrophages (adherent cell population) in the process of the antigenic activation of immune lymphocytes for the production of migration inhibitory factor (MIF) has been demonstrated previously. It was found, in the present study, that the culture supernatant of peritoneal macrophages, which had been pulse-stimulated with a bacterial lipopolysaccharide (LPS), could be substituted for live macrophages. Fractionation of the supernatant by gel filtration revealed its activity in the fraction of 15,000–100,000 MW and the activity was completely abolished by heat treatment at 85°C for 30 min. These results distinguished the nature of the active component from that of LPS which was found to be present in a trace amount in the supernatant and strongly suggested the presence of a factor(s) derived from macrophages which effects, in some way, the process of the antigenic activation of immune lymphocytes. Our experiment showed that the cooperating function of macrophages was inhibited by the treatment of macrophages with vinblastine. It may be that vinblastine affects the release of an active factor(s) from macrophages although other possibilities remain that the inhibition resulted from its effects on other functioning processes of macrophages.     

Staphylococcal enterotoxin B induced release of macrophage migration inhibition factor from normal lymphocytes

Staphylococcal enterotoxin B (SEB), a potent lymphocyte mitogen, inhibits migration of peritoneal exudate cells from most guinea pigs but does not inhibit migration of purified macrophages. Experiments were designed to test the ability of highly purified SEB to induce normal lymphocytes to release migration inhibition factor (MIF). Supernatants of lymph node lymphocytes cultured with SEB inhibited the migration of purified macrophages, indicating the release of a migration inhibition factor. Mitomycin-C blocked the SEB-induced release of MIF. SEB-induced MIF localized in the albumin fraction on Sephadex G-200 chromatography. Antibody to SEB specifically blocked the inhibitory effect of SEB on migration of normal guinea pig peritoneal exudate cells.     

Cell-mediated and humoral immune responses to aspermatogenic antigen in experimental allergic orchitis in the guinea-pig

Guinea-pigs were immunized with a defined and highly potent aspermatogenic antigen, G75m, and the occurrence of orchitis was correlated with (1) cell-mediated immune response to G75m, determined by lymph node cell proliferation and by secretion of macrophage migration inhibitory factor (MIF) by peritoneal exudate cells, and (2) humoral antibodies to G75m and to cell surface antigens of guinea-pig testicular cells, by radioimmunometric assays. A consistent temporal relationship between cell-mediated immune responses and disease was found: lymph node cell proliferation was positive by Day 4, followed 3 days later by maximum secretion of MIF, and orchitis lesions were manifest on Day 10. In contrast, maximal IgG antibodies to G75m or to the surface antigens of spermatozoa/testicular cells were detected at a time when cell-mediated immune responses and active testicular lesions had subsided. In individual animals, lymph node cell proliferation increased with severity of orchitis, while MIF secretion by peritoneal cells increased with orchitis only late in the disease. Early in disease, MIF response showed a negative correlation with orchitis. Moreover, peritoneal injection of oil reduced the incidence of early lymph node cell proliferative responses, and delayed the onset of testicular disease. These findings are consistent with competition between different inflammatory sites for recently antigen-activated T lymphocytes. We conclude that (1) the development of orchitis correlates with cell-mediated immune responses to purified aspermatogenic antigens but not with IgG antibody responses, and (2) when the same animal is used to assess different aspects of cellular immunity and autoimmune disease, one study may significantly influence the other.     

Suppression of immune response by lung cells in experimental tuberculosis

In vitro proliferative response of lung cells from mice infected with Mycobacterium tuberculosis H37Rv against PPD and Con A was studied. It was shown that the infected lung contained immune T cells, but their response in vitro was totally inhibited by plastic and nylon wool adherent suppressor cells. The whole population of lung cells from infected, but not intact mice, efficiently suppressed the proliferative response of immune lymph node cells against various antigens (non-specific suppression). The inhibition of response again depended on the presence of plastic adherent lung cells. Our data suggest that at least two suppressor pathways are induced in the course of tuberculosis infection: one being specific for mycobacterial antigens and other non-specific. Both types of suppressor pathways depend on the plastic adherent lung cells from tuberculosis lesion.     

Eosinophils and immune mechanisms. III. Production of the lymphokine eosinophil stimulation promoter by mouse T lymphocytes.

The lymphoid cell population responsible for production of eosinophil stimulation promoter (ESP), a lymphokine which increases migration of eosinophils, was investigated in murine Schistosoma mansoni infection. Con A challenge induced ESP production, whereas LPS did not. Prior treatment with anti-thetaC3H alloantiserum plus complement in vitro eliminated ESP production; in vivo treatment with rabbit anti-mouse thymocyte serum consistently reduced ESP production by splenic lymphoid cells, but affected lymph node cell ESP production only after exceptionally large doses. Thymocytes did not produce significant amounts of ESP; nor did lymphoid cells from congenitally athymic mice. Depletion of B lymphocytes and macrophages by nylon fiber adherence eliminated antigen-induced ESP production; this was partially restored by addition of non-immune, 72-hr peritoneal exudate cells. Con A-induced ESP production was not affected by nylon fiber treatment. These results demonstrate that ESP is produced by an ATS-sensitive, peripheralized T lymphocyte population, and suggest a macrophage requirement for antigen-induced production of this lymphokine.     

Enhancement of macrophage bactericidal capacity by antigenically stimulated immune lymphocytes

The ability of antigenically stimulated immune lymphocytes to influence the bactericidal capacity of normal macrophages was studied in vitro. Purified lymphocytes were obtained from the lymph nodes and peritoneal exudates of guinea pigs immunized with bovine gamma globulin (BGG) and from control animals. Immune and control lymphocytes were added to normal macrophages and incubated overnight in the presence or absence of BGG. After washing, the macrophage monolayers were infected with Listeria monocytogenes; 4 hr later, the cells were lysed and the surviving intracellular bacteria quantitated. The macrophages which had been incubated with BGG-immune lymphocytes in the presence of BGG displayed a markedly enhanced listericidal capacity. In parallel experiments, these same antigen-stimulated lymphocytes were shown to inhibit the migration of normal macrophages. Lymphocytes derived from peritoneal exudates were more active than lymph node lymphocytes in both assays.     

Functional activation of immune lymphocytes by antigenic stimulation in cell-mediated immunity. III. A soluble factor released from LPS-stimulated peritoneal adherent cells effective in antigenic activation of sensitized lymphocytes.

Antigen-induced production of migration inhibitory factor (MIF) by sensitized lymphocytes requires macrophages to effectively stimulate lymphocytes with soluble antigen in vitro. The present study showed that macrophage-depleted lymphocytes of sensitized guinea pigs could be activated with antigens when the culture supernatant of peritoneal adherent cells pulse-stimulated with a macromolecular fraction of bacterial lipopolysaccharide (LPS) was added to the lymphocyte culture. The apparent macrophage-replacing activity was found in the fraction which emerged slightly ahead of serum albumin upon gel filtration of the culture supernatant, and the activity was shown to be destroyed by heating at 65 °C for 30 min or by trypsin digestion. These results appeared to show that the activity was due to a protein component, most probably released from macrophages. Two-step culture experiments revealed that the soluble factor should be present in the early stage of the culture to activate the macrophage-depleted immune lymphocytes with antigen, as well as in the later stage when the presence of antigen in the medium is no longer required. Furthermore, the factor was shown to act in the activation of a T-cell-enriched fraction of immune lymphocytes. The factor appeared to be playing some essential role in making an antigenic stimulus effective for the activation of immune lymphocytes.     

Stimulation of mouse migration inhibitory factor (MIF) production form MSV-immune lymphocytes by soluble tumor-associated antigen: requirement for histocompatible macrophages.

Spleen cells from C57BL/6 mice immunized with murine sarcoma virus (MSV) are capable of producing migration inhibition factor (MIF) in response to stimulation with a specific tumor-associated antigen prepared by solubilization with 3 M KCL. We have previously demonstrated that this response is T cell-dependent. Further investigations into the effector cells involved in the production of MIF have revealed that spleen cells from mice immunized with MSV cannot produce MIF when stimulated with tumor extract if the population has been previously depleted of macrophages. However, the response can be restored by adding nonimmune syngeneic macrophages but not by allogeneic macrophages. The inability of allogeneic macrophages to provide this function was not due to their increased suppressor activity since in mixing experiments they did not interfere with the ability of immune spleen cells to produce MIF. Furthermore, they were not defective since they could supply this "cooperative function" to appropriate F1 mice. The results indicate that macrophages are required for stimulation of MIF by soluble tumor antigens and that for efficient interaction the macrophages and lymphocytes must share some genetic similarities.     

Macrophage-lymphocyte clusters in the immune response to soluble protein antigen in vitro: III. DNA synthesis of lymphocytes in clusters

The lymphocytes which engage in DNA synthesis during the in vitro immune response to PPD (purified protein derivative of tuberculin) were studied by scintillation counting and in autoradiographs prepared from cultures of macrophages and immune T-lymphocyte-enriched lymphocytes. The lymphocytes in these cultures were located in three compartments: lymphocytes in macrophage-lymphocyte clusters, lymphocytes attached to macrophages but not involved in clusters, and not macrophage-attached lymphocytes. One of the cluster cells, the central lymphocyte, which is attached directly to the macrophage, was identified as the only DNA-synthesizing lymphocyte in the cluster early in the culture period. In cultures extended beyond 20 hr the increase in percentage of DNA-synthesizing lymphocytes in the cluster and macrophage-attached compartments exceeded the increase in the compartment of not macrophage-attached lymphocytes. The total amount of radiolabeled thymidine incorporated into lymphocytes in a blast transformation assay was directly proportional to the number of macrophage-lymphocyte clusters produced by the same lymphocytes in a cluster assay.     

Studies on macrophage-activating factor (MAF) in antitumor immune responses. I. Tumor-specific Lyt-1+2- T cells are required for producing MAF able to generate cytolytic as well as cytostatic macrophages

In the present study we investigated some of the cellular mechanisms for the generation of macrophage-activating factor(s) (MAF) in immune responses to tumor antigens. C3H/HeN mice were immunized to syngeneic MH134 hepatoma or MCH-1-A1 fibrosarcoma by intradermal inoculation of viable tumor cells, followed by the surgical resection of the tumor. Spleen and lymph node cells from these tumor-immune mice were stimulated in vitro with the corresponding tumor cells, and supernatant from such a culture was tested for an ability to activate macrophages to exert their cytostatic and cytolytic activities as detected on tumor cells unrelated to immunizing tumors. Peritoneal adherent cells as a macrophage source, which were preincubated with supernatant from co-culture of tumor-unimmunized normal spleen and lymph node cells plus tumor cells, failed to exhibit any significant antitumor effect on unrelated X5563 tumor cells, whereas the addition of supernatant from cultures containing immune lymphocytes to adherent cells resulted in appreciably potent cytostatic and cytolytic effects on X5563 tumor cells, indicating the generation of MAF in culture supernatant. The activation of tumor-immune spleen and lymph node cells for MAF generation was tumor-specific, because anti-MH134- and anti-MCH-1-A1-immune lymphocytes produced MAF by the stimulation with the respective but not with the other alternative tumor cells. Such MAF production was abolished by treatment of tumor-immune spleen and lymph node cells with anti-Thy-1.2 or anti-Lyt-1.1 but not with anti-Lyt-2.1 antibody plus complement before culturing. These results indicate that the tumor-specific Lyt-1+2- T cell subset has a crucial role in generating MAF by which an adherent cell population as a source of macrophages acquires the potential for inducing a cytolytic as well as a cytostatic effect on tumor cells.     

Macrophage-lymphocyte interaction in migration inhibition factor (MIF) production against soluble or cellular tumor-associated antigens. I. Characteristics and genetic control of two different mechanisms of stimulating MIF production.

For MIF production in response to 3 M KCl extracts of tumor, viable and metabolically active macrophages have been shown to be required to interact with the soluble tumor antigens and then come in contact with immune lymphocytes. The Mphi-lymphocyte interaction for MIF production was found to be under the control of genes mapping in the IA subregion of the H-2 complex. However, when intact tumor cells were used as antigen, Mphi were not required for immune lymphocytes to produce MIF. In addition, the interaction of immune lymphocytes with tumor cells for MIF production did not require H-2 compatibility. These and other observations strongly suggest that there are two different mechanisms for MIF production and that these may be mediated by two separate subpopulations of immune lymphocytes.     

Antigen-specific murine T-cell proliferation: role of macrophage surface Ia and factors.

The proliferation of Mycobacterium-primed murine lymph node T cells to purified protein derivative of tuberculin (PPD), as measured by the uptake of tritiated thymidine, requires the obligatory participiation of macrophages which stimulate the T cells either directly with antigen in association with cell surface Ia (I region-defined antigens), or indirectly by means of soluble factors. We have examined the possibility that this functional dichotomy is due to heterogeneity within the macrophage population. Since the maturation of macrophages from the precursor monocytes is associated with cell enlargement, macrophage subpopulations differing in developmental stage are obtained by cell fractionation according to size by velocity sedimentation. Nylon-wool-purified T cells which have been depleted of macrophages and B cells are stimulated with PPD either in a free form or bound to macrophages which have been incubated for a short time (i.e., pulsed) with PPD. We found that for PPD-pulsed macrophages, only the smallest (and probably the most immature) are capable of inducing T-cell proliferation. This antigen presentation function is mediated by cell surface Ia since it is abolished by pretreatment of the macrophages with anti-Ia serum and complement. On the other hand, all macrophages, irrespective of sensitivity to anti-Ia serum, secrete factors which will stimulate T-cell proliferation in the presence of free PPD. Thus the maturation of macrophages is accompanied by a shift from Ia-dependent to Ia-independent mechanisms of immunostimulation.     

The biochemistry and in vitro activity of soluble factors of activated lymphocytes

Summary Activated lymphocytes release numerous products which are either synthesized de novo or in increased amounts; some of these products play a role in the regulation of the immune response and are designated as mediators of cellular immune reactions or lymphokines. The first lymphokine described was the macrophage migration inhibitory factor (MIF) which has been studied most extensively with regard to its chemical and biological properties. Using sensitive radiolabelling techniques and an antiserum against highly purified fractions of MIF we were able to identify several products of activated guinea pig lymphocytes with different molecular weights of 15.000, 30.000, 45.000, 60.000 which all had an isoelectric point of 5.2 and were all inhibitory to macrophage migration. It is suggested, that these molecules are oligomers of a common subunit of molecular weight 15.000. It was further shown, that molecules of the same physical-chemical and serological characteristics are produced by activated B-cells, L₂C leukemia cells and growing fibroblasts, thus further substantiating earlier reports on the production of MIF by lymphoid and non-lymphoid cells. The described molecules were also shown not to contain determinants of the major histocompatibility complex and to be distinct from lymphotoxin, another lymphocyte activation product. It is concluded, that MIF is not sa single molecule but rather a system of structurally related molecules. Thier interaction with macrophages and possible relationship to macrophage activating factor is discussed.     

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.     

Human Antigen-Presenting Cells: Characterization of the Cells in the T-Lymphocyte Proliferative Response

Human antigen-presenting cells (APC) which present the antigen to T lymphocytes resulting in a T-lymphocyte proliferative response were found among peripheral mononuclear cells (MNC), by employing purified protein derivative (PPD) as soluble antigen. To assess the adherence capacity of human antigen-presenting cells, MNC were separated by plastic Petri dishes or nylon wool columns. Plastic nonadherent cells were almost equivalent to unseparated cells in antigen-presenting ability. Plastic adherent cells, however, showed better antigen-presenting ability than unseparated cells. On the other hand, cells passed over nylon wool columns showed essentially no ability to present PPD to T lymphocytes. Removal of phagocytic cells by carbonyl iron resulted in about 50–70% reduction in antigen-presenting ability. Carrageenan, which is known to be toxic to macrophages, had no effect on APC. By using both rabbit anti-human Ia-like antiserum and alloantiserum specific for HLA-DR phenotype and complement, it was shown that APC possessed Ia-like antigens, whereas they did not bear surface immunoglobulins. These results indicate that the human APC is probably a cell in the monocyte-macrophage lineage. Allogeneic MNC were used as APC in order to determine whether any genetic restriction exists between MNC as APC and responding T lymphocytes. Optimal stimulation was shown to require identity of mixed leukocyte reaction (MLR)-activating determinants between APC and T lymphocytes. It is, however, obscure whether an HLA-D region restriction exists in these combinations because PPD-pulsed allogeneic MNC lost their ability to elicit even MLR. It is possible that this failure to elicit MLR was caused by T lymphocytes among the MNC used as APC.     

Adherent cell function in murine T lymphocyte antigen recognition. I. A. macrophage-dependent T cell proliferation assay in the mouse.

The data in this report describe a T cell proliferation assay with nylon wool column-purified murine lymph node lymphocyte from animals immunized by footpad injection of antigen in CFA. It was found that the in vitro immune response of sensitized T cells to soluble protein antigens was functionally dependent on the presence of adherent cells, more specifically macrophages, at all concentrations of in vitro antigen challenge. The response was due to T cells in that cytotoxic treatment of the immune lymphocyte cells with anti-Thy 1.2 serum and complement effectively eliminated the antigen-specific DNA synthetic responses. The antigen-specific proliferation of murine lymphocytes depleted of adhereent cells could not be reconstituted with either guinea pig macrophages nor murine fibroblasts, indicating the existence of species and cell type specificity. In contrast to previous observations in the guinea pig, soluble products of cultured adherent cells could at least partially replace the function of intact macrophages in the response to antigen.