Studies on the regulation of lymphocyte reactivity by normal and activated macrophages.

To determine the potential role of macrophages as regulators of the immune response, the effect of mouse peritoneal macrophages on transforming mouse spleen lymphocytes was investigated. Mitogen and antigen stimulated lymphocyte transformation, as measured by DNA synthesis, was enhanced by all concentrations of normal macrophages tested, but only by low concentrations of activated macrophages. High concentrations of activated macrophages markedly inhibited lymphocyte transformation. This inhibition occurred whether lymphocyte DNA synthesis was measured by incorporation of [³H]TdR or of ³²P. Activated macrophages cultured with lymphocytes within 4 hr of being removed from the peritoneal cavity inhibited lymphocyte transformation. When activated macrophages were cultured alone for 24 or more hours before addition of lymphocytes, enhancement of transformation was noted. Once lymphocytes were exposed to activated macrophages, they could not be induced to undergo transformation in the presence of Con A. Whereas heat-killed activated macrophages, which appeared intact morphologically, lost their capacity to inhibit lymphocyte transformation, macrophages treated with mitomycin C to inhibit DNA synthesis retained this capacity. Syngeneic and allogeneic macrophages had similar inhibitory ability. Supernatants from cultures of many cell types (including normal or activated macrophages, lymphocytes, lymphocytes plus macrophages, and L cells) inhibited [³H]TdR incorporation by both mitogen stimulated lymphocytes and tumor cells. These studies demonstrate the capacity of macrophages to regulate lymphocyte transformation in vitro and suggest a role for these cells as regulators of cell-mediated immunity in vivo.     

Immunological depression produced in vitro by the action of normal RNA on mice spleen cells

Macrophages and lymphocytes from normal mice spleens were treated separately with isologous RNA, pooled again and then stimulated with antigen (rat red blood cells). The number of rosette-forming cells was used as a measure of the antibody production. A low immunological response was obtained when macrophages were incubated with normal RNA. No effect was observed when macrophages were previously sensitized with the antigen and then incubated with normal RNA or when the antigen was pretreated with RNA. On the other hand, RNA had apparently no effect on lymphocytes. Macrophage phagocytosis was markedly diminished by the addition of RNA to the culture.It can be concluded that the effect of normal RNA is exerted on the macrophages by depressing their ability either to recognize or process the antigen or both. The capacity of macrophages to transfer the immunological information to lymphocytes after it has been acquired does not seem to be altered. It can be reasonably assumed, moreover, that RNA has no effect on the antigen.     

Suppressor macrophages in lymphocyte proliferation of cynomolgus monkeys

The present study demonstrated the presence of cells belonging to monocyte/macrophage lineage which suppressed mitogen-induced blastogenesis of peripheral blood lymphocytes in cynomolgus monkeys. Depletion of adherent or phagocytic cells from peripheral mononuclear cells caused a substantial increase in the blastogenic response of cynomolgus monkey lymphocytes whereas the same treatment led to marked reduction rather than enhancement in human lymphocyte blastogenesis. Addition of thioglycollate-elicited peritoneal exudate adherent cells as macrophages suppressed the blastogenic response of nonadherent lymphocytes in a dose-dependent manner. The suppressive effect was observed not only in autologous but also in allogeneic macrophages to the responder lymphocytes. Treatment of macrophages with silica, carrageenan or freezing-thawing reduced their suppressive effect but there was no reduction with mitomycin C or indomethacin. No suppressive activity was detected in the cell-free supernatant of macrophages cultured in the presence or absence of mitogens for up to 4 days. From these findings, it appeared that monocyte/macrophage lineage might be responsible for the observed suppressive effect on mitogen-induced blastogenesis of cynomolgus monkey lymphocytes.     

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.     

Expression of Inducible Nitric Oxide Synthase and Fas/Fas Ligand Correlates with the Incidence of Apoptotic Cell Death in Atheromatous Plaques of Human Coronary Arteries

It was recently reported that inducible nitric oxide synthase was expressed in advanced atheromatous plaques. So we investigated the effect of NO or peroxynitrite reactive product of NO or O(2)(-) released by iNOS induced in macrophages or T lymphocytes on inflammatory cells in atheromatous plaques of human coronary arteries by immunohistochemistry. We found that iNOS was expressed in T lymphocytes and macrophages in T lymphocytes and macrophages coexisted advanced atheromatous areas. Most of the smooth muscle cells are not coexisted with T lymphocytes. We could not find iNOS in those smooth muscle cells. Only a small number of iNOS-positive smooth muscle cells were found close to T lymphocytes and macrophages. Markers for apoptotic cells induced in situ terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) showed that many apoptotic T lymphocytes and macrophages existed near iNOS induced cells. Fas and Fas ligand were expressed in almost same areas that iNOS was expressed. By double-label immunostaining, Fas was expressed in T lymphocytes but Fas ligand was expressed in macrophages and in some T lymphocytes. These results suggest that NO from iNOS induces Fas and Fas ligand-mediated apoptosis and associates with regression of atherosclerosis. On the other hand, nitrotyrosine was detected wider areas than iNOS. So peroxynitrite from iNOS damages cells and tissues widely and may associate with progression of atherosclerosis. These results suggest an important role of iNOS in mediating both regressive changes and progressive change in atheromatous plaques.     

Soluble suppressor of T cell proliferation in primary in vitro response to murine minor histocompatibility antigens

Normal mouse lymphocytes are not capable of mounting a primary cytotoxic T cell response to Mls encoded, non H-2, allodeterminants, although a strong lymphoproliferative response is observed in primary MLR between Mls incompatible cells. In this study it is reported that in the supernatant of primary cultures between AKR macrophages and CBA/H lymphocytes (H-2 identical, incompatible for Mls and other minor antigens) a suppressor of T cell proliferation in MLR is detected. By contrast, a suppressor is not detected in supernatants from primary cultures between BALB/C macrophages and CBA/H lymphocytes (H-2 incompatible, Mls identical), B10.BR macrophages and CBA/H macrophages and CBA/H lymphocytes (syngeneic) suggesting that the production of the suppressor factor occurs only when an Mls incompatibility exists. The suppressive activity of the Mls incompatible culture supernatant upon MLR between incompatible macrophages and lymphocytes is neither antigen specific nor Mls or H-2 restricted, nor is it due to an irreversible toxic effect on T lymphocytes or macrophages. The inhibition of T cell proliferation could be explained by inhibition of IL 2 production, by blocking its union to T cells or by a combination of both effects. Our findings could help explain previous observations that lymphocytes from mice preimmunized with Mls incompatible cells have a depressed proliferative response as well as depressed cytotoxicity against alloantigens.     

The requirement for macrophage-lymphocyte interaction in T lymphocyte proliferation induced by generation of aldehydes on cell membranes.

Guinea pig T lymphocyte proliferation induced by sodium periodate (NaIO4) or neuraminidase-galactose oxidase (NG) occurs when lymphocytes and macrophages are cultured together after treatment of either purified T lymphocytes or macrophages with these agents. Regardless of which cell initially bears the modified surface carbohydrate, lymphocyte proliferation requires the presence of viable homologous macrophages and fails to occur when they are replaced with fibroblasts, erythrocytes, L2C leukemia cells, thymocytes, PMN, line I hepatoma cells, or murine macrophages. Lymphocyte proliferation resulting from NaIO4 or NG treatment of lymphocytes is diminished when these cells are treated with proteolytic enzymes or aged in in vitro culture for 48 hr. By contrast, proteolytic enzyme treatment or in vitro aging has no effect on the ability of NaIO4 or NG-treated macrophages to induce lymphocyte proliferation. The requirement for macrophage-lymphocyte interaction in NaIO4 or NG-induced lymphocyte proliferation is indicative of a central role for the macrophage in the initiation of T lymphocyte proliferation.     

Modulation of lymphocyte proliferation by macrophages and macrophages loaded with arachidonic acid

Arachidonic acid (AA) is incorporated and exported by macrophages. This fatty acid is also transferred from macrophages (Mphi) to lymphocytes (LY) in co-culture. This observation led us to investigate the effect of macrophages pre-loaded with AA on concanavalin A (Con A)-stimulated lymphocyte proliferation. The experiments were performed in co-culture. This condition reproduces the in vivo microenvironment in which the modulation of lymphocyte proliferation is dependent on the interaction with macrophages. Lymphocytes obtained from untreated rats or from intraperitoneally thioglycolate-injected rats (THIO-treated) were co-cultured with macrophages from the same rats. Firstly, macrophages were co-cultured for 48 h with Con A-stimulated lymphocytes in different proportions: 0.5, 1, 2.5, 5, 10, 20 and 30% of 5 x 10(5) lymphocytes per well. At 1% proportion, macrophages caused maximum stimulation of lymphocyte proliferation; a four- to five-fold increase, for cells from both thioglycolate-treated and untreated rats, respectively, whereas at 20% it caused maximum inhibition. In addition, 1 or 20% macrophages were pre-loaded with several AA concentrations during a period of 6 h and co-cultured with lymphocytes. At 180 microM AA and 1% macrophages, lymphocyte proliferation was inhibited (by 25%), whereas at 20% macrophages, proliferation was increased, by 25- and three-fold, respectively, for cells from untreated and THIO-treated rats. AA added directly to the medium reduced lymphocyte proliferation, also being toxic to these cells at 100 microM. No toxic effects of AA were observed on macrophages. Additional evidence suggests that nitric oxide production is involved in the modulation of lymphocyte proliferation by AA-pre-loaded macrophages. These findings support the proposition that AA can directly modulate lymphocyte proliferation and the interaction between macrophages and lymphocytes.     

Tumour necrosis factor-alpha and lipopolysaccharide enhance interferon-induced tryptophan degradation and pteridine synthesis in human cells

The capacity of recombinant interferon-alpha, -beta and -gamma, of bacterial lipopolysaccharide and of recombinant tumour necrosis factor-alpha to induce indoleamine 2,3-dioxygenase and synthesis of pteridines was studied in human peripheral blood mononuclear cells, human macrophages and normal dermal fibroblasts. The action of interferon-alpha and -beta on macrophages was supported by lymphocyte factors as indicated by the effect of these mediators in the absence or presence of lymphocytes. Tumour necrosis factor-alpha alone was ineffective in peripheral blood mononuclear cells and macrophages, but it significantly increased the action of all three interferon species on macrophages and fibroblasts. Lipopolysaccharide directly affected macrophages or dermal fibroblasts and enhanced the effect of interferon-gamma. However, in the presence of lymphocytes, the action of lipopolysaccharide was mediated via interferon-gamma.     

Effects of immune lymphocyte products and serum antibody on the multiplication of Toxoplasma in murine peritoneal macrophages.

In vitro studies on cell-mediated immunity against Toxoplasma infection were carried out by estimating the ability of antigenically stimulated lymphocytes. Splenic lymphocytes from normal mice and from hyper-immunized mice were cultured in the presence or absence of Toxoplasma lysate antigen. The cell-free supernatant fluids from the lymphocyte cultures were assassed for their ability to alter the functional capacities of normal macrophages. When glycogen-induced peritoneal macrophages were incubated with the culture supernatant from immune lymphocytes reacting with specific angigen, the intracellular multiplication of the organisms was inhibited remarkably. In contrast, the addition of antitoxoplasma antibody to culture medium had no effect on the enhancement of phagocytic activity of culture macrophages. However, when extra-cellular organisms were exposed to fresh or heat-inactivated immune serum just before infection of the monolayers, the intracellular multiplication of Toxoplasmas was inhibited significantly by either activated or normal macrophages.     

Augmentation of impaired tumoricidal function in alveolar macrophages from lung cancer patients by cocultivation with allogeneic, but not autologous lymphocytes

 It has been reported that the in vitro development of tumoricidal function in alveolar macrophages from lung cancer patients is reduced significantly when compared to that in peripheral blood monocytes from the same patients or alveolar macrophages from control patients. In the present investigation, a method for potentiating the development of tumoricidal function in alveolar macrophages from lung cancer patients is described. This method, which relies on priming the macrophages with purified, allogeneic peripheral blood lymphocytes from normal donors, could not be demonstrated when autologous lymphocytes from lung cancer patients were used in the priming coculture. The augmentation of tumoricidal function appears to be mediated by one or more soluble factors, since supernatants from cocultures of alveolar macrophages and allogeneic peripheral blood lymphocytes could enhance the cytotoxic function of freshly obtained alveolar macrophages. Furthermore, it appears that NK cells are necessary for this effect, since depletion of CD56⁺/CD57⁺ cells from allogeneic lymphocytes eliminated their capacity to enhance alveolar macrophage cytotoxic function. The augmentation of cytotoxic function elicited in alveolar macrophages by this method was not associated with changes in the secretion of tumor necrosis factor α, or interleukin 1β. Received: 15 March 1997 / Accepted: 11 June 1997     

Effect of LDL-In, a normal immunoregulatory human serum low density lipoprotein, on the interaction of macrophages with lymphocytes proliferating in response to mitogen and allogeneic stimulation.

The immunoregulatory properties of LDL-In, a normal species of human serum low density lipoprotein which suppresses indictive events involved in triggering of lymphoid cells by lectins and antigens, were analyzed in order to distinguish between a primary effect on macrophages and lymphocytes. LDL-In was found to be equally effective in suppression of the response of human lymphocytes to tpha at concentrations of lectin demonstrated to impart apparent macrophage-independence or macrophage-dependence to the culture system. Exposure of only the macrophages to LDL-In was shown to be without effect on subsequent in vitro lymphocyte responses to either PHA or allogeneic cells, whereas exposure of only the lymphocytes to LDL-In was fully effective. The cellular locus was further identified by demonstrating that the responder lymphocytes, but not the stimulator lymphocytes, were the target of the suppressive activity in mixed lymphocyte reactions. These data considered in conjunction with previous studies suggest that the primary untriggered lymphocyte is the most probable cellular target for the bioregulatory effects of LDL-In.     

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.     

Changes in the composition of canine respiratory cells obtained by bronchial lavage following irradiation or drug immunosuppression.

Canine respiratory cells, obtained by bronchial lavage, and blood leukocytes were monitored to observe cellular changes following acute and chronic immunosuppression. Irradiation (350 R) produced bone marrow suppression and prompt peripheral blood leukopenia, but did not affect recovery of pulmonary alveolar macrophages or lymphocytes for 12 days after. Treatment for 6 weeks with daily methylprednisolone (1 mg/kg) caused a progressive decrease in the number of recoverable respiratory lymphocytes, whereas alternate day methylprednisolone (2 mg/kg) had less effect. Cyclophosphamide in combination with steroids generally augmented the progressive loss of blood and respiratiory lymphocytes. Recovery of alveolar macrophages was not changed appreciably. Thus, the population of lung macrophages, sampled by pulmonary lavage, withstood acute and chronic forms of immunosuppression very well. In contrast, canine lymphocytes seem more susceptible to injury, especially to drug regimens containing steroids.     

The role of macrophages in thymocyte mitogenesis.

The thymic lymphocytes of CBA/J mice respond to the mitogen Concanavalin A (Con A) only in the presence of adherent cells of the monocyte-macrophage series. Depletion of adherent cells abrogates the response, and macrophage-rich population of cells restore it. The need for macrophages and mitogen is completely provided by irradiated splenic macrophages which have been exposed to Con A and washed free of the soluble mitogen. The mitogenmacrophage effect in this case is apparently not due to soluble factors. Even more striking than the effect of macrophages on fresh cultures of thymic lymphocytes is their ability to restimulate quiescent cells 72 hr after their first stimulation with Con A. The quiescent cells respond immediately and quantitatively to Con A in the presence of fresh macrophages. This stimulation, like that of fresh thymocytes, is also controlled by a lymphokine ("costimulator") produced by mixing macrophages, mitogen, ant T lymphocytes. Our data suggest a model in which two signals are required for mitogenesis. First, the interaction of macrophage, T cell, and mitogen elicits a soluble costimulator, which is itself not mitogenic. Secondly, in the presence of costimulator, the mitogen (either soluble, or, more efficiently, bound to macrophages) induces a proliferative response in the T cell.     

Leishmania enriettii: Immune induction of macrophage activation in an experimental model of immunoprophylaxis in the mouse

This study describes some of the parameters of the cellular immune response elicited in mice by inoculation of the nonpathogenic protozoan parasite, Leishmania enriettii. Incubation in vitro of leishmania-infected mouse peritoneal macrophages with spleen cells from syngeneic leishmania-immune animals resulted in activation of the phagocytes, leading to intracellular parasite destruction. Activation required interaction of sensitized lymphocytes with parasite antigen released or displayed by infected macrophages. The effect was dependent both on the dose of parasites used for in vivo priming and on the number of spleen cells cocultivated with parasitized macrophages. The activating capacity of lymphocytes was abrogated by anti-Thy-1 antiserum treatment and was retained in the effluent cells after nylon-wool separation. Activation was followed by lysis of part of the macrophage monolayer. Destruction of the phagocytes did not appear to result from the activation process per se and may represent a cytotoxic activity of sensitized lymphocytes for macrophages bearing parasite antigen on their surface.     

Suppressor cell infleunce in selected strains of inbred rats. II. Macrophage-associated suppression of cell-mediated immune responsiveness.

Responses to concanavalin A or antigen by allogeneic combinations of Lewis (LE) and Brown-Norway (BN) rat derived lymphocytes and macrophages were of comparable magnitude to responses by the syngeneic combinations. But at increased concentrations of macrophages to lymphocytes, suppression of lymphocyte reactivity, as assayed by incorporation of tritiated thymidine, was evident. Suppression associated with BN derived macrophages alone, or in combination with LE or Buffalo derived macrophages, was consistently of a greater magnitude. Possible explanations of the macrophage associated suppression are discussed.     

Functional activation of immune lymphocytes by antigenic stimulation in cell mediated immunity: I. Requirement for macrophages in antigen-induced MIF production by guinea pig immune lymphocytes in vitro

The role of macrophages in the process of antigen-induced production of mediators in cellular immune response was studied, using the antigen-induced production of migration inhibitory factor (MIF) as a measure of the activation of immune lymphocytes.The production of MIF by guinea pig immune lymph node cells in response to the stimulation with PPD was abolished when the lymph node cells were depleted of adherent cell population by passing the cells through a Tetron fiber column and incubating the effluent cells in plastic dishes. These purified immune lymphocytes did not respond to particle-bound PPD, either. However, the response was obviously restored by the addition of a small number of the purified peritoneal adherent cells (macrophages) which had been pulse-treated with PPD. The PPD-pulsed macrophages produced no MIF by themselves. Thus, the results clearly indicated the requirement for macrophages in the process of antigen-induced MIF production by immune lymphocytes. Destruction of PPD-pulsed macrophages by freezing and thawing or by homogenization abrogated their ability to stimulate immune lymphocytes. Attempts to restore the response of the purified immune lymphocytes to PPD by adding 2-mercaptoethanol or the culture supernatant of macrophages to the medium have so far been unsuccessful.     

Species-dependent regulation of monocyte/macrophage Ia antigen expression and antigen presentation by prostaglandin E

The expression of Ia antigen by various murine and human macrophage populations and the ability of prostaglandins of the E series to regulate Ia antigen expression were explored. Monocytes and macrophages from human and murine populations demonstrated a dichotomy in the expression of Ia antigen. Both human monocytes and macrophages expressed elevated levels of Ia antigen compared to their murine counterpart. Murine macrophages appear to express elevated levels of Ia antigen only when actively interacting with T lymphocytes in vivo or with lymphokines in vitro. Prostaglandins of the E series can suppress murine macrophage Ia antigen expression, but have little effect on the expression of Ia antigen by human monocytes and macrophages. Also, prostaglandins of the E series do not modulate the ability of human monocytes to present antigen to autologous lymphocytes when studied over a broad concentration range. These data suggest that prostaglandin E compounds do not profoundly affect human monocyte/macrophage Ia antigen expression or human monocyte antigen presenting activity.     

Macrophage-mediated suppression of immune responses in Toxoplasma-infected mice. I. Inhibition of proliferation of lymphocytes in primary antibody responses

The suppressor cells induced by Toxoplasma infection were shown to be macrophages, since they adhered to plastic, and their suppressive activity in anti-sheep erythrocytes (SRBC) antibody responses was abrogated by treatment with silica or carrageenan, which are selectively cytotoxic for macrophages. The suppressor macrophages strongly inhibited the uptake of tritiated thymidine ( [3H]TdR) by normal mouse spleen cells in the responses to SRBC and Toxoplasma antigens. Supernatant fluids from the suppressor macrophages could not passively transfer the suppressive effect on anti-SRBC antibody responses. Furthermore, when the suppressor macrophages were isolated by a cell-impermeable membrane from normal mouse spleen cells, the antibody responses of normal spleen cells were not suppressed. These results indicate that suppression of antibody responses in Toxoplasma-infected mice is caused by an inhibitory effect of the suppressor macrophages upon proliferation of lymphocytes via direct contact with responder target cells. The suppressive effect of the macrophages was not counteracted by indomethacin, a potent inhibitor of prostaglandin synthesis, or catalase, a catabolic enzyme for hydrogen peroxide (H2O2).