Suppression of lymphokine production: I. Macrophage-mediated inhibition of MIF production

Macrophages have been found to suppress the in vitro production by stimulated T lymphocytes of a lymphokine, migration inhibitory factor. When macrophages isolated from primary MSV-induced tumors were added to antigen-stimulated MSV-immune spleen cells, a complete suppression of MIF production was observed. This suppression was nonspecific, since MIF production by antigen-stimulated alloimmune spleen cells and by PHA-stimulated normal spleen cells was also inhibited. Suppressor macrophages could also be induced by inoculation with Corynebacterium parvum, whereas light mineral oil-induced peritoneal macrophages had no detectable effect on MIF production. The failure to detect MIF in the supernatants of stimulated cultures containing activated macrophages appeared to be due to inhibition of lymphokine production rather than to absorption or inactivation of MIF or to interference with the assay for detection of MIF. Macrophages were able to suppress MIF production only when added during the first 4–5 hr of culture and they had no effect when added later. These data show that activated macrophages can nonspecifically suppress lymphokine production and that this appears to be due to inhibition of an early step in lymphocyte stimulation.     

Changes in macrophage status in vivo during infection with and immunity to Leishmania enriettii

The changing status of peritoneal macrophages in guinea pigs infected with Leishmania enriettii has been examined. It was possible to demonstrate that, at certain times during a primary infection and following attempted reinfection of immune animals, the response of peritoneal macrophages to lymphokine contact in vitro was altered. At these times the harvested cells appeared to behave in vitro as if they had been at least partially activated in vivo before removal. They were unresponsive to lymphokine in the migration inhibition assay, and contact with lymphokine in culture caused a rapid increase in the level of glucose oxidation in these cells. It is suggested that changes in the response of macrophages to lymphokine in vitro may be one way of monitoring activation in vivo.     

Changes in macrophages in vivo induced by desensitization.

Peritoneal exudate macrophages were removed from animals sensitized to horse cytochrome c and from similar animals which had been desensitized with this antigen. The ability of lymphokine to induce migration inhibition and also alterations of the level of glucose oxidation in these cells has been examined. It was found that for a transient period after the desensitization, macrophages removed from the peritoneum were unresponsive to lymphokine in the migration inhibition assay. At the same time, culturing these cells with lymphokine for 1 hr caused a significant rise in their glucose oxidation activity. It is suggested from these results that desensitization may result in macrophage activation in vivo. This is discussed in relation to current concepts of the mechanisms of desensitization.     

N-Acetylcysteine inactivates Migration Inhibitory Factor and Delayed Hypersensitivity Reactions

In vitro studies suggest that delayed hypersensitivity follows the production of migration inhibitory factor (MIF) by sensitive lymphocytes in the presence of specific antigen. This factor arrests the migration of macrophages in vitro and in vivo. After attraction, aggregation and activation in vivo, these bystander cells produce toxic substances which induce the local reaction¹. When lymphocytes from tuberculin (PPD) sensitized guinea-pigs were incubated with PPD, cell-free supernatant fluids of the cultures contained MIF². Such migration inhibitory fluids injected intradermally with PPD, into PPD-sensitive animals, enhanced the delayed hypersensitivity reaction³. Concentrated migration inhibitory supernatant fluids injected intradermally into unsensitized animals produced local reactions of induration and erythema within 6 h; reactions reached a maximum after 16 h. Histologically there was an infiltrate of mononuclear cells at the site of injection and neutrophils and eosinophils were also present¹.     

Macrophage activation for tumor cytotoxicity: genetic variation in macrophage tumoricidal capacity among mouse strains.

Development of activated tumoricidal macrophages following Mycobacterium bovis, strain BCG infection in vivo or lymphokine treatment in vitro was examined with more than 20 mouse strains. Peritoneal macrophages from 8 of 22 strains failed to develop tumoricidal capacity by 7 days after intraperitoneal BCG infection. Macrophages from 6 of 6 in vivo nonresponder strains also failed to develop tumoricidal capacity after in vitro treatment with lymphokines. Identification of nonresponder mouse strains should provide a useful resource for analysis of intermediary reactions in macrophage activation.     

Dissociation of in vitro lymphocyte transformation from production of a mononuclear leucocyte chemotactic factor in agammaglobulinemic chickens

Cultures of splenic and peripheral lymphocytes from normal chickens immunized intravenously with Brucella abortus organisms were stimulated by this antigen to incorporate significantly greater amounts of ³H-thymidine and ¹⁴C-leucine than lymphocytes from unimmunized animals. Lymphocytes from immunized agammaglobulinemic chickens were unresponsive to Brucella. This defect could not be corrected by the addition of either normal nonimmune or irradiated normal immune spleen cells to cultures of ag chicken lymphocytes which suggests that normally B cells transform in vitro in response to this antigen. In contrast, cultured peripheral blood leucocytes from both immunized normal and agammaglobulinemic chickens produce significantly more monocyte chemotactic factor in response to Brucella than leucocytes from nonimmune chickens. This indicates that the production of this mediator is a B cell independent function and suggests that T cells are the producers of this lymphokine.     

Induction of protective immunity against Schistosoma mansoni by a nonliving vaccine. III. Correlation of resistance with induction of activated larvacidal macrophages

Mice protected against Schistosoma mansoni infection by intradermal (i.d.) immunization with nonliving larval or adult worm antigens plus bacterial adjuvant developed 24-hr skin test responsiveness to schistosome antigens with the histologic features of delayed hypersensitivity. Intraperitoneal antigen injection elicited a mononuclear cell-enriched exudative population containing macrophages activated for direct cytotoxicity against schistosomula and tumor cell targets. This was likely to be due to in vivo exposure to macrophage-activating lymphokines, since these cells were unresponsive to further lymphokine stimulation in vitro and splenocytes from immunized mice reacted to specific in vitro antigen challenge by production of lymphokines capable of conferring larvacidal activity upon control macrophages. In contrast, mice treated with schistosome antigens by i.v. injection, which were not protected against challenge infection, failed to develop delayed hypersensitivity or activated macrophages in response to specific antigen challenge in vivo, and the titers of macrophage-activating lymphokine produced by in vitro antigen-stimulated splenocytes from these mice were threefold to fourfold lower than those produced by cells from animals immunized by the i.d. route. Thus, sensitization for cell-mediated immune responses including lymphokine production and macrophage activation correlated with induction of resistance to S. mansoni in this model of vaccination.     

Lectin induction of lymphokines in cultured murine leukocytes

Antigens induce sensitized lymphocytes to undergo mitosis and to secrete soluble products, termed lymphokines, which modulate the immune response. Plant lectins are known to act as polyclonal lymphocyte mitogens and, in some cases, stimulate lymphocytes to produce lymphokines. In an effort to explore the relationship of specific cell surface glycoconjugates to the induction of mitosis and the production of lymphokine activities we have examined the ability of the mitogenic lectins, concanavalin A and Wistaria floribunda mitogen, and the nonmitogenic hemagglutinin from Wistaria floribunda seeds to stimulate the production of macrophage migration inhibition factor (MIF), macrophage chemotactic factor (CF), and lymphotoxin (LT). Concanavalin A causes lymphocytes to produce MIF and LT but no detectable CF activities. W. floribunda mitogen induces lymphocytes to produce soluble substances which exhibit all three lymphokine activities. The nonmitogenic W. floribunda agglutinin causes lymphocytes to produce MIF and CF but no observable LT activity. Within the sensitivity of the assays employed, the results indicate that mitogenesis is not a corequisite of the expression of either macrophage migration inhibition factor or lymphocyte-derived chemotactic factor but it may be associated with the induction of lymphotoxin. It is also apparent that the expression of each lymphokine activity is independent of the expression of the other lymphokines studied.     

Functional subsets of human T cells defined by "active" rosette formation

Experiments were conducted defining the possible basis for increased susceptibility of alloxan-treated and genetically diabetic C57Bl/KsJ mice to infections with Candida albicans. Alloxan monohydrate (175 mg/kg) produced a prolonged state of hyperglycemia, which persisted through 31 days. Parameters of immune responses varied depending upon the interval between alloxan administration and testing. In the period immediately following alloxan treatment (1–14 days), the numbers of lymphocytes in the thymus and spleen were reduced, the numbers of recoverable peritoneal macrophages were decreased, and the mice showed an increased susceptibility to intravenous infection with C. albicans. In contrast, splenic lymphocytes responded normally to stimulation with Con A, and in vitro phagocytosis of yeast cells by peritoneal macrophages was normal. Also, in vivo production of such lymphokines as migration inhibitory factor (MIF) and macrophage activating factor (MAF), as well as delayed hypersensitivity footpad responses, was generally within the normal range. In the later phase of alloxan diabetes (21–28 days) after administration of alloxan, lymphoid cellularity recovered progressively and the numbers of recoverable peritoneal macrophages were normal. However, these mice still showed an increased susceptibility to C. albicans infection. Genetically diabetic mice (C57Bl/KsJ, db/db) were abnormal in virtually all the assays involving cell-mediated immunity. The numbers of lymphocytes and peritoneal macrophages were markedly decreased, lymphoid cells responded poorly to Con A, and the phagocytosis of yeast cells by macrophages was depressed. The in vivo production of lymphokines and footpad responses of the delayed-type hypersensitivity were depressed. In addition, these mice were highly susceptible to intravenous infection with C. albicans.     

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.     

Biologic activity of extracts of delayed hypersensitivity skin reaction sites

Extracts obtained from skin sites of delayed hypersensitivity reactions show chemotactic activity for monocytes and lymphocytes but not neutrophils. The soluble extractable factors present at these sites have in vivo activity as well; they promote the accumulation of monocytes in peritoneal exudates and cause inflammatory reactions in the skin of nonimmunized animals. The skin inflammatory infiltrates are predominantly mononuclear and are similar to those of delayed hypersensitivity reactions in actively immunized guinea pigs. The extracts which produced these effects had no detectable MIF activity, nor permeability inducing activity in excess of that obtainable from normal skin.These monocyte and lymphocyte chemotactic factors were analyzed by sucrose density ultracentrifugation. By this technique the distribution of monocyte factors corresponded rather closely with that of the monocyte chemotactic factors obtained from an antigen-activated lymphocyte culture. Similar correspondence was obtained for the bulk of the lymphocyte chemotactic activity present in skin extracts and in culture supernatants. This suggests the possibility that the lymphokine-like substances in the skin extracts might in fact represent lymphokines. Further documentation of this point will provide a link between in vitro and in vivo manifestations of delayed hypersensitivity.     

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.     

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.     

Activity and Properties of Macrophage Migration Inhibitory Factor produced by Mixed Lymphocyte Cultures

THE macrophage migration test is an in vitro demonstration of delayed hypersensitivity. Supernatant fluids of sensitive lymphocytes cultured for 24 h in the presence of specific antigen contain migration inhibitory factor (MIF) that arrests the migration of macrophages of unsensitized animals in vitro¹,². In vivo, it induces delayed skin reactions³. The use of the macrophage migration test, based on differences of transplantation antigens in donor and recipient, to show histocompatibility has been suggested⁴. The test was also recommended as an indicator of immunological reactivity after organ transplantation, to demonstrate impending rejection⁵. It can demonstrate homograft sensitivity, for migration of peritoneal exudate cells (containing lymphocytes and macrophages) of CBA mice previously sensitized by grafts from A/Jax donors was inhibited when they were mixed with peritoneal exudate cells of the donor strain. However, histocompatibility was not demonstrated, for mixtures of peritoneal exudate cells of ungrafted CBA mice and A/Jax mice migrated regularly during the 24 h test⁶.     

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.     

Lymphocyte and macrophage adenyl cyclase activity in animals with enhanced cell-mediated resistance to infection and tumors.

As cyclic AMP has been associated with the inhibition of lymphocyte cytotoxicity, studies were performed to investigate adenyl cyclase activity in lymphocytes and macrophages of Toxoplasma-infected mice in which the efferent limb of the cell-mediated immune response had previously been found to be activated. In peritoneal or splenic lymphocytes from $\text{Balb}\text{c}$ mice chronically infected with Toxoplasma in which growth of an isogeneic bladder tumor was found to be inhibited, adenyl cyclase activity was significantly less than in lymphocytes from uninfected control mice. Stimulation by prostaglandin E₁ or NaF in vitro led to higher levels of adenyl cyclase activity in lymphocytes from unifected animals than in cells from Toxoplasma-infected animals. Similar observations were made with peritoneal macrophages from Toxoplasma-infected and uninfected mice. Lower levels of adenyl cyclase activity were also found in lymphocytes from tumor-bearing mice than in lymphocytes from nontumor-bearing controls. These data suggest that production of cyclic AMP by lymphocytes is inhibited with activation of certain cell-mediated immune functions.     

Resistance of dogs to Echinococcus granulosus

Further studies on the immunization of dogs against E. granulosus indicated that certain dogs have a natural resistance, which is not mediated by specific antibodies or sensitized lymphocytes to tapeworm secretory antigens. In a pilot experiment with two pups, E. granulosus recovered from an immunized pup were arrested in their development and exhibited 100% inhibition of egg production compared to 100% egg production in mature worms recovered from a control pup. When the experiment was repeated with 10 pups, three of five control pups given Freud's adjuvant and Bordetella pertussis carried E. granulosus which were arrested in their development and showed 100% inhibition of egg production. Two of five immunized pups also exhibited signs of resistance. Arrested worms recovered 40 days after infection appeared to be at a stage equivalent to about 18 days of optimal development. Although immunized pups exhibiting resistance showed strong positive in vitro and in vivo immune responses, all control pups gave negative reactions to tapeworm secretory antigens.     

Lymphokine production in mouse mixed lymphocyte reaction (MLR)

We have investigated alloantigen differences which stimulate lymphokine release and³H-TdR uptake in primary ‘one-way’ MLC among allogeneic mice. When mice differing at the wholeH-2 region were tested, MIF and immune IF release was observed, along with a marked³H-TdR uptake. Differences atK, D, orI-S-G regions stimulate both lymphokine release and³H-TdR uptake, though stronger immune IF and³H-TdR responses were observed with differences atI-S-G regions. On the other hand, when mice differing in their minor histocompatibility antigens, and notably at theMls locus, were tested, lymphokine release took place even in the absence of proliferation. Lastly, in MLC between mice differing at multiple minor loci, butH-2 andMls matched, MIF release only, and not immune IF and³H-TdR responses were observed in a few combinations. These findings show that T lymphocytes can recognize alloantigens by releasing lymphokines even without going through proliferation. Moreover, different levels of T-lymphocyte activation exist, depending on the kind of stimulating alloantigens present.     

The effect of levamisole on human lymphocyte mediator production in vitro

Levamisole has previously been demonstrated to increase delayed hypersensitivity reactions in anergic patients. In order to elucidate the mechanism by which levamisole stimulates the immune response in vivo, we have studied the effect of this substance on both human lymphocyte proliferation and lymphocyte-mediator production in vitro. Our results indicate that in vitro levamisole augments the production of soluble mediators by mitogen-stimulated lymphocytes, while having no effect on lymphocyte proliferation.