Interactions Between Macrophages of Guinea Pigs and Salmonellae III. Bactericidal Action and Cytophilic Antibodies of Macrophages of Infected Guinea Pigs

The fate of virulent Salmonella typhimurium within macrophages of guinea pigs was assessed by a suspended cell culture procedure. The present study confirmed that macrophages of normal guinea pigs were capable of inactivating the ingested salmonellae. Macrophages of previously infected guinea pigs were not endowed with any significant increase in their ability to eliminate the ingested pathogen. However, the immune macrophages were observed to clump together tightly when they were exposed to salmonellae. This phenomenon was attributed to the presence of specific cytophilic antibodies on the immune macrophages. When immune macrophages were inactivated with Merthiolate, they agglutinated with both the H and the O antigens of S. typhimurium, but not with the O antigens of other species of Salmonella nor with the O antigens of Escherichia coli. Cytophilic antibodies could be eluted from immune macrophages by incubation in the absence of immune serum. Conversely, cytophilic antibodies could be passively transferred onto normal macrophages by incubation in the presence of immune serum. Furthermore, using immune serum previously adsorbed with the O antigens of S. typhimurium, cytophilic antibodies against the H antigens alone could be transferred onto normal macrophages, or those against the O antigens alone could be eluted from immune macrophages. These data suggest that immune macrophages possess specific cytophilic antibodies against both the H and the O antigens of S. typhimurium. It is proposed that the presence of cytophilic antibodies on immune macrophages represents an expression of antibacterial cellular immunity by enhanced clumping and phagocytic activities of the macrophages.     

Migration Inhibitory Factor and Macrophage Bactericidal Function

A homogeneous population of immunologically active lymphocytes was obtained from peritoneal exudates of guinea pigs with delayed hypersensitivity to bovine gamma globulin (BGG). The lymphocytes were cultured with and without BGG for 24 hr, and cell-free supernatant fluids were then assayed simultaneously for their ability to influence two in vitro parameters of macrophage function: migration from capillary tubes and bactericidal capacity. In four consecutive experiments, supernatants from antigenically stimulated lymphocytes exhibited substantial migration-inhibitory-factor activity without enhancing the ability of macrophages to kill Listeria monocytogenes. Lymphocyte lysates were inactive in both assays. Possible mechanisms of lymphocyte-macrophage interactions are discussed.     

Inhibition of macrophage migration by muramyl peptides.

In the capillary tube migration system a synthetic muramyl dipeptide (MDP; N-acetylmuramyl-L-alanyl-D-isoglutamine), a part of bacterial cell wall peptidoglycans, inhibited the migration of peritoneal exudate macrophages from normal guinea pigs or rats. The migration inhibition was also caused by some MDP-containing peptidoglycan fragments from cell walls of Lactobacillus plantarum and Staphylococcus epidermidis. The migration inhibition could not be explained on the basis of macrophage migration inhibitory factor. A stereochemically highly specific structure of MDP required for its adjuvant activity was also required for the macrophage migration inhibition. These findings suggest that MDP and MDP-containing cell wall fragments may activate macrophages and that this activation may be important in the exertion of their adjuvant activity.     

Establishment of cutaneous Leishmania enriettii infection in hamsters.

A model of a self-healing type of cutaneous leishmaniasis was established in hamsters using the guinea pig parasite Leishmania enriettii. L. enriettii was passaged several times in hamsters without losing its infectivity for guinea pigs or for hamsters. The course of the infection in hamsters resembled that of guinea pigs, with the exception that the lesion at the site of parasite inoculation did not ulcerate and no metastatic lesions developed spontaneously. Moreover, unlike guinea pigs, infected or recovered hamsters were skin test unresponsive to various preparations of L. enriettii antigens. However, histological examination of draining lymph nodes showed features of a cell-mediated immune response, and in vitro inhibition of macrophage migration was demonstrable using peritoneal exudate cells from recovered animals and specific leishmanial antigen. Antibody was demonstrable by indirect immunofluorescence starting 1 week after infection. Recovered animals were immune to reinfection; however, the passive transfer of peritoneal exudate cells or serum from recovered animals did not confer protection against L. enriettii infection in normal animals.     

In Vitro Induction of Nonspecific Resistance in Macrophages by Specifically Sensitized Lymphocytes

Experiments were carried out to determine whether macrophages can be activated in vitro to resist challenge with heterologous microorganisms. Sensitized spleen cells from guinea pigs chronically infected with Toxoplasma gondii were cultured with normal guinea pig peritoneal macrophages in the presence and absence of Toxoplasma antigen. Macrophage monolayers incubated with sensitized spleen cells and antigen were markedly resistant to challenge from Listeria monocytogenes. Resistance was manifested by prolonged survival of the monolayers and rapid intracellular killing of the bacteria. Macrophages incubated with sensitized spleen cells but in the absence of antigen, as well as macrophages cultured with normal spleen cells, in the presence or absence of antigen, were rapidly destroyed. Sensitized spleen cells responded to the presence of Toxoplasma antigen by increased uptake of tritium-labeled thymidine. Supernatant fluid medium obtained from cultures of macrophages, sensitized spleen cells, and antigen contained a macrophage migration inhibitory factor(s). In addition, these supernatant fluids were capable of inducing increased resistance to Listeria in normal macrophages.     

Fat of Coxiella burnetti in macrophages from immune guinea pigs.

The interaction between Coxiella burnetii and peritoneal macrophages obtained from immune guinea pigs was studied by transmission electron microscopy. Phagocytosis and subsequent fate of ingested phase I and II rickettsiae were compared. Phase I rickettsiae were more resistant to phagocytosis than were phase II organisms. Macrophages from phase I- and II-immunized animals were equally capable of phagocytizing rickettsiae. Phase I and II rickettsiae previously treated with normal serum multiplied and destroyed macrophages from guinea pigs that had been immunized with phase II rickettsiae. Phase II organisms were initially suppressed in macrophages from phase I-immunized animals, but eventually multiplied in these cells. In contrast, only phase I organisms were destroyed by macrophages from phase I-immunized animals. Treatment of rickettsiae with immune serum enhanced ingestion by macrophages and potentiated the destruction of organisms by both types of macrophages. The macrophage migration inhibition assay was performed on peritoneal exudate cells from immune animals. Migration of peritoneal macrophages from phase I-immunized guinea pigs was inhibited, whereas macrophages from phase II-immunized animals migrated when cells were cultured in the presence of killed, intact phase I or II C. burnetii.     

In vitro assessment of cellular immunity to vaccinia virus: contribution of lymphocytes and macrophages

The possible contributions of lymphocytes and macrophages to immunity to vaccinia virus were examined in vitro by determination of antigen-specific inhibition of migration of macrophages and the replication of the virus in purified macrophages. Lymphocytes from various anatomic sites of vaccinia virus-immune rabbits mixed with macrophages from nonimmune rabbits resulted in antigen-specific inhibition of macrophage migration; purified lymphocytes of peritoneal exudates were found to be the most potent inhibitors. Macrophages from peritoneal exudates of immune rabbits purified to contain less than 1% lymphocytes were sensitive to inhibition of migration by vaccinia antigens. Virus grew in macrophages from nonimmune rabbits, but failed to replicate in macrophages from the peritoneal cavity of immune rabbits. Alveolar maracrophages from immune rabbits were not inhibited from migrating in the presence of the viral antigen, and the virus replicated in these cells. The data suggest, but do not prove, that cellular immunity to vaccinia virus in rabbits may be mediated both by lymphocytes and by macrophages.     

Comparison of the capillary tube and explant methods for the in vitro assay of tuberculin

Purified antigens prepared from mycobacteria were tested for nonspecific toxicity and tuberculin activity by two in vitro methods. One technique utilized measurements of macrophage migration in 4-day-old cultures of spleen from normal and tuberculin-sensitive rabbits. The other method was a modification of the capillary tube technique. To obtain enough peritoneal macrophages for good quantitation, changes were made in the method of harvesting cells, and in some instances exudates from two or more Wright strain no. 13 guinea pigs were combined. The capillary tube method was as sensitive as the explant method for detecting nonspecific toxicity. Each tuberculin assay included a group of control cultures of cells from sensitive animals, test groups containing two widely spaced concentrations of a standard tuberculin, PPD-S, and one or more concentration of the tuberculin to be tested. Macrophages from tuberculin-sensitive animals were regularly inhibited by 0.25 μg of PPD-S per ml with both in vitro methods. The potency of the test tuberculins relative to that of PPD-S was somewhat greater in capillary tube assays than in explant cultures. A purified tuberculopolysaccharide was equally inhibitory for both normal and tuberculin-sensitive cells in both culture systems.     

Direct Activation of Guinea Pig Macrophages by a B-Cell Mitogen

Since polyclonal B-cell activators (PBA) require macrophages to induce modifications in the lymphocyte in vitro primary response to thymus-dependent antigens, we have investigated whether PBA act directly on macrophages. [14C]glucosamine uptake by guinea pig peritoneal adherent cells after stimulation with purified protein derivative (PPD), lipopolysaccharide (LPS), and dextran sulfate (DxS) was tested. PPD produced an increased [14C]glucosamine uptake, whereas LPS and DxS did not. According to our experiments, (a) PPD does not require the presence of lymphocytes to stimulate macrophages; furthermore, when lymphocytes were present in a concentration higher than 5%, a suppressor effect in the glucosamine uptake was found, and (b) there was no significant difference between the findings when peritoneal adherent cells were cultured in normal medium and in supernatant of lymphocyte cultures stimulated with PPD.     

Lymphokine-mediated stimulation of eosinophil migration in the guinea-pig.

Guinea-pig lymphokines were shown to stimulate the migration of eosinophils from capillary tubes. Eosinophil migration stimulatory activity was produced by Freund's complete adjuvant (FCA)-sensitized lymph node or peritoneal exudate lymphocytes in the presence of purified protein derivative (PPD), as well as by Con A-stimulated lymph node lymphocytes. Like murine and human eosinophil-stimulation promoter lymphokine (ESP), the guinea-pig lymphokine activity is T cell-derived, non-dialysable and resistant to heating at 56 degrees. In contrast to the migration inhibition factor (MIF) which could be adsorbed by macrophages, eosinophil migration stimulatory activity could not be removed by pre-adsorption to macrophages or eosinophils.     

Differential growth of Legionella pneumophila in guinea pig versus mouse macrophage cultures.

Legionella pneumophila is a facultative intracellular bacterium which replicated well in inbred guinea pig strain 2 peritoneal macrophages at a low infectivity ratio. In contrast, the growth of this organism was markedly restricted in mouse (BDF1) peritoneal macrophages, even at a relatively high infectivity ratio. The initial uptake of L. pneumophila organisms by macrophages was similar in both animal species, and both groups of macrophages supported the growth of Listeria monocytogenes. Treatment of L. pneumophila with immune guinea pig serum did not result in restriction of bacterial growth in macrophages, but guinea pig macrophages were readily induced to suppress the growth of L. pneumophila by preincubation with supernatants obtained from mitogen-activated normal guinea pig splenocyte cultures. Thus, lymphokines generated from mitogen-stimulated guinea pig lymphocytes induced a restriction of growth of these organisms similar to that observed naturally with macrophages from mice, which are considered highly resistant to these bacteria. Although guinea pigs are considered highly susceptible to L. pneumophila infections and mice are considered relatively resistant, the mechanism of this difference is not clear. The results of the present study suggest that the restriction of L. pneumophila growth by macrophages relates to host susceptibility to infection and that cell populations permissive for L. pneumophila can be transformed to nonpermissive by products from stimulated lymphocytes but not by opsonization with immune serum.     

Alterations in mouse macrophage migration: a function of assay systems, lymphocyte activation product preparation, and fractionation.

Supernatants from mouse spleen cell and peritoneal cell cultures were tested for the presence of lymphocyte activation products. Supernatants from mouse spleen cell and peritoneal cell cultures incubated with brucella antigens contained a macrophage migration inhibition factor(s) and a macrophage spreading factor(s) only if the cells were harvested from Brucella-infected mice. After dialysis and freeze-drying, the supernatants were fractionated by preparative acrylamide-gel electrophoresis. Three fractions with lymphocyte activation product activity were obtained from the fractionated supernatants of mouse spleen cells and peritoneal cells harvested from Brucella-infected mice and cultured with brucella antigen. One fraction inhibited mouse macrophage migration from capillary tubes but not from agarose wells. A second fraction not only inhibited macrophage migration from both agarose wells and capillary tubes, but also contained an activity(s) that stimulated macrophage migration through Nuclepore filters and induced macrophage spreading. A third fraction timulated macrophage migration from agarose wells and also contained an activity(s) that stimulated macrophage migration through Nuclepore filters. Fractionated supernatants of mouse spleen cells and peritoneal cells harvested from uninfected mice incubated with and without brucella antigen, as well as of cells harvested from infected mice and not incubated with antigen, did not contain detectable lymphocyte activation products.     

Formed blood elements in peritoneal effusion and the macrophage migration inhibition test in healthy guinea pigs and in guinea pigs with experimental allergic encephalomyelitis.

An attempt was made to correlate the percentages of macrophages, lymphocytes and granulocytes in the peritoneal effusion in healthy guinea pigs and guinea pigs with experimental allergic encephalomyelitis (EAE), with the macrophage migration inhibition (MMI) test. Varying percentages of the cells had no influence on values of MMI. Similarly, in guinea pigs with EAE, percentages of formed elements in peritoneal effusion were not correlated with intensity of MMI or with histopathologic lesions in the brain and spinal cord. It is suggested that the observed differences are due to individual immunologic responsiveness of animals and, probably, to other hitherto unknown mechanisms.     

Macrophages are stimulated by muramyl dipeptide to induce polymorphonuclear leukocyte accumulation in the peritoneal cavities of guinea pigs.

N-Acetylmuramyl-L-alanyl-D-isoglutamine (muramyl dipeptide [MDP]) injected intraperitoneally significantly increased the number of cells entering the peritoneal cavity of guinea pigs primed with liquid paraffin or thioglycollate. There was a close relationship between peritoneal polymorphonuclear leukocyte (PMN) accumulation and the uptake of glucosamine by macrophages in guinea pigs treated with a variety of bacterial cell surface components such as cell wall peptidoglycan subunits and bacterial or synthetic lipid A. The PMN accumulation was also facilitated by the intraperitoneal transfer of the peritoneal macrophages that had been stimulated by MDP in vitro. Furthermore, cell-free lavage fluids taken from the peritoneum of MDP-treated guinea pigs also initiated the influx of PMNs when introduced into the peritoneal cavities of liquid paraffin-pretreated guinea pigs. These results suggest that a soluble factor which attracts neutrophils is produced by MDP-treated macrophages. Partial characterization of the factor is described.     

Species-restricted effects of human and mouse lymphokines on macrophages

Lymphokines produced by antigenic or mitogenic stimulation of human, guinea pig and mouse lymphocytes were tested for their effects on monocytes or macrophages of the same and heterologous species, to determine whether there is any species restriction in their reactivity. Supernatants from lymphocyte cultures were tested for migration inhibitory activity in an indirect agarose microdroplet assay and for their ability to augument cytolytic activity of macrophages or monocytes in a [3H]thymidine-release assay. Supernatants of human peripheral blood mononuclear cells, stimulated with phytohemagglutinin or purified protein derivative of tuberculin (PPD) were able to strongly inhibit the migration of human monocytes and guinea pig peritoneal exudate cells (PEC), but had no detectable effect on mouse PEC. The human supernatants could also significantly augment the cytolytic activity of human monocytes, but had no effect on cytotoxicity by mouse peritoneal macrophages. Conversely, supernatants of PPD-stimulated spleen cells from mice immune to bacillus Calmette-Guérin (BCG) strongly inhibited the migration of, and significantly augmented cytolysis by, mouse PEC, but had no detectable effects on human monocytes. Moreover, supernatants of concanavalin A-stimulated lymph node cells from guinea pigs inhibited the migration of guinea pig PEC and human monocytes, but had no effects on mouse PEC. The migration inhibitory effects of the human and mouse supernatants did not appear to be mediated by interferon (IF), since partially purified type-1 IF had no detectable effect. In addition, supernatants of human lymphocytes stimulated by Corynebacterium parvum strain 5888, that induced little or no IF, were able to inhibit the migration of, and augmented cytolysis by, human monocytes. These C.parvum supernatants also showed migration inhibitory activity on mouse PEC but did not induce cytolytic activity in these cells.     

Cytokine production by blue tongue virus-infected fetal sheep cells.

The migration inhibition of guinea pig peritoneal macrophages by a factor(s) from media obtained from blue tongue virus-infected monolayer cultures was studied. Medium from blue tongue virus-infected sheep fetal cell cultures inhibited migration of guinea pig macrophages from agarose droplets. Medium from control cultures and stock virus did not inhibit macrophage migration. Medium containing migration inhibiting factor(s) in vitro induced an inflammatory reaction in the skin of a newborn sheep. The inflammatory reaction was observed 20 h after intradermal inoculation. The skin reaction consisted of infiltrates of mononuclear leukocytes in the superficial dermis. Control medium and stock virus caused no skin reaction.     

Role for Macrophages and Thymus-Derived Lymphocytes in Cholera Toxin-Induced Immunosuppression

The exo-enterotoxin derived from Vibrio cholerae bacilli has marked immunomodulating activities, both in vivo and in vitro. In the present study, the mechanism whereby cholera toxin depresses the antibody-forming ability of murine splenocytes was investigated by in vitro reconstitution experiments. Spleen cells derived from mice treated with cholera toxin 2 days earlier were markedly deficient in their ability to respond to sheep erythrocytes upon challenge immunization in vitro. Addition of graded numbers of normal spleen cells to spleen cell cultures from toxin-treated mice partially restored the antibody response. Adherent splenocyte populations were even more effective in restoring antibody formation. Normal peritoneal exudate cells rich in macrophages were also capable of restoring the antibody-forming ability of toxin-pretreated splenocytes. Furthermore, thymus (T)-derived spleen cells from normal mice, as well as sheep erythrocyte "educated" T cells, were capable of restoring antibody formation to normal levels. The importance of T lymphocytes in restoring immune competence of spleen cell cultures from toxin-treated mice was shown by additional experiments in which T-depleted cell preparations were found to be ineffective in restoring antibody activity. These studies point to macrophages and T-derived lymphocytes as a major target for cholera toxin-induced immunosuppression.     

Effect of age, culture medium and lymphocyte presence on ascorbate content of peritoneal macrophages from mice and guinea pigs during phagocytosis

Changes in ascorbate content have been measured by high-performance liquid chromatography in peritoneal macrophages from mice and guinea pigs stimulated by latex particles, in the presence and absence of peritoneal lymphocytes. There was a significant decrease in ascorbate content in murine macrophages 5 min after stimulation that did not occur in controls. The rate of ascorbate consumption was nearly identical with murine macrophages incubated in three different ascorbate-free culture media (phosphate-buffered saline, Hanks' solution and RPMI) studied, as well as with macrophages from young and old mice. In contrast to mice, both control and stimulated peritoneal macrophages from guinea pigs showed significant decreased contents of ascorbate in the absence of lymphocytes compared with those in the presence of lymphocytes. Moreover, stimulated macrophages showed significantly decreased ascorbate contents with respect to controls. These results indicate that ascorbic acid plays an important role in macrophages and that peritoneal lymphocytes also seem to play a significant role in the maintenance of ascorbate content in macrophages during phagocytosis in guinea pigs.     

Macrophage-bound C3 fragments as adhesion molecules modulate presentation of exogenous antigens.

The involvement of complement in the response to T cell dependent antigens is generally accepted, however the mechanism has not been clarified. We compared the T cell response in vitro, using antigen-pulsed macrophages from normal and genetically C3-deficient guinea pigs, and show, that C3-fragments fixed covalently to the surface of the antigen-presenting cells are involved in the triggering of responder T cells. Binding of guinea pig C3-specific mAb to oil-elicited, OVA- and PPD-pulsed macrophages of C3D guinea pigs is reduced compared to normal cells, while the expression of Ia antigens is the same. C3-like peptides can be immunoprecipitated only from the lysate of oil-elicited normal cells. These C3-fragments are fixed to the cell-membrane via ester-bonds, since they are released upon treatment with hydroxylamine. In comparison with normal cells, the antigen-presenting capacity of macrophages derived from C3D animals is strongly impaired in cultures containing 10% normal guinea pig serum. A further impairment is observed in cultures with 10% C3D guinea pig serum. Two of the tested C3-specific mAb inhibited antigen-induced T cell proliferation in a dose-dependent manner. Our data point to the importance of C3, as a bivalent molecule, having the capacity to facilitate the cooperation between the antigen-presenting cell and the responder T lymphocytes.     

Relationship Between Tuberculin Hypersensitivity and Cellular Immunity to Infection in Mice Vaccinated with Viable Attenuated Mycobacterial Cells or with Mycobacterial Ribonucleic Acid Preparations

The migration inhibition technique has been used to study delayed hypersensitivity in vitro by using peritoneal exudate cells and splenic lymphocytes from mice vaccinated with viable cells of the attenuated H37Ra strain of Mycobacterium tuberculosis and from mice vaccinated with ribonucleic acid (myc RNA) preparations obtained from viable mycobacterial cells of the same strain. Inhibition of macrophage migration was noted when purified protein derivative (PPD) or viable H37Ra cells were added to peritoneal exudate cells obtained from mice immunized with viable H37Ra cells and not from mice immunized with myc RNA. Splenic lymphocyte cultures were exposed to the same antigens in vitro. Filtered supernatant fluids from these lymphocyte cultures, when added to peritoneal exudate cells obtained from nonimmunized mice, inhibited migration only when they were obtained from lymphocytes which came from mice immunized with viable H37Ra cells. Injection of PPD intravenously into vaccinated mice resulted in inhibitory supernatant fluids from splenic lymphocyte cultures only when the lymphocytes came from mice immunized with viable H37Ra cells. However, intravenous injection of either viable H37Ra cells or of myc RNA preparations into mice vaccinated with myc RNA occasionally produced inhibitory supernatant fluids when lymphocytes were obtained from these mice. On the other hand, mice vaccinated with myc RNA or viable H37Ra cell preparations were consistently and equally protected against intravenous challenge with the virulent H37Rv strain. Thus, although some evidence was obtained for a delayed type hypersensitivity in mice vaccinated with H37Ra cells or with myc RNA to ribosomal proteins or other proteins associated with the RNA preparation, no evidence of tuberculin hypersensitivity could be detected in any mice vaccinated with the myc RNA. These results argue against a role for tuberculin hypersensitivity in immunity to tuberculous infection.