Effect of local and systemic macrophage activation in hamsters on infection with Treponema pertenue and Treponema pallidum Bosnia A

The role of nonspecific macrophage activation in the destruction of treponemes needs to be defined. Studies have been hindered by an inability to confirm that macrophages have enhanced bactericidal activity at the site of treponemal infection. We show that subcutaneous and intravenous vaccination with BCG (Mycobacterium bovis) induces macrophage activation in hamsters, as determined by an enhanced ability to suppress the growth of Listeria monocytogenes in the livers, spleens, and inguinal lymph nodes. However, hamsters challenged in the inguinal region with Treponema pertenue during periods of enhanced microbial resistance (3 to 8 weeks after BCG vaccination) developed lesions faster and with more necrosis. Increased numbers of treponemes were recovered from the regional lymph nodes of BCG-vaccinated hamsters than from nonvaccinated controls, although the differences were not statistically significant. No pathological differences were detected in BCG-vaccinated and non-vaccinated hamsters challenged with Treponema pallidum Bosnia A. These studies demonstrate that BCG vaccination influences the pathogenesis of some treponemal diseases without inducing macrophage-mediated treponemicidal activity.     

Treponemal infection specifically enhances node T-cell regulation of macrophage activity.

Hamsters experimentally inoculated in the inguinal region with Treponema pallidum subsp. endemicum develop considerable pathology at that site. We examined the cell populations from these inguinal lymph nodes to determine their intercellular responses to infection. In vitro, syphilitic-node T cells markedly suppressed C3b receptor-mediated ingestion (C3bMI) in syphilitic macrophages derived from sites both proximal and distal to the inoculation. This activity was more pronounced when node T cells rather than peritoneal T cells were used. When treponemal preparations or live treponemes were added to the coculture system, the suppression was specifically enhanced, whereas the addition of heterologous agents did not promote this effect. Syphilitic macrophages from either compartment cultured alone showed no significant inhibition of C3bMI. In parallel studies on syphilitic macrophages, we observed that the expression of Ia quickly became elevated and was sustained throughout the infection. Moreover, in vitro culturing of the syphilitic-node T cells with these macrophages did not alter this function. These observations suggest that the syphilitic node contains a subpopulation of T cells that can selectively suppress macrophage C3bMI activity and concurrently regulate their cellular response to treponemal infection.     

Ability of Macrophages to Process and Present Treponema pallidum Bosnia A Strain Antigens in Experimental Syphilis of Syrian Hamsters

The ability of macrophages to process and present treponemal antigens to T-lymphocytes was studied in early stages of experimental syphilis produced by Treponema pallidum Bosnia A strain (the causative agent of endemic syphilis) infection of inbred Syrian hamsters (LSH/Ss Lak strain). A difference was noticed in the response of macrophages obtained from the peritoneal cavity, lymph nodes, and spleens of the infected animals. In all of these locations, a general increase in the population of Ia^k-positive macrophage was seen during the entire period of infection, i.e., 3 to 18 weeks after inoculation. Peritoneal cavity-derived macrophages showed no difference in antigen presentation to sensitized and nonsensitized T-lymphocytes for the first 7 weeks of infection. However, at 18 weeks after infection, peritoneal macrophages lost their ability to process treponema antigens. Spleen- and lymph node-derived macrophages did not exhibit a parallel loss in their ability to process treponema antigens. A fluctuation without a consistent pattern was noticed in the antigen processing and presentation by macrophages from the spleen and lymph nodes. In general, the sensitized T-lymphocytes responded to treponema antigen presented by macrophages more vigorously than the nonsensitized T-lymphocytes. An increased ability of spleen-derived macrophages to process and present antigens was noticed throughout the entire period of infection. The macrophages from the lymph nodes showed such an increase only temporarily at 3 weeks after infection. These data suggest that the processing and presentation of treponema antigens by macrophages in acute syphilitic infection fluctuates considerably and depends on the source of macrophages and the duration of the infection. The differences in the response of peritoneal cavity-, spleen-, and lymph node-derived macrophages probably reflect the complex interactions between the macrophage and other cells involved in the immune response to treponema infection.     

Tumor necrosis factor and macrophage activation are important in clearance of Nocardia brasiliensis from the livers and spleens of mice.

The roles of tumor necrosis factor (TNF) and macrophage activation in clearance of Nocardia brasiliensis from BALB/c mouse livers and spleens were evaluated. TNF activity was detectable in sera from animals at all stages of infection. Treatment of infected mice with an antiserum against TNF significantly enhanced the experimental infection as judged by enumeration of CFU in the spleens and livers of infected mice. In another set of experiments, a population of activated macrophages from the peritoneal cavities of N. brasiliensis-infected mice was studied by using a cytostatic assay. The observed cytotoxic activity of these activated macrophages against L929 cells was mediated by TNF, since this activity was inhibited by anti-TNF antiserum treatment. The level of TNF activity generated in vitro in the presence of lipopolysaccharide (LPS) by peritoneal macrophages from infected mice was higher than that of adherent peritoneal cells obtained from normal mice after challenge with LPS. When the nocardiacidal activity of peritoneal cells from N. brasiliensis-infected mice was estimated in vitro, a significant decrease in the number of CFU recovered was observed. Moreover, nocardiacidal activity of peritoneal cells obtained from N. brasiliensis-infected mice previously treated with anti-TNF antiserum was significantly reduced compared with the activity of cells obtained from infected mice previously treated with normal rabbit serum and that of cells from uninfected mice. These data suggest a role for TNF in resistance to N. brasiliensis infection.     

Interleukin-17 is not required for classical macrophage activation in a pulmonary mouse model of Cryptococcus neoformans infection

Cryptococcus neoformans is an opportunistic fungal pathogen that causes disease in individuals with suppressed cell-mediated immunity. Recent studies in our laboratory have shown that increases in pulmonary Th1-type and interleukin-17A (IL-17A) cytokine production, classical macrophage activation, and sterilizing immunity are elicited in response to infection with a gamma interferon (IFN-γ)-producing C. neoformans strain, H99γ. IL-17A-treated macrophages, compared to IL-4-treated macrophages, have been demonstrated to exhibit increased microbicidal activity in vitro, a characteristic consistent with classical macrophage activation. The purpose of these studies is to determine the role of IL-17A in the induction of classically activated macrophages following infection with C. neoformans. Immunohistochemistry and real-time PCR were used to characterize the macrophage activation phenotype in lung tissues of mice treated with isotype control or anti-IL-17A antibodies and given an experimental pulmonary infection with C. neoformans strain H99γ. The pulmonary fungal burden was resolved, albeit more slowly, in mice depleted of IL-17A compared to the fungal burden in isotype control-treated mice. Nonetheless, no difference in classical macrophage activation was observed in IL-17A-depleted mice. Similarly, classical macrophage activation was evident in mice deficient in IL-17A or the IL-17 receptor A, which mediates IL-17A signaling, following pulmonary infection with wild-type C. neoformans strain H99 or H99γ. These studies suggest that IL-17A may play a role in the early immune response to C. neoformans but is not required for classical macrophage activation in mice experimentally infected with C. neoformans.     

Effect of acute nutritional deprivation on immune function in mice. I. Macrophages.

This study was designed to explore the effects of acute nutritional deprivation (starvation) on macrophage function in mice. In vivo macrophage activity was increased by starvation, as determined by multiplication of Listeria monocytogenes in both spleens and livers after intravenous injection. Similarly, in vitro studies revealed that the capacity of peritoneal macrophages to kill listeria was enhanced by starvation. This function was increased further by the addition of small concentrations of lipopolysaccharide (LPS; 10-100 ng/ml). The bactericidal activity of macrophages from starved mice, however, did not reach the levels observed with macrophages from BCG-infected mice. Furthermore, LPS did not appear to be an important second signal for macrophage activation in vivo, as LPS-unresponsive mice (C3H/HeJ and A/J) were protected by starvation. In contrast to these results we found that starved mice were not protected against Toxoplasma gondii infection and that macrophages from starved mice were unable to prevent multiplication of toxoplasma trophozoites in vitro. In toto, these experiments suggest that macrophage function is enhanced by starvation, but that this enhancement is not sufficient to fulfill all criteria for macrophage activation.     

Reassessment of the role of splenic leukocyte oxidative activity and macrophage activation in expression of immunity to malaria.

The role of splenic leukocyte oxidative activity and macrophage activation in the development of protective immunity was examined during acute Plasmodium chabaudi adami malaria. Splenic leukocyte oxidative activity was compared in infected BALB/c and P/J mice; the latter are known to suffer from defects in macrophage function. Phorbol myristate acetate-stimulated chemiluminescence and superoxide anion production by splenic leukocytes from infected BALB/c mice were found to be increased dramatically, while the response of splenic leukocytes from infected P/J mice was elevated only minimally. Hydrogen peroxide release was slightly increased in splenic leukocytes from infected BALB/c mice but remained essentially unchanged in those from infected P/J mice. Macrophage function was assessed on the basis of measurements of tumoricidal activity. Splenic macrophages from uninfected BALB/c mice displayed significant tumoricidal activity against L929 target cells. As a result of splenomegaly during infection, tumoricidal activity, when calculated on a per-spleen basis, was increased further in infected BALB/c mice. In contrast, the tumoricidal activity of splenic macrophages from P/J mice was minimal, regardless of infection. Despite these differences, both strains of mice developed malarial infections that resolved within 16 days. Thus, while the production of reactive oxygen radicals by splenic leukocytes and the phenomenon of macrophage activation have traditionally been associated with the resolution of malarial infection, this study failed to establish a correlation between these parameters and the development of protective immunity to blood-stage infection with P. chabaudi adami.     

Induction of unresponsiveness to gamma interferon in macrophages infected with Mycobacterium leprae.

We have previously demonstrated that Mycobacterium leprae-burdened granuloma macrophages isolated from infected nude mice are refractory to activation by gamma interferon (IFN-gamma). To explore further both the afferent and efferent functional capacity of M. leprae-infected macrophages, we examined the IFN-gamma-mediated activation of resident mouse peritoneal macrophages infected in vitro with live or dead M. leprae. When IFN-gamma was administered within 24 h of M. leprae infection, macrophages were fully activated. However, defective activation was evident at 3 to 5 days postinfection in macrophages that were heavily burdened with viable M. leprae. This defect was evident by four parameters of activation in which IFN-gamma failed to stimulate the enhancement of microbicidal activity, cytotoxicity for tumor target cells, O2- production, and surface Ia antigen expression. The development of defective activation closely followed an increase in macrophage production of prostaglandin E2. Defective activation of M. leprae-burdened macrophages was reversible by indomethacin, and a similar block in IFN-gamma activation was observed in three of these four parameters in normal macrophages treated with exogenous prostaglandin E2. Thus, infection of mouse macrophages with M. leprae appears to restrict IFN-gamma-mediated activation at least in part by induction of inhibitory levels of prostaglandin E2.     

Differential regulation of the mitogen-activated protein kinases by pathogenic and nonpathogenic mycobacteria

Mycobacteria are the etiologic agents of numerous diseases which account for significant morbidity and mortality in humans and other animal species. Many mycobacteria are intramacrophage pathogens and therefore the macrophage response to infection, which includes synthesis of cytokines such as tumor necrosis factor alpha (TNF-alpha) and production of nitric oxide, has important consequences for host immunity. However, very little is known about the macrophage cell signaling pathways initiated upon infection or how pathogenic mycobacteria may modulate the macrophage responses. Using primary murine bone marrow macrophages, we established that p38 and extracellular signal-regulated kinases 1 and 2 of the mitogen-activated protein kinase (MAPK) pathways are activated upon infection with different species of mycobacteria. However, we observed decreased MAPK activity over time in macrophages infected with pathogenic Mycobacterium avium strains relative to infections with nonpathogenic mycobacteria. Furthermore, macrophages infected with M. avium produced lower levels of TNF-alpha, interleukin 1beta, and inducible nitric oxide synthase 2 than macrophages infected with nonpathogenic species. Inhibitor studies indicate that the MAPKs are required for the Mycobacterium-mediated induction of these effector proteins. Our data indicate that MAPKs are activated in macrophages upon invasion by mycobacteria and that this activation is diminished in macrophages infected with pathogenic strains of M. avium, resulting in decreased production of important immune effector proteins. The decreased MAPK activation associated with M. avium infections suggests a novel point of immune intervention by this mycobacterial species.     

Intracellular tubercle bacilli-alveolar macrophage lysosomal enzymes interaction in experimental tuberculosis

This study is an attempt to understand the mechanism of macrophage activation and its effect on the microbicidal properties of the macrophage. Alveolar macrophages (AM) from normal and BCG-vaccinated guinea pigs were harvested at intervals of 1, 7, 14, 21, and 28 days. Half of the guinea pigs from each group were challenged intratracheally with Mycobacterium tuberculosis H37Rv. In AM, the levels of three lysosomal enzymes, beta-galactosidase (beta-gal), N-acetylglucosaminidase (N-ac), and lysozyme (lyso), were measured histochemically. The percentage of AM staining positively for these enzymes and the intensity of this staining were estimated as parameters of AM activation, along with the number of intracellular bacilli in these cells. Histochemical methods are preferred to biochemical methods as only the former indicate activation in individual cells. The enzymatic responses of AM depend on the type of vaccination and infection. Thus, beta-gal activity was significantly enhanced in immune animals whereas no such enhancement of activity was observed in the case of N-ac and lyso. The N-ac content was higher in infected animals and in the immune group, whereas lyso fluctuated at different time intervals after infection.     

Canine distemper virus increases procoagulant activity of macrophages.

Inflammatory demyelination in canine distemper has been proposed to be due to a "bystander" mechanism, in which macrophages play an important role. In the present work we studied whether infection of macrophages by canine distemper virus (CDV) results in changes of macrophage functions, including Fc receptor-dependent and -independent phagocytosis, release of reactive oxygen species (ROS), and procoagulant activity (PCA). As a source of macrophages, dog bone marrow cells were seeded in teflon bags and grown for 1-2 weeks, at which time a marked enrichment of macrophages was noted. These cells were infected with the A75/17 strain of CDV. We could not detect any significant difference between uninfected and CDV-infected macrophages with respect to Fc receptor-dependent or -independent phagocytosis or with respect to the release of ROS. However, from Day 4 p.i. to the end of our observation period (10 days p.i.), PCA was up to 10-fold higher in CDV-infected unstimulated macrophage cultures than in uninfected unstimulated cultures of the same age. Increase in PCA was not due to the inoculation procedure by itself nor to components of the inoculum other than CDV; in particular, PCA was not due to contaminating endotoxin. Thus, several important macrophage functions do not appear to be impaired by CDV infection. The marked increase of macrophage PCA expression suggests that certain macrophage functions may even be enhanced as a result of infection. Such macrophage activation might contribute to the pathogenesis of the disease.     

Intracellular acid phosphatase content and ability of different macrophage populations to kill Nocardia asteroides.

It has been reported that the activity of lysosomal acid phosphatase decreases inversely with numbers of ingested virulent Nocardia spp. in normal murine peritoneal and alveolar macrophages. These studies suggested that this relationship correlated with the effectiveness of these macrophage populations in killing Nocardia asteroides. Experiments were designed to determine if acid phosphatase activity is affected by infection with N. asteroides in four different macrophage populations isolated from normal and nocardia-immunized mice. Macrophages were also tested simultaneously for their ability to kill N. asteroides. Peritoneal, alveolar, and splenic macrophages and Kupffer cells were infected in vitro with strains of N. asteroides of differing virulence. Uptake and killing assays were performed. Acid phosphatase levels and numbers of intracellular nocardiae were quantitated in the same macrophages, using a computer-assisted cytophotometry system. Acid phosphatase activity decreased inversely with numbers of intracellular nocardiae in macrophages that could not kill or inhibit this pathogen. Acid phosphatase activity was not significantly changed in macrophages that inhibited growth of, but did not kill, N. asteroides, whereas activity was increased or enhanced in macrophages that killed most of the ingested nocardiae. The order of nocardicidal effectiveness (and resistance to enzyme activity reduction with infection) for normal macrophages was splenic greater than peritoneal greater than alveolar greater than Kupffer. In contrast, the order of these two parameters for macrophages isolated from immunized mice was Kupffer greater than peritoneal greater than alveolar greater than splenic. These results demonstrate that lysosomal acid phosphatase activity is an effective marker of the ability of macrophages to inhibit growth of and kill N. asteroides and that macrophages isolated from different anatomical sites differ functionally from each other with respect to nocardicidal and acid phosphatase activities.     

Activation of p38 mitogen-activated protein kinase attenuates Leishmania donovani infection in macrophages

Leishmania-induced macrophage dysfunctions have been correlated with altered signaling events. In this work, we report that SB203580, a specific inhibitor of p38 mitogen-activated protein kinases (MAPK), increases Leishmania donovani survival in human peripheral blood mononuclear macrophages. Consistent with this finding, activation of p38 and c-jun N-terminal kinase (JNK) MAPK signaling pathways by anisomycin significantly reduced parasite survival within these cells. However, the majority of the effect was seen in a 50% reduction in the percentage of macrophages infected, with little effect on the highly infected macrophages. The observed effect was likely to be due to the p38 MAPK pathway since SB203580 was able to completely reverse the effect of anisomycin. These findings suggest that the previously reported p38 MAPK inhibition by Leishmania infection may be partially overcome by anisomycin. Similar effects were observed in pretreated macrophages or in treatment of infected macrophages. These results suggests that p38 MAPK activation may have a potential therapeutic value in the treatment of visceral leishmaniasis.     

Haemophilus ducreyi-Induced Interleukin-10 Promotes a Mixed M1 and M2 Activation Program in Human Macrophages

During microbial infection, macrophages are polarized to classically activated (M1) or alternatively activated (M2) cells in response to microbial components and host immune mediators. Proper polarization of macrophages is critical for bacterial clearance. To study the role of macrophage polarization during Haemophilus ducreyi infection, we analyzed a panel of macrophage surface markers in skin biopsy specimens of pustules obtained from experimentally infected volunteers. Lesional macrophages expressed markers characteristic of both M1 and M2 polarization. Monocyte-derived macrophages (MDM) also expressed a mixed M1 and M2 profile of surface markers and cytokines/chemokines upon infection with H. ducreyi in vitro. Endogenous interleukin 10 (IL-10) produced by infected MDM downregulated and enhanced expression of several M1 and M2 markers, respectively. Bacterial uptake, mediated mainly by class A scavenger receptors, and activation of mitogen-activated protein kinase and phosphoinositide 3-kinase signaling pathways were required for H. ducreyi-induced IL-10 production in MDM. Compared to M1 cells, IL-10-polarized M2 cells displayed enhanced phagocytic activity against H. ducreyi and similar bacterial killing. Thus, IL-10-modulated macrophage polarization may contribute to H. ducreyi clearance during human infection.     

Effect of insulin-like growth factor-I on Leishmania amazonensis promastigote arginase activation and reciprocal inhibition of NOS2 pathway in macrophage in vitro

We showed previously that insulin-like growth factor (IGF)-I induces an exacerbation of the lesion development in experimental cutaneous leishmaniasis favouring parasite growth within host macrophages. Here we studied the effect of IGF-I in vitro in BALB/c mouse peritoneal macrophages infected with stationary phase Leishmania amazonensis promastigotes. IGF-I was used to pre-incubate either macrophage or parasite before infection of the macrophages or adding it at the start of the Leishmania-macrophage culture and maintaining it throughout the experimental period. Independent of stimulation protocol, IGF-I induced significantly increased parasite growth within macrophages. Arginase activation considered as a key factor in Leishmania growth was studied, and its expression and activity were increased in Leishmania-infected macrophages but significantly more in infected cells upon IGF-I stimulus, an effect specifically inhibited by NOHA. Arginase known to be present on Leishmania was also studied, and its expression and activity were seen in the absence of any stimulus but significantly increased after 5 min of incubation with IGF-I. In addition, Leishmania was pre-incubated with NOHA for 5 min, washed, then macrophages infected observing a significantly reduced parasite burden in both IGF-I-stimulated and non-stimulated macrophages. Reciprocal decrease in the nitric oxide (NO) level and inhibition of nitric oxide synthase (NOS2) expression were also observed in IGF-I-stimulated infected macrophages. Our data strongly suggest that IGF-I induces preferential expression and activation of Leishmania promastigote arginase, contributes to the alternative activation of macrophages in the context of innate immunity and interferes with NOS pathway in infected macrophages probably as a reciprocal effect.     

Macrophage Migration Inhibitory Factor contributes to drive phenotypic and functional macrophages activation in response to Toxoplasma gondii infection

Cytokines are small molecules secreted by numerous cells. Macrophage Migration Inhibitory Factor (MIF) is a cytokine initially described due to its function of inhibiting random macrophage migration. Currently, new functions have been described for MIF, such as stimulating inflammatory functions in response to infections by microorganisms including, Toxoplasma gondii. However, the primordial MIF function related to macrophages has been little addressed. The main purpose of the study was to recapitulate MIF function on macrophages in response to T. gondii infection. To achieve this goal, peritoneal macrophages were collected from C57BL/6WT and Mif1^-/- mice after recruitment with thioglycolate. Macrophages were cultured, treated with 4-Iodo-6-phenylpyrimidine (4-IPP), and infected or not by T. gondii for 24 h. Following this, the culture supernatant was collected for cytokine, urea and nitrite analysis. In addition, macrophages were evaluated for phagocytic activity and T. gondii proliferation rates. Results demonstrated that T. gondii infection triggered an increase in MIF production in the WT group as well as an increase in the secretion of IL-10, TNF, IFN-γ, IL-6 and IL-17 in the WT and Mif1^-/- macrophages. Regarding the comparison between groups, it was detected that Mif1^-/- macrophages secreted more IL-10 compared to WT. On the other hand, the WT macrophages produced greater amounts of TNF, IFN-γ, IL-6 and IL-17. Urea production was more pronounced in Mif1^-/- macrophages while nitrite production was higher in WT macrophages. T. gondii showed a greater ability to proliferate in Mif1^-/- macrophages and these cells also presented enhanced phagocytic activity. In conclusion, T. gondii infection induces macrophage activation inciting cytokine production. In presence of MIF, T. gondii infected macrophages produce pro-inflammatory cytokines compatible with the M1 activation profile. MIF absence caused a dramatic reduction in pro-inflammatory cytokines that are balanced by increased levels of urea and anti-inflammatory cytokines. These macrophages presented increased phagocytic capacity and shared features activation with the M2 profile.     

Macrophages in resistance to rickettsial infections: early host defense mechanisms in experimental scrub typhus.

Several early nonspecific host defense mechanisms were examined in resistant (BALB/c) and susceptible (C3H/He) mice after intraperitoneal inoculation with Rickettsia tsutsugamushi strain Gilliam. Inflammatory exudates were formed in both mouse strains in response to rickettsial inoculation, but the inflammatory response of C3H animals was delayed several days, and influx of peroxidase-positive macrophages occurred late in infection. Peritoneal cells of C3H mice became progressively infected, with 40% of both macrophages and lymphocytes containing intracellular rickettsiae by day 10. The early flammatory response of BALB/c mice was unexpectedly associated with a low percentage of infected peritoneal cells (1 to 2%). In vitro, no difference was detected in ability of resident macrophages of either strain to support the growth of R. tsutsugamushi or to become activated by treatment with lymphokines for rickettsiacidal activity. In vivo, however, macrophages from C3H mice inoculated with Gilliam were not activated on days 6 and 7 after infection, whereas BALB/c macrophages were continuously activated beginning on day 4. The lack of in vivo C3H macrophage activation was not secondary to deficient lymphokine production by infected lymphocytes, as levels of lymphokines produced by peritoneal lymphocytes of both strains were similar and peaked on day 7 after infection. Susceptibility to infection appears to be related to defective regulation of macrophage responses rather than to defects in macrophage function.