Macrophage Dependence of Polyriboinosinic Acid-Polyribocytidylic Acid-Induced Resistance to Herpes Simplex Virus Infection in Mice

The relative contributions of macrophages and lymphocytes to the induction of resistance to primary herpes simplex virus type 1 (HSV-1) infection by polyriboinosinic-polyribocytidylic acid complex [poly (I:C)] were investigated in C58 mice. The induction of resistance was found to be strongly dependent on macrophages compared to lymphocytes. Macrophage-deficient (silica-treated) mice produced less interferon and were not as responsive to prophylactic treatment of HSV-1 infections with poly (I:C) as were either normal, lymphocyte-deficient (cyclophosphamide-treated), or T-lymphocyte-deficient (anti-thymocyte serum-treated, adult-thymectomized) mice. Silica and cyclophosphamide treatments reduced the therapeutic activity of poly (I:C), whereas T-cell depletion did not have a significant effect. Similarly, the protection of mice with exogenous interferon was markedly reduced in silica-treated mice and moderately reduced in cyclophosphamide-treated mice, but unaffected in T-cell-deficient mice. Furthermore, suppression of HSV-1 plaque formation was obtained by cocultivation of infected mouse fibroblast monolayers with peritoneal (macrophage-rich) cells, but not with splenic (lymphocyte-rich) cells, from poly (I:C)-treated mice. Peritoneal cells did not protect heterologous (human) fibroblasts, suggesting that the protection of mouse embryo fibroblasts is mediated by interferon. Collectively, the data indicate that macrophages are required for the production of poly (I:C)-induced interferon and that macrophages and perhaps B-lymphocytes are important for mediating the protection against HSV-1 infection after interferon has been produced.     

Macrophage extrinsic antiviral activity during herpes simplex virus infection

Peritoneal macrophages from mice infected with herpes simplex virus type 2 (HSV-2) exhibited extrinsic antiviral resistance. When the macrophages were co-cultivated in vitro with virus-infected cells the yield of virus was reduced markedly. Activity was not present 1 to 2 days p.i., peaked at 3 to 4 days, declined by 7 days and was absent at 14 days after HSV-2 infection. The extrinsic antiviral activity was limited to the adherent peritoneal macrophage population. The macrophage antiviral activity was also dose-dependent, with approx. 10(6) macrophages (macrophage: host cell ratio of approx. 2:1) reducing virus plaques by greater than 90% and virus yield 1.5 to 3.0 log10. Comparable extrinsic antiviral activity was also exhibited by Corynebacterium parvum- or thioglycollate-elicited peritoneal macrophages. The macrophage activity was not species-specific, activity on Vero cells or syngeneic mouse embryo fibroblasts being comparable. Activity was also not virus-specific, as the active macrophages also inhibited vesicular stomatitis virus (VSV). The antiviral effects required viable macrophages; cell lysates did not inhibit virus growth.     

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.     

Role of activated macrophages in resistance of congenitally athymic nude mice to hepatitis induced by herpes simplex virus type 2.

Congenitally athymic nude (nu/nu) mice of a BALB/c genetic background were found considerably more resistant to the induction of focal necrotic hepatitis by herpes simplex virus type 2 (HSV-2) tha, were phenotypically normal littermates (nu/+) or BALB/c mice. The augmented resistance was age dependent, as it was only manifested in mice from 4 to 5 weeks of age. Studies of the course of infection showed that nude mice were able to restrain virus multiplication in the liver far better than normal mice in the early phase of infection. However, they seemed inferior to normal mice in eliminating the infectious process. In vitro investigation of peritoneal macrophages revealed that macrophages from 6-week-old nude mice exhibited accelerated spreading and were three times as restrictive in the replication of HSV-2 as macrophages from normal mice. However, no difference was found in the efficiency of adsorption/phagocytosis between macrophages from nude and normal mice. The increased resistance of nude mice could be abolished by blockade of the microphage function of the mice by silica. Nude mice reconstituted at birth with thymus cells were just as susceptible to infection as normal mice. These data suggest that the increased resistance of nude mice to HSV-2 hepatitis is due to the presence of nonspecifically activated macrophages before infection.     

Role of macrophages in hepatitis induced by Herpes simplex virus types 1 and 2 in mice.

A marked difference was found between herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) in the induction of hepatic necrotic lesions in mice inoculated intraperitoneally. Although HSV-2 produced many large, progressive liver lesions in 4-week-old BALB/c mice, HSV-1 only occasionally induced a few, self-limiting foci, which eventually healed. This was reflected in the isolation of HSV from the liver and spleen, two organs that are rich in macrophages. Although HSV-1 could be only temporarily isolated, HSV-2 was found in the two organs until the mice died. On the other hand, no such difference was found in the isolation of virus from the brain, which contains no macrophages, and the mice eventually died from encephalitis. This difference in hepatic involvement caused by the two virus types was found to parallel a marked difference in the restriction of HSV-1 and HSV-2 replication by macrophages as measured by an infectious center assay in vitro. HSV-2 produced 17 times as many infectious centers in infected peritoneal macrophage cultures as did HSV-1. Furthermore, the HSV-2 plaques in the cell overlay were large and increasing in size, whereas the HSV-1 plaques were small and showed regression on prolonged incubation. It was shown that this diversity was unique to the macrophage population and not caused by differences in the uptake of virus by macrophages. This model involving two closely related virus types shows the importance of tissue macrophages in the primary host defense against virus infections.     

Analysis of Ia-antigen(s) positive macrophages in experimental murine toxoplasmosis

The present study has demonstrated marked increase in the relative percentage of Ia-positive macrophages among the peritoneal macrophage population(s) of BALB/c mice infected intraperitoneally with Toxoplasma gondii or when chronically infected mice were rechallenged with Toxoplasma. The observed increase in Ia-positive peritoneal macrophage numbers coincided with the appearance of interferon-gamma (IFN-gamma) in the sera of infected mice. The appearance of elevated serum IFN-gamma titer was a temporary event but the increased percentage of Ia-positive macrophages persisted. The data presented have documented that antigen-specific proliferation response of vigorously purified Toxoplasma-sensitized T-cells is dependent upon Ia-bearing macrophages. This model is useful for future studies into various aspects of the interaction(s) between parasite antigens and Ia-antigen(s) at the macrophage surface for successful recognition of antigen-specific T-cells.     

Macrophages and age-dependent resistance to hepatitis induced by herpes simplex virus type 2 im mice.

An age-dependent increase in the resistance of BALB/c mice to induction of focal necrotic hepatitis by herpes simplex virus type 2 was demonstrated. In 3-week-old mice inoculated intraperitoneally with virus, numerous necrotic foci developed in the liver. As the mice matured, the number of lesions declined until the age of 8 weeks, when no further increase in resistance appeared. Corresponding to this, the virus titers of livers and spleens of 3-week-old mice were higher than in 8-week-old animals throughout the infection, and the infection was apparently terminated in these organs of the adult mice by day 5. In vitro infection of peritoneal macrophages from 3-week-old and 8-week-old mice showed that this age-related resistance was concomitant with an increased restriction of virus replication in peritoneal macrophages from adult mice. Since, furthermore, the resistance of adult mice could be abolished by intravenous inoculation of the macrophage-toxic agent silica before infection, and since adoptive transfer of 2 X 10(6) syngeneic macrophages from adult mice to young ones conferred to the latter a resistance comparable to that of the adult mice, it is concluded that macrophage maturation is responsible for the age-dependent resistance seen in this infection.     

Role of immune cells in animal models for inherited neuropathies: facts and visions

Mice heterozygously deficient in the peripheral myelin adhesion molecule P0 (P0+/- mice) are models for some forms of Charcot-Marie-Tooth (CMT) neuropathies. In addition to the characteristic hallmarks of demyelination, elevated numbers of CD8-positive T-lymphocytes and F4/80-positive macrophages are striking features in the nerves of these mice. These immune cells increase in number with age and progress of demyelination, suggesting that they might be functionally related to myelin damage. In order to investigate the pathogenetic role of lymphocytes, the myelin mutants were cross-bred with recombination activating gene 1 (RAG-1)-deficient mice, which lack mature T- and B-lymphocytes. The immunodeficient myelin mutants showed a less severe myelin degeneration. The beneficial effect of lymphocyte-deficiency was reversible, since demyelination worsened in immunodeficient myelin-mutants when reconstituted with bone marrow from wild-type mice. Ultrastructural analysis revealed macrophages in close apposition to myelin and demyelinated axons. We therefore cross-bred the P0+/- mice with spontaneous osteopetrotic (op) mutants deficient in the macrophage colony-stimulating factor (M-CSF), hence displaying impaired macrophage activation. In the corresponding double mutants the numbers of macrophages were not elevated in the peripheral nerves, and the demyelinating phenotype was less severe than in the genuine P0+/- mice, demonstrating that macrophages are also functionally involved in the pathogenesis of genetically mediated demyelination. We also examined other models for inherited neuropathies for a possible involvement of immune cells. We chose mice deficient in the gap junction component connexin 32, a model for the X-linked form of CMT. Similar to P0-deficient mice, T-lymphocytes and macrophages were elevated and macrophages showed a close apposition to degenerating myelin. We conclude that the involvement of T-lymphocytes and macrophages is a common pathogenetic feature in various forms of slowly progressive inherited neuropathies.     

Activity of the keggin polyoxotungstate pm-19 against herpes simplex virus type 2 infection in immunosuppressed mice: Role of peritoneal macrophage activation

The in vivo antiviral activity of the Keggin polyoxotungstate PM-19 [K₇(PTi₂W₁₀O₄₀) · 6H₂O] against herpes simplex virus type 2 (HSV-2) was investigated in mice immunosuppressed by cyclophosphamide (CY). When PM-19 was administered intraperitoneally to immunosuppressed mice for 3 days (once daily) starting at the time of infection, it prevented death due to HSV-2 encephalitis in a dose-dependent manner (10–25 mg/kg). The in vivo anti-HSV-2 activity of PM-19 was superior to that of acyclovir. Intraperitoneal administration of PM-19 to the immunosuppressed mice significantly increased the number of peritoneal cells, especially macrophages. PM-19 did not stimulate interferon-inducing activity or natural killer cell activity, but markedly enhanced peritoneal macrophage functions: (1) phagocytic activity as assessed by measuring the amount of⁵¹Cr-labeled sheep red blood cells taken into the macrophages, and (2) extrinsic antiviral activity as monitored by reduction in the numbers of plaque formed upon cocultivation of HSV-2-infected HEL cells with the macrophages. These results point to the role of peritoneal macrophage activation in the activity of PM-19 against HSV-2 infection in immunosuppressed mice.     

Effects of granulocyte/macrophage colony-stimulating factor on the development and differentiation of CD5-positive macrophages and their potential derivation from a CD5-positive B-cell lineage in mice.

In co-cultures of either the murine pre-B cell line J13, fetal liver cells, or adult peritoneal or bone marrow cells with ST2 mouse bone marrow stromal cells in the presence of granulocyte/macrophage colony-stimulating factor (GM-CSF), the development of CD5+ macrophages was demonstrated by immunohistochemical staining and flow cytometry. Although CD5+ macrophages were not present in the peritoneal cavities of normal mice, approximately 30% of the peritoneal macrophages in viable motheaten (mev/mev) mice, deficient in SHP-1 protein tyrosine phosphatase, expressed cell surface CD5 and B220, markers for B cells. In the mev/mev mice, GM-CSF level in peritoneal fluid was increased significantly. At 5 days after daily intravenous injection with GM-CSF, many CD5+ macrophages appeared in the peritoneal cavity and in omental milky spots of normal mice but fewer in osteopetrosis (op) mutant mice, deficient in macrophage (M)-CSF. These results indicate that GM-CSF, in combination with M-CSF, induces the development and differentiation of CD5+ macrophages in the peritoneal cavity, particularly in the omental milky spots of mice. In the peritoneal cavity of GM-CSF-treated mice, the percentages of hematopoietic progenitor cells doubly positive for CD5 and CD34 or c-kit and of macrophage precursor cells doubly positive for CD5 and ER-MP58 or ER-MP20 were increased significantly during the development of CD5+ macrophages and CD5 B cells, suggesting that CD5+ macrophages and B cells may share a bipotential progenitor in vivo.     

Role of Macrophages in Resistance to Murine Cytomegalovirus

The role of macrophages in protecting mice from murine cytomegalovirus (MCMV) was studied in Swiss, CBA/J, and C57BL/6J mice. CBA/J mice were more resistant to virus than were C57BL/6J mice at all ages tested. Prior treatment of adult Swiss mice with 60 mg of silica, a dose selectively toxic to macrophages, increased mortality due to MCMV infection. Transfer of syngeneic adult macrophages to suckling mice significantly increased their resistance to subsequent MCMV infection. Transfer of syngeneic, nonimmune adult lymphocytes to suckling mice also had a lesser but significant protective effect against subsequent MCMV challenge. In vitro infection of adult CBA/J and C57BL/6J macrophages with virulent and attenuated MCMV resulted in productive infection in only a small percentage of cells and recovery of very little virus from the extracellular fluid. Infection of CBA macrophages was no less productive than C57BL/6J nor was infection with virulent virus more productive than with attenuated virus. Histological examination of the livers of MCMV-infected CBA/J and C57BL/6J mice suggested that divergent cellular immune responses to infection might account for differences in susceptibility. It is postulated that the macrophage may facilitate the inductive phase of cellular immunity, one possible explanation for its demonstrated importance in host defenses against MCMV.     

The initial suppression of bacterial growth in a salmonella infection is mediated by a localized rather than a systemic response

Mice were infected intravenously with two antibiotic resistance tagged variants of the same S. typhimurium strain given in close succession, or simultaneously with strains of different virulence. The first manifestation of acquired resistance--suppression of exponential bacterial growth in liver and spleen--occurred independently for the different strains in the same individuals, implying that it is due to localized rather than systemic events. This early suppression of bacterial growth was ablated by whole body X-irradiation (800R), whereas the immediately preceding phase of exponential growth (Ity controlled innate resistance) was not affected. Transfer of spleen cell suspensions from infected mice into syngeneic recipients conferred protection by suppressing the growth of an intravenous challenge. Pre-treatment of the suspensions to deplete them of macrophages abolished their protective capacity, while depletion of T-cells did not. Mice deficient in T-cells by adult thymectomy and anti-T-cell monoclonal antibody treatment were able to suppress the growth of an intravenous challenge. Taken collectively, the present data show that the very early phase of acquired resistance to salmonellae, essential for survival, is not the result of systemically developing resistance but a localized event at the site of infection.     

In vitro reaction to orthopaedic biomaterials by macrophages and lymphocytes isolated from patients undergoing revision surgery

Periprosthetic tissues observed at sites of loose total joint implants exhibit abundant macrophages, lymphocytes, fibroblasts and particulate debris. Macrophages phagocytose orthopaedic debris and release proinflammatory cytokines, chemokines, matrix metalloproteinases and other substances. In addition, other cell types present in tissues harvested from the bone-implant interface are thought to influence periprosthetic bone resorption. The present study examined the effects of polymethylmethacrylate (PMMA), cobalt chrome molybdenum alloy (CoCr), and titanium-alloy particle challenge on macrophages co-cultured with lymphocytes in vitro. Potential synergistic effects of lymphocytes on macrophage activation were determined by measuring interleukin-6 and tumor necrosis factor-alpha release following exposure to orthopaedic biomaterial particles. Exposure of macrophages or macrophages co-cultured with lymphocytes to all three types of particles resulted in increased release of interleukin-6 and tumor necrosis factor-alpha at 48 h, when compared to macrophages or macrophages co-cultured with lymphocytes, respectively, cultured in the absence of particles. Lymphocytes isolated from periprosthetic tissues secreted increased basal levels of cytokines relative to peripheral blood lymphocytes. Higher doses of PMMA and titanium-alloy particles stimulated increased levels of cytokine release in the macrophage and macrophage/lymphocyte groups. In contrast, a higher dose of CoCr particles (0.075% v/v) was not as effective as the 0.015% v/v dose, indicating probable CoCr toxicity. The macrophage/lymphocyte co-culture did not show synergism between the two types of cells with respect to cytokine release. T-cells at the bone-implant interface may alter the biological response to particulate debris.     

Herpes simplex virus type 2 infection induced apoptosis in peritoneal macrophages independent of Fas and tumor necrosis factor-receptor signaling.

Freshly isolated macrophages from mature mice are poorly or nonpermissive for infections with HSV. However, despite lack of significant viral replication, HSV infection has been demonstrated to induce substantial cell death among macrophages. To determine if HSV-induced cytotoxicity of macrophages is due to apoptosis, peritoneal macrophages were obtained from C57BL/6 (B6) mice, and apoptosis was analyzed following HSV-2 infection in vitro. Macrophages underwent apoptosis upon HSV-2 infection indicated by annexin V staining, labeling of DNA strand breaks and electronmicroscopy. Apoptosis was associated with macrophage activation demonstrated by upregulation of MHC class II and Mac-1 surface expression. Though there was also an upregulation of Fas (Apo-1/CD95) and tumor necrosis factor (TNF)-receptor 1 (TNF-R1) pathways, inhibition of Fas by soluble Fas and blocking of TNF-alpha using a TNF-binding protein did not prevent HSV-induced apoptosis. Moreover, apoptosis was not impaired in HSV-2 infected macrophages from Fas-deficient B6-lpr/lpr mice suggesting involvement of other apoptosis pathways, or activation of Fas or TNF-R pathways downstream of the receptor level. The present results demonstrate that HSV-2 infection leads to activation and subsequent apoptosis in peritoneal macrophages independent of Fas or TNF-R1 signaling.     

Experimental histoplasmosis in the beige mouse

Mice carrying the beige mutation (bg/bg) on a C57Bl/6 background were challenged with Histoplasma capsulatum. bg/bg mice had higher mortality and higher lung tissue fungal counts in their lungs than either bg/+ or C57Bl/6 mice challenged with equal inocula. Immunologic studies showed that bg/bg mice developed normal delayed-type hypersensitivity (DTH) reactions to histoplasmin, but had deficient NK cell cytotoxic activity against YAC-1 target cells. Studies of macrophage killing of H. capsulatum in vitro showed that T lymphocytes of either bg/+ or bg/bg mice were able to activate fungal killing by bg/+ but not by bg/bg macrophages. These studies, while not excluding a role for the NK cell, suggest that macrophage dysfunction may be critical in the greater susceptibility of the bg/bg mouse and, by extension, that macrophage function is of major importance in host defense against H. capsulatum.     

Analysis of age-dependent resistance to murine coronavirus JHM infection in mice.

Resistance to intraperitoneal murine coronavirus JHM infection in mice develops with age. C3H mice were found to be fully susceptible up to the age of 20 days and resistant after 23 days of age. Protection of susceptible animals from death due to infection could be achieved by maternal antibodies or by transfer of spleen cells from immunized, but not from nonimmunized, donor mice. Lack of protection by transfer of unprimed adult spleen cells was not related to immunosuppression by the host. Moreover, resistance of adult mice could not be abrogated by application of lymphocytes from suckling mice, although immune suppression by other means did affect the resistance of adult animals. On the other hand, spleen cells from nonimmunized mice could be primed with inactivated JHM virus in suckling mice and protected these mice from death due to a subsequent virus infection. Thus, the outcome of infection with JHM virus in suckling and adult mice can be influenced by immunological events, but is not exclusively due to the different stages of immune competence.     

Effect of silica on the pathogenic distinction between herpes simplex virus type 1 and 2 hepatitis in mice.

The role of macrophages in the difference in liver pathogenicity between herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) in mice was investigated by selectively blocking the macrophage function of the mice by silica. Intravenous administration of 3 mg of silica 2 h before virus inoculation partially abolished the difference between the two virus types, as judged by macroscopic and microscopic examination of the livers and by virus isolation studies. Intraperitoneal inoculation of 50 mg of silical before virus seemed more effective in suppressing the macrophage function, since this treatment almost completely eliminated the difference in hepatotropism between HSV-1 and HSV-2 as assessed by the number and size of the lesions appearing in the liver. The final outcome of the infection, death from encephalitis, was, however, not influenced by macrophage blockade.     

Resistance to listeriosis in mice that are deficient in the fifth component of complement.

Infection with Listeria monocytogenes was studied in strains of mice with genetic absence of the fifth component of complement (C5). Mice deficient in C5 consistently showed an increased growth of Listeria in their spleens as compared to normal mice. This increased growth was not corrected by administration of plasma containing C5. Furthermore, depletion of C5 and terminal complement components by administration of cobra venom factor did not impair the resistance to Listeria infection of normal mice. No phagocytic defect could be detected in macrophages from strains lacking C5. Transfer of bone marrow cells from C5+ but not from C5- mice corrected the marked increase of Listeria growth in mice having blockade of the reticuloendothelial system. We hypothesize that the defect of mice lacking C5 lies not in the absence of serum C5 but somewhere at the level of the macrophage.     

CD40-CD40 Ligand Interactions Augment Survival of Normal Mice, but Not CD40 Ligand Knockout Mice, Challenged Orally with Salmonella dublin

Interactions between CD40 expressed on macrophages and CD40 ligand expressed on T lymphocytes can be an important signal for optimal macrophage activation. Previous studies have demonstrated that the optimal response against certain intracellular pathogens (e.g., Crytosporidium and Leishmania spp.) by macrophages requires CD40-CD40 ligand interactions. However, this finding is not universal, since two recent reports utilizing CD40 knockout mice have shown no such contribution to the protective immune response against Mycobacterium tuberculosis or Histoplasma capsulatum. We demonstrate here that CD40-CD40 ligand interactions are significant events in the protective response against the intracellular pathogen Salmonella dublin in normal mice but not for animals genetically deficient in CD40 ligand expression. Treating BALB/c mice exogenously with a CD40 agonist (i.e., soluble trimeric CD40 ligand) increased resistance against a lethal, orally administered dose of S. dublin. Conversely, in vivo administration of a monoclonal antibody against CD40 ligand to block endogenous CD40-CD40 ligand interactions resulted in a decreased resistance to salmonellosis. In contrast, CD40 ligand knockout mice demonstrated no increased susceptibility to salmonellosis. In vitro treatment of Salmonella-infected macrophages from BALB/c mice with soluble trimeric CD40 ligand resulted in an elevated production of interleukin 12p70 by these cells, suggesting a mechanism whereby CD40-CD40 ligand interactions might enhance protective immune responses to this pathogen. Taken together, these studies strongly suggest that CD40-CD40 ligand interactions in normal mice play an important protective role in immune responses against the gram-negative, intracellular pathogen S. dublin.