Infection Of Peritoneal Macrophages Research Articles

Protein phosphorylation in murine peritoneal macrophages induced by infection with Salmonella species

Infection of peritoneal macrophages from C3H/HeN and C3H/HeJ mice with Salmonella typhimurium or S. enteritidis induced extensive phosphorylation in a set of proteins with molecular masses of 85, 72, 35, 30, and 23 kDa, which were different from those induced by bacterial lipopolysaccharide. The phosphorylated proteins of 35, 30, and 23 kDa (pp35, pp30, and pp23, respectively) originated from the infecting bacteria, because living bacteria could induce these phosphorylated proteins themselves, and no induction of the proteins occurred in macrophages after phagocytosis of heat-killed or UV-irradiated organisms. When the infected macrophages were disrupted and separated into bacterial and macrophage debris fractions, pp85 and pp72 remained in the macrophage debris fraction, with none in the bacterial fraction. Induction of pp85 and pp72 in infected macrophages was inhibited in the presence of chloramphenicol but not cytochalasin D, suggesting that bacterial growth in the macrophages is necessary for induction of both proteins. Neither of these proteins could be detected in macrophages infected with Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, or Listeria monocytogenes. These results support the view that phosphorylation of the 85- and 72-kDa proteins occurs in the macrophages during the early phases of the interaction between Salmonella organisms and macrophages. The functions of specific proteins remain to be clarified.

Infection of macrophages with Legionella pneumophila induces phosphorylation of a 76-kilodalton protein.

Infection of peritoneal macrophages from susceptible A/J mice with Legionella pneumophila induced phosphorylation of a 76-kDa protein. The phosphorylation occurred when macrophages were infected with a virulent strain of L. pneumophila but did not occur when they were infected with an avirulent strain or with other bacteria such as either Pseudomonas aeruginosa or Salmonella typhimurium. Also, no phosphorylation of this protein was observed when macrophages were stimulated with either lipopolysaccharide or phorbol myristate acetate. However, phosphorylation did occur in macrophages infected with a virulent strain of L. pneumophila and treated with either erythromycin to inhibit growth or with cytochalasin D to inhibit uptake of L. pneumophila by macrophages. These results support the view that phosphorylation of this protein occurs during the early phases of interaction between L. pneumophila and macrophages. The role of this specific protein in the recognition, intracellular uptake, and growth of L. pneumophila in permissive macrophages remains to be clarified.

Trypanosoma cruzi: effect of phenothiazines on the parasite and its interaction with host cells.

Phenothiazines were observed to have a direct effect on Trypanosoma cruzi and on its in vitro interaction with host cells. They caused lysis of trypomastigotes (50 uM/24 h) and, in axenic medium, dose-dependent inhibition of amastigote and, to a lesser extent, epimastigote proliferation. Treatment of infected peritoneal macrophages with 12.5 uM chlorpromazine or triflupromazine inhibited the infection; this effect was found to be partially reversible if the drugs were removed after 24 h of treatment. At 60 uM, the drugs caused damage to amastigotes interiorized in heart muscle cells. However, the narrow margin of toxicity between antitrypanosomal activity and damage to host cells mitigates against in vivo investigation at the present time. Possible hypotheses for the mechanism of action of phenothiazines are discussed.

Monoclonal antibodies to Leishmania mexicana promastigote antigens II. Cellular localization of antigens in promastigotes and infected macrophages

In the accompanying paper by Ilg et al., it was shown that Leishmania mexicana promastigotes covalently modify a secreted acid phosphatase and other proteins by carbohydrate epitopes characteristic for lipophosphoglycan (LPG). In this study, the reaction of the anti-LPG monoclonal antibodies (mAbs AP3 and L7.25) and of mAb L3.13, an antibody directed against an epitope present on the secreted acid phosphatase but not on LPG, with promastigotes and infected peritoneal macrophages is studied by immunofluorescence and immunoelectron microscopy. AP3 labels the surface, the flagellar pocket and intracellular structures in promastigotes, while L3.13 reacts predominantly with an antigen located in the flagellar pocket. Early after infection with promastigotes, but not amastigotes, AP3 or L7.25 transiently label epitopes at the surface of live macrophages. No L3.13-reactive material is detected at the surface of infected macrophages. In permeabilized, infected macrophages, AP3 heavily labels the surface of amastigotes and the lumen of the parasitophorous vacuole, while L3.13 reveals antigen in the flagellar pocket, intracellular vesicles of amastigotes, and components in the lumen of the parasitophorous vacuole. Possible mechanistic implications for Leishmania-macrophage interaction raised by these findings are discussed.

Infection of peritoneal macrophages in vitro and in vivo with feline immunodeficiency virus

Macrophages were harvested from the peritoneal cavities of healthy specific-pathogen-free cats by saline lavage. Three days before collection, the peritoneal cavities were stimulated with glutaraldehyde-fixed Saccharomyces cerevisiae cells to induce greater numbers of macrophages and to begin the activation sequence. Peritoneal macrophages from cats stimulated once with yeast consisted mainly of small macrophages and a smaller number of larger activated macrophages. After several days in culture, many of the small macrophages became activated and a portion of the activated macrophages developed into multinucleated giant cells. Peritoneal cells from cats that were stimulated twice or three times with yeast at 3-week intervals consisted of a higher proportion of activated macrophages initially and produced more and larger multinuclear giant cells with time. Cultures of peritoneal cells stimulated once with yeast were easily infected in vitro with feline immunodeficiency virus (FIV) and produced a transient burst of reverse transcriptase activity. After the initial burst of virus replication, the infection became latent. Even more multinucleated giant cells appeared after infection, and many of these cells fused with each other. Replicating virus could be rescued from the latently infected macrophages after 2 to 3 weeks of phorbol myristate acetate stimulation and cocultivation with T-lymphocyte-enriched peripheral blood mononuclear cells. Multiply stimulated peritoneal cells, which contained a much higher proportion of activated macrophages, could also be infected in vitro with FIV. The infection usually became latent, however, without going through an initial replicative stage. Peritoneal cells from chronically FIV-infected specific-pathogen-free cats contained a higher proportion of activated macrophages and were latently infected with FIV from the outset.

Poly I:C-induced antiviral activity to HSV-1 infection in peritoneal macrophages

Poly I:C-induced antiviral activity to HSV-1 infection in peritoneal macrophages

Molecular localization of abortive infection of resident peritoneal macrophages by herpes simplex virus type 1

Mononuclear phagocytes exhibit different patterns of intrinsic resistance to herpes simplex virus type 1 (HSV-1) that are related to the heterogeneity of macrophage populations and may reflect the particular differentiation or maturation state of the macrophages. In this study, we characterized the molecular basis for the block in HSV-1 replication in resident peritoneal macrophages from B6C3F1 mice. Infected resident peritoneal macrophages were analyzed for the presence of virus-specific mRNA by Northern (RNA) blotting and in situ hybridization and for proteins by immunofluorescence. The data were compared with those obtained in HSV-1-infected permissive Vero cells. The immediate-early genes ICP4, ICP0, ICP22, and ICP27 were transcribed in resident peritoneal macrophages, as was the early gene tk. Virus-specific mRNA for the major DNA-binding protein ICP8 was barely detectable, and that for another early gene, the viral DNA polymerase, was not detected. In addition, transcripts for the delayed-early gene glycoprotein D and the true late gene glycoprotein C (gC) were not detectable in resident peritoneal macrophages. In situ hybridization and immunofluorescence studies confirmed that transcripts and proteins for the immediate-early and some early HSV-1 genes were present. These data also established that 14% of the resident peritoneal macrophages were positive for RNA and polypeptide specific for the immediate-early gene ICP4 and that 7 to 11% were positive for RNA or polypeptides specific for the early genes tk and ICP8. The fact that only a few cells expressed viral products emphasizes the heterogeneity that exists even in this relatively homogeneous resident peritoneal macrophage population. Consistent with the Northern blot analysis, no RNA specific for the early DNA polymerase gene or the late gC gene was detected by in situ hybridization nor could the polypeptide for the gC gene be seen by immunofluorescence. Thus, while early transcriptional events were initiated in some resident peritoneal macrophages, there was a block in replication localized at the level of expression of the early to delayed-early viral genes.

Decreased resistance to Listeria Monocytogenes in mice following vanadate exposure: Effects upon the function of macrophages

We have previously demonstrated that ammonium metavanadate has a broad immuno-modulating effect in mice after subchronic exposure. Because host resistance to pathogenic Listeria monocytogenes and in vitro phagocytic activity of harvested peritoneal macrophages (PEM) were strongly affected by vanadate treatment, we investigated the effect of vandate on the functional role of resident PEM in active listeriosis in the mouse. Vanadium treatment results in altered patterns of clearance of the organism from the peritoneal cavity, liver and spleen. The total in vitro phagocytic uptake of Listeria by PEM was consistently decreased as a function of infection period. Similarly, intracellular killing of Listeria was decreased although the PEM from the vanadate-treated and control mice were more bacteriostatic than bactericidal. Population distributions of Listeria within infected PEM were not affected by host pretreatment with vanadate. Vanadate exposure interferes with both the uptake and ultimate intraphagolysosomal killing of Listeria. These results were expected in light of our previous studies of the effects of vanadate on PEM superoxide production, and hexose monophosphate shunt and glutathione redox cycle activity. The results provide additional information for the development of a mechanism to explain why workers exposed to vanadium-containing dusts have increased susceptibility to bacterial respiratory diseases.

Early and late heat-induced proteins during Leishmania mexicana transformation

During in vitro transformation of L. mexicana from promastigotes to amastigotes at 37 degrees C, a higher growing temperature, transforming parasites showed two different sets of polypeptides. One set of proteins was synthesized at the beginning of temperature-shift and corresponded to the so called "heat shock proteins" (hsp) being expressed by promastigotes mostly. The second set was expressed later on was specifically associated with the amastigotes stages and was strongly similar to the pattern of polypeptides synthesized by amastigotes from infected peritoneal macrophages. These results suggest hsp may play a function at a defined time in the transformation of the parasite.

Primaquine Is Lethal for Intracellular but Not Extracellular Trypanosoma cruzi

Primaquine has been used to treat Chagas' disease in humans and has been reported to be active against extracellular Trypanosoma cruzi. Experiments were designed to evaluate the relative activity of primaquine against extra- and intracellular T. cruzi and to determine if primaquine might be combined advantageously with ketoconazole. Primaquine at 0.5 micrograms/ml significantly inhibited T. cruzi replication in infected mouse peritoneal macrophages and also effectively treated infected L929 cells. To examine the effect of primaquine on extracellular organisms, tissue culture T. cruzi were incubated with primaquine for different periods of time and then used to infect macrophages. Incubation with 10 micrograms/ml for 14 hr but not 8 hr significantly inhibited but did not eradicate replication. Incubation of spleen amastigotes or blood trypomastigotes for 2 hr with 10 micrograms/ml did not inhibit replication. Incubation of extracellular tissue culture T. cruzi with primaquine for 2 hr did not potentiate the activity of ketoconazole against intracellular organisms. The combination of primaquine and ketoconazole administered to acutely infected mice significantly decreased parasitemias in comparison to treatment with primaquine or ketoconazole alone. Thus primaquine acts primarily on intracellular rather than extracellular T. cruzi. Primaquine and ketoconazole appear to have additive activity in vivo.

Vaccinia virus proteins on the plasma membrane of infected cells III. Infection of peritoneal macrophages

Primary macrophage cultures were prepared from the peritoneal exudate cell population harvested from mice challenged intraperitoneally with saline, thioglycollate, or vaccinia virus. Vaccinia virus was adsorbed and penetrated into primary macrophages and L-cells with similar kinetics. As evidenced by the expression of some “early” virus-specified proteins, partial uncoating and activation of the virion-associated DNA-dependent RNA polymerase occurred in the infected macrophages. Subsequently, the viral replication cycle in macrophages was aborted; with time after infection, viral DNA and virion proteins initially associated with infected cells could be detected in an acid-soluble form in the medium harvested from infected macrophage cultures. The results suggest that at the time that the final stages of virus uncoating should have occurred, intracellular subviral particles were, instead, degraded in the infected, primary macrophages. Viral DNA synthesis could not be measured in vaccinia virus-infected macrophages, no “late” virus functions were expressed, and progeny virions were not assembled. As measured by the binding of antiviral-antibody- 125I-protein A complexes to the surface of vaccinia virus-infected cells, the expression of virus-specified antigens on the surfaces of infected macrophages was significantly reduced and never exceeded that measured at 2 hr after infection on the surfaces of infected L-cells. The expression of virus-specified polypeptides with mol mass of 48–50, 45–46, 36–37, and 25 kDa on the plasma membranes of vaccinia virus-infected, thioglycollate-elicited macrophages, rendered the infected macrophages susceptible to lysis by vaccinia virus-specific cytotoxic T-cells.

Induction by specific T lymphocytes of intracellular destruction of Leishmania major in infected murine macrophages.

The following cell populations derived from lymph nodes of mice primed in vivo with living Leishmania major promastigotes were tested for their capacity to induce parasiticidal activity in L. major-infected macrophages: a L. major-primed lymph node cells, draining lymph node cells from mice primed by a subcutaneous injection of living L. major in Freund's Complete Adjuvant; b L. major-specific T blasts, i.e. blast T cells resulting from in vitro challenge of primed lymph node cells with L. major, c propagated L. major specific T blasts, i.e. blast T cells after propagation in vitro in antigen-free medium containing interleukin-2. Results indicate that cocultivation of these L. major specific lymphocyte populations with infected peritoneal exudate macrophages induced progressive destruction of intracellular L. major. This effect was antigen specific since similar populations obtained from mice primed either with ovalbumin or bovine serum albumin did not induce significant parasite killing. The various lymphocyte populations examined did not express cytolytic activity for syngeneic macrophages infected with L. major when tested in a short-term 51Cr release assay. These negative results could not be attributed to an inability of infected macrophages to be lysed by cytolytic lymphocytes since cytolytic T lymphocytes directed to H-2 alloantigens present on macrophages were perfectly capable of lysing these infected macrophages as revealed in a 4 h 51Cr release assay. Interestingly, infected macrophages from either BALB/c (H-2d), NZB (H-2d) or CBA (H-2k) mice were lysed by cytolytic T lymphocytes specific for their respective H-2 alloantigens as well as uninfected macrophages. These results suggest that H-2 expression on the surface of infected macrophages from either L. major susceptible or resistant mouse strains is sufficient to be detected by allogeneic cytolytic T lymphocytes.

Abortive ectromelia virus infection in peritoneal macrophages activated by Corynebacterium parvum.

We have previously demonstrated that peritoneal macrophages (M phi S) from C3H mice were resistant to in vitro infection by ectromelia virus, following activation by intraperitoneal injection of the immunomodulator Corynebacterium parvum. In contrast, resident and mineral oil-elicited M phi S were fully susceptible to virus infection. This report analyzes the infectious cycle of ectromelia virus in C parvum-activated and mineral oil-elicited M phi S and demonstrates that an abortive infection occurred in the activated M phi S that blocked the infectious cycle prior to the release of DNA from the infecting virions. The kinetics of adsorption of radiolabeled virus were similar in both susceptible and resistant M phi cultures; however, viral-induced incorporation of uridine and thymidine occurred only in the mineral oil-elicited and not the C parvum-activated M phi S. In addition, the late protein hemagglutinin was only detected in infected cultures of susceptible mineral oil-elicited M phi S. An electron micrographic analysis of the infectious cycle indicated that the adsorption of virus to the plasma membrane, uptake into lysosomes, and the primary undercoating and release of viral cores into the M phi cytoplasm were identical in both M phi types. In contrast, secondary uncoating (release of genomic DNA from the viral cores into the cytoplasm) was never detected in infected C parvum M phi S. These data are consistent with our previous findings and with the hypothesis that activation of M phi S by C parvum induces an interferon-mediated resistance to ectromelia virus 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.

T-Antigen and DNA Synthesis in Macrophages infected with Polyoma Virus

CELLS infected with polyoma virus may undergo a cytocidal interaction or a moderate one in which a fraction of the population is transformed. In either case a new complement fixing antigen1, known as T-antigen2, appears in the nucleus of the infected cells and cellular DNA synthesis is stimulated3–6. A requirement for DNA stimulation is that cells are not actively dividing7. It has been suggested that a correlation exists between appearance of T-antigen and stimulation of host DNA synthesis1. The use of cells which are incapable of synthesizing DNA would facilitate study of this relationship because in the available systems mitotic activity and DNA synthesis of contact-inhibited cells are only temporarily and partially repressed. The experiments reported here show that polyoma virus induces an abortive infection in peritoneal macrophages, a system in a steady state situation8, and that synthesis of T-antigen and DNA appears as two unrelated processes.

Effect of chloroquine on lysosomes and on growth of mouse hepatitis virus (MHV-3)

After a 6-hour treatment with chloroquine, infected mouse peritoneal macrophages produce markedly less mouse hepatitis virus than untreated cells. Macrophages maintained in culture for 72 hours after the treatment produce a higher virus yield. Cytochemical investigations immediately and 3 days after chloroquine treatment show an increased permeability of the lysosomes of the latter. Variations of the enzymes escaping from the lysosomes are thought to be responsible for differences in virus uncoating and consequent virus yield.