In vivo opsonization of Japanese encephalitis virus by peritoneal macrophages in infant mice

Anti-JE antibody in nonneutralizable concentration and Concanavalin A are synergistic in protecting 10-day-old mice from lethal JE virus challenge by i.p. route.     

In vivo particle uptake by airway macrophages in healthy volunteers

We combined two techniques, radiolabeled aerosol inhalation delivery and induced sputum, to examine in vivo the time course of particle uptake by airway macrophages in 10 healthy volunteers. On three separate visits, induced sputum was obtained 40, 100, and 160 min after inhalation of radiolabeled sulfur colloid (SC) aerosol (Tc99 m-SC, 0.2 microm colloid size delivered in 6-microm droplets). On a fourth visit (control) with no SC inhalation, induced sputum was obtained and SC particles were incubated (37 degrees C) in vitro with sputum cells for 40, 100, and 160 min (matching the times associated with in vivo sampling). Total and differential cell counts were recorded for each sputum sample. Compared with 40 min (6 +/- 3%), uptake in vivo was significantly elevated at 100 (31 +/- 5%) and 160 min (27 +/- 4%); both were strongly associated with the number of airway macrophages (R = 0.8 and 0.7, respectively); and the number and proportion of macrophages at 40 min were significantly (P < 0.05) elevated compared with control (1,248 +/- 256 versus 555 +/- 114 cells/mg; 76 +/- 6% versus 60 +/- 5%). Uptake in vitro increased in a linear fashion over time and was maximal at 160 min (40 min, 12 +/- 2%; 100 min, 16 +/- 4%; 160 min, 24 +/- 6%). These data suggest that airway surface macrophages in healthy subjects rapidly engulf insoluble particles. Further, macrophage recruitment and phagocytosis-modifying agents are factors in vivo that likely affect particle uptake and its time course.     

In vivo Junin virus-mouse macrophages interaction

The role of mononuclear phagocytic cells in extraneural infection of the mouse with Junin virus (JV) was studied. Endpoint susceptibility (4 days of life) was evaluated by intraperitoneal (i.p.) inoculation of suckling mice. By means of immunofluorescence (IF) and C3 receptor assays, it was found that macrophages were permissive to viral replication in vivo and fostered the recruitment of inflammatory cells as evidenced by the absence of C3 marker. In support, in vitro infection failed to induce alterations of this receptor. Throughout, both in vivo and in vitro, there were no signs of C3-mediated phagocytosis. Silica treatment had no effect on either resistance or susceptibility, suggesting that the "macrophage-barrier" failed to hinder or favour the course of disease. Differences with other JV models are discussed.     

In vivo labeling of resident peritoneal macrophages

A novel method for labeling resident peritoneal macrophages (M phi) by injection of a dye into the peritoneal cavity is described. The dye, which fluoresces green, is selectively taken up by the resident M phi. Dye labeled cells can be further characterized by labeling of cell surface antigens with monoclonal antibodies (Mabs) and phycoerythrin conjugated second antibody. After such labeling with the Mabs F4/80 or Mac 1 the resident M phi were labeled by both the green dye and the red Mab markers, while recruited M phi or neutrophils were labeled with just the red Mab; the two populations of cells were readily distinguished by two-color flow cytometry. This technique enabled identification of resident and recruited M phi in each animal without the use of radioisotopes, irradiation, or bone marrow ablation. Sufficient numbers of cells can be analyzed from each animal so that individual animals could be evaluated. We found no adverse effects of this labeling technique on expression of cell surface antigens or M phi mediated cytotoxicity. We did find evidence that the i.p. injection induced a mild inflammation in the peritoneal cavities of animals injected with either the dye or the balanced salt solution vehicle. Examination of the intracellular staining pattern indicated that the label rapidly sequestered in the cytoplasm of the M phi, possibly in the lysosomes. Dye solubility studies showed that the dye was partially soluble at the concentration used for in vivo labeling. We hypothesize that the M phi labeling occurred by a combination of phagocytosis of dye aggregates and endocytosis of labeled plasma membrane.     

Repopulation of macrophages in popliteal lymph nodes of mice after liposome-mediated depletion

Macrophages lining the subcapsular sinus (SCS) and those located in the medulla of popliteal lymph nodes (PLN) of mice were eliminated after subcutaneous (s.c.) injection of dichloromethylene diphosphonate (Cl2MDP)-containing liposomes. No effect of liposome-entrapped Cl2MDP could be seen on nonphagocytic cells, e.g., interdigitating cells (IDC) and B- and T-lymphocytes. One month after injection the eliminated subsets of macrophages were still absent. After 2 mo a small number of macrophages had reappeared along the SCS and in the medulla of the PLN of a few animals. Complete repopulation of the PLN with macrophages was observed only after 5 mo. This extremely long repopulation time could be shortened drastically by local administration of Freund's complete adjuvant (FCA). A small number of macrophages reappeared along the SCS and in the medulla 1 wk after FCA and after 3 wk the repopulation of the PLN with macrophages was complete. Such rapid repopulation of macrophages was not achieved after s.c. injection of paraffin oil or paratyphoid vaccine. These results indicate that the normal rate of influx of mononuclear phagocytes into the PLN is low, but that it can be sped up after administration of FCA.     

Antigen uptake in vivo by peritoneal macrophages from mice exhibiting antigenic competition

The in vivo uptake of human serum albumin and ferritin by peritoneal macrophages has been studied. Peritoneal macrophages were obtained from normal mice, mice undergoing primary and secondary responses and mice exhibiting serological evidence of antigenic competition. The antigens were conjugated with two different fluorochromes and traced by fluorescence microscopy. Both antigens were ingested concurrently by the macrophages regardless of the immunological state of the mice.     

In vivo dendritic cell depletion reduces breeding efficiency, affecting implantation and early placental development in mice

Implantation of mammalian embryos into their mother's uterus ensures optimal nourishment and protection throughout development. Complex molecular interactions characterize the implantation process, and an optimal synchronization of the components of this embryo-maternal dialogue is crucial for a successful reproductive outcome. In the present study, we investigated the role of dendritic cells (DC) during implantation process using a transgenic mouse system (DTRtg) that allows transient depletion of CD11c(+) cells in vivo through administration of diphtheria toxin. We observed that DC depletion impairs the implantation process, resulting in a reduced breeding efficiency. Furthermore, the maturity of uterine natural killer cells at dendritic cell knockout (DCKO) implantation sites was affected as well; as demonstrated by decreased perforin expression and reduced numbers of periodic-acid-Schiff (PAS)-positive cells. This was accompanied by disarrangements in decidual vascular development. In the present study, we were also able to identify a novel DC-dependent protein, phosphatidylinositol transfer protein beta (PITPbeta), involved in implantation and trophoblast development using a proteomic approach. Indeed, DCKO mice exhibited substantial anomalies in placental development, including hypocellularity of the spongiotrophoblast and labyrinthine layers and reduced numbers of trophoblast giant cells. Giant cells also down-regulated their expression of two characteristic markers of trophoblast differentiation, placental lactogen 1 and proliferin. In view of these findings, dendritic cells emerge as possible modulators in the orchestration of events leading to the establishment and maintenance of pregnancy.     

In vivo activation of murine peritoneal macrophages by intraperitoneal administration of cisplatin

A single intraperitoneal injection of cisplatin (10 mg/kg body weight) in C3H/He mice increases the total number of peritoneal exudate cells (PEC) and macrophages (m phi) within 24 to 48 h. The total number of PEC from untreated mice ranged from 4 to 5 x 10(6) cells/ml containing 2.5 to 3 x 10(6) macrophages, whereas in cisplatin treated mice total number of PEC ranged up to 25 x 10(6) cells/ml. These PEC contained up to 16 x 10(6) m phi. The macrophages obtained from cisplatin injected mice show enhanced cytotoxicity, cytostasis and binding to Dalton's lymphoma cells in vitro. These activated macrophages release into the culture medium factors having cytolytic and cytostatic effect on Dalton's lymphoma cells. The activated macrophages also show enhanced capacity to release superoxide anions, hydrogen peroxide, lysozyme, arginase and interleukin-1.     

In vivo hyporesponsiveness induced by Sendai virus in CFLP mice

Sendai and Semliki Forest viruses (SFV) raised the interferon (IFN) level in blood and suppressed the acute inflammatory response induced by carrageenan in CFLP mice. After Sendai virus had been inoculated, unresponsiveness developed to repeated challenge either with the same virus or with SFV. The hyporeactive state culminated 48 hr after first virus inoculation. It was characterized (1) by absence of IFN induction and (2) by disappearance of the virus-induced anti-inflammatory effect. In contrast, the anti-inflammatory effect of indomethacin and dexamethasone remained unchanged. In addition, peripheral white blood cells were counted upon Sendai virus inoculation either in normal or in hyporesponsive mice. Six hr after inoculation, Sendai virus induced a marked granulocytosis with lymphopenia. In hyporesponsive mice leukocytosis was observed. Repeated Sendai virus injection was followed by a less pronounced granulocytosis, while the decreased number of mononuclear cells remained unchanged. These alterations in mice inoculated with Sendai virus offers a model of hyporesponsiveness established in vivo.     

In vivo depletion of natural killer cell activity leads to enhanced multiplication of Mycobacterium avium complex in mice.

Earlier investigations have shown that murine natural killer (NK) cells inhibit the growth of fungal and bacterial pathogens in vivo and in vitro. In order to define the role of NK cells in Mycobacterium avium complex infection, in vivo depletion of NK cells by using anti-NK1.1 monoclonal antibody and conventional anti-asialo-GM1 antibody has been attempted. Repeated injection of 200 micrograms of anti-NK1.1 or 50 micrograms of anti-asialo-GM1 antibody effectively depleted NK activity in the spleens of C57BL/6 mice. The growth kinetics of M. avium complex over a period of 4 weeks showed that the colony counts in the spleens of the antibody-treated group were significantly (P less than 0.01) higher than those of the control group and compared well with those of the genetically NK cell-deficient C57BL/6 bg/bg mutant. The alternate strategy of in vivo stimulation of NK activity by poly(I:C) administration did not show a similar reduction in CFU in the spleen compared with the untreated control. The in vivo antibody depletion of NK activity provides direct evidence on the role of NK cells in the control of intracellular mycobacterial pathogens such as M. avium complex.     

In vivo targeting of alveolar macrophages via RAFT-based glycopolymers

Targeting cell populations via endogenous carbohydrate receptors is an appealing approach for drug delivery. However, to be effective, this strategy requires the production of high affinity carbohydrate ligands capable of engaging with specific cell-surface lectins. To develop materials that exhibit high affinity towards these receptors, we synthesized glycopolymers displaying pendent carbohydrate moieties from carbohydrate-functionalized monomer precursors via reversible addition-fragmentation chain transfer (RAFT) polymerization. These glycopolymers were fluorescently labeled and used to determine macrophage-specific targeting both in?vitro and in?vivo. Mannose- and N-acetylglucosamine-containing glycopolymers were shown to specifically target mouse bone marrow-derived macrophages (BMDMs) in?vitro in a dose-dependent manner as compared to a galactose-containing glycopolymer (30- and 19-fold higher uptake, respectively). In addition, upon macrophage differentiation, the mannose glycopolymer exhibited enhanced uptake in M2-polarized macrophages, an anti-inflammatory macrophage phenotype prevalent in injured tissue. This carbohydrate-specific uptake was retained in?vivo, as alveolar macrophages demonstrated 6-fold higher internalization of mannose glycopolymer, as compared to galactose, following intratracheal administration in mice. We have shown the successful synthesis of a class of functional RAFT glycopolymers capable of macrophage-type specific uptake both in?vitro and in?vivo, with significant implications for the design of future targeted drug delivery systems.     

In vivo arming of cutaneous mast cell receptors by IgE released from macrophages

Intracutaneous injection of purified peritoneal macrophages harvested from ovalbumin (OVA)-hypersensitive high-IgE-responder BN rats into naive animals sensitised the injection sites for subsequent OVA-specific passive cutaneous anaphylaxis (PCA) reactions. The underlying mechanism(s) were investigated using a macrophage cell line (WEHI 265.1), which exhibited comparable sensitising activity in rat or mouse skin, after initial pulsing in vitro with antiserum rich in OVA-specific IgE. Transfer of OVA-hypersensitivity by the cell line (1) was IgE-dependent and did not occur when the cells were pre-exposed to antiserum containing OVA-specific IgG alone, (2) was blockable by saturation of cell surface receptors in the recipient with myeloma IgE (but not myeloma IgG), and (3) did not occur in mast cell-deficient mice carrying the W/Wv mutation, in contrast to their normal heterozygous littermates which developed marked OVA-hypersensitivity at the injection site. These results are consistent with arming of IgE-receptors on cutaneous mast cells by IgE antibody released from macrophages, and hint at a possible role for phagocytes in amplifying IgE-mediated reactions in tissues.     

In vivo production of heat shock protein in mouse peritoneal macrophages by administration of lipopolysaccharide.

The in vivo production of heat shock protein was studied by administration of bacterial lipopolysaccharide (LPS) into mice. Heat shock protein 70 was detected in the extract of adherent peritoneal cells from mice injected intraperitoneally with LPS by using the immunoblotting method. The expression of heat shock protein 70 was found 2 days after injection of LPS and reached its peak 4 days after injection. The intraperitoneal injection of LPS induced the expression of heat shock protein 70, whereas its subcutaneous injection did not. The in vivo production of heat shock protein 70 was inhibited by administration of LPS together with quercetin, an inhibitor of accumulation of heat shock protein 70 mRNA. Tumor necrosis factor alpha enhanced LPS-induced heat shock protein production in vivo. There was a decrease of gamma delta T cells in the peritoneal cavity of mice injected intraperitoneally with LPS. It was suggested that bacterial LPS is a stressful agent which induces the in vivo heat shock protein response, and its administration leads to the production of heat shock protein 70 in peritoneal macrophages.     

In vivo efficacy of albendazole againstGiardia duodenalis in mice

The antigiardial effects of albendazole were demonstrated in vivo using experimental infections ofGiardia duodenalis in mice. These results complement previous in vivo studies in which albendazole was shown to have more potent antigiardial action than the currently applied antigiardial drugs. In mice, 2–4 doses (>100 mg/kg twice daily) were required for complete inhibition of cyst excretion and full elimination of trophozoites from the small intestine. The high doses necessary in mice were not unexpected and are discussed in light of the possible pharmacokinetics of albendazole in the animal model used in this study.Dedicated to Prof. Dr. J. Eckert (Zürich) on the occasion of his 60th birthday