Phagocytosis of latex beads is defective in cultured human retinal pigment epithelial cells with persistent rubella virus infection.

Phagocytosis, a secondary function of retinal pigment epithelial (RPE) cells essential to sight, was significantly decreased, when measured with latex beads, during persistent rubella virus (RV) infection of human cultured RPE cells. A target for RV in vivo, RPE cells infected with RV (RPE/RV) ingested fewer fluorescent microspheres (26%) than did uninfected RPE cells (68%) (P < 0.001), as measured by flow cytometry. In RPE/RV cells, with characteristic RPE monolayer appearance and normal growth during subculturing over 6 months, persistent RV infection was shown by specific RV antigen immunofluorescence, by the presence of the RV genome in RPE/RV cell messenger RNA, and by recovery of cell-free RV after cocultivation with Vero cells. The adhesion of latex beads to apical cell surfaces of RPE/RV and uninfected RPE cells appeared similar, as imaged by scanning electron microscopy. Cytoskeletal actin, a component of phagocytosis in RPE, appeared altered in 60 to 75% of RPE/RV cells by antiactin immunofluorescence staining, as previously described in other RV-infected cells, but its role in the disturbed phagocytosis of latex beads was not determined. Persistently RV-infected human RPE is an additional example of RV-associated secondary cellular dysfunction in the absence of cytopathic effects.     

Flow cytometric analysis of immunophagocytosis using sensitized fluorescent microspheres bearing C3b

We analyzed the phagocytic activity of purified human monocytes using fluorescent latex beads sensitized with IgG or IgG.C3 by flow cytometry. To prepare IgG-sensitized latex beads (BA), BSA-coated latex beads (B) were incubated with diluted rabbit IgG anti-BSA. To bind complement components, BA were incubated with whole serum pretreated with K-76 monocarboxylic acid (K-76COOH). K-76COOH inhibits the activity of factor I and C5, resulting in deposition of C1, C4b, C2a, C3b on BA (BAC). Phagocytic activity was assessed by percent phagocytosis and phagocytic index (PI). To eliminate the effects of non-phagocytosed latex beads, subtraction of the data at 4 degrees C from 37 degrees C was performed. Percent phagocytosis for 60 min. was B 5.0%, BA 18.3%, and BAC 57.5%, and PI (ingested latex beads/100 cells) was B 7.9, BA 36.8, and BAC 152.7, respectively. In addition, K-76COOH caused dose dependent inhibition on IgG.C3 mediated phagocytosis. Comparison of inhibition pattern on BAC and BA indicated that K-76COOH directly inhibited C3.C3-receptor binding.     

Acid phosphatase localization in normal and dystrophic retinal pigment epithelium

Summary In this study acid phosphatase (ACPase) was localized in the retinal pigment epithelium (RPE) of normal and Royal College of Surgeons (RCS) rats pink-eyed and pigmented with inherited retinal dystrophy to determine differences in staining during the post-engulfment stages of phagocytosis using two substrates, Na--glycerophosphate and cytidine-5-monophosphate. Staining was similar using either substrate and in the normal RPE the Golgi system, lysosomes and phagosomes were ACPase-positive. In the dystrophic RPE, which has a diminished capacity to phagocytose photoreceptor rod outer segments, ACPase staining was localized on melanosomes in the pigmented dystrophic and on the apical microvillous membranes in the pink-eyed dystrophic, but was not localized on phagosomes in either the pink-eyed or pigmented dystrophic RPE. Since only a few phagosomes were seen at any given time in dystrophic RPE in vivo, a tissue expiant system was used to examine the number of latex beads phagocytosed by normal and RCS RPE, as well as the number of phagosomes containing both beads and ACPase activity in the normal and mutant RPE. Our findings indicate that in the dystrophic, fewer phagosomes are ACPase-positive than in the normal, and that some enzyme may be inappropriately shunted to either the apical microvilli or to melanosomes instead of to phagolysosomes.     

Impaired macrophage phagocytosis of apoptotic neutrophils in patients with human immunodeficiency virus type 1 infection

Dysfunction of neutrophils (polymorphonuclear leukocytes [PMNL]) and macrophagic cells occurs as a consequence of human immunodeficiency virus type 1 (HIV-1) infection. Macrophages contribute to the resolution of early inflammation ingesting PMNL apoptotic bodies. This study investigated macrophage ability to phagocytose PMNL apoptotic bodies in patients with HIV-1 infection in comparison with uninfected individuals and the effect of HIV Nef protein on apoptotic body phagocytosis to determine if phagocytic activity is impaired by HIV infection. Monocytes/macrophages were isolated from 10 HIV-1-infected patients and from five healthy volunteers, whereas PMNL were isolated from healthy volunteers. Macrophage phagocytosis of apoptotic PMNL was determined by staining of apoptotic bodies with fluorescein-conjugated concanavalin A or with fluorescein-labeled phalloidin. Our data show significant impairment of PMNL apoptotic body macrophage phagocytosis in subjects with HIV-1 infection presenting a concentration of CD4(+) T lymphocytes of >200/mm(3) and in particular in those with <200 CD4(+) T lymphocyte cells/mm(3). In addition, HIV-1 recombinant Nef protein is able to decrease phagocytosis of apoptotic PMNL from normal human macrophages in a dose-dependent manner. The results of our study suggest that impaired macrophage phagocytosis of PMNL apoptotic bodies may contribute to the persistence of the inflammatory state in HIV-infected subjects, especially during opportunistic infections that are often favored by defective phagocytic activity.     

Coxsackie B1 virus infection enhances the bacterial invasiveness, the phagocytosis and the membrane permeability in HEp-2 cells

To analyze the effect of coxsackie B1 virus infection on bacterial invasiveness, phagocytosis and cytoplasma membrane permeability, we have studied invasiveness of Shigella flexneri, unspecific phagocytosis of latex particles and release of the non-metabolizible amino acid, alpha-aminoisobutyric acid (AIB). Virus infection enhanced invasiveness of S. flexneri and phagocytosis of latex beads and increased plasma membrane permeability as measured by release of AIB. The effect on all three functions increased with virus concentration, but the kinetics were different. During the early phase of virus infection there was no difference between the effect on invasiveness, phagocytosis and permeability in cell cultures pretreated with viable or with UV-inactivated virus. However, after 6 h, 5 h and 2 h respectively, there was an increased response in cell cultures pretreated with viable virus compared to cells inoculated with UV-inactivated virus. The results indicate that the virus effect on bacterial invasiveness is a function of several parameters, including phagocytosis and membrane function changes.     

Interleukin-12 secretion by Mycobacterium tuberculosis-infected macrophages.

Infection with Mycobacterium tuberculosis or phagocytosis of large latex beads induced interleukin-12 (IL-12) production in macrophages. In contrast, tumor necrosis factor (TNF) was produced only in response to M. tuberculosis infection, not after phagocytosis of latex beads. Comparable results were obtained with cells from immunocompetent C57BL/6 and gamma interferon receptor-deficient mutant mice. Thus, phagocytosis by mechanisms not specific for M. tuberculosis was a sufficient trigger for IL-12 secretion, emphasizing the central role of this cytokine in the initiation of anti-infective immunity.     

Enhanced luminol-dependent chemiluminescence of stimulated rat alveolar macrophages by pretreatment with alveolar lining material

Previous reports indicate that the in vitro bactericidal activity of rat alveolar macrophages (AM) is dependent on the lipid fraction (ALM-L) of the alveolar lining material (ALM). The present study demonstrates that luminol-dependent chemiluminescence of stimulated rat AM is increased when rat AM are preincubated in the ALM or in the ALM-L. Evidence is presented that oxidation of the unsaturated lipids is responsible for the increase. In addition to pretreatment with the ALM, cells were also pretreated in commercial preparations of several lipids found to be present in the ALM. Preincubation in these lipids produced a significant increase in the luminol-dependent chemiluminescence response. However, when a saturated lipid, dipalmitoyl phosphatidylcholine, was used no increase was found. Pretreatment in ALM did not increase the nitro blue tetrazolium dye reduction by the AM, nor was the phagocytosis of latex beads by the AM altered by the addition of the ALM.     

Rhinovirus replication causes RANTES production in primary bronchial epithelial cells.

The mechanisms by which rhinovirus (RV) infections produce lower airway symptoms in asthmatic individuals are not fully established. To determine effects of RV infection on lung epithelial cells, primary human bronchial epithelial (BE) cells were infected with either RV16 or RV49, and viral replication, cell viability, and cell activation were measured. Both viral serotypes replicated in BE cells at 33 degrees C (DeltaTCID50 / ml = 2 to 2.5 log units) and at 37 degrees C (DeltaTCID50 /ml = 1.6 log units), but only high doses of RV49 (10(6) TCID50 /ml) caused cytopathic effects and reduced cell viability. In addition, regulated on activation, normal T cells expressed and secreted (RANTES) secretion was increased in epithelial cells infected with RV16 or RV49 (243 and 398 pg/ml versus 13 pg/ml uninfected control cells), and a similar pattern was seen for RANTES messenger RNA. RV infection also caused increased secretion of interleukin-8 and granulocyte macrophage colony-stimulating factor, but did not alter expression of either intercellular adhesion molecule-1 or human leukocyte-associated antigen-DR. These observations suggest that RVs can replicate in lower airway cells in vivo, and support epidemiologic studies that link RV with lower respiratory illnesses. Further, RV-induced secretion of RANTES and other cytokines could trigger antiviral immune responses in vivo, but these effects could also contribute to the pathogenesis of respiratory symptoms in subjects with asthma.     

The role of Fc and C3b receptors in phagocytosis by inflammatory polymorphonuclear leucocytes in man.

Polymorphonuclear leucocytes from the gingival crevice (CREV-PMN) in man have a defective capacity to phagocytose Candida albicans blastospores. Phagocytosis of zymosan particles, which detect C3b receptors, is also impaired but ingestion of latex beads coated with heat-aggregated IgG, which detects Fc receptors, is normal compared to peripheral blood polymorphonuclear leucocytes (PB-PMN). If phagocytosis is inhibited by Cytochalasin B, fewer CREV-PMN bind Candida and zymosan but the binding of IgG-coated latex beads remains unchanged. CREV-PMN have IgG (88%), IgM (45%) and C3 (48%) on their cell membrane, whilst less than 5% of PB-PMN have any of these components. Incubation of PB-PMN in fluid from the gingival crevice confers surface IgG and C3 to the cells. Such treatment also inhibits the subsequent binding of IgG coated latex beads. The results suggest that the deficiency of phagocytosis by CREV-PMN is due to decreased binding of particles to the C3b receptor of PMN, whilst the Fc receptor system remains intact.     

Impaired Macrophage Phagocytosis of Apoptotic Neutrophils in Patients with Human Immunodeficiency Virus Type 1 Infection

Dysfunction of neutrophils (polymorphonuclear leukocytes [PMNL]) and macrophagic cells occurs as a consequence of human immunodeficiency virus type 1 (HIV-1) infection. Macrophages contribute to the resolution of early inflammation ingesting PMNL apoptotic bodies. This study investigated macrophage ability to phagocytose PMNL apoptotic bodies in patients with HIV-1 infection in comparison with uninfected individuals and the effect of HIV Nef protein on apoptotic body phagocytosis to determine if phagocytic activity is impaired by HIV infection. Monocytes/macrophages were isolated from 10 HIV-1-infected patients and from five healthy volunteers, whereas PMNL were isolated from healthy volunteers. Macrophage phagocytosis of apoptotic PMNL was determined by staining of apoptotic bodies with fluorescein-conjugated concanavalin A or with fluorescein-labeled phalloidin. Our data show significant impairment of PMNL apoptotic body macrophage phagocytosis in subjects with HIV-1 infection presenting a concentration of CD4⁺ T lymphocytes of >200/mm³ and in particular in those with <200 CD4⁺ T lymphocyte cells/mm³. In addition, HIV-1 recombinant Nef protein is able to decrease phagocytosis of apoptotic PMNL from normal human macrophages in a dose-dependent manner. The results of our study suggest that impaired macrophage phagocytosis of PMNL apoptotic bodies may contribute to the persistence of the inflammatory state in HIV-infected subjects, especially during opportunistic infections that are often favored by defective phagocytic activity.     

In vitro study of the phagocytic processes in splenic granulocytes of the tench (Tinca tinca, L.).

The different stages of the phagocytic process by splenic granulocytes of Tinca tinca were studied. Adherence capacity to both endothelium and tissue substrate, mobility rate, the phagocytosis capacity for both cells (Candida albicans) and inert particles (latex beads), candidicide power, and capacity of digestion measured by nitroblue tetrazolium (NBT) reduction were evaluated in splenic granulocytes of healthy adult tench. The capacity of adherence to nylon fiber was possessed by 51% of the granulocytes. The percentage capable of adherence to smooth plastic surfaces rose with incubation time. Casein, an effective chemoattractant, increased the random mobility of the granulocytes. Phagocytosis was greater for opsonized C. albicans than for nonopsonized. However, the number of phagocytosed yeast cells destroyed by the granulocytes did not depend on whether or not the C. albicans had been previously opsonized. The phagocytosis indices and the percent phagocytosis of latex beads were greater than those obtained for the phagocytosis of C. albicans in the absence of serum. Finally, the metabolic activity in these cells following the digestion of ingested material showed a 148 ± 31% stimulation. The results show that splenic cells of tench have the capacity to make a phagocytic response against both cells (C. albicans) and inert particles (latex beads).     

Phagocytosis of Leishmania mexicana amastigotes by macrophages leads to a sustained suppression of IL-12 production

Healing of leishmaniases is dependent on activation of parasitized macrophages (Mϕ) by IFN-γ, which is secreted by Leishmania-specific Th1 cells. IL-12 enhances IFN-γ production by Th1 cells and is crucial for cure. The host cells of Leishmania sp., Mϕ, are a main source of IL-12 in vivo. We report that infection of quiescent murine Mϕ with L. mexicana or L. major amastigotes does not induce IL-12 production. Moreover, infection suppresses IL-12 secretion by Mϕ activated by LPS, by CD40 cross-linking or cognate interaction with Th1 cells. IL-12 secretion is also suppressed in Mϕ activated after phagocytosis of latex beads. Suppression is independent of engagement of CR3 or FcγR during phagocytosis, is not mediated by IL-10 and does not alter steady state IL-12p40 mRNA levels. In addition, suppression of IL-12 secretion does not depend on Mϕ activation concurrent to infection. In contrast, NO production was not inhibited. Thus, Mϕ effector functions are differentially affected and this may be a general effect of phagocytosis of non-activating particles. The possible implications of this effect on the infection are discussed.     

Effect of environmental particulates on cultured human and bovine endothelium. Cellular injury via an oxidant-dependent pathway

The effects of respirable environmental fibers on cultures of human umbilical vein and bovine pulmonary artery endothelial cell monolayers were studied. Interaction among endothelial cell monolayers and amosite and chrysotile asbestos, attapulgite, fiberglass, or latex beads resulted in rapid phagocytosis of the particulates. A gradient of time-dependent and concentration-dependent endothelial cell injury (measured by specific 51Cr release) was observed with amosite and attapulgite being markedly toxic. Chrysotile and fiberglass were much less toxic, and latex beads were not significantly injurious at any time or dose examined. Responses of bovine pulmonary artery and human endothelial vein endothelial cells to fiber phagocytosis and fiber-induced injury were similar. In human umbilical cell monolayers, fiber-mediated stimulation of the arachidonate metabolite prostacyclin paralleled endothelial cell injury; i.e. amosite and attapulgite were stimulatory, whereas fiberglass (0-500 micrograms/ml) and latex beads (10(9) beads/ml) did not significantly increase prostacyclin generation. Although chrysotile was only weakly cytotoxic, significant stimulation of prostacyclin was observed at the highest dose tested (500 micrograms/ml). To investigate whether toxic oxygen species may be involved in fiber-induced cytotoxicity, oxidant scavengers or inhibitors were used in injury studies. Both superoxide dismutase (a scavenger of O2-) and catalase (an inhibitor of H2O2) produced significant protection against fiber-mediated endothelial cell injury. In addition, chelation by deferoxamine of elemental Fe present in the fiber preparations was also protective, suggesting Fe, via the modified Haber-Weiss reaction, may promote hydroxyl radical formation and contribute to endothelial cell injury induced by these particulates. These results suggest that the interaction within the interstitial environment between endothelial cells and occupationally relevant dusts may be important in fiber-mediated inflammatory processes in the lung.     

Streptococcus suis capsular polysaccharide inhibits phagocytosis through destabilization of lipid microdomains and prevents lactosylceramide-dependent recognition

Streptococcus suis type 2 is a major swine pathogen and a zoonotic agent, causing meningitis in both swine and humans. S. suis infects the host through the respiratory route, reaches the bloodstream, and persists until breaching into the central nervous system. The capsular polysaccharide (CPS) of S. suis type 2 is considered a key virulence factor of the bacteria. Though CPS allows S. suis to adhere to the membrane of cells of the immune system, it provides protection against phagocytosis. In fact, nonencapsulated mutants are easily internalized and killed by macrophages and dendritic cells. The objective of this work was to study the molecular mechanisms by which the CPS of S. suis prevents phagocytosis. By using latex beads covalently linked with purified CPS, it was shown that CPS itself was sufficient to inhibit entry of both latex beads and bystander fluorescent beads into macrophages. Upon contact with macrophages, encapsulated S. suis was shown to destabilize lipid microdomains at the cell surface, to block nitric oxide (NO) production during infection, and to prevent lactosylceramide accumulation at the phagocytic cup during infection. In contrast, the nonencapsulated mutant was easily internalized via lipid rafts, in a filipin-sensitive manner, leading to lactosylceramide recruitment and strong NO production. This is the first report to identify a role for CPS in lipid microdomain stability and to recognize an interaction between S. suis and lactosylceramide in phagocytes.     

Stimulatory effect of latex and zymosan particles on cyclic adenosine 3',5'-monophosphate content in human granulocytes

Human polymorphonuclear leukocytes respond to latex beads and opsonized zymosan particles with increased cyclic AMP formation. The enhancement of cyclic AMP content in cells is related to particle concentration in the incubation medium and to the time of exposure. The temporary stimulation declines after 15 min of phagocytosis, that is in good relation to the uptake of latex particles which reaches saturation point 10 min after the addition of the beads. The cyclic AMP content in granulocytes decreases after between 15 and 30 min of incubation with latex particles. The decrease is not caused by the loss of the granulocyte viability for: (a) the incorporation of labeled methyldeoxythymidine and uridine into macromolecules continues, (b) the sensitivity of phagocytosing granulocytes to prostaglandin E₁ stimulation of cyclic AMP production persists, (c) less than 10% of cells absorbs trypan blue dye after phagocytosis of latex beads. The prostaglandin synthesis inhibitors, aspirin and indomethacin, have no effect on cyclic AMP content in phagocytosing granulocytes.     

Establishment of a Phagocytic Cell Line from Bombina orientalis

Continuous serum-free culture of Bok-2 cells was generated from primary culture of the tail bud stage embryos of the toad, Bombina orientalis. Bok-2 cells can be maintained in modified L-15 serum-free medium prepared by mixing L-15 medium, lactalbumin enzymatic hydrolysate, sucrose and sodium bicarbonate. Bok-2 cells have an ameboid behavior and morphology. When Bok-2 and viable Candida albicans were co-cultured, Bok-2 cells showed an immune response characterized by chemotaxis, phagocytosis and partial clearing activity of Candida cells and colonies. And Bok-2 cells also showed phagocytosis of latex beads without serum treatment and displayed numerous pseudopodia, membrane evagination for phagocytosis and phagosome activity. On the basis of these results, Bok-2 cells were identified as professional phagocytes.     

Functional assays for evaluation of serogroup B meningococcal structures as mediators of human opsonophagocytosis

Functional flow cytometry and chemiluminescence (CL) assays have been modified to identify serogroup B meningococcal structures that mediate anti-meningococcal opsonophagocytosis. Serogroup B meningococcal outer membrane vesicles (OMV) were adsorbed to fluorescent latex beads (OMV-beads) and opsonized with acute phase and convalescence sera from patients with serogroup B meningococcal disease. Phagocytosis of these beads by human monocytes and polymorphonuclear leukocytes (non-lymphocytes) was dependent on both antigen exposure on the bead surface and on serum opsonization. OMV-beads opsonized with serum from a patient recovering from meningococcal disease, caused 97% of the non-lymphocytes to phagocytose an average of 15.8 beads per cell with a CL response of 46550 mVs, whereas opsonized control beads were phagocytosed by 19% of the non-lymphocytes with 1.1 beads per cell and a CL response of 53 mVs. Increased amounts of functional, anti-OMV opsonins were detected during infection, and opsonized OMV-beads elicited phagocyte responses of similar magnitude to those of opsonized whole meningococci. Phagocyte internalization of OMV-beads was confirmed by confocal laser scanning microscopy. We conclude that epitopes on the meningococcal outer membrane are recognized by anti-meningococcal opsonins in these functional phagocytosis assays, which provide a basis for subsequent evaluation of various purified bacterial components as mediators of human opsonophagocytic responses and hence future vaccine constituents.     

Asbestos-induced alteration of human peripheral blood monocyte activity

Incubation of chrysotile and anthophyllite asbestos fibers with normal human peripheral blood monocytes resulted in significant suppression of monocyte metabolic activity as measured by chemiluminescence. Both fiber types were cytotoxic to monocytes and depressed monocyte phagocytosis of latex beads. We conclude that asbestos-induced monocyte cytotoxicity could result in release of lysosomal enzymes and/or degradation products which contribute to fibrosis in asbestosis. The depression of phagocytosis and microbicidal function may contribute to the increased incidence of carcinogenesis observed in asbestosis.     

Phagocytosis by hemolymph cells of the land slug, Incilaria fruhstorferi Collinge (Gastropoda: Pulmonata)

Phagocytosis by hemolymph cells of the land slug, Incilaria fruhstorferi Collinge, were studied with the scanning electron microscopy (SEM) and the transmission electron microscopy (TEM). The fate of foreign materials, i.e., sheep red blood cells (SRBC) and latex beads, introduced into the hemocoel of the slug were followed. Certain hemocytes named as Type I cell were involved in spontaneous cyto-adherence while both Type II and III cells were not observed to adhere to foreign materials. SRBC and latex beads (luminal diameter 0.79 micron) were phagocytosed and latex beads (luminal diameter 15.8 micron) were engulfed by Type I cells. In vitro phagocytosis experiments showed that SRBC formed the rosette structure surrounding a Type I cell and both SRBC and latex beads (luminal diameter 0.79 micron) were swallowed by Type I cells in vivo. The plate-like structures with long fine fibers attached the latex beads. The latex beads were often seen deep in the aggregates of plate-like structures.